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Endolysosomal effectors and their relevance for antiviral activity against the Hepatitis E virus
(2021)
Mit über 20 Millionen registrierter Fälle pro Jahr, repräsentiert das Hepatitis-E-Virus (HEV) eine Hauptursache einer viralen Hepatitis weltweit und stellt ein erhebliches Risiko insbesondere für Schwangere und Immunsupprimierte dar. Jedoch sind Behandlungsoptionen stark limitiert und mit teils schweren Nebenwirkungen verbunden. Neue Erkenntnisse des Wechselspiels zwischen Wirtszelle und HEV werden deshalb benötigt, um neue antivirale Wirkstoffe zu entwickeln. Der Fokus der Arbeit wurde hierbei auf Effektoren des endosomalen Systems gesetzt, welches von HEV zur Freisetzung von Virionen genutzt wird.
Eine virale Infektion führt in der Zelle zur Produktion von Interferonen (IFNs) und weiters zu einer IFN-Antwort. Ein essenzielles Effektormolekül, welches HEV nachweislich effizient repressiert, ist die GTPase guanylate binding protein 1 (GBP1). In dieser Studie wurde beleuchtet, dass Letztere durch eine HEV-Infektion induziert wird. Zusätzlich reduziert die ektopische Expression von GBP1 sowohl die intrazelluläre Menge des HEV Kapsidproteins als auch die Menge freigesetzter Virionen. Mechanistisch liegt diesem Sachverhalt die GBP1-induzierte Inkorporation von Virionen in Lysosomen zugrunde, was schlussendlich deren Abbau nach sich zieht. Erkenntnisse über die Rolle verschiedener GBP1 Proteindomänen innerhalb des Mechanismus wurden unter Verwendung ektopischer Expression von GBP1-Mutanten erlangt. Inkorporation der Mutation R48A führt zum Verlust der GTPase-Aktivität. Andererseits führt eine Inkorporation der Mutation S73A zum Verlust der Homodimerisierung, was die nachfolgende Farnesylierung und gekoppelte Membranassoziation reduziert. Hierbei behält GBP1-R48A Fähigkeiten zur Induktion lysosomalen Abbaus von HEV bei, GBP1-S73A jedoch nicht. Dies wiederum bedeutet, dass eine GBP1 Homodimerisierung notwendig für den antiviralen Mechanismus ist, was eine Adapterfunktion des Moleküls für lysosomale Inkorporation nahelegt. Die Relevanz von GBP1 während einer IFNγ-Antwort wurde deshalb mittels siRNA-basiertem Silencing untersucht. Ähnlich der ektopischen Expression von GBP1 induziert IFNγ die lysosomale Degradation von HEV. In Abwesenheit von GBP1 jedoch, ist dieser Effekt signifikant geringer ausgeprägt, was zu einem Effizienzverlust von IFNy in Bezug auf dessen antiviralen Effekt bedeutet. Dies führte schlussendlich zur Identifizierung von GBP1 als essenziellen Restriktionsfaktor gegen HEV, was seine Rolle in Abhängigkeit seiner Homodimerisierung via Induktion lysosomalen Abbaus erfüllt.
Nebst der Induktion von GBP1, konnte eine Akkumulation von Cholesterin in Lysosomen durch IFNy nachgewiesen werden. Da dieses Lipid einen essenziellen Faktor für endosomale Reifung, Transport und Funktionalität darstellt, wurden Cholesterinspiegel und verbundene transkriptionelle Fußabdrücke im Kontext einer HEV Infektion untersucht. Letztere führt zu einer Dysregulation Cholesterin-assoziierter Genexpression, was eine Reduktion intrazellulären Cholesterins nach sich zieht. Auch in HEV infizierten Patienten liegt eine Abnahme des Serumcholesterins vor. Unter Modulation intrazellulären Cholesterins, wurde deutlich, dass die Inhibition der Cholesterinsynthese durch Simvastatin eine verstärkte Freisetzung von Virionen nach sich zieht, was ebenso in HEV infizierten Patienten nachweisbar war. Im Gegensatz hierzu zieht eine Erhöhung intrazellulären Cholesterins via Supplementierung von Lipoproteinpartikeln niedriger Dichte (LDL) oder 25-Hydroxycholesterin eine signifikante Reduktion des viralen Kapsidproteins und freigesetzter Virionen nach sich. Dem liegt eine verstärkte Inkorporation von HEV in Lysosomen mit anschließender Degradation zugrunde. Ob dieser Mechanismus pharmakologisch nutzbar ist, wurde mittels eines Screenings Lipid modulatorischer Medikamente untersucht. Der p-Glykoprotein Inhibitor PSC833 und besonders der PPARα-Agonist Fenofibrat stellten sich als äußerst effiziente Inhibitoren des HEV heraus. Beide führen zu einer Erhöhung und Akkumulation zellulären Cholesterins in vesikulären Strukturen. Dies zieht eine dramatische Erhöhung lysosomaler Lokalisation von HEV nach sich und führt letzten Endes zu einer signifikanten Reduktion freigesetzter Virionen.
Zusammenfassend konnten in dieser Studie essenzielle Funktionen von GBP1 in Bezug auf dessen restriktiven Effekt gegen HEV identifiziert werden. Weiters wurde dieses als entscheidender Wirtsfaktor für die IFNγ-Antwort gegen das Virus identifiziert. Andererseits legt diese Studie nahe, dass HEV niedrige Cholesterinspiegel innerhalb infizierter Zellen für die Freisetzung von Virionen benötigt. Andererseits sind erhöhte intrazelluläre Cholesterinspiegel schädlich für die virale Freisetzung, da der lysosomale Abbau von Virionen induziert wird. Dies führte zur erfolgreichen Entdeckung eines neuartigen antiviralen Wirkstoffes, welcher diesen cholesterinabhängigen Effekt effizient induziert: Fenofibrat.
Fokus meiner Doktorarbeit ist die Anwendung und Entwicklung NMR-spektroskopischer Methoden zur Charakterisierung zeitabhängiger Strukturänderungen von Biomolekülen – von lokalen dynamischen Veränderungen bis zur vollständigen Rückfaltung von Proteinen – und fasst die Ergebnisse meiner drei wichtigsten PhD-Projekte zusammen.
In meinem ersten Projekt habe ich die Leistung eines Temperatursprung-Probenkopfs – mit dem Proben mit hoher Salzkonzentration schnell erwärmt werden können – mithilfe einer Hochfrequenzspule technisch optimiert. Die optimierten Radiofrequenz-Bestrahlungsparameter, Lösungsmittel-bedingungen und der reduzierte Arbeitszyklus führten zu einem Temperatursprung von 20 °C in 400 ms. Ich habe eine Cystein-freie Mutante von Barstar hergestellt, die nach Zugabe von Harnstoff bei 0 °C kalt denaturiert werden kann, während sie ihren gefalteten Zustand bei 30 °C hält. Dadurch wurde auch ermöglicht, dass der Rückfaltungsprozess hunderte Male ohne Abbau oder Aggregation wiederholt werden kann. Die Kombination von reversibler Rückfaltung und rascher Temperaturänderung des kalt denaturierten Barstars ermöglichte die Entwicklung eines neuen kinetischen Experiments, bei dem der Rückfaltungsprozess von Barstar mit einem zweidimensionalen Echtzeit-NMR in hoher Zeitauflösung untersucht wird. Die vollständige Rückgratresonanzzuweisung wurde sowohl für den gefalteten als auch für den kalt denaturierten Zustand von Barstar durchgeführt und ergab, dass in der denaturierten Form beide Prolin-Reste einen gemischten Konformationszustand aufweisen. Dabei befindet sich die Tyr47-Pro48-Amidbindung im ungefalteten Zustand hauptsächlich in trans-, während im gefalteten Zustand in der seltenen cis-Konformation. Das neue hochauflösende kinetische Experiment zeigte, dass die Rückfaltung von Barstar durch die trans-cis-Isomerisierung der Tyr47-Pro48-Amidbindung verlangsamt wird, was sowohl die Sekundärstruktur als auch die Bildung der Tertiärstruktur beeinflusst. Basierend auf diesen Ergebnissen konnte ich einen plausiblen Faltungsmechanismus für den langsamen Faltungsweg von kalt denaturiertem Barstar skizzieren. Durch Änderung der Zeitparameter des Heizungszyklus wurde erreicht, dass die Tyr47-Pro48-Amidbindung im ungefalteten Zustand in der cis-Konformation bleibt und daher der schnelle Faltungsweg dominant wird. Das Starten des Magnetisierungstransfers vor der Temperaturänderung ermöglichte die Aufzeichnung eines Spektrums, das den entfalteten Zustand mit dem gefalteten Zustand korreliert. Dieses Spektrum ermöglichte quantitative Analysen des schnellen Faltungsweges und lieferte sogar indirekte Hinweise auf einen Zwischenzustand. Diese Methode aus Kombination von schnellem Temperatursprung und Kaltdenaturierung zeigt ein hohes Potenzial, Proteinfaltung auf atomarer Ebene experimentell zu untersuchen und ein tieferes Verständnis verschiedener Faltungswege zu erlangen.
In meinem zweiten Projekt – das Teil einer interdisziplinären Forschung war – konzentrierte ich mich auf die NMR-spektroskopische Charakterisierung von Nukleinsäuren, die mit einer photolabilen Schutzgruppe modifiziert wurden. Zuerst wurde mithilfe homonuklearer Korrelationsexperimente eine vollständige Protonresonanzzuweisung erreicht. Danach wurde die relative Konfiguration der photolabilen Schutzgruppen bestimmt basierend auf einer dreidimensionalen Modellstruktur und spezifischer NOE-Korrelationen. Des Weiteren wurde ein Strukturmodell unter Verwendung von NOE-Einschränkungen berechnet. Dieses Strukturmodell zeigte eine eingeschränkte Rotation um die CN-Bindung zwischen dem Käfig und der Nukleobase. Das Modell zeigte auch, dass der Käfig in der Hauptrille positioniert ist und nicht in das Lösungsmittel herausklappt. Im Vergleich zu einem zuvor charakterisierten NPE-Käfig führte die erhöhte Größe zu einer weiteren Senkung des Schmelzpunkts, zeigte jedoch einen geringeren Schmelzpunktunterschied zwischen der S- und der R-Konfiguration des Käfigs, wobei die S-Konfiguration zu einer größeren Reduktion des Schmelzpunktes führt. Dieser Trend wurde weiter untersucht und durch ein Screening unterstützt. Durch selektive Wasserinversions-Rückgewinnungsexperimente konnte ich auch zeigen, dass der Käfig die lokale Stabilität nur bis zu einer Entfernung von zwei benachbarten Basenpaaren von der Modifikationsstelle verringert. Die NOE-Daten dienten auch als guter Bezugspunkt, um die Qualität molekulardynamischer Simulationen zu testen, mit denen zusätzliche Käfigdesigns untersucht wurden. Die Kombination aus Synthese, NMR-Spektroskopie und MD-Simulationen ermöglichte bis jetzt die detaillierteste Untersuchung des Effekts vom Einbau eines einzelnen Käfigs zur Destabilisierung der DNA-Sekundärstruktur. Dabei wurden Einschränkungen des möglichen Designs aufgedeckt, aber auch die Entwicklung einer neuen, effizienteren Struktur ermöglicht.
Mein drittes Projekt konzentrierte sich auf die Charakterisierung eines RNA-Modellsystems. NMR-spektroskopische Daten von kleinen RNA-Modellsystemen – wie NOE, skalare Kopplungen, kreuzkorrelierte Relaxationsraten und RDC – sind eine unschätzbare Referenz für MD-Simulationen, obwohl die Menge der verfügbaren Literaturdaten – bis jetzt – sehr begrenzt ist. ...
Autophagy, together with the ubiquitin-proteasome system, is the main quality control pathway responsible for maintaining cell homeostasis. There are several types of autophagy distinguished by cargo selectivity and means of induction. This thesis focuses on macroautophagy, hereafter autophagy, where a double-layered membrane is formed originating from the endoplasmatic reticulum (ER) engulfing cargo selectively or unselectively. Subsequently, a vesicle forms around the cargo, an autophagosome, and eventually fuses with the lysosome leading to degradation of the vesicle content and release of the cargo “building blocks”. Basal autophagy continuously occurs, unselectively engulfing a portion of the cytoplasm. However, autophagy can also be induced by stress such as starvation, protein aggregation, damaged organelles, intracellular pathogens etc. In this case, the cargo is selectively targeted, and the fate of the autophagosome is the same as in basal autophagy. In recent years, interest in identifying mechanisms of autophagy regulation has risen due to its importance in neurodegenerative diseases and cancer. Given the complexity of the process, its execution is tightly regulated from initiation, autophagosome formation, expansion, closure, and finally fusion with the lysosome. Each of the steps involves different protein complexes, whose timely activity is orchestrated by post-translational modifications. One of them is ubiquitination. Ubiquitin is a small, 76-amino acid protein conjugated in a 3-step reaction to other proteins, in a reversible manner, meaning undone by deubiquitinases. Originally described as a degradation signal targeting proteins to the proteasome, today it is known it has various additional non-proteolytic functions, such as regulating a protein’s activity, localization, or interaction partners. The role of ubiquitin in autophagy has already been shown. However, given the reversibility and fine-tuning of the ubiquitin signal, many expected regulators remain unidentified. This work aimed to identify novel deubiquitinating enzymes that regulate autophagy. We identified ubiquitin-specific protease 11 (USP11) as a novel, negative regulator of autophagy. Loss of USP11 leads to an increase in autophagic flux, whereas overexpression of USP11 attenuates it. Moreover, this observation was reproducible in model organism Caenorhabditis elegans, emphasizing the importance of USP11 in autophagy regulation. To identify the mechanism of USP11-dependent autophagy regulation, we performed a USP11 interactome screen after 4 hour Torin1 treatment and identified a plethora of autophagy-related proteins. Following the most prominent hits, we have investigated versatile ways in which USP11 regulates autophagy. USP11 interacts with the PI3KC3 complex, the role of which is phosphorylating lipids of the ER, thereby initiating the formation of the autophagosomal membrane. Phosphorylated lipids serve as a recruitment signal for downstream effector proteins necessary for the membrane expansion. The core components of the complex are VPS34, the lipid kinase, ATG14, the protein responsible for targeting the complex to the ER, VPS15, a pseudokinase with a scaffolding role, Beclin1, a regulatory subunit, and NRBF2, the dimer-inducing subunit. We have found USP11 interacts with the complex and, based on its activity, USP11 influences post-translational status of all the aforementioned subunits, except for ATG14. Moreover, we have found that loss of USP11 leads to an increase in NRBF2 levels, whereas it does not change the levels of the other proteins. Given that the dimerization of the complex leads to an increase in complex activity, we investigated if the complex is more tightly formed in the absence of USP11, and if it is more active. We have found both to be the case. Although the exact mechanism of USP11-dependent PI3KC3 complex regulation remains to be identified, we found that loss of USP11 stimulates the complex formation and activity, likely contributing to the general effect of USP11 on autophagy flux. Additionally, we found that USP11 modulates levels of mTOR, the most upstream kinase in autophagy initiation steps and general multifaceted metabolism regulator. Loss of USP11 led to downregulation of mTOR levels, suggesting USP11 may rescue mTOR from proteasome-mediated degradation. Furthermore, we found mTOR to be differentially modified depending on the activity of USP11. However, it remains to be shown if USP11-dependent mTOR regulation contributes to the observed autophagy phenotype. Taken together, USP11 is a novel, versatile, negative regulator of autophagy, and an important addition to our knowledge on the regulation of autophagy by the ubiquitin system.
The aim of this work was to establish a new way of predicting novel dual active compounds by combining classical fingerprint representation with state-of-the-art machine learning algorithms. Advantages and disadvantages of the applied 2D- and 3D-fingerprints were investigated. Further, the impact of various machine learning algorithms was analyzed. The new method developed in this work was used to predict compounds, which inhibit two different targets (LTA4H and sEH) involved in the same disease pattern (inflammation). The development of multitarget drugs has become more important in recent years. Many widespread diseases like metabolic syndrome, or cancer are of a multifactorial nature, which makes them hard to be treated effectively with a single drug. The new in silico method presented in this work can help to accelerate the design and development of multitarget drugs, saving time and efforts.
The nowadays readily available access to a large number of 3D-structures of biological targets and published activity data of millions of synthesized compounds enabled this study and was used as a starting point for this work. Four different data sets were compiled (crystalized ligands from the PDB, active and inactive compounds from ChEMBL23, newly designed compounds using a combinatorial library). Those data sets were collected and processed using an automated KNIME workflow. This automation has the advantage of allowing easy change and update of compound sources and adapted processing ways.
In a next step, the compounds from the compiled data sets were represented using a variety of well-established 2D- and 3D-fingerprints (PLIF, AtomPair, Morgan, FeatMorgan, MACCS). All those fingerprints share the same underlying bit string scheme but vary in the way they describe the molecular structure. Especially the difference between 2D- and 3D-fingerprints was investigated. 2D-fingerprints are solely based on ligand information. 3D-fingerprints, on the other hand, are based on X-ray structure information of protein-ligand complexes. One major difference between 2D- and 3D-fingerprints usage is the need for a 3D-conformation (pose) of the compound in the targets of interest when using 3D-fingerprints. This additional step is time-consuming and brings further uncertainties to the method.
Based on the calculated fingerprints state-of-the-art machine learning algorithms (SVC, RF, XGB and ADA) were used to predict novel dual active compounds. The models were evaluated by 10-fold cross validation and accuracy as the primary measure of model performance was maximized. Second, individual parameters of the four machine learning algorithms were optimized in a grid search to achieve maximal accuracy using the optimized partitioning scheme. Overall accuracies, regardless of fingerprint and machine learning algorithm, are slightly better for LTA4H than for sEH.
The goal to predict dual active compounds was realized by comparing the set of predicted to be active compounds for LTA4H and sEH. For the 3D-fingerprint PLIF the machine learning algorithm Random Forest was chosen, from which compounds for synthesis and testing were selected. Of 115 predicted to be active compounds, six compounds were cherry picked. Two compounds showed very good/moderate dual inhibitory activity. Of the 2D-fingerprints, the AtomPair fingerprint in combination with the machine learning algorithm Random Forest was chosen from which compounds were selected for synthesis and testing. 116 compounds were predicted to be dual active against LTA4H and sEH. One of those compounds showed good dual inhibitory activity.
In this work it was possible to show advantages and disadvantages of using 2D- and 3D-fingerprints in combination with machine learning algorithms. Both strategies (2D: ligand-based, 3D: structure-based) lead to the prediction of novel dual active compounds with moderate to very good inhibitory activity. The method developed in this work is able to predict dual active compounds with very good inhibitory activity and novel (previously unknown) scaffolds inhibiting the targets LTA4H and sEH. This contribution to in silico drug design is promising and can be used for the prediction of novel dual active compounds. Those compounds can further be optimized regarding binding affinity, solubility and further pharmacological and physicochemical properties.
The fact that the interaction of oligonucleotides follows strict rules has been utilized to create two- or three-dimensional objects made of DNA. With computer-assisted design of DNA sequences, any arbitrary structure on the nanometer- to micrometer-scale can be generated just by hybridization of the needed strands. As astonishing these structures are, without any modification of the DNA strands involved no function can be assigned to them. Many different ways of functionalizing DNA-nanostructures have been developed with light-responsive nanostructures having a rather subordinated role. Almost all light responsive DNA-nanostructures involve the acyclic azobenzene-linking system tAzo based on D-threoninol which is known to work best at elevated temperatures to ensure optimal switching. As the structure of DNA-constructs is mainly maintained by hydrogen-bonding, variation of the temperature should be avoided in order to keep the structure intact.
To develop a light-responsive nanostructure model system with low-temperature operating azobenzene C-nucleosides, DNA-minicircles have been utilized. Those minicircles bear a lariat-like protrusion with a 10 base long single-stranded overhang, which is responsible for the dimerization with a ring bearing a complementary binding region. DNA-minicircles have been produced in a sequential manner by building and purifying the single stranded minicircle first by splint ligation and prepratative PAGE or RP-HPLC, followed by annealing it to the outer ring and subsequent purification by molecular-weight cut-off. Imaging of DNA-minicircles by atomic force microscopy (AFM) was possible with several methods of sample preparation leading to images of varying quality. With the help of AFM, qualitative analysis of the minicircles was possible. It could be shown, that theoretical and empirical size dimensions of the rings and their interactions were in great accordance. Designing the interaction site of the minicircles proved to be the main task in this project. The amount of C-nucleosidic modifications was identified by screening, followed by a screening of their optimal position and binding partners in the counterstrand. Two azobenzene C-nucleosides in a 10mer binding region and abasic sites opposing them appeared to give the best compromise between absolute dimerization ratio and photocontrolled change of it, as identified by native PAGE. In the following, the dimerization ratios of minicircles containing azobenzene C-nucleosides were compared with minicircles containing tAzo and unmodified minicircles. It could be shown, that the tAzo-modification leads to an elevated binding affinity compared to the unmodified minicircles, but the change upon irradiation is relatively humble compared to the C-nucleosides. For the C-nucleosidic modifications dimerization ratios reached a maximum of 40% in favored trans-state, but could be almost completely turned-off when switching into cis-state. In addition, arylazopyrazole-modified C-nucleosides could be switched into trans-state by irradiating at 530 nm, which is an improvement compared to standard azobenzene, as it shifts irradiation wavelength closer to the phototherapeutic window.
The utilization of DNA-analogous C-nucleosides bring two drawbacks with them: the ribose units include the flexibility of the sugar conformation and it is reasonable to think, that upon isomerization of the azobenzene, part of the steric stress generated is compensated by the sugar reconfiguration, which is lost for duplex
destabilization. In addition, the combination of the ribosidic linker end the end-to-end distance of trans-azobenzene causes the chromophore to penetrate deep into the base stack of the opposing strand, causing a serious destabilization even in favored trans-state. The goal was to find a linker system, that combines the benefits of the azobenzene C-nucleoside without the possibility to change sugar conformation and the strong destabilization in the trans-state. For this reason locked azobenzene C-nucleosides in analogy to LNA nucleosides have been synthesized. The synthesis of LNA analogous azobenzene C-nucleosides (LNAzo) was possible over a 16-step synthesis, with the critical step being the addition of in situ lithiated azobenzene to protected sugar aldehyde. Both anomers of LNAzo and mAzo as reference where incorporated into different oligonucleotide test systems by solid phase synthesis for thorough evaluation. It could be shown, that LNAzo β has a similar performance to mAzo in DNA with overall slightly increased TM- and ΔTM-values. Performance of LNAzo β was similar to mAzo even if steric stress is reduced by using abasic sites in the counterstrand opposing the azobenzene. Only in a RNA context, the true potential of LNAzo β could be observed. In a DNA/RNA duplex, photocontrol could be improved by almost 50%, in a RNA/RNA duplex even by over 100%. Although the primary goal was the improvement of the azobenzene C-nucleoside for a DNA-nanostructure context, LNAzo β proved not to give a sufficient improvement in regard to the cost-value ratio. Never the less, the invention of the locked azobenzene C-nucleoside was a huge success for reversible photoregulation of RNA hybridization. With this, a new way to regulate RNA hybridization has been found, which could be used to create RNA therapeutics in an antisense-approach.
As LNAzo β improved duplex stability only in a limited amount in DNA, further improvements on the backbone have been declared futile and focus shifted onto optimization of the chromophore. First, the azobenzene as it is installed on the ribosidic linker decreases duplex stability by forcing its distal aromat deep into opposing base stacking region. It would be an improvement, if in favored trans-state the distal aromat would be positioned in the less confined space of either major or minor groove and only upon isomerization would shift into base pairing region. Second, the azobenzene itself is not able to contribute to attractive interactions aside from relatively weak π-interactions to adjacent nucleobases, which could be improved, if it could partake in hydrogen bonding. For those apparent reasons, 2-phenyldiazenyl-modified purines have been selected as targets. They combine the ability to contribute to hydrogen bonding of nucleobases with the photochomicity of azobenzenes. Both 2’-deoxyadenosine- and 2’-deoxyguanosine-analogue photoswitches dAAzo and dGAzo have been synthesized and incorporated into 10mer DNA test systems by solid phase synthesis. It could be shown, that duplex stability could be increased compared to established azobenzene C-nucleoside. The improvement was stronger for dAAzo than for dGAzo as in the case for guanosine the amino function on the C2-position had to be replaced by the phenyldiazenyl function, reducing its ability to form hydrogen bonds. Unfortunately, photocontrol of duplex stability caused by 2-phenyldiazenyl purines was rather limited. A reason for this could be the positioning of the distal aromat within the duplex, which can be close to the opposing nucleobase (endo-helical) or in greater distance (exo-helical). The exo-helical conformation of the trans-isomer can only switch to the exo-P-cis-conformation, which relocates the distal aromat in the minor groove, without significant impact on duplex stability.
Inhibition of F1Fo ATP synthases by bacterial
virulence factors and photoswitchable azopolyphenols
(2019)
F1Fo ATP synthases are important membrane-embedded nano-machines which are conserved among all three kingdoms of life. They use a proton or sodium gradient across the membrane to drive ATP synthesis, which is the major source of energy for the cell. As ATP synthases are essential for pathogens such as mycobacteria, they are important drug targets for the treatment of infectious diseases. In this work, structural studies on the E. coli ATP synthase are performed. Furthermore, bacterial virulence MgtC proteins are investigated. Additionally, photo-switches are used to spatiotemporally control yeast ATPase activity...
NADH:ubiquinone oxidoreductase (Complex Ⅰ) is the first and largest enzyme in the respiratory chain. It catalyzes the transfer of two electrons from NADH to ubiquinone via a series of enzyme-bound redox centers - Flavin mononucleotide (FMN) and iron-sulfur (Fe-S) clusters – and couples the exergonic reaction with the endergonic translocation of four protons across the membranes. Bacteria contain the minimal form of complex I, which is composed of 14 conserved core subunits with a molecular mass of around 550 kDa. Complex Ⅰ has an L-shaped structure which can be subdivided into two major parts (arms). The hydrophilic arm protruding into the bacterial cytosol (or mitochondrial matrix) harbors the binding site for the substrate NADH, the two- to one-electron switch FMN and all one-electron transferring Fe-S clusters and therefore considered as the catalytic unit. The membrane arm consists of the membranespanning subunits and conducts the proton pumping process. The Quinone binding site is located at the interface of both arms. ...
Die Tumorprotein-Familie des Proteins p53 besteht aus drei Familienmitgliedern p53, p63 und p73 mit diversen Funktionen als Transkriptionsfaktoren. p53 war das erste Mitglied dieser Familie, das im Jahre 1979 entdeckt wurde und wurde zunächst als krebsverursachendes Protein eingeordnet, weil es in vielen Tumorgeweben in erhöhter Menge vorgefunden wurde. Es wurde allerdings festgestellt, dass der Großteil dieser gefundenen p53-Proteine funktionsunfähig durch Mutationen in ihrer Aminosäuresequenz waren. Unmutiertes p53 hingegen führt zu einem Stopp von Zellteilung oder sogar Zelltod, sofern die Zellen genetischem Stress durch Strahlung oder mutagene Chemikalien ausgesetzt sind. Heute wird p53 als eines der wichtigsten Tumor-Unterdrückungsproteine betrachtet. Die beiden anderen Familienmitglieder p63 und p73 existieren in einer Vielzahl von Isoformen. Neben carboxyterminaler alternativer mRNA-Prozessierung (α, β, γ, usw. Isoformen) führen zwei unabhängige Promotoren auch zu zwei unterschiedlichen Aminotermini. Hier wird zwischen ΔN- und TA-Isoformen unterschieden. Im Falle von p63 treten zwei dominante Isoformen auf, ΔNp63α und TAp63α. Während ΔNp63α eine Rolle in der Differenzierung von Haut spielt, wurde TAp63α bisher ausschließlich in Eizellen gefunden. Dort hat es die Funktion eines Sensors, der die genetische Integrität der weiblichen Keimbahn sicherstellt. Es liegt in Eizellen in hoher Konzentration vor, allerdings in einer komplett inaktiven Form. Werden Schäden im der Erbgut der Eizelle festgestellt, so wird das Protein aktiviert und kann so den Prozess des Zelltods der Eizelle einleiten. Mutationen oder das Fehlen des p63-Genes führen zu Missbildungen während der Entwicklung und zu unvollständig ausgebildeter Haut. Im Falle von p73 gibt es ebenfalls mehrere Isoformen, wobei die Funktionen und Relevanzen der einzelnen Isoformen bisher nicht komplett geklärt werden konnten. Eine p73-negative Maus hat einen diffusen Phänotyp, der sich durch niedrige Intelligenz, fast sterile Männchen und chronische bronchiale Infektion auszeichnet. Generell sind alle Mitglieder der p53-Familie tetramere Proteine und sind nur in diesem Zustand auch aktiv. Die einzige Ausnahme stellt, wie oben beschrieben, TAp63α dar, das in einem inaktiven dimeren Zustand vorliegt und nur durch Modifikation durch zwei unabhängige Kinasen aktiviert werden kann. Dabei geht es in den tetrameren Zustand über und ist daraufhin aktiv.
Alle drei Proteine haben (anhand ihrer längsten Isoform beschrieben) eine konservierte Domänenstruktur. Am Aminoterminus befindet sich zunächst die transaktivierende-Domäne (TAD), die für Interaktionen mit transkriptionellen Koaktivatioren relevant ist. Danach folgt die stark konservierte Desoxyribonukleinsäure (DNA) bindende Domäne (DBD). Sie stellt sicher, dass der Transkriptionsfaktor sequenzspezifisch an der richtigen Stelle auf die DNA bindet. Weitergehend folgt die Tetramerisierungsdomäne (TD), welche den oligomeren Zustand des Proteins herstellt. Im Falle von p53 endet das Protein an dieser Stelle, bei p63 und p73 folgen noch das Sterile-Alpha-Motiv (SAM) und die Transkription-inhibierende Domäne (TID). Die SAM Domäne wird generell als Interaktionsdomäne beschrieben, es konnte allerdings bis dato kein Interaktionspartner gefunden werden. Die TID hat einen negativen Einfluss auf die transkriptionelle Aktivität der Proteine. Im Falle von TAp63α interagiert sie zusätzlich mit der TAD um den Dimeren Zustand zu stabilisieren.
Histon Acetylasen
Die Acetylierung von Histonen ist neben deren Methylierung die wichtigste Modifikation. Sie ist essenziell für die Transkription innerhalb aller eukaryontischen Lebewesen, da sie durch die Modifikation von Histonen die DNA für die DNA-Polymerase II zugänglich macht. Es gibt insgesamt fünf verschiedene, nicht näher miteinander verwandte Familien von Histonacetylasen. Diese Studie beschäftigt sich ausschließlich mit der KAT3 Familie, bestehend aus den Proteinen p300 und CBP. Beide sind hochgradig konserviert, in gefalteten Bereichen der Proteine erreicht die Sequenzidentität fast 100%. Beide Proteine scheinen sehr ähnliche Aufgaben zu erfüllen, die jedoch nicht komplett identisch sind. Die Fehlfunktion von einem Allel von CBP führt zum Krankheitsbild des Rubinstein-Taybi-Syndrom (RTS), während ein Mangel an p300 sich in Mäusen auf das Gedächtnis auswirkt. Der komplette Verlust beider Allele eines der Proteine ist immer tödlich, genauso wie auch Verlust jeweils eines Allels bei beiden Proteinen. Insgesamt vier unabhängige Domänen in p300/CBP sind in der Lange die transaktivierende Domänen der p53-Familie zu binden. Bei zwei der Domänen handelt es sich um Zinkfinger-Proteine (Taz1 und Taz2), die anderen beiden sind kleine, ausschließlich α-helikale Domänen (Kix und IBiD).
Diese Studie beschäftigt sich mit der Lösung von Strukturen von der transaktivierenden Domäne von p63 und p73 mit der p300-Domäne Taz2. Außerdem wurden die Auswirkungen von direkten Acetylierungen von TAp63α charakterisiert und der Effekt von einem potenten p300/CBP Inhibitor auf Oozyten unter genotoxischem Stress analysiert. Zusätzlich wurde die Phosphorylierungskinetiken von Tap63α wärend der Aktivierung durch Kinasen untersucht.
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Infections with multidrug resistant bacterial strains like Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa or Acinetobacter baumanii that can accumulate resistance mechanisms against different groups of drugs cause increasing problems for the health care system. Multidrug efflux pumps are able to transport different classes of substances, providing a basic resistance to different antibiotics. Especially when they are overexpressed they can keep bacterial cells alive under antibiotic pressure unless other high level resistance mechanisms like expression of β-lactamases are established. One example for a clinically relevant multidrug efflux pump is the AcrAB/TolC tripartite system of E. coli, that transports a variety of different substrates, including besides antibiotics dyes, detergents, bile salts and organic compounds from the periplasm or the inner membrane out of the cell. AcrB is the inner membrane component of the protein complex that determines not only the substrate specificity of the tripartite system but energises the transport through the whole system process via proton transduction as well. TolC is the outer membrane spanning protein that forms a pore in the outer membrane enabling the system to transport drugs over the latter out of the cell. The periplasmic membrane fusion protein AcrA connects AcrB and TolC in the periplasm completing the channel from the periplasm, respective the inner membrane to the extracellular space. AcrB assembles as trimers, in asymmetric crystal structures each of the protomers adapts a different conformation designated L(oose), T(ight) and O(pen). In the protomers tunnels open up and collaps in different conformations. In the L protomer a periplasmic cleft opens up that can initially bind substrates to the periplasmic part of AcrB. In the T conformation the deep binding pocket opens that is assumed to bind substrates tightly that were bound to the access pocket before. As well in the T conformation a second pathway leading to the deep binding pocket opens that can guide substrates from a groove between transmembrane helices TM7, TM8 and TM9, the TM8 groove, that is connected with socalled tunnel 1 that ends in the deep binding pocket. In the O conformation a new tunnel opens that connects the collapsing deep binding pocket with the periplasmic space, respective the channel through the periplasmic space formed from AcrA and TolC. Substrates were cocrystallised in access and deep binding pocket verifying their role in substrate transport. In the TM8 groove in high resolution crystal structures DDM molecules were cocrystallised in L and T conformation, indicating that the AcrB substrate DDM may utilise this entrance to the deep binding pocket. The asymmetry observed in the AcrB trimers trongly suggests a peristaltic pump mechanism. The functional rotation cycle demands communication between the subunits and tight control of substrate load of protomers during the transport to optimise the ration between protons that are transduced and substrates transported. Indeed it was shown that AcrB transport mechanism is positively cooperative for some β-lactam substrates. For the communication between the subunits it was assumed that ionic interaction between ion pairs established between charged amino acids at the interfaces of protomers in different conformations are of special importance. Thus the amino acids engaged in ionic interactions, respective ion pairs D73-K131, E130-K110, D174-K110, R168, R259-E734 were substituted with non-charged amino acids pairwise and phenotypes were determined in plate dilution assays and MIC experiments. No evidence for a general, substrate independent, reduction of AcrB activity, that would be expected when the ionic residues are of special importance for AcrB function, could be found with the methods applied. Substitutions were not only combined pairwise according to the putative ion pairs but as well in combinations of R168A with D174N, E130Q and K131M. AcrB activity is reduced for the variant R168A_D174N significantly, activity decreases further for quadruple variant E130Q_K131M_ R168A_D174N. Because the reduced activity is only observed in this combination of substitutions the phenotype must result from accumulation of small effects of the single substitutions. R168A may destabilise the protomer interfaces, as its side chain is oriented in direction to the neighbouring protomer at all interfaces, enhancing substratespecific effects of substitutions E130Q, K131M, D174N that are not in all conformations oriented towards the neighbouring protomer but as well along the substrate transport pathway. Further investigations to figure out the details of the effects observed were not conducted because fluctuating expression of the variants hindered experimental procedures.
In another approach TM8 was in focus of the interest. As mentioned above it is a possible substrate entrance in the inner membrane. The linker between TM8 and the periplasmic PC2 subdomain undergoes a coil-to-helix transition when AcrB cycles through L, T and O conformations. Linking the transmembrane part of AcrB that provides the energy for the transport process via proton transduction with the periplasmic part harbouring the major part of the substrate pathway assignes TM8 and the periplasmic linker (859-876) an important role in the function of AcrB. Thus it was investigated with an alanine-scan of residues 859 to 884 and G/P respective P/G exchange followed by phenotype characterisation in growth curve and plate dilution assays of selected variants. In the phenotype determinations none of the variants, except G861P that seems to cause massive sterical restriction in an α-helical region, displayed a general, substrate independent decrease of AcrB activity. Thus it is concluded that the individual properties of amino acids in TM8 and the periplasmic linker are not of general importance for the mechanism of AcrB. The substitution of individual amino acids had impact on uptake of different substrates in plate dilution assays in a substrate dependent manner. The uptake of some substrates, like erythromycin or chloramphenicol is more affected than that of others with rhodamine 6G resistance being only reduced for the G861P variant. A relation between the PSA of substrates and reduced activity of AcrB was observed. in Substrates with higher PSA values are more affected by substitutions in TM8 or periplasmic linker, resulting in the conclusion that substrates with higher PSA are more likely to be taken up via the TM8 groove/tunnel 1 pathway than those with lower PSA values.
Retroviral vectors are powerful tools in clinical gene therapy as they integrate permanently into the target cell genome and thus guarantee long-term expression of transgenes. Therefore, they belong to the most frequently used application platforms in clinical gene therapy involving a broad range of different target cells and tissues. However, stable genomic integration of retroviral vectors can be oncogenic, as reported in several animal models and in clinical trials. In particular, γ-retroviral vectors, which derive from naturally mutagenic γ-retroviruses, integrate semirandomly into the host genome with regard to the target sequence, but have a preference for regions of active transcription and regulatory elements of transcriptionally active genes. The integration can result in overexpression of adjacent genes or disruption of ‘target’ gene expression. Moreover, γ-retroviral integration can cause modified transcripts and proteins through alternative or aberrant splicing or through premature termination of transcription.
Initially, the event of insertional mutagenesis and subsequent induction of leukemia by the genotoxicity of a γ-retroviral vector was described in a mouse model after genetic modification of hematopoietic stem cells (HSCs). Vector-related activation and overexpression of the oncogene ecotropic viral integration site-1 (Evi1) fostered clonal outgrowth and leukemogenesis. Additional genotoxic events of γ-retroviral vectors were observed in clinical HSC gene therapy trials for X-linked severe combined immune deficiency (SCID-X1), chronic granulomatous disease (X-CGD), and Wiskott-Aldrich Syndrome (WAS). But, genotoxicity induced by γ-retroviral vectors has never been described in clinical gene therapy trials involving adoptive transfer of genetically modified mature T lymphocytes. This fact is surprising, since T cells are long-lived and have a high capacity of self-renewal.
In a previous study, the susceptibility towards oncogenic transformation of mature T cells and HSCs after genetic modification was compared. It could be demonstrated that T-cell receptor (TCR)-polyclonal mature T cells are far less prone to transformation after γ-retroviral transfer of (proto-)oncogenes in vivo than HSCs. Additional experiments revealed that TCR-oligoclonal (OT-I and P14) mature T cells are transformable in the same setting and give rise to mature T-cell lymphomas (MTCLs).
In the present thesis, the susceptibility of mature T cells towards insertional mutagenesis was investigated. Within the first part of the thesis, retroviral integration sites (RISs) from 33 murine MTCLs were retrieved and subsequently analyzed in terms of integration pattern, detection of common integration sites (CIS) and gene ontology (GO). As these bioinformatic results demonstrated that insertional mutagenesis most likely contributed to mature T-cell lymphomagenesis, the susceptibility of mature T cells was directly assessed in a mouse model. Therefore, murine TCR-oligoclonal OT-I T cells were transduced with an enhanced green fluorescent protein (EGFP) encoding γ-retroviral vector and gene-modified T cells were transplanted into RAG1-/- mice. After 16 months, including one round of serial transplantation, a case of MTCL emerged. Tumor cells were characterized by CD3, CD8, TCR and ICOS expression. Integration site analysis via ligation-mediated polymerase chain reaction (LM-PCR) revealed a proviral insertion in the Janus kinase 1 (Jak1) gene. Subsequent overexpression of Jak1 could be demonstrated on transcriptional and protein level. Furthermore, T-cell lymphoma cells were characterized by an activated Jak/STAT-pathway as signal transducer and activator of transcription 3 (STAT3) was highly phosphorylated. The overexpression of Jak1 was causally implicated in tumor growth promotion as specific pharmacological inhibition of Jak1 using Ruxolitinib significantly prolonged survival of mice transplanted with these Jak1-activated tumor cells. A concluding systematic metaanalysis of available gene expression data on human mature T-cell lymphomas/leukemias confirmed the relevance of Jak/STAT overexpression in sporadic human T-cell tumorigenesis.
This was the first reported case of an insertional mutagenesis event in mature T cells in vivo. Thus, the results obtained in this thesis underline the importance of long-term monitoring of genetically modified T cells in vivo and the evaluation of vector toxicology and safety in T-cell based gene therapies. In particular, the transduction of T cells with a recombinant TCR or CAR (chimeric antigen receptor) bears a risk enhancement, as normal T-cell homeostasis is perturbed besides the general risk of insertional mutagenesis.
Rhabdomyosarcoma is the most common paediatric soft-tissue sarcoma, and for tumour recurrence, the prognosis is still unfavourable. The current standard therapy consisting of surgery, radiation and combined chemotherapy does not consider the specific biology of this tumour.
Histone deacetylases (HDACs) and the Lysine-specific demethylase-1 (LSD1) are two epigenetic modifiers which are both part of repressor complexes leading to transcriptional silencing of target genes. Whereas HDACs lead to deacetylation of several lysine-residues within the histone tail, LSD1 is specific for demethylation of H3K4me2 and H3K4me1, as well as in a different context for H3K9me2. Rhabdomyosarcoma is reported to harbour high levels of LSD1, but the functional relevance is yet unclear. HDAC inhibition proved to be effective as single agent treatment, however, the proximity of HDAC1/2 and LSD1 in repressor complexes at the DNA implies a suitable rationale for a combination therapy potentially leading to cooperative effects on target gene transcription. In this study, we aimed to evaluate the potential of a combined LSD1 and HDAC inhibition for cell death induction in rhabdomyosarcoma cell lines. Whereas LSD1 inhibitors failed to induce cell death on their own, the combined inhibition of HDACs and LSD1 resulted in highly synergistic cell death induction. This effect extended to several combinations of LSD1 and HDAC inhibitors as well as to four different rhabdomyosarcoma cell lines, two of embryonal and two of alveolar histology.
With the use of the HDAC inhibitor JNJ-26481585 and the reversible LSD1 inhibitor GSK690, we demonstrated that the cell death induced by the combination matches with the details of intrinsic mitochondrial apoptosis. JNJ-26481585/GSK690-induced cell death is partially caspase-dependent and leads to caspase cleavage, followed by substrate cleavage as shown for PARP, as well as loss of the mitochondrial membrane potential.
Furthermore, JNJ-26481585 and GSK690 acted together to transcriptionally upregulate the proapoptotic proteins NOXA, BIM and BMF, which resulted in respective changes on protein level for both cell lines. However, the antiapoptotic BCL-2 family proteins BCL-2, MCL-1 and BCL-xL displayed only minor changes in protein levels upon treatment with GSK690 and JNJ-26481585, which did not rely on transcriptional activity. Therefore, the increase in proapoptotic proteins induces a shift towards proapoptotic signalling at the mitochondrial membrane. This shift is functionally relevant since knockdown of a proapoptotic protein or overexpression of one of the antiapoptotic proteins BCL-2 and MCL-1, as well as a stabilized mutant MCL-1, can significantly protect from GSK690/JNJ-26481585-induced cell death.
Knockdown of the mitochondrial membrane protein BAK, which is directly guarding the mitochondrial membrane integrity, potently protected from GSK690/JNJ-26481585- induced cell death, directly linking the shift in the BCL-2 family proteins to the observed loss of mitochondrial membrane potential and the further downstream activation of caspases. Furthermore, treatment with JNJ-26481585 and GSK690 resulted in a cell cycle arrest in G2/M phase, indicating additional effects on the tumour cells beside apoptosis induction. Taken together, the combined inhibition of LSD1 and HDACs is a promising strategy for rhabdomyosarcoma treatment.
Pulsed electron-electron double resonance (PELDOR), also called Double Electron-Electron Resonance, (DEER) is a pulsed EPR technique that can provide structural information of biomolecules, such as proteins or nucleic acids, complementary to other structure determination methods by measuring long distances (from 1.5 up to 10 nm) between two paramagnetic labels. Incorporation of the rigid Ç-label pairwise into DNA or RNA molecules enables the determination not only of the distance but also of the mutual orientation between the two Ç-labels by multi-frequency orientation-selective PELDOR data (X-, Q- and G-band frequencies). Thus, information about the orientation of secondary structure elements of nucleic acids can be revealed and used as additional angular information for structure determination. Since Ç does not have motion independent from the helix where it resides, the conformational flexibility of the nucleic acid molecule can be directly determined. This thesis demonstrates the advancement of PELDOR spectroscopy, beyond its original scope of distance measurements, to determine the mutual orientation between two rigid spin labels towards the characterization of the conformational space sampled by highly flexible nucleic acid molecules. Applications of the methodology are shown on two systems: a three-way junction, namely a cocaine aptamer in its bound-state, and a two-way junction, namely a bent DNA.
More in detail, the conformational changes of the cocaine aptamer upon cocaine binding were investigated by analysis of the distance distributions. The cocaine-bound and the unbound states could be differentiated by their conformational flexibility, which decreases in the presence of the ligand. Moreover, the obtained distance distributions revealed a small change in the mean distance between the two spin labels upon cocaine binding. This indicates a ligand-induced conformational change, which presumably originates at the junction where cocaine is known to bind. The investigation of the relative orientation between the two spin-labeled helices of the aptamer revealed further structural insights into the conformational dynamics of the cocaine-bound state. The angular information from the orientation-selective PELDOR data and the a priori knowledge about the secondary structure of the aptamer were helpful in obtaining a molecular model describing its global folding and flexibility. In spite of a large flexible aptamer, the kink angle between the Ç-labeled helices was found to be rather well-defined.
As for the bent DNA molecule, a two-step protocol was proposed to investigate the conformational flexibility. In the first step, a database with all the possible conformers was created, using available restraints from NMR and distance restraints derived from PELDOR. In a second step, a weighted ensemble of these conformers fitting the multi-frequency PELDOR data was built. The uniqueness of the obtained structural ensemble was checked by validation against an independent PELDOR data set recorded at a higher magnetic field strength. In addition, the kink and twist angle pairs were determined and the resulting structural ensemble was compared with the conformational space deduced both from FRET experiments and from the structure determined by the NMR restraints alone.
Overall, this thesis underlines the potential of using PELDOR spectroscopy combined with rigid spin labels in the context of structure determination of nucleic acids in order to determine the relative orientation between two helices, the conformational flexibility and the conformational changes of nucleic acid molecules upon ligand binding.
Cancer cells, in general and especially Rhabdomyosarcoma (RMS) cells have been reported to be highly susceptible to oxidative stress. Based on this knowledge we examined whether the inhibition of the two main antioxidant defense pathways, i.e. the thioredoxin (TRX) and the glutathione (GSH) system, represents a possible new strategy to induce cell death in RMS. To do so, we combined the -glutamylcysteine synthetase (γGCL) inhibitor buthionine sulfoximine (BSO) or the cystine/glutamate antiporter (xc-) inhibitor erastin (ERA), both GSH depleting enzymes, with the thioredoxinreductase (TrxR) inhibitor auranofin (AUR) to evaluate synergistic cell death in the alveolar RMS (ARMS) cell line RH30 and the embryonal RMS (ERMS) cells RD.
Furthermore, we tried to unravel the underlying molecular mechanisms of AUR/BSO or AUR/ERA treatment in RMS cells. Thereby we showed that AUR/BSO as well as AUR/ERA treatment leads to proteasome inhibition characterized by the accumulation of ubiquitinated proteins, which is in agreement with the already published ability of AUR to inhibit proteasomeassociated deubiquitinases (DUBs) aside from TrxR. As a consequence, the protein levels of ubiquitinated short-lived proteins, like NOXA and MCL-1, increase upon treatment with AUR/BSO or AUR/ERA. Consistently, we could detect an increased binding of NOXA to MCL-1. Interestingly, not only NOXA protein levels but also mRNA levels rise upon treatment, pointing to a transcriptional regulation of pro-apoptotic NOXA through AUR/BSO or AUR/ERA combination treatment. The fact that siRNA mediated knockdown of NOXA rescues cells from combination treatment-induced cell death strengthens the role of NOXA as an important regulator of cell death induction. Apart from proteasome inhibition and subsequent NOXA accumulation, AUR cooperates with BSO or ERA to trigger BAX/BAK activation, which is needed for cell death induction, too. Additionally, loss of mitochondrial membrane potential (MMP) as well as caspase activation and PARP cleavage is detected after treatment of RMS cells with AUR/BSO or AUR/ERA.
Except of apoptotic cell death we also detected features of iron-dependent ferroptosis after treatment with AUR/BSO or AUR/ERA. This is not surprising, since BSO and ERA already have been described to induce ferroptotic cell death. Although lipid peroxidation takes place in both cell lines, only in RH30 cells, cell death seems to be partially ferroptosis-dependent, since especially in this cell line AUR/BSO- or AUR/ERA-induced cell death can be rescued with different ferroptosis inhibitors.
Although both combination treatments, AUR/BSO as well as AUR/ERA, induce production of reactive oxygen species (ROS), only the thiol-containing ROS scavengers GSH and its precursor N-acetylcysteine (NAC), but not the non-thiolcontaining antioxidant α-Tocopherol (α-Toc), consistently prevent proteasome inhibition, NOXA accumulation and cell death.
Additionally, we demonstrated that BSO and ERA abolish AUR-mediated upregulation of GSH thereby releasing the AUR cytotoxic effect on RMS cells, in line with the described ability of cysteines to inhibit the function of AUR. Together, this points to the conclusion that GSH depletion, rather than an increase in ROS levels, is important for AUR/BSO- or AUR/ERA-induced cell death.
In conclusion, through revealing that the antitumor activity of AUR is enhanced in combination with GSH depleting agents, we identified redox homeostasis as a new and promising target for the treatment of RMS cells.
Bacteria are highly organized organisms which are able to adapt to and propagate under a multitude of environmental conditions. Propagation hereby requires reliable chromosome replication and segregation which has to occur cooperatively with other cellular processes such as transcription, translation or signaling. Several mechanisms were proposed for segregation of the Escherichia coli (E. coli) chromosome, for example a mitotic-like active segregation model or entropy-based passive chromosome segregation. Another segregation model suggests coupled transcription, translation and insertion of membrane proteins (termed "transertion"), which links the replicating chromosome (nucleoid) to the growing cell cylinder.
Fluorescence microscopy was widely used to provide evidence for a distinct segregation model. However, the dynamic nature of bacterial chromosomes, the small bacterial size and the optical resolution limit of ~ 200-300 nm impair unveiling the underlying mechanisms. With the emergence of super-resolution fluorescence microscopy techniques and advanced labeling methods, a new toolbox became available enabling scientists to visualize biomolecules and cellular processes in unprecedented detail. Single-molecule localization microscopy (SMLM) represents a set of super-resolution microscopy techniques which relies on the temporal separation of the fluorescence signal and detection of single fluorophores. Separation can be achieved using photoactivatable or -convertible fluorescent proteins (FPs) in photoactivated localization microscopy (PALM), photoswitchable organic dyes in direct stochastic optical reconstruction microscopy (dSTORM) or dynamically binding fluorescent probes in point accumulation for imaging in nanoscale topography (PAINT). In all these techniques, the fluorescence emission pattern of single fluorophores is spatially localized with nanometer-precision. An artificial image is finally reconstructed from the coordinates of all single fluorophores detected. This provides a spatial resolution of ~ 20 nm, which is perfectly suited to investigate cellular processes in bacteria. In this thesis, different SMLM techniques were applied to study fundamental processes in E. coli. This includes determination of protein copy numbers and distributions as well as the nanoscale organization of nucleic acids and lipids.
A novel labeling approach was applied and used for super-resolution imaging of the E. coli nucleoid. It is based on the incorporation of the modified thymidine analogue 5-ethynyl-2’- deoxyuridine (EdU) into the replicating chromosome. Azide-functionalized organic fluorophores can be covalently attached to the ethynyl group of incorporated EdU bases using a copper-catalyzed "click chemistry" reaction. Under the investigated growth condition, E. coli cells exhibited overlapping replication cycles, which is commonly referred to as multi-fork replication and enables cells to divide faster than they can replicate the entire chromosome. dSTORM imaging of such labeled nucleoids revealed chromosome features with diameters of 50 - 200 nm, representing highly condensed DNA filaments. Sorting single E. coli cells by length allowed visualizing structural changes of the nucleoid throughout the cell cycle. Replicating nucleoids segregated and expanded along the bacterial long axis, while constantly covering the entire width of the cell. Measuring cell and nucleoid length revealed a relative nucleoid expansion rate of 78 ± 6 %. At the same time, nucleoids populated 63 ± 8 % of the cell length, almost exclusively being localized to the cylindrical part of the cell. This value was hence normalized to the cylindrical fraction of the cell, yielding a value of 79 ± 10 % (nucleoid-populated fraction of the cell cylinder), which is in good agreement with the observed relative nucleoid expansion rate. These results therefore support a growth-mediated segregation model, in which the chromosome is anchored to the inner membrane and passively segregated into the prospective daughter cells upon cell growth. 3-dimensional dSTORM imaging of labeled nucleoids confirmed that compacted nucleoids helically wrap along the inner membrane. Similar results were obtained by imaging orthogonally aligned E. coli cells using a holographic optical tweezer approach.
In order to visualize particular proteins together with the nucleoid, several correlative imaging workflows were established, facilitating multi-color SMLM imaging in single E. coli cells. These workflows bypass prior limitations of SMLM, including destruction of FPs by reactive oxygen species in copper-catalyzed click reactions or incompatibility of PALM imaging with dSTORM imaging buffers. A sequential SMLM imaging routine was developed which is based on postlabeling and retrieval of previously imaged cells. Optimal imaging conditions can be maintained for each fluorophore, enabling to extract quantitative information from PALM measurements while correlating the protein distribution to the nucleoid ultrastructure within the highly resolved cell envelope. Applying this workflow to an E. coli strain carrying a chromosomal rpoC - photoactivatable mCherry (PAmCh) fusion, transcribing RNA polymerase (RNAP) was found to be localized on the surface of nucleoids, where active genes are exposed towards the cytosol. During growth in nutrient-rich medium, the majority of RNAP molecules was bound to the chromosome, thus ensuring that the RNAP pool is equally distributed to the daughter cells upon cell division. This work represented the first triple-color SMLM study performed in E. coli cells. ...
A great challenge in life sciences remains the site-specific modification of proteins with minimal perturbation for in vitro as well as in vivo studies. Therefore, different chemoselective reactions and semi-synthetic techniques such as native chemical ligation or intein-mediated protein splicing have been established. They enable a site-specific incorporation of chemical reporters into proteins, such as organic fluorophores or unnatural amino acids. In this PhD Thesis, protein trans-splicing was guided by minimal high-affinity interaction pairs to trace proteins in mammalian cells. In addition, the temporal modulation of cellular processes by photo-cleavable viral immune evasins was achieved.
Protein trans-splicing mediated by split inteins is a powerful technique for site-specific and 'traceless' protein modifications. Despite recent developments there is still an urgent need for ultra-small high-affinity intein tags for in vitro and in vivo approaches. So far, only a very few in-cell applications of protein trans-splicing are reported, all limited to C-terminal protein modifications. Here, a strategy for covalent N-terminal intein-mediated protein labeling at sub-nanomolar probe concentrations was developed. Combined with the minimalistic Ni-trisNTA/His-tag interaction pair, the affinity between the intein fragments was increased 50-fold (KD ~ 10 nM). Site-specific and efficient 'traceless' protein modification by high-affinity trans-splicing is demonstrated at nanomolar concentrations in mammalian cells.
High background originating from non-reacted, 'always-on' fluorescent probes still is a crucial issue in life sciences. Covalent labeling approaches with simultaneous activation of fluorescence are advantageous to increase sensitivity and to reduce background signal. Therefore, high-affinity protein trans-splicing was combined with fluorophore/quencher pairs for online detection of covalent N-terminal protein labeling in cellular environments. Substantial fluorescence enhancement at nanomolar probe concentrations was achieved. This ultra-small fluorogenic high-affinity split intein system is an unprecedented example for real-time monitoring of the trans-splicing reaction in cell-like environments as well as for protein labeling with fluorogenic probes at nanomolar concentrations.
To extend the field of chemical immunology and to address spatiotemporal aspects in adaptive immune response, new tools to control antigen processing are required. Therefore, synthetic photo-conditional viral immune evasins were designed to modulate antigen processing on demand. By using light, the time and dose controlled antigen translocation by the transporter associated with antigen processing (TAP) was triggered with response in the second regime. Peptide delivery and loading by the peptide-loading complex (PLC) was rendered inactive, whereas blocking was abolished in a light-controlled fashion to inactivate the synthetic viral immune evasin ICP47 along with simultaneous activation of the antigen presentation pathway. Lightresponsive peptide translocation by the TAP complex was assayed in vitro by utilizing microsomes isolated from professional antigen presenting B-cell lymphomas (Raji). To extend these studies, suppression and photo-controlled rescue of antigen presentation was examined at single-cell resolution in human primary immune cells.
Native chemical ligation interconnects peptide chemistry with recombinantly expressed proteins. This technique was applied to generate the semi-synthetic full-length ICP47. Although this approach was realized, the low product yield was not sufficient for further functional studies. Therefore, full-length ICP47 was consecutively generated by utilizing a full synthetic four-fragment ligation approach. However, this synthetic viral immune evasin was not able to block peptide translocation in a robust way.
This thesis is concerned with quantum dynamical propagation methods suitable for high-dimensional systems, and their application to excitation energy transfer (EET), electron transfer (ET), and intra-molecular vibrational redistribution (IVR) in molecular aggregates. The theoretical description of these processes, which are often ultrafast – with time scales in the range of femtoseconds to picoseconds – is challenging, both with regard to quantum dynamical simulations and electronic structure calculations.
The present thesis comprises two parts. The first part concerns the implementation of a novel quantum dynamical method based on Gaussian Wavepackets (GWPs): the 2-Layer Gaussian-MCTDH (2L-GMCTDH) method. This method, which has recently been proposed in [S. Römer, M. Ruckenbauer, I. Burghardt, The Journal of Chemical Physics, 2013, 138, 064106] was implemented in a Fortran90 code and applied to various high-dimensional test systems. The second part of the thesis addresses the combined electronic structure and dynamical study of a novel type of donor-acceptor systems that have been investigated in a joint project with experimental collaboration partners at Strasbourg University. In both parts, numerical applications focus on high-dimensional model Hamiltonians for EET and ET processes.
Regarding the first part, the interest of using GWP-based methods is two-fold: First, GWPs represent spatially localized basis sets that are useful for on-the-fly dynamics in conjunction with electronic structure calculations. Second, they are naturally suited for the explicit representation of quantum mechanical system-bath type problems where a large number of vibrational bath modes are weakly perturbed from equilibrium. In this context, various methods exist that are based upon classically evolving GWP bases. A major improvement results from variational methods which involve optimized, non-classical GWP trajectories. In particular, the variational Gaussian-based Multi-Configuration Time-Dependent Hartree (GMCTDH) and its variational Multi-Configurational Gaussians (vMCG) variant were originally derived as semiclassical variants of the Multi-Configuration Time-Dependent Hartree (MCTDH) method. However, the G-MCTDH and vMCG methods mostly use Frozen Gaussian (FG) basis sets that are far less flexible than the single-particle (SPF) representation of standard MCTDH. As a consequence, a significantly larger number of GWPs are generally required to reach convergence. To remedy the lack of flexibility of the FG basis sets, the abovementioned two-layer (2L-G-MCTDH) approach has been introduced: Here, the first layer is composed of flexible SPFs, while the second layer is composed of low-dimensional FGs. The numerical scaling properties are significantly improved as compared with the conventional G-MCTDH and vMCG schemes. The first implementation of the method in an in-house Fortran90 code is presented, along with applications to (i) a model of site-to-site vibrational energy flow in the presence of intra-site vibrational energy redistribution (IVR) and (ii) a multidimensional donor-acceptor electron transfer system described within a linear vibronic coupling model. The second system relates to a model for ET at an oligothiophene-fullerene interface relevant to organic photovoltaics. Besides the description of the implementation, a detailed assessment of the convergence properties and comparison with multi-layer MCTDH (ML-MCTDH) benchmark calculations is presented. Finally, a perspective is given on the future combination with the existing ML-MCTDH scheme; indeed, such a combination is straightforward since the first layer of the 2L-G-MCTDH approach can be chosen to be orthogonal.
Regarding the second part of the thesis, two generations of a novel donor-acceptor (DA) system for organic photovoltaics applications, involving self-assembled block co-oligomers DA dyads and triads with perylene-diimide (PDI) accepter units, are addressed within a collaborative project with S. Haacke and S. Mery (University of Strasbourg). Based upon detailed excited-state electronic structure investigations along with quantum dynamical and kinetic studies, the relevant ET formation and recombination steps are characterized quantitatively, in view of optimizing the chemical design and reducing recombination losses.
In a first-generation variant of the abovementioned DA systems, which involves liquid-crystalline triads, we were able to show that a highly efficient inter-chain ET process prevails over intra-molecular ET, leading to fast recombination. Due to the latter, this system turns out to be inefficient for photovoltaic applications. To fully understand the elementary steps, high-dimensional quantum dynamics simulations were carried out using the ML-MCTDH method, in collaboration with Matthias Polkehn from our group. In the second-generation variant, which is in the focus of the present thesis, both the nanomorphology and the chemical design were modified. The present work, focuses upon the aspect of chemical design, by characterizing a series of modified DA’s, with donor units of varying length while the PDI accepter units remain unchanged. The intra-molecular ET is observed in these systems, but the processes are comparatively slow, of the order of tens to hundreds of picoseconds. Hence, a kinetic analysis using the Marcus-Levich-Jortner rate theory is employed. Among the main results of the study is that addition of an electron donating amine unit strongly increases the lifetime of the charge-separated state, and therefore reduced recombination losses.
Overall, the present thesis shows how a combination of high-dimensional quantum dynamics, electronic structure calculations, and vibronic coupling model Hamiltonians can be employed to obtain an accurate picture of EET, ET, and IVR in high-dimensional molecular assemblies. Furthermore, the 2L-GMCTDH method paves the way for accurate and efficient on-the-fly calculations; a suitable set-up for such calculations is currently in progress.
In dieser Dissertation wurde die Rolle des Proteins Carboxypeptidase E (CPE) im Glioblastom (GBM) untersucht. Ursprünglich wurde CPE in der neuroendokrinen Regulation beschrieben, wo es die Reifung der meisten Neuropeptide und Hormone reguliert und somit Einfluss auf Stoffwechsel und humorale Effekte hat (Fricker et al., 1982; Fricker & Snyder, 1982 and 1983; Davidson & Hutton, 1987; Shen & Loh, 1997; Lou et al., 2005). Ab 1989 wurde CPE in unterschiedlichen Tumorentitäten nachgewiesen (Grimwood et al., 1989; Manser et al., 1991), jedoch ohne Hinweise, welche Bedeutung das Protein dort haben könnte. Erst im letzten Jahrzehnt konnten sowohl pro- als auch anti-tumorigene Wirkungen von CPE gezeigt werden. Die beschriebenen Wirkungen von CPE sind jedoch von dessen Isoform abhängig. Das ∂(delta)N-trunkierte CPE zeigte sich mit erhöhtem Tumorwachstum und schlechter Überlebensprognose in verschiedenen Krebsentitäten assoziiert (Murthy et al., 2010; Lee et al., 2011; Zhou et al., 2013). Im Gegensatz dazu verringerte sezerniertes CPE (sCPE) im Fibrosarkom und Glioblastom die Zellmigration, was einen anti-tumorigenen Effekt suggeriert (Höring et al., 2012; Murthy et al., 2013a). Die Molekularmechanismen, die für die Regulation der Migration zuständig sind, sind jedoch kaum untersucht. Die meisten Untersuchungen von sCPE in Normal- und Tumorgewebe beschränken sich hauptsächlich auf Apoptose und Zellüberleben (Skalka et al., 2013; Murthy et al., 2013b; Cheng et al., 2013; Selvaraj et al., 2015; Cheng et al., 2015). Die vorliegende Arbeit ist demzufolge die erste Studie, die sich dem Mechanismus der Migrationsregulation durch sCPE im Glioblastom widmet.
Humane Gliome stellen die größte und bösartigste Gruppe hirneigener Tumore dar. Bösartige Gliome sind höchst resistent gegen alle zurzeit verfügbaren Behandlungsmethoden. Einer der Hauptgründe dafür ist, dass die Tumorzellen durch diffuse Infiltration in das Gehirn einwandern können. Ferner sind Gliomzellen metabolisch sehr aktiv und können sich dadurch an schnell verändertes Milieu anpassen (Fack et al., 2015; Demeure et al., 2016). Über die grundlegenden Mechanismen für diese Art des infiltrierenden Tumorwachstums ist bisher noch nicht viel bekannt. Zurzeit sind nur wenige Schlüsselfaktoren beschrieben, die den sogenannten Mechanismus der Migration oder Proliferation ("go or grow") in bösartigen Tumoren beeinflussen: wenige Transkriptionsfaktoren, miRNAs sowie metabolische Faktoren. Interessanterweise, sind miRNAs zum Teil mit der Regulation des Metabolismus in Tumorzellen assoziiert. Eine vorangehende Studie aus unserem Labor hat sCPE aufgrund seines Potentials, Zellwanderung zu verringern, als einen weiteren Schlüsselfaktor identifiziert. Wir konnten zeigen, dass sCPE in der Gliomzelllinie LNT-229 zur einer differentiellen Regulation von Migration und Proliferation führt (Höring et al., 2012). Die vorliegende Arbeit widmet sich nun der Frage nach den genauen zugrundeliegenden Mechanismen, wie sCPE seine Effekte auf molekularer Ebene vermittelt. Darüber hinaus soll geklärt werden, ob sCPE auch in der metabolischen Adaptation eine Rolle spielt und dadurch ebenfalls die Gliomzellmigration beeinflußen kann.
Proteinen die ExHepatitis C ist eine entzündliche Erkrankung der Leber, die durch das Hepatitis-C-Virus (HCV) verursacht wird. Trotz vieler Bemühungen ist heutzutage immer noch keine prophylaktische Vakzinierung verfügbar. Neuartige Therapien versprechen eine hohe Heilungsrate, sind aber mit hohen Kosten verbunden. HCV induziert oxidativen Stress, welcher für das Auftreten und die Progression der Pathogenese eine zentrale Rolle spielt. Um zellulären Stress (z.B. durch ROS) entgegenzuwirken, haben Zellen cytoprotective und detoxifizierende Mechanismen entwickelt, die die zelluläre Homöostase aufrechterhalten. Dabei kontrolliert der redoxsensitive Transkriptionsfaktor Nrf2 als Heterodimer zusammen mit sMaf- pression von cytoprotective und ROS-detoxifizierenden Genen. Vorherige Studien haben gezeigt, dass HCV den Nrf2/ARE-Signalweg beeinträchtigt. Dabei induziert HCV eine Translokation der sMaf-Proteine aus dem Zellkern in das Cytoplasma, wo diese das virale Protein NS3 binden. Im Cytoplasma lokalisierte sMaf-Proteine verhindern dadurch eine Translokation von Nrf2 in den Zellkern. Folglich ist die Expression von Nrf2/ARE-abhängigen cytoprotective Genen inhibiert und intrazelluläre ROS-Spiegel dauerhaft erhöht. Ein weiterer zentraler cytoprotective Mechanismus ist die Autophagie. Sie dient der Aufrechterhaltung der zellulären Homöostase durch den Abbau von defekten Proteinen und Organellen. Des Weiteren ist bekannt, dass Autophagie nicht nur im Laufe von Nährstoffmangel induziert wird, sondern auch durch erhöhte Mengen an ROS. In sämtlichen Studien konnte beobachtet werden, dass Autophagie für die Aufrechterhaltung des viralen Lebenszyklus eine wesentliche Rolle spielt, da sie mit der Ausbildung des membranous web, der Translation, der Replikation und der Freisetzung des Virus interferiert. Ausgehend davon sollte in dieser Arbeit zunächst die Relevanz von HCV-induziertem oxidativen Stress, resultierend aus der Nrf2/ARE-Signalweginhibition, als möglicher Aktivator der Autophagie untersucht werden. Dabei wurde in HCV-positiven Zellen eine Akkumulation von LC3-II beobachtet, was auf eine Induktion der Autophagie schließen lässt. In Übereinstimmung damit wurde eine erhöhte Expression von Autophagie-Markerproteinen in HCV-infizierten PHHs detektiert. Im Laufe der Autophagie wird p62 abgebaut. Somit sollte eine Induktion der Autophagie in einer Verminderung der Menge an p62 resultieren. Nichtsdestotrotz ist eine Akkumulation von p62 in HCV-positiven Zellen nachzuweisen. Dies erscheint zunächst widersprüchlich. Aufgrund der Tatsache, dass die Expression der katalytischen Untereinheit des Proteasoms (PSMB5) Nrf2-abhängig ist, führt die beeinträchtigte Nrf2-Aktivität in HCV-positiven Zellen jedoch zu einer verringerten Aktivität des konstitutiven Proteasoms. Dieser Befund kann auch die erhöhte Halbwertzeit von p62 in HCV-positiven Zellen erklären. Kürzlich wurde ein Zusammenspiel des Nrf2/ARE-Signalwegs und der Autophagie beobachtet. Dabei kann Nrf2 nicht nur über den kanonischen Signalweg aktiviert werden, sondern auch durch eine direkte Interaktion des phosphorylierten Autophagie-Adaptorproteins p62 (pS[349] p62) mit Keap1. In HCV-positiven Zellen können nicht nur eine Zunahme der Gesamtmenge von p62 beobachtet werden, sondern auch erhöhte Mengen an pS[349] p62. Die Berechnung des Quotienten aus pS[349] p62 und p62 zeigt in etwa eine Verdopplung der Menge an pS[349] p62 , was auf eine vermehrte Phosphorylierung von p62 in HCV-positiven Zellen rückschließen lässt. Des Weiteren konnte beobachtet werden, dass erhöhte Mengen an ROS, wie sie auch in HCV-positiven Zellen vorkommen, Autophagie induzieren können, die durch eine Akkumulation von LC3-II und die Zunahme von LC3 Puncta charakterisiert ist. Auch eine Zunahme von pS[349] p62 konnte beobachtet werden. Ferner resultierte die Überexpression der phosphomimetischen Mutante (p62 [S351E]) in einer Akkumulation von LC3-II, was auf die Fähigkeit von pS[349] p62 rückschließen lässt, Autophagie zu induzieren. Eine Modulation der Autophagie mittels der Inhibitoren 3-Methyladenin und Bafilomycin führte zu einer inhibierten Freisetzung von infektiösen viralen Partikeln und unterstreicht damit, dass der Autophagie eine essentielle Bedeutung bei der Freisetzung viraler Partikel zukommt. Eine HCV-Infektion wird sowohl von erhöhten Mengen an ROS als auch von einer Induktion der Autophagie begleitet. Dementsprechend führte eine Verminderung des intrazellulären Radikalspiegels durch eine Inkubation mit den Radikalfängern PDTC und NAC zu geringeren Mengen an LC3-II und pS[349] p62. Dabei konnte auch eine Abnahme der freigesetzten infektiösen viralen Partikel beobachtet werden, was ein Zusammenspiel zwischen erhöhten Mengen an ROS, Induktion der Autophagie und Virusfreisetzung nahelegt. Vorschlag: Erhöhte Mengen an ROS werden durch eine Aktivierung des Nrf2/ARE-Signalwegs detoxifiziert und würden somit den zuvor beschriebenen viralen Mechanismus verhindern. HCV die Aktivierung Nrf2/ARE-regulierter Gene beeinträchtigt, wurde die Hypothese aufgestellt, dass in HCV-positiven Zellen dieser komplexe Mechanismus dazu dient, die Translokation des pS[349] p62-abhängig freigesetzte Nrf2 in den Zellkern zu verhindern. Das wiederum hat eine eingeschränkte Expression von Nrf2/ARE-abhängigen Genen und Detoxifizierung von ROS zur Folge. Um diese Hypothese experimentell zu untersuchen, wurden HCV-positive und negative Zellen cotransfiziert mit dem p62 Wildtyp (p62 [wt]), der p62 phosphomimetischen Mutante (p62 [S351E]) oder einem Kontrollplasmid in Kombination mit einem Reporterkonstrukt, welches die Nrf2-Aktivierung darstellt (OKD48). Während in HCV-negativen Zellen im Vergleich zum p62 [wt] eine Transfektion mit p62 [S351E] zu einer signifikanten Aktivierung des Nrf2-abhängigen Reportergens führt konnte dies in HCV-positiven Zellen nicht beobachtet werden. Zusammengenommen beschreiben diese Ergebnisse einen neuartigen Mechanismus wie HCV das Zusammenspiel zwischen dem Nrf2/ARE-Signalweg, erhöhten Mengen an ROS und Autophagie beeinflusst. Dabei übt HCV einen negativen Effekt auf den Nrf2/ARE-Signalweg aus, um dem pS[349] p62-abhängig freigesetzten Nrf2 zu entkommen. Folglich werden erhöhte Mengen an ROS aufrechterhalten, die eine Induktion der Autophagie ermöglichen, welche für die Freisetzung viraler Partikel essentiell ist.
Riboswitches are an important class of regulatory RNA elements that respond to cellular metabolite concentrations to regulate gene expression in a highly selective manner. 2’-deoxyguanosine-sensing (2’dG) riboswitches represent a unique riboswitch subclass only found in the bacterium Mesoplasma florum and are closely related to adenine- and guanine-sensing riboswitches. The I-A type 2’dG-sensing riboswitch represses the expression of ribonucleotide reductase genes at high cellular concentrations of 2’dG as a result of premature transcription termination.
Increasing evidence within the last decade suggests that transcriptional regulation by riboswitches is controlled kinetically and emphasizes the importance of co-transcriptional folding.2–4 Addition of single nucleotides to nascent transcripts causes a continuous shift in structural equilibrium, where refolding rates are competing with the rate of transcription.5,6
For transcriptional riboswitches, both ligand binding and structural rearrangements within the expression platform are precisely coordinated in time with the rate of transcription. The current thesis investigates the mechanistic details of transcriptional riboswitch regulation using the I-A 2’dG-sensing riboswitch as an example for a riboswitch that acts under kinetic control.
Das Hauptziel dieser Dissertation lag in der Verbesserung einzelner Schritte im Prozess der automatischen Proteinstrukturbestimmung mittels Kernmagnetischer Resonanz (NMR). Dieser Prozess besteht aus einer Reihe von sequenziellen Schritten, welche zum Teil bereits erfolgreich automatisiert wurden. CYANA ist ein Programmpaket, welches routinemäßig zur automatischen Zuordnung der chemischen Verschiebungen, der Nuclear Overhauser Enhancement (NOE) Signalen und der Strukturrechnung von Proteinen verwendet wird. Einer der Schritte, der noch nicht erfolgreich automatisiert wurde, stellt die Signalidentifizierung von NMR Spektren dar. Dieser Schritt ist besonders wichtig, da Listen von NMR-Signalen Grundlage aller Folgeschritte sind. Fehler in den Signallisten pflanzen sich in allen Folgeschritten der Datenauswertung fort und können am Ende in falschen Strukturen resultieren. Daher war ein Ziel dieser Arbeit, einen robusten und verlässlichen Algorithmus zur Signalidentifizierung von NMR Spektren in CYANA zu implementieren. Dieser Algorithmus sollte mit dem in FLYA implementierten Ansatz zur automatischen Resonanzzuordnung, der automatischen NOE-Zuordnung und der Strukturrechnung mit CYANA kombiniert werden. Der in CYANA implementierte CYPICK Algorithmus ahmt den von Hand durchgeführten Ansatz nach. Bei der manuellen Methode schaut sich der Wissenschaftler zweidimensionale Konturliniendarstellungen der NMR Spektren an und entscheidet anhand verschiedener Geomtrie- und Ähnlichkeitskriterien, ob es sich um ein Signal des Proteins oder um einen Artefakt handelt. Proteinsignale sind ähnlich zu konzentrischen Ellipsen und erfüllen bestimmte geometrische Kriterien, wie zum Beispiel ungefähr kreisförmiges Aussehen nach entsprechender Skalierung der spektralen Achsen und gänzlich konvexe Formen, die Artefakte nicht aufzeigen. CYPICK bewertet die Konturlinien lokaler Extrema nach diesen Bedingungen und entscheidet anhand dieser, ob es sich um ein echtes Signal handelt oder nicht. Das zweite Ziel dieser Arbeit war es ein Maß zur Quantifizierung der Information von strukturellen NMR Distanzeinschränkungen zu entwickeln. Der sogenannte Informationsgehalt (I) ist vergleichbar mit der Auflösung in der Röntgenkristallographie. Ein weiteres Projekt dieser Dissertation beschäftigte sich mit der strukturbasierten Medikamentenentwicklung (SBDD). SBDD wird meist von der Röntgenkristallographie durchgeführt. NMR hat jedoch einige Vorteile gegenüber der Röntgenkristallographie, welche interessant für SBDD sind. Daher wurden Strategien entwickelt, die NMR für SBDD zugänglicher machen sollen.
Respiration is one of the key processes of energy transduction used by the cell. It consists of two components: electron transfer and ATP production. The electron transfer chain converts the energy released from several biochemical redox reactions into an electrochemical proton gradient across membranes. This stored energy is used as the driving force for the production of ATP by the ATP synthase. The mitochondrial electron transfer chain contains four major protein complexes called complexes I-IV, with counting starting at the lower side of the redox potentials. It has been discussed for a long time how these protein complexes are organized in the membranes. Do they diffuse freely in the membrane? Alternatively, do they form a supercomplex built up of several neighboring complexes? The evidence supporting the free diffusion mode is that both electron transfer intermediates (cytochrome c and quinone) behave as “pool”. However, respiratory supercomplexes have been detected in membranes from bacteria, fungi, yeast, plant and animal during the last decade, and sometimes the respiratory complexes are only stable inside a supercomplex. Therefore, the idea of supercomplex formation has become more popular. The argument that the supercomplex arises from solubilization and is a detergent artifact could be rejected because: 1) supercomplexes can be isolated from many organisms in an active form; 2) supercomplexes have been proven to stabilize the individual complexes in some cases; 3) supercomplexes can be very stable after chromatographic isolation in some cases....
Biophysical studies of the translation-regulating add adenine riboswitch from Vibrio vulnificus
(2017)
Bacterial gene expression can be regulated at mRNA level by cis-acting mRNA elements termed riboswitches. Riboswitches operate by conformational switching between a ligand-free and a ligand-bound state with different structures that either activate or inhibit gene expression. This PhD thesis contributes to the molecular level understanding of full-length purine riboswitches. It presents biophysical investigations on the ligand-dependent folding of the full-length translation-regulating add adenine riboswitch from the gram-negative human pathogenic marine bacterium Vibrio vulnificus (Asw). Asw has the typical bipartite riboswitch architecture with a 5’ ligand-sensing aptamer domain and a 3’ regulatory domain termed expression platform. According to the working hypothesis, Asw employs a unique thermodynamically-controlled 3-state conformational switching mechanism between an apoB, an apoA and a holo conformation to regulate translation initiation in a temperature-compensated manner. The two apo conformations are the putative translation-OFF states and the holo conformation is the putative translation-ON state of Asw. In the main project of this PhD thesis, an integrated nuclear magnetic resonance (NMR) and smFRET spectroscopic study of the full-length 112-nucleotide Asw (112Asw) was performed. The adenine-dependent folding of 112Asw was monitored at the level of base pairing interactions by NMR of the RNA imino protons, and at the level of three long-range intramolecular distances by smFRET of immobilized molecules. The integrated NMR and smFRET spectroscopic study of 112Asw yielded two major findings. First, NMR and smFRET both revealed that adenine binding to 112Asw impedes apoB formation by stabilizing the apoA secondary structure in the holo conformation without modulating tertiary structural interactions between the two riboswitch domains. This highlights the central role of competitive P1 and P4 helix formation at the interface of the aptamer and the expression platform for switching the accessibility of the ribosome binding site of 112Asw. Moreover, it strongly corroborates the hypothesis that purine riboswitches in general operate according to the key principle of a spatially decoupled secondary structural allosteric switch that proceeds without ligand-induced tertiary structural interactions between the aptamer domain and the expression platform. Second, it was uncovered by smFRET that the apoA and the holo conformation of 112Asw do not adopt a single folding state at near-physiological Mg2+ concentration. Instead, apoA and holo exhibit a persistent dynamic equilibrium between substates with an undocked (U), a short-lived docked (D1; ~s) and a Mg2+-bound long-lived docked (D2; ~10 s) aptamer kissing loop motif. In the holo conformation, the fractional population of the long-lived docked substate is ~2-fold increased compared to the apoA conformation, but undocked and docked substates are still comparably stable. The here described multiple folding states of the apoA and the holo conformation might have regulatory properties that are in between the apoB translation-OFF state and the holo-D2 translation-ON state. Additonally, an integrated NMR and smFRET analysis of 127-nucleotide Asw (127Asw) is presented. Compared to 112Asw, 127Asw is 3’-elongated by 15 nucleotides of the adenosine deaminase encoding sequence of the add gene from Vibrio vulnificus. 127Asw was chosen as mRNA template for future investigations of the interaction between Asw and the 30S ribosomal subunit. The NMR spectra of 127Asw demonstrated that 127Asw has the same overall secondary structure as 112Asw. Like for 112Asw, the combined NMR and smFRET analysis of 127Asw showed that adenine binding impedes apoB formation and stabilizes a long-lived docked aptamer kissing loop fold. However, compared to 112Asw, 127Asw has a destabilized aptamer kissing loop motif and a stabilized P4 helix in the expression platform. Finally, ligand-observed studies of the transient encounter complex between Asw and the near-cognate ligand hypoxanthine are described. By competition binding WaterLOGSY NMR experiments with hypoxanthine and the adenine analogue 2,6-diaminopurine, it could be shown that hypoxanthine binds to the same binding site of 112Asw as the cognate ligand adenine. The hypoxanthine binding constant measured with the WaterLOGSY method is in the low mM range (1.8 mM) and substantially exceeds the physiological hypoxanthine concentration in E. coli (~0.3 mM), thus ruling out that hypoxanthine binding can significantly impact the translational regulation of Asw in vivo. Also, preliminary FTIR difference spectra of 13C,15N-labelled and unlabelled hypoxanthine in complex with the pbuE adenine riboswitch aptamer and the xpt guanine riboswitch aptamer are discussed. These spectra showed a pattern of multiple IR bands that appeared to be characteristic for the respective complex.
Membrane proteins are biological macromolecules that are located in a cell’s membrane and are responsible for essential functions within an organism, which makes them to prominent drug targets. The extraction of membrane proteins from the hydrophobic membrane bilayer to determine high-resolution crystal structures is a difficult task and only 2% of all solved proteins structures are membrane proteins. Computational methods may help to gain deeper insights into membrane protein structures and their functions. This study will give an overview of such computational methods on a representative set of membrane proteins and will provide ideas for future computational and experimental research on membrane proteins.
In a first step (chapter 2), I updated an earlier, manually-curated data set of homologous membrane proteins (HOMEP) to more recent versions in 2010 (HOMEP2) and 2013 (HOMEP3) using an automated clustering approach. High-resolution structures of membrane proteins listed in the PDB_TM database were structurally aligned and subsequently clustered using structural similarity scores. Both data sets were used as a standard gold reference set for subsequent work.
Subsequently, I have updated and applied the sequence alignment program AlignMe to determine protein descriptors that are suitable for detecting evolutionary relationship between homologous a-helical membrane proteins. Single input descriptors were tested alone and in combination with each other in different modes of AlignMe by optimizing gap penalties on the HOMEP2 data set. Most accurate alignments and homology models on the HOMEP2 data set were observed when using position-specific substitution information (P), secondary structure propensities (S) and transmembrane propensities (T) in the AlignMe PST mode. An evaluation on an independent reference set of membrane protein sequence alignments from the BAliBASE collection showed that different modes of AlignMe are suitable for different sequence similarity levels. The AlignMe PST mode improved the alignment accuracy significantly for distantly related proteins, whereas for closely-related proteins from the BAliBASE set the AlignMe PS mode was more suitable. This work was published in March 2013 in PLOS ONE. In order to allow also an easier usage of the AlignMe program, I have implemented a web server of AlignMe (chapter 4) that provides the optimized settings and gap penalties for the AlignMe P, PS and PST modes. A comparison to other recent alignment web server shows that the alignments of AlignMe are similar or even more accurate than those of other methods, especially for very distantly related proteins for which the inclusion of membrane protein information has been shown to be suitable. This work was published in the NAR web server issue in July 2014.
Although membrane-specific information has been shown to be suitable for aligning distantly related membrane proteins on a sequence level, such information was not incorporated into structural alignment programs making it unclear which method is the most suitable for aligning membrane proteins. Thus, I compared 13 widely-used pairwise structural alignment methods on an updated reference set of homologous membrane protein structures (HOMEP3) and evaluated their accuracy by building models based on the underlying sequence alignments and used scoring functions (e.g., AL4 or CAD-score) to rate the model accuracy (chapter 5). The analysis showed that fragment-based approaches such as FR-TM-align are the most useful for aligning structures of membrane proteins that have undergone large conformational changes whereas rigid approaches were more suitable for proteins that were solved in the same or a similar state. However, no method showed a significant higher accuracy than any other. Additionally, all methods lack a measure to rate the reliability of the accuracy for a specific position within a structure alignment. In order to solve these problems, I propose a consensus-type approach that combines alignments from four different methods, namely FR-TM-align, DaliLite, MATT and FATCAT and assigns a confidence value to each position of the alignment that describes the agreement between the methods. This work has been published 2015 in the journal “PROTEINS: structure, function and bioinformatics”.
Consensus alignments were then generated for each pair of proteins of the HOMEP3 data set and subsequently analyzed for single evolutionary events within membrane spanning segments and for irregular structures (e.g., 310- and p-helices) (chapter 6). Interestingly, single insertions and deletions could be observed with the help of consensus alignments in the conserved membrane-spanning segments of membrane proteins in four protein families. The detection of such single InDels might help to identify crucial residues for a proteins function.
If the biotechnological production of chemicals can further replace or support regular synthetic chemistry, industry will be able to move away from fossil oils towards renewable sources. However, in many cases the much needed adaptation of biotechnological production systems is not yet developed to the necessary level.
For processes where short fatty acids (FA) are needed, as for example in the microbial production of biofuels in the gasoline range, protein engineering had not yet delivered feasible solutions. In this thesis, several approaches to introduce chain length control on type I fatty acid synthases (FAS) were established and made available in a publication and two patents. Therein, engineering was focused on rational design based on available structural information.
First, the type I FAS from C. ammoniagenes was used as a model enzyme to probe modifications on FAS in a low complex in vitro environment in order to gain information about structure-function relationships. At this stage, engineering was conducted in several rounds, first addressing possible ways to alter product distributions by changing substrate affinities through concise mutations in binding channels. Several FAS constructs were generated ranging from first successes, where short FA were produced as side products, to FAS where native chain length programming was overwritten and only short FA were produced.
Furthermore, another engineering target was addressed with the modification of domain-domain interactions on FAS. For its exploitation to direct synthesis, contact surfaces on catalytic domains were changed to interfere with acyl carrier protein binding. This channeling of the kinetic process on the enzyme led to similar successes and short FA became the primary product.
The two approaches have proven to be potent tools to introduce systems of chain length control in FAS. This rational engineering has the big advantage that it is mostly minimally invasive and due to the high conservation of de novo FA synthesis, individual mutations could easily be used in other FAS (and their organisms) as well. Even heterologous expression of modified FAS genes is feasible.
Engineering was not only tested in a defined in vitro environment and but also in S. cerevisiae as an exemplary in vivo system. The results eventually confirmed the in vitro findings and proved that the chosen engineering could be transferred to more complex systems. Even before any optimization for highest output, the titers of short FA from S. cerevisiae fermentation matched previous reports with 118 mg/L.
In sum, this work covers several layers from basic research to preliminary applications. The presented modifications to create short FA producing FAS can be a key step in synthesis pathways and will likely enable a whole range of new succeeding research. It can be seen as a valuable contribution towards establishing novel ways for the production of chemicals from renewable sources.
FUSE Binding Protein 1 (FUBP1) is a transcriptional regulator, which is overexpressed in various cancer entities, including hepatocellular carcinoma (HCC) and colorectal cancer (CRC). It fulfills pro-proliferative and anti-apoptotic functions in cancer cells, resulting in increased proliferation and reduced sensitivity towards apoptotic stimuli.
Previously, camptothecin (CPT) and its clinically used analog 7-ethyl 10hydroxycamptothecin (SN-38) were shown to inhibit FUBP1 in biophysical interaction displacement assays (AlphaScreen; surface plasmon resonance, SPR), and first insights into the cellular effects of FUBP1 inhibition were obtained. CPT and SN-38 are known to potently inhibit topoisomerase 1 (TOP 1), and until today, these inhibitors were thought to be specific for this target. This could be disproved by our FUBP1 binding studies. An open issue, which is addressed in this thesis, was the contribution of FUBP1 inhibition to SN-38-mediated apoptosis apoptosis.
During this thesis, a low micromolar efficacy of CPT/SN-38-induced inhibition of FUBP1 binding to the Far Upstream Sequence Element (FUSE) oligonucleotide of p21 was determined. Furthermore, FUBP1 was for the first time shown to directly interact with a potential FUSE sequence upstream of the transcription start in pro-apoptotic gene BIK. In proof of-principle experiments, an effective inhibition of the binding of FUBP1 to the FUSE BIK DNA by CPT/SN-38 was verified.
One of the main goals of this thesis was to further elucidate the contribution of cellular FUBP1-inhibition by CPT/SN-38 to the anti-cancer potential of these substances. For this purpose, the TOP 1 mutant and TOP 1 wild type colorectal cancer sub-cell lines HCT116 G7 and HCT116 S were used. CPT/SN-38 was shown to induce apoptosis in single and combinatorial treatments with mitomycin c (MMC), independently of the TOP 1 mutation status of the cells. Furthermore, a prominent induction of a FUBP1 target gene signature was observed upon treatment of both cell lines with CPT/SN-38. Consequently, CPT/SN-38 was able to fulfill its anticancer effects in these cells, although TOP 1 could not be the main target in the mutant cell line.
In a second approach to gain indirect evidence for FUBP1 dependent effects of CPT/SN-38, the TOP 1-specific inhibitors topotecan (TTN) and β lapachone (BL) were used for the treatment of HCC and CRC cell lines. Interestingly, the TOP 1 inhibitors TTN and BL exhibited a reduced potency in apoptosis induction compared to the dual (FUBP1 and TOP 1) inhibitor SN-38.
Finally, two independent screens for a specific FUBP1 inhibitor were performed. In the first approach, a small number of structural and functional CPT-derivatives that exhibited a reduced inhibitory potential against TOP 1, were tested for their ability to interfere with the FUBP1/FUSE binding. Two particular indenoisoquinoline derivatives revealed potent in vitro inhibition of FUBP1 with low micromolar IC50 values.
In a second approach, previously identified candidate FUBP1 inhibitors that had been isolated from the Maybridge Hit Finder library served as lead structures for a structure activity relationship (SAR) study of the inhibition of FUBP1 binding to the FUSE oligonucleotide. After two cycles of optimization, a medium-potent FUBP1 inhibitor was obtained that induced effective deregulation of FUBP1 target genes in cell culture experiments.
Protein synthesis is a central process within every living cell, where information embodied in the nucleotide sequence of the mRNA is translated into the primary sequence of proteins. The translation procedure comprises four steps: initiation, elongation, termination, and recycling. Ribosome recycling orchestrated by the ATP‐binding cassette (ABC) protein ABCE1, renders mRNA translation into a cyclic process, connecting termination with re initiation. In Archaea and Eukarya, the ABC protein ABCE1 catalyzes ribosome recycling by splitting the ribosome (80S/70S) into the small 40S/30S and large 60S/50S subunits, providing them for the next translation round.
The ABC‐type ATPase one of the most conserved proteins, present in all Archaea and Eukarya, but not in Bacteria, is essential for life in all organisms examined so far. ABCE1 was initially identified as RNase L inhibitor (Rli1), involved in the antiviral RNA immunity, and as host protein 68 (HP68) playing a role in HIV capsid assembly. However, the strong sequence conservation of ABCE1 points towards a more fundamental function within cell homeostasis, which was found by its involvement in various translation processes. ABCE1 turned out to be the major ribosome recycling factor indispensable for life in Eukarya and Archaea, being involved in canonical translation, mRNA surveillance, ribosome biogenesis, and translation initiation.
Recent functional and structural data provided first insights into the mechanism of ABCE1 in ribosome recycling. The nucleotide‐binding domains (NBDs) sandwich two ATP molecules in the NBD1‐NBD2 interface causing an NBD engagement, which is released upon ATP hydrolysis. In case of ABCE1, this ATP‐dependent tweezer‐like motion of the NBDs transfers mechanical energy to the ribosome and tears the subunits apart. The FeS‐cluster domain may swing out of the NBD cleft into the inter‐subunit space of the ribosome, which drives the subunits apart either directly or via the bound a/eRF1. Hence, the subunits are released and the post‐splitting complex (PSC, 40S/30S∙ABCE1∙ATP) is available for re‐initiation events, presumably occurring via the known interactions of ABCE1with initiation factors.
One of the most crucial aspects of this model is the nucleotide‐dependent conformational switch of ABCE1, which drives ribosomal subunit splitting. However, the conformational states, which ABCE1 undergoes during ribosome recycling, including their mechanistic importance for its diverse functions, remain unknown. Further, the exact role and movement of the essential FeScluster domain during ribosome recycling are not yet understood. Additional, it remains elusive where ABCE1 is bound in the post‐splitting complex and how the splitting mechanism is regulated concerning the asymmetric NBDs and the coupling of nucleotide binding with NBD closing and ATP hydrolysis.
Thus, in order to monitor the conformational dynamics of the ribosome recycling factor ABCE1 two complementing methods in structural biology, namely single‐molecule based Förster resonance energy transfer (smFRET) and pulsed electron‐electron double resonance (PELDOR) spectroscopy were applied.
Single‐molecule FRET as an integrated biophysical approach based on Förster resonance energy transfer and single‐molecule detection was used to understand the fundamental molecular principles of ABCE1. Contrary to the anticipated two‐state model of ABC proteins, it was shown in this thesis that both nucleotide‐binding sites of ABCE1 are always in a dynamic equilibrium between conformational states with distinct properties: open, intermediate, and closed. The equilibrium in the two nucleotide‐binding sites is distinctly affected when ABCE1 interacts with ribosomal subunits and nucleotides. While ABCE1 can adopt all three conformational states in its free or 30S bound situation, the closed state has the highest affinity for 30S subunit. Further, dissociation of ABCE1 from the small ribosomal subunit, a step that completes the recycling process, is followed by the opening of the NBSs. Hence, the current findings have important implications not only for ribosome recycling but represent a new paradigm for the molecular mechanisms of twin‐ATPases.
The complementing PELDOR measurements provide the advantage of high distance precision and reliability studying macromolecular complexes. Distance distributions of a number of ABCE1 variants even bound to the 1‐MDa post‐splitting complex (30S∙ABCE1∙AMP‐PNP), composed of the 16S rRNA, 28 ribosomal proteins, and ABCE1, was analyzed. Thus, the available crystal structures of ABCE1 in the open state were validated, since all distances of ABCE1 measured in this study perfectly correspond to this crystallized state. Unfortunately, ABCE1 could not be trapped in the closed state under the experimental conditions applied, although plenty different approaches to stabilize this state were performed.
In the second part of this study the architecture yet unknown of the 1‐MDa post splitting complex (40S/30S∙ABCE1∙ATP), concerning especially the ABCE1 binding site and its interactions with translational proteins, was probed by a method, which combines chemical cross linking with mass‐spectrometry (XL‐MS). Following this approach, it was demonstrated that ABCE1 remains bound at the translational GTPase‐binding site after ribosome splitting, contacting the S24e protein of the small subunit. The platform for the intensive contacts to the small ribosomal subunit is thereby provided by the unique helix‐loop‐helix motif of ABCE1. Notably, the FeScluster domain of ABCE1 undergoes a large rotational and translational rearrangement towards the small ribosomal subunit S12 upon nucleotide‐dependent closure of the NBDs. Thus, a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling processes, was reconstituted and structurally analyzed.
In view of the diverse functionalities of RNA, the search for tools suitable for regulating and understanding RNA grows continuously. Dysfunction of RNA controlled processes can lead to diseases, calling for external regulation mechanisms – a difficult task in view of the complexity of biological systems. One of the recently developed methods that aim to systematically control RNA relates to photoregulation. Here, the RNA functions are triggered by photochromic molecules – for example, azobenzene or spiropyran – which are bound either covalently or non-covalently to the target RNA. This is a flexible approach, which can be improved by using suitably substituted chromophores. However, many issues regarding the details of photocontrol are still open. A detailed understanding of the mechanism of photocontrol is therefore of crucial importance.
The present thesis explores theoretical approaches to the photocontrol of RNA, focussing upon azobenzene chromophores covalently bound to RNA. The aim of the thesis is to characterize, at a molecular level, the effect of trans-to-cis isomerization of the azobenzene chromophore on RNA, and thus understand the mechanism of RNA unfolding triggered by azobenzene isomerization. In particular, we attempt to answer the following questions:
How does azobenzene isomerization happen in an RNA environment, i.e., how is
the isomerization influenced by the local RNA environment?
Conversely, how is RNA dynamics, on a longer time scale, affected by azobenzene attachment and photoisomerization?
Further, can regulation be enhanced by substituted azobenzenes? And, does simulation yield a picture that is consistent with experiment?
Due to the very different times scales of azobenzene isomerization (femtoseconds to picoseconds) and the much slower RNA response (nanoseconds to milliseconds), complementary techniques have been chosen: (i) hybrid quantum-classical approaches, i.e., on-the-fly Quantum Mechanics/Molecular Mechanics (QM/MM), to characterize the isomerization and RNA response on an ultrafast time scale, and (ii) molecular dynamics with enhanced sampling techniques, in particular, Replica Exchange MD (REMD), to explore longer time scales where the effect of RNA unfolding becomes manifest. Furthermore, substituent effects on azobenzene were separately investigated, in collaboration with two experimental groups.
The first part of this thesis is focused on the conformational influence of azobenzene on a small RNA hairpin on longer time scales using REMD simulations. In accordance with experiment, it is found that both the trans and cis form of azobenzene destabilize the RNA system. Trans azobenzene stays stacked in the double strand, whereas the cis form flips out of the RNA. These stacking interactions are the main reason why a trans azobenzene-RNA-complex is more stable than a cis-azobenzene-RNA-complex. Furthermore, the loop region of the RNA hairpin is highly destabilized by the intercalation of azobenzene.
In the second part, on-the-fly QM/MM simulations of the same azobenzene substituted hairpin are undertaken. These simulations use a surface hopping (SH) algorithm in conjunction with hybrid QM/MM electronic structure calculations to give a complete picture of the isomerization process on a picosecond time scale. It is shown that, due to the constraints of the RNA environment, the isomerization time of the azobenzene chromophore is significantly increased (from 300 femtoseconds in the gas phase to around 20 picoseconds in the RNA environment), and the isomerization yield is low. To the best of our knowledge, these are the first QM/MM simulations reported for azobenzene in a nucleic acid environment.
In the third and final part of this thesis, the properties of substituted azobenzenes have been explored, in collaboration with two experimental groups at the department. In particular, para- and meta-hydroxy substituted azobenzenes were suggested as improved photoswitches for the photoregulation of RNA, but spectroscopic investigations showed that isomerization was inefficient in some of the investigated species. Therefore, we investigated the photoisomerisation pathway of the keto/enol-form of para- and meta-hydroxy-azobenzenes by Time-Dependent Density Functional Theory (TDDFT) calculations. These calculations show that the competing keto/enol-tautomerism can result in an unstable cis form, making these substituted chromophores unsuitable as photoswitches.
Overall, the present thesis has contributed to obtaining a molecular-level understanding of photocontrol in azobenzene substituted RNAs, showing that theory and simulations can provide useful guidance for new experiments.
The transporter associated with antigen processing (TAP) is a heterodimeric ATP-binding cassette (ABC) transport complex, which selects peptides for export into the endoplasmic reticulum (ER) and subsequent loading onto major histocompatibility complex class I (MHC I) molecules to trigger adaptive immune responses against virally or malignantly transformed cells. Due to its pivotal role in adaptive immunity, TAP is a target for infectious diseases and malignant disorders, such as bare lymphocyte syndrome type I and cancer. A detailed knowledge about the TAP structure and transport mechanism is fundamental for the development of therapies or drugs against such diseases, but numerous aspects are insufficiently determined to date. The aim of this PhD thesis was to elucidate several structural details of TAP using powerful biochemical and biophysical methods and thereby to contribute to the understanding of the translocation machinery functionality.
High protein yields, an efficient isolation from the lipid environment and subsequent purification of a stoichiometric, stable, and functional TAP complex are prerequisites to get detailed insights into TAP functionality. The natural product digitonin is typically used as detergent to isolate TAP, but suffered from fluctuating purity and high costs. The novel detergent GDN was selected from a number of potential detergents upon their ability to isolate and purify TAP overcoming the limitations of digitonin without compromising on functional integrity. State-of-the-art biophysical techniques, such as solid-state nuclear magnetic resonance (NMR), require highly concentrated protein samples. A new and mild procedure to concentrate TAP was established within this thesis. Freeze drying is superior to conventional concentration techniques, such as ultrafiltration, resulting in TAP inactivation and aggregation already at concentrations of 10 mg/mL. This new procedure enables stabilizing TAP in a condensed glycerol matrix and to concentrate the transport complex up to 30 mg/mL active transporter. The functional integrity of the freeze-dried TAP complex was verified by determining equilibrium dissociation constants, peptide dissociation and ATP-hydrolysis rates as well as long-term stabilities identical to untreated TAP. The combined application of the detergent GDN and the freeze drying procedure facilitates the cost-efficient isolation of functional and highly concentrated TAP and enables to study the structure and mechanism of the peptide transporter TAP using modern analyses methods.
Information on peptide-TAP interactions at atomic level have not been obtained so far. This lack of knowledge hampered the mechanistic understanding of the initial steps of substrate translocation catalyzed by TAP. Dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS) solid-state NMR on highly concentrated TAP samples prepared with the freeze-drying procedure was used within this thesis to study this challenging membrane protein-substrate complex. The affinity and specificity of peptide binding by TAP are mediated by multiple recognition sites in the N- and C-terminal regions. Side-chains of positions 1, 3, and 9 are most substantially affected upon binding to TAP, revealing recognition principles of the translocation machinery. The nonamer peptide binds to TAP in an extended conformation with an N-to-C terminus distance of ~2.5 nm. Molecular docking revealed that the peptide substrate is locked with its N and C termini between TAP1 and TAP2 and adopts a tilted pose with respect to the membrane plane. The identified contact sites of TAP are consistent with results from earlier crosslinking and mutational analyses on the TAP complex.
The inadequate structure determination and insufficient knowledge about the dynamics of substrate translocation impedes a detailed comprehension of the TAP transport mechanism. Advanced biophysical methods, such as pulsed electron paramagnetic resonance (EPR) or single-molecule Förster resonance energy transfer (FRET), enable to locate the peptide-binding pocket and to elucidate dwell-times, conformational states and dynamics within the translocation cycle of TAP. The specific introduction of spin or fluorescent labels via single cysteines for such studies requires a cysteine-less TAP complex. The endogenous cysteine 213 in TAP2 remained to create a pseudo Cys-less TAP complex within this thesis due to its altered substrate repertoire when mutated to serine as shown in previous studies. Latter complex was used to introduce single-Cys mutations in the cytosolic extensions of transmembrane helices of TAP1. Their functional integrity with respect to peptide binding and translocation was comparable to pseudo Cys-less TAP. All pseudo single cysteines were efficiently labeled, but unintentionally C213TAP2 was labeled as well and TAP concomitantly inactivated. These unsatisfactory initial experiments required the generation of a functional, entirely Cys-less TAP transporter within this thesis. Therefore, C213TAP2 was replaced by all 19 proteinogenic amino acids. All analyzed mutants were capable to bind a high-affinity peptide of TAP, but with varying affinities and binding capacities. The replacement of C213 by isoleucine enabled the generation of a cysteine-less TAP complex with functional characteristics similar to the wild-type transporter and will promote the elucidation of the translocation mechanism of the peptide transporter TAP in future studies using pulsed EPR and single-molecule FRET.
The centerpiece of all neuronal processes is the synaptic transmission. It consists of a complex series of events. Two key elements are the binding of synaptic vesicles (SV) to the presynaptic membrane and the subsequent fusion of the two membranes. SV are neurotransmitter-filled membranous spheres with many integral and peripheral proteins. The synaptic SNARE complex consists of three interacting proteins, which energize and regulate the fusion of the SV membrane with the presynaptic membrane. Both processes are closely orchestrated to ensure a specific release of neurotransmitter. Already many experiments have been performed, such as genetic screens and proteome analysis of SV, to determine the functions of the various proteins involved. Nevertheless, the functions of the identified proteins are still not fully elucidated. The aim of this thesis was initially applying a tandem affinity purification (TAP) of SV to identify unknown interaction partner of SV and to determine their role. This was supposed to be performed in the model organism Caenorhabditis elegans (C. elegans). The underlying mechanisms are conserved throughout the phylogentic tree and identified interaction partners will help to understand the processes in the mammalian brain. Although there is no neuron-rich tissue in C. elegans as in other model organisms, the diverse genetic methods allows a rapid creation of modified organisms and a prompt determination of the function of identified proteins. The integral SV protein synaptogyrin has been fused to a TAP-tag. The TAP-tag consists of a ProteinA, a TEV protease cleavage site and a calmodulin binding peptide (CBP). Both affinity purification steps are performed sequentially and allow a highly specific native purification of proteins and their interaction partners. Due to technical difficulties the purification strategy was modified several times during the course of this thesis and then finally abandoned for a more promising project, the SNARE complex purification. In conclusion, one of the reasons was the necessary lack of detergent.
The amended aim of this thesis has been the TAP of solubilized SNARE complex to identify unknown interaction partner and to determine their role. In order to increase the specificity of the purification, in terms of formed complexes, the two SNARE subunits, synaptobrevin (SNB-1 in C. elegans) and syntaxin (UNC-64 in C. elegans), were separately fused to the different affinity tags. As the modifications of the proteins could impair their function and lead to false interaction partners, their functionality was tested. For this purpose, the corresponding fusion constructs were expressed in strains with mutated snb¬1 and unc-64. Non-functional synaptic proteins display an altered course of paralysis in an aldicarb assay. The fusion proteins which were expressed in their respective mutant strains displayed a near to wild-type (WT) behavior in contrast to the naive mutant strains. Multiple TAP demonstrated SNB-1 signals in Western blot analysis and complex sets of proteins in the final elution step in a silver staining of SDS-PAGEs. These samples were sent with negative control (WT purification) for MS analysis to various cooperation partners. 119 proteins were identified which appeared only in data sets with SNARE proteins and not in WT samples. If proteins were detected in ≥ 2 SNARE positive MS analysis and had known neural functions or homologies to neuronal proteins in other species, they were selected for further analysis. These candidates were knocked down by RNAi and tested for synaptic function in a following aldicarb assay. The treatment with their specific RNAi resulted for mca-3 in a strong resistance, while frm-2, snap-29, ekl-6, klb-8, mdh-2, pfk-2, piki-1 and vamp-8 resulted in hypersensitivity. The most responsive genes frm-2, snap-29 and mca-3 were examined, whether they displayed a co-localization together with synaptobrevin in promoter fusion constructs or functional fusion constructs. In fluorescence microscopy images only MCA-3::YFP demonstrated neuronal expression.
In order to substantiate the synaptic nature and functionality of the MCA-3::YFP a swimming assay was performed. Here, fusion construct expressing strains, which contained mutated mca-3, were compared with untreated mutant strains and WT strains according to their behavior. In this swimming assay a partial restoration of WT behavior was shown in the MCA-3::YFP expressing mutant strains. Based on these data, we discovered with MCA 3 a new interaction partner of the SNARE complex. MCA-3 is a plasma membrane Ca2+-ATPase and was initially seen only in their role in the endocytosis. Its new putative role is the reduction of Ca2+ concentration at the bound SNARE complex. Since an interaction of syntaxin with Ca2+ channels has been demonstrated, it would be comprehensible to reduce the local concentration of Ca2+ to a minimum by tethering Ca2+ transporters to the SNARE complex.
Development and implementation of novel optogenetic tools in the nematode Caenorhabditis elegans
(2016)
Optogenetics, though still only a decade old field, has revolutionized research in neurobiology. It comprises of methods that allow control of neural activity by light in a minimally-invasive, spatio-temporally precise and genetically targeted manner. The optogenetic actuators or the genetically encoded light sensitive elements mediate light driven manipulation of membrane potential, intracellular signalling, neuronal network activity and behaviour (Fenno et al. 2011; Dugué et al. 2012). These techniques have been particularly useful for dissecting neural circuits and behaviour in the transparent and genetically amenable nematode model system Caenorhabditis elegans (Husson et al. 2013; Fang-yen et al. 2015).
In fact, C. elegans was the first living organism in which microbial rhodopsin based optogenetic tools (Channelrhodopsin-2 or ChR2, and Halorhodopsin or NpHR) were successfully implemented and bimodal 'remote' control of behaviour was achieved (Nagel et al. 2005; Zhang et al. 2007). Since then it has been a prominent model for the development and application of novel optogenetic tools and techniques, especially in the nervous system which comprises of 302 neurons and is organised in a hierarchical organization. The environmental stimuli are sensed by the sensory neurons, leading to the processing of information by the downstream interneurons, that relay to motor neurons which in-turn synapse onto muscles that drive the movement-based responses.
The microbial rhodopsins like ChR2 and NpHR mediate light driven depolarization and hyperpolarization, respectively and thereby activate or inhibit neural activity. However, they do not allow local control of membrane potential as they are expressed all over the plasma membrane of the cell rather than being restricted to specific domains, for example synaptic sites. Moreover, they completely over-ride the intrinsic activity of the cell, completely bypassing the signal transduction processes inside the cell. Thus, in order to study intracellular signalling and to answer questions pertaining to the endogenous role of receptors and channels in an in-vivo context, the optogenetic tool-kit needs to be expanded.
This thesis aimed at developing and implementing novel optogenetic tools in C. elegans that allow for sub-cellular signalling control as well as endogenous receptor control. These are: two light activated guanylyl cyclases (bPGC and BeCyclOp) to modify cyclic guanosine monophosphate (cGMP) mediated signalling in the sensory neurons, as well as attempts towards rendering endogenous C. elegans receptors - glutamate receptor (GLR-3/-6), acetylcholine receptor (ACR-16), glutamate gated chloride channel (GLC-1) light switchable and to understand their biological function in-vivo.
Organisms respond to sensory cues by activation of a primary receptor followed by relay of information downstream to effector targets by secondary signalling molecules. cGMP is a widely used 2nd messenger in cellular signaling, acting via protein kinase G or cyclic nucleotide gated (CNG) channels. In sensory neurons, cGMP allows for signal modulation and amplification, before depolarization. Chemo-, thermo-, and oxygen-sensation in C. elegans involve sensory neurons that use cGMP as the main 2nd messenger. For example, ASJ is the pheromone sensing neuron regulating larval development, AWC is the chemosensory neuron responding to volatile odours and BAG senses oxygen and carbon dioxide in the environment. In these neurons, cGMP acts downstream of the GPCRs and functions by activating cationic TAX-2/-4 CNG channels, thereby depolarising the sensory neuron. Manipulating cGMP levels is required to access signalling between sensation and sensory neuron depolarization, thereby provide insights into signal encoding. We achieve this by implementing two photo-activatable guanylyl cyclases - 1) a mutated version of Beggiatoa sp. bacterial light-activated adenylyl cyclase, with specificity for GTP (Ryu et al. 2010), termed BlgC or bPGC (Beggiatoa photoactivated guanylyl cyclase) and 2) guanylyl cyclase rhodopsin (Avelar et al. 2014) from Blastocladiella emersonii (BeCyclOp).
bPGC is a BLUF (blue light sensing using flavin) domain containing cyclase which uses FAD as the co-factor and catalyses the synthesis of cGMP from GTP upon activation by blue light. Prior to implementation in sensory neurons, a simpler heterologous system with co-expression of the TAX-2/-4 CNG channel in C. elegans body wall muscle (BWM) was used. The cGMP generated by the light activated cyclases activates the CNG channel leading to the muscle depolarization, thereby causing changes in body length which can be easily scored.
Für die Optimierung sowie Entwicklung lichtsteuerbarer Systeme für biologische Anwendungen oder neue Materialien ist ein detailliertes Verständnis der zugrunde liegenden komplexen, lichtinduzierten Prozesse eine Voraussetzung. Die Verwendung von Photoschaltern in Makromolekülen ermöglicht eine zeitliche und örtliche Kontrolle über strukturelle Änderungen sowie die entsprechend folgenden (biologischen) Funktionen durch die Verwendung von Licht als externem Auslöser.
Ein wichtiger Bestandteil dieser Arbeit befasst sich mit der Entwicklung eines auf Licht reagierenden Riboschalters, welcher die gezielte Kontrolle über Genexpression ermöglicht. Hierzu wurde eine spektroskopische Charakterisierung von verschiedenen Photoschaltern bezüglich einer Verwendung als biologischer Ligand sowie der Wechselwirkungen zwischen Azobenzolen und RNA, auch hinsichtlich ihrer Bindungsdynamiken durchgeführt. Zunächst wurde die hohe Abhängigkeit der (photo-)chemischen Eigenschaften der Azobenzole von der Wahl der Substituenten untersucht, wobei besonders die Anwendung in wässrigem Milieu betrachtet wurde. In einer detaillierten (zeitaufgelösten) Studie wurde der positionsabhängige Einfluss der Hydroxy-Substitution von Azobenzolen auf die Photoisomerisierung in wässriger Lösung untersucht. Für eine ortho-Substitution ergab sich hierbei ein alternativer Deaktivierungskanal nach Photoanregung, welcher stärker ausgeprägt ist als die Isomerisierung. Hierbei wird ein intramolekularer Protontransfer im angeregten Zustand (ESIPT) beobachtet, welcher mit einer Zeitkonstante von 0.3 ps beschrieben werden kann und in einer Keto-Spezies resultiert. Eine Keto-Enol-Tautomerie konnte für die para-Hydroxy-Substitution schon im Grundzustand beobachtet werden. Somit können beide Spezies gezielt adressiert werden. Durch Acetylierung der Hydroxygruppe verlangsamt sich die thermische Relaxation des cis-Isomer zu dem entsprechenden trans-Isomer signifikant ohne die Isomerisierung zu beeinträchtigen. Dementsprechend ermöglicht eine solche Acetylierung die Verwendung von bekannten Azobenzolderivaten als Photoschalter.
Zudem werden in dieser Arbeit zwei verschiedene Herangehensweisen in der Entwicklung eines Riboschalters beschrieben, welcher sich durch Licht regulieren lässt.
Diese sind durch kovalentes bzw. nicht-kovalentes Einbringen eines Azobenzolderivats in die RNA Struktur charakterisiert. Ein neuer Linker, welcher auf einer Desoxyribose-Struktur beruht, wird für die kovalente Anbindung des Azobenzols an den RNA Strang präsentiert, welcher eine licht-induzierte Dehybridisierung ermöglichen soll. Eine außergewöhnlich hohe Schaltamplitude mit einem cis-Gehalt von etwa 90% konnte für das Azobenzol im RNA Einzelstrang schon bei Raumtemperatur ermittelt werden. Zudem wurde der Einfluss des Photoschalters sowie der benachbarten Nukleotide in der RNA auf die Stabilität der RNA Doppelhelix untersucht. Die zweite Vorgehensweise beruht auf einer nicht-kovalenten Bindung zwischen einem Azobenzolderivat und einem RNA-Aptamer, welche lediglich für eines der Photoisomere ermöglicht wird, wodurch eine örtliche und zeitliche Kontrolle der Ligandenbindung der RNA erfolgt. Im Rahmen dieser Arbeit war es möglich zwei verschiedene photoschaltbare RNA Aptamere zu identifizieren und zu untersuchen, welche eine hohe Spezifität und Affinität aufweisen. Zudem wurde die Photoisomerisierung des Azobenzols innerhalb der RNA-Struktur sowie daraus resultierende lichtinduzierte Konformationsänderungen der RNA mittels zeitaufgelöster Anreg-/Abtastspektroskopie untersucht. Die daraus resultierende Dynamik der photoinduzierten Ligandenbindung sollte eine weitere gezielte Optimierung lichtschaltbarer biologischer Systeme erlauben.
Der zweite Teil dieser Arbeit beschäftigt sich mit der zeitaufgelösten Untersuchung eines photoschaltbaren Foldamers. Speziell wurde der strukturelle Übergang des OmPE-Foldamers 10-5 zwischen einer definierten helikalen und einer ungefalteten Konformation auf Grund der Photoisomerisierung der, in das Rückgrat integrierten, Azobenzole untersucht.
Dabei konnten die frühen (Ent-)Faltungsmechanismen des Foldamers im sub-Nanosekunden-Zeitbereich beobachtet werden, welche durch quantenmechanische Rechnungen unterstützt werden konnten. Darüberhinaus, war es möglich einen Anregungsenergietransfer vom PE-Rückgrat des Foldamers auf die Azobenzole nachzuweisen, welcher die Lebensdauer der angeregten Zustände des Systems signifikant verkürzt.
Diese Arbeit liefert wichtige Informationen zu den Reaktionspfaden, den gezielten Wechselwirkungen zwischen Photoschaltern und größeren organischen Molekülen, sowie den daraus resultierenden lichtinduzierten strukturellen Änderungen durch die Anwendung einer Vielzahl an (zeitaufgelösten) spektroskopischen Methoden. Diese Ergebnisse tragen zum weiteren Verständnis komplexer Prozesse in biologischem sowie nicht-biologischem Zusammenhang und somit zu einer weiterführenden Entwicklung neuer Systeme bei.
Metal-organic frameworks (MOFs) have emerged as a promising class of crystalline porous inorganic-organic hybrid materials showing a wide range of applications. In order to realize the integration of MOFs into specific devices, this thesis mainly focuses on the controlled growth and the properties of highly oriented surface-mounted metal-organic frameworks (SURMOFs).
The stepwise layer-by-layer (LbL) growth method exhibits vast advantages for the controllable growth of SURMOFs regarding the crystallite orientation, film thickness and homogeneity. However, up to date, only a few MOFs have been demonstrated to be suited for this protocol. So the first project of this thesis was designed to extend the applicability of the LbL growth. To this end, a semi-rigid linker based [Cu2(sdb)2(bipy)] (sdb = 4,4’-sulfonylbiphenyl dicarboxylate; bipy = 4,4’-bipyridine) MOF was chosen. Employing the LbL growth, [Cu2(sdb)2(bipy)] SURMOFs were successfully grown onto both pyridyl- and carboxyl-terminated surfaces at the temperature range of 15-65 °C. Interestingly, the orientation of the SURMOFs largely depends on temperature on both surfaces. At low temperatures (below 40 °C), SURMOFs with exclusive [010] orientation are obtained. In contrast, at high temperatures (40-65 °C), [001] oriented SURMOF growth is favored. A novel growth mode was demonstrated, which is, instead of surface chemistry, the temperature-induced ripening processes and the tendency to minimize surface energies can dominate the SURMOF growth.
Inspired by the advantages of LbL deposition of isoreticular SURMOFs, the second project was conceived to grow multivariate SURMOFs (MTV-SURMOFs) using mixed dicarboxylate linkers. We advance a hypothesis that linker acidity (expressed by the pKa values) may have an influence on the oriented growth of MTV-SURMOFs. To test the hypothesis, seven isoreticular [Cu2L2(dabco)] (L = single kind of dicarboxylate linker; dabco = 1,4-diazabicyclo[2.2.2]octane) SURMOFs were grown onto pyridyl-terminated surfaces at 60 °C. The quality of [001] orientation is greatly affected by the acidity of the linkers. With this observation, we deposited a series of [Cu2Lm2(dabco)] (Lm = mixed dicarboxylate linkers) SURMOFs under the same conditions. [Cu2Lm2(dabco)] SURMOFs with exclusive [001] orientation are obtained when the growth solution contains two linkers of relatively high pKa value or more than two kinds of linkers (independent of the pKa values), while the mixtures of ligands with relatively low pKa values or a high content of low pKa valued linkers can result in mis-oriented growth of SURMOFs with unexpected [100] orientation.
Moreover, the LbL growth shows enormous potential in the rational construction of functional SURMOFs. Therefore, the third project of this thesis was devised to deposit SURMOFs containing redox-active species. For this, the 4,4’-biphenyldicarboxylic acid (H2(bpdc)) linker was functionalized with ferrocene (Fc) and dimethyl ferrocene (Me2Fc) moieties. [Cu2(bpdc-amide-Fc)2(dabco)] SURMOF (Fc-SURMOF) is perfectly grown along the [100] direction, while mis-oriented growth of [Cu2(bpdc-amide-Me2Fc)2(dabco)] SURMOF (Me2Fc-SURMOF) was observed. Surprisingly, Fc-SURMOF shows excellent electrochemical properties due to the reversible oxidation and reduction of the ferrocene moieties in the oriented pores, while the Me2Fc-SURMOF was found to be a closely packed insulating layer since no extensive charge transfer is observed. A diffusion controlled mechanism of redox reaction is proposed, where the diffusion of the counter anions in the pores limits the current.
Besides the LbL growth protocol, the spin-coating technique is also promising for the oriented growth of SURMOFs. Driven by the specific applications, the fourth project of this thesis was planned to grow functional SURMOFs containing catalytically active units. The Keggin-type polyoxometalates (POMs) with high catalytic activities were chosen to functionalize the HKUST-1 SURMOFs. Combining the technique with methanol vapor induced growth, a series of POM functionalized HKUST-1 SURMOFs (denoted as POM@HKUST-1 SURMOFs) were controllably deposited onto pyridyl-terminated surfaces. The SURMOFs exhibit great potential as electrocatalysts in electrochemical devices due to the excellent redox properties of POMs. In addition, the PTA@HKUST-1 (PTA = phosphotungstic acid) SURMOF can be employed as an ideal platform for the selective loading of methylene blue (MB) dye with high efficiency. Owing to the strong binding between the dye molecules and the framework, the MB dye cannot be desorbed by ion exchange and MB loaded PTA@HKUST-1 SURMOF shows reliable redox properties under inert conditions, further confirming the application potential in electrochemical devices.
Ribosomes are the central cellular assembly lines for protein synthesis. To cope with the translational needs, a proliferating mammalian cell can produce up to 7500-ribosomes per minute. However, under growth limiting conditions, such as nutrient depletion, ribosome synthesis is rapidly shut down exemplifying the importance of a tight coordination between ribosome supply and cellular energy status. In addition to the quantitative regulation, a strict quality control of ribosome synthesis is equally important, because alterations in the composition or function of ribosomes can lead to a variety of pathologies. To cope with these challenges a highly regulated, multi-step pathway of ribosome biogenesis has evolved. In mammals this pathway generates the mature 80S ribosomes that comprise the large 60S and the small 40S subunits. Together they contain around 80 ribosomal proteins and the 28S, 18S, 5.8S and 5S rRNAs. The 28S, 5.8S and 5S rRNAs are assembled into the large subunit, while the 18S rRNA is part of the small subunit. The pathway of ribosome biogenesis is a multi-step cellular process, where specific stages occur in distinct subcellular compartments. Transcription of the 47S rRNA, which is the precursor for the 28S, 18S and 5.8S species, occurs in the nucleolus. Modification of distinct bases and early processing of this precursor also take place in the nucleolus. Subsequently, the 40S and 60S pre-ribosomes take separate maturation routes through the nucleoplasm before their export and final assembly in the cytoplasm. The various stages of preribosomal maturation require the constant and sequential action of a large number of non-ribosomal proteins, known as trans-acting factors. These factors coordinate the delicate remodeling of the pre-ribosomal intermediates and thereby ensure proper progression of the maturation process. The remodeling events largely depend on the dynamics of post-translational modifications, such as phosphorylation or SUMOylation. This requires that the enzymes controlling these modifications are properly targeted to their sites of activity as they fulfill their functions within specific compartments. Here we studied the regulatory principles that govern the subcellular partitioning of the SUMO-specific isopeptidase SENP3 and its associated factor PELP1. Previous work from our laboratory has delineated the importance of the SUMO system for proper ribosome biogenesis in mammalian cells. In particular, we have shown that SENP3 is critically involved in 28S rRNA formation, which is a key step for pre-60S subunit maturation. A critical involvement of SENP3 at this stage of the maturation process is in agreement with the observed enrichment of SENP3 in the nucleolus, since 28S rRNA processing is considered to occur in the nucleolus. Our subsequent work identified the nucleolar scaffold protein NPM1 and the ribosomal trans-acting factor PELP1 as bona fide substrates of SENP3. For both proteins we could demonstrate modification by SUMO2/3 and define SENP3 as the demodifying enzyme. Depletion of SENP3 enhanced the conjugation of SUMO to both proteins and concomitantly reduced conversion of the 32S pre-rRNA to the mature 28S rRNA. PELP1 is part of a larger protein complex consisting of the core components PELP1, TEX10 and WDR18. We could show that the balanced SUMOylation/deSUMOylation of PELP1 controls the nucleolar/nucleoplasmic distribution of this complex. Enhanced SUMOylation, which is observed in the absence of SENP3, triggers the nucleolar release of the complex suggesting that SENP3-mediated deSUMOylation controls the dynamics of nucleolar trans-acting factors. Based on these findings we first wanted to understand, in which cellular compartment(s) SENP3 exerts its function on 28S maturation. Next, we wanted to tackle the question how the subcellular distribution of SENP3 is controlled. Finally
we addressed the question how the SUMOylation of PELP1 determines the subnuclear distribution of the PELP1 complex. This work initially revealed that the nucleolar localization of SENP3 is crucial for proper 28S rRNA formation and 60S ribosome maturation. Importantly, we could demonstrate that the nucleolar compartmentalization of SENP3 depends on its direct physical interaction with NPM1. Further, we could show that the amino-terminal region of SENP3 is necessary for its binding to NPM1 and nucleolar recruitment. Strikingly, this interaction requires the phosphorylation of SENP3, which is brought about by the mTOR kinase. By in-vitro kinase assays and mass-spectrometric approaches we identified five serine/threonine residues within the amino-terminal region of SENP3 that are targeted by mTOR (S/T 25, 26, 141, 142, 143). We could further demonstrate by mutagenesis that these sites in SENP3 are in fact critical for the phospho-dependent binding of SENP3 to NPM1 and its nucleolar recruitment.
Consistent with these data, we found that chemical inhibitors of the mTOR kinase trigger the nucleolar release of SENP3 and impair its interaction with NPM1. Strikingly, this goes along with severe 28S rRNA maturation defects demonstrating the physiological importance of mTOR signaling in the regulation SENP3 function and rRNA processing. By specifically depleting components of the either mTORC1 or mTORC2, we could attribute the observed effects to signaling by mTORC1 rather than mTORC2. In an attempt to find the negative regulators of SENP3 phosphorylation, we identified PP1-γ as the candidate phosphatase in this pathway. We found a strong physical interaction of SENP3 with PP1-γ and observed a loss of SENP3 nucleolar localization upon ectopic expression of PP1-γ. Thus we could define mTOR/PP1-γ mediated phosphorylation/dephosphorylation of SENP3 as an important
mechanism in the control of ribosome maturation. Given that mTOR activity is controlled by nutrient availability, SENP3 functions as a sensor that couples ribosome synthesis with nutrient availability. The second part of this work delineated the role of SUMOylated PELP1 in nucleoplasmic partitioning of the SENP3-PELP1 complex. It was revealed that the AAA-ATPase MDN1 binds preferentially to SUMO modified PELP1 and likely segregates SUMOylated PELP1 from nucleolar pre-60S particles. We initially found that the PELP1 complex associates with MDN1, a factor known to be involved in the 28S rRNA maturation. Notably, depletion of MDN1 led to an enhanced accumulation of the PELP1 complex in the nucleolus and a strong association of PELP1 with pre-60S particles, suggesting that MDN1 is required for the release of this complex from the pre-ribosomes. Intriguingly, the interaction of PELP1 with MDN1 requires SUMO2/3 and SUMOylated PELP1 shows enhanced binding to MDN1 when compared to unmodified PELP1. Taken together this work provides new insights in the control of the SENP3-PELP1 complex dynamics. We could define several layers for the coordinated spatial regulation of SENP3 and the PELP1 complex. This work therefore underscores the crucial importance of dynamic post-translational modifications for the control of ribosome maturation.
Cells perform a wide range of functions such as signalling, transportation, immunoprotection and metabolism. Unravelling the molecular mechanism behind those processes will provide a platform for more targeted and rational drug design. This is achieved by discerning the structural and functional aspects of the biological macromolecules involved. This thesis discusses about the biophysical characterization of protein structures and the biological importance of protein dynamics. Membrane receptors and enzymes which are ubiquitously present in our biological systems and regulate wide variety of functions are excellent choice for such study. From a pharmaceutical point of view, receptor and enzymes are exceptionally important drug targets as they represent the major share (receptor, 30% and enzymes, 47%) of all marketed drugs. Therefore, apart from biological insights, the detailed study of receptors and enzymes will provide the basis for new pharmaceutical applications. Most information about receptor activation and enzyme activity come from the structural and functional analysis of target members of the above mentioned systems.
In “Chapter 1 – General Introduction” the readers are introduced to the world of proteins with special focus on G-protein coupled receptors (GPCRs) and methyltransferases. The first part of this chapter discusses about GPCRs with emphasis on their classification, structural features and functions. GPCRs are the most abundant membrane receptors present in mammalian cells, accounting for almost 15% of all membrane proteins. The GPCR superfamily consists of ~800 members and can be subdivided into six classes (A-F). Class A containing rhodopsin, peptide hormones, olfactory GPCRs, is the most abundant with a large share of 85% of GPCR protein family. GPCRs share a common architecture of 7 transmembrane a-helices, with different ligand binding sites. Although a variety of ligands ranging from subatomic particles (a photon) to large proteins can activate a GPCR, their mechanism of signal transduction is almost similar. There are two major signal transduction pathways identified for GPCRs: the cAMP pathway and the phosphatidylinositol pathway. The therapeutic relevance of GPCRs has also been pointed out here since a large share (30%) of modern marketed drugs target GPCRs.
In the second part of this chapter, the structural and functional characterizations of methyltransferases (MTs) are discussed in detail. Several important biological processes in cells e.g. drug metabolism, gene transcription, epigenetic regulations are modulated by methylation of targets ranging from small biomolecules to large proteins. MTs are the proteins which catalyze this methylation reaction and transfer the methyl group to an acceptor molecule through SN2 like nucleophilic substitution reaction. The MTs can be classified on the basis of the substrate atoms they methylate: O (54% of all MTs), N (23%), C (18%), S (3%) and other acceptors (such as halides; 2%). They can also be categorized into five different classes (Class I-V) depending upon distinctive structural features facilitating substrate binding or catalytic activity. Rossmann fold and SET (acronym acquired from the Drosophila Su(var)3-9 and 'Enhancer of zeste' proteins) domain are the two characteristic structural motifs commonly found in MTs. Similar to GPCRs, MTs dysfunction has been shown to be involved in various diseases including neuropsychiatric diseases and cancer. Therefore they are also interesting targets for drug development. The final part of this chapter discusses the importance of structural biology in gathering information related to structure and conformational dynamics of proteins. The two prominent biophysical techniques used in structural biology, X-ray crystallography and NMR, are discussed with focus on their advantages and limitation. The importance of NMR spectroscopic techniques to investigate different dynamic processes of protein at atomic resolution under physiological conditions is also discussed. Real time NMR spectroscopy required for the analysis of slow protein dynamic processes (protein folding, enzyme catalysis, domain rearrangement) has been explained in detail.
The second part of the thesis (Chapters 3-4), which is the cumulative part, comprises the original publications grouped into 2 chapters according to their topic:
• NMR-spectroscopic characterization of the transiently populated photointermediates of bovine rhodopsin and it’s interaction with arrestin (Chapter 3)
• Structural and biophysical characterization of PaMTH1, a putative SAM dependent O-methyltransferase from filamentous fungi Podospora anserina (Chapter 4)
Each chapter is initiated by a detailed introduction to the topic, providing the framework for the following papers. The personal contribution of this thesis’ author to each publication is stated in the introduction to the respective article.
In dieser Arbeit wurden die Strukturen von drei Membranproteinen mittels Einzelpartikel-Kryo‑Elektronenmikroskopie (Kryo‑EM) gelöst. Bei den Membranproteinen handelt es sich um den humanen TRP-Kanal Polycystin‑2, den sekundär-aktiven Transporter BetP aus Corynebacterium glutamicum und den Rotor-Ring der N‑Typ ATPase aus Burkholderia pseudomallei.
Kanäle sind Membranproteine, die Ionen durch eine Pore über die Membran diffundieren lassen. Durch einen präzisen, kanalabhängigen Regulationsmechanismus wird die Pore nur bei Bedarf geöffnet. TRP (transient receptor potential) Kanäle sind anhand von DNA-Sequenzvergleichen identifiziert worden und kommen ausschließlich in Eukaryonten vor. In dieser Arbeit lag der Fokus auf der Strukturbestimmung des humanen TRP Kanals Polycystin‑2 (PC‑2). PC‑2 wurde in einer Studie entdeckt, in der Patienten mit der autosomal dominanten Erbkrankheit „polyzystische Nierenerkrankung“ untersucht wurden. Patienten mit dieser Krankheit tragen eine Mutation in einem der beiden Gene PKD1 oder PKD2, welche für die Proteine Polycystin‑1 und ‑2 kodieren. In dieser Arbeit wurden verschiedene Deletionsmutanten von PC‑2 hergestellt und in das Genom menschlicher HEK293 GnTI‑ Zellen inseriert. Die Zellen, die PC‑2 bzw. die Deletionskonstrukte am stärksten synthetisierten, wurden isoliert und für die rekombinante Proteinherstellung verwendet. Die Expression von PC‑2 führte zu der Entstehung von kristalloidem endoplasmatischem Retikulum. Mutationsstudien in dieser Arbeit zeigen, dass diese morphologische Veränderung durch die Akkumulation von Membranproteinen, die mit sich selbst interagieren, begünstigt wird. Weiter ist es in dieser Arbeit gelungen, PC‑2 zu reinigen und die Struktur des Proteins mit Hilfe von Einzelpartikel Kryo-EM mit einer Auflösung von 4.6 Å zu bestimmen. Die Membrandomäne von PC‑2 ist sehr ähnlich zu den bekannten TRP Kanal Strukturen. Ein Vergleich der PC‑2 Struktur mit dem offenen und geschlossenen TRPV1 Kanal legt nahe, dass PC‑2 in seiner offenen Konformation gelöst wurde.
Der sekundär aktive Transporter BetP von C. glutamicum gehört zu der Familie der BCC- (betaine-carnitine-choline) Transporter und wird durch osmotischen Schock aktiviert. Nach seiner Aktivierung importiert BetP zwei Natriumionen und ein Glycinbetain Molekül. Durch die Akkumulierung von Glycinbetain in der Zelle steigt das osmotische Potential des Zytoplasmas, was den Wasserausstrom aus der Zelle stoppt. Viele Strukturen, die BetP in unterschiedlichen Stadien des Transportprozesses zeigen, konnten bereits mittels Röntgenkristallographie gelöst werden. Allerdings ist die N‑terminale Domäne für die Kristallisation entfernt worden und die C‑terminale Domäne, die komplett aufgelöst ist, ist an einem wichtigen Kristallkontakt beteiligt. Um strukturelle Informationen über die N‑ und C‑terminale Domäne ohne Kristallisationsartefakte zu erhalten, wurde in dieser Arbeit die Struktur von BetP mittels Einzelpartikel Kryo‑EM bestimmt. Die Struktur mit einer Auflösung von 6.8 Å zeigt BetP in einem zum Zytoplasma geöffneten Zustand. Der größte Unterschied zu allen Kristallstrukturen ist die Position der C‑terminalen α‑Helix, die um ~30° rotiert ist und dadurch deutlich enger am Protein zu liegen kommt. Da BetP in Abwesenheit von aktivierenden Stoffen analysiert wurde, wird vermutet, dass es sich bei der gelösten Struktur um den inaktiven Zustand von BetP handelt.
Rotierende ATPasen sind membrangebunden Enzymkomplexe, die bei der zellulären Energieumwandlung eine entscheidende Rolle einnehmen. Sie bestehen aus einem löslichen und einem membrangebundenen Teil. Während in dem löslichen Teil der zelluläre Energieträger Adenosintriphosphat (ATP) entweder synthetisiert oder hydrolysiert wird, baut der membrangebundene Teil entweder einen Ionengradienten auf oder nutzt die Energie eines existierenden Gradienten für die ATP Synthese. Ein wesentlicher Bestandteil des membrangebundenen Teils einer rotierenden ATPase ist der Rotor-Ring. Dieser transportiert Ionen über die Membran und rotiert dabei um seine eigene Achse. In dieser Arbeit wurde eine Studie fortgesetzt, die den Rotor-Ring der N‑Typ ATPase von B. pseudomallei mittels Kryo‑EM untersuchte und zeigte, dass der Rotor-Ring aus 17 identischen Untereinheiten aufgebaut ist. Damit hat die N‑Typ ATPase das größte Ionen-zu-ATP-Verhältnis aller bisher charakterisierten ATPasen. In dieser Arbeit wurde die c17 Stöchiometrie des N‑Typ ATPase Rotor-Rings bestätigt und die Struktur mittels Kryo‑EM bestimmt. Im besonderen Fokus lag dabei der Einfluss von Detergenzien auf die Strukturbestimmung. Es konnte gezeigt werden, dass die beiden Parameter Dichte und Mizellengröße der verwendeten Detergenzien ausschlaggebend für den Erfolg der Strukturbestimmung dieses sehr kleinen Membranproteins sind.
Das Enzym 5-Lipoxygenase (5-LO) spielt eine entscheidende Rolle in der Generierung von Leukotrienen. Diese fungieren als wichtige proinflammatorische Mediatoren. Darüber hinaus ist die 5-LO anhand ihrer N-terminalen Domäne in der Lage mit verschiedenen Proteinen zu interagieren. Unter den Interaktionspartnern befindet sich Dicer, ein Enzym welches für den finalen Schritt der microRNA (miRNA)-Biosynthese verantwortlich ist. MiRNA sind kurze, nicht kodierende RNA Stränge mit einer typischen Länge von etwa 23 Nukleotiden, die an der posttranskriptionalen Regulierung der Proteinbiosynthese beteiligt sind.
Ziel dieser Arbeit war es den Einfluss der 5-LO auf die miRNA-Prozessierung im zellulären Kontext zu untersuchen. Als Modellsystem wurde die MonoMac6 (MM6) Zelllinie ausgewählt. MM6-Zellen exprimieren im undifferenzierten Grundzustand nur geringe Mengen an 5-LO. Erst nach Differenzierung mittels transformierenden Wachstumsfaktors ß (TGFß) und Calcitriol kommt es zur Induktion der 5-LO Proteinbiosynthese. Darüber hinaus war es Basavarajappa et al. möglich die 5-LO-Expression in diesen Zellen mittels RNA-Interferenz stark herunter zu regulieren (Δ5-LO).
Um die Frage der Auswirkungen des 5-LO knockdowns auf die miRNA-Expression analysieren zu können, wurde ein Microarray in differenzierten Kontroll-und Δ5-LO-Zellen durchgeführt.Es wurden 37 miRNAs identifiziert deren Expression 5-LO abhängig ist. Dabei war das Niveau von 30 Vertretern in Abwesenheit der 5-LO erhöht, wohingegen die Expression von sieben miRNAs reduziert war. Unter diesen sieben herunter regulierten miRNAs befanden sich miR-99b-5p und miR-125a-5p, die einem gemeinsamen Cluster entstammen. Als Cluster wird eine Gruppe von miRNAs bezeichnet, die aus einem gemeinsamen primären Transkript (pri-miRNA) hervorgeht. Diese Eigenschaft führte zur Vermutung, dass bereits die Expression dieser pri-miRNA durch die 5-LO reguliert wird. Allerdings zeigte sichim Verlauf dieser Arbeit, dass die Expression der pri-miRNA 5-LO unabhängig verläuft. Im Gegensatz dazu wies die Zwischenstufe zwischen pri-miRNA und reifer miRNA eine reduzierte Expression in Δ5-LO Zellen auf. Für die Prozessierung dieser sogenannten precursor miRNAs (pre-miRNA) ist die Ribonuklease III Drosha verantwortlich, welche die pre-miRNA aus der jeweiligen pri-miRNAs chneidet. Das verringerte pre-miR-99b-und pre-miR-125a-Niveau ist daher ein Hinweis darauf, dass überDicerhinausmöglicherweise ebenfalls die Drosha Aktivität mittels 5-LO reguliert wird.
Des Weiteren wurde untersucht iniefern Leukotriene beziehungsweise 5-LO-Inhibitoren die Expression von miR-99b-5p und miR-125a-5p beeinflussen. Dabei stellte sich heraus, dass das miRNA-Niveau unabhängig von der vorhandenen Leukotrien-Menge ist. Das 5-LO aktivierende Protein (FLAP) besitzt dahingegen einen mit der 5-LO vergleichbaren Einfluss auf die reife miRNA. FLAP ist ein weiterer Interaktionspartner der 5-LO und essentiell für die Leukotrien-Biosynthese in vivo. Anhand von Protein-Lokalisationsstudien mittels Immunofluoreszenz konnte gezeigt werden, dass FLAP außerdem in der Lage zu sein scheint die Relokalisation der 5-LO aus dem Zytoplasma in den Nukleus einzuschränken. Im Zytoplasma ist die 5-LO in der Lage mit Dicer zu interagieren. Daten bezüglich einer Interaktion zwischen Drosha und 5-LO im Zellkern liegen bisher nicht vor. Eine etwaige Interaktion könnte allerdings helfen die reduzierten pre-miRNA Spiegel in Abwesenheit der 5-LO zu erklären.
Im Laufe dieser Arbeit wurden weiterhin die Auswirkungen von proinflammatorischen Lipopolysacchariden (LPS) auf die Prozessierung von miR-99b-5p und miR-125a-5p analysiert. Ausschließlich in Anwesenheit von 5-LO zeigte sich eine differenzierungsunabhängig gesteigerte Biosynthese der pri-und der reifen miRNA. Allerdings konnte kein Einfluss von LPS auf die 5-LO-Lokalisation beziehungsweise Expression festgestellt werden. Aufgrund dessen sind weiterführende Studien, die den Zusammenhang zwischen LPS induzierter miR-99b-5p- beziehungsweise miR-125a-5p-Biosynthese und 5-LO herstellen, nötig.
Abschließend hat sich diese Arbeit mit den Zielgenen der durch 5-LO regulierten miRNAs auseinandergesetzt. Es konnte gezeigt werden, dass in Abwesenheit von miR-99b-5p und miR-125a-5p die Freisetzung der beiden durch LPS stimulierten Zytokine Interleukin 6 (IL-6) und Tumornekrosefaktor α (TNFα) gesteigert ist. Interessanterweise besitzt TNFα einen stimulierenden Effekt auf die Leukotrien-Biosynthese. Allerdings konnte kein direkter Zusammenhang zwischen miR-99b-5p/miR-125a-5p Expression, TNFα und der 5-LO Aktivität hergestellt werden. Der Einsatz von miR-99b-5p-und miR-125a-5p-Inhibitoren zeigte keine Auswirkungen auf die Leukotrien-Biosynthese nach LPS Stimulation. Im Gegensatz dazu konnte in unstimulierten Zellen eine signifikante Aktivitätssteigerung in Abwesenheit von miR-125a-5p festgestellt werden. Diese Beobachtungen legen nahe, dass miR-125a-5p einen TNFα unabhängigen Einfluss auf die 5-LO Aktivität besitzt. In LPS stimulierten Zellen kommt es möglicherweise zu Überlagerungen dieses Effektes.
Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass 5-LO eine regulierende Funktion auf die Reifung der beiden miRNAs miR-99b-5p und miR-125a-5p aufweist. Dieser Effekt könnte einer direkten Interaktion zwischen 5-LO und Dicer zuzuschreiben sein. Des Weiteren konnte gezeigt werden, dass die Regulierung der Expression bestimmter miRNAs mittels 5-LO nicht auf deren kanonischer enzymatischer Aktivität beruht. Diese Ergebnisse schlagen eine neue Richtung der 5-LO-Forschung ein und können in Zukunft dazu beitragen 5-LO vermittelte Effekte besser charakterisieren zu können.
HDAC inhibitors (HDACI), a new class of anticancer agents, induce apoptosis in many cancer entities. JNJ-26481585 is a second generation class І HDACI that displays improved efficacy in preclinical studies compared to the established HDACI SAHA (Vorinostat). Therefore, this study aims at evaluating the effects of JNJ-26481585 on human rhabdomyosarcoma (RMS) and at identifying novel synergistic interactions of JNJ-26481585 or the more common HDACI SAHA with different anticancer drugs in RMS cells. Indeed, we show that JNJ-26481585 and SAHA significantly increase chemotherapeutic drug-induced apoptosis in embryonal and alveolar RMS cell lines, when used in combination with chemotherapeutic agents (i.e. doxorubicin, etoposide, vincristine, and cyclophosphamide) which are currently used in the clinic for the treatment of RMS.
We demonstrate that JNJ-26481585 as single agent and in combination with doxorubicin induces apoptosis, which is characterized by activation of the caspase cascade, PARP cleavage, and DNA fragmentation. Induction of caspase-dependent apoptotic cell death is confirmed by the use of the broad-range caspase inhibitor zVAD.fmk, which significantly decreases both JNJ-26481585-triggered and combination treatment-mediated DNA fragmentation, and in addition completely abrogates loss of cell viability. Importantly, JNJ-26481585 significantly inhibits tumor growth in vivo in two preclinical RMS models, i.e. the chicken chorioallantoic membrane (CAM) model and a xenograft mouse model, supporting the notion that JNJ-26481585 hampers tumor maintenance. Also, in combination with doxorubicin JNJ-26481585 significantly reduces tumor growth in in vivo experiments using the CAM model.
Mechanistically, we identify that JNJ-26481585-induced apoptosis is mediated via the intrinsic apoptotic pathway, since we observe increased loss of mitochondrial membrane potential and activation of the proapoptotic Bcl-2 family members Bax and Bak. Interestingly, we find that JNJ-26481585 triggers induction of Bim, Bmf, Puma, and Noxa on mRNA level as well as on protein level, pointing to an altered transcription of BH3-only proteins as important event for the Bax/Bak-mediated loss of mitochondrial membrane potential as well as mitochondrial apoptosis induction upon JNJ-26481585 treatment. JNJ-26481585-initiated activation of Bax and Bak is not prevented with the addition of zVAD.fmk, suggesting that JNJ-26481585 first disrupts the mitochondria and subsequently activates the caspase cascade. When JNJ-26481585 is used in combination with doxorubicin, we observe not only an increase of proapoptotic Bcl-2 proteins, but also a decrease in the level of the antiapoptotic mitochondrial proteins Bcl-2, Mcl-1, and Bcl-xL. This indicates that Bax, Bak, Bim, and Noxa are crucial for JNJ-26481585-induced as well as JNJ/Dox treatment-induced apoptosis, since RNAi mediated silencing of Bax, Bak, Bim, and Noxa significantly impedes DNA fragmentation upon those treatments.
Furthermore, ectopic overexpression of Bcl-2 profoundly impairs both JNJ-26481585 and combination treatment-mediated apoptosis, abrogates caspase cleavage, and reduces activation of Bax and Bak, underlining the hypothesis that JNJ-26481585 initially targets the mitochondria and then activates caspases.
With the more commonly used HDACI SAHA we confirm the results obtained with the HDACI JNJ-26481585, since combination treatment with SAHA and doxorubicin also induces intrinsic apoptosis, which can be significantly diminished by zVAD.fmk or ectopic overexpression of Bcl-2. Treatment with SAHA and doxorubicin also affects expression levels of pro- and antiapoptotic mitochondrial proteins, thus shifting the balance towards the proapoptotic mitochondrial machinery, resulting in Bax/Bak activation, caspase activation, and subsequently apoptosis.
Taken together, we provide evidence that the HDACIs JNJ-26481585 and SAHA are promising therapeutic agents for the treatment of RMS and that combination regimens with HDACIs represent an efficient strategy to prime RMS cells for chemotherapy-induced apoptosis. These findings have important implications for mitochondrial apoptosis-targeted therapies of RMS.
The ability of endothelial cells to properly adapt to changes in a dynamic blood perfused environment is essential to maintain the physiological function of the vascular system and of the organs. Epigenetic control of gene expression is believed to be the mechanism controlling cell-fate determination and cell-phenotype maintenance. In the thesis, two JmjC demethylases were screened for their function in endothelial biology. Both of them were proved to play a central role in angiogenesis.
The histone 3 lysine 4 demethylase JARID1B was identified as the most highly expressed demethylase in endothelial cell. Knockdown of JARID1B in human umbilical vein endothelial cells (HUVEC) attenuated cell migration, angiogenic sprouting and tube formation. Jarid1b null mice exhibited attenuated retinal angiogenesis and reduced endothelial sprout outgrowth from aortic segments. Microarray data identified that the antiangiogenic transcription factor HOXA5 was suppressed by JARID1B. Consistently, chromatin immunoprecipitation experiment revealed that JARID1B occupies and reduces the histone 3 lysine 4 methylation levels at the HOXA5 promoter, demonstrating a direct function of JARID1B in endothelial HOXA5 gene regulation. Hence, as a highly expressed JmjC protein in endothelial cells, JARID1B fundamentally maintains endothelial angiogenic phenotypes perhaps through suppression of HOXA5.
As second enzyme it was identified that the histone plant homeodomain finger protein 8 (PHF8) plays a role in endothelial angiogenic sprouting as well as tube formation and cell migration. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. To characterize the underlying mechanism, E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4, but not E2F1, expression in endothelial cells. Likewise, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Thus, it is proposed that PHF8 maintains endothelial function by controlling E2F4 expression. On the other hand, microarray and subsequent qPCR validation revealed that the expressions of small nuclear RNAs (snRNAs) were regulated by PHF8. Co-immunoprecipitation experiment demonstrated that PHF8 interacts with spliceosome related proteins SNRP70 and SRPK1 as well as snRNA. Indeed, PHF8 contributed to splicing: GLS and VEGF-A displayed alternative splicing in PHF8 depleted cells. In addition, c-FOS introns were showed to be retained after knockdown of PHF8 in endothelial cells. These results demonstrated that, by controlling angiogenic mRNA splicing, PHF8 could affect endothelial properties.
Collectively, the results uncover the important roles of JARID1B and PHF8 in endothelial cells in the control of angiogenesis. Changing histone modifiers appears as an attractive concept for pro- and antiangiogenic therapy. The present work adds JARID1B and PHF8 as novel potential targets to this emerging field.
G protein coupled receptors (GPCRs) constitute the largest family of cell-surface receptors in mammals and are key players in signal transduction. By responding to a plethora of extracellular stimuli ranging from photons to amines to fatty acids to peptides and proteins, these receptors trigger intracellular signalling cascades and regulate a variety of cellular responses. Approximately 800 genes in humans encode GPCRs which are classified according to sequence conservation into rhodopsin-like, glutamate, adhesion, frizzled/taste2 and secretin receptors. GPCRs share a seven transmembrane domain fold undergoing a conformational change upon ligand binding which is translated to the intracellular surface of the receptor thereby allowing a heterotrimeric G protein to couple. Heterotrimeric G proteins consist of a Ga, Gb and Gg subunit and dissociate into their Ga and Gbg entities upon activation by a GPCR. Subsequently, distinct signalling cascades are triggered by each G protein protomer.
Membrane proteins and GPCRs in particular, are highly important targets in drug design and development as currently approximately 60% of all marketed drugs target membrane proteins. Although these classes of proteins are of high therapeutic interest, our understanding of their mechanism of action and structure remains limited. The first structure of a human GPCR was determined in 2007 and required the development of protein engineering and innovative crystallisation techniques. Since then, approximately 130 GPCR structures of less than 40 individual receptors have been determined providing insights into the structural arrangement of the transmembrane helices, ligand binding pockets and G protein interactions. Combined with spectroscopic methods, these studies allowed a more detailed understanding of the molecular aspects of GPCR activation and signalling. Despite the tremendous advances in GPCR structural biology, certain aspects of GPCR function still remain poorly understood. Due to their size and inherent flexibility, the interaction of protein and peptide ligands with their receptors remains a challenging aspect in the structural characterisation of GPCRs. Moreover, structural information on subtype selectivity of peptide ligands continues to be scarce. To contribute functional and structural information on the molecular mechanisms of peptide interactions with GPCRs, this thesis focused on characterising receptors from the chemoattractant cluster using radioligand binding assays as well as NMR spectroscopy.
The chemoattractant cluster mainly groups the kinin, angiotensin, anaphylatoxin chemotactic complement and apelin receptors according to conserved residues in their ligand binding cavities. All receptors in this cluster bind to peptide ligands deriving from high molecular weight protein precursors upon proteolytic processing. Comparable to the conserved binding pocket of the chemoattractant receptors, the peptide ligands display a certain sequence conservation although they differ strongly in size. The largest ligands used in this thesis are the anaphylatoxins complement 3a and 5a, comprising 77 or 74 residues, respectively. Due to their size and complex fold involving three intramolecular disulphide bonds, solid phase synthesis is impossible, which prompted us to develop a modified cell-free expression system to produce these ligands in tritiated form for subsequent functional characterisation of the complement receptors. To demonstrate the versatility of the developed system, it was applied to another disulphidebond containing peptide ligand, the 21 amino acid endothelin-1. We describe a reliable and multifaceted tool to generate custom labelled peptide ligands for the structural and functional characterisation of GPCRs. The system allows the production of custom radioligands, peptides labelled for NMR studies or with fluorescent amino acids.
Apart from the modulation of GPCR activity by orthosteric ligands, GPCR signalling has long been described to be regulated by allosteric ligands including peptides, small molecules and ions. In this thesis, the influence of sodium ions on the activity state of the chemoattractant cluster receptors and in particular on the apelin, bradykinin 2 and angiotensin II type 1 receptors was examined. In recent high resolution crystal structures an allosteric sodium ion pocket beneath the orthosteric ligand binding cavity was identified and residues contributing to the coordination of sodium ions are conserved throughout the chemoattractant cluster receptors. This allosteric sodium ion coordinated within the transmembrane domain bundle has been described to negatively influence the affinity of agonists but not of antagonists. It was found that sodium ions have distinct influences on the affinity state as well as the available number of binding sites of the chemoattractant receptors. In case of the apelin and bradykinin 2 receptors, sodium ions drastically reduced the number of available binding sites whereas the affinity of peptide ligands to the bradykinin 2 receptors remained constant and the ligand binding affinities to the apelin receptor were completely abolished. In contrast, the angiotensin II type 1 receptor affinity state towards the endogenous peptide ligand angiotensin II is highly dependent on the presence of sodium ions, whereas binding of the synthetic peptide antagonist Sar1-Ile8-angiotensin II remained unaffected by the sodium ion concentration. As differential effects irrespective of the efficacy class but dependent on the amino acid composition of the applied ligands are observed, it can be concluded that electrostatic interactions between charged residues of the peptide ligands and amino acids on the extracellular surface of the receptors are influenced by sodium ions thereby adding another layer of complexity on GPCR signalling.
To elucidate the structure-function relationship of ligand selectivity between the kinin receptors, the structure of desArg10-kallidin (DAK) bound to the bradykinin 1 receptor was determined using solid state NMR (SSNMR) in the course of this thesis. The kinin peptides DAK and bradykinin bind with high affinity and high selectivity to either the bradykinin 1 or bradykinin 2 receptor, respectively. The binding pockets of the receptors are highly conserved and the two peptide ligands only differ in one amino acid at their N- and C-termini whereas the remaining eight amino acids are fully conserved. DAK adopts a U-shaped structure when bound to the bradykinin 1 receptor which resembles a horse shoe-like conformation. Using 2D TEDOR spectroscopy it could furthermore be demonstrated that positively charged residues at the N-terminal part of the peptide engage in ionic interactions with negatively charged amino acids on the extracellular surface of the bradykinin 1 receptor. In contrast, bradykinin displays a distinct b-turn at the C-terminus and an S-shaped conformation of the N-terminal segment when bound to the bradykinin 2 receptor. By using SSNMR to study the binding mode of DAK on the bradykinin 1 receptor we could determine that subtype selectivity between the kinin receptors is conferred by distinct conformational restraints within the peptide ligands and by the formation of specific ionic interaction between charged residues on the peptide and receptor, respectively.
In brief, this thesis contributes structural and functional data on the binding mechanisms and binding mode of different peptide-ligand GPCRs helping to understand subtype selectivity and allosteric modulation of the chemoattractant cluster receptors. In addition, a versatile cell-free expression system was developed that allows the custom synthesis of isotopically labelled peptides containing disulphide bonds for the functional characterisation of GPCRs.
Acute myeloid leukemia is a hematopoietic stem cell disorder and a type of acute leukemia which is characterized by clonal proliferation of myeloid precursors with a reduced capacity to differentiate into more mature cellular elements. Clinically AML is characterized by a high degree of heterogeneity with respect to chromosome abnormalities, gene mutations, and changes in expression of multiple genes and microRNAs. Cytogenetic abnormalities can be detected in approximately 50% to 60% of newly diagnosed AML patients. Majority of AML cases are associated with chromosomal aberrations, more specifically translocations that often result in gene arrangements and expression of aberrant fusion proteins. This study was carried out with two fusion proteins: PML/RARα and DEK/CAN which results from the translocations t(15;17) and t (6,9) respectively. PML/RARα is the most common translocation (97%) and the main driver in Acute Promyelocytic Leukemia (APL), a wellcharacterized and well treatable subtype of AML. In contrast, DEK/CAN occurs in 1-5% of AML, associated with poor prognosis and defines a high risk group in AML. The expression of PML/RARα results in a fusion protein that acts as a transcriptional repressor by interfering with gene expression programs involved in differentiation, apoptosis, and selfrenewal. Current therapy focused on the targeting of PML/RARα fusion protien. Success has been achieved by using either ATRA, anthracyclines and Arsenic trioxide or their combinations. These agents induce differentiation in PML/RARα positive AML and hence called differentiation therapy. In comparison with ATRA, ATO and anthracyclines are poor cellular differentiation agents. Despite early promise, several studies have reported that differentiation therapy is unable to target/eradicate leukemic stem cells or eradicate the disease. Therefore current therapeutic focus is to eliminate leukemic stem cells and achieve complete molecular remission not only in APL but also in acute lymphoblastic leukemia and chronic myeloid leukemia as well. Key enzymes of the eicosanoid pathways in the arachidonic acid metabolism, such as COX1/2 as well as the 5-LO have been shown to be good targets for leukemic stem cell therapy approach in AML by interfering with the Wntsignaling which is known to be indispensable for the pathogenesis of AML. Recently it was reported that the third eicosanoid pathway based on the cytochrome P450 (CYP) enzymes interferes with Wnt-signaling as well as with the proliferation and mobilization of hematopoietic stem cells...
Structural characterization of stressosome complexes by single-particle cryo-electron microscopy
(2015)
The stressosome is a Mega Dalton macromolecular complex involved in stress adaptation in bacteria. Stressosomes are considered as stress signaling hubs. They are able to perceive a variety of different stress stimuli and transduce them into one single cellular answer, which is the initialization of a transcriptional up-regulation of hundreds of different genes encoding for universal but also very specific stress response proteins.
The stressosome of Bacillus subtilis became a prime example for this intriguing stress-triggered transcriptional regulation when its architecture was determined by Single-particle cryo-electron microscopy (cryo-EM) in 2008. In Gram-positive Bacillus species, the stressosome complex senses changes in salt concentration, ethanol content, blue-light, heat or acid stress contributing to the general stress response by activation of the alternative σB factor. σB is a transcriptional promoter that initiates the transcription of over 150 general stress genes, e.g., genes that encode osmolyte transporters to counteract osmotic and chill stress. The B. subtilis stressosome (stressosome_Bc) is composed of multiple copies of the 3 proteins: RsbR, RsbS and RsbT. These three Rsb proteins (Regulator of Sigma B) are found clustered in one operon forming the conserved RST module. RsbS and RsbR are scaffold proteins comprising a STAS domain, respectively. Because these domains are dominantly associated to sulfate transporters and anti-sigma antagonist they were named STAS domains, however, they were also identified in other sensor proteins. In the stressosome they form the internal ball-shaped core, while the N-terminal globin-fold sensor domain of RsbR, protruding to the outside, facilitates stress sensing. It is assumed that the stress signal is transduced to the stressosome core via the STAS domain resulting in conformational changes of the core. These changes affect the binding of the third protein, RsbT, a serin-threonine kinase. As a direct consequence of stress sensing the RsbT kinase is released from the complex to start an activation cascade involving the stepwise activation of RsbU, V, W, and X, which are all part of the same operon, and finally of σB. In Bacillus species, several RsbR orthologs were identified varying mainly in the sequence of the N-terminal sensor domains. It is assumed that the stressosome_Bc assembles with a still unknown combination of RsbR orthologs allowing for the broad spectrum of stress stimuli that can be processed in vivo. The pathogenic bacteria Listeria monocytogenes is a close relative of Bacillus. Its potent stress response allows Listeria to survive the harsh environmental conditions during host infection and therefore the stress regulation machinery is contributing heavily to the virulence of this pathogen. In Listeria the Rsb operon is conserved and highly homologous to the Bacillus one. In the frame of this thesis, the in vitro assembly of Listeria innocua stressosomes was shown for the first time by Single-particle (SP) negative stain EM. Moreover, binding of Listeria RsbT to the assembled RsbR-RsbS complex was demonstrated biochemically.
Despite the conservation of the RST-module the entire Rsb operon is not conserved in the bacterial kingdom suggesting that signal transduction and regulation of gene expression might occur by very different mechanisms in stressosomes of different species. We have focused here on a stressosome type from the Gram-negative pathogen Vibrio vulnificus that is quite distinct from the Bacillus ones with respect to (1) the missing conservation of the Rsb operon, (2) the role of RsbT, (3) the activation of a different transcriptional promoter, and (4) the absence of additional RsbR orthologs. Interestingly, there is only one RsbR protein encoded in the genome. This one contains a Haem-group in its N-terminal domain being oxygen sensitive. It is assumed that the Vibrio stressosome perceive only oxidative stress and that regulation occurs via a diguanylate cyclase with a GAF domain that synthesizes the second messenger c-di-GMP from GTP.
We have started a structure determination of the Vibrio vulnificus stressosome by SP cryo-EM to elucidate the differences in the molecular mechanism of stress sensing in divers stressosome types. A 3D map of the oxidized (activated) Vibrio vulnificus stressosome was determined to 7.6 Å resolution revealing an increased flexibility of both the core and the N-terminal sensor domains in comparison to the Bacillus stressosome suggesting that our structure has trapped for the first time an active state of a stressosome complex. A 3D map of the stressosome core to 7 Å resolution allowed fitting of a homology model of the Vibrio stressosome based on the Bacillus stressosome as template. The conformational changes could be attributed to the entire core, which was confirmed by MD simulations.
Hepatitis B caused by infection with the hepatitis B virus (HBV) still ranks among the most challenging infectious diseases of our time. Despite the availability of an effective prophylactic vaccine, 240 million people worldwide are estimated to be chronically infected with HBV and are at risk of developing life-threatening liver diseases, including cirrhosis and liver cancer. The underlying pathogenic mechanisms of HBV-associated liver diseases are only incompletely understood. It is widely accepted that liver pathology results from long-term immune-mediated liver injury and inflammation as a consequence of inefficient viral elimination. This injury can be naturally compensated by liver regeneration. However, chronic liver damage and permanent inflammation debilitates the regenerative capacity of the liver and fosters fibrosis as well as accumulation of chromosomal aberrations, which both contribute to cirrhosis and liver cancer. Liver regeneration requires the presence of the redox-sensitive transcription factor Nrf2 and intact insulin receptor signaling. A lack of Nrf2 causes increased intracellular levels of reactive oxygen species (ROS) that inactivate insulin receptor signaling and induce insulin resistance. Interestingly, HBV was observed to activate Nrf2 and the expression of Nrf2-regulated genes. This argues against an inhibitory effect of HBV on insulin receptor signaling by increased ROS levels. However, chronic HBV infection is associated with dysregulation of hepatocyte proliferation and retardation of liver regeneration. Hence, the aim of this thesis was to investigate the influence of HBV on the process of liver regeneration with respect to the insulin receptor signaling pathway. After short-term carbon tetrachloride (CCl4)-induced liver damage, HBV transgenic mice present prolonged liver damage and impaired liver regeneration as reflected by reduced hepatocyte proliferation and increased apoptosis. Impaired hepatocyte proliferation in HBV transgenic mice correlates with diminished activation of the insulin receptor. It was further observed in vitro that the activation of Nrf2 by HBV induces increased levels of the insulin receptor mRNA and protein in HBV-expressing cells. Strikingly, stably HBV-expressing cells as well as primary mouse hepatocytes from HBV transgenic mice bind less insulin due to reduced amounts of insulin receptor on the cell surface. This is caused by intracellular retention of the insulin receptor in HBV-expressing cells as a consequence of increased amounts of the cellular trafficking factor α-taxilin. The reduced amounts of insulin receptor on the cell surface impair insulin sensitivity in HBV-expressing cells and inactivate downstream signaling cascades that initiate insulin-dependent gene expression and glucose uptake. As a consequence of impaired hepatocyte proliferation and liver regeneration, HBV transgenic mice exhibit increased development of fibrosis after long-term CCl4-induced liver damage. Taken together, in this thesis, a novel pathomechanism could be uncovered that includes inactivation of insulin receptor signaling by HBV via intracellular retention of the insulin receptor leading to impaired liver regeneration after liver damage and promotion of liver fibrosis. These findings significantly contribute to an enhanced understanding of HBV-associated liver pathogenesis.
Molecular signaling networks, organized in discrete subsets of proteins in space and time, represent the major principle by which the cell achieves its functional specificity and homeostasis. Complex network organization is preserved by numerous mechanisms, including sequestration of proteins into specific subcellular compartments (eg. organelles), post-translational modifications and most importantly by balanced timing of their biosynthesis and turnover. Two routes of protein degradation, which are fundamentally quite different, are proteasomal and lysosomal-mediated destruction. The latter not only governs degradation of molecules that passed through endocytic or secretory process (trafficking from plasma membrane or Golgi compartment), but also the degradation of cytoplasmic molecules that have been sequestered by a process called macroautophagy (henceforth autophagy). Recently our understanding of autophagic regulatory mechanisms has increased significantly, as molecular details of how autophagy contributes to the degradation of proteins (old, misfolded or aggregated), damaged organelles or pathogens have been deciphered. Initially described as bulk, nonspecific membrane sequestration process induced primarily by nutrient deprivation, autophagy is now known to be selective in terms of cargo recognition and integration into dynamic cellular membrane trafficking system.
My work has addressed the fundamental question of how small ubiquitin-like modifiers LC3/GABARAP, that are conjugated to the autophagic membranes, function within the process of cargo selection and crosstalk between autophagic and endocytic membrane trafficking events. We have employed an initial yeast twohybrid screen to identify LC3/GABARAP interacting partners. Using this technique, we have identified several novel autophagy receptor proteins, mitochondrial protein Nix (BNIP3L), and adaptor proteins, including Rab GTPase activating proteins (TBC family of proteins). Through a conserved LC3 interacting region (LIR), Nix, Rab GAPs and other autophagy adaptor/receptor molecules share a common mode of binding to LC3/GABARAP. However, in contrast to Nix, which specifically facilitates removal of mitochondria in maturing erythrocytes, Rab GAP proteins preferably regulate the dynamics of autophagosome formation and maturation as well as sorting of cargo. Fourteen out of 36 screened Rab GAPs interacted with LC3/GABARAPs. Importantly, identified Rab GAPs are clustered in different regulatory nodes according to the conservation of their GAP domain hence they impact various cellular membrane compartments and organelles, marked by specific subsets of small Rab GTPases. Identification of Rab GAPs that are directly involved in autophagy via binding to LC3 was the first report that clearly pointed to a broader implication of autophagy in all aspects of cellular membrane trafficking. Currently, only few of Rab GAPs are studied in context of autophagy regulation, while large number of them requires further functional characterization.
I have identified two LIR motifs in TBC1D5, Rab7 GAP. LIR1 has also the ability to interact with retromer complex subunit, Vps29. Using several functional assays I have shown that this motif, as well as catalytic Arg within GAP domain are particularly important for function of TBC1D5 in retrograde transport of CI-M6PR from endosomes to the trans-Golgi network (TGN). I have also shown that TBC1D5 binds to LC3 and Vps29 in mutually exclusive way and that Thr at the position 1 and Phe at position 5 of LIR1 motif are both required for TBC1D5 interaction with Vps29. Upon autophagy induction TBC1D5 dissociates from retromer, and associates with autophagic vesicles, while silencing of TBC1D5 significantly impairs autophagic flux. These findings led to the hypothesis that LIR interacting surface on TBC1D5 acts as molecular switch for dual function of TBC1D5. This also indicated that similar surfaces for LIR interaction (similarly to ubiquitin-like domains) are present on proteins other than LC3, and pointed to a dual functionality of the LIR sequence within both endocytic and autophagic pathways.
Following these initial studies, I have also shown that TBC1D5 interacts with AP2 complex subunit AP2M1, and that this interaction plays critical role in TBC1D5-dependent trafficking of Atg9. It is known that Atg9, the only trans-membrane autophagic protein, plays essential role in initiation of autophagy and growth of nascent phagophore membranes. However, machinery that specifically recruits Atg9 traffic carriers to the site of autophagosomes was not known. I subsequently demonstrated that TBC1D5 associates not only with LC3, but also with Atg9 traffic carriers and major initiatory kinase ULK1 during autophagy, while retromer failed to do so. Association of TBC1D5 with Atg9 was dependent on presence of AP2 complex, and on functional clathrin-mediated endocytosis (CME). Based on these and previous findings, model was proposed, that upon induction of autophagy TBC1D5 re-routes Atg9-containing clathrin vesicles from plasma membrane to the site of autophagosome. This led us to the better understanding of TBC1D5 function, but also to the first molecular cue that Atg9 traffics within clathrin-coated vesicles (CCVs). In fact, mutation of Leu-Leu motif within N terminus of Atg9, that potentially mediates interaction with adaptor protein complexes, led to enrichment of Atg9 on plasma membrane and in TGN. This suggested that the sorting motif could be important for interaction of Atg9 with AP2 and AP1 complex, as well. More importantly, TBC1D5 and Atg9 could be directly involved in dynamic regulation of growth factor receptor sorting during autophagy, thus explaining vital role of autophagy in organism development and pathogenesis.
In summary, the work contained within my thesis provides data on the mechanism by which autophagy adaptor proteins participate in cargo selection and regulation of trafficking during autophagy. Firstly, the LIR motif can target proteins or organelles for autophagic degradation (eg. Nix). Secondly, specific LIR motifs can play essential function in recruiting membrane trafficking regulatory proteins that subsequently facilitate phagophore expansion (eg. TBC1D5). Thirdly, by means of reorganization of different protein assemblies (eg. TBC1D5-VPS29 vs. TBC1D5-LC3-Atg9), dynamics of membrane remodeling mediated by Rab GTPases is kept in control during autophagy, thus keeping the organelle integrity and balance within cellular lipid sources unaffected.
Im Rahmen dieser Arbeit wurden zum Vergleich die Strukturen der ATP-Synthasen von Arabidopsis thaliana, Asparagus officinalis, Allium cepa, Helianthus annus, Solanum tuberosum, Bos taurus und Saccharomyces cerevisiae gelöst. Die ATP-Synthase von S. cerevisiae konnte mit einer Auflösung von 19 Å gelöst werden. Der Winkel zwischen den zwei ATP-Synthase-Monomeren in dem ATP-Synthase-Dimer hatte für jede Spezies einen bestimmten Wert. Dieser Winkel änderte sich innerhalb einer Spezies nur wenig im Gegensatz zu Untersuchungen mit Einzelpartikelanalyse.
Die ATP-Synthase-Dimere aus den untersuchten Spezies besitzen unterschiedliche Winkel zwischen 78˚ und 122˚. Der Winkel des ATP-Synthase-Dimers aus S. tuberosum (122˚) viel größer als der in anderen Pflanzen (~98˚), B. taurus (105˚) und S. cerevisiae (78˚). Die Proben von S. tuberosum und B. taurus waren jedoch dünner, was den Winkel eventuell beeinflussen könnte. Um dies auszuschließen müssen in Zukunft weitere Untersuchungen durchgeführt werden.
Des Weiteren wurde im peripheren Stiel der ATP-Synthasen von allen Pflanzenspezies eine Dichte entdeckt, die in B. taurus und S. cerevisiae nicht vorhanden ist. Die Dichte könnte durch eine zusätzliche Untereinheit oder veränderte Untereinheit im Vergleich zu B. taurus und S. cerevisiae kommen.
Weiterhin wurde die Bildung von Reihen aus ATP-Synthase-Dimeren untersucht. Es wurden ATP-Synthase-Dimere von Polytomella sp. gereinigt und in Lipid rekonstituiert. Es wurde das ATP-Synthase-Dimer von Polytomella sp. verwendet, da dieses besonders stabil ist und während der Reinigung nicht zum ATP-Synthase-Monomer zerfällt. Zur Rekonstitution wurde die milde GRecon-Methode verwendet. Hierbei werden Membranproteine in einem Zuckergradienten gleichzeitig in Lipid rekonstituiert und nach ihrer Dichte getrennt. Abhängig von der Dichte der Proteoliposomen ist die Konzentration an Membranproteinen unterschiedlich. In Proteoliposomen mit einer hohen Konzentration bilden sich dünne Schichten in denen die ATP-Synthase-Dimeren Zickzack-Muster formen. Dies deutet darauf hin, dass das ATP-Synthase-Dimer die Membran verformt. In Proteoliposomen mit einer niedrigeren Konzentration an ATP-Synthase-Dimeren wurden runde Vesikel detektiert, in denen die ATP-Synthase-Dimere lange Reihen bilden und die Membran innerhalb jedes ATP-Synthase-Dimer ebenfalls verformt ist. Molekulare Simulationen bestätigen dieses Ergebnis.
Zudem wurde das ATP-Synthase-Dimer in zwei verschiedene Lipide ohne Cardiolipin rekonstituiert, da Cardiolipin ein Lipid ist welches in der bakteriellen und mitochondrialen Membran gefunden wurde und in hohen Konzentrationen in Membrankrümmungen lokalisiert ist (Huang et al., 2006), wie auch die ATP-Synthase-Dimere. Ohne Cardiolipin ist die Rekonstitution nicht geglückt beziehungsweise sind die ATP-Synthase-Dimere weniger gut zueinander angeordnet. Das deutet auf die Wichtigkeit von Cardiolipin in der Stabilisierung der Reihen von ATP-Synthase-Dimeren hin. Weitere Experimente mit verschiedenen ATP-Synthase-Dimeren in verschiedenen Lipiden sind nötig um dies zu untermauern.
Ein weiteres Ziel dieser Arbeit war es ein klonierbares Label zu etablieren, um ein bestimmtes Protein in Kryo-Elektronentomogramme zu identifizieren. Das Label sollte klein sein, um das zu identifizierbare Protein nicht zu beeinflussen und groß genug um in Kryo-Elektronentomogramme identifizierbar zu sein. In Einzelbildern wurde das 6 kDa große Metallothionein gebunden mit Gold identifiziert, wenn zwei Metallothioneine an dem gewünschten Protein kloniert wurden. Metallothionein besteht zu 33 % aus Cysteinen, welche Schwermetalle binden.
In meinen Studien habe ich bewiesen, dass drei Metallothioneine, gebunden mit Gold, in Kryo-Elektronentomogramme detektiert werden können. Jedoch tritt bei der Verwendung von Metallothionein durch die hohe Anzahl an Cysteinen vermehrt Aggregation auf. Bei meinen Untersuchungen fand ich heraus, dass auch das Maltose-Binde-Protein (MBP) ein Signal gleicher Intensität erzeugt. Durch Verwendung von MBP tritt aber keine Aggregation auf und man kann MBP auch zum Reinigen des Proteins verwenden.
This thesis is concerned with protein structures determined by nuclear magnetic resonance (NMR), and the text focuses on their analysis in terms of accuracy, gauged by the correspondence between the structural model and the experimental data it was calculated from, and in terms of precision, i.e. the degree of uncertainty of the atomic positions. Additionally, two protein structure calculation projects are described...
Photoinduzierte Energietransferprozesse und -reaktionen spielen in vielen Gebieten von Chemie, Physik und Biologie eine wichtige Rolle. Zu den prominentesten Beispielen zählen der Lichtsammelprozess in der Photosynthese und der Anregungsenergietransfer in funktionellen Materialien. Der Fokus dieser Arbeit liegt auf letzterem Bereich, genauer auf organischer Elektronik und flexiblen Donor-Akzeptor-Bausteinen und Schaltern. Im Besonderen werden hier zwei verschiedene Typen von funktionellen organischen Systemen betrachtet: zum einen oligomere Fragmente organischer halbleitender Polymere wie Oligo-p-Phenylen-Vinylen (OPV) und Oligo-Thiophen (OT), welche als Bausteine für neuartige organische Solarzellen dienen, und zum anderen kleine funktionelle Donor-Akzeptor-Einheiten wie Dithienylethen-Bordipyrromethen (DTE-BODIPY). Letzteres wurde in Kooperation mit den experimentellen Gruppen von K. Rück-Braun (TU Berlin) und J. Wachtveitl (Goethe Universität) untersucht. Um die relevanten Energietransfermechanismen genauer zu verstehen, wurden an diesen Systemen elektronische Strukturrechnungen und quantendynamische Untersuchungen durchgeführt. Hierzu wurden mittels ab initio-Methoden Modell-Hamiltonians parametrisiert und mit hochdimensionalen quantendynamischen oder semiklassischen Methoden kombiniert. Während die Parametrisierung für kleinere Fragmente durchgeführt wurde, lässt sich der so parametrisierte Hamiltonian ohne Weiteres auf größere Systeme erweitern. Die dynamischen Studien der betreffenden Systeme wurden mittels der Multikonfigurationellen Zeitabhängigen Hartree (MCTDH) Methode durchgeführt, welche eine vollständige quantendynamische Beschreibung des Systems zulässt. Für größere Systeme wurde die semiklassische Ehrenfest Methode in Verbindung mit dem Langevin-Ansatz zur Beschreibung von Umgebungseffekten genutzt. Hierzu wurde ein eigens für diese Methode und Systeme geschriebenes Programm eingesetzt. Im Falle der OT- und OPV-Oligomere wurde die Dynamik bei Vorliegen eines strukturellen Defekts untersucht. Ziel war es hierbei, die dynamischen Phänomene, welche durch die Photoanregung induziert werden, zu untersuchen. Des Weiteren wurde untersucht, ob das Konzept von „spektroskopischen Einheiten“, welche die Lokalisierung der Anregung durch strukturelle Defekte beschreibt, in diesen Systemen zutrifft. Hierzu wurden die Systeme in einer Frenkel-Basis definiert, welche ein auf einem Monomer lokalisiertes Elektron-Loch-Paar beschreibt. Delokalisierte elektronische Anregungen können somit als Superposition solcher Frenkel-Zustände beschrieben werden. Neben der Frenkel-Basis wurde aber auch eine verallgemeinerte Elektron-Loch-Basis verwendet, welche über zusätzliche Ladungstransferzustände eine räumliche Separation von Elektronen und Löchern erlaubt.Die Parametrisierung des OPV- und OT-Hamiltonians erfolgte mittels der Algebraischen Diagrammatischen Konstruktions (ADC(2))-Methode, welche in Kombination mit einer Übergangs-Dichte-Matrix-Analyse eine sehr akkurate Beschreibung der Frenkel- und Ladungstransferzustände basierend auf den supermolekularen Zuständen erlaubt. Um vibronische Effekte auf die Dynamik miteinzubeziehen,wurden nieder- und hochfrequente Torsions- und alternierende Bindungslängenmoden des Systems im Hamiltonian berücksichtigt. Hierzu wurden eindimensionale Schnitte der Potentialflächen entlang dieser Koordinaten berechnet und mittels einer Transformation in diabatische Potentialflächen überführt. Mit diesem Setup wurden die quantendynamischen und semiklassischen Simulationen für ein OPV/OT-Hexamer und ein 20-mer durchgeführt. Die Ergebnisse dieser Simulationen zeigen, dass der Energietransfer auf einer Subpikosekunden-Zeitskala stattfindet und eine starke Abhängigkeit vom Vorliegen eines strukturellen Defekts aufweist. Des Weiteren konnte auf einer Zeitskala von 100 Femtosekunden eine Lokalisierung des Exzitons beobachtet werden. Fluktuationseffekte werden zudem über Quantenfluktuationen im Falle von MCTDH bzw. über thermische Fluktuationen im Falle des Ehrenfest-/Langevin-Ansatzes berücksichtigt. Letzterer ist jedoch nicht in der Lage, die kohärente Charakteristik der mit den Schwingungsmoden gekoppelten Exziton- und Lokalisierungsdynamik wiederzugeben. Dagegen kann dieser Ansatz erfolgreich genutzt werden, um eine fluktuationsgetriebene „Hopping“-Dynamik des quasi- stationären Zustandes auf einer längeren Zeitskala in Abhängigkeit von der Temperatur zu beschreiben. Die Beschreibung der Photodynamik der DTE-BODIPY-Dyade zielt darauf ab, experimentell beobachtete vibrationelle Schwingungen des BODIPY-Fragments zu erklären, die ohne eine direkte Anregung dieses Fragments zustande kommen. Diese wurden nach einer selektiven Anregung des DTE-Fragments in zeitaufgelösten UV/Vis Anreg-Abtast-Experimenten beobachtet. Der Fokus der Untersuchung liegt daher auf der Beschreibung der photoinduzierten intramolekulare Energieumverteilung (IVR) auf einer Subpikosekunden-Zeitskala. Die DTE-BODIPY Dyade wurde mittels eines Hamiltonians, welcher durch TDDFT Rechnungen parametrisiert wurde, dargestellt. Basierend auf den Normalmoden des Systems, wurden lokale DTE- und BODIPY-Moden konstruiert, wobei einige dieser Moden miteinander gekoppelt sind und die Photoanregung des DTE auf das BODIPY-Fragment übertragen. Hierbei zeigte sich, dass die Zeitskala und die charakteristischen Frequenzen des Experiments mittels der hochdimensionalen MCTDH-Methode gut reproduziert wurden. Aus den Simulationen ergab sich zudem, dass der beobachtete Energietransfer stark von einem Reservoir von vibrationell angeregten lokalen DTE-Moden beeinflusst wird. Der untersuchte IVR- Prozess zeigt zudem eine ausgeprägte Abhängigkeit von lokalen Kopplungen und der Kopplung an eine Umgebung.
Recently, two of the most common types of bone cancers in children and young adults have been proven to exhibit vulnerability to poly(ADP)-ribose polymerase, (PARP) inhibitors (e.g. olaparib, talazoparib). Ewing’s sarcoma (ES) are reported to harbor a fusion gene EWS-FLI1 (85%), inducing tumorigenesis. Additional, as the fusion gene acts as aberrant transcription factor, it similarly induces elevated PARP expression levels sensitizing ES to PARP inhibition. Second, by an exome sequencing approach in a set of primary osteosarcomas (OS) we identified mutation signatures being reminiscent of BRCA deficiency. Therefore, the sensitivity of a panel of OS cell lines to either talazoparib single treatment or in combination with several chemotherapeutic drugs was investigated.
To screen ES tumor cell lines against PARP inhibitors we applied four different PARP inhibitors (talazoparib, olaparib, niraparib and veliparib) that are frequently being used for clinical studies. We combined those PARP inhibitors with a set of chemotherapeutics (temozolomide (TMZ), SN-38, etoposide, ifosfamide, doxorubicin, vincristine and actinomycin D) that are part of the first-line therapy of ES patients. Here, we demonstrate how PARP inhibitors synergize with TMZ or SN-38 to induce apoptosis, whereas the combination of PARP inhibitors with the other drugs are not favorable. By investigation of key checkpoints in the molecular mechanisms of cell death, the pivotal role of the mitochondrial pathway of apoptosis mediating the synergy between olaparib and TMZ was revealed.
Employing talazoparib monotherapy in combination with or without several chemotherapeutic drugs (TMZ, SN-38, cisplatin, doxorubicin, methotrexate and etoposide/carboplatin), the correlation between homologous recombination (HR) repair deficiency (BRCAness) and the response to talazoparib as prototypical PARP inhibitor was validated in different OS cell lines. By calculation of combination indices (CI) and fraction affected (Fa) values, we identified TMZ as the most potent chemotherapeutic drug in combination with talazoparib inducing the mitochondrial apoptotic pathway in OS.
In our studies of two independent tumor entities with contrary genetic background we identified the combination of PARP inhibitor and TMZ as being most effective. Our studies point out that after TMZ induced DNA methylation and concomitant PARP trapping, DNA damage-imposed checkpoint kinase activation consequently induces G2-cell cycle arrest. Subsequent, PARP inhibitor/TMZ causes MCL-1 degradation, followed by activation of BAK and BAX, succeeding in loss of mitochondrial outer membrane potential (LMMP) and activation of downstream effector-caspases in mitochondrial apoptosis. Our findings emphasize the importance of PARP inhibition in order to chemosensitize ES, which express high PARP levels, or OS that bear features of BRCAness.
Functional dynamics of ribonucleic acids : development and application of spectroscopic tools
(2016)
Im Rahmen der vorliegenden Dissertation wird der Aufbau eines zeitaufgelösten Fluorimeters, die photophysikalische Grundcharakterisierung der drei 2-(Pyrenylethinyl)-Adenosine (PyAs) und das Wechselwirkungsgeflecht des tetracyclinbindenden Aptamers (TC-Aptamer) mit seinem Liganden Tetracycyclin (TC) und Mg2+ dargestellt.
Das zeitaufgelöste Fluorimeter basiert auf der experimentellen Technik des zeitkorrelierten Einzelphotonenzählens. Es verfügt über zwei Anregungsquellen: gepulste UV-LEDs und einen frequenzverdoppelten titandotierten Saphirlaser. Diese Quellen Decken einen Wellenlängenbereich von (310 - 550) nm ab. Das Spektrometer kann unter günstigen Umständen eine Zeitauflösung von 50 ps erreichen bei einer zeitlichen Messungenauigkeit von weniger als 0,02 %.
Die Leistungsfähigkeit des Aufbaus wird anhand einer umfangreichen Studie an den drei PyAs demonstriert.
Die drei PyAs 2-(1-Pyrenylethinyl)-Adenosine (1PyA), 2-(2-Pyrenylethinyl)-Adenosine (2PyA) und 2-(4-Pyrenylethinyl)-Adenosine (4PyA) sind eine Gruppe fluoreszierender RNA-Nukleosidanaloga, welche die Gesamtheit aller möglichen Konfigurations-isomere der Grundverbindung PyA umfassen. Ihre zeitabhängigen Fluoreszenzzerfallseigenschaften werden ergänzt von stationären Absorptions- und Fluoreszenzspektren, ultraschneller transienter Absorptionsspektroskopie und quantenchemischen Rechnungen. Die Fluoreszenz von 1PyA und 4PyA gehorchen der Regel von Kasha, wohingegen 2PyA einen triexponentiellen Zerfall mit ausgeprägter Abhängigkeit von der Anregungswellenlänge zeigt. Die transienten Absorptionsspektren aller drei Isomere weisen im gesamten Spektrum dominante, wenig strukturierte Absorptionsbanden des ersten angeregten Zustands auf, welche im nahen UV in unterschiedlichem Maße vom Grundzustandsbleichen und stimulierter Emission überlagert werden. 2PyA zeigt eine deutlich ausgeprägte Signatur für eine interne Umwandlung hin zum S1, wenn es in höhere angeregte Zustände angeregt wird.
Das Fluoreszenzverhalten von 2PyA wird mithilfe eines lokal angeregten (LE) und zweier intramolekularer Ladungstransferzustände, von denen einer der koplanaren Orientierung von Pyren und Adenin (MICT) und der andere einer um 90 ° verdrehten Orientierung (TICT) entspricht. Der LE-Zustand ist hierbei verknüpft mit dem S2 von 2PyA, welcher einer rein pyrenlokalisierten Anregung entspricht. Dieser Zustand existiert so in 1PyA und 4PyA nicht. Der verdrehte TICT-Zustand ist nur in 2PyA bevölkerbar, weil für 2PyA die Barriere zur Bildung von Rotameren am niedrigsten ist und das Molekül nach Anregung daher in diese Geometrie kommen kann und dann durch die stärkere elektrostatische Anziehung stabilisiert wird. 1PyA und 4PyA emittieren hingegen nur aus dem MICT-Zustand.
Die Komplexbildung des TC-Aptamers mit seinem Liganden TC in Lösung wird empfindlich beeinflusst durch die-Konzentration von Magnesiumkationen. Dies wird untersucht durch Bindungs- und Faltungs- und Denaturierungsstudien mit verschiedenen Mg- und Harnstoffkonzentrationen. Als experimentelle Observable dienen hierbei die konformationsabhängige Nukleobasenabsorption und ihr Zirkulardichroismus im fernen UV, die Fluoreszenz des Liganden TC und die freiwerdende Wärme der exothermen Bindungsreaktion des Aptamers mit Mg in An- und Abwesenheit von TC.
Ohne Mg ist eine Interaktion des TC-Aptamers mit TC nicht nachweisbar. Dies liegt daran, dass Mg die notwendige elektrostatische Abschirmung der negativen elektrischen Ladung am RNA-Rückgrat zur Verfügung stellt. Die Abschirmung erlaubt es dem Aptamer kompakte Strukturen mit tertiären Kontakten auszubilden. Wenn die Mg-Konzentration die Faltung des Aptamers vollständig unterstützt (> 1 mM), so befindet sich das Aptamer weitgehend in einer vorgefalteten Konformation, welche der bindungskompetenten stark ähnelt. In diesem Zustand kann das Aptamer seinen Liganden extrem schnell, nämlich annähernd diffusionslimitiert binden. Unter diesen Bedingungen hat TC kaum Einfluss auf die Konformation seines Aptamers.
Bei physiologischen Mg-Konzentrationen (0,2 - 0,8 mM) kann das Aptamer kompakte Konformationen mit tertiären Strukturen einnehmen. Diejenige Konformation, welche der bindenden sehr stark ähnelt, dominiert das konformationelle Gleichgewicht jedoch noch nicht vollständig, es ist lediglich eine Konformation von vielen möglichen. Daher eröffnen physiologische Mg-Konzentrationen dem TC-Aptamer Teile des Konformationsraumes, welche andernfalls nicht zugänglich wären und TC stabilisiert selektiv die native Konformation. Diese konformationelle Verschiebung liefert kann hierbei zur robusten Signalgebung für die Funktion als Riboschalter dienen.
This thesis deals with the NMR characterization of the structure and the folding dynamics of DNA G quadruplexes as potential therapeutic target in cancer therapy and building block for DNA based nanotechnology.
The first part of this thesis (Chapters 1-5) introduces the reader to the world of G quadruplexes.
The main features of the classic Watson Crick double helix and alternative non B DNA structures are illustrated in Chapter 1. Many different base pairing schemes are possible, besides the canonical Watson Crick motif, thereby expanding the structural complexity of DNA. Non canonical base pairing, such as Hoogsteen hydrogen bonding, enables the assembly of triplets and quartets, which are the building blocks of triplex and quadruplex structures, respectively.
The structural characteristics of DNA G quadruplexes are delineated in detail in Chapter 2.
G quadruplex structures are extremely polymorphic, in terms of strands orientation, loops geometry, grooves width and arrangement of the glycosidic torsion angles. The various structural elements as well as the different cation coordination geometries are here presented, with a special emphasis on the diversity of conformations reported for the telomeric DNA G quadruplexes.
Chapter 3 describes the biological roles of G quadruplex structures in the genome. After introducing the architecture of the telomeric DNA and its interacting proteins, the mechanism of the telomeres elongation catalysed by the telomerase enzyme and its implications for cancer are discussed. The occurrence of G quadruplex structures in functional regions of the genome, such as promoter regions of oncogenes, and their possible roles in regulating the gene transcription are then outlined in the second part of the chapter.
The potential of G quadruplex as a novel anti cancer target is examined in Chapter 4 and the proposed anti cancer mechanisms for a ligand stabilizing G quadruplex structures are discussed.
RNA G quadruplexes and their putative role in gene regulation at the level of translation are briefly illustrated at the end of the chapter.
A general overview on the NMR methods to investigate the G quadruplex structures is presented in Chapter 5. The experimental set up used for the real time NMR studies of the G quadruplex folding is also described.
The second part of the thesis (Chapters 6-8), which is the cumulative part, comprises the original publications grouped in three Chapters according to the topic.
The state of the art on small molecules targeting G quadruplex structures is given at the beginning of Chapter 6, including a summary of the experimental structures of G quadruplexes in complex with ligands available up to date. The publications presented in Chapters 6.1-6.3 are concerned with the elucidation of the interaction modes between DNA G quadruplexes and selected ligands with potential therapeutic applications.
The binding ability of two natural alkaloids (berberine and sanguinarine) to telomeric G quadruplexes is examined in Chapter 6.1. The ability of carbazole and diguanosine derivatives (synthetized in the group of Prof. Dash, IISER, Kolkata) to interact with c-MYC G quadruplex and down regulate c-MYC expression is explored in Chapter 6.2 and Chapter 6.3, respectively.
The energy landscape of human telomeric G quadruplex structures is discussed in Chapter 7, in light of the experimental kinetic studies as well as molecular dynamics simulations reported in literature until now. Up to date there is no general consensus regarding the folding pathway of unimolecular human telomeric G quadruplex, in particular due to the lack of atomic resolution data on the species involved in the folding. Chapter 7.1 presents the first real time NMR study of the human telomeric G quadruplex folding kinetics.
The final chapter of this thesis (Chapter 8) outlines the potential of G-quadruplex structures as building blocks in nanotechnology. After illustrating briefly the additional possibilities offered by alternative non B DNA structures to programme nanomaterials, a number of applications employing G quadruplex structures in different fields of nanotechnology are described. The article presented in Chapter 8.1 investigates the structural and photoswitching properties of a novel intermolecular azobenzene containing G quadruplex synthetized in the group of Prof. Heckel (Goethe University, Frankfurt).
Tectonin β-propeller containing protein 2 (TECPR2) was first identified in a mass- spectrometric approach as an interactor of GABARAP, an ATG8-family protein playing a role in autophagy. The mammalian ATG8 protein family consists of seven members, namely MAP1LC3A (LC3A), MAP1LC3B (LC3B), MAP1LC3C (LC3C), GABARAP, GABARAPL1 and GABARAPL2. All share an ubiquitin-like core and possess two additional N-terminal α-helices, which are important for the distinct functions of the proteins. First determined in various organelles the ATG8 proteins are shown to be involved in autophagy, supporting the formation and cargo recruitment of autophagosomes, the vesicles transporting cargo for autophagic degradation.
Autophagy is the process of recycling cytoplasmic contents by degradation of misfolded proteins or damaged organelles in order to supply nutrients. Also clearance of pathogens can be achieved via autophagy. Importantly, LC3B is incorporated into the autophagosomal membrane and is therefore used as the main marker for autophagosomes. Previous studies exhibited that depletion of TECPR2 leads to a loss of LC3B-positive structures in cells, which suggests TECPR2 to positively regulate autophagic processes.
A frame shift deletion in the gene encoding for TECPR2 causes the generation of a premature stop codon and subsequent an unstable version of the protein, which is then degraded. Mutation in the TECPR2 gene triggers a neurodegenerative disorder termed hereditary spastic paraparesis (HSP). HSPs are a diverse group of neurodegenerative diseases that are characterized by spasticity in prevalent lower extremities and were mediated by a loss of axonal integrity of the corticospinal motor neurons. In the context of HSP more than 50 gene loci were identified by now. While TECPR2 is a human ATG8 binding protein and positive regulator of autophagy causing a form of HSP, the exact function of TECPR2 is unknown.
This study primarily focused on the determination of TECPR2’s binding mode to ATG8 proteins in vitro and in cells. The association of TECPR2 to all ATG8-family proteins was confirmed in in vitro pulldown experiments. Following fragment-based binding and peptide array experiments, the LC3-interacting region (LIR) of TECPR2 could be verified with mutants of TECPR2 lacking the LIR motif. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) were conducted to gain deeper insights into the binding preference to the different ATG8-family members. Moreover, the crystal structure of TECPR2-LIR was solved. In cells colocalization studies with overexpressed ATG8 proteins unraveled a preferential binding to the LC3-subfamily.
Further, mass spectrometric analysis revealed novel association partners of TECPR2: SEC24D, HOPS and BLOC-1, all of those participating in different endomembrane trafficking pathways. Interaction and colocalization of TECPR2 with these components was validated with several immunoprecipitation experiments and the N-terminal part of the protein comprising the WD40-domain could be defined as the binding site for all three of the association partners. In further approaches, the requirement of the LIR-motif and the necessity of the availability of LC3 protein for the particular interactions were determined. Interestingly, in the absence of LC3C the binding of TECPR2 to SEC24D was completely disrupted whereas a loss of LC3B only resulted in a decreased association. Notably, the binding proteins were not subjected to autophagosomal degradation, indicating that TECPR2 may operate as a multifunctional scaffold protein. While depletion of TECPR2 destabilized HOPS and BLOC-1, the autophagy defect observed in TECRP2-deficient cells could not be attributed to functional impairment of these two complexes.
Moreover, loss of TECPR2 led to a decline in protein levels of SEC24D and of its heterodimer partner SEC23A. Thus, TECPR2 is required to regulate the protein levels of SEC23A and SEC24D and subsequently the formation of the heterodimers. Together, SEC24D and SEC23A form the inner coat of COPII vesicles. These vesicles are responsible for the anterograde transport of cargo from the ER toward the Golgi compartment. COPII-coated vesicles are secreted form ER at distinct sites, termed ER exit sites (ERES). The small GTPase SAR1A maintains the vesicle budding, coating and secretion at the ERES. Together with SEC13, SEC31 forms the outer coat of the COPII vesicles and therefore serves as a general ERES marker.
Consistent with a defect in COPII coat assembly, the number of ERES diminished in the absence of TECPR2. These phenotypes could be rescued by the wildtype TECPR2 protein but not by the LIR-mutant. Intriguingly, these results were mimicked by depletion of LC3C, which localized to ERES. By monitoring the release of various cargos from ER in dependency of TECPR2 or LC3C, a role of both proteins in ER export was determined. These facts indicated that TECPR2 cooperates with LC3C to facilitate COPII assembly, ERES maintenance and ER export. Notably, fibroblast derived from a HSP patient carrying mutated TECPR2 showed diminished SEC24D protein levels and delayed ER export.
Concurrent with emerging evidence for a role of ERES in autophagosome formation, depletion of TECPR2 or LC3C or overexpression of a constitutive inactive SAR1 mutant reduced puncta formation of the early autophagosomal protein WIPI2.
In summary, this study uncovered a role for TECPR2 in ER export at ERES through interaction and stabilization of SEC24D, a COPII coat protein. This process also depended on ATG8-family protein LC3C, which is localized at ERES. Both proteins are required for correct COPII-mediated secretion. Moreover, the presence of TECPR2 and LC3C on ER allows development of omegasomes, membranous structures budding ER to form autophagosomes, by stabilization of WIPI2 and therefore contribute to autophagosome formation.
Transport processes across the membrane are essential to ensure survival of every living cell. Therefore, the exchange of membrane impermeable molecules is mediated by specific transport proteins, which are embedded in the lipid bilayer.
One important class comprises secondary active transporters, which couple very efficiently the uphill transport of the main substrate against its concentration gradient to the downhill transport of an additional substrate. These transporters are widely distributed among all kingdoms of life and accomplish many crucial functions. One function is to counteract the deleterious effect of hyperosmotic stress in bacteria. Several members of the BCCT (betaine-choline-carnitinetransport) family of secondary transporters mediate osmostress protection by the accumulation of the compatible solute betaine or its precursor choline (Lamark et al., 1991; Peter et al., 1996; Ziegler et al., 2010). Besides osmo-dependent sodium or proton-coupled symporters, the BCCT family includes few rare representatives of osmo-independent transporters such as the substrate:product antiporter CaiT from E. coli (Jung et al., 2002; Ziegler et al., 2010).
The best-characterized member of the BCCT family is the sodium-coupled betaine transporter BetP from Corynebacterium glutamicum. BetP together with the ABCtransporter OpuA and the H+-solute symporter ProP, became a paradigm for osmoregulated osmolyte transport. Although, all three transporters were extensively studied, the general mechanism of osmoregulation is still far from being understood. Thus, one task of this thesis was to elucidate further the regulatory properties of BetP.
BetP is tightly regulated by osmotic stress and is able to increase its basal betaine uptake activity dramatically upon elevated osmolalities within one second (Peter et al., 1998a). The osmotic stress is sensed by BetP via two stimuli, one is the increase of the internal K+ concentration above a threshold of 220 mM (Rübenhagen et al., 2001), the second is related to a change in the physical state of the membrane (Maximov et al., 2014). So far, several solved crystal structures in combination with functional and computational analysis provided insights into the coupling mechanism of betaine and its co-substrate sodium (Khafizov et al., 2012; Perez et al., 2012). Despite the wealth of data, the precise regulatory mechanism of trimeric BetP is still unclear.
Channelrhodopsin-2 (ChR2) is a light-gated cation selective channel from the unicellular alga Chlamydomonas reinhardtii, which is involved in phototaxis and photophobic responses. As other rhodopsins, ChR2 comprises a seven-transmembrane helix (TMH) motif and a retinal as the light-sensitive chromophore. The chromophore is covalently attached via a protonated Schiff base to the conserved lysine residue Lys257 located in TMH7. Based on its primary sequence and the all-trans configuration of the retinal in the ground state, ChR2 is assigned to the type I rhodopsins, also referred to as microbial-type rhodopsins. Upon light activation, the retinal isomerizes from the all-trans to the 13-cis form. This photoisomerization, which is accompanied by conformational changes of the protein, eventually leads to the opening of the channel and cation translocation. Cation flux during the conductive state leads to depolarization of the cell membrane and subsequent triggering of action potentials when expressed in neurons. Therefore, ChR2 has become the most versatile optogenetic tool, enabling a non-invasive investigation of neural circuits at high spatial and temporal resolution. With the rapidly increasing importance of ChR2 as a tool in neurobiology and cell biology, structural information is the prerequisite to an unambiguous understanding of the molecular mechanisms of this unique light-activated ion channel. The coupling between isomerization and structural alterations is well understood for other microbial-type rhodopsins, like bacteriorhodopsin (bR), halorhodopsin (HR) and sensory rhodopsin II (SRII). In case of ChR2, the first data on light-induced conformational changes came from spectroscopic studies and structural information is still missing. However, in order to fully understand the mechanism of light transduction by ChR2, it is necessary to determine the changes in the protein structure at specific steps in the photocycle.
By the time I started my PhD thesis, there was no structural information of ChR2 available. Therefore, the objective of this thesis was to obtain structural information of the transmembrane domain containing the first 315 amino acids of ChR2 by cryo electron crystallography. Besides revealing the structure of membrane proteins, cryo-EM of two-dimensional (2D) crystals is ideal for investigating conformational changes in membrane proteins induced by different stimuli. Therefore, the second objective of my thesis was the investigation of light-induced conformational changes in the slow C128T ChR2 mutant. The ~1,000 times longer lifetime of the open state of the C128T mutant compared to the wild-type allowed to trap different intermediates that accumulate during the photocycle.
In 2012, the X-ray structure of a channelrhodopsin-1/channelrhodopsin-2 chimaera (C1C2) at 2.3 Å resolution in the closed dark-adapted state was published (Kato et al., 2012). The structure revealed the essential molecular architecture of C1C2, including the retinal-binding pocket and the putative cation conduction pathway. Together with biochemical, spectroscopic, mutagenesis experiments, and the high-resolution model, some functionally important residues of ChR2 have been identified. However, unambiguous explanation of the molecular determinants that contribute to activation (gating) and transport were still mostly unknown.
RESULTS AND CONCLUSIONS
The first half of my theses dealt with 2D crystallization of ChR2. I succeeded in obtaining 2D crystals of ChR2 of four different types, which differed in size, crystal packing, crystal contacts and resolution, yielding structure factors up to 6 Å resolution. The crystals were grown by reconstituting the protein with different lipids at various lipid-to-protein ratios. The best crystals formed with the synthetic lipid DMPC and EPL upon detergent removal by dialysis. The projection maps calculated from these crystals revealed the overall structure of C128T ChR2 at 6 Å resolution and were published in 2011 (Müller et al., 2011). Surprisingly, ChR2 was found to be a dimer in all crystal types. The ChR2 dimer was stable both in detergent solution and in the presence of lipids for 2D crystallization. The monomers clearly showed the expected densities for the seven TMHs.
The arrangement of the ChR2 dimers on the four 2D lattices was different. However, comparison of the individual rojection maps revealed no significant differences within the ChR2 interface in the four crystal forms. The observation that the structure of the dimer was the same in all four crystal forms and in different lipids suggested strong specific contacts between the two protomers and implied that the protein was also dimeric in the native membrane. These findings were in agreement with Western blot analysis of plasma membranes from oocytes expressing ChR2 and laser-induced liquid bead ion desorption mass spectrometry, which both showed ChR2 as a dimer. The unusual stability of the ChR2 dimer contrasts with other microbial rhodopsins, which exist in different oligomeric states, i.e. monomers, trimers or dimers. These observations raised the question whether the functional unit is the monomer or the dimer.
The comparison of the projection map of the light-driven proton pump bR at the same resolution showed similar overall dimensions. Based on this comparison, the densities which became evident in the ChR2 projection maps could be assigned to the corresponding seven densities in bR. The shape of the densities near the dimer interface suggested that TMHs 2, 3, and 4 are oriented more or less perpendicular to the membrane plane, while the other four helices appear to be more tilted, as in bR.
Based on the high-resolution bR structure and the projection structures obtained, I have built a homology model. On the basis of this homology model, several residues found in the dimer interface were selected for mutational studies in order to disrupt the dimer interface.
The investigation of light-induced conformational changes in C128T ChR2 was the second part of my thesis. I designed an experimental setup for trapping light-induced conformational changes in C128T ChR2. In addition, I optimized the sample preparation in a way that the different illumination conditions did not alter the quality of the crystals. I have trapped two different functional states, namely the conductive open state and the non-conductive closed dark-adapted state.
In order to visualize the location and the extent of conformational changes, projection difference maps were calculated between the open and the closed state. Visual inspection of the difference maps between the open and the two closed states revealed three difference peaks that map to the TMHs 2, 6, and 7, indicating significant and specific rearrangements of these helices. The strong pair of positive/negative peaks at TMH6 suggests an outward tilt movement of approximately 2 Å. Close comparison of similar work on bR revealed that this movement is likely to occur at the cytoplasmic end of TMH6. A second highly significant negative peak is observed at TMH7, indicating a less pronounced tilt compared to TMH6. The third negative peak at TMH2 indicates a loss of density in this region. No significant differences were recorded at the TMH1, 5 and at the dimer interface formed by TMH3 and 4.
I succeeded in trapping and characterizing the open and closed state in the photocycle of ChR2 and could demonstrate that the transition from the closed to the open state is linked to significant light-induced tilt movements of TMH6 and 7, plus a loss of order in TMH2. These conformational changes are likely to create a large water-filled conducting pore, which seems to be required for the conductance of up to 2,000 ions per photocycle. The previously mentioned spectroscopic studies support the difference structures I obtained. This approach sets the stage for studying structural changes accompanying the formation and decay of other photocycle intermediates in ChR2. Future studies will aim at three-dimensional maps of the open and closed state at higher resolution.
The high selectivity of biological transformations taking place in Nature have long inspired synthetic chemists to develop analogous chemical processes. Similarly, transient intermediates identified in chemical transformations often provide a basis to understand biological processes. Therefore, new insights gained in biological studies are often useful for chemistry and vice versa.
Proteins, and catalytically active enzymes, are among the most essential units of living cells. Metalloproteins or -enzymes, i.e., proteins or enzymes that contain transition metal ions such as copper, nickel, iron or zinc are often involved in processes like (1) metal-ion storage and transport, (2) exchange of electrons with the environment in catalysis and electron transfer reactions, and (3) dioxygen storage, transport, and metabolization.
For decades, copper-mediated biological oxidations have spurred a great deal of interest among synthetic and catalytic chemists. Copper enzymes such as dopamine β-monooxygenase (DβM), peptidylglycine α-hydroxylating monooxygenase (PHM),particulate methane monooxygenase (pMMO) and tyrosinase activate molecular oxygen (O2) and incorporate one of the oxygen atoms selectively into C−H bonds yielding hydroxylated organic substrates. Remarkable progress in bioinorganic research has led to the development of a large number of copper-based model systems supported by various nitrogen donor ligands that bind O2, cleave the O−O bond, and/or afford hydroxylation reactions similar to copper enzymes. These synthetic model systems have helped to understand the structureactivity relationships of their biological role models and supporting theoretical studies have contributed substantially to the development of the field. Specifically, several density functional theory (DFT) studies have provided detailed mechanistic insights into coppermediated aliphatic and aromatic hydroxylation reactions. Until to date, however, pertinent quantum chemical research still suffers from severe problems as to identify sufficiently accurate and efficient methods for mechanistic studies, and conflicting literature reports have created confusions within the scientific community. Therefore, the first aim of this thesis is to identify a DFT method well suited to describe copper-mediated hydroxylation reactions. With this method at hand a number of interesting hydroxylation reactions is investigated aiming at a detailed understanding of the underlying reaction mechanisms.
The thesis is divided into four chapters of which the first, the introductory chapter, is further divided into three sections (1) copper proteins and enzymes, (2) copper-O2 reactivity in enzymes and (3) biomimetic Cu/O2 chemistry. The first section gives a brief overview of a number of copper enzymes. The second section provides a concise introduction to the biochemical transformations brought about by those copper enzymes that perform aliphatic and aromatic hydroxylation reactions. It is shown that such copper enzymes carry different types of active sites which are responsible for their specific biological functions. These copper enzymes with their biological function are the role models for synthetic chemistry. In the third section, biomimetic Cu/O2 chemistry, the insights gathered in the past 35 years of extensive research on copper-based synthetic model systems that mimic various aspects of copper-enzyme reactivity are reviewed. Various types of active copper sites have been realized in these synthetic model systems and a brief introduction to the respective reactivities towards C−H bonds is presented. We will specifically focus on isomerization processes of dinuclear active Cu2O2 sites and the specific reactivity aspects of these isomers, as these phenomena have been the subject of enormous research efforts aiming at the understanding of the function of the enzyme tyrosinase.
Theory has been integral part of this research and density functional theory (DFT) has effectively taken over the role as a working horse in most studies. Therefore, the second chapter is devoted to an exposition of earlier DFT applications in mechanistic studies of Cu/O2 chemistry. We specifically highlight the problems related to the use of DFT in this field and illustrate the present state of knowledge.
The third chapter of this thesis provides results and discussion of (1) DFT benchmark studies and (2) mechanistic studies. In the first section, the results of a careful benchmark study on the performance of various DFT methods to study the μ-η2:η2-peroxodicopper(II)/bis(μ-oxo)dicopper(III) core isomerization and the C–H hydroxylation processes are compared with available experimental reference data. We provide an assessment of the effects of relativity, counteranions, and dispersion on the reference reactions. The most suitable DFT method evolving from this study, BLYP-D/def2-TZVP including solvent and relativistic corrections, is applied in the next sections to investigate the mechanistic scenario underlying three copper-dioxygen mediated hydroxylation reactions of aliphatic and aromatic C–H bonds. Our mechanistic studies show that bis(μ-oxo)dicopper(III) complexes are capable of achieving selective aliphatic and aromatic C–H hydroxylations. The study of substituent effects in these reactions has further shown that the bis(μ-oxo)dicopper complex acts as an electrophile in hydroxylation.
The fourth chapter presents the conclusions of our investigations. Part of the work presented in this thesis has been published in a peer reviewed journal and enclosed in appendix 1. Further research work, not presented in chapters 1-4, was conducted during my PhD time. This has led to two publications which are added in the appendix.
Small molecule inhibitors sensitize neuroblastoma cells for chemotherapeutic drug-induced apoptosis
(2015)
Neuroblastoma (NB) is one of the most common solid extracranial pediatric tumors, deriving from undifferentiated cells of the peripheral nervous system. It accounts for approximately 10% of all childhood cancers. High stage tumors usually show poor prognosis despite aggressive treatment such as radiotherapy or chemotherapy. Therefore, it is of utmost importance to find novel treatment strategies in order to improve existing chemotherapy protocols. Combination treatment offers advantages, as chemotherapeutic drugs can be applied in low and subtoxic doses, reducing possible side-effects. Here, we report in a two-part study that small molecule inhibitors (SMI), namely BI 2536, a PLK1 inhibitor and BV6, a SMAC mimetic (SM), sensitize neuroblastoma cells for chemotherapeutic drug-induced cell death. By using i) BI 2536 in combination with vinca alkaloids and ii) BV6 in combination with either doxorubicin or vinca alkaloids, we show that cell death is synergistically enhanced compared to monotherapy. Furthermore, combination treatment significantly reduces survival of NB cells in long-term assays, compared to single treatment. We identify that vinca alkaloid/SMI combinations induce mitotic arrest, as shown by phosphorylation of histone H3, which results in the induction of intrinsic apoptosis and inhibition of CDK1 by RO-3306 could abolish these findings. Mechanistically, upon vinca alkaloid/SMI-induced mitotic arrest, anti-apoptotic BCL-2 proteins such as MCL-1, BCL-2 or BCL-XL are degraded or inactivated by phosphorylation, which induces the activation of the proapoptotic BCL-2 family proteins BAX and BAK. The importance of the mitochondrial apoptosis pathway in vinca alkaloid/SMI-induced cell death was further highlighted by the fact that ectopic expression of BCL-2 inhibits vinca alkaloid/SMI-induced DNA fragmentation and BAK- and caspase-activation. In contrast to the vinca alkaloid/SMI cotreatment, DOX/SMI (DOX/BV6)-induced apoptosis only partially involves the mitochondrial pathway. Instead, we clarify that RIP1 is required for DOX/BV6-induced apoptosis, as pharmacological and genetic inhibition of RIP1 rescues from apoptosis induction. Although it has been shown in previous studies that SM-treatment (e.g. BV6) can induce the NF-κB pathway and auto-/paracrine TNFα production through cIAP1/2 depletion, DOX/BV6-induced apoptosis is completely independent of NF-κB activation in our setting, despite fast cIAP1 depletion. This conclusion is based on the fact that inhibition of the NF-κB pathway by exogenously expressed dominant-negative IκBα as well as application of a TNFα blocking antibody does not reduce DOX/BV6-induced cell death. In summary, we unravel two new promising treatment strategies for neuroblastoma patients by using a combination treatment of two different small molecule inhibitors, combined with well-characterized chemotherapeutic agents. Furthermore we give detailed insights into cell death pathways induced by these combination treatments, in which mitochondria and RIP1 have a differential role in chemotherapeutic drug-induced apoptosis.
Cardiac progenitor cells hold great potential for regenerative therapies in heart disorders. However, the molecular mechanisms regulating cardiac progenitor cell expansion and differentiation remain poorly defined. Here we show that the multi- adaptor protein Ldb1, which mediates interactions between different classes of LIM domain transcription factors, is a multifunctional regulator of cardiac progenitor cell differentiation. Ldb1-deficient embryonic stem cells (ESCs) show a markedly decreased expression of second heart field (SHF) marker genes and subsequently impaired cardiomyocyte differentiation. Conditional ablation of Ldb1 in the early SHF using an Isl1-Cre driver led to embryonic lethality at Embryonic day (E)10.5 with cardiac abnormalities including a significantly smaller right ventricle and a shortened outflow tract, supporting a crucial role of Ldb1 in the SHF. Mechanistically we show that the importance of Ldb1 for SHF development is two-fold: On the one hand, Ldb1 binds to Isl1 and protects it from proteasomal degradation, as a consequence of which Ldb1-deficiency leads to an almost complete loss of Isl1+ cardiovascular progenitor cells. On the other hand the Isl1/Ldb1 complex promotes long-range promoter-enhancer interactions at the loci of the core cardiac transcription factors Mef2c and Hand2. Chromosome conformation capture followed by sequencing (3C- seq) identified specific Ldb1-mediated interactions of the Isl1/Ldb1 responsive Mef2c anterior heart field enhancer with genes which play key roles in cardiac progenitor cell function and cardiovascular development. These interactions are of critical importance to regulate the expression of the downstream target genes since their expression levels are strongly dependent on the Ldb1/Isl1 levels. Overexpression of an Ldb1 mutant, which contains the LIM interaction domain and thereby can protect Isl1 protein from degradation, but lacks the dimerization domain and thus cannot promote long-range interactions, does not collaborate with Isl1 to regulate the expression of their common targets and results in defects in Isl1+ cardiac progenitor differentiation. In this thesis we show one of the first examples of genome-wide chromatin reorganization mediated by a developmental regulated, cell type specific, transcription complex. Ldb1 in concert with Isl1 promotes long range promoter- enhancer and enhancer-enhancer interactions in order to create active chromatin hub where gene important for heart development can be co-regulated. Moreover, Isl1 and Ldb1 genetically interact during heart development, as Isl1/Ldb1 haplodeficient embryos show various cardiac anomalies. The dosage-sensitive interdependence between Isl1 and Ldb1 in the expression of these key factors in cardiogenesis, further supports a key role of the Isl1/Ldb1 complex in coordinating a three dimensional genome organization, upstream of a regulatory network driving cardiac differentiation and heart development.
In conclusion, the Isl1/Ldb1 complex orchestrate a genome-wide three dimensional chromatin reorganization resulting in a transcriptional program responsible for the differentiation of multipotent cardiac progenitor cells into cardiomyocytes.
Small molecule drug discovery is strongly supported by biophysical data. In the reach of this thesis, cell free protein expression was used to produce human target proteins for ligand binding assays using Surface Plasmon Resonance spectroscopy (SPR). In the second step the binding and interaction characteristics of small molecules and fragments were analyzed using Nuclear Magnetic Resonance spectroscopy (NMR).
The first target protein was the human acid sensing channel 1 (ASIC1a). ASIC1a was expressed in a cell free expression system based on E.coli lysate. To optimize the expression, several parameters including fusion tags, ion concentrations and different hydrophobic environments were tested.
The adaption of the folding environment for ASIC1a needed more optimization, because it is a very challenging target to express in an in vitro system. Three different expression modes were employed to find a suitable folding environment.
SPR binding studies with ASIC1a were performed with chicken ASIC1a expressed in insect cells. The immobilization of cASIC1a and the used buffer conditions were tested using Psalmotoxin 1, a naturally occurring peptide venom which binds strong to the trimeric form of ASIC1a. Compound characterization experiments were performed with a variety of different ligands including amiloride, a general blocker of the whole ENaC protein family. None of the used ligands showed titration curves that would match a simple 1:1 binding model. The experiments either show no binding signal or signal that could be interpreted as unspecific binding. Even amiloride that should be binding the protein shows no signals that fit a simple binding model.
Another target protein that was investigated is the soluble prolyl cis/trans isomerase Cyclophilin D (or peptidyl prolyl isomerase F – PPIF). This protein is involved in the regulation of the mitochondrial permeability transition pore and therefore a potential drug target to treat neurodegenerative diseases. Small molecule binding was tested with CypD using SPR. Following the kinetic analysis of small molecule ligands, the binding position of different binding fragments was analyzed. These fragments originated from a SPR based fragment screen and gave no co-crystal structures with CypD. Therefore NMR was used to investigate the binding position of these fragments. An analysis of the chemical shift perturbations upon ligand addition revealed that the NMR analysis was in line with the results gathered by x-ray crystallography. The fragments with unknown binding position however, all bind to a specific patch slightly outside the binding pocket.
The ligand CL1 showed a special behavior in the NMR experiments. Upon addition to CypD, it produced large shifts on many signals of the protein, accompanied by a severe line broadening. The shift perturbations were so numerous and large that the spectrum had to be reassigned in complex with the ligand. Triple selective labeling was applied to allow a fast and nearly complete signal assignment. The possibility to use highly sophisticated labeling schemes, is one of the advantages of cell free protein expression. After the assignment of the complex spectrum, the chemical shift perturbations were analyzed and quantified. The residues showing the strongest CSPs are also identified in the crystal structure to be involved in the binding of CL1, giving a consistent picture. The numerous and large shift perturbations, produced by CL1 led to the assumption, that the ligand induces a conformational change in CypD, which is not represented in the co-crystal structure. This conformational change was characterized by a NMR based structure determination. CypD apo yielded a defined bundle, whose folded regions overlap well with the corresponding crystal structure.
For the calculation of the CypD-CL1 complex structure, the sidechain resonances were assigned using an automated assignment approach with the software FLYA. The calculation of the CypD-CL1 complex structure did not result in a defined bundle. While parts of the protein converge in a well folded state, the region around the active site shows no defined folding. Careful analysis of the structure calculation suggests that the problems during structure calculation did not originate from an incorrect resonance assignment, but rather from a lack of NOE crosspeaks. This might be due to a broadening of the corresponding NOE crosspeaks or the coexistence of many different conformations. This leads to the conclusion, that the protein conformation is not defined by the NMR data and could be in a dynamic interchange between multiple structures.
This hypothesis is supported by other observations. The line broadening of the signals in the complex is pronounced in the area around the active site and the substrate binding pocket, hinting to a connection between catalytic activity and protein dynamics. In addition many NMR signals are sensitive to changes in the measurement field strength and the temperature. This field dependent signal splitting suggests dynamic conformational changes in the protein between at least two different conformations on a millisecond timescale.
The current working model is that CL1 binds to CypD and induces the catalytic cycle and the connected conformational changes in CypD. As a result the proline like moiety in CL1 is constantly switching between the cis and the trans conformation. Due to the high affinity of CL1, the inhibitor does not leave the binding pocket after successful catalysis, but stays bound in the pocket stimulating further catalytic cycles. These findings as well as the working model are well in line with data published for Cyclophilin A, another member of the cyclophilin family, thereby supporting the model.
During the last decade of the 20th century, the field of mass spectrometry has seen a revolutionary change in its application and scope. The introduction of soft ionization methods for the analysis of biological molecules has expanded the area of mass spectrometry from its early roots in the analysis of inorganic and organic species into the fields of biology and medicine.
Today, the use of the mass spectrometry is extended to a wide range of applications in biotechnology and pharmaceutical industry, in geological, environmental and clinical research. In biochemistry, the principles of mass spectrometry are, however, broadly applicable in accurate molecular weight determination, reaction monitoring, amino acid sequencing, oligonucleotide sequencing and protein structure.
In order to carry out their biological activities, proteins interact most often to each other and form transient or stable complexes. In addition, some proteins specifically interact also with other proteins or with non-protein molecules, such as DNA, RNA or metabolites, these interactions being critical for their function. Hence, defining the composition of protein complexes, as well as understanding how protein complexes are assembled and regulated yield invaluable insights into protein function. Coupled with an isolation technique to purify a specific protein complex of interest, mass spectrometry can rapidly and reliably identify the components of complexes. In addition, quantitative MS techniques offer the possibility of studying dynamically regulated interactions....
Rotary adenosine triphosphate (ATP)ases are ubiquitous, membrane-bound enzyme complexes involved in biological energy conversion. The first subtype, the so-called F1Fo ATP synthase, predominantly functions as an ATP synthesizing machinery in most bacteria, mitochondria and chloroplasts. The vacuolar subtype of enzyme, the V1Vo ATPase, operates as an ATP driven ion pump in eukaryotic membranes. The subtype found in archaea and some bacteria is called A1Ao ATP (synth)ase and is capable of working in both directions either to synthesize ATP or to generate an ion motive force by consuming the same.
All the three above-mentioned subtypes of rotary ATPases work as nanomolecular machines sharing a conserved mechanism to perform the energy conservation process. The simplest form of these enzymes is the bacterial F1Fo ATP synthase. Here, ions are channelled via the membrane stator subunit a to the rotor ring of the enzyme. After almost a complete rotation of the ring the ions are released again on the other side of the membrane. This rotation is further transmitted via the central stalk to the soluble part of the enzyme, the F1-complex, where conformational changes within the nucleotide binding sites result in the synthesis of ATP from ADP and Pi.
The rotor or c-ring of the enzyme is the key protein complex in mediating transmembrane ion translocation. Several structural and biochemical methods have been applied in the past years to study the rotor rings from many different organisms. The results revealed that the stoichiometry of a c-ring of a given species is constant while it can vary between different species within a range of 8 to 15 c subunits. The c-ring stoichiometry determines directly the number of ions transported through Fo per rotation whereby three molecules of ATP are concurrently synthesized in the water-soluble F1 headgroup. Hence the number of c subunits has an important influence on the bioenergetics of the corresponding enzyme and thus the entire organism.
The c-ring of a rotary ATPase is able to specifically bind either protons (H+) or sodium ions (Na+) as the coupling ion for the enzyme. Several structures are already available revealing the coordination network of both types of rotor rings. In each case ion binding includes a highly-conserved carboxylic acid residue (glutamate or aspartate), in addition to a more varying combination of amino acid residues, whereby Na+ coordination is structurally more demanding than H+ binding.
In the first part of my PhD thesis, I aimed to characterize the F1Fo ATP synthase rotor ring of the opportunistic pathogenic bacterium Fusobacterium nucleatum on a functional and structural level. F. nucleatum is an anaerobic bacterium which uses peptides and amino acids as a primary energy source. It is one of the most frequently occuring bacteria in human body infections and involved in human periodontal diseases.
The protein complex was heterologously expressed within a hybrid ATP synthase in Escherichia coli and purified without an affinity tag for further analysis. Two high resolution X-ray structures of the c-ring were solved at low (5.3) and high (8.7) pH to 2.2 and 2.64 Å, respectively. In both structures, the conserved glutamate is in an ion-locked conformation, revealing that the conformational state of the ion binding carboxylate is not depending on the pH of the crystallization condition, which is in good agreement with previous structural and biochemical studies of other c-rings.
A Na+ ion is present within the c-ring binding site and directly coordinated by four amino acid residues and a structural water molecule. Remarkably, the Na+ is bound by two glutamate residues instead of one as is the case in the I. tartaricus Na+ binding c-ring, of which the first high resolution X-ray structure of a c-ring has been solved in 2005. Thus, a new type of Na+ coordination in an ATP synthase rotor ring with a two-carboxylate ion binding motif is described here, which also occurs in other bacteria, including several pathogens. Na+ specificity of the investigated c-ring was further confirmed by a competitive biochemical labeling reaction performed with a fluorescent ATP synthase inhibitor molecule (N-cyclohexyl-N`-[4(dimethylamino)-α-naphtyl] carbodiimide, NCD-4).
We furthermore complemented our functional and structural data of the F. nucleatum c-ring by computational studies to explore the ion translocation mechanism of this enzyme in more details. We therefore analyzed the protonation state of the second, additional glutamate in the ion binding site. Molecular dynamics (MD) simulations and free-energy calculations indicated that this glutamate is constitutively protonated, in the ion-locked as well as in a simulated, more hydrated open-conformation of the ion binding glutamate as when it is travelling through the a/c-ring interface upon c-ring rotation.
During my thesis, I worked on two different membrane proteins. One is a bacterial secondary transporter and the second is a human mitochondrial calcium channel.
The first part of my thesis involves the structural and biochemical characterization of an L-carnitine/ γ-butyrobetaine antiporter from bacteria called CaiT. The aim of the project was to understand the Na+ independence of CaiT and to determine the crystal structures of CaiT in different conformations to expand the mechanistic understanding of substrate/ product antiport in CaiT.
The study revealed how a positively charged amino acid side chain (arginine 262) in CaiT could structurally and functionally mimic a sodium ion. Additionally, various crystal structures of CaiT obtained in this study demonstrate that the central substrate-binding site is highly dynamic and can accommodate the substrate in various orientations.
In the second part of my thesis, I was able to optimize the expression and purification conditions for the human mitochondrial calcium uniporter or the MCU. Understanding how this channel functions can help us unravel the mechanism of calcium uptake by mitochondria. Secondary structure prediction analysis in combination with mass spectrometry of degraded MCU products obtained during the purification of the full-length protein led to the identification of a stable MCU construct. This study resulted in the successful purification of milligram quantities of stable MCU protein for the first time. Further optimization may be required to obtain more homogenous protein that is amenable for crystallization.
Disturbances in lipid metabolism are responsible for many chronic disorders, such as type 2 diabetes and atherosclerosis. Regulation of lipid metabolism occurs by activated transcription factors peroxisome proliferator-activated receptor δ (PPARδ) and liver X receptor α (LXRα) mediating transcription of different target genes involved in regulation of fatty acid uptake and oxidation or cellular cholesterol homeostasis. This is especially relevant for the macrophages, since pathways regulated by PPARδ and LXRα affect foam cell formation, a process driving the progression of atherosclerotic lesion. AMP-activated protein kinase (AMPK) plays a central role in energy homeostasis in every type of eukaryotic cell, but its role in human macrophages, particularly with regard to lipid metabolism, is not precisely defined yet. Thus, I investigated the impact of AMPK activity on PPARδ and LXRα and the expression of their target genes involved in fatty acid oxidation (FAO) and cholesterol metabolism.
As PPARδ has been described as a potential target for prevention and treatment of several disorders and AMPK as interesting drug target for diabetes and metabolic syndrome, the aim of the first part of my studies was to investigate their interaction in primary human macrophages. Completing the first challenge successfully, I was able to establish a lentiviral transduction system for constitutively active AMPK (consisting of a truncated catalytic AMPKα1 subunit bearing an activating T198D mutation) in primary human macrophages.
Using genome-wide microarray analysis of gene expression, I demonstrate FAO as the strongest affected pathway during combined AMPKα1 overexpression and PPARδ activation.
The most influenced genes were validated by quantitative PCR as well as by Western analysis. I found that AMPK increases the expression of FAO-associated genes targeted by PPARδ. Corroborating the results obtained using AMPKα1 overexpression, PPARδ target gene expression was increased not only by PPARδ agonist GW501516, but also by pharmacological allosteric AMPK activator A-769662. Additional enhancement of target gene mRNA expression was achieved upon co-activation of PPARδ and AMPK. Silencing PPARδ expression increased basal expression of target genes, confirming the repressive nature of ligand-free PPARδ, abolishing the increased target gene expression upon AMPK or PPARδ activation. Measurements of triglyceride contents of human macrophages incubated with VLDL following PPARδ activation demonstrated a reduction of intracellular triglyceride accumulation in cells, which may reflect the enhancement of fat catabolism.
In the second part of my studies, I concentrated on the regulation of cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) expression by AMPK. ABCA1 facilitates
cholesterol efflux from macrophages thus, preventing atherosclerosis progression. For the first time, AMPK implication in the regulation of the ABCA1 pathway could be presented. Both AMPK overexpression and activation lead to significantly increased ABCA1 expression, whereas AMPKα1 knock-down strongly reduced this effect. Besides, I was able to prove an enhanced activity of ABCA1 during AMPK activation in human THP-1 macrophages by measuring cholesterol efflux into apolipoprotein AI-containing medium.
Previous findings showed regulation of ABCA1 by LXRα. I confirmed these results by silencing experiments indicating an essential role of LXRα in ABCA1 regulation pathway.
Here, ABCA1 mRNA as well as protein expression were positively mediated by LXRα. LXRα activation elevated ABCA1 levels, whereas its silencing down-regulated this effect.
Interestingly, ABCA1 was found to be regulated only by LXRα and not through LXRα. At the same time, knock-down of PPARδ, -γ or -δ, which may be also involved in the regulation of LXR/ABCA1 axis, did not influence the activation of ABCA1 expression by an AMPK activator. To confirm that LXRE on Abca1 promoter is essential for ABCA1 regulation, I performed luciferase reporter assay using constructs based on Abca1 promoter with or without LXRE mutation. Mutation of LXRE abolished reporter activity, whereas AMPK activation increased luciferase activity of wild-type LXRE construct. Furthermore, I demonstrate AMPK-dependent LXRα binding to the LXRE site of Abca1 promoter using the method of chromatin immunoprecipitation. AMPK activation significantly increased, whereas silencing of AMPK significantly attenuated LXRα binding, indicating AMPK as one of the most important regulators of ABCA1 expression.
In summary, I provided an evidence for AMPK involvement into lipid and cholesterol metabolism in human macrophages showing the regulation of PPARδ and LXRα target genes. The understanding of AMPK and PPARδ interaction allows the development of new approaches for treatment of metabolic syndrome and related diseases. Increased FAO during the activation of both proteins may exhibit better therapeutic benefit. On the other hand, I have shown the impact of AMPK activation on ABCA1 via LXRα up-regulation leading to increased cholesterol efflux in human macrophages for the first time. These findings thus may impact future improving of anti-atherosclerosis therapies.
Since Inhibitor of Apoptosis (IAP) proteins are frequently dysregulated in different cancer entities and contribute to apoptosis resistance, pharmacological IAP antagonists are considered to be promising agents for the future development of cancer treatment strategies. IAP antagonists are small-molecule drugs that have been designed to mimic the interaction site of IAP proteins with their endogenous inhibitor Second mitochondrial activator of caspases (SMAC). Thus, they are frequently referred to as SMAC mimetics. Treatment with SMAC mimetics engages an apoptotic program in cancers by affecting different components of the apoptotic machinery. Besides disinhibition of caspases, SMAC mimetics trigger non-canonical nuclear factor-κB (NF-κB) signaling, which induces upregulation of tumor necrosis factor (TNF) α and other NF-κB target genes. In particular, TNFα production has been closely linked to the induction of SMAC mimetic-mediated cell death. The TNFα-dependent para/autocrine loop facilitates the formation of a cytosolic complex consisting of caspase-8, Fas-associated death domain (FADD) and Receptor-interacting protein (RIP) 1, which serves as caspase-8 activation platform and ultimately triggers induction of apoptosis. In the present study, we use the small-molecule bivalent SMAC mimetic BV6 to analyze SMAC-stimulated NF-κB signaling in cancer cell lines of different entities. Interestingly, we identify two novel NF-κB-regulated factors that are both required for SMAC mimetic-induced apoptosis in a context-dependent manner. First, we show that NF-κB-dependent upregulation of death receptor 5 (DR5) can serve as an alternative mechanism of BV6-mediated cell death. We demonstrate that BV6 treatment induces NF-κB-dependent but largely TNFα -independent apoptosis in A172 glioblastoma cells. By using an unbiased whole genome expression analysis approach, we identify DR5 as a critical NF-κB target gene, which substitutes TNFα and is indispensable for BV6-initated cell death in A172 cells. Second, we demonstrate that Interferon regulatory factor (IRF) 1 is required for BV6-induced TNFα production and apoptosis. Our study provides evidence that IRF1 closely cooperates with the NF-κB network in BV6-mediated cell death and additionally alters expression of selective SMAC mimetic-induced target genes. Furthermore, we show that BV6 treatment triggers secretion of a set of proinflammatory cytokines and increases attraction of monocytes to BV6-treated tumor cells in an IRF1-dependent manner. In summary, our work supports the notion that NF-κB-regulated factors are critically required for SMAC mimetic-initiated apoptosis. We show that IRF1 is indispensable for TNFα production and cell death in BV6-sensitive cell lines and that also DR5 can serve as a proapoptotic NF-κB-controlled factor in BV6-induced apoptosis besides TNFα. Furthermore, this study contributes to an improved understanding on non-apoptotic functions of SMAC mimetics, as IRF1 additionally influences expression levels of proinflammatory cytokines and attraction of immune cells. Thus, our work provides novel insights into the regulation of SMAC mimetic-induced signaling events, which is crucial for the translation of SMAC mimetics for use in clinical application.
In mitochondria, biogenesis of oxidase is a crucial process involving the participation of an array of assembly factors. Studying the process of biogenesis in eukaryotes is highly complicated due to the presence and partaking of two genetic systems. Employing a bacterial model such as Paracoccus denitrificans that utilizes only one genetic system enables easy studying of the assembly process. The aa3 cytochrome c oxidase of P. denitrificans shows high structural and functional homology to its mitochondrial counterpart despite its simple subunit composition. The assembly of the core subunits I and II that house the active redox centers (heme a, and heme a3.CuB centre in subunit I; and the binuclear CuA centre in subunit II) along with the chaperons responsibly for their incorporation form the crux of this work. This work concentrates particularly on CtaG, a chaperone previously speculated to be involved in the delivery of copper to the CuB center in subunit I. As the full length structure of CtaG or its structural homologues have not been solved, attempts were made to obtain high-diffracting crystals of CtaG by heterologously expressing it in E. coli. Growth media, expression strains and induction parameters were some of the conditions screened in order to obtain optimal yield. Additives, pH and detergent were screened to yield a homogeneous preparation of CtaG. Crystallization trials were conducted by employing the sitting drop, vapour diffusion, method and later the bicelles were employed. Preliminary crystals obtained were further optimized employing seeding, detergent and additives, to improve diffraction. The diffraction improved from 30 Å to 15 Å. BN PAGE (Blue Native Polyacrylamide Gel Electrophoresis) analysis and cross-linking studies were undertaken to decipher the oligomeric condition of CtaG. Both the methods indicate that the protein is a dimer under native conditions. To study the importance of CtaG in the process of oxidase assembly, two deletion mutants were obtained from the lab; one with only ctaG deleted and the other with ctaG and most of the upstream ORF. The effect of the deletion was assayed on the assembly and activity of oxidase. The deletion mutants showed residual activity of approx. 20 %, while displaying a very low heme signal (both in membranes and in purified COX). In order to exclude polar effects arising due to gene manipulation, complementation strains were prepared, reintroducing ctaG alone into both the deletion strains. Complementation strains, where only ctaG was deleted and re-introduced assayed for COX activity showed a restoration in activity to approx. 70 %. Further, calculating the heme:protein ratio, the deletion strains displayed a value of 7 nmol/mg of oxidase which was increased to wild type levels of 16 nmol/mg in the complementation strains. To further confirm the absence of the copper in subunit I, total reflection X-ray fluorescence spectroscopy analysis was carried out, which showed a decrease in the copper content in the deletion strain, restored on complementation. The strain lacking in the ORF and ctaG when complemented with ctaG alone illustrated no increase in activity or heme signal in comparison to that of the deletion strain. These point at a possible role for ORF in the assembly of COX, which is still absent in the complementation strains. To further characterize the ORF, a series of bioinformatical analysis was carried out, the results from which were insufficient to characterize the ORF conclusively. In order to enlist the proteins involved in the biosynthesis of COX, two independent approaches were employed. Two-dimensional gel examinations of solubilised membranes from untreated and cross-linked cells were analyzed by Western blotting. The CtaG-COX interaction was observed in untreated membranes, which was additionally strengthened by cross-linking. To further confirm this association, pull-down assays were done employing protein A coated magnetic beads coated with different antibodies and incubated with solubilised membranes derived from untreated or cross-linked cells. The elutions were assayed by Western blotting and confirmed for the CtaG-COX interaction. These fractions were further analysed by mass spectrometry to identify other chaperons involved in biogenesis of oxidase. Along with CtaG, I also noticed Sco, Surf1c and other factors involved in the recruitment and transport of heme (CtaB, CtaA, and Ccm proteins). Interestingly, protein components of both ribosomal subunits and protein translocation factors were observed, which indicated a co-translational approach for co-factor insertion into COX.
Necroptosis is a programmed cell death pathway that is implicated in a variety of human diseases. In recent years, increasing knowledge has been gained on the necroptotic signaling cascade. Nevertheless, the role of reactive oxygen species (ROS) in necroptosis is still ambiguous. In this study, we reveal that ROS critically regulate BV6/TNFα-induced necroptotic signaling in FADD-deficient Jurkat cells and in zVAD-treated MV4-11 cells. We show that several ROS scavengers such as butylated hydroxyanisole (BHA), N-acetylcysteine (NAC), α-tocopherol (αToc) and ethyl pyruvate (EP) significantly reduce ROS production and BV6/TNFα–induced cell death. Importantly, ROS are produced prior to cell death induction and promote the assembly of the Receptor-interacting protein kinase (RIP)1/RIP3 necrosome complex via a potential positive feedback loop since on the one hand radical scavengers diminish RIP1/RIP3 necrosome formation and since on the other hand RIP1 or RIP3 silencing attenuates ROS production. Furthermore, the deubiquitinase CYLD contributes to BV6/TNFα-induced ROS generation, necrosome assembly and cell death since CYLD knockdown attenuates all these events. Of note, knockdown of the downstream effector protein mixed lineage kinase domain like (MLKL) only partly reduces BV6/TNFα-triggered ROS production and cell death and does not affect necrosome formation. Contrary to expectations, the MLKL inhibitor Necrosulfonamide (NSA) not only decreases BV6/TNFα-stimulated ROS production and cell death but also attenuates RIP1/RIP3 necrosome assembly pointing to additional and MLKL-independent anti-necroptotic effects of NSA. Interestingly, silencing of the potential necroptotic excecutors mitochondrial proteins phosphoglycerate mutase family member 5 (PGAM5) or Dynamin-related protein 1 (Drp1) does not affect BV6/TNFα-induced cell death. Consistently, mitochondrial perturbations are not implicated in BV6/TNFα-induced cell death since mitochondrial membrane potential and respiration remain stable along with to BV6/TNFα-triggered necroptosis induction. Interference with the mitochondrial potential by depolarizing agents such as FCCP reduces BV6/TNFα-induced necroptosis indicating that proper mitochondrial function or a well-defined redox status is required for necroptotic cell death execution. This study demonstrates that ROS are critically involved in BV6/TNFα-induced necroptosis and thus provides novel insights into the redox regulation of necroptotic signaling.
To overcome poor treatment response of pediatric high-risk acute lymphoblastic leukemia (ALL), novel treatment strategies are required to reactivate programmed cell death in this malignancy. Therefore, we take advantage of using small-molecule antagonists of Inhibitor of apoptosis (IAP) proteins, so called Smac mimetics such as BV6, which are described to overcome apoptosis resistance and thereby sensitize tumor cells for several apoptotic stimuli. To address the question whether redox alterations can sensitize leukemic cells for Smac mimetic-mediated cell death, we interfered with the cellular redox status in different ALL cell lines. Here, we show for the first time that redox alterations, mediated by the glutathione depleting agent Buthioninesulfoximine (BSO), prime ALL cells for BV6-induced apoptosis. Besides ALL cell lines, BV6/BSO cotreatment similarly synergizes in cell death induction in patient-derived primary leukemic samples. In contrast, the combination treatment does not exert any cytotoxicity against peripheral blood lymphocytes (PBLs) or mesenchymal stroma cells (MSCs) from healthy donors, suggesting some tumor selectivity of this treatment. We also identify the underlying molecular mechanism of the novel synergistic drug interaction of BSO and BV6. We demonstrate that both agents act in concert to increase reactive oxygen species (ROS) production, lipid peroxidation and finally apoptotic cell death. Enhanced ROS levels in the combination treatment account for cell death induction, since several ROS scavengers, like NAC, MnTBAP and Trolox attenuate BSO/BV6-induced apoptosis. BSO/BV6-induced ROS can be mainly classified as lipid peroxides, since the vitamin E derivate α-Tocopherol as well as Glutathione peroxidase 4 (GPX4), which both specifically reduce lipid-membrane peroxides, prevent lipid peroxidation, caspase activation and cell death induction. Vice versa, GPX4 knockdown and pharmacological inhibition of GPX4 by RSL3 or Erastin enhance BV6-induced cell death. Importantly, cell death induction critically depends on the formation of a complex consisting of RIP1/FADD/Caspase-8, since all complex components are required for ROS production, lipid peroxidation and cell death induction. Taken together, we demonstrate that BSO and BV6 cooperate to induce ROS production and lipid peroxidation which are eventually required for caspase activation and cell death execution. Collectively, findings of this study indicate that BV6-induced apoptosis is mediated via redox alterations offering promising new treatment strategy to overcome apoptosis resistance in ALL.
Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor and third leading cause of cancer-related death worldwide. Most cases arise as a consequence of underlying liver disease, e.g. developed from chronic hepatitis B or C infectionsalcohol abuse or obesity, and are most often associated with liver cirrhosis. Hypoxiand the hypoxia inducible factors (HIF)-1α and -2α promote tumor progression of HCC, not only affecting tumor cell proliferation and invasion, but also angiogenesis and lymphangiogenesis and thus, increasing the risk of metastasis.
HCC is characterized as one of the most vascularized solid tumors. While HIF-1α and HIF-2α are frequently up-regulated in HCC only HIF-2α is correlated with high patientlethality. HIF-dependent regulation of HCC angiogenesis is controversially discussed.VEGFA, for example, as the most prominent factor inducing tumor angiogenesis represents not only a HIF-1 target, but also a HIF-2 target gene in HCC. This questions whether both isoforms have overlapping functions in regulating the angiogenic switch in HCC.
Besides angiogenesis also tumor-associated lymphangiogenesis significantly influences patient survival in HCC. Lymphatic spread is an important clinical determinant for the prognosis of HCC, but little is known how lymphangiogenesis is controlled in this context. To date, mainly HIF-1α was positively correlated with olymphatic invasion and metastasis in HCC, while a defined role of HIF-2α is missing. Thus, although HIF-1α and HIF-2α are structurally alike and regulate overlapping but not identical sets of target genes, they promote highly divergent outcomes in cancer progression and may even have counteracting roles. The aim of my work was to characterize the specific role of HIF-1α and HIF-2α in the angiogenic switch and lymphangiogenesis induction during HCC development.
Therefore, I created a stable knockdown of HIF-1α and HIF-2α in HepG2 cells and generated cocultures of HepG2 spheroids and embryonic bodies derived from embryonic mouse stem cells as an in vitro tumor model mimicking the cancer microenvironment to analyze which HIF isoform has key regulatory functions in HCC (lymph)angiogenesis. In cocultures with a HIF-2α knockdown angiogenesis was attenuated but lymphangiogenesis increased, while the knockdown of HIF-1α was without effect. Microarray analysis identified plasminogen activator inhibitor 1 (PAI-1)and insulin-like growth factor binding protein 1 (IGFBP1) as HIF-2 target genes.However, prominent angiogenic and lymphangiogenic factors such as VEGFs, PDGFB, ANG and their receptors were not regulated in a HIF-dependent manner. As PAI-1 was linked to angiogenesis in literature and IGF-signaling, which is negatively regulated by IGFBP-1, was correlated with lymphangiogenesis, I decided to investigate their HIF-2α-dependent influence on HCC (lymph)angiogenesis. The knockdown of PAI-1 in HepG2 cells also lowered angiogenesis in PAI-1k/d cocultures similar to the HIF-2α k/d phenotype. PAI-1 as the potent inhibitor of tPA and uPA, both inducing the conversion of plasminogen to plasmin, also inhibits plasmin directly. Therefore, I assumed an increase of plasmin in HIF-2α k/d and PAI-1 k/d cocultures as a result of the reduced PAI-1 levels. Blocking plasmin with aprotinin in HIF-2α k/d cocultures restored angioge nesis, suggesting that HIF-2α increases PAI-1 to lower concentrations of active plasmin, thereby supporting angiogenesis. In further experiments I could exclude PAI-1 to reduce angiogenesis by inducing plasmin-mediated apoptosis of differentiating stem cells in PAI-1 k/d and HIF-2α k/d cocultures, but demonstrated an increase of VEGFA165 degradation in these cocultures, suggesting plasmin-catalyzed proteolysis of VEGF as an additional layer of regulation required to explain the angiogenic phenotype. Besides the pivotal role of PAI-1 in angiogenesis I also investigated its potentialinfluence in lymphangiogenesis. Indeed, the knockdown of PAI-1 reduced lymphaticstructures and implied an important but opposing role in lymphangiogenesis comparedto induced lymphangiogenesis in HIF-2α k/d cocultures. However, blocking plasmin again with aprotinin in HIF-2α k/d cocultures restored lymphangiogenesis to the level of control virus, which indicates a divergent lymphangiogenic role of plasmin in PAI-1 k/d and HIF-2α k/d cocultures, possibly because of other essential pathways masking the lymphangiogenic effects of PAI-1 in HIF-2α k/d cocultures.
HIF-2α resulting in reduced IGFBP1 expression induced the differentiation of stem cells toward a lymphatic cell type and significantly enhanced the assembly of human dermal lymphatic endothelial cells into tubes. These data point the first time to an important impact of HIF-2 in the regulatin of lymphangiogenesis in vitro by inducing IGFBP1 and thus, scavenging IGF-1. Furthermore, matrigel plug assays to investigate the in vivorelevance of these observations confirmed HIF-2α as a crucial factor in the regulation of lymphangiogenesis in vivo
In conclusion, this work provides evidence that HIF-2α is a key regulator of angiogenesis and lymphangiogenesis in HCC by regulating PAI-1 and IGFBP1. HIF-2α positively influences the angiogenic switch via PAI-1 and negatively affects lymphangiogenesis via IGFBP1 expression. Targeting HIF-2α in HCC to reduce tumor angiogenesis should be approached carefully, as it might be overcome by induced lymphangiogenesis and metastasis.
The RAF family of kinases constitutes the members A, B and CRAF. They mediate RAS signaling by linking it to the MEK/ERK transduction module, which regulates cellular processes such as cell proliferation, migration, survival and cell death. As the RAS/RAF/MEK/ERK (MAPK) pathway is found to be activated in human cancers, the RAF kinases have been exploited as valuable therapeutic targets and RAF inhibitors show promising results in the clinic, esp. with tumors harboring an activating BRAFV600E mutation. However, RAF inhibitors paradoxically accelerate metastasis in RAS mutant and BRAF wildtype tumors. They also become ineffective over time in BRAFV600E tumors because of reactivation of downstream mitogen-activated protein kinase (MAPK) signaling by promoting RAF dimerization. Aims of the present work were 1) to investigate the role of ARAF kinase in the paradoxical activation of the enzymatic cascade by RAF inhibitors downstream of mutated RAS and 2) to study the consequences of the loss of ARAF function on signal transduction in vitro and in vivo (nude mice). We have engineered several cell lines that would allow the study of basal and RAF inhibitor induced effects on MAPK activation, tumor cell migration and invasion.
In summary, we were able to show that the RAF isoform ARAF has an obligatory role in promoting MAPK activity and tumor cell invasion in a cell type dependent manner. In these cell types, ARAF depletion prevented the activation of MAPK kinase 1 (MEK1) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) and led to a significant decrease of protrusions growing out of tumor cell spheroids in a three-dimensional (3D) culture that were otherwise induced by BRAFV600E-specific or BRAF/CRAF inhibitors (GDC-0879 and sorafenib, respectively). RAF inhibitors stimulated homodimerization of ARAF and heteromerization of BRAF with CRAF and the scaffolding protein KSR1. However, induced oligomerization was not sufficient to activate MAPK signaling if ARAF was depleted. By employing full length recombinant kinases, we were able to show for the first time that the three RAF isoforms competed for the binding to MEK1. In cell culture models, the overexpression of dimer-deficient ARAF mutants impaired the interaction between ARAF and endogenous MEK1 and thus prevented the subsequent phosphorylation of MEK1 and ERK1/2. Our findings reveal a new role for ARAF in directly activating the MAPK cascade through homodimerization and thereby promoting tumor cell invasion, suggesting the conserved RAF-dimer interface as a target for RAS- and RAF mediated cancer therapy.
Collectively, we provide evidence for the dual role ARAF plays in controlling MAPK signaling and cancer as loss of ARAF promoted strong lung metastasis formation in nude mice. Preliminary data describing the underlying mechanisms behind ARAF-regulated metastases have been presented and discussed.
The four subunit (SU) aa3 cytochrome c oxidase (CcO) from Paracoccus denitrificans is one of the terminal enzymes of the respiratory chain. It uses electrons from cytochrome c to reduce molecular oxygen to water. Its binuclear active center, residing in SU I, contains hemeÊa3 and CuB, the latter being liganded by three histidine residues. Apart from its oxygen reductase activity, the protein possesses a peroxidase and a catalase activity.
To compare variants and the wild type (WT) protein in a more stringent way, a recombinant (rec.) WT CcO was constructed, carrying the gene for SUÊI on a low copy number plasmid. This rec. WT showed, as expected, no difference in oxygen reductase activity compared to the American Type Culture Collection (ATCC) WT CcO but surprisingly its catalase activity was increased by a factor of 20. The potential overproduction of SUÊI due to plasmid coding and the resulting deficiency in metal inserting chaperones might impair the correct insertion of hemeÊa3 and CuB because of a deficiency in metal inserting chaperones. This in turn might lead to differences in side chain orientation and to changes in the water network. However, slight changes might cause an increased accessibility of the active center for hydrogen peroxide, resulting in an increased catalase activity. The availability of chaperones and therefore the proposed structural reasons for the difference was improved by cloning the genes for the two metal inserting chaperones CtaG and Surf1c on the same plasmid together with SUÊI. This new rec. WT CcO showed in fact a reduced catalase activity. Another WT with a deletion in the chromosomal second, non expressing gene of SU I was analysed to prove plasmid coding as the reason for the difference of the ATCC WT and the rec. WT. This strain showed an increased kcat of the catalase activity as well, additionally pointing to a regulatory effect of the non expressed gene for SU I in the chromosome. To fathom the structural difference of the increased catalase activity, differential scanning calorimetry was used, but no significant difference in thermal stability between the ATCC WT CcO and the rec. WT CcO was detected. However, upon aging, the thermal stability of the rec. WT CcO declined faster than that of the ATCC WT CcO pointing to a decreased structural stability of the rec. WT CcO.
To characterize the catalase reaction, several known inhibitors were used to probe the contribution of the different metal cofactors in the catalase reaction. In addition variants in aromatic amino acids near the active center were constructed to conclude on a possible reaction mechanism of the catalase activity of CcO. These variants in combination with the wild type forms were analysed for radical signals by EPR-spectroscopy. A radical relevant for the catalase reaction of CcO was found in the F-intermediate of all variants and all wild type forms. This narrow 12 G radical signal was assigned to a porphyrine radical probably involved in the catalase reaction of CcO. Moreover, gas chromatography-mass spectrometry measurements were used to analyse isotopically labelled oxygen produced in the catalase reaction.
As a result of these experiments, a reaction cycle of the catalase activity of CcO is postulated and the structural difference between the ATCC and rec. WT CcO is outlined. The catalase activity appears to be a true catalase activity and not a "pseudocatalase" activity.
The knowledge of three-dimensional structures of biomolecules is fundamental for the understanding of their function. Nuclear magnetic resonance (NMR) spectroscopy represents besides X-ray crystallography one of the two most widely used techniques to study macromolecules at atomic resolution. Its application has long been a laborious task that could take months and required the expertise of an experienced scientist, however, owing to the tremendous effort that has been put into the development of respective computer algorithms, structure determination by NMR spectroscopy of small- to medium sized proteins is nowadays routinely performed. CYANA is one widely used software package, which combines the majority of individual steps towards a three-dimensional structure. The most common application of the program, however, restricts to the combined automated NOE assignment and structure calculation based on NOESY peak lists and an existing chemical shift assignment. Completely automated structure determination starting from NMR spectra is to date technically possible with CYANA, however, not yet routinely applied. In order to achieve this long-term goal, the individual steps need to become more robust with regard to data imperfections such as peak overlap, spectral artifacts or a limited amount of NMR data. The work presented in this thesis should be placed within the context of increasing the reliability and improving the accuracy of structures determined by CYANA on the basis of solution- as well as solid-state NMR data.
The chapter “Systematic evaluation of combined automated NOE assignment and structure calculation with CYANA” comprises an extensive study on the robustness of the combined automated NOE assignment and structure calculation algorithm based on experimental solution NMR data sets that were modified in multiple ways to mimic different kinds of data imperfections. The results show that the algorithm is remarkably robust with regard to imperfections of the NOESY peak lists and the chemical shift tolerances but susceptible to lacking or erroneous resonance assignments, in particular for nuclei that are involved in many NOESY cross peaks.
In the chapter “Peakmatch – A simple and robust tool for peaklist matching” a method to achieve self-consistency of the chemical shift referencing among a set of peak lists is presented. The Peakmatch algorithm matches a set of peak lists to a specified reference peak list, neither of which have to be assigned, by optimizing an assignment-free match-score function. The algorithm has been extensively tested on the basis of experimental NMR data sets of five different proteins. The results show that peak lists from many different types of spectra can be matched reliably as long as they contain at least two corresponding dimensions.
NMR structures are represented by bundles of conformers whose spread indicates the precision of the atomic coordinates. However, there is as yet no reliable measure of structural accuracy, i.e. how close NMR conformers are to the “true” structure. Instead, the precision of structure bundles is widely (mis)interpreted as a measure of structural quality. Attempts to increase the precision thus often yield tight structure bundles where the precision overestimates the accuracy. To overcome this problem, the chapter “Increased reliability of NMR protein structures by consensus structure bundles” introduces a new protocol for NMR structure determination with the software package CYANA that produces bundles of conformers with a realistic precision that is throughout a large number of test data sets a much better estimate of the structural accuracy than the precision of conventional structure bundles.
Solid-state NMR is a powerful technique to study molecules which are not amenable to either solution NMR or X-ray crystallography. Despite the reporting of individual atomic resolution structures of membrane proteins and amyloid fibrils based on solid-state NMR data, the application is far from routine. One major obstacle that hinders structure determination by solid-state NMR is the overall lower quality of the solid-state NMR spectra. It is therefore necessary to increase the robustness of the computer algorithms in order to improve the results when using lower quality solid-state NMR spectra. The chapter “Structure calculations of the model protein GB1 from solid-state NMR data” presents structure calculations on the basis of a set of two-dimensional solid-state NMR experiments of the model protein GB1. The most important result obtained from these test calculations is that the limitation of structural accuracy can be attributed to inaccurate distance information resulting from the limited correlation between peak intensities and distance, which is especially severe in spin diffusion-based solid-state NMR experiments.
The chapter “Full relaxation matrix-based correction of relayed polarization transfer for solid-state NMR structure calculation” therefore introduces a method which corrects experimental peak intensities for spin diffusion in order to improve the distance information from solid-state NMR spectra. The results show that the structural accuracy can be significantly improved when using the corrected distance information, however, strongly dependent on the preliminary structural model that is required as input for the method.
Die Sulfonyl-Gruppe (-SO2-) ist ein weit verbreitetes Strukturmotiv in der organischen Chemie und Bestandteil vieler biologisch aktiver Moleküle, insbesondere Arzneistoffen. Zwei der am häufigsten auftretenden Gruppen sind Sulfone und Sulfonamide, die in über 100 zugelassenen Medikamenten und 10% der meistverkauften Medikamente sind. Insofern kommt der Entwicklung neuer Synthesemethoden eine große Bedeutung zu. Dabei stehen besonders einfache, wirtschaftliche und zeitsparende Vorgehensweisen im Vordergrund, die eine große Bandbreite an neuen Substanzen generieren können. Ein Ansatz hierfür sind Multikomponenten- oder Eintopfreaktionen.
Aufgrund der Wichtigkeit dieser zwei Strukturklassen, sollen im Rahmen der hier vorliegenden Doktorarbeit neue Syntheserouten für Sulfone und Sulfonamide entwickelt werden. Besonderes Augenmerk wird auf die die Einführung der SO2-Einheit während der Reaktionsführung gelegt. Im Vergleich zu bereits existierenden Verfahren ist dies ein enormer Fortschritt, da die Mehrheit der bekannten Routen auf Schwefel- oder Schwefeldioxid-haltige Startmaterialien zurückgreift.
In der vorliegenden Arbeit gelang es, einen synthetischen Zugang zu Arylsulfonen basierend auf von Natrium-, Lithium-, Magnesium- und Zinksulfinaten zu finden. Diese Reaktion besitzt eine sehr große Anwendungsbandbreite und setzt sowohl Aryl- als auch Alkylsulfinate effizient um. Außerdem weisen Reaktionen mit unsymmetrischen Diaryliodoniumsalzen hohe Chemoselektivitäten auf.
Auf der Grundlage auf der Reaktion zwischen Natriumsulfinaten und Iodoniumsalzen wurde eine simple Route zur Synthese von Diarylsulfonen abgeleitet, jedoch war hierbei die Sulfonylgruppe noch Bestandteil eines der Edukte. Um die SO2-Einheit während der Reaktion einführen zu können, wurde ein praktisches Eintopf-Protokoll entwickelt, welches die direkte Umsetzung von (hetero)aromatischen und alkylischen Halogeniden zu Arylsulfonen gestattet. Diese innovative Methode besteht aus folgenden vier Schritten: (1) Generierung des Organometallreagenzes via Halogen-Metall-Austausch, direkte Metallinsertion oder Deprotonierung; (2) Reaktion des Organometallreagenzes mit SO2 zum Sulfinat; (3) Entfernen des SO2-Überschusses und flüchtiger Komponenten und (4) Umsetzung des nicht aufgereinigten Sulfinates mit einem Iodoniumsalz.
Desweiteren wird in dieser Arbeit ein neuartiger Übergangsmetall-katalysierter Ansatz zur Darstellung von Diarylsulfonen ausgehend von Arylhalogeniden und Sulfinaten diskutiert. Erste Experimente deuten auf Nickel-Katalysatoren als gute Wahl für die Reaktion. Optimierungsreaktionen zeigten eine starke Abhängigkeit der Ausbeute in Hinsicht auf die Bisswinkel der an das zentrale Nickelatom koordinierten Liganden. Da die bis dato besten Ergebnisse mit dem Komplex [o-tol-Ni(PPh2Me)2Cl] erzielt wurden, wird der [o-tol-Ni(PMe3)2Cl]-Komplex momentan in unserem Labor weiteren Studien unterzogen. Bislang ist davon auszugehen, dass dieser Katalysator hervorragende Ergebnisse liefert und zu einer allgemein gültigen Methode führt.
In weiteren Kapiteln wird die Anwendbarkeit von SO2-Surrogaten, Metabisulfiten „S2O52-„ oder DABSO; untersucht; mit dem Ziel eine Eintopf- oder Multikomponentenreaktion zu entwickeln.
Zum einen wird die Entwicklung einer Ein-Topf-Reaktion von Alkylhalogeniden mit Metabisulfiten und Organozinkreagenzien zur Darstellung von Alkylarylsulfonen vorgestellt. Darüber hinaus wird eine Übergangsmetall-katalysierte Multikomponenten Reaktion zur Synthese von Sulfonsäureamiden vorgestellt. Eine Reaktion zwischen Aminen, Arylhalogeniden und DABSO als SO2-Quelle wurde in Form einer Palladium-katalysierten Aminosulfonylierung entwickelt.
In Reaktion auf zellulären Stress wie etwa Schädigungen der DNA oder die vermehrte Aktivität von Onkogenen aktivieren vorgeschaltete Signalkaskaden den Transkriptionsfaktor (TF) p53. Dieser kann über die Aktivierung der Expression von Zielgenen wiederum die Zellteilung stoppen, die Reparatur von DNA Schäden initiieren oder in schweren Fällen die Eliminierung der Zelle durch Apoptose einleiten. Ist p53 durch Mutationen deaktiviert, können sich entartete somatische Zellen vermehren und in der Folge Krebs entstehen.
In Wirbeltieren finden sich neben p53 mit p63 und p73 zwei weitere TFs, welche während der Evolution aus dem gleichen gemeinsamen Vorläufer durch Genduplikationen hervorgegangen sind. Die drei TFs sind modular aufgebaut und alle Isoformen verfügen jeweils minimal über eine DNA Bindungsdomäne (DBD) und eine Tetramerisierungsdomäne (TD). Werden die p53 ähnlichen TFs aktiviert, lagern sie sich über die TD vermittelt zu Tetrameren zusammen, wodurch ihre DBDs kooperativ an DNA Sequenzmotive binden können. Die DBD ist auch über große phylogenetische Abstände hinweg hoch konserviert, wodurch bereits gezeigt werden konnte, dass auch primitive vielzellige Tiere bereits Homologe dieser TF Familie besitzen. Im Vergleich zur DBD variiert die Proteinsequenz der TD deutlich stärker, was andeutet, dass deren Struktur im Laufe der Evolution erhebliche Veränderungen durchlaufen hat. Diese Veränderungen aufzuklären ist das übergeordnete Forschungsvorhaben zu dem diese Dissertationsschrift beiträgt.
Ciona intestinalis (C.int.) ist eine Spezies aus dem Unterstamm der Manteltiere. Diese sind die engsten lebenden Verwandten der Wirbeltiere und C.int. ist ein populärer Modelorganismus für die Erforschung der Embryonalentwicklung. Sein Genom kodiert für zwei p53 ähnliche TFs, welche mit p53/p73-a und p53/p73-b bezeichnet werden. Die Struktur ihrer TDs wurde im Rahmen der vorliegenden Arbeit mittels Kernspinresonanz (NMR) Spektroskopie untersucht.
Die TD von menschlichem p53 (hp53) ist ein Dimer aus Dimeren. Jedes Monomer formt einen beta-Strang und eine alpha-Helix. Im primären Dimer lagern diese sich so zusammen, dass ein beta-Faltblatt entsteht und die alpha-Helices mit entgegen gesetzter Orientierung der Länge nach aneinander packen. Zwei dieser Dimer lagern sich dann so zum Tetramer zusammen, dass zwischen pol-ständigen beta-Faltblättern ein Bündel aus vier Helices entsteht. Dieses Motiv ist auch in den TDs der Ciona Proteine hochkonserviert und wird im Folgenden als Kern?TD bezeichnet. In den TDs von menschlichem p63 und p73 (hp63 und hp73) verfügt jedes Monomer an seinem C-terminus noch über eine zweite Helix. Die zweiten Helices eines jeden Dimers greifen wie Klammern um das jeweils andere primäre Dimer und stabilisieren so das Tetramer. Entscheidend für die stabile Anbindung an die Kern?TD ist dabei ein charakteristisches Tyrosin-Arginin (YR) Motiv in der zweiten Helix, welches sich auch in der Sequenz der TD von C.int. p53/p73-a wiederfindet. Analysen der Sekundärstruktur auf Basis von NMR Experimenten ergaben jedoch, dass die TD von C.int. p53/p73-a bei 25°C keine zweite Helix ausbildet. Mit Hilfe von chimären TD Peptiden, in denen Teile der Ciona Sequenz gegen die entsprechenden Abschnitte von hp73 ausgetauscht wurden, konnte gezeigt werden, dass die Kern TD von C.int. p53/p73-a fähig ist eine zweite Helix zu stabilisieren und hierfür neben dem YR Motiv auch der Sequenzabschnitt zwischen erster und zweiter Helix entscheidend ist. Stabilisierende Substitutionen in diesem Bereich bewirkten ebenso wie ein Absenken der Temperatur die Ausbildung einer zweiten Helix, welche jedoch im Gegensatz zu jener in hp73 nur transient faltet und auch nicht essentiell für die Bildung des Tetramers ist, wohl aber dessen Stabilität erhöht.
Spezifisch in der Entwicklungslinie von Ciona kam es dazu, dass eine, für eine entsprechende Vorläuferversion von C.int. p53/p73-a kodierende, mRNA spontan zurück in DNA übersetzt und ins Genom eingefügt wurde. Die durch diese Retrotransposition erzeugte neue Genkopie C.int. p53/p73-b muss demnach ursprünglich einmal für die gleiche Proteinsequenz kodiert haben, innerhalb der TD finden sich konservierte Reste jedoch nur im Bereich der Kern TD.
Von der TD von C.int. p53/p73-b wurde die molekulare Struktur in freier Lösung mittels NMR ermittelt. Diese zeigte, dass interessanterweise in der TD von C.int. p53/p73-b jedes Monomer am C-terminus eine stabil gefaltete, zweite Helix besitzt. Obwohl diese zweite Helix sich aus einer Sequenz faltet, die keinerlei Sequenzhomologie zu homologen Proteinen aus Wirbeltieren aufweist, lagert sie sich in einer Position auf die Kern TD, welche der in hp73 sehr nahe kommt. Da die primären Dimere der Kern TD aber anders als in hp63 und hp73 durch Salzbrücken miteinander verbunden sind, ist die zweite Helix jedoch nicht essentiell, um das Tetramer zu stabilisieren. Vermutlich kommt der zweiten Helix von C.int. p53/p73-b vielmehr u.a. die Aufgabe zu die Bildung von Heterotetrameren aus C.int. p53/p73-a und –b zu unterbinden.
Zusammengenommen zeigen die Ergebnisse, dass die Architektur der TD mit zweiter Helix bereits der Prototyp für die TDs aller p53 ähnlichen Proteine der Wirbel- und Manteltiere war und die als eine Art Klammer das Tetramer stabilisierende zweite Helix sich nicht erst während der Evolution der Wirbeltiere entwickelt hat.
ATP synthases are multi-subunit membrane enzymes, which utilize the energy stored in a transmembrane electrochemical ion gradient to produce adenosine-5´-triphosphate (ATP), the universal energy carrier in biological systems. Research on these important enzymes goes back more than 50 years and has produced innumerable studies. The F-type ATP synthase consists of two functionally distinct, but tightly coupled subcomplexes, the water-soluble F1 and the membrane-embedded Fo complex. In its simplest form, F1 consists of five different subunits with a stoichiometry of α 3β3γδε, and harbors three catalytic centers in the α 3β3-headpiece, while Fo consists of three different subunits in a stoichiometry of ab2cn, where n varies between 8 to 15 depending on the species. From a mechanistic standpoint, the complex can also be divided into two different units, namely a stator, α3β3δ-ab2, and a rotor, γε-cn. The enzyme utilizes the energy stored in a transmembrane electrochemical gradient of protons, or in some cases Na+, to drive ATP synthesis. In particular, the downhill translocation of these ions across the Fo complex drives rotation of the γε-cn unit, which is then transduced to the active centers, catalyzing the phosphorylation of adenosine-5`-diphosphate (ADP) with inorganic phosphate (Pi), and the release of ATP....
So far clinical human immunodeficiency virus (HIV) therapy is limited to non-curative treatments. However, as recently shown, alternative approaches such as HIV gene therapy have the potential to functionally cure the disease (e.g. the hematopoietic stem cell (HSC)-transplantation with a CCR5Δ32 homozygous transplant) (1). In contrast to the highly personalized medical treatment applied in the ‘Berlin case’, more broadly applicable approaches are currently under intensive investigation.
One example is the adeno-associated-virus (AAV)-mediated delivery of in vivo secreted antiviral entry inhibitors (iSAVE), the concept of which is based on the direct in vivo administration of a broadly applicable highly potent antiviral gene (here: a C46-derived entry inhibitory peptide interfering with HIV-1 membrane fusion). The AAV-based gene delivery is believed to overcome several limitations of gene therapeutic treatments based on ex vivo lentiviral trials in the past. It is (i) targeting differentiated HIV target cells (i.e. liver and differentiated lymphatic cells) reducing the risk of genotoxicity compared to stem cell-based trials, (ii) overcoming the limitation of a low number of genetically modifiable cells as in lentivirally based ex vivo transduction strategies (i.e. limited modifiable cell number due to culture conditions and lower vector titers) and (iii) using the safe AAV vector system, which has not been associated with major genotoxicity in men. (iv) Most importantly, the concept of secretable entry inhibitors does not require transduction of large amounts of cells due to the protective bystander effect. Thus, iSAVE might be a treatment principle for HIV infection that might be able to cure patients irrespective of their viral isolates or adherence.
Accordingly, the iSAVE concept could aim at two different sites in the patient for the production of antiviral transgenes, either the systemic production via suitable producer cells (e.g. hepatocytes) or the local production in the lymphatic system.
In a first approach, we are able to efficiently target hepatocytes using the natural AAV serotype 8 to express high plasma levels of secretable antiviral entry inhibitors in order to systemically suppress viral replication. In this setting we could show that iSAVE peptides are highly expressed in hepatocytes. However, plasma levels of iSAVE were insufficient when using a secretable peptide as sole antiviral transgene.
As a second treatment strategy, the iSAVE project aimed to deliver antiviral genes directly to the site of viral replication, the lymphatic system. Here, (i) a panel of naturally occurring AAV serotypes as well as (ii) AAV retargeting approaches were employed to design a highly efficient and selective AAV vector variant for gene delivery into the lymphatic system after intravenous vector administration.
In detail, (i) screening of the natural occurring serotypes revealed that the AAV serotype 1 (AAV-1) was best in targeting splenic tissue in two humanized mouse models, however at a very low level. After systemic AAV-1 vector administration neither transduction of human lymphocytes did occur nor was iSAVE expressed in the lymphatic system in a humanized mouse model.
(ii) In a second approach, we modified the well-characterized AAV-2 serotype in a tropism-defining region of its capsid gene by insertion of human peripheral blood lymphocytes (hPBL)-tropic peptide ligands. These in turn were selected by M13 in vivo phage display and by in vivo AAV peptide display. Selected variants were cloned and tested for hPBL transduction in vitro. Although the selected variants did not show increased expression efficacies compared to AAV-2 WT, it still might be possible that the selected variant are more specific for hPBLs as these conditions have not been tested.
As these selection processes required a humanized mouse model that comprises a functional lymphatic system, we established the previously described Trimera mouse model in our lab (2). We found that this mouse model could be further improved to allow engraftment of a lower number of gene-modified (gm) human T cells as in the classical Trimera model. These modified Trimera mice (mT3 mice) were conditioned by inclusion of cyclophosphamide (CTX) to the irradiation-conditioning scheme of the classical Trimera model.
Comparison of mT3 mice with established NSG and DKO mice in an adoptive gm T cell transplantation setting revealed that NSG mice were the most robust model providing high reproducibility in human T cell engraftment. MT3 mice allowed a substantial, yet more variable engraftment of gm T cells. Besides comparing engraftment kinetics, the graft quality (i.e. clonality and cytokine milieu) was analyzed. Again, NSG mice showed the most balanced homeostatic repopulation three weeks after transplantation, while mT3 mice were prone to Th1-type, oligloclonal repopulation, indicating an early onset of xenograft-versus-host disease. Finally, the lymphatic infiltration was analyzed. As expected, mT3 mice provided the most intact lymphatic structures, although the normal lymphatic morphology was not restored.
In conclusion, it was demonstrated in this work that AAV-mediated iSAVE gene therapy faces specific limitations depending on the respective targeting approach
In the systemic approach, iSAVE peptides have to be further optimized in terms of transgene design itself, as high-level accumulation in murine plasma was not feasible for the short iSAVE precursor. In the local, lymphatic targeting approach, AAV-mediated expression faces its limits in targeting specificity but foremost expression efficacy. Thus, the AAV vector itself needs further optimization for sufficient local iSAVE expression levels. Independently from the AAV-related approaches, a novel humanized mouse model was established in this work. Despite drawbacks regarding repopulation variability and set-up complexity, the novel mT3 mouse model comprised improved secondary lymphatic structures for adoptive T cell transfer, which might be an interesting platform for studies in lymphoma or leukemia therapy.
Nichtribosomale Peptid Synthetasen sind Quelle für eine Vielzahl an Sekundärmetaboliten mit antibiotischer Wirkung. Jede Synthetase besteht aus einer Abfolge von Modulen, wobei jedes Modul die nötigen Domänen für den Einbau eines Bausteins in das gebildeten Peptids enthält. Ein Ansatz zur Gewinnung neuer Peptidantibiotika, die angesichts der steigenden Zahl multiresistenter Keime dringend benötigt werden, ist der Austausch von Domänen oder Modulen. Aufgrund bisher noch nicht verstandener Selektivitäten, entweder zwischen den Domänen oder zwischen einzelnen Domänen und Zwischenstufen des gebildeten Peptids, führt dieser Ansatz jedoch in der Praxis oft zu keiner oder nur geringer Ausbeute.
Ziel der vorgelegten Arbeit war es, einige dieser Selektivitäten zu untersuchen, wobei der Fokus auf Peptidyl Carrier Proteinen Domänen (PCPs) lag. An diese Domänen sind alle Intermediate während der Reifung des Peptids kovalent über einen Phosphopantethein-Kofaktor (Ppan-Arm) gebunden.
Im ersten Teil der Arbeit sollte die Struktur einer mit einem Heptapeptid beladenen PCP mittels Lösungs-Kernspinresonanzspektroskopie (NMR) bestimmt werden. Hierbei konnte die natürliche Verknüpfung zwischen Ppan-Arm und Peptid über einen Thioester nicht verwendet werden, da diese Bindung zu Hydrolyse-anfällig war. Es konnte jedoch gezeigt werden, dass die Substitution des Thioesters durch eine nicht hydrolysierbare Amidbindung keinen Einfluss auf die Struktur hat, wodurch die Strukturbestimmung möglich war. Hierbei zeigte sich, dass die Peptid-beladene PCP in der sogenannten A/H state Konformation vorliegt, wobei das an sie gebundene Peptid frei beweglich ist. Somit scheint es wahrscheinlich, dass die PCP keine Selektivität für das an sie gebundene Peptid aufweist. Dies ist ein Unterschied zu den strukturell ähnlichen Acyl Carrier Proteinen (ACPs) aus der bakteriellen Fettsäurebiosynthese, da diese eine Bindungstasche für die an sie gebundenen Fettsäuren ausbilden.
Untersuchungen der Selektivität der Kondensationsdomäne (C Domäne) für das PCP gebundene Peptid mittels NMR-Titrationen und biochemischer Analysen konnten nicht durchgeführt werden, da sich im Laufe des Projekts zeigte, dass die aus der Synthetase herausgetrennte C Domäne katalytisch nicht aktiv war. Stattdessen sollte die Kristallstruktur einer Peptid-beladenen PCP-C Bidomäne, für welche eine katalytische Aktivität bereits gezeigt worden war, gelöst werden. Da aber bereits ein signifikanter Anteil der Bidomäne während der Expression mit dem Ppan-Arm beladen wurde, war die nötige quantitative Beladung mit dem Peptid gekoppelten Ppan-Arm in vitro nicht möglich. Eine quantitative Modifizierung mit dem Ppan-Arm in vitro war hingegen erfolgreich, und die Struktur der Ppan-beladenen Bidomäne konnte gelöst werden. Aufgrund des großen Abstands zwischen den aktiven Zentren der beiden Domänen kann es sich bei der beobachteten Orientierung nicht um jene handeln, die die beiden Domänen zueinander annehmen, wenn die C Domäne das PCP-gebundene Peptid bindet.
Im zweiten Teil der Arbeit wurde die Modifizierung einer PCP durch eine Gruppe II Phosphopantetheintransferase (PPT) untersucht. PPTs katalysieren die Übertragung des Ppan Arms auf die Seitenkette eines in PCPs konservierten Serins. In dieser Magnesium-abhängigen Reaktion dient Coenzym A (CoA) als Quelle für den Ppan-Arm. Durch Mutation des konservierten Serins in der PCP zu Alanin konnte ein stabiler Komplex aus PCP und PPT in Anwesenheit von CoA und Magnesium kristallisiert und seine Struktur bestimmt werden.
In einem Strukturmodell für den PCP/PPT Komplex war eine andere Konformation für die PCP postuliert worden, als sie in der Kristallstruktur des Komplexes zu beobachten ist. Durch Strukturbestimmung der PCP mittels Lösungs-NMR und anschließender Titrationsexperimente konnte jedoch gezeigt werden, dass sowohl die freie als auch die komplexierte PCP in Lösung ebenfalls die in der Kristallstruktur beobachtete Konformation einnehmen.
Aufgrund der gelösten Kristallstruktur konnten zwei Bereiche identifiziert werden, in denen die beiden Proteine im Komplex in direktem Kontakt zueinander stehen. Der eine Bereich ist durch eine intermolekulare Wasserstoffbrücke, der andere durch hydrophobe Wechselwirkungen zwischen den Proteinen gekennzeichnet. Durch ortsspezifische Mutagenese konnten beide Wechselwirkungen gestört werden, was sich in einer Abnahme der Komplexstabilität und einer veränderten Geschwindigkeit der Übertragung des Ppan-Arms äußerte.
Die große strukturelle Ähnlichkeit zwischen dem in dieser Arbeit untersuchten Komplex aus zwei in Bacillus vorkommenden Proteinen und einem humanen ACP/PPT Komplex legt die Vermutung nahe, dass die beobachteten Wechselwirkungen in vielen Organismen konserviert sind.
Pulsed Electron Paramagnetic Resonance (EPR) spectroscopy is the most powerful tool to investigate structural properties and dynamics of paramagnetic substances. Up to date the electron spin is almost exclusively manipulated by rectangular shaped microwave pulses generated with switches. These pulses are unselective which means they excite outside their nominal bandwidth which is in most cases shallow compared to the overall spectral width of the spin system. Shaped pulses which are widely applied in NMR promise higher bandwidth and selectivity. The use of amplitude and phase modulated pulses was not possible for EPR due to the three orders of magnitude faster timescale compared to NMR. In this work, for the first time, an AWG (arbitrary waveform generator) operating with a 1 ns time resolution and 14 bit amplitude resolution was implemented into a commercial Bruker pulsed EPR spectrometer.
First results were obtained with broadband excitation pulses derived by optimum control theory (OCT). The OCT-pulse used excites transverse magnetization with 98% efficiency over a more than four times larger bandwidth than common rectangular pulse generating the same 1 B field. The benefit of such a pulse was demonstrated for magnitude FT-EPR spectroscopy on organic radicals in liquid phase.
Due to Spectrometer deadtime an FID cannot be observed for most inhomogeneous spin systems. For that reason prefocused pulses have been evaluated for their applicability to EPR spectroscopy. OCT-derived prefocused pulses can be understood as a compact Hahn Echo sequence in one monolithic pulse. Here, two problems have been encountered. 1) The limited bandwidth of the active and passive microwave components in the excitation path as well as microwave resonator cause linear distortions of the pulse shape which results in inferior pulse performance. This could be circumvented by measuring the impulse response function of the whole spin excitation path and including this information in the pulse optimization procedure. 2) Anisotropic hyperfine interaction which was not taken into account during the pulse optimization also caused efficiency losses.
PELDOR spectroscopy is a valuable tool to measure distance distributions between two or more paramagnetic centers in the range from 2-8 nm. It is demonstrated that the S/N ratio of PELDOR experiments can be substantially increased by substituting the rectangular shaped pump pulse by an adiabatic inversion pulse. The damping of the dipolar oscillations introduced by the prolonged pump pulse towards shorter distances could be circumvented by introducing a second time reversed pump pulse.
By substituting the refocused echo of the well-known 4-pulse PELDOR with a CPMG sequence the dipolar evolution time and thus the validity of PELDOR experiments would be increased. To achieve the maximum dipolar evolution time in a CPMG PELDOR for each refocusing pulse one pump pulse has to be applied. This could only be achieved with the new adiabatic inversion pulses since multiple inversions with efficiency close to one are not possible with rectangular pulses. Even with adiabatic pump pulses a reduced efficiency was observed due to hardware limitations thus limiting the sequence to three refocusing pulses. An iterative method was developed to remove the residual dipolar signals attributed to the reduced inversion efficiency.
The new 7-pulse CPMG PELDOR sequence enabled measuring reliable distance distributions between the protomers of the trimeric betaine transporter BetP. With these it could be shown that the asymmetries found for the 2 and 3-dimensional crystal structures are even larger in frozen detergent.
Heme-copper oxidases (HCOs) are the terminal enzymes of the aerobic respiratory chain in the inner mitochondrial membrane or the plasma membrane in many prokaryotes. These multi-subunit membrane protein complexes catalyze the reduction of oxygen to water, coupling this exothermic reaction to the establishment of an electrochemical proton gradient across the membrane in which they are embedded. The energy stored in the electrochemical proton gradient is used e.g. by the FOF1-ATP synthase to generate ATP from ADP and inorganic phosphate. The superfamily of HCOs is phylogenetically classified into three major families: A, B and C. The A-family HCOs, represented by the well-studied aa3-type cytochrome c oxidases (aa3-CcOs), are found in mitochondria and many bacteria. The B-family of HCOs contains a number of bacterial and archaeal oxidases. The C-family comprises only the cbb3-type cytochrome c oxidase (cbb3-CcO) and is most distantly related to the mitochondrial respiratory oxidases.
Physical Biology is a field of life sciences dealing with the extraction of quantitative data from biophysical or molecular biological experiments with different levels of complexity. Such data are further used as parameters for mathematical models of the biological system. These models allow to predict reactions on external stimuli by describing the relevant molecular interactions and are therefore used for example to generate a deeper comprehension of complex human diseases. An essential technique in biophysical research on human diseases is fluorescence microscopy. This is a constantly developed toolbox comprising a large number of specific labeling strategies, as well as a broad spectrum of fluorescent probes. It is further minimal invasive and therefore suitable for measurements in living cells or organisms. The sensitivity of modern photo-detectors even allows for the detection of a single fluorescent probe with an accuracy of approximately 10 nm.
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The model-prediction was further verified by two color SMLM experiments. In this work the development and application of imaging-systems are described which provide quantitative data with single-molecule resolution for systems biological model approaches with a low degree of abstractness. In the near future, the impact of mathematical models in the research field of complex human diseases will increase. The predictions of these models will be more exact, the more detailed and accurate the input parameters will become. This work gives an impression of how quantitative data obtained by SMLM may serve as input parameters for mathematical models at the single-cell level.
Characterization of mouse NOA1 : subcellular localizaion, G-Quadruplex binding and proteolysis
(2013)
Mitochondria contain their own protein synthesis machinery with mitoribosomes that are similar to prokaryotic ribosomes. The thirteen proteins encoded in the mitochondrial genome are members of the respiratory chain complexes that generate a proton gradient, which is the electromotoric force for ATP synthesis.
NOA1 (Nitric Oxide Associated Protein-1) is a nuclear encoded GTPase that positively influences mitochondrial respiration and ATP production. Although a role in mitoribosome assembly was assigned to NOA1 the underlying molecular mechanism is poorly understood. This work shows that the multi-domain protein NOA1 serves multiple purposes for the function of mitochondria. NOA1 is a dual localized protein that makes a detour through the nucleus before mitochondrial import. The nuclear shuttling is mediated by a nuclear localization signal and the now identified nuclear export signal. SELEX (Systemic Evolution of Ligands by Exponential Enrichment) analysis revealed a G-quadruplex binding motif that characterizes NOA1 as ribonucleoprotein (RNP). G-quadruplex binding was coupled to the GTPase activity and increased the GTP hydrolysis rate. The sequence of localization events and the identification of NOA1 being a RNP lead to the discussion of an alternative import pathway for RNPs into mitochondria. The short-lived NOA1 contains ClpX recognition motifs and is specifically degraded by the mitochondrial matrix protease ClpXP. NOA1 is the first reported substrate of ClpXP in higher eukaryotes and augments the contribution of the ClpXP protease for mitochondrial metabolism. To assess the direct action of NOA1 on the mitoribosome co-sedimentation assays were performed. They showed that the interaction of NOA1 and the mitoribosome is dependent on the GTPase function and the nascent peptide chain. In vitro, NOA1 facilitated the membrane insertion of newly translated and isotope labeled mitochondrial translation products into inverted mitochondrial inner membrane vesicles. In conclusion, NOA1 is a G-quadruplex-RNP that acts as mitochondrial membrane insertion factor for mtDNA-encoded proteins.
This thesis provides a comprehensive model of the molecular function of NOA1 and is the basis for future research. The identification of NOA1 as ClpXP substrate is a major contribution to the field of mitochondrial research.
The human endothelin receptors, ETA and ETB, are two members of the G-protein coupled receptors family (GPCRs) and they are key players in cardiovascular regulation. The characterization of their functionality in vitro has been limited by the possibility to obtain high quality samples using conventional expression systems. The Cell-Free expression system is an alternative technique for the production of membrane protein as well as GPCRs and can overcome some of the limitations that are commonly encountered using an in vivo approach. Cell-Free expression protocols for the two receptors ETA and ETB have been optimized by implementing post- and co-translational association to lipid bilayers. The efficiency of the reconstitution or association to liposomes and nanodiscs has systematically been studied and the ligand binding properties of the two receptors have been analyzed using a set of different complementary techniques. In several different conditions a high affinity binding of the peptide ligand ET-1 to both endothelin receptors could be obtained and the highest activity values were detected in sample prepared using a co-translational approach in presence of nanodiscs. Furthermore, the characteristic differential binding pattern of selected agonists and antagonists to the two receptors was confirmed. In samples obtained from several Cell-Free expression conditions, two intrinsic properties of the functionally folded ETB receptor, such as the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were detected. ETA and ETB are able to induce in vivo the activation of hetrotrimeric G proteins upon stimulation with an agonist, leading to the dissociation of the heterotrimeric complex and the exchange of GDP to GTP in the Galpha subunit. The Cell-Free expression system was chosen for the production of two G alpha subunit, Galpha s and Galpha q. Soluble expression of the two proteins was achieved and the production of active Galpha s was confirmed using fluorescent as well as radioactive assays. In conclusion, the obtained results document a new process for the production of ligand binding competent endothelin receptors, as well as Galpha proteins, using a Cell-Free expression system. The combination of this expression system and the nanodiscs technology appears to be a promising tool for the further characterization of membrane proteins as well as GPCRs.
5-lipoxygenase (5-LO) is an enzyme with a substantial role in inflammatory processes. In vitro kinase assays using [32P]-ATP in combination with mutagenesis have revealed that serine residues 271, 523 and 663 can be phosphorylated by MK2, PKA and ERK2 kinases, respectively. A few available reports regarding 5-LO protein sequence have covered up to 30% of the sequence after amino acid sequencing including Ser663. In LCMS/MS analyses of 5-LO tryptic digests from different cellular sources different peptides have been detected; however, none of the three phosphorylations has been detected and only Ser663 was included in the covered sequence.
As there was no comprehensive mass spectrometric analysis of 5-LO, the purpose of this study was to optimize the experimental conditions under which detection of the aforementioned phosphorylation events, as well as other possible post-translational modifications (PTMs), would be feasible. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was used for peptide analysis of 5-LO cleaved either by chemical reagents or by proteases. Sequence coverage of 5-LO could be enhanced to be close to completion by combination of results from digestions by trypsin, AspN and chymotrypsin. In-gel trypsin digestion followed by in-solution AspN digestion proved to be a useful sample treatment for reproducible detection of the Ser271-containing peptide.
Nevertheless, in none of the examined cleavage protocols the sequence around Ser523 was detected reproducibly or with acceptable signal intensity for subsequent peptide fragmentation. Propionic anhydride and sulfo-NHS-SS-biotin cross-linker (EZ-linkTM), were used for derivatization of lysine side chains and hindrance of lysine residue recognition by trypsin. Phosphopeptide enrichment became possible after tryptic digestion of these samples, not only due to formation of an individual Ser523-containing peptide, but also because TiO2-mediated enrichment, which is performed in acidic pH, was not impaired by positively charged free lysine side chains. Additionally, biotinylation of lysine residues was exploited for an intermediate enrichment step of the lysine containing peptides, prior to TiO2 phosphopeptide enrichment.
MALDI-MS analysis after in-vitro phosphorylation of 5-LO by the three kinases showed that Ser271 was phosphorylated in the MK2 and PKA kinase assays, while Ser523 was phosphorylated only in the PKA kinase assay. Surpisingly, no phosphopeptides were detected in the in-vitro kinase assays with ERK2, even though the unmodified counterpart of the Ser663-containing peptide was easily detected. The detection limit for each of the three phosphorylation sites was determined by the use of custom made phosphopeptides and an amount of 0.06 pmol of phosphopeptide in 1 μg 5-LO (representing 0.5% phosphorylation rate) was sufficient in all cases for successful enrichment and detection by MS.
In-vitro kinase assays with [32P]-ATP were performed for some kinases that were expected to phosphorylate 5-LO according to in-silico data. Three members of the Src tyrosine kinase family (Fgr, Hck and Yes) and the Ser/Thr specific kinase DNA-PK used 5-LO as their substrate and mainly residues at the N-terminal part of 5-LO were detected phosphorylated by MS (e.g. Y42, Y53). Additional in-vitro assays for recombinant 5-LO modification included incubation with glutathione or compound U73122, previously described as inhibitor of 5-LO.
Since in-vitro assays might have generated artifacts, a method for 5-LO purification from human cells was sought, in order to examine the modification state of the protein in the cellular context. ATP-agarose affinity purification and anti-5-LO immunoprecipitation proved inappropriate for sample purification for MALDI-MS analysis. Consequently, two human cell lines that are able to express 5-LO (Rec-1 Blymphocytes and MM6 monocytes) were transduced with a DNA cassette that contained recombinant human 5-LO sequence with an attached N-terminal FLAG-tag. Anti-FLAG immunoprecipitation was then performed effectively in cell lysates and the precipitated FLAG-5-LO was separated by SDS-PAGE before MALDI-MS analysis.
The examined cell stimuli were expected to result to phosphorylation of 5-LO at Ser523 by PKA in Rec-1 cells and to phosphorylation of Ser271 and/or Ser663 in MM6 cells by activated MK2 and ERK2, respectively. Additionally, under the conditions of MM6 cell stimulation, Fgr, Hck and Yes kinases, which phosphorylated 5-LO in vitro, were expected to be activated and the possibility of 5-LO phosphorylation on tyrosine was investigated. Although immunoblotting results indicated that all the aforementioned phosphorylation events existed in the examined samples, MALDI-MS analysis verified only phosphorylation on Ser271 in differentiated MM6 cells, interestingly regardless of cell stimulation.
Finally, the primary amine derivatization procedure by EZ-linkTM was utilized for MS analysis of lysine rich proteins. In the past, chemical propionylation of histones had been employed prior to trypsin digestion; however it was easily confused in MS with combinations of other PTMs (e.g. acetylation, methylation). Moreover, propionylation is a PTM for histone H3 and this information was lost. Consequently, the EZ-link reagent was more useful for analysis of histones, as unambiguous assignment of PTMs and detection of native propionylation on bovine H3 became possible.
Tumor development usually follows predictable paths where tumor cells acquire common characteristics and features known as the hallmarks of cancer. Recently, additional characteristics have been added to these hallmarks since solid tumors are composed of a very heterogeneous population of transformed, formerly normal tissue cells and stromal cells, e.g. immune cells and fibroblasts. Compelling evidence suggests that stromal cells and tumor cells maintain a symbiotic relationship to build up the tumor microenvironment and to fuel tumor growth. In cancer therapies, common features of tumors such as unrestricted cell growth, suppression of immunological responses, and the ability to form new blood vessels (angiogenesis) have emerged as the main targets of interest. The lipid mediator prostaglandin E2 (PGE2) is known to promote all these features and thus, is connected to cancer progression in general. Its synthesis is triggered in response to stress factors or during inflammation. Inducible PGE2 production relies on the enzymes cyclooxygenase 2 (COX-2) and microsomal prostanglandin E synthase 1 (mPGES-1), which are simultaneously expressed in response to a variety of different stimuli and are functionally coupled. Inhibition of COX-2 with non-steroidal antiinflammatory drugs (NSAIDs) for cancer treatment is, however, limited by cardiovascular risks, since selective COX-2 inhibition disrupts the prostacyclin/thromboxane balance. Therefore targeting mPGES-1 downstream of COX-2 for PGE2 inhibition was evaluated in this work in different steps of carcinogenesis. Knockdown of mPGES-1 in DU145 prostate cancer cells revealed that the mPGES-1 status did not affect growth of monolayer tumor cells, but significantly impaired 3D growth of multi-cellular tumor spheroids (MCTS). Spheroid formation induced COX-2 in DU145 and other prostate cancer spheroids. High levels of PGE2 were detected in supernatants of DU145 MCTS as opposed to monolayer DU145 cells. Pharmacological inhibition of COX-2 and mPGES-1 confirmed the pivotal role of PGE2 for DU145 MCTS growth. Besides promoting spheroid growth, MCTS-derived PGE2 also inhibited cytotoxic T lymphocyte (CTL) activation. When investigating the mechanisms of COX-2 induction during spheroid formation, the typical tumor microenvironmental factors such as glucose deprivation, hypoxia or tumor cell apoptosis failed to enhance COX-2. Interestingly, when interfering with apoptosis in DU145 spheroids, the pan-caspase inhibitor Z-VAD-FMK triggered a Summary 12 shift towards necrosis, thus enhancing COX-2 expression. Coculturing viable DU145 monolayer cells with isolated heat-shocked-treated necrotic DU145 cells, but not with necrotic cell supernatants, induced COX-2 and PGE2, confirming the impact of necrosis for MCTS growth and CTL inhibition. As mentioned, in vivo tumors are very heterogenous mixtures of tumor cells and stromal cells e.g. immune cells. Hence, the interaction of the immune system with tumors was investigated in further experiments. When coculturing MCF-7 breast cancer spheroids with human peripheral blood mononuclear cells (PBMCs), only low levels of PGE2 were detected, since MCF-7 cells did not upregulate COX-2 during spheroid formation and did not induce PGE2 production by PBMCs. Under inflammatory conditions, by adding the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) to cocultures, PGE2 production was triggered, spheroid sizes were reduced, and numbers of high levels of granzyme B expressing (GrBhi) CTLs were increased, while CD80 expression by tumor-associated phagocytes was also elevated. Inhibition of CD80 but not CD86 diminished numbers of GrBhi CTLs and attenuated spheroid lysis. To determine the role of ctivation-induced PGE2 production, use of the COX-2 inhibitor celecoxib and the experimental mPGES-1 inhibitor C3 further increased CD80 expression. Addition of PGE2, the prostaglandin E2 (EP2) receptor agonist butaprost, and the phosphodiesterase 4 (PDE4) inhibitor rolipram reduced LPS/C3-triggered CD80 expression, confirming the impact of COX- 2/mPGES-1-derived PGE2 on shaping phagocyte phenotypes in an EP2/cAMP-dependent manner. In a spontaneous breast cancer model (MMTV-PyMT), mPGES-1-deficiency significantly delayed tumor growth in mice, confirming an overall protumorigenic role of mPGES-1 in breast cancer development in vivo. However in tumors of mPGES-1-/- mice, tumor-infiltrating phagocytes expressed low levels of CD80 similar to their wildtype counterparts. These data suggest that the immunosuppressive microenvironment does not allow for immunostimulatory effects by mPGES-1 inhibition without an activating stimulus. Evidences in this study recommend the application of mPGES-1 inhibitors for treating cancer diseases, since mPGES-1 promotes tumor growth in multiple steps of carcinogenesis, ranging from well-characterized effects of tumor cell growth to immune suppression of CTL activity and phagocyte polarization. Regarding the latter, blunting PGE2 during immune activation may limit the tumor-favoring features of inflammation and improve the efficiency of TLR4 based immune therapies.
In Nervensystemen werden zahlreiche Informationen wahrgenommen und verarbeitet um ein adäquates Verhalten hervorzurufen. Für die Untersuchung der funktionellen Zusammenhänge hierbei wurden verschiedene Methoden entwickelt, die eine gezielte Manipulation neuronaler Prozesse ermöglichen. Durch Analyse der resultierenden Effekte können dabei synaptische Proteine, einzelne Neuronen oder neuronale Netzwerke funktionell charakterisiert werden. Bisherige Ansätze verfügen jedoch nur über eine geringe zeitliche und räumliche Auflösung oder erlauben lediglich eine eingeschränkte Anwendung im frei beweglichen Tier.
Diese Nachteile können durch die heterologe Expression von lichtgesteuerten, mikrobiellen Rhodopsinen zur gezielten Manipulation des Membranpotentials umgangen werden. So induziert die Photoaktivierung des Kationenkanals Channelrhodopsin 2 (ChR2; (Nagel et al., Curr Biol 2005)) eine Depolarisation, während die Chloridpumpe Halorhodopsin (NpHR; (Zhang et al., Nature 2007)) für die Hyperpolarisation verwendet werden kann. Dabei ermöglichen die schnellen Kinetiken der Rhodopsine eine zeitlich präzise Steuerung des Membranpotentials. Durch Auswahl geeigneter Promotoren ist zudem oftmals eine zell spezifische Expression möglich. Dieser Ansatz wird daher allgemein als Optogenetik bezeichnet.
In der vorliegenden Arbeit wurden zunächst konventionelle Techniken genutzt, um die Funktion von zwei assoziierten Proteinen eines Acetylcholin Rezeptors in C. elegans zu untersuchen. Des Weiteren wurden verschiedene Methoden für den Fadenwurm entwickelt und angewendet, die die Vorteile optogenetischer Techniken für die funktionelle Charakterisierung synaptischer Proteine und neuronaler Netzwerke nutzbar machen. Hierbei erlaubt die Transparenz von C. elegans die optogenetische Stimulation im lebenden Organismus unter nicht invasiven Bedingungen. Weitere Vorteile von C. elegans als neurobiologischem Modellorganismus liegen in seiner einfachen Handhabung (Hope, 1999) und der stereotypen Entwicklung seines Nervensystems mit bekannten anatomischen Ausprägungen (Sulston and Horvitz, Dev Biol 1977; Varshney et al., PLoS Comput Biol 2011; White et al., Philos Trans R Soc Lond B Biol Sci 1986). Durch ihre Häufigkeit und die experimentelle Zugänglichkeit wird hierbei die neuromuskuläre Synapse oftmals zur Erforschung der synaptischen Reizweiterleitung genutzt (Von Stetina et al., Int Rev Neurobiol 2006). Durch pharmakologische (Lewis et al., Neuroscience 1980; McIntire et al., Nature 1993; Miller et al., Proc Natl Acad Sci U S A 1996; Richmond and Jorgensen, Nat Neurosci 1999) und elektrische Stimulation (Richmond and Jorgensen, Nat Neurosci 1999) können dabei Defekte der Transmission hervorgehoben werden, während Verhaltensexperimente oder elektrophysiologische Messungen der post synaptischen Ströme in Muskelzellen eine quantitative Analyse ermöglichen (Richmond and Jorgensen, Nat Neurosci 1999).
Diese Methoden wurden für die funktionelle Charakterisierung von NRA 2 und NRA 4 verwendet, die beide als akzessorische Proteine zusammen mit dem Levamisol sensitiven Acetylcholin Rezeptor der Körperwandmuskelzellen aufgereinigt wurden (Gottschalk et al., EMBO J 2005). Dabei konnte gezeigt werden, dass NRA 2 und NRA 4 im Endoplasmatischen Retikulum (ER) der Muskelzellen einen Komplex bilden, der die Sensitivität von beiden nikotinischen Acetylcholin Rezeptoren gegenüber verschiedenen cholinergen Agonisten verändert. In diesem Zusammenhang wurde auch nachgewiesen, dass die Oberflächenexpression einzelner Untereinheiten der beiden Rezeptoren durch NRA 2/4 beeinflusst wird. Diese Resultate legen die Vermutung nahe, dass beide Proteine die Zusammensetzung der Rezeptoren und somit ihre pharmakologischen Eigenschaften modulieren. Denkbar ist dabei eine regulatorische Funktion bei der Assemblierung verschiedener Untereinheiten zu einem funktionellen Rezeptor oder bei der Kontrolle des ER Austritts von Rezeptoren mit bestimmter Zusammensetzung. In dieser Hinsicht konnte jedoch keine Interaktion von NRA 2/4 mit der Notch Signalkaskade nachgewiesen werden, wie sie für die homologen Proteine nicalin und NOMO in Vertebraten gezeigt wurde (Haffner et al., J Biol Chem 2007; Haffner et al., EMBO J 2004).
Für die Untersuchung synaptischer Proteine durch optogenetische Techniken wurde ChR2(H134R) selektiv in cholinergen oder GABAergen Motorneuronen exprimiert, um die akute und lichtgesteuerte Freisetzung des jeweiligen Neurotransmitters zu ermöglichen. Die resultierende Stimulation bzw. Inhibition von Muskelzellen wurde hierbei durch elektrophysiologische Messungen der post synaptischen Ströme und durch Analyse von Kontraktionen respektive Relaxationen untersucht. Dabei wurde gezeigt, dass Störungen der synaptischen Reizweiterleitung die Ausprägung und Dynamik dieser lichtinduzierten Effekte beeinflussen und dadurch charakterisiert werden können. So zeigten beispielsweise Mutanten von Synaptojanin und Endophilin nachlassende Effekte bei anhaltender oder wiederholter Stimulation, was durch die gestörte Regeneration synaptischer Vesikel erklärt werden kann (Harris et al., J Cell Biol 2000; Schuske et al., Neuron 2003; Verstreken et al., Neuron 2003).
Die hohe Sensitivität dieser Methode wurde im Nachfolgenden dazu verwendet, die Inhibition cholinerger Motorneuronen durch den metabotropen GABAB Rezeptor zu untersuchen, der in C. elegans aus den beiden Untereinheiten GBB 1 und GBB 2 gebildet wird (Dittman and Kaplan, J Neurosci 2008; Vashlishan et al., Neuron 2008). Dabei konnte zunächst gezeigt werden, dass diese heterosynaptische Inhibition verschiedene lokomotorische Verhaltensweisen der Tiere beeinflusst. Für die mechanistische Untersuchung wurden anschließend cholinerge Motorneuronen durch ChR2(H134R) photoaktiviert, während resultierende Kontraktionseffekte in Abhängigkeit von GBB 1/2 analysiert wurden. Um hierbei die Funktion von GBB 1/2 durch erhöhte GABA Konzentrationen hervorzuheben, wurden zusätzlich GABAerge Motorneuronen optogenetisch stimuliert oder die Wiederaufnahme von GABA aus dem synaptischen Spalt durch Mutation des Membran ständigen GABA Transporters blockiert. So konnte gezeigt werden, dass GBB 1/2 eine akute Inhibition der cholinergen Motorneuronen bewirken, was vermutlich für die Regulation von Bewegungsabläufen eine wichtige Rolle spielt. Die geringe Dynamik der GBB 1/2 induzierten Effekte deutet allerdings darauf hin, dass die synaptische Aktivität durch den metabotropen Rezeptor kaum nachhaltig moduliert wird.
In nachfolgenden Versuchen wurde die optogenetische Stimulation von Motorneuronen außerdem mit der elektronenmikroskopischen Analyse der präsynaptischen Feinstruktur kombiniert. Dadurch konnte die Dynamik der Exozytose und Endozytose synaptischer Vesikel (SV) in Abhängigkeit von neuronaler Aktivität untersucht werden. So wurde gezeigt, dass synaptische Vesikel nahe der aktiven Zone während einer 30 sekündigen Hyperstimulation nahezu komplett aufgebraucht waren. Die vollständige Regeneration der SV Pools benötigte anschließend etwa 12 Sekunden und erfolgte zunächst in der Peripherie der aktiven Zone, was auf eine laterale Heranführung der Vesikel schließen lässt. Nach etwa 20 Sekunden erholte sich ebenfalls die Wirksamkeit der Stimulation von Muskelzellen durch die Motorneuronen, was durch elektrophysiologische Messungen der photo induzierten post synaptischen Ströme gezeigt wurde. Während der Hyperstimulation bildeten sich außerdem große vesikuläre Strukturen, die sich anschließend nach etwa acht Sekunden wieder aufgelöst hatten. In Analogie zu vergleichbaren Experimenten in anderen Organismen liegt die Vermutung nahe, dass es sich dabei um Zwischenprodukte der so genannten Bulk Phase Endozytose handelt, die das Clathrin abhängige Recycling von synaptischen Vesikeln bei starker neuronaler Aktivität ergänzt (Heuser and Reese, J Cell Biol 1973; Miller and Heuser, J Cell Biol 1984; Richards et al., Neuron 2000). Bemerkenswerterweise war der Abbau der vesikulären Strukturen in Synaptojanin und Endophilin defizienten Tieren stark verzögert. Denkbar ist, dass beide Proteine für die Synthese von synaptischen Vesikeln aus den vesikulären Zwischenprodukten der Bulk Phase Endozytose wichtig sind, analog zur ihrer Funktion bei der Clathrin abhängigen Endozytose an der Plasmamembran.
Durch die zielgerichtete Manipulation der Zellaktivität ermöglichen optogenetische Techniken außerdem die funktionelle Charakterisierung von Neuronen und neuronalen Netzwerken. Um die zelluläre Spezifität dieses Ansatzes zu erhöhen, wurde ein Tracking System entwickelt das die Position frei beweglicher Tiere in Echtzeit bestimmt und nachverfolgt. Dadurch konnte die Photoaktivierung optogenetischer Proteine auf definierte Bereiche der Fadenwürmer und somit auf ausgewählte Neuronen innerhalb der Expressionsmuster von verwendeten Promotoren eingeschränkt werden. Des Weiteren ermöglichte hierbei die Auswertung translatorischer Parameter die Analyse verschiedener lokomotorischer Merkmale wie Geschwindigkeit, Bewegungsbahn oder Ausprägung der Körperbiegungen. Dieses System wurde beispielhaft für die konzertierte Photoaktivierung durch ChR2(H134R) bzw. Photoinhibition durch MAC von zwei verschiedenen Gruppen von Neuronen angewendet, um die Integration mechanosensorischer Informationen durch Command Interneuronen zu untersuchen. In diesem Zusammenhang wurde zudem eine Rekombinase basierte Methode für optogenetische Proteine adaptiert, die die Transkription auf die zelluläre Schnittmenge von zwei verschiedenen Promotoren einschränkt und somit die Spezifität der Expression erhöht. Idealerweise kann dieser Ansatz außerdem mit der gezielten Photoaktivierung kombiniert werden, um die zelluläre Selektivität optogenetischer Anwendungen weiter zu verbessern.
Weiterhin ist die Anwendung optogenetischer Techniken bisher durch intrinsische Eigenschaften der verwendeten Rhodopsine auf die relativ kurzzeitige Manipulation des Membranpotentials von Zellen beschränkt. So benötigt ChR2 durch die schnelle Schließung seines offenen Kanals eine kontinuierliche Photoaktivierung, um eine andauernde Depolarisation hervorzurufen. Dies ist jedoch potentiell mit phototoxischen und – besonders bei C. elegans – phototaktischen Nebeneffekten verbunden. Deswegen wurden diverse Mutanten von ChR2 mit stark verlangsamter Inaktivierung (Berndt et al., Nat Neurosci 2009) für ihren Nutzen zur Langzeit Stimulation von erregbaren Zellen im Nematode getestet. Dabei wurde gezeigt, dass ChR2(C128S) durch einen kurzen Photostimulus mit vergleichsweise niedriger Intensität eine anhaltende Depolarisation über mehrere Minuten auslösen kann. Die wiederholte Stimulation in ASJ Neuronen ermöglichte zudem eine langzeitige Depolarisation über mehrere Tage, wodurch die genetisch veranlagte Entwicklung von Tieren manipuliert werden konnte. Durch gezielte Punktmutation konnten außerdem relevante Eigenschaften von ChR2(C128S) für die Langzeit Stimulation weiter verbessert werden.
Als weiteres optogenetisches Werkzeug wurde zudem die Photoaktivierbare Adenylatzyklase alpha (PACa) aus Euglena gracilis (Iseki et al., Nature 2002; Ntefidou et al., Plant Physiol 2003; Schroder-Lang et al., Nat Methods 2007) für die akute und lichtgetriebene Synthese des sekundären Botenstoffs cAMP in C. elegans etabliert. Die Photoaktivierung von PACa in cholinergen Motorneuronen verstärkte dabei die Neurotransmitterfreisetzung und induzierte hyperlokomotorische Phänotypen, vergleichbar zu Mutanten mit erhöhten cAMP Konzentrationen.
Zusammengefasst wurden diverse optogenetische Techniken für C. elegans entwickelt und optimiert, die die zellspezifische und nicht invasive Manipulation des Membranpotentials beziehungsweise die Synthese des sekundären Botenstoffs cAMP durch Licht im frei beweglichen Tier ermöglichen. Diese Methoden können zur gezielten Störung neuronaler Aktivität angewendet werden, um dadurch neurobiologische Fragestellungen im Fadenwurm zu untersuchen. Dies wurde beispielhaft für die Erforschung der synaptischen Reizweiterleitung und die funktionelle Analyse neuronaler Netzwerke demonstriert. Denkbar ist außerdem, diese für C. elegans etablierten Methoden vergleichbar in anderen Modellorganismen anzuwenden. So sind die Fruchtfliege ebenso wie der Zebrafisch Embryo bereits für optogenetische Techniken erprobt (Arrenberg et al., Proc Natl Acad Sci U S A 2009; Schroll et al., Curr Biol 2006). Für Säugetiere wie die Maus, die Ratte und den Makaken wurden zudem bereits Ansätze entwickelt, die die gezielte Photostimulation in lebenden und frei beweglichen Tieren ermöglichen (Han et al., Neuron 2009; Wentz et al., J Neural Eng 2011; Yizhar et al., Nature 2011; Zhang et al., Nat Rev Neurosci 2007).
Structural determinants for substrate specificity of the promiscuous multidrug efflux pump AcrB
(2013)
Opportunistic Gram-negative pathogens such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter Baumanii and Pseudomonas aeruginosa are becoming more and more multiresistant against many commonly available antibiotics [39, 40]. An important resistance mechanism of Gram-negative bacteria is the efflux of noxious compounds by tripartite systems [39, 41-44]. The best studied and most clinically relevant tripartite system is the AcrA-AcrB-TolC system of Escherichia coli, where substrate recognition and energy transduction takes place in the inner membrane protein AcrB. AcrB has a remarkably huge substrate spectrum and can recognize structurally diverse molecules, such as hexan in contrast to erythromycin, as its substrates [45]. Therefore, overproduction of the tripartite system can render a Gram-negative pathogen resistant against multiple antibiotics at once. The mechanisms of how AcrB is able to recognize such an enormous spectrum of molecules as substrates, without compromising its specificity (e.g. by neglecting essential compounds like lipids or gluclose as its susbtates), remained puzzling. Structural insight into substrate specificity was so far limited to two co-crystal structures of AcrB, where minocycline and doxorubicin, respectively, were identified bound to an internal binding pocket of AcrB. This binding pocket is particularly deeply buried into internal parts of the T monomer of AcrB and was, therefore, denoted deep binding pocket (DBP). Analysis of several AcrB co-crystal structures with substrate molecules bound to the DBP [4, 23, 25] indicated that the substrate promiscuity involved multisite binding modes within the DBP. Multisite binding modes, where different substrate molecules can bind to slightly different positions and orientations to the same binding pocket, is a common feature of multidrug recognizing proteins such as QacR or BmrR [27-29]. Nevertheless, AcrB's substrate spectrum is much broader than substrate spectra of most other multidrug recognizing proteins. Therefore, it is likely that additional mechanisms are involved in mediating the observed high substrate promiscuity of AcrB. In our recently published high-resolution AcrB/doxorubicin co-crystal structure (pdb entry: 4DX7 [23]) we were able to identify two additional substrate binding pockets in the L monomer of AcrB: i) the access pocket (AP), with an opening towards the periplasm, and ii) a putative binding site in a groove between transmembrane helices 8 and 9 (TM8/TM9 groove), accessible from the lipid layer of the inner membrane. Both binding pockets are likely to be access sites for substrates towards AcrB. Furthermore, each of the binding pockets are possibly specialized to recognize a specific subset of the entire substrate spectrum of AcrB, i.e. highly hydrophobic substrates (e.g. n-dodecyl-ß-d-maltoside or sodium dodecylsulfate) might access AcrB towards the TM8/TM9 groove and water soluble substrates (e.g. berberine) might access AcrB towards the AP. Since substrates will accumulate in the membrane or the periplasm according to their hydrophilic or hydrophobic nature, substrates will be "pre-selected" by the medium, rather than by the protein itself, and guided to their appropriate access site. This process is proposed to be called "medium- mediated pre-selection". The AcrB/doxorubicin co-crystal structure (pdb entry: 4DX7 [23]) furthermore revealed that the AP and DBP are in next neighborhood to each other and are separated by a switch loop. This switch loop adopts distinct conformations in the L, T and O monomers. Specific switch loop conformations are strongly involved in coordinating the selective occupation of both binding pockets, the AP and the DBP. The conformation of the switch loop in the L monomer (L-switch loop) opens the AP and closes the DBP, whereas the conformation of the switch loop in the T monomer (T-switch Loop) opens the DBP and closes the AP. An analysis of all asymmetric AcrB structures indicated that the L-switch loop is able to adopt multiple distinct conformations, whereas the conformation of T-switch loop remained largely congruent in all crystal structures. Moreover, each distinct switch loop conformation, observed in co-crystal structures of AcrB with occupied AP [4, 23], was perfectly adapted to the bound substrate molecule. Therefore, the putatively flexible switch loop is likely to act as an adaptive module and mediates a high binding pocket plasticity without altering the global protein structure. This binding mode is called adaptor-mediated binding mechanism, where an flexible adaptive module (like the switch loop) is able to adapt the surface shape of an binding pocket to different substrate molecules. Furthermore, structural and biochemical analyses of an AcrB G616N variant, revealed the involvement of specific switch loop conformations in the substrate specificity of AcrB. A substitution of G616, located on the switch loop, to N616 was able to alter the conformation of the switch loop exclusively in the L monomers of AcrB, whereas the switch loop conformations in T and O monomers remained congruent to the conformations observed in crystal structures of wildtype AcrB. Moreover, cells producing the AcrB G616N and MexB, both bearing the G616N amino acid substitution, exhibited a reduced resistance against certain substrates, whereas the resistance against most other substrates remained on the level of wildtype AcrB. Correlations of the phenotypes with minimal projection areas, a novel 2-spatiodimensional parameter which approximates the size of a substrate molecule, revealed that AcrB variants with a G616N substitution have a reduced efflux activity for exclusively large substrate molecules. The rejection of large substrates is most likely connected with altered L-switch loop conformations....
In the past century, scientists have realized that venoms are a source of a number of natural substances presenting a wide range of pharmacological properties and often displaying a high specificity for their targets. Thus, the field of toxinology came into being, which is defined as the study of toxic substances of biological origin. Toxins are found in a wide variety of animals, including fish, cone snails, scorpions, snakes, and even some mammals. To be classified as venom, these must contain substances, i.e. toxins, which disturb physiological processes and must be deliberately delivered to the target animal. Snakes have evolved one of the most sophisticated mechanisms for venom delivery. Envenomation by snakebite can induce and inhibit aggregation/agglutination of platelets as well as inhibit/activate hemostasis, but also disrupt other physiological functions via neurotoxins and angioneurin growth factors. Snake venoms contain a substantial amount of C-type lectin-related proteins (CLRPs) which are known to function, notably, as integrin inhibitors. CLRPs are heterodimers composed of homologous α and β subunits which can assemble either covalently or noncovalently to oligomers, resulting in αβ, (αβ)2 and (αβ)4 structures. Some of the main targets of CLRPs are membrane receptors, coagulation factors, and proteins essential to hemostasis. The platelet collagen receptors GPVI and α2β1 integrin as well as the von Willebrand factor receptor GPIb play important roles in platelet activation and aggregation and are considered main targets of antithrombotic drugs. In this thesis, the integrin α2β1 is particularly considered as it is the sole collagen-binding integrin on platelets. Reduced expression of this platelet receptor results in dysfunction of platelet responses. Equivalently, overexpression of α2β1 integrin results in an increased risk of thrombosis. As a result, selective inhibitors of the collagen-α2β1 interaction could give rise to effective antithrombotic drugs. Integrins are large receptors which mediate cell-cell contacts and the binding of cells to the extracellular matrix (ECM). Therefore, they play a role in physiological processes, e.g. hemostasis and immunity, as well as in pathological processes, e.g. tumor angiogenesis and atherosclerosis. 18 α and 8 β integrin subunits, with nine α subunits containing an additional A domain, associate non-covalently to form 24 heterodimers with distinct binding specificities. Integrin collagen receptors are a subclass of four receptors which all utilize the β1 subunit. The α2β1 integrin is a collagen-binding receptor expressed not only on platelets, but also on endothelial and epithelial cells. Consequently, this integrin is also essential for cell adhesion and migration playing a role in angiogenesis as well as tumor metastasis. To date, there are five known antagonists of α2β1 integrin: EMS16, rhodocetin, vixapatin, and most recently rhinocetin and flavocetin-A. The first four have been shown to be specific for the integrin α2A domain, the major collagen-binding domain. All these antagonists are CLRPs and present new leads for drug design. In the past few years, many insights into the structure and function of rhodocetin were obtained. Monoclonal antibodies proved to be advantageous in disclosing this information, making them not only useful as therapeutic agents, but also as tools for protein characterization. The venom of the Vipera palaestinae snake was recently shown to contain an α2β1 integrin inhibitor, which prevented the integrin from binding collagen. This inhibitor, called vixapatin, was the initial focus of this dissertation. Vixapatin’s interaction with the α2β1 integrin needed further characterization on a molecular and cellular level to assess its medical potential and monoclonal antibodies were to be used as a tool. Originally, vixapatin had been isolated by reversed-phase high-performance liquid chromatography. To avoid the stringency of this method, for this study, it was replaced with gentler chromatographic methods. First, the α2β1 integrin inhibitor was isolated from the crude snake venom with affinity chromatography using the α2A domain as bait, establishing a method to quickly screen venoms for α2β1-binding proteins which affect the collagenintegrin interaction. The applicability of this method to other snake venoms was shown by isolating an α2A domain-specific toxin from the venom of Trimeresurus flavoviridis. To allow further characterization of both these toxins, gel filtration and ion exchange chromatography were employed to purify the protein without the α2A domain. These classical protein purification methods resulted in similar separation patterns of both the V. palaestinae and T. flavoviridis venom proteins. Purified proteins exhibiting the potential of inhibiting integrinbinding to collagen were analyzed by two-dimensional gel electrophoresis. Both VP-i and flavocetin-A, the integrin inhibitors from V. palaestinae and T. flavoviridis, respectively, were shown to have more complex structures than was evident from the purification. Each consisted of four low-molecular-weight proteins which assembled into two bands (for VP-i) or one single band (for flavocetin-A) under non-reducing conditions. Mass spectrometry analyses revealed VP-i to belong to the family of CLRPs, just like vixapatin does. However, these two proteins differed in their primary sequences and only showed homology to one another. The toxin purified from T. flavoviridis revealed this toxin to be flavocetin-A, a heterodimeric CLRP which had so far only been shown to have GPIb-binding activity. At the time of flavocetin-A’s purification, flavocetin-B was co-purified; flavocetin-B consists of the same two α and β subunits, plus an additional γ subunit. As no sequence information is known to date for the γ subunit, it may be one of the additional proteins purified here, along with an additional δ subunit. Therefore, the toxin isolated here may actually consist of four different subunits forming a tetramer of two different heterodimers, generating an (αβ)2(γδ)2 structure. This proposed (αβ)2(γδ)2 flavocetin-A structure has binding sites for both α2β1 integrin and GPIb, with no sterical overlap, as shown by affinity chromatography using the α2A domain and the extracellular domain of the GPIb receptor. The potential of VP-i and flavocetin-A to inhibit integrin-binding to type I collagen was shown during purification: Both toxins efficiently bind to the integrin α2A domain; also, VP-i and vixapatin bind to the A domain with the same affinity. Surface plasmon resonance showed the interaction of flavocetin-A with the α2β1 integrin to be extremely strong and association to be very fast. Furthermore, both toxins were shown to inhibit binding of the wildtype integrin to collagen: VP-i and flavocetin-A acted antagonistically on cell adhesion and cell migration. Initially, the interaction between VP-i and α2β1 integrin was to be further characterized with the help of monoclonal antibodies. However, this proved problematic, the procedure requiring various optimizations. Although, after expert consultation, some monoclonal antibodies could be obtained, the cells were extremely sensitive and gave unsatisfactory results when tested as detection tools in Western blot and immunoassays. Concluding, two novel α2β1 integrin inhibitors were discovered: VP-i and flavocetin-A, which were purified using the same procedure and which have similar functions. Both are Ctype lectin-related proteins which effectively inhibit cell adhesion and migration. This underlines that nature has instrumentalized CLRPs to specifically inhibit α2β1 integrin. Further characterization of VP-i and flavocetin-A will be able to provide leads for future drug development.
Plants absorb sunlight via photosynthetic pigments and convert light energy intochemical energy in the process of photosynthesis. These pigments are mainly bound to antenna protein complexes that funnel the excitation energy to the photosynthetic reaction centres. The peripheral antenna of plant photosystem II (PSII) consists of the major light-harvesting complex of PSII (LHC-II) and the minor LHCs CP29, CP26 and CP24. Light intensity can change frequently and plants need to adapt to high-light conditions in order to avoid photodamage. When more photons are absorbed than can be utilised by the photosynthetic machinery, excessive excitation energy is dissipated as heat by short-term adaptation processes collectively known as non-photochemical quenching (NPQ). A decrease in PSII antenna chlorophyll (Chl) fluorescence yield and a reduction in the average Chl fluorescence lifetime are associated with NPQ. The main component of NPQ is the so-called energy-dependent quenching (qE), and it is triggered by the rapid drop in thylakoid lumenal pH resulting from the plant’s photosynthetic activity. This process is thought to take place at the PSII antenna complexes, which therefore not only capture and transfer light energy but are also involved in balancing the energy flow. The decrease in lumenal pH acivates the enzyme violaxanthin de-epoxidase (VDE), which converts the xanthophyll violaxanthin (Vio) into zeaxanthin (Zea) in the xanthophyll cycle. In addition, the PSII subunit PsbS was discovered to be essential for qE by screening qE-deficient Arabidopsis thaliana mutants. This membrane protein is considered a member of the LHC superfamily, which also includes LHC-II and the minor LHCs. Previous studies on PsbS isolated either from native source or refolded in vitro have produced inconsistent results on its pigment binding capacity. Interestingly, a pH-dependent change in the quaternary structure of PsbS under high light conditions has been reported. This observed dimer-tomonomer transition very likely follows the protonation of lumenal glutamates upon the drop in pH and is accompanied by a change in PSII supercomplex localisation. PsbS dimers are preferentially found in association with the PSII core, whereas PsbS monomers co-localise with LHC-II.Despite the identification of !pH, Zea and PsbS as key players in qE, both the nature of the quencher(s) as well as the underlying molecular mechanism leading to excess energy dissipation still remain unknown. Several models have been put forward to explain the reversible switch in the antenna from an energy-transmitting to a quenched state. Proposals include a simple pigment exchange of Vio for Zea, and aggregation or an internal conformational change of LHC-II. Charge transfer (CT)quenching in the minor LHCs or quenching by carotenoid dark state (Car S1)-Chl interactions have also been suggested. However, none of these qE models has so far been capable of accommodating all the physiological observations and available experimental data. Most importantly, the function of PsbS remains an enigma. A recent qE model suggested that monomerisation of PsbS enables the protein to transiently bind a carotenoid and form a quenching unit with a Chl of a PSII LHC. In view of the various proposed qE mechanisms, this thesis aimed at understanding the interplay of the different qE components and the contribution of the PSII subunits LHC-II, the minor LHCs and PsbS to qE. The initial approach was to investigate the properties of the PSII subunits in the most simple in vitro model system, namely in detergent solution. For this purpose, LHC-II was isolated either from native source or refolded from recombinantly produced protein. Investigation of the minor LHCs and PsbS required heterologous expression and refolding. In addition, experiments were performed on aggregated LHC-II. Aggregates of LHC-II have been used as a popular model system for qE because they exhibit highly quenched Chl fluorescence. At the final stage of this doctoral work, a more sophisticated model system to approximate the thylakoid membrane was developed by reconstitution of the PSII subunits LHC-II and PsbS into liposomes. This system not only allowed for investigation of these membrane proteins in their native environment, but also for mimicking the xanthophyll cycle by distribution of Zea within the membrane as well as !pH by outside buffer exchange. The role of Zea in qE was first investigated with detergent solubilised antenna proteins. The requirement of this xanthophyll for qE is well-known, but the specific contribution to the molecular quenching mechansim is unclear. Previous work had shown that replacement of Vio for Zea in LHC-II was not sufficient to induce Chl fluorescence quenching in Zea-LHC-II, as suggested by the so-called molecular gearshift mechanism. However, by means of selective two-photon excitation spectroscopy, an increase in electronic interactions between Car S1 and Chls was observed for LHC-II upon lowering the pH of the detergent buffer. Electronic Car S1-Chl coupling became even stronger when Zea-LHC-II was probed. The extent of Car S1-Chl coupling correlated directly with the extent of Chl fluorescence quenching, in a similar way as observed previously in live plants under high-light conditions. However, very similar results were obtained with LHC-II aggregates. This implied that the increase in electronic interactions and fluorescence quenching was independent of Zea and low pH. Further experiments on aggregates of LHC-II Chl mutants indicated that the targeted pigments were also not essential for the observed effects. It is proposed that the same molecular mechanism causes an increase in electronic Car S1-Chl interactions and Chl fluorescence quenching in Zea-LHC-II at low pH as well as in aggregated LHC-II. Most likely, surface exposed pigments form random quenching centres in both cases. On the other hand, it was possible that Zea could act as a direct quencher of excess excitation energy in the minor LHCs. However, enrichment of refolded CP29, CP26 and CP24 with Zea did not lead to a change in the Chl excited state lifetime. Formation of a carotenoid radical cation, previously implied in CT quenching, was also not observed, although artificial generation of such a radical cation was principally possible as shown for CP29. During the course of this work, a study reporting the formation of Zea radical cations in minor LHCs was published. Therefore, Zea-enriched minor LHCs were again investigated on the experimental apparatus used in the reported study. Indeed, the presence of at least one carotenoid radical cation for each minor complex was detected. It is suggested that either the preparation method of incubating the refolded minor LHCs with Zea in contrast to refolding the complexes with only Zea and lutein causes the observed differences or that the observed spectral radical cation signatures are due to experimental artifacts. While the experiments with LHC-II and the minor LHCs gave useful insights into the putative qE mechanism, the quencher site and the mode of action of Zea could still not be unambiguously identified. Most importantly, these studies could not explain the function of the qE keyplayer PsbS. Therefore, the focus of the work was shifted to PsbS protein production, purification and characterisation. In view of inconsistent reports on the pigment binding capacity of this PSII subunit, refolding trials with and without photosynthetic pigments were conducted. The formation of a specific pigmentprotein complex typical for other LHCs was not observed and neither was the earlier reported “activation” of Zea for qE by binding to this protein. Nevertheless, PsbS refolded without pigments displayed secondary structure content in agreement with previous studies, indicating pigment-independent folding. Reconstitution of pigmentfree, refolded PsbS into liposomes confirmed that the protein is stable in the absence of pigments. Zea distributed in PsbS-containing liposomes also showed no spectral alteration that would indicate its “activation”. With the ability to reconstitute PsbS, it was then possible to proceed to modelling qE in a proteoliposome system. For this purpose, PsbS was co-reconstituted with LHC-II, which has been reported to interact with PsbS. One-photon excitation (OPE) and two-photon excitation (TPE) spectroscopy measurements were performed on LHC-II- and LHC-II/PsbS-containing liposomes. This enabled both quantification of Chl fluorescence quenching as well as determination of the extent of electronic Car S1-Chl interactions. The effect of Zea was investigated by incorporating it in the proteoliposome membrane. It was shown that Zea alone was not able to induce significant Chl fluorescence quenching when only LHC-II was present. However, when LHC-II and PsbS were co-reconstituted, pronounced Chl fluorescence quenching and an increase in electronic Car S1-Chl interactions were observed and both effects were enhanced when Zea was present. Western blot analysis indicated the presence of a LHC-II/PsbS-heterodimer in these proteoliposomes. In addition to the OPE and TPE measurements, the average Chl fluorescence lifetime was determined in detergent-free buffer at neutral pH and directly after buffer exchange to low pH. No significant changes in the average lifetime were observed for LHC-II proteoliposomes when either Zea was present or after exchange for low pH buffer. This indicated that Zea alone cannot act as a direct quencher, which concurs with the OPE measurements. Moreover, the complex was also properly reconstituted as no aggregation or significant Chl fluorescence quenching were observed. The average lifetime was not significantly affected in LHC-II/PsbS-proteoliposomes, independent of Zea or pH. However, a shortlived component in the presence of a long-lived component was not resolvable with the time resolution of the fluorescence lifetime apparatus.
Implications for qE model systems and the in vivo quenching mechanism are discussed based on the experiments in detergent solution, on LHC-II aggregates and with the proteoliposome model system.
Biological membranes separate the cell interior from the outside and have diverse functions from signal transduction, apoptosis to transportations of ions and small molecules in and out of the cell. Most of these functions are fulfilled by proteins incorporated in the membrane. However, lipids as the main component of membrane not only serve as structural element for bilayer formation but they are also directly involved e.g. signalling processes and bilayer properties are important to mediate protein interactions. To fully understand the role of lipids, it is necessary to develop a molecular understanding of how certain membrane components modify bulk bilayer structure and dynamics. Membranes are known to have many different motions in different conditions and time scales. Temperature, pH, water content and many other conditions change membrane dynamics in a high degree. In addition to this, time scales of motions in membranes vary from ns to ms range corresponding to fast motion and slow motion, respectively. Therefore, membranes are needed to be studied systematically by varying the conditions and using methods to investigate motions in various time scales separately. The aim of this study was therefore perform a combined solid-state NMR / molecular dynamics study on model membranes. Different substrates, such as potential drugs, polarizing agents and signaling lipids were incorporated into bilayers and their location within the membrane and their effect onto the membrane was probed. NSAIDs (non-steroidal anti-inflammatory drugs), pirinixic acid derivatives, ceramides and polarizing agents were the substrates for membranes in this study. There were several experimental methods that were applied in order to investigate effects of these substrates on membrane dynamics. Different kind of phospholipids including POPC, DMPC and DPPC were used. In addition to experimental work, with the information gathered from solid state NMR experiments molecular dynamics simulations were performed to obtain more information about the membranes at the molecular level. As a result, combination of solid-state NMR with molecular dynamics simulations provides very systematic way of investigating membrane dynamics in a large range of time scales.
Pirinixic acid derivatives were special interest of this study because of their activity on peroxisome proliferator-activated receptor (PPAR) as an agonist as well as on enzymes of microsomal prostaglandin E2 synthase-1 (PGE2s) -1 and 5-lipoxygenase (5-LO) as dual inhibitor. Two potent pirinixic acid derivatives, 2-(4-chloro-6-(quinolin-6-ylamino)pyrimidin-2-ylthio)octanoic acid (compound 2) and 2-(4-chloro-6-(quinolin-6-ylamino)pyrimidin-2-ylthio)octanoate (compound 3), have been worked and their insertion depts were investigated by combining of solid state NMR and molecular dynamics simulations. Both experimental and theoretical results pointed out that compound 3 was inserted the phospholipid bilayer more deeply than 2. NSAIDs – lipid mixtures have been also studied here. It is known that consumption of NSAIDs as in mixture with lipids results much fewer side effects than consumption of the drugs alone. Thus, it is crucial to understand interactions of NSAIDs with lipids and investigate the possible complex formation of drugs with lipids. In this study, interactions of three widely used NSAIDs, ibuprofen, diclofenac and piroxicam, with DPPC were investigated by solid-state NMR. 1H and 31P NMR results depicted that ibuprofen and diclofenac had interactions with lipids, which is an indication of drug-lipid complex formation whereas piroxicam didn’t show any interactions with lipids suggesting that no complex formation occurred in the case of piroxicam. Ceramides are known to play key roles in many cell processes and many studies showed that the functions of ceramides are related with the ceramide effects on biological membranes. Therefore, in this study, influences of ceramides on biophysics of lipid bilayers were investigated by using various solid state NMR techniques and molecular dynamics simulations. Results from molecular dynamics simulations clearly showed that ceramide and lipids have strong interactions. More evidences about ceramide-lipid interactions were provided from 1H and 14N NMR results. In addition, it was indicated by both simulation and experimental methods that ceramide increased the rigidity of DMPC by increasing chain order parameters. BTbk is a biradical, which is used as polarizing agent for dynamic nuclear polarization (DNP) experiments and found to be more efficient than other widely used polarizing agents such as TOTAPOL. Since it is a hydrophobic compound, which prefers to stay inside lipid bilayer it is important to investigate the location and orientation of bTbk along the bilayer in order to understand its enhancement profile in DNP measurements. In this study, both NMR relaxation time measurements and molecular dynamics simulations revealed that bTbk tends to stay more close to hydrophobic chain of lipids than the interfacial part of lipids at bilayer surface.
In the first part of this work, a brief introduction on lipid membranes as well as a theoretical summary on both methods of solid-state NMR and molecular dynamics simulations is given. Then, in the second part methodology is introduced for both solid-state NMR spectrometer and theoretical calculations. Afterwards, results of different membrane systems are discussed in the following parts for both solid state NMR and MD. Finally, in the last part, a summary and the conclusion of the overall results together with some future plans are explained.
In this thesis the integral membrane protein diacylglycerol kinase (DAGK) from E.coli is investigated with solid-state NMR. The aim is to gain an insight into the enzyme’s mechanism through integration of kinetic, structural and dynamic data. The biological function of DAGK is the transfer of the γ-phosphate group from Mg*ATP to diacylglycerol (DAG) building phosphatidic acid (PA)[6] as port of the membrane-derived oligosaccharide cycle[31,34]. Surprisingly, DAGK does not share structural or sequential similarities with other kinases[12]. Typical sequence motives found in other kinases, which catalyze phosphoryl transfer reactions, are not found[13]. In its physiological form DAGK is a homo-trimer with nine transmembrane helices, three catalytic centers and a size of 39.6 kDa.
First, the set-up of a real-time 31P MAS NMR experiment is shown. This experiment allows measuring in real-time the simultaneous ATP hydrolysis in the aqueous phase and lipid substrate phos-phorylation in the membrane phase with atomic resolution under magic angle spinning[56]. After fast transfer of the sample into the NMR spectrometer the enzymatic reaction is started with a temperature jump. This approach of real-time MAS NMR in a dual-phase system was demonstrated for the lipid substrate analogs dioleoyl- (DOG) and dibutyrylglycerol (DBG), with a C8 and C4 aliphatic chain, respectively. The combination of 31P direct and cross polarization functions as a dynamic filter. In the 31P direct polarized experiment nuclei in both phases are detected, while in the 31P cross polar-ized experiment, only nuclei in the membrane phase are detected. Rates for substrate turnover, i.e. degradation of γP-, βP, αP-ATP and build-up of βP-, αP-ADP, free phosphate as side reaction, and PA are obtained, which reveal a Michaelis-Menten behavior with regard to Mg*ATP and DBG. Here Mg*ATP and DBG follow a random-equilibrium model, where every substrate can bind indepen-dently from the other substrate. Analyses of the peak integrals from educts and products of the enzymatic reaction, revealed the stoichiometry of the reaction: 1.5 ATP molecules are used to phos-phorylate one DBG molecule. The excess of ATP is attributed to the basal ATPase activity. Further-more, experiments with ATPγS, usually regarded as a non-hydrolysable ATP-analog, where carried out. Surprisingly, DAGK hydrolyzes ATPγS and also transfers the thio-phosphate group to the lipid acceptor DBG, which points to a certain degree of plasticity in the active center. A phosphorylated enzyme intermediate was not detected. These results suggest the building of a ternary complex of Mg*ATP, DBG and DAGK performing a direct-phosphoryl transfer reaction, without passing through a phosphorylated enzyme intermediate. Experiments with the transition state analog ortho-vanadate (Vi) showed a decoupling of the ATP hydrolysis activity from lipid substrate phosphorylation. This indicates a specific transfer site for the γ-phosphate group from ATP to DAG, which can be blocked by Vi.
A general disadvantage of NMR spectroscopy compared to other spectroscopic methods is its inherent low sensitivity. One possible starting point for the improvement of signal-to-noise per unit time is the reduction of the spin-lattice relaxation time of protons[209]. Usually 95 % of the experi-mental time is required for the relaxation of the 1H to equilibrium. The addition of paramagnetic species can be used to reduce the 1H T1[233]. In a comprehensive study four different paramagnetic agents were tested: Cu2+-EDTA, Cu2+-EDTA-tag, Gd3+-TTAHA and Gd3+-DOTA. The titration of these paramagnetic complexes showed the principle feasibility of this approach, but differences between the tested species exist. The most promising complex is Gd3+-DOTA which, at a concentration of 2 mM, causes a 10-time improvement of signal-to-noise ratio per unit time. This allowed measuring 2D 13C-13C correlation spectra of proteoliposomes in one tenth of the usual required experimental time (i.e. 10 hours vs. 4 days) with good signal-to-noise.
For the investigation of structural or dynamic changes in the protein upon substrate interaction with MAS NMR, the spectral properties CP efficiency and resolution of the DAGK in liposomes needed to be improved. The most critical step during sample preparation is the reconstitution of the membrane protein from detergent micelles into a membrane of synthetic lipids under detergent removal. For this procedure the important criteria are enzymatic activity, measured in a coupled ATPase assay[55], and homogeneity of the proteoliposomes, which was tested e.g. on a discontinuous sucrose step gradient. Therefore an extensive study was carried out, in which different detergents, lipids and lipid mixtures, techniques for detergent removal and different protein-to-lipid ratios were tested. A direct correlation between high ATPase activity and good resolution was not found. Moreover, active DAGK in a mixture of DMPC and cholesterol, which emulates the membrane features of a membrane containing DAG, showed the best CP efficiency and resolution.
The assignment of the protein backbone and amino acid side chains the first mandatory step towards the investigation of structural and dynamical features influencing and defining the enzymatic mechanism by MAS NMR. As the assignment procedure is very time consuming for a total protein, a special labeling scheme for DAGK was developed, which allows assigning most of the protein areas presumably involved in enzyme catalysis. The assignment of DAGK with solution NMR[132] was not transferable to the MAS NMR spectra. Most important for the assignment process were the unique pairs[335], two consecutive amino acids which only appear once in the amino acid sequence. These unique pairs served as anchor points. Five different multinuclear MAS NMR experiments (DARR, NCO, NCA, NCACX, NCOCX) were required for the sequential assignment. It was possible to assign 35 % of the total amino acid sequence with one sample and 8 experiments acquired at 850 MHz. The secondary structure analysis showed subtle differences to the DAGK assignment with solution NMR[132], which can be attributed to the different environment in lipid bilayers and detergent micelles.
Data about structural and dynamical changes under substrate interaction can reveal details about the enzymatic mechanism. Therefore changes in chemical shift in 2D heteronuclear correlation experiments in the apo-state and under substrate saturated conditions with the substrates Mg*AMP-PNP, a non-hydrolysable ATP-analog, DOG, a mixture of Mg*AMP-PNP and DOG as well as inhibited by Vi were recorded. The most significant peak changes were observed at the interface membrane-cytoplasm as well as the the N-terminal amphipathic helix. The residues revealing chemical shift perturbations correlate with conserved residues or such residues, for which importance for catalysis and/or folding could be shown in mutation studies[8]. Especially noticeable were the changes at the amino acids Asn 72, Lys 64, His 87, Tyr 86 and Asp 95.
Beside changes of the chemical shift, changes of line width or signal doubling were observable. These changes can point to a correlation with dynamic reorientations in the μs-ms time regime, which are most relevant for enzymatic processes. The protein backbone dynamics in the apo-state as well as saturated with the substrates or inhibited with Vi were investigated with a 15N-CODEX experiment, which is based on the reorientation of the CSA tensor upon dynamical changes[350]. Specific effects of the different substrates or analogs on the protein backbone dynamic were revealed complementing the structural data and the chemical shift perturbation experiments.
This work presents a biochemical, functional and structural characterization of Aquifex aeolicus F1FO ATP synthase obtained using both a native form (AAF1FO) and a heterologous form (EAF1FO) of this enzyme.
F1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane and therefore play a key cellular function. Because of their central role in supporting life, F1FO ATP synthases are ubiquitous and have been remarkably conserved throughout evolution. For their biological importance, F1FO ATP synthases have been extensively studied for many decades and many of them were characterized from both a functional and a structural standpoint. However, important properties of ATP synthases – specifically properties pertaining to their membrane embedded subunits – have yet to be determined and no structures are available to date for the intact enzyme complex. Therefore, F1FO ATP synthases are still a major focus of research worldwide. Our research group had previously reported an initial characterization of AAF1FO and had indicated that this enzyme presents unique features, i.e. a bent central stalk and a putatively heterodimeric peripheral stalk. Based on such a characterization, this enzyme revealed promising for structural and functional studies on ATP synthases and became the focus of this doctoral thesis. Two different lines of research were followed in this work.
First, the characterization of AAF1FO was extended by bioinformatic, biochemical and enzymatic analyses. The work on AAF1FO led to the identification of a new detergent that maintains a higher homogeneity and integrity of the complex, namely the detergent trans-4-(trans-4’-propylcyclohexyl)cyclohexyl-α-D-maltoside (α-PCC). The characterization of AAF1FO in this new detergent showed that AAF1FO is a proton-dependent, not a sodium ion-dependent ATP synthase and that its ATP hydrolysis mechanism needs to be triggered and activated by high temperatures, possibly inducing a conformational switch in subunit γ. Moreover, this approach suggested that AAF1FO may present unusual features in its membrane subunits, i.e. short N-terminal segments in subunits a and c with implications for the membrane insertion mechanism of these subunits.
Investigating on these unique features of A. aeolicus F1FO ATP synthase could not be done using A. aeolicus cells, because these require a harsh and dangerous environment for growth and they are inaccessible to genetic manipulations. Therefore, a second approach was pursued, in which an expression system was created to produce the enzyme in the heterologous host E. coli. This second approach was experimentally challenging, because A. aeolicus F1FO ATP synthase is a 500-kDa multimeric membrane enzyme with a complicated and still not entirely determined stoichiometry and because its encoding genes are scattered throughout A. aeolicus genome, rather than being organized in one single operon. However, an artificial operon suitable for expression was created in this work and led to the successful production of an active and fully assembled form of Aquifex aeolicus F1FO ATP synthase. Such artificial operon was created using a stepwise approach, in which we expressed and studied first individual subunits, then subcomplexes, and finally the entire F1FO ATP synthase complex. We confirmed experimentally that subunits b1 and b2 form a heterodimeric subcomplex in the E. coli membranes, which is a unique case among ATP synthases of non-photosynthetic organisms. Moreover, we determined that the b1b2 subcomplex is sufficient to recruit the soluble F1 subcomplex to the membranes, without requiring the presence of the other membrane subunits a and c. The latter subunits can be produced in our expression system only when the whole ATP synthase is expressed, but not in isolation nor in the context of smaller FO subcomplexes. These observations led us to propose a novel mechanism for the assembly of ATP synthases, in which first the F1 subcomplex attaches to the membrane via subunit b1b2, and then cring and subunits a assemble to complete the FO subcomplex. Furthermore, we could purify the heterologous ATP synthase (EAF1FO) to homogeneity by chromatography and electro-elution. Enzymatic assays showed that the purified form of EAF1FO is as active as AAF1FO. Peptide mass fingerprinting showed that EAF1FO is composed of the same subunits as AAF1FO and all soluble and membrane subunits could be identified. Finally, single-particle electron microscopy analysis revealed that the structure of EAF1FO is identical to that of AAF1FO. Therefore, the EAF1FO expression system serves as a reliable platform for investigating on properties of AAF1FO.
Specifically, in this work, EAF1FO was used to study the membrane insertion mechanism of rotary subunit c. Subunits c possess different lengths and levels of hydrophobicity across species and by analyzing their N-terminal variability, four phylogenetic groups of subunits c were distinguished (groups 1 to 4). As a member of group 2, the subunit c from A. aeolicus F1FO ATP synthase is characterized by an N-terminal segment that functions as a signal peptide with SRP recognition features, a unique case for bacterial F1FO ATP synthases. By accurately designing mutants of EAF1FO, we determined that such a signal peptide is strictly necessary for membrane insertion of subunit c and we concluded that A. aeolicus subunit c inserts into E. coli membranes using a different pathway than E. coli subunit c. Such a property may be common to other ATP synthases from extremophilic organisms, which all cluster in the same phylogenetic group.
In conclusion, the successful production of the fully assembled and active F1FO ATP synthase from A. aeolicus in E. coli reported in this work provides a novel genetic system to study A. aeolicus F1FO ATP synthase. To a broader extent, it will also serve in the future as a solid reference for designing strategies aimed at producing large multi-subunit complexes with complicated stoichiometry.
Silicon wafers such as Silicon on Insulator (SOI) and strained silicon on Insulator (sSOI) are the essential and basic materials of advanced microelectronic devices. However, they often show various kinds of crystal defects which impair the function of these devices. The most efficient method to date, for detecting such defects and for determining their density, is to delineate them by etching the wafers with a suitable etching solution and characterise them via light optical microscopy. Etch pits are formed at defect sites which are etched at a faster rate than at the perfect lattice. The standard etching solution used for SOI and sSOI is a dilute version of Secco. As Secco contains carcinogenic and environmentally hazardous chromium (VI), the use of which is or will be restricted by law in many countries, suitable chromium (VI)-free etching solutions like Organic Peracid Etches (OPE), modified Chemical Polishing Etches (CP) like CP4 mod and mixtures with organic oxidizing agents like chloranil (CA) have been developed for the successful delineation of various types of crystal defects.
However there are still nanometer-sized defects which are hard to detect or escape detection by this method. Copper decoration is a well known method to magnify these defects. It consists in applying a copper nitrate solution to the back of the SOI or sSOI wafer. On annealing, copper diffuses through the substrate and the BOX (buried oxide) to the SOI/sSOI film and on quenching to room temperature, copper precipitates as copper silicide, SiCu3, foremost at crystal defects where the lattice strain is greater than at perfect lattice sites. These silicides increase the volume in these parts of the crystal lattice and defect magnification occurs. A considerable disadvantage of this method is its tendency for artefact formation, when the copper concentration used is too high, with the copper precipitating at the film surface. The consequence is a higher density of etch pits whereby true defect etch pits cannot be differentiated from those caused by artefacts.
The aim of this thesis is to show that the processes of decorating and etching can be combined successfully to delineate all crystal defects in SOI and sSOI. An ideal result would have been to find a copper decoration procedure that decorates all existing crystal defects at a copper concentration that avoids artefact formation.
Modern computational molecular quantum chemical studies, such as the present one, typically employ a wide range of theoretical techniques. The latter are often rather complicated and one should not generally expect that an experimental scientist in the area of physical chemistry, a potential reader of this work, should be familiar with all these techniques. To simplify the reading of the Thesis and to make it self-sufficient, it is supplied with an overview of the employed theoretical methodologies (Chapter 1). The overview explains basic quantum-chemical terminology referred to throughout the Thesis, introduces theoretical foundations of the methods and outlines their properties and limitations. In Part 1.1 of Chapter 1, methods for the solution of the molecular Schrödinger equation are introduced, while in the subsequent Parts 1.2 and 1.3 methods for the solution of the electronic Schrödinger equation are presented to find the ground and excited states, respectively. Part 1.4 is dedicated to basis-set effects which are omnipresent in electronic-structure calculations. It contains a number of unusual insights and concepts proposed by the author and, thus, may be insightful also to experts in quantum chemistry.
In Chapter 2, the phenomenon of acetone-water proton exchange catalyzed by tubular as well as amorphous aggregates of calix[4]hydroquinone (CHQ) macromolecules, which has been observed previously in NMR experiments (Ref. D1D), is investigated by means of correlated quantum-chemical methods. The first part of the study (Section 2.3-2.7) considers concerted proton transfer, assisted by several initially neutral OH-groups in the hydrogen-bonded networks of CHQ aggregates. The second part of the study (Section 2.8-2.13) is dedicated to a second mechanism of proton exchange: step-wise proton transfer via formation of ionic intermediates resulting from CHQ pre-dissociation. CHQ application-specific as well as general conclusions, relevant to the main topic of the Thesis (i.e. influence of specific microsolvation on the considered proton transfer processes), are presented in Section 2.14.
The phenomenon of dual fluorescence observed in clusters of methyl 4-N,N-dimethylaminobenzoate ester (DMABME) and two water molecules in the gas phase, is studied in Chapter 3. Experimentally, the dual fluorescence was detected in experiments combining optical and ground-state ion-depletion infrared spectroscopies in ultracold molecular beams (Ref. D2D). In Section 3.3, calculated ground-state infrared spectra are presented that allow to identify the structures of those isomers, which are present in the gas-phase, as well as the structure of the isomer responsible for dual fluorescence. To further understand the reaction mechanism of dual fluorescence, excited-state potential energy surfaces of the identified isomers were computed along the relevant twisted intermolecular charge-transfer formation coordinate and the mechanism of energy dissipation in these complexes was investigated (Section 3.4-3.5) (Ref. D3D). A brief summary of the main results of this chapter and conclusions are given in Section 3.6. Finally, in Section 3.7 a complementary benchmark study of the quality of ground-state potential energy surfaces of prototypical hydrogen-bonded systems (ammonia-water and formic acid-water dimers) obtained at the level of BSSE-corrected MP2 combined with moderate basis sets, has been conducted. The quality of potential energy surfaces was tested with respect to basis-set size, level of electron correlation and anharmonicity effects and the applied methodology to identify the IR spectrum of hydrated DMABME complexes (Section 3.3) has been found to be sufficient to uniquely assign the IR spectra.
Alzheimer’s disease (AD), which was first reported more than a century ago by Alhzeimer, is one of the commonest forms of dementia which affects >30 million people globally (>8 million in Europe). The origin and pathogenesis of AD is poorly understood and there is no cure available for the disease. AD is characterized by the accumulation of senile plaques composed of amyloid beta peptides (Ab 37-43) which is formed by the gamma secretase (GS) complex by cleaving amyloid precursor protein. Therefore GS can be an attractive drug target. Since GS processes several other substrates like Notch, CD44 and Cadherins, nonspecific inhibition of GS has many side effects. Due to the lack of crystal structure of GS, which is attributed to the extreme difficulties in purifying it, molecular modeling can be useful to understand its architecture. So far only low resolution cryoEM structures of the complex has been solved which only provides a rough structure of the complex at low 12-15 A resolution Furthermore the activity of GS in vitro can be achieved by means of cell-free (CF) expression.
GS comprises catalytic subunits namely presenilins and supporting elements containing Pen-2, Aph-1 and Nicastrin. The origin of AD is hidden in the regulated intramembrnae proteolysis (RIP) which is involved in various physiological processes and also in leukemia. So far growth factors, cytokines, receptors, viral proteins, cell adhesion proteins, signal peptides and GS has been shown to undergo RIP. During RIP, the target proteins undergo extracellular shredding and intramembrane proteolysis.
This thesis is based on molecular modeling, molecular dynamics (MD) simulations, cell-free (CF) expression, mass spectrometry, NMR, crystallization, activity assay etc of the components of GS complex and G-protein coupled receptors (GPCRs).
First I validated the NMR structure of PS1 CTF in detergent micelles and lipid bilayers using coarse-grained MD simulations using MARTINI forcefield implemented in Gromacs. CTF was simulated in DPC micelles, DPPC and DLPC lipid bilayer. Starting from random configuration of detergent and lipids, micelle and lipid bilyer were formed respectively in presence of CTF and it was oriented properly to the micelle and bilyer during the simulation. Around DPC molecules formed micelle around CTF in agreement of the experimental results in which 80-85 DPC molecules are required to form micelles. The structure obtained in DPC was similar to that of NMR structure but differed in bilayer simulations showed the possibility of substrate docking in the conserved PAL motif. Simulations of CTF in implicit membrane (IMM1) in CHAMM yielded similar structure to that from coarse grained MD.
I performed cell-free expression optimization, crystallization and NMR spectroscopy of Pen-2 in various detergent micelles. Additionally Pen-2 was modeled by a combination of rosetta membrane ab-initio method, HHPred distant homology modeling and incorporating NMR constraints. The models were validated by all atom and coarse grained MD simulations both in detergent micelles and POPC/DPPC lipid bilayers using MARTINI forcefield.
GS operon consisting of all four subunits was co-expressed in CF and purified. The presence of of GS subunits after pull-down with Aph-1 was determined by western blotting (Pen-2) and mass spectrometry (Presenilin-1 and Aph-1). I also studied interactions of especially PS1 CTF, APP and NTF by docking and MD.
I also made models and interfaces of Pen-2 with PS1 NTF and checked their stability by MD simulations and compared with experimental results. The goal is to model the interfaces between GS subunits using molecular modeling approaches based on available experimental data like cross-linking, mutations and NMR structure of C-terminal fragment of PS1 and transmembrane part of APP. The obtained interfaces of GS subunits may explain its catalysis mechanism which can be exploited for novel lead design. Due to lack of crystal/NMR structure of the GS subunits except the PS1 CTF, it is not possible to predict the effect of mutations in terms of APP cleavage. So I also developed a sequence based approach based on machine learning using support vector machine to predict the effect of PS1 CTF L383 mutations in terms of Aβ40/Aβ42 ratio with 88% accuracy. Mutational data derived from the Molgen database of Presenilin 1 mutations was using for training.
GPCRs (also called 7TM receptors) form a large superfamily of membrane proteins, which can be activated by small molecules, lipids, hormones, peptides, light, pain, taste and smell etc. Although 50% of the drugs in market target GPCRs , only few are targeted therapeutically. Such wide range of targets is due to involvement of GPCRs in signaling pathways related to many diseases i.e. dementia (like Alzheimer's disease), metabolic (like diabetes) including endocrinological disorders, immunological including viral infections, cardiovascular, inflammatory, senses disorders, pain and cancer.
Cannabinoid and adrenergic receptors belong to the class A (similar to rhodopsin) GPCRs. Docking of agonists and antagonists to CB1 and CB2 cannabinoid receptors revealed the importance of a centrally located rotamer toggle switch, and its possible role in the mechanism of agonist/antagonist recognition. The switch is composed of two residues, F3.36 and W6.48, located on opposite transmembrane helices TM3 and TM6 in the central part of the membranous domain of cannabinoid receptors. The CB1 and CB2 receptor models were constructed based on the adenosine A2A receptor template. The two best scored conformations of each receptor were used for the docking procedure. In all poses (ligand-receptor conformations) characterized by the lowest ligand-receptor intermolecular energy and free energy of binding the ligand type matched the state of the rotamer toggle switch: antagonists maintained an inactive state of the switch, whereas agonists changed it. In case of agonists of β2AR, the (R,R) and (S,S) stereoisomers of fenoterol, the molecular dynamics simulations provided evidence of different binding modes while preserving the same average position of ligands in the binding site. The (S,S) isomer was much more labile in the binding site and only one stable hydrogen bond was created. Such dynamical binding modes may also be valid for ligands of cannabinoid receptors because of the hydrophobic nature of their ligand-receptor interactions. However, only very long molecular dynamics simulations could verify the validity of such binding modes and how they affect the process of activation.
Human N-formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) involved in many physiological processes, including host defense against bacterial infection and resolving inflammation. The three human FPRs (FPR1, FPR2 and FPR3) share significant sequence homology and perform their action via coupling to Gi protein. Activation of FPRs induces a variety of responses, which are dependent on the agonist, cell type, receptor subtype, and also species involved. FPRs are expressed mainly by phagocytic leukocytes. Together, these receptors bind a large number of structurally diverse groups of agonistic ligands, including N-formyl and nonformyl peptides of different composition, that chemoattract and activate phagocytes. For example, N-formyl-Met-Leu-Phe (fMLF), an FPR1 agonist, activates human phagocyte inflammatory responses, such as intracellular calcium mobilization, production of cytokines, generation of reactive oxygen species, and chemotaxis. This ligand can efficiently activate the major bactericidal neutrophil functions and it was one of the first characterized bacterial chemotactic peptides. Whereas fMLF is by far the most frequently used chemotactic peptide in studies of neutrophil functions, atomistic descriptions for fMLF-FPR1 binding mode are still scarce mainly because of the absence of a crystal structure of this receptor. Elucidating the binding modes may contribute to designing novel and more efficient non-peptide FPR1 drug candidates. Molecular modeling of FPR1, on the other hand, can provide an efficient way to reveal details of ligand binding and activation of the receptor. However, recent modelings of FPRs were confined only to bovine rhodopsin as a template.
To locate specific ligand-receptor interactions based on a more appropriate template than rhodopsin we generated the homology models of FPR1 using the crystal structure of the chemokine receptor CXCR4, which shares over 30% sequence identity with FPR1 and is located in the same γ branch of phylogenetic tree of GPCRs (rhodopsin is located in α branch). Docking and model refinement procedures were pursued afterward. Finally, 40 ns full-atom MD simulations were conducted for the Apo form as well as for complexes of fMLF (agonist) and tBocMLF (antagonist) with FPR1 in the membrane. Based on locations of the N- and C-termini of the ligand the FPR1 extracellular pocket can be divided into two zones, namely, the anchor and activation regions. The formylated M1 residue of fMLF bound to the activation region led to a series of conformational changes of conserved residues. Internal water molecules participating in extended hydrogen bond networks were found to play a crucial role in transmitting the agonist-receptor interactions. A mechanism of initial steps of the activation concurrent with ligand binding is proposed.
I accurately predicted the structure and ligand binding pose of dopamine receptor 3 (RMSD to the crystal structure: 2.13 Å) and chemokine receptor 4 (CXCR4, RMSD to the crystal structure 3.21 Å) in GPCR-Dock 2010 competition. The homology model of the dopamine receptor 3 was 8 th best overall in the competition.
Pulsed electron–electron double resonance (PELDOR) spectroscopy is a powerful tool for measuring nanometer distances in spin-labeled systems and recently is increasingly applied to membrane proteins. However, after reconstitution of labeled proteins into liposomes, spin labels often exhibit a much faster transversal relaxation (Tm) than in detergent micelles, thus limiting application of the method in lipid bilayers. In the first part of the thesis, optimization of transversal relaxation in phospholipid membranes was systematically investigated by use of spin-labeled derivatives of stearic acid and phosphatidylcholine as well as spin-labeled derivatives of the channel-forming peptide gramicidin A under the conditions typically employed for PELDOR distance measurements. Our results clearly show that dephasing due to instantaneous diffusion that depends on dipolar interaction among electron spins is an important contributor to the fast echo decay in cases of high local concentrations of spin labels in membranes. The main difference between spin labels in detergent micelles and membranes is their local concentration. Consequently, avoiding spin aggregation and suppressing instantaneous diffusion is the key step for maximizing PELDOR sensitivity in lipid membranes. Even though proton spin diffusion is an important relaxation mechanism, only in samples with low local concentrations does deuteration of acyl chains and buffer significantly prolong Tm. In these cases, values of up to 7 μs have been achieved. Furthermore, our study revealed that membrane composition and labeling position in the membrane can also affect Tm, either by promoting the segregation of spin-labeled species or by altering their exposure to matrix protons. Effects of other experimental parameters including temperature (<50 K), presence of oxygen, and cryoprotectant type are negligible under our experimental conditions.
In the second part of the thesis, inhomogeneous distribution of spin-labels in detergent micelles has been studied. A common approach in PELDOR is measuring the distance between two covalently attached spin labels in a macromolecule or singly-labeled components of an oligomer. This situation has been described as a spin-cluster. The PELDOR signal, however, does not only contain the desired dipolar coupling between the spin-labels of the molecule or cluster under study. In samples of finite concentration the dipolar coupling between the spin-labels of the randomly distributed molecules or spin-clusters also contributes significantly. In homogeneous frozen solutions or lipid vesicle membranes this second contribution can be considered to be an exponential or stretched exponential decay, respectively. In this study, it is shown that this assumption is not valid in detergent micelles. Spin-labeled fatty acids that are randomly partitioned into different detergent micelles give rise to PELDOR time traces which clearly deviate from stretched exponential decays. As a main conclusion a PELDOR signal deviating from a stretched exponential decay does not necessarily prove the observation of specific distance information on the molecule or cluster. These results are important for the interpretation of PELDOR experiments on membrane proteins or lipophilic peptides solubilized in detergent micelles or small vesicles, which often do not show pronounced dipolar oscillations in their time traces.
In the third part, PELDOR has been utilized to study the structural flexibility of the Toc34 GTPase homodimer, a preprotein receptor of the translocon of the outer envelope of chloroplasts (TOC). Toc34 belongs to GAD subfamily of G-proteins that are regulated and activated by nucleotide-dependent dimerization. However, the function of Toc34 dimerization is not yet fully understood. Previous structural investigations of the Toc34 dimer yielded only marginal structural changes in response to different nucleotide loads. PELDOR revealed a nucleotide-dependent transition of the dimer flexibility from a tight GDP to a flexible GTP-loaded state. Substrate-binding stabilizes the dimer in the transition state mimicked by GDP-AlFx, but induces an opening in the GDP or GTP-loaded state. Thus, the structural dynamics of bona fide GTPases induced by GTP hydrolysis is replaced by substrate-dependent dimer flexibility, which represents the regulatory mode for dimerizing GTPases.
In the fourth part of the thesis, conformational flexibility and relative orientation of the N-terminal POTRA domains of a cyanobacterial Omp85 from Anabaena sp. PCC 7120, a key component of the outer membrane protein assembly machinery, were investigated by PELDOR spectroscopy. Membrane proteins of the Omp85-TpsB superfamily are composed of a C-terminal β-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. It has been suggested that the N-terminal POTRA domains (P1 and P2) might have functions in substrate recognition. Molecular dynamics (MD) simulations predicted a fixed orientation for P2 and P3 and a flexible hinge between P1 and P2. The PELDOR distances measured between the P2 and P3 POTRA domains are in good agreement with the structure determined by X-ray, and compatible with the MD simulations suggesting a fixed orientation between these domains. PELDOR constraints between the P1 and P2 POTRA domains imply a rather rigid structure with a slightly different relative orientation of these domains compared with the X-ray structure. Moreover, the large mobility predicted from MD is not observed in the frozen solution. The PELDOR results further highlight the restricted relative orientation of the POTRA domains of the Omp85-TpsB proteins as a conserved characteristic feature that might be important for the processive sliding of the unfolded substrate towards the membrane.
Hepatitis C virus (HCV) assembly and production is closely linked to lipid metabolism. Indeed, lipid droplets (LD) have been shown to serve as a platform for HCV assembly. To investigate the effect of HCV on the host cell proteome, 2D-gelelectrophoresis with subsequent MALDI-TOF mass spectrometry of HCV replicating and the corresponding control cells were done. Based on this analysis, it was found out that HCV-replicating Huh7.5 cells revealed lower amounts of TIP47 (tail interacting protein of 47kD) compared to HCV-negative cells. TIP47, a cytoplasmic sorting factor, has been shown to be associated with lipid droplets. As it is known that HCV-replication and assembly takes place at the so called ”membranous web” that is composed of LDs and rearranged ER-derived membranes, it was tempting to investigate the role of TIP47 in HCV life-cycle. Western blot analysis did reveal that overexpression of TIP47 in HCV replicating Huh7.5 cells leads to decreased amounts of the HCV core protein while the levels of non-structural protein (NS)5A and intracellular HCVgenomes are increased. Moreover, in TIP47 overproducing cells higher amounts of infectious HCV particles are secreted. Vice versa, inhibition of TIP47 expression by siRNA results in a decreased level of intracellular NS5A, increased amounts of intracellular core and less infectious viral particles in the supernatant. In addition, complete silencing of TIP47 by lentiviral transduction abolishes HCV replication that can be restored by transfection of these cells with a TIP47 expression construct. It has been shown recently that apoE binds to NS5A and that this interaction plays an important role for the HCV life cycle (Benga et al., 2010). The C-terminal part of TIP47 harbours a 4 helix bundle motif and displays high homology to the N-terminus of apoE. Therefore, we investigated the interaction of NS5A and TIP47. Confocal double immunofluorescence microscopy revealed that a fraction of NS5A colocalizes with TIP47. Coimmunoprecipitation experiments and a yeast-two-hybrid screening confirmed the interaction between NS5A and TIP47 and deletion of the N-terminal-TIP47-PAT domain abolishes this interaction. From this we conclude that the TIP47-NS5A interaction is required for virus morphogenesis. Moreover, TIP47 can bind to Rab9 and this is relevant for targeting the viral particle out of the cell. In accordance to this, TIP47 was identified to be associated to the viral particle. Mutants of TIP47 that fail to bind Rab9 reveal lower amounts and a changed distribution of the HCV core protein. Furthermore, we could see that the core staining colocalizes with subcellular structures that were identified as autophagosomes using a p62-specific antibody which is a specific autophagosome-marker. Based on this, we hypothized that destruction of the Rab9 binding domain misdirects the viral particle towards the lysosomal compartment.
For the first time it could be shown that TIP47 interacts with NS5A and is associated to the viral particle, therefore plays a crucial role for the virus morphogenesis and secretion of the viral article.
Taken together, these results indicate that TIP47 is an essential cellular factor for the life cycle of HCV Abstract and might be used as target for antiviral treatment, e.g. by targeting the NS5A-TIP47 interaction, based on small molecules that mimic the NS5A-specific sequence that binds to TIP47 which might result in a competition of the TIP47/NS5A interaction.
The universal biological energy currency adenosine triphosphate (ATP) is synthesized by the F1Fo-ATP synthase in most living organisms. The overall structure and function of F-type ATPases is conserved in the different organisms. The F1Fo-ATP synthase consist of two domains; the soluble F1 complex has the subunit stoichiometry α3β3γδε and the membrane embedded Fo complex consists of subunits ab2c10-15 in its simplest form found in bacteria. F1 and Fo both function as reversible rotary motors that are connected by a central stalk (γε) and a peripheral stalk (b2δ).
For ATP synthesis, the electrochemical energy formed by a proton or sodium ion gradient is required. The ion translocation across the Fo subcomplex induces torque in the motor part of the enzyme (cnγε), which causes conformational changes in the α3β3 domain leading to ATP synthesis from ADP and inorganic phosphate (Pi) catalyzed in the β-subunits. ATP hydrolysis causes a reverse torque in the Fo subcomplex triggering uphill ion translocation from cytoplasm to periplasm, and the enzyme functions as an ion pump.
The ATP synthesis mechanism is well understood, since several high-resolution structures of F1 are available. In contrast, the ion translocation mechanism across the membrane, mediated by the Fo subcomplex, is not understood in its structural detail.
Subunit a and the c-ring form an ion pathway, but subunit b is needed to form an active ion translocation pathway in both H+- and Na+-dependent systems. Several high-resolution structures of c-rings have provided insights in the ion translocation mechanism. The different ion translocation models based on biochemical, biophysical and structural analysis are in agreement in the fact that ions are translocated through a periplasmic ion access pathway in subunit a to the middle of the membrane and there to the binding site of a c-subunit. After almost a whole rotation of the c-ring the ion returns into the a-c interface, where it can be released to the cytoplasm. In the different models the cytoplasmic access pathway has been proposed to be located in subunit a, at the a-c interface or within the c-ring. The driving force of torque generation has been proposed to be the pH gradient or membrane potential. Several biochemical studies show that a conserved arginine in helix four of subunit a (R226 in Ilyobacter tartaricus or R210 in Escherichia coli)plays a critical role in the ion translocation. The arginine has been proposed to function as an electrostatic separator between the cytoplasmic and periplasmic pathways and as a mediator of the ion exchange into the c-ring ion-binding site.
Structural data of a related enzyme (V1Vo-ATPase from Thermus thermophilus) has provided insight into the helical arrangement of the ion translocating subunits I and Lring (related to subunit a and the c-ring). These structures indicated a small interface between subunit I and the L-ring, and two four-helix bundles in the N-terminal domain of subunit I were proposed to build the periplasmic and cytoplasmic ion pathways. To comprehend the ion-translocation and torque generation mechanism in F1Fo-ATP synthase, structural data of an intact a-c complex is needed.
The goal of this work was to obtain structural data of subunit a, most preferably in a complex with the c-ring or additionally with subunit b. Therefore, a new purification procedure for the I. tartaricus Fo-subcomplex, heterologously expressed in E. coli cells, was established. The purified Fo was characterized biochemically and by Laserinduced liquid bead ion desorption mass spectrometry (LILBID-MS). These analyses showed that pure and completely assembled Fo containing all its subunits in the correct stoichiometry (ab2c11) was obtained. The purified Fo complex was stable at 4°C for several months and at room temperature in the presence of lipids for several weeks. A lipid analysis was performed by thin-layer chromatography (TLC) to investigate the qualitative lipid composition of I. tartaricus whole lipid extract and various I. tartaricus F1Fo isolates. The whole lipid extract contained PC, PG and PE lipids and probably cardiolipin. PC, PG and PE lipids were bound to wild type I. tartaricus F1Fo, whereas recombinant I. tartaricus F1Fo did not have any bound lipids, but was able to bind the synthetic lipids POPC and POPG if they were provided during the purification.
For subsequent structural studies the purified Fo was subjected to two-dimensional (2D) crystallization trials. Vesicles and sheets tightly packed with protein and crystals with a rare plane group for I. tartaricus c11 (p121) were obtained. The c-ring was visible in the CCD images, and immunogold-labeling revealed the presence of the His-tagged a-subunit in the reconstituted vesicles. Furthermore, atomic force microscopy (AFM) imaging showed protein densities next to the c-rings, which protruded less from the membrane (0.4±0.1 nm) than the c-ring (0.7±0.1 nm). These protein densities presumably belonged to subunit a.
Cryo-electronmicroscopy (cryo-EM) was used to collect data of the p121 crystals and a merged projection density map was calculated to 7.0 Å resolution. The unit cell of the crystals (81 × 252 Å) contained two asymmetric units with three c-rings in each and next to the c11-rings new prominent densities were visible. In each extra density up to 7 transmembrane helices were visible, belonging to the stator subunit a and/or subunit b. To elucidate whether there are conserved elements in the three extra densities non-crystallographic averaging was applied using a single-particle approach.
Six possible arrangements for the c-rings and the extra densities were identified and used for the averaging. The extra densities were enhanced only in one of the possible arrangements. The average showed a four-helix bundle and a fifth helix in close proximity to the c-ring. Two more helices were present in each position but their position was ambivalent. The data obtained in this work provides the first insight in the helical arrangement in the a-c interface of F1Fo-ATP synthase.
In the absence of apparent mutations, alteration of gene expression patterns represents the key mechanism by which normal cells evolve to cancer cells.
Gene expression is tightly regulated by posttranscriptional processes. Within this context, RNA-binding proteins (RBPs) represent fundamental factors, since they control mechanisms, such as mRNA-stabilization, -translation and -degradation. Human antigen R (HuR) was among the first RBPs that have been directly associated to carcinogenesis. HuR modulates the stability and translation of mRNAs which encode proteins facilitating various ‘hallmarks of cancer’, namely proliferation, evasion of growth suppression, angiogenesis, cell death resistance, invasion and metastasis. Furthermore, it is well established that tumor-promoting inflammation contributes to tumorigenesis. In this process, monocytes are attracted to the site of the tumor and educated towards a tumor-promoting macrophage phenotype. While HuR has been extensively studied in various tumor cell types, little is known about HuR in hepatocellular carcinoma (HCC). Thus, the aim of my work was to characterize the contribution of HuR to the development of cancer characteristics in HCC. I was particularly interested to investigate if HuR facilitates tumor-promoting inflammation, since a role for HuR has not been described in this context. To this end, I depleted HuR in HepG2 cells (HuR k/d) and used a co-culture model of HepG2 tumor spheroids and infiltrating monocytes to study the impact of HuR on the tumor microenvironment. I could show that depletion of HuR resulted in the reduction of cell numbers. Additionally, the expression of proliferation marker KI-67 and proto-oncogene c-Myc was reduced, supporting a proliferative role of HuR. Furthermore, exposure to cytotoxic staurosporine elevated apoptosis in HuR k/d cells compared to control cells. Concomitantly, the expression of the anti-apoptotic mediator B-cell lymphoma protein-2 (Bcl-2) was markedly reduced in the HuR k/d cells, pointing to an involvement of HuR in cell survival processes.
Accordingly, a pro-survival function of HuR was also observed in tumor spheroids, since HuR k/d spheroids exhibited a larger necrotic core region at earlier time points and showed elevated numbers of dead cells compared to control (Ctr.) spheroids. Interestingly, HuR k/d spheroids isplayed reduced numbers of infiltrated macrophages, suggesting that HuR contributes to a tumor-promoting, inflammatory microenvironment by recruiting monocytes/macrophages to the tumor site. Aiming at identifying HuR-regulated factors responsible for the recruitment of monocytes, I found reduced levels of the chemokine interleukin 8 (IL-8) in supernatants of HuR k/d spheroids, supporting a critical involvement of HuR in the chemoattraction of monocytes. Analyzing supernatants of co-cultures of macrophages and HuR k/d or Ctr. spheroids revealed additional differences in chemokine secretion patterns. Interestingly, protein levels of many chemokines were elevated in co-cultures of HuR k/d spheroids compared to control co-cultures. Albeit enhanced chemokine secretion was observed, less monocytes are recruited into HuR k/d spheroids, further underlining the necessity of HuR in cancer related monocyte/macrophage attraction and infiltration. Differences between chemokine profiles of mono- and co-cultured spheroids could be attributable to changes in spheroid-derived chemokines as a result of the crosstalk with the immune cells. Provided the chemokines originate from monocytes/macrophages, the different secretion patterns suggest that HuR contributes to the modulation of the functional phenotype of infiltrated macrophages, since the tumorenvironment is critically involved in the shaping of macrophage phenotypes. Regions of low-oxygen (hypoxia) represent another critical feature of tumors. Therefore, I next analyzed the impact of HuR on the hypoxic response. Loss of HuR attenuated hypoxia-inducible factor (HIF) 2α expression after exposure to hypoxia, while HIF-1α protein levels remained unaltered. Considering previous results of our group, showing that HIF-2α depletion (HIF-2α k/d) resulted in the enhanced expression of HIF-1α protein, I aimed to determine the involvement of HuR in the compensatory upregulation of HIF-1α protein in HIF-2α k/d cells. I could demonstrate that not only total HuR protein levels, but specifically cytoplasmic HuR was elevated in HIF-2α depleted cells pointing to enhanced HuR activity. Silencing HuR in HIF-2α deficient cells attenuated enhanced HIF-1α protein expression, thus confirming a direct role of HuR in the compensatory upregulation of HIF-1α. This as also reflected on HIF-1α target gene expression. I further investigated the mechanism underlying the compensatory HIF-1α expression in HIF-2α deficient cells. Analyzing HIF-1α mRNA expression, I excluded enhanced HIF1-α transcription and stability to account for elevated HIF-1α expression in HIF-2α k/d cells. HIF-1α promoter activity assays confirmed the mRNA data. Furthermore, HIF-1α protein half-life was not elevated in HIF-2α k/d cells compared to control cells, indicating that HIF-1α protein stability is not altered in HIF-2α k/d cells. Analysis of the association of HIF-1α with the translational machinery using polysomal fractionation finally revealed an increased istribution of HIF-1α mRNA in the heavier polysomal fractions in HIF-2α k/d cells compared to control cells. Since augmented ribosome occupancy is an indicator for more efficient translation, I propose enhanced HIF-1α translation as underlying principle of the compensatory increase in HIF-1α protein levels in HIF-2α k/d cells. In summary, my results demonstrate that HuR is critical for the development of cancer characteristics in HCC. Future work analyzing the impact of HuR on tumor-promoting inflammation, specifically macrophage attraction and activation could provide new trategies to inhibit macrophage-driven tumor progression. Furthermore, I provide evidence that HuR contributes to the hypoxic response by regulating the expression of HIF-1α and HIF-2α. Targeting single HIF-isoforms for tumor therapy should be carefully considered, because of their compensatory regulation when one α-subunit is depleted. Thus, therapeutic strategies targeting factors such as HuR that control both α-subunits and at the same time prevent compensation might be more promising.
The tumor suppressor programmed cell death 4 (Pdcd4) exerts its function by inhibiting protein translation initiation. Specifically, it displaces the scaffold protein eukaryotic initiation factor 4G (eIF4G) from its binding to the eukaryotic initiation factor 4A (eIF4A). Thereby, Pdcd4 inhibits the helicase activity of eIF4A, which is necessary for the unwinding of highly structured 5’ untranslated regions (UTRs) of messenger RNAs (mRNAs) often found in oncogenes like c-myc to make them accessible for the translation machinery and subsequent protein production. Overexpression of Pdcd4 inhibits tumorigenesis in vitro and in vivo and inversely, Pdcd4 knockout mice show enhanced tumor formation. In line, Pdcd4 is lost in various tumor types and proposed as prognostic factor in colon carcinomas. Unlike most other tumor suppressors that are rendered nonfunctional by mutations (e.g., p53), Pdcd4 loss is not attributable to mutational inactivation. It is regulated via translational repression by microRNAs and increased degradation of the protein under tumor promoting, inflammatory conditions and mitogens. Specifically, proteasomal degradation of Pdcd4 is controlled by p70 S6 Kinase (p70S6K)-mediated phosphorylation in its degron sequence (serines 67, 71 and 76). Stimulation of the PI3K-AKT-mTOR pathway by growth factors, hormones and cytokines initiates p70S6K activity. Phosphorylated Pdcd4 is subsequently recognized by the E3 ubiquitin ligase beta-transducin repeats-containing protein (β-TrCP) and marked with a polyubiquitin tail to be detected by the 26S proteasome for degradation. β-TrCP represents the substrate specific recognition subunit of the ubiquitin ligase complex responsible for protein-protein interaction with Pdcd4 as substrate for ubiquitin transfer and subsequent proteasomal disassembly.
The first part of the present work aimed at identifying novel stabilizers of the tumor suppressor Pdcd4 in a high throughput screen (HTS). As assay design, a fragment of Pdcd4 from amino acid 39 to 91, containing the phosphorylation sensitive degron sequence, was fused to a luciferase reporter gene construct. Stable expression of this Pdcd4(39-91)luciferase (Pdcd4(39-91)luc) fusion protein in HEK 293 cells served as read-out for the Pdcd4 protein amount to be detected in a high throughput compatible cell-based assay. Loss of Pdcd4(39-91)luc was induced by treatment with 12-O-
tetradecanoylphorbol-13-acetate (TPA), a phorbolester, which activates the PI3K signaling cascade leading to degradation of Pdcd4. The cut-off for hit definition was set at >50% activity in rescuing the Pdcd4(39-91)luc signal from TPA-induced degradation. Activity was calculated relative to the difference of DMSO- and TPA-treated cells (ΔDMSO-TPA = RLUDMSO-RLUTPA). Initial screening of a protein kinase inhibitor library (PKI) revealed hit substances expected to show Pdcd4 stabilizing activity by inhibition of kinases involved in Pdcd4 downregulation, e.g., the mTOR inhibitor rapamycin, the PI3K inhibitors wortmannin and LY294002 and the PKC inhibitors GF 109203X and Ro 31-8220.
The Molecular Targets Laboratory (MTL) of the National Cancer Institute (NCI) in Frederick, USA, hosts one of the largest collections of crude natural product extracts as well as a big substance libraries from pure synthetic sources. Screening of over 15 000 pure compounds and over 135 000 natural product extracts identified 46 pure and 42 extract hits as Pdcd4 stabilizers. For nine synthetic and six natural product derived compounds (after bioassay-guided fractionation), dose-dependent activities for recovering the TPA-induced Pdcd4(39-91)luc loss defined IC50s in the low micromolar range. Most importantly, these compounds were confirmed to stabilize endogenous Pdcd4 protein levels from forced degradation as well. This result proved the assay design to be highly representative for endogenous cellular mechanisms regulating Pdcd4 protein stability. The next step was to stratify the hit substances according to their likely mechanism of action to be located either up- or downstream of the p70S6K-mediated phosphorylation of Pdcd4. Therefore, phosphorylation of S6, as proto-typical p70S6K target, was analyzed and uncovered two natural derived compounds to influence p70S6K activity. Four substances did not affect p70S6K phosphorylation activity and were therefore considered to stabilize Pdcd4 by acting downstream, i.e. on the β-TrCP-mediated proteasomal degradation.
In the second part of this work, one of these compounds, namely the sesquiterpene lactone erioflorin, isolated by bioassay-guided fraction from the active extract of Eriophyllum lanatum, Asteraceae, was further characterized in detail with respect to its molecular mechanism of action. Erioflorin dose-dependently protected both Pdcd4(39-91)luc and endogenous Pdcd4 protein from TPA-induced degradation with IC50s of 1.28 and 2.64 μM, respectively. Pdcd4 stabilizing activity was maximal at 5 μM erioflorin. Up to this concentration, erioflorin was verified not to inhibit p70S6K activity. In addition, it was observed that erioflorin rescued Pdcd4(39-91)luc from both, wild type and constitutively active p70S6K-mediated downregulation. Only wild type p70S6K was inhibitable by the mTOR inhibitor rapamycin which served as an upstream acting control. To study the next section of Pdcd4 regulation, i.e. recognition by the E3 ubiquitin ligase β-TrCP, Pdcd4(39-91)luc and endogenous Pdcd4 were immunoprecipitated from whole cell extracts with the corresponding antibodies. In this key experiment, treatment with TPA increased overexpressed β-TrCP binding to both and this coimmunoprecipitation could be strongly reduced by erioflorin treatment. This result strongly pointed to an inhibitory mechanism of the β-TrCP specific binding to Pdcd4 by erioflorin. In addition, erioflorin disrupted the binding of in vitro transcribed/translated β-TrCP to Pdcd4 in an in vitro interaction assay to exclude nonspecific intracellular signals. Furthermore, polyubiquitination of Pdcd4 was decreased by erioflorin treatment as well. To clarify questions regarding specificity of erioflorin for the E3 ubiquitin ligase β-TrCP, stability of another important β-TrCP target was explored, i.e. the tumor suppressor inhibitor of kappa B alpha (IκBα). Indeed, the tumor necrosis factor alpha (TNFα)-mediated loss of IκBα could be prevented by erioflorin cotreatment. On the other hand, the E3 ubiquitin ligase von Hippel Lindau protein (pVHL) was left unaffected as its target hypoxia inducible factor 1 alpha (HIF-1α) could not be stabilized from oxygen-dependent degradation by erioflorin treatment. These results argued strongly for erioflorin being a specific inhibitor of β-TrCP-mediated protein degradation. Functional consequences of erioflorin treatment were investigated by observing its influence on the transcriptional activities of the transformation marker activator protein 1 (AP-1, an indirect downstream target of Pdcd4) and nuclear factor κB (NF-κB which is directly inhibited by IκBα). Indeed, erioflorin showed significant inhibition of AP-1 and NF-κB reporter constructs at 5 μM, a concentration for which an impact on cell viability was excluded. Finally to characterize the significance of erioflorin in a cell-based tumorigenesis assay, the highly invasive colon carcinoma cell line RKO was tested in a two dimensional migration assay. Erioflorin was discovered to significantly lower cell migration in a wound closure assay.
In conclusion, development of a high throughput compatible cell-based reporter assay successfully identified novel substances from pure synthetic and natural product derived background as potent stabilizers of the tumor suppressor Pdcd4. In addition, this work aimed at elucidating the detailed mechanism of action of the sesquiterpene lactone erioflorin from Eriophyllum lanatum, Asteraceae. Erioflorin was discovered to inhibit the E3 ubiquitin ligase β-TrCP, thereby preventing protein degradation of tumor suppressors like Pdcd4 and IκBα. This may offer the possibility to more specifically target protein degradation and generate less adverse side effects by blocking a particular E3 ubiquitin ligase compared to general proteasome inhibition.
The adaptive immune system protects against daily infections and malignant transformation. In this, the translocation of antigenic peptides by the transporter associated with antigen processing (TAP) into the ER lumen is an essential step in the antigen presentation by MHC I molecules. The heterodimeric ATP-binding cassette transporter (ABC) TAP consist of the two halftransporters TAP1 and TAP2. Each monomer contains an N-terminal transmembrane domain (TMD) and a conserved C-terminal nucleotide-binding domain (NBD). Together, the TMDs build the translocation core and the NBDs bind and hydrolyze ATP, energizing the peptide transport. TAP features an asymmetry in the two ATP-binding sites that are built of several conserved motifs. One motif is the D-loop with the consensus sequence SALD. The highly conserved aspartate of the D-loop of TAP1 reaches into the canonic ATP-binding site and contacts the Walker A motif and the H-loop of the opposite NBD, while the Asp of D-loop of TAP2 is part of the non-canonic ATP-binding site.
To examine this ABC transport complex in mechanistic detail, a purification and reconstitution procedure was established with the function of TAP being preserved. The heterodimeric TAP complex was purified via a His10-tag at TAP1 in a 1:1 ratio of the subunits. Nucleotide binding to the purified transporter was elucidated by tryptophan quenching assays and the affinity constants for MgADP and MgATP were determined to be 1.0 μM and 0.7 μM, respectevely. In addition, the TAP complex shows strict coupling between peptide binding and ATP hydrolysis, revealing no basal ATPase activity in the absence of peptides. Furthermore, TAP was reconstituted into proteoliposomes and the activity was tested by peptide transport and ATP hydrolysis. Interestingly, the kinetic parameters of the transporter in the reconstituted state are comparable to the data gained for TAP in microsomes.
To characterize the functional importance of the D-loop, D-loop mutants of either TAP1 or TAP2 were analyzed. Strikingly, TAP containing a mutated D-loop in TAP1 (D674A) shows an ATP-hydrolysis independent peptide translocation. Accordingly, the MHC I surface expression is similar to the wildtype situation. However, the same mutation in TAP2 (D638A) results in an ATPase dependent peptide transport similar to wildtype, whereas TAP containing mutations in both subunits leads to an inactive transporter. Although all D-loop mutants showed no altered peptide binding activity, the TAP1 mutant is inactive in peptide-stimulated ATPase activity. Strikingly, ATP or ADP binding is strictly required for the peptide translocation. Experiments carried out in proteoliposomes demonstrate that wildtype TAP can export peptides against their gradient when low peptide concentrations are offered. In contrast, the D674A mutant can facilitate peptide translocation along their concentration gradient in the two directions. At high peptide concentrations, TAP is trapped in a transport incompetent state induced by trans-inhibition. In conclusion, a TAP mutant that uncouples solute translocation from ATP hydrolysis was created. Since this passive substrate movement is strictly dependent on binding of ATP or ADP, an active transporter was turned into a “nucleotide-gated facilitator”.
In a cysteine cross-linking approach the conformational changes of TAP during peptide transport and the flexibility of the nucleotide binding domains were examined. Single cysteines were introduced in the D-loops of TAP1 and TAP2. Cross-linking by copper-phenantroline (CuPhe) was possible for all combinations. However, by adding ATP, ADP or peptide to the TAP complex no differences in the cross-linking efficiency were detected. By CuPhe cross-linking TAP was trapped in a conformation, in which the peptide binding site was not accessible. To complete a transport cycle, a flexibility of at least 17.8 Å of the NBDs is needed, since TAP cross-linked by CuPhe (2.0 Å) or bismaleimidoethane (BMOE, 8.0 Å) was transport inactive but when TAP was cross-linked by 1,11-bismaleimido-triethyleneglycol (BM[PEG]3, 17.8 Å) transport activity was preserved.
5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes, a group of inflammatory lipid mediators derived from arachidonic acid. Here, the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD) was investigated. In the present study, the identification of two truncated transcripts and four novel 5-LO splice variants containing premature termination codons (PTC) was reported. The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression.
RT-PCR analysis of different cell types revealed the existence of a large number of 5-LO splice variants. The most interesting splice variants were observed in BL41-E95A cells, which give a raise to novel 5-LO protein isoforms. This leads to the hypothesis of a novel regulatory mechanism in which the dimerization of 5-LO with 5-LO isoforms might regulate the 5-LO activity.
The 5-LO protein expression was reduced on translational level in UPF1 knock down cells, suggesting that UPF1 has a positive influence on 5-LO translation. Therefore, a mass spectrometry based proteomics study was started to identify compartment specific protein expression changes upon UPF1 knockdown in differentiated and undifferentiated MM6 cells. The proteomics analysis demonstrated that the knockdown of UPF1 results in numerous protein changes in the microsomal fraction (~ 21%) but not in the soluble fraction (< 1%). Western blot data confirmed the trend of the proteomics analysis. This data suggest that UPF1 is a critical gene expression regulator in a compartment specific way. During differentiation by TGFβ and calcitriol the majority of UPF1 regulated proteins was adjusted to normal level. It appears that that not only the NMD mechanism alters its composition during differentiation. Also the gene expression regulation on translational level by UPF1 seems to be also cell differentiation dependent. An interesting group of UPF1 target genes represent the downregulated proteins. qRT-PCR analysis of randomly chosen genes revealed no effect on mRNA expression upon UPF1 knockdown, suggesting that UPF1 positively influences the translation of these genes. Computational sequence analysis identified a conserved C-rich sequence which might be a hnRNP E2-binding site. hnRNP E2 has been characterized as a translational repressor in myeloid cells. Western blot analysis revealed a differentiation independent up regulation of hnRNP E2 by UPF1 knockdown. Additionally, microRNA-328 (miR-328) has been described as an RNA decoy modulating hnRNP E2 regulation. Due to this, stem loop qRT-PCR showed an up regulation of miR-328 in TGFβ and calcitriol differentiated MM6 cells. Based on this data we suggest a model in which downregulation of UPF1 increases hnRNP E2 expression, leading to translation inhibition. During differentiation, miRNA-328 is upregulated thereby competing with hnRNP E2 leading to an efficient translation
Seit einigen Jahrzehnten ist Lysozym eines der am meisten erforschten Proteine in der Literatur und wird hauptsächlich als Modell Protein zur Aufklärung der Faltungs- und Entfaltungsprozesse genutzt. Da die Frage nach Fehlfaltung und deren Verknüpfung mit neurodegenerativen Krankheiten bis zum heutigen Tag nicht vollständig geklärt ist, besteht hier ein großer Spielraum für weitere Forschungsansätze. In der vorliegenden Arbeit wurden daher zwei Modellsysteme verwendet, Hühereiweiß-Lysozym und menschliches Lysozym, jeweils in ihrem nicht-nativen ungefalteten Zustand. Diese ungefalteten Ensembles wurden mit Hilfe NMR spektroskopischer Methoden untersucht und ergaben sehr detaillierte, zum Teil auch überraschende neue Einblicke in Struktur und Dynamik der beiden Proteine und liefern somit wichtige Erkenntnisse zu Faltungs- und Aggregationsprozessen. ...
The display of foreign polypeptides and proteins on the surface of viruses or cells provides an important tool for the engineering of biomolecules and the analysis of their interactions with binding partners. The most extensively used display platform is the coat protein of the filamentous bacteriophage (Smith, 1985). Phage display libraries have often been selected for polypeptides, e.g. single chain (sc) antibodies that bind to a protein of interest, but in vivo selection could only be demonstrated for peptides so far. An alternative display platform is the retrovirus murine leukemia virus (MLV). Here, polypeptides are displayed at the N-terminus of the viral envelope glycoprotein. Proof of principle for this platform was demonstrated for protease substrate libraries, which can be selected through coupling proteolytic activation with viral infectivity (Buchholz et al., 1998). Selection of the library CX4A on living cells resulted in viruses with more than three orders of magnitude improved spreading efficiency through tumor cells (Hartl et al., 2005). Also scAb libraries have recently been displayed and selected using retroviruses (Urban et al., 2005). The library scFvlibxMo displays the repertoire of phage display preselected sc antibodies for laminin-1 binding. The retrovirus based selection process resulted in laminin-specific sc antibodies with improved expression levels in mammalian cells.
This thesis describes the in vivo (i.e. in mouse tumor models) selection of the C-X4-A and scFvlibxMo for tumor homing upon systemic delivery.
For selection of the protease substrate library C-X4-A a subcutaneous tumor was induced in SCID mice followed by three systemic injections of the library. The selection process was monitored over a period of 34 days. After the incubation period mice were sacrificed and virus load in organs and tumor determined. PCR analysis after 34 days showed that virus from the library had preferentially infected the tumor. Sequence analysis showed the selection of protease substrates with the most prominent one with a frequency of over 65%. The four most prominent protease substrate variants where reconstituted into the original viral backbone for further investigation (C-SK-A, C-HI-A, C-HM-A and C-HS-A). Interestingly, these viruses exhibited a reduced spreading capacity in vitro on HT1080 cells as compared to the C-AK-A virus, which had previously been selected on HT1080 cells. When assayed for tumor homing, however, viruses C-HI-A and C-HS-A had clearly improved in comparison to C-AK-A. Tumor tissue had been infected at rates of over 55% while virus load of extratumoral organs was very low (infection rates <0.7 for C-HS-A and <0.02 for C-HI-A). Tumor targeting capacity had thus been improved over 10-fold by the in vivo selection of the C-X4-A library.
The experimental set up for the in vivo selection of the scFvlibxMo library was performed according to that of the C-X4-A library. Fingerprint analysis of the selected viruses that infected tumor tissue resulted in the identification of seven antibody variants showing unique CDR3 sequences. Two prominent clones (M49T-A and M49T-B) were cloned back into the MoMLV genome for further analysis of the reconstituted viruses. While variant B bound laminin-1 efficiently, variant A was unable to do so, although it was selected at highest frequency (76%). Both reconstituted viruses were equally well infectious and spread through HT1080rec1 cells at a similar efficiency as MoMLV. In an in vivo competition experiment the selected viruses clearly out-competed a laminin-1 binding reference virus L36xMo for tumor homing. To understand the molecular driving forces behind the in vivo selection process the epitope of the selected scFv M49T-A was identified using a phage peptide library approach. In silico analysis led to the identification of a small group of possible antigens, including tenascin, fibronectin and collagen.
The data described in this thesis demonstrate that the retrovirus display platform is capable of allowing the in vivo selection of protease substrates and scFvs. Notably, the replication competence of the system introduced an additional level of complexity to the library. The performed in vivo selections significantly enhanced tumor tropism. Selective infection of tumor cells combined with transfer of anti-tumoral genes is an attractive strategy for cancer therapy being in focus of current research. The viruses selected in this thesis build prime candidates for targeted retrovirus based tumor therapy.
Nuclear Magnetic Resonance ("NMR") is a powerful and versatile technique relying on nuclei that posses a spin. Since its discovery more than 6 decades ago, NMR and related techniques has become a tool with innumerable applications throughout the fields of Physics, Chemistry, Biology and Medicine. Numerous Nobel Prizes have been awarded for work in the field and a multi billion dollar industry has developed on its basis.
One of NMR's major shortcomings is its inherent lack of sensitivity. Because it relies on the Boltzmann populations of spin states with a minuscule Zeeman splitting, this is particularly true for room temperature experiments.
As a result, in an enormous technological effort to enlarge the Zeeman splitting NMR magnets have been moving to higher and higher magnetic fields. However, even for proton spins possessing the largest magnetic moment of all nuclei, the degree of polarization that can be achieved in the strongest spectroscopic magnets available today (~24 T) at room temperature is merely ~ 8*(10 exp (-5)). In other words, this low polarization theoretically allows a sensitivity enhancement of 104 towards full polarization.
Since Magnetic Resonance Imaging ("MRI") is based on the same principle, it shares this problem with NMR. Furthermore, for technical and physiological reasons full body MRI tomographs do not reach the magnetic field strengths of spectroscopic NMR magnets, making this even more of an issue for MRI.
In consequence, MRI is chiefly restricted to detecting protons, while both MRI and NMR detection of 13C (or other low nuclei) under physiological conditions, i.e. low natural abundance of 13C and a low concentration of the respective substance, suffer from long acquisitions times that are necessary to obtain adequate signal to noise ratios ("SNR").
However, this drawb of NMR can be overcome. The enormous potential sensitivity increase of four orders of magnitude can - at least partially - be exploited by several hyperpolarization techniques, creating entirely new applications and fields of research.
These hyperpolarization techniques comprise chemical approaches like Parahydrogen Induced Polarization ("PHIP") or Photochemically Induced Dynamic Nuclear Polarization ("Photo-CIDNP"), as well as physical techniques like optically pumped (noble) gases13, 14 or Dynamic Nuclear Polarization ("DNP"), which will be the focus of this work. A hyperpolarized substance will render a larger signal without being physically or chemically altered in any other way. It is therefore "marked" without any marker, making it an agent free contrast agent for MRI.
DNP is a technique, in which hyperpolarization of nuclear spins is achieved by microwave (\MW") irradiation of unpaired electron spins in radicals, which are coupled to these nuclei, e.g. 1H, 13C or 15N. The electron spin population is perturbed if the microwave irradiation is resonant with the electron spin transition, which affects the polarization of hyperfine-coupled close nuclei. For large microwave power (i.e. saturating the electron spin transition) the orders of magnitude larger thermal electron spin polarization is effectively transferred to these nuclear spins in the sample. For proton spins the maximum polarization gain amounts to 660, whereas for 13C the sensitivity gain can be as large as 2600. In contrast to e.g. PHIP, which is restricted to specific reaction precursors, DNP is not limited to specific nuclei or hyperpolarization target molecules, making it a very versatile technique. DNP has been first proposed by Overhauser in 1953,15 and experimentally observed shortly thereafter in metals16 and liquids,17 both being systems with mobile electrons. In the 1960s and 70s, DNP was used as a spectroscopic tool in liquids, thoroughly mapping the effect in the low field regime. As well, several other transfer mechanisms were discovered, which are active in the solid state with localized electrons, namely the solid effect the cross effect and thermal mixing. The theory for all three of these mechanisms predicts reduced transfer efficiencies at higher magnetic fields. This fact and the lack of high frequency microwave sources to excite electron spins at magnetic field strengths above 1 T, effectively relegated DNP to a position of an interesting scientifi curiosity.
In the early 1990s, DNP came to a renaissance, when DNP was performed at high field in solid state magic angle spinning ("MAS") experiments using high power gyrotron microwave sources. This pioneering work sparked a surge of new developments and applications.
As well, this success triggered attempts to investigate also the potential of DNP in the liquid state at high magnetic fields, e.g. at 3.4 T35{38 and 9.2 T. To date, DNP can be considered one of the "hot topics" in the field of magnetic resonance, bringing about special issue in magnetic resonance journals and DNP sections on magnetic resonance conferences.
This thesis deals with the development of an in-bore liquid state DNP polarizer for MRI applications operating in ow through mode at a magnetic field strength of 1.5 T. Following this introductory chapter, the theoretical background necessary to understand and interpret the experimental results is explained in chapter 2. Subsequently, chapter 3 deals with the issue of performing liquid state DNP at high magnetic fields and its challenges. The chapter comprises a quick overview of the necessary hardware, the experimental findings for various samples and the interpretation of these findings. along with the ramifications for the aim of this work. Chapter 4 deals with the issue of increasing sensitivity and contrast in MRI, in particular by means of DNP. The chapter illustrates the development of our polarizer by presenting the hardware that was developed and demonstrating its performance under various conditions. As well, several alternative approaches are introduced and compared to our approach. Finally, chapter 5 summarizes the findings and gives an outlook on further developments.