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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.
Cardiac arrhythmias are often associated with mutations in ion channels or other proteins. To enable drug development for distinct arrhythmias, model systems are required that allow implementing patient-specific mutations. We assessed a muscular pump in Caenorhabditis elegans. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human cardiac physiology. To yield precise rhythmicity, we optically paced the pharynx using channelrhodopsin-2. We assessed pharynx pumping by extracellular recordings (electropharyngeograms--EPGs), and by a novel video-microscopy based method we developed, which allows analyzing multiple animals simultaneously. Mutations in the L-type VGCC (voltage-gated Ca(2+)-channel) EGL-19 caused prolonged pump duration, as found for analogous mutations in the Cav1.2 channel, associated with long QT syndrome. egl-19 mutations affected ability to pump at high frequency and induced arrhythmicity. The pharyngeal neurons did not influence these effects. We tested whether drugs could ameliorate arrhythmia in the optogenetically paced pharynx. The dihydropyridine analog Nemadipine A prolonged pump duration in wild type, and reduced or prolonged pump duration of distinct egl-19 alleles, thus indicating allele-specific effects. In sum, our model may allow screening of drug candidates affecting specific VGCCs mutations, and permit to better understand the effects of distinct mutations on a macroscopic level.
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.
The c-MYC proto-oncogene is a regulator of fundamental cellular processes such as cell cycle progression and apoptosis. The development of novel c-MYC inhibitors that can act by targeting the c-MYC DNA G-quadruplex at the level of transcription would provide potential insight into structure-based design of small molecules and lead to a promising arena for cancer therapy. Herein we report our finding that two simple bis-triazolylcarbazole derivatives can inhibit c-MYC transcription, possibly by stabilizing the c-MYC G-quadruplex. These compounds are prepared using a facile and modular approach based on Cu(I) catalysed azide and alkyne cycloaddition. A carbazole ligand with carboxamide side chains is found to be microenvironment-sensitive and highly selective for "turn-on" detection of c-MYC quadruplex over duplex DNA. This fluorescent probe is applicable to visualize the cellular nucleus in living cells. Interestingly, the ligand binds to c-MYC in an asymmetric fashion and selects the minor-populated conformer via conformational selection.
Mechanism of Na+-dependent citrate transport from the structure of an asymmetrical CitS dimer
(2015)
The common human pathogen Salmonella enterica takes up citrate as a nutrient via the sodium symporter SeCitS. Uniquely, our 2.5 Å x-ray structure of the SeCitS dimer shows three different conformations of the active protomer. One protomer is in the outside-facing state. Two are in different inside-facing states. All three states resolve the substrates in their respective binding environments. Together with comprehensive functional studies on reconstituted proteoliposomes, the structures explain the transport mechanism in detail. Our results indicate a six-step process, with a rigid-body 31° rotation of a helix bundle that translocates the bound substrates by 16 Å across the membrane. Similar transport mechanisms may apply to a wide variety of related and unrelated secondary transporters, including important drug targets.
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.