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Eukaryotische Zellen sind durch, aus Lipiddoppelschichten bestehenden, Membranen in Kompartimente mit unterschiedlichen Funktionen eingeteilt. Um einen Transport von Molekülen über die Membranen hinweg zu gewährleisten, werden Kanälen und Transporter benötigt. Eine Familie von Transportern sind die ATP-binding cassette (ABC) Transporter, die in allen Lebewesen, von Bakterien bis zum Menschen, vorkommen. Ein Mitglied dieser Familie ist der transporter associated with antigen processing-like (TAPL oder ABCB9). TAPL ist ein lysosomaler Polypeptidtransporter der per ATP-Hydrolyse Peptide von 6 – 59 Aminosäuren Länge vom Zytosol in das Lumen der Lysosomen transportiert. Hierbei kann TAPL, das ein Homodimer ist, in zwei funktionale Domänen geteilt werden. Der Teil des Komplexes, der für den Transport zuständig ist, wird als coreTAPL bezeichnet. Dieser beinhaltet die zytosolischen nucleotide binding domains (NBDs), die ATP binden und hydrolysieren können, und die Transmembrandomänen (TMDs), die Peptide binden und sie durch konformationelle Änderungen auf der anderen Membranseite freilassen. Die zweite Domäne ist eine N-terminale TMD, die als TMD0 bezeichnet wird. Dieser, aus vier Transmembranhelices (TMHs) bestehende Teil des Proteins, ist für die Lokalisation von TAPL in der lysosomalen Membran verantwortlich, sowie für die Interaktion mit den dort lokalisierten Membranproteinen LAMP-1 und LAMP-2. CoreTAPL ohne die TMD0s erreicht nicht die Lysosomen, sondern liegt in der Plasmamembran (PM) der Zelle vor. Die TMD0 hingegen benötigt coreTAPL nicht um korrekt in der lysosomalen Membran lokalisiert zu sein.
Die korrekte Lokalisation in der Zelle ist ein kritischer Punkt für ein Protein, um seine Funktion ausüben zu können. Die Transportprozesse vom Ort der Synthese des Proteins, dem Endoplasmatischem Reticulum (ER), zum Organell wo es seine Funktion ausüben soll, umfassen dutzende Proteine und Proteinkomplexe und ein komplexes Zusammenspiel zwischen Proteinen und den einzigartigen Lipidzusammensetzungen der Membranen verschiedener Organellen. Auf das Einfachste heruntergebrochen benötigt ein Transmembranprotein eine kurze Aminosäuresequenz auf der zytosolischen Seite, die Signalsequenz. Diese Sequenz wird von sogenannten Adapterproteinen erkannt, die wiederum andere Bestandteile der zellulären Maschinerie rekrutieren, die letztlich Vesikelbildung, Transport und Fusion mit der Zielorganelle vermitteln. Allerdings weisen nicht alle lysosomalen Transmembranproteine eine solche Signalsequenz auf, sondern besitzen unkonventionelle Zieldeterminanten, wie posttranslationale Modifikationen, oder sie interagieren mit anderen Proteinen, die wiederum die Interaktion mit den Adapterproteinen vermitteln.
Der Fokus der vorliegenden Arbeit liegt in der erfolgreichen Entwicklung von vier neuen Methoden zur Darstellung von Sulfonen und von einer neuen Methode zur Synthese von N-Aminosulfonamiden. Dabei sollen die Strukturmotive von Sulfonen und Sulfonamiden aus stabilen Startmaterialien in einer einfachen Durchführung, vorzugsweise in einer Eintopf-Synthese oder Multikomponenten-Reaktion, aufgebaut und der Reaktionsmechanismus weitestgehend experimentell aufgeklärt werden. In diesem Rahmen konnte die Lücke einer Nickel-katalysierten Darstellung von Diarylsulfonen sowohl unter thermischen als auch unter photochemischen Bedingungen gefüllt werden. Zusätzlich konnten im Bereich der SO2-Fixierung Sulfonylradikale mittels Diaryliodoniumsalzen und sichtbaren Licht erzeugt werden, die mit dem entsprechenden Quencher zum Sulfonamid oder Sulfon weiter reagieren konnten.
Protein quality control (PQC) machinery is in charge of ensuring protein homeostasis in the cell, i.e. proteostasis. Chaperones assist polypeptides throughout their maturation until functionality is achieved. This process might be disrupted in the presence of mutations or external damaging agents that affect the folding and stability of proteins. In this case, proteins can be efficiently recognized and targeted for degradation in a controlled manner. Ubiquitylation refers to the covalent attachment of one or more ubiquitin moieties to faulty proteins, thus triggering their degradation by the 26S proteasome.
More than 30% of proteins need cofactor molecules. Lack of cofactors renders proteins non-functional. We wanted to understand how the PQC deals with wild-type proteins in the absence of their cofactors. Several studies have indicated the importance of the riboflavin-derived cofactor FAD in the stability of individual flavoproteins, and hence we assumed that loss of flavin should mediate a targeted degradation of this group of proteins. Indeed, our mass spectrometry experiments showed that flavoproteome levels decreased under riboflavin starvation. The oxidoreductase NQO1 was used as a model enzyme to further investigate the mechanism of flavoproteome targeting by the PQC. We showed that cofactor loading determines ubiquitylation of NQO1 by the co-chaperone CHIP, both in vivo and in vitro. Furthermore, subtle changes in the C-terminus of NQO1 in the absence of FAD seemed to be crucial for this recognition event. ApoNQO1 interactome differed from holoNQO1. Chaperones and degradation factors were enriched on NQO1 upon cofactor withdrawal, probably to support maturation and prevent aggregation of the enzyme.
Loss of protein folding and stability, even to a small extent, can enhance the aggregating behavior of proteins. Proper loading with FAD reduced the co-aggregation of NQO1 with Aβ1-42 peptide. We assumed that the flavoproteome might represent aggregating-prone species under riboflavin deprivation. Supportingly, reversible apoNQO1 aggregates were observed in vivo in the absence of cofactor. General amyloidogenesis in vivo also increased under these conditions, apparently as a result of flavoproteome destabilization. In this context, we think that our data might have important implications considering the onset and development of conformational diseases.
This work has shed some light on the therapeutic implications of riboflavin deficiency as well. The sensitivity of melanoma cells towards the alkylating agent methyl methanesulfonate (MMS) increased under riboflavin starvation. Subsequent analyses indicated that a complex metabolic reorganization, mostly affecting proliferation and energy metabolism, occurs in response to starvation. What we suggest to call “flavoaddiction” can be understood as the dependence of melanoma cells on the flavoproteome structural and functional intactness to survive chemotherapy. Understanding this cellular reprogramming in detail might reveal new possibilities for future therapies.
Epigenetic mechanisms largely influence how genetic information on DNA level is translated into different phenotypes. DNA methylations and histone post-translational modifications make up what is referred to as "epigenetic landscape", an interconnected pattern that regulates access to genes and serves as platform for specific binding partners. The epigenetic landscape is maintained by "writers", which add the modifications, "erasers", which delete the modifications and "readers" which specifically bind modifications and mediate their location to other proteins connected to transcription. In the context of acetylations, which are the focus of this thesis, the writers are called histone acetyl transferases (HATs), the erasers are called histone deacetylases (HDACs) and the readers comprise Bromodomains (BRDs) as well as Yaf9, ENL, AF9, Taf14, Sas5 (YEATS) domains. An aberrant epigenetic landscape and mutated forms of epigenetic readers can lead to diseases including cancer and inflammatory diseases, making epigenetic reader domains attractive drug targets.
The focus of this thesis were YEATS domains and the development of inhibitors for this new class of epigenetic readers. Eleven-nineteen-leukemia protein (ENL) and ALL1-fused gene from chromosome 9 protein (AF9) are also part of the super elongation complex and are common fusion partners of mixed lineage leukemia protein (MLL) in acute myeloid leukemia (AML) (Wan et al., 2017, Erb et al., 2017). In this thesis, the first ligand-free crystal structure of ENL YEATS revealed an inherent flexibility of the Y78 side chain in the aromatic triad and two conserved water molecules. Soaking experiments led to the first co-crystal structures between a YEATS domain and small molecule inhibitors and defined prerequisites for ENL YEATS inhibitor scaffolds. The discovered inhibitory fragments had a central amide bond in common, which replaced one of the two conserved water molecules to form beta-sheet-like hydrogen bonds between the loop 6 backbone and the S58 side chain. The amide bond was flanked by two aromatic moieties, of which one stacks with H56 in the front pocket and the other interacts with the aromatic triad in the rear pocket. The development of the first chemical probe for ENL/AF9, SGC-iMLLT, show that the affinity is increased to low nanomolar levels if the rear flanking aromatic moiety forms additional hydrogen bonds with loop 6 and the side chain of E75 (Moustakim et al., 2018). In case of the probe, this is achieved with a 2-methyl-pyrrolidine-benzimidazole moiety. The probe binds with high affinity to ENL (129 nM) and AF9 (77 nM) and shows no significant affinity towards other human YEATS domains or BRDs. Target engagement was shown by fluorescence recovery after photobleaching (FRAP), cellular thermal shift assay (CETSA) and in case of AF9 also with NanoBRET. The probe changed the expression of three AML-related genes (MYC, dendrin and CD86) in MV4;11 cells, encouraging application of this probe in more AML cell lines.
This doctoral thesis deals with the structural and dynamical NMR characterization of biomolecules, covering a broad range of proteins, from small peptides to large GPCRs proteins. This work consists of two projects, which are presented in chapter II and III. Chapter II is focused on the structural screening of peptides and small proteins ranging from 14 to 71 amino acids, while chapter III describes the structure and light dynamics of the disease relevant rhodopsin G90D mutant. The main method used to investigate both types of proteins is NMR spectroscopy. Both chapters comprise individual general introduction, materials and methods, results and discussion sections, and a final conclusion paragraph.
‘Chapter I: Methodological aspects of protein NMR spectroscopy’ presents an overview of different NMR methods developed for the rapid characterization of protein structure and dynamics. Multidimensional NMR, which is routinely used in structural biology, is indispensable for protein structure determination in solution. However, detailed information with resolution at the atomic level is time consuming and requires weeks of expensive measurement time, followed by the manual data analysis. Therefore, the development of time-saving NMR techniques is highly required for screening studies of a large amount of proteins, and can be also helpful for studying unstable biomolecules, as their short lifetime often restricts the experimental procedure.
This chapter covers the two main approaches to accelerate a multidimensional NMR experiment: fast-pulsing techniques that aim to reduce the duration of an individual measurement, and non-uniform sampling technique (NUS), which was developed to reduce the overall number of increments in virtual time domains. A combination of both approaches, fast-pulsing and non-uniform sampling, allows speeding up the measurement time by 2-3 orders of magnitude. Furthermore, recently developed software called TA (targeted acquisition) combines various time-saving approaches, including fast-pulsing, non-uniform sampling and targeted acquisition. Targeted acquisition algorithm records a set of multidimensional NMR spectra in semi-interleaved incremental mode. This provides the ability to monitor the quality of the recorded spectra in real-time and therefore enables the completion of the experiments after the desired quality is achieved. Using this approach will greatly reduce the measurement time without losing important structural information. The implemented automated FLYA assignment further contributes to the rapid and simplified readout of the chemical shift assignment progress of the TA program. During this doctoral dissertation, the scientific collaboration with the TA software developer Prof. Vladislav Orekhov (Sweden) took place, and resulted in the successful establishing of this new NMR technology in the Schwalbe laboratory. TA is now routinely applied in Prof. Schwalbe group for the structure elucidation of small proteins.
‘Chapter II: Rapid NMR and biophysical characterization of small proteins’ describes the structural analysis of peptides and small proteins, which were recently identified within the framework of the Priority Program (SPP 2002). Due to technical limitations in detections of small systems and strict assumptions concerning the smallest size of the gene that can be translated, small open reading frames (sORFs) were excluded from the automated gene annotation for a very long time. Thanks to the newly developed computational and experimental approaches, the ability to identify and detect the small proteins consisting of less than approximately 70 amino acids sparked a growing scientific interest by microbiologist. In the past years, hundreds of new short protein sequences were discovered. Although some peptides were found to be involved in diverse essential biological processes, the functional elucidation of a large number of recently discovered peptides and small proteins remains a challenging task. It is well established that the structure of proteins is often linked to their function. However, the size of small constructs often restricts the possible diversity of secondary structure elements that might be adopted by a protein. Furthermore, as was shown for intrinsic discorded proteins (IDPs), the absence of a well-defined three-dimensional structure does not necessarily mean lack of function. Moreover, peptides, which are initially unstructured in the isolated form can fold in a stable structured conformation upon interaction with their biological partners. Solution state NMR spectroscopy is perfectly amenable for the structural characterization of systems of this size. It provides a rapid readout about the conformational state of small peptides unambiguously, distinguishing between folded, molten globule and unstructured conformations.
During this doctoral thesis the workflow protocol for fast screening of peptides and small proteins was established and applied to 20 candidates ranging from 14 to 71 amino acids, which were identified and selected by six microbiological groups, all members of the Priority Program on small proteins (SPP2002) funded by the German research foundation (DFG). The screening protocol includes sample preparation and biochemical characterization. Peptides containing less than 30 amino acids were synthesized by solid phase synthesis (SPPS), while small proteins containing more than 30 amino acids were heterologously expressed in E. coli.
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Die Kenntnis der Struktur von Biomolekülen und der biologischen Abläufe, in welche diese involviert sind, ist grundlegend für die Entwicklung von medizinischen Behandlungen. Im Rahmen dieser Arbeit wurden Systeme zur Untersuchung von Biomolekülen, insbesondere Proteinen, hergestellt. Im Mittelpunkt stand die Entwicklung von Materialien, welche neue Möglichkeiten zur Präparation von Proteinen zur Untersuchung derer Struktur mittels Kryo-Transmissionselektronenmikroskopie (Kryo-TEM) eröffnen. In zwei weiteren Projekten wurden biomimetische Systeme aufgebaut, welche die Oberfläche eines Biomoleküls oder biologischen Ensembles nachahmen und hierdurch deren Untersuchung ermöglichen. Hier wurden Systeme zur einfachen Nachbildung biologischer Membranen oder Proteinoberflächen betrachtet.
Eine wichtige Methode zur Untersuchung der dreidimensionalen Struktur von Biomolekülen ist die Kryo-TEM. Zur Mikroskopie werden die Biomoleküle in wenige Mikrometer großen Löchern eines amorphen Kohlenstofflochfilms mittels einer wenige Nanometer dicken Schicht aus amorphem Eis fixiert. Hierfür wird ein dünner Film einer wässrigen Probe auf den Kohlenstofflochfilm aufgebracht und gefroren. Insbesondere für Membranproteine ist die Herstellung derartiger Proben schwierig, da die Proteinpartikel zur Aggregation und Adsorption an dem Kohlenstofflochfilm neigen, wodurch keine Partikel in den Löchern des Kohlenstofffilmes auftreten, welche mikroskopiert werden können.
In dieser Arbeit wurden Materialien zur Verbesserung der Präparation von Proteinen für die Kryo-TEM entwickelt. Es wurden hierfür verschiedene biorepulsive Materialien, auch solche, welche eine spezifische Anbindung der Biomoleküle erlauben, untersucht. Da in der TEM die Probe durchstrahlt wird, eignen sich Nanometer dünne Membranen dieser Materialien als Trägermaterial für die Biomoleküle, da sie nur zu einem geringen Hintergrund führen. Zum einen wurden Nanomembranen durch die chemische Quervernetzung von Nanometer dicken Hydrogelfilmen mit verschiedenen quervernetzenden Molekülen hergestellt. Zum anderen wurden Trägerfilme, wie amorphe Kohlenstofffilme oder Kohlenstoffnanomembranen (engl. carbon nanomembranes, CNM) biorepulsiv funktionalisiert. Darüber hinaus wurde eine Nitrilotriessigsäure(NTA)-funktionalisierte Hydrogel-beschichtete Nanomembran entwickelt, welche markierte Proteine selektiv über einen His-Tag bindet.
Neben der Entwicklung von Materialien zur Untersuchung von Proteinen mittels Kryo-TEM wurden Beschichtungen hergestellt, welche die Oberfläche eines Biomoleküls oder eines Ensembles von Biomolekülen nachahmen. Diese Modelloberflächen sollten ebenfalls die Untersuchung von Eigenschaften der biologischen Systeme ermöglichen. Biologische Membranen bestehen aus einem Ensemble von Biomolekülen. Eine Vielzahl verschiedener Biomolekülen tritt in einer komplexen Anordnung in diesen dünnen Membranen auf. Es wurde versucht, strukturierte Membranen mit lokalen Variationen der physikalischen und chemischen Eigenschaften, jedoch weitaus weniger komplexen Aufbau, herzustellen. Die hergestellten Membranen mit biologisch relevanten Strukturen im Mikrometer- bis Zentimeterbereich, können nach weiterer Forschung als einfache Modellsysteme zur Nachahmung ihrer komplexen biologischen Vorbilder dienen.
In einem weiteren Projekt wurde eine Modelloberfläche für die Bindungstasche des Proteins FimH, welches eine wichtige Rolle in der bakteriellen Adhäsion spielt, entwickelt. In dem Kooperationsprojekt mit der Arbeitsgruppe Lindhorst wurde ein Modellsystem entwickelt, welches dazu dient, herauszufinden, inwiefern eine Funktionalisierung einer Aminosäurevon FimH über eine vorgeschlagenen Ligationsstrategie möglich ist. Das Modellsystem besteht aus einer biorepulsiven Hydrogel-Matrix, aus welcher die Seitenkette der Aminosäure Tyrosin in die Lösung exponiert ist. Die Substrat-katalysierte Reaktion der Aminosäuren-Seitenkette mit dem Photoschalter wurde mithilfe eines Bakterienadhäsionstests untersucht. Es konnte gezeigt werden, dass sich die vorgeschlagene Ligationsstrategie unter Berücksichtigung von Nebenreaktionen zur Modifizierung des Proteins eignet.
Es konnten vier neuartige Systeme, welche die Probenpräparation zur Untersuchung von Proteinen mittels Kryo-TEM vereinfachen, entwickelt werden. Die Ergebnisse sind von wissenschaftlicher Relevanz, da sie die Strukturbestimmung vieler Proteine deutlich vereinfachen und hierdurch beschleunigen können. Außerdem wurden biomimetische Beschichtungen entwickelt, welche entweder Proteinoberflächen oder Biomembranen nachahmen. Die entwickelten Modellsysteme erweitern das Spektrum an Möglichkeiten, Biomoleküle oder biologische Ensembles zu untersuchen.
An essential part of the animal survival strategy comprises the ability to control body movement and coordinate long-term navigational strategies, in order to maintain locomotion towards a nutrition source and stay in its vicinity. In the nematode Caenorhabditis elegans (C. elegans) this function is carried out by neuronal circuits, that vary their activity in response to diverse environmental condition.
This comprises different classes of neurons, acting together in a sensory, signaling and modulatory system to control body posture and induce behavioral responses. For this reason, one particular goal in the field of neuroscience research is to elucidate the mechanisms of how neuronal circuits integrate multiple sensory cues to navigate the environment. Aim of this study was to analyze the function of a neuronal network comprising the interneurons AVK, as well as the identification of signaling molecules, controlling body posture during food related locomotory behavior. This should be achieved by establishing optogenetic approaches, which provide a non inversive and temporally precise control of neuronal activity and drives the activation or silencing of individual neurons, to alter the neuronal basis of behavior. Animals exposed to food perform a dwelling-like behavior, characterized by a slowing of locomotion with a reduced crawling distance and an irregular movement, accompanied by a high frequency of pauses, reversals and directional changes. Upon food-removal, they initiate a local-search behavior with the same behavioral characteristics, but with a more pronounced sinusoidal movement. After a prolonged period of unsuccessful food finding, animals exhibited long runs with reduced pauses, reversals and turnings, increasing their maximal covered distance, indicated as dispersal behavior. Acute photoinhibition of AVK neurons, mediated by cell-specific expression of halorhodopsin (NpHR) caused the animals to perform a dwelling-like locomotory state with increased bending angles, as seen during local-search behavior. Thus, food-induced behavioral effects are mimicked by the optogenetic manipulation of AVK interneurons.
In this study, signaling molecules were ascertained by cell specific mRNA profiling of AVK neurons, mediating these behavioral responses. It was able to demonstrate, that flp-1, coding for a FMRFamidelike neuropeptide, is one of the genes with the highest distribution in AVK. In the absence of food, AVK neurons continuously release the FMRFamide-like neuropeptide FLP-1 to inhibit a subset of target motoneurons, leading the animals to maintain a low body curvature to promote dispersing behavior.
Conversely, if AVK was inhibited by NpHR or the presence of food, less FLP-1 was secreted to the body fluid, indicated by reduced intracellular fluorescence levels of mCherry-tagged FLP-1 proteins in the scavenger cells. The search of a FLP-1 receptor was successful by in vitro investigation on G protein-coupled receptors (GPCRs) and neuropeptide ligands, revealing NPR-6 to be activated by FLP-1 neuropeptides, but with a low potency. Expression pattern of the NPR-6 receptor indicated receptor localization in in the VC ventral cord and SMB head motoneurons, as well as in a subset of other neurons required for chemosensation and feeding. AVK interneurons are highly coupled to SMB head motoneurons, forming electrical synapses composed of the gap junction protein subunits UNC-7 and UNC-9. Elimination of SMB or gap junction genes using cell ablation and RNA interference, respectively, phenocopied effects of AVK inhibition on bending angles. Furthermore, this study was able to demonstrate that these neurons get inhibited during FLP-1 transmission to the NPR-6 receptor, which was required to mediate AVK effects on crawling behavior. Consequently, photoinhibition of AVK caused disinhibition of VC and SMB neurons, in order to enhance sinusoidal movement and to induce a local-search related locomotory behavior.
Thereby, FLP-1 neuropeptide transmission is the preferred used signaling pathway over direct gap junction coupling. Additional neuropeptides and receptors were identified to be essential downstream to AVK neurons to mediate effects on body curvature and locomotory behavior as well. The high-potency FRPR-7 receptor was shown to mediate FLP-1 peptide effects on undulatory motion during swimming in a liquid environment, rather than crawling locomotion on a solid surface. This result suggests that the receptor NPR-6 is required for FLP-1 peptide effects on bending and crawling locomotion, whereas conversely the receptor FRPR-7 is addressed by FLP-1 peptides to exclusively regulate swimming behavior. The FRPR-7 receptor is expressed in the AIM and NSM motoneurons, which are suggested to be the primary neuronal candidates mediating swimming behavior. Furthermore, this study provides evidence, that FRPR-7 acts in the DVC interneuron to control spontaneous reversal behavior, most probably by inhibitory FLP-1 signaling from the AVK neurons. Among other neuropeptides, the FMRFamide-like peptide FLP-26 binds with higher affinity to NPR-6 receptors than FLP-1 peptides. FLP-26 peptides are expressed in the SMB motoneurons, where they are able to further potentiate FLP-1 inhibitory effects by simultaneous binding to NPR-6.
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Rhabdomyosarcoma (RMS) is the most frequent pediatric soft-tissue sarcoma comprising two major subtypes – the alveolar and the embryonal rhabdomyosarcoma. The current therapeutic regime is multimodal including surgery, radiation and chemotherapy with cytostatic drugs. Although the prognosis for RMS patients has steadily improved to a 5-year overall survival rate of 70% for ERMS and 50% for ARMS, prognosis for subgroups with primary metastases or relapsed patients is still less than 25%, highlighting the need for development of new therapies for these subgroups. Since cancer cells are addicted to their cancer promoting transcriptional program, remodeling transcription by targeting bromodomain and extraterminal (BET) proteins has emerged as compelling anticancer strategy. However, in many cancer types BET inhibition was proved cytostatic but not cytotoxic emphasizing the need for combination protocols.
In this study we identify a novel synergistic interaction of the BET inhibitor JQ1 with p110α-isoform-specific Phosphoinositid-3-Kinase (PI3K) inhibitor BYL719 (Alpelisib) to induce mitochondrial apoptosis and global reallocation of BRD4 to chromatin. At first, we showed that JQ1 single treatment had cytostatic effects at nanomolar concentrations and inhibited MYC and Hedgehog (Hh) signaling in RMS known to promote proliferation of RMS. However, JQ1 single treatment barely induced cell death in RMS cells even at concentrations of up to 20 µM (< 20% cell death). Thus, we next tested combination approaches to elicit cell death. Since we previously identified synergistic cell death induction of Hh inhibition and PI3K inhibition in RMS cells we tested JQ1 in combination with the pan-PI3K/mTOR inhibitor PI-103 and the p110α-isoform-specific PI3K inhibitor BYL719. In addition, we tested JQ1 in combination with distinct HDAC inhibitors namely JNJ-26481585, SAHA (Vorinostat), MS-275 (Entinostat) and LBH-589 (Panobinostat) since the synergistic interaction of BET and HDAC inhibition has previously been described for other tumor entities.
Interestingly the synergism of cell death induction of JQ1/BYL719 co-treatment is superior to the synergism of JQ1 with pan-PI3K/mTOR inhibitor PI-103 or the tested HDAC inhibitors as confirmed by calculation of combination index. To investigate the molecular mechanisms underlying the synergy of JQ1/BYL719 co-treatment, we performed RNA-Seq and BRD4 ChIP-Seq experiments. RNA-Seq exhibited, that JQ1/BYL719 co-treatment shifted the overall balance of BCL-2 family gene expression towards apoptosis and increased gene expression of proapoptotic BMF, BCL2L11 (BIM) and PMAIP1 (NOXA) while decreasing gene expression of antiapoptotic BCL2L1 (BCL xL). These changes were verified by qRT-PCR and Western blot. Notably, BRD4 is phosphorylated upon JQ1/BYL719 co-treatment and globally reallocates BRD4 to chromatin. This BRD4 reallocation includes enrichment of BRD4 at the super-enhancer site of BMF, at the super-enhancer, typical enhancer and promoter regions of BCL2L11 (BIM) and at the PMAIP1 (NOXA) promoter, while JQ1 alone, as expected, reduces global chromatin binding of BRD4. Integration of RNA-Seq and BRD4 ChIP-Seq data underlines the transcriptional relevance of reallocated BRD4 upon JQ1/BYL719 co-treatment. Immunopreciptation studies showed, that RMS cells are initially primed to undergo mitochondrial apoptosis since BIM is constitutively bound to antiapoptotic BCL-2, BCL xL and MCL-1. JQ1/BYL719 co-treatment increased BIM expression and its neutralization of antiapoptotic BCL-2, BCL-xL and MCL-1 thereby rebalancing the ratio of pro- and antiapoptotic BCL-2 proteins in favor of apoptosis. This promotes activation of BAK and BAX resulting in caspase-dependent apoptosis. The functional relevance of proapoptotic re-balancing for the execution of JQ1/BYL719-mediated apoptosis was confirmed by individual silencing of BMF, BIM, NOXA or overexpression of BCL-2 or MCL-1, which all significantly rescued JQ1/BYL719-induced cell death. Execution of cell death by mitochondrial caspase-dependent apoptosis was veryfied by individual knockdown of BAK and BAX or caspase inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (zVAD.fmk), which all significantly rescued JQ1/BYL719-induced cell death.
In summary, combined BET and PI3Kα inhibition cooperatively induces mitochondrial apoptosis by proapoptotic re-balancing of BCL-2 family proteins accompanied by reallocation of BRD4 to transcriptional regulatory elements of BH3-only proteins.
Die Plasmamembran eukaryotischer Zellen dient als Barriere zwischen dem Inneren einer Zelle und ihrer Umgebung. Eine wichtige Aufgabe von Proteinen, die sich in der Plasmamembran befinden, besteht in der Erkennung der Umgebung, der Übermittlung dieser Informationen über die Plasmamembran in das Innere einer Zelle und der Einleitung einer zellulären Antwort. Membranrezeptoren binden Liganden, was zu ihrer Aktivierung und der Rekrutierung von intrazellulären Proteinen führt. Funktionelle Signalkomplexe werden gebildet und leiten einen Informationstransfer durch die Zellmembran ein, so dass die Expression bestimmter Gene stimuliert oder unterdrückt wird. Eine Störung der Signalinitiierung und -übertragung tritt bei vielen Krankheiten auf, so dass Membranproteine ein wichtiges Ziel in der Medikamentenentwicklung sind.
In dieser Arbeit wird die Fragestellung bearbeitet, wie der Tumornekrosefaktor-Rezeptor 1 (TNFR1) in funktionelle Komplexe in der Plasmamembran einer intakten Zelle organisiert ist. TNFR1 besitzt vier cysteinreiche Domänen (CRDs) in seiner extrazellulären Region. Die erste und von der Plasmamembran am weitesten entfernte CRD ist die Pre-Ligand Assembly Domain (PLAD). Kristallstrukturen zeigten, dass sich in einem TNFR1-Dimer zwei PLAD in unmittelbarer Nähe befinden. Crosslinking-Experimente berichteten über mehrere oligomere Zustände von TNFR1; die Ergebnisse unterschieden sich nach Art und Konzentration des Crosslinkers. In der nativen Umgebung einer intakten Zelle wurde der oligomere Zustand von TNFR1 bisher nicht bestimmt. Der kanonische Ligand für TNFR1 ist der Tumornekrosefaktor alpha (TNF), ein Homotrimer, welches in löslicher oder membrangebundener Form vorliegt. Nach der Bindung von TNF an TNFR1 bilden sich Rezeptortrimere. Diese Proteinkomplexe rekrutieren intrazellulär Proteine und bilden einen funktionellen Membrankomplex, der intrazelluläre Signalkaskaden aktiviert. Die kanonische Signalweiterleitung erfolgt durch den nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-B), welcher Zellteilung oder Entzündung induziert. TNFR1 kann auch andere Signalwege wie beispielsweise Apoptose durch einen zytosolischen Komplex und die Procaspase-8, oder Nekroptose durch das Nekrosom und die mixed lineage kinase domain-like (MLKL)-Domäne einleiten. Die Dysregulation von TNFR1 ist bei einer Vielzahl von Krankheiten zu finden. Erhöhte TNFR1-Expressiosraten treten bei acquired immune deficiency syndrome (AIDS), multipler Sklerose und verschiedenen Krebsarten auf.
In einem zweiten Projekt wurde in Zusammenarbeit mit Prof. Dr. Michael Lanzer (Heidelberg, Germany) der Expressionsgrad des Proteins VAR2CSA in membranassoziierten knobs bestimmt, welche in Erythrozyten vorkommen, die mit dem Parasiten Plasmodium falciparum infizierten wurden. VAR2CSA gehört zur Proteinfamilie des Plasmodium falciparum erythrocyte membrane protein 1 (pfEMP1). Nach einer Infektion wird VAR2CSA zur Wirtszellmembran transportiert und in knobs eingelagert. Patienten, die Sichelzellenanämie-Erythrozyten (HbAS) aufweisen, sind im Gegensatz zu Patienten mit gesunden Erythrozyten (HbAA) immun gegen Malaria. Während die beiden Erythrozytentypen eine unterschiedliche Morphologie der knobs aufweisen, blieb ihre Zusammensetzung in Bezug auf VAR2CSA bisher ungeklärt.
Das Verständnis der Proteinfunktion erfordert eine Beschreibung der molekularen Organisation funktioneller Einheiten in der zellulären Umgebung. Hierfür ist die Fluoreszenzmikroskopie eine geeignete Methode, da sie eine gezielte Markierung von Zielproteinen ermöglicht. Die hohe Sensitivität ermöglicht die Visualisierung einzelner Proteine. Eine Einschränkung in der konventionellen Fluoreszenzmikroskopie ist die Auflösungsgrenze. Strukturelle Elemente, die kleiner als etwa die halbe Anregungswellenlänge sind (für die meisten Anwendungen 200 bis 300 nm) können nicht aufgelöst werden. Die Entwicklung der hochauflösenden Fluoreszenzmikroskopie ermöglichte es, diese Auflösungsgrenze zu umgehen und eine räumliche Auflösung von wenigen Nanometern zu erreichen, was die Visualisierung und Charakterisierung einzelner Proteinkomplexe ermöglichte. Eine Art der hochauflösenden Fluoreszenzmikroskopie ist die single-molecule localization microscopy (SMLM), die auf der Detektion einzelner Fluorophore, einer genauen Bestimmung ihrer Position (Lokalisation) und der Erzeugung eines rekonstruierten Bildes unterhalb der optischen Auflösungsgrenze basiert. Da die meisten Proben in der Fluoreszenzmikroskopie eine zu hohe räumliche Dichte an Fluorophoren aufweisen, um den Nachweis von einzelnen Fluorophoren zu ermöglichen, werden Verfahren zur Kontrolle der Emission von Fluorophoren eingesetzt. Eine Möglichkeit ist der Einsatz von Fluorophoren, die optisch zwischen einem nicht-fluoreszierenden und einem fluoreszierenden Zustand geschaltet werden können, z.B. photoschaltbare fluoreszierende Proteine in photoactivated localization microscopy (PALM) oder organische Farbstoffe in (direct) stochastic optical reconstruction microscopy ((d)STORM). SMLM erreicht eine räumliche Auflösung von 20 nm, was in den meisten Fällen ausreicht, um einzelne Proteinkomplexe in einer Zelle aufzulösen. Diese räumliche Auflösung ist jedoch nicht ausreichend, um Untereinheiten innerhalb eines Proteinkomplexes zu visualisieren. Zu diesem Zweck wurde SMLM erweitert und die verfügbare kinetische Information genutzt, die bei der Detektion einzelner Fluorophore ausgelesen wird. Viele Fluorophore weisen metastabile Dunkelzustände auf, die eine Lebensdauer von bis zu Sekunden aufweisen. Diese Übergänge erscheinen als "Blinken" der Fluoreszenzemission. In Kombination mit kinetischen Modellen kann aus der Anzahl an Blink-Ereignissen die Anzahl der Fluorophore ermittelt werden. Angewendet auf hochaufgelöste Proteinkomplexe kann die Auflösungsgrenze von hochauflösender Mikroskopie umgangen werden, und die Anzahl der Protein-Untereinheiten in einem hochaufgelösten Proteincluster ermittelt werden. Hierzu wird beispielsweise das photoschaltbare fluoreszierende Protein mEos2 an ein Zielprotein funsioniert (quantitative PALM (qPALM)).
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Electron microscopy (EM) demarcates itself from other structural biology techniques by its applicability to a large range of biological objects that spans from whole cells to individual macromolecules. In single-particle cryo-EM, frozen-hydrated samples, prepared by vitrification with liquid ethane, retain macromolecules in a medium that approximates their natural aqueous environment and that, in this way, preserves high-resolution structural information. Nonetheless, the sensitivity of biological specimens to the high-energy electron beam introduces restrictions on the total dose that can be used during imaging while avoiding significant radiation damage. Consequently, the signal-to-noise ratio attained in each individual image is very low, and structures with high-resolution detail must be recovered by averaging thousands of projections in random orientations. This is achieved through the use of image processing algorithms capable of aligning and classifying particle images through the evaluation of cross-correlation functions between each particle and a reference.
In recent years, several innovations took place in the field of single-particle cryo-EM, among which the development of direct electron detectors must be highlighted. Direct electron detectors have a better detective quantum efficiency (DQE) than both photographic film and CCD cameras, and offer a fast readout, compatible with the acquisition of movie stacks. Additionally, new image processing software has become available, with more sophisticated algorithms and designed to take advantage of the specific characteristics of the movies produced with direct electron detectors. These technological advances in both hardware and software catalyzed a revolution in single-particle cryo-EM, which is now routinely used for the determination of near-atomic structures. As a result, the range of macromolecules accessible to cryo-EM has increased drastically, as targets that were unsuitable before for imaging due to their small dimensions can now be adequately visualized and refined to high-resolution.
During my doctoral work, I have used single-particle cryo-EM to structurally characterize challenging membrane proteins, with a strong emphasis on protein complexes from aerobic respiratory chains. In chapter I of this thesis, I present my results on the bovine respirasome, a mitochondrial supercomplex composed of complexes I, III and IV. Chapter II is dedicated to the analysis of the structure of alternative complex III (ACIII) from Rhodothermus marinus, a bacterial quinol:cytochrome c/HiPIP oxidoreductase unrelated to the canonical cytochrome bc1 complex (complex III). In addition, in chapter III I describe the structure of KimA, a high-affinity potassium transporter that drives the transport of its substrate by using the energy stored in the form of a proton gradient. These three membrane proteins, with molecular weights ranging from 140 kDa to 1.7 MDa, illustrate the possibilities and limitations faced in single-particle cryo-EM.
The aerobic respiratory chain is responsible for the generation of a transmembrane difference of electrochemical potential that is then used by ATP synthase for the production of ATP or for driving solute transport over the membrane. They catalyze the transfer of electrons from a substrate, such as NADH or succinate, to molecular oxygen and use the chemical energy released in these redox reactions to drive the translocation of protons, or in some cases sodium ions, to the intermembrane space in mitochondria or the periplasm in bacteria.
In mitochondria, the respiratory chain is composed of four complexes: complex I (NADH:ubiquinone oxidoreductase), complex II (succinate dehydrogenase), complex III (cytochrome bc1 complex) and complex IV (cytochrome c oxidase). While it was for a long time believed that these complexes existed as single entities in the membrane, the use of milder procedures for protein purification and analysis revealed that respiratory complexes associate into well-ordered structures, known as supercomplexes. These have been proposed to offer different structural and functional advantages that are still controversial, including substrate channeling, stabilization of individual complexes and reduction of reactive oxygen species (ROS) production. The most thoroughly studied respiratory supercomplex has been the respirasome, conserved in higher eukaryotes and composed of one copy of complex I, a complex III dimer and one complex IV. By single-particle cryo-EM analysis, I retrieved a 9 Å map of the respirasome from Bos taurus, which allowed the accurate docking of atomic models of the three component complexes. The structure shows that complex III associates to the concave side of the membrane arm of complex I, while complex IV is located between the end of the complex I hydrophobic arm and complex III. Several defined protein-protein contacts are observed between the component complexes, which are mediated predominantly by supernumerary subunits and close to the membrane surfaces. The interactions established between complex I and complex III are extensive and may support the argument that the association of complex I into supercomplexes is required for the stabilization or even the biogenesis of this complex.
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Polyketide synthases (PKSs) are large megaenzymes that occur in bacteria, fungi, and plants and produce polyketides, a class of secondary metabolites. Many polyketide natural products exhibit high biological activities e.g. as antibiotics or anti-fungal compounds. The modular architecture of assembly line PKSs makes them exciting targets for engineering approaches via the exchange of whole modules or single domains. Although many engineering attempts have been pursued over the last three decades, the resulting chimeric PKSs often exhibit decreased turnover rates or diminished product yields.
In this thesis, new approaches to engineer chimeric PKSs were explored, each targeting a different aspect of the chimeric system: First the relative contribution of protein-protein and protein-substrate recognition on the turnover of chimeric PKS was assessed, revealing the importance of protein-protein interactions between the acyl carrier protein (ACP) and the ketosynthase (KS) domain in the chain translocation step. Directed evolution experiments followed to optimize the protein-protein interaction across a chimeric interface. Additionally, different junction sites for the generation of chimeric PKSs were compared, showing the ability for recombination without interfering with the chain translocation reaction, and highlighting the use of SYNZIP domains to bridge PKS modules. To optimize chimeric PKSs even further, multipoint mutagenesis of KS domains was established, with positive effects on the activity of chimeric systems.
To support engineering attempts, several structure elucidation techniques were combined with in silico modeling to characterize the architecture of a PKS module and the domain-domain interactions within it. Preliminary results show a strong conformational flexibility of the PKS module and the great potential of these techniques to define the multitude of transient interactions in PKS modules.
Transport mechanism of a multidrug resistance protein investigated by pulsed EPR spectroscopy
(2019)
In human several diseases result from malfunctions of ATP-binding cassette (ABC) systems, which form one of the largest transport system superfamily. Many ABC exporters contain asymmetric nucleotide-binding sites (NBSs) and some of them are inhibited by the transported substrate.1 For the active transport of diverse chemically substrates across biological membranes, ABC transport complexes use the energy of ATP binding and subsequent hydrolysis. In this thesis, the heterodimeric ABC exporter TmrAB2,3 from Thermus thermophilus, a functional homolog of the human antigen translocation complex TAP, was investigated by using pulsed electron-electron double resonance (PELDOR/DEER) spectroscopy. In the presence of ATP, TmrAB exists in an equilibrium between inward- and outward-facing conformations. This equilibrium can be modulated by changing the ATP concentration, showing asymmetric behaviour in the open-to-close equilibrium between the consensus and the degenerate NBSs. At the degenerate NBS the closed conformation is more preferred and closure of one of the NBSs is sufficient to open the periplasmic gate at the transmembrane domain (TMD).3 By determining the temperature dependence of this conformational equilibrium, the thermodynamics of the energy coupling during ATP-induced conformational changes in TmrAB were investigated. The results demonstrate that ATP-binding alone drives the global conformational switching to the outward-facing state and allows the determination of the entropy and enthalpy changes for this step. With this knowledge, the Gibbs free energy of this ATP induced transition was calculated. Furthermore, an excess of substrate, meaning trans-inhibition of the transporter is resulting mechanistically in a reverse transition from the outward-facing state to an occluded conformation predominantly.3 This work unravels the central role of the reversible conformational equilibrium in the function and regulation of an ABC exporter. For the first time it is shown that the conformational thermodynamics of a large membrane protein complex can be investigated. The presented experiments give new possibilities to investigate other related medically important transporters with asymmetric NBSs or other similar protein complexes.
Many processes in living cells involve interaction and cooperation of multiple proteins to fulfill a specific function. To understand biological processes in their full complexity, it is not sufficient to only identify the molecules being involved but also to understand the kinetic aspects of a reaction. Mass spectrometry (MS) is a very powerful tool which allows to precisely identify the molecules of a reaction. Usually this is done with tandem-MS experiments for purpose of de-novo peptide sequencing. However, since this involves protein digestion, a statement of the in-vivo constitution of non-covalently bound protein complexes is not possible. In order to detect an intact protein complex it is necessary to analyze the biological system softly and in a near-native environment with native MS. Native MS allows the non-destructive analysis of these non-covalent protein complexes as well as to detect their components. However, up to now native MS does not offer a possibility to resolve the timing of the constitution of protein complexes on a fast time-scale. Therefore, the progress of reactions on fast time-scales is invisible. However, a method which delivers both types of information - identification of the components of a protein complex, as well as time-resolving their interaction - would be of high interest.
A suitable ionization technique for native MS is laser-induced liquid-bead ion desorption (LILBID). LILBID employs well-defined droplets which are irradiated by IR laser pulses to generate gas phase ions. The not-continuous, repetitive nature of ion generation offers itself to the development of a time-resolved (TR) native MS system which is able to investigate protein complexes on a fast time scale. The LILBID-droplets can serve as reaction vessels if they are levitated in an electrodynamic Paul-trap. This new setup would allow sample manipulation and MS analysis on precise and fast reaction time-scales. The first part of this dissertation presents the construction and characterization of a setup for TR-LILBID-MS.
An example for a complex biological system is the self-assembly of beta-amyloid (Aβ). This small peptide is the major component in plaques related to Alzheimer’s disease. Clinically relevant is especially the 42 amino acid peptide Aβ42 which aggregates from monomers to oligomers through to fibrils. The oligomers are the neurotoxic species in this process and thus of high interest. Nevertheless, standard analytical techniques are unable to detect those oligomers which makes MS an optimal tool to study the oligomerization process of Aβ with the focus on disease relevant oligomers. TR-LILBID-MS allows to follow the oligomerization of Aβ enabling to study molecules which influence this kinetic. Combining MS with ion-mobility spectrometry adds an additional dimension - the collision cross section - to the mass-to-charge ratio obtained from MS. Therewith structural alterations induced by ligands can be correlated to differences in the aggregation kinetic. This allows to draw a picture of the aggregation process of Aβ for the development of disease-relevant small oligomers on a molecular level.
Die in der vorliegenden Arbeit gewonnenen Erkenntnisse zur Reaktivität zweifach reduzierter 9,10-Dihydro-9,10-diboraanthracene [A]2– erweitern das Einsatzspektrum von Hauptgruppenverbindungen im Hinblick auf die Aktivierung kleiner Moleküle. Komplementär zu Übergangsmetallkomplexen und FLPs ermöglichen die Salze M2[A] (M+ = Li+, Na+, K+) die Entwicklung neuartiger Synthesestrategien. Als besondere Herausforderung gilt die Aktivierung des stabilen H2-Moleküls, dessen Bindung die Dianionen [A]2– homolytisch in einer konzertierten Reaktion spalten.
Untersuchungen zur Kinetik der H2-Addition an M2[A] stellten die Abhängigkeit dieses Reaktionsschritts vom borgebundenen Substituenten und vom Kation heraus. Eine geringe sterische Abschirmung der Boratome durch kleine borgebundene Substituenten (C≡CtBu, Me, H) begünstigt die H2-Aufnahme gegenüber großen Substituenten (pTol, Xyl, Et). Die maximale Ausbeute an M2[A-H2] wird für M+ = Li+ erst nach mehreren Tagen bei 100 °C erhalten, während einige Stunden bei nur 50 °C für die quantitative Bildung von K2[A-H2] ausreichen.
Unter den Salzen M2[A] eignet sich Li2[68] mit borgebundenen Me-Substituenten besonders gut für den Einsatz als Hydrierungskatalysator. Mit Li2[68] konnten das Imin Ph(H)C=NtBu, das terminale Alken Ph2C=CH2 und Anthracen erfolgreich im NMR-Maßstab hydriert werden (Katalysatorladung 37 mol%, THF-d8, 1 atm H2-Initialdruck, 100 °C, 16 h). Im Reaktionsautoklaven war für die Hydrierung von Ph(H)C=NtBu eine Verringerung der Katalysatorladung auf 10 mol% Li2[68] möglich (THF, 7 atm H2-Initialdruck, 100 °C, 18 h). Konkurrenzreaktionen begründen Einschränkungen in Bezug auf die Substratpalette, da M2[68] (M+ = Li+, Na+) mit elektronenarmen ungesättigten Verbindungen, die C=C-, C≡C-, C=O- oder C=N-Bindungen enthalten, [4+2]-Cycloadditionsprodukte bilden können. Die Reversibilität dieser Reaktion entscheidet, ob Li2[68] als Katalysator fungiert oder irreversibel in den Strukturen gebunden bleibt.
Vielseitiger sind die H2-Aktivierungsprodukte M2[A-H2] als H–-Donoren geeignet: Na2[68-H2] ersetzt Halogenid- durch H–-Substituenten in Bromethan, sowie in Chlorsilanen und PCl3; CO2 wird in Natriumformiat überführt. Unabhängig von der Anzahl der Chlorliganden werden die Produkte immer vollständig hydriert. Eine erneute Reduktion von 68 kann wieder Na2[68] bereitstellen, das H2 aufnimmt und Na2[68-H2] regeneriert, welches für neue H–-Abgaben zur Verfügung steht. Bei der experimentellen Umsetzung des Kreislaufs ist es wichtig, die beschriebenen Reaktionsschritte nacheinander auszuführen und jeweils nur stöchiometrische Mengen des Elektrophils zuzugeben. Bei Abweichungen vom schrittweisen Syntheseprotokoll finden formale nukleophile Substitutionen mit M2[68] statt und monoanionische Spezies entstehen, z. B. wenn Et3SiBr als Elektrophil anwesend ist.
Gegenüber CO2 zeigt Li2[68] eine hohe Reaktivität, durch die selektiv CO und [CO3]2– gebildet werden. Wie zuvor bei den H–-Transferreaktionen ermöglicht die Reduktion der Neutralverbindung 68 die Regeneration von Li2[68].
Die Dianionen [A]2– stechen unter anderen cyclischen Borverbindungen in niedrigen Oxidationsstufen heraus, da mit [A]2– nicht nur die Aktivierung von H2 oder CO2 gelang, sondern erstmalig über die Einbindung der Additionsprodukte in zum Teil katalytische Folgereaktionen berichtet werden konnte.
Multidomain enzymes, such as fatty acid synthases (FASs) or polyketide synthases (PKSs), play a crucial role in the biosynthesis of important natural products. They have a high significance in the development of new pharmaceuticals and various research approaches focus on the engineering of these proteins. For example, human type I FAS is an interesting therapeutic target. Owing to its importance in lipogenesis, upregulation of human type I FAS expression has been observed in numerous cancers. Type I FAS is also regarded as important target in antiobesity treatment. Both multidomain enzyme classes - FASs and PKSs - show high structural and functional similarities. Particularly animal type I FAS is most relevant as evolutionary precursor of the PKS family. Therefore, the well characterized FASs are suitable model proteins for the poorly characterized PKSs, to gain deeper understanding in these megasynthases.
Furthermore, fatty acids are considered to be strategically important platform chemicals accessible through sustainable microbial approaches. The recently acquired structural information on FASs provides an excellent understanding of the molecular basis of fatty acid synthesis. The specific understanding of chain-length control, the characterization of a multitude of substrate-specific thioesterases, and the emerging tools and means for metabolic engineering have fostered targeted approaches for modulating chain length. There is large interest in short-chain fatty acids, since these compounds are biotechnologically valuable platform chemicals and biofuel precursors, and attempts on the synthesis of short-chain fatty acids have been reported during the last years.
Primary focus of this thesis lies on the animal type I FASs, which exhibit large conformational variety, as seen in electron microscopy and high-speed atomic force microscopy. Conformational dynamics facilitate productive protein-protein interactions between catalytic domains within the enzyme and aid acyl carrier protein (ACP)-mediated substrate shuttling during the catalytic cycle of fatty acid biosynthesis. To gain deeper insight into the fundamental processes of ACP-mediated substrate shuttling and the underlying conformational dynamics, spectroscopic methods like Förster resonance energy transfer and electron paramagnetic resonance spectroscopy shall be employed. These spectroscopic methods demand site-specific labeling of proteins with fluorophore or spin labels, which can be accomplished with the amber codon suppression technology. Through amber codon suppression, a non-canonical amino acid (ncAA) with an orthogonal functional group is incorporated site-specifically into the protein sequence, which can be used in chemoselective reactions for protein labeling.
This thesis is at the forefront of employing the technology of amber codon suppression for addressing complex biological questions on megasynthases. The successful production of ncAA-modified FASs is challenging. With the aim of incorporating ncAAs into the multidomain 540 kDa large murine FAS, we by far exceed boundaries of documented application of amber codon suppression. Most of the proteins that are reported by Liu & Schultz in applications of amber codon suppression are in the range of 30kDa - for example the TE domain of human FAS. In the same review, the largest protein amber codon suppression was applied to is a potassium channel with roughly 80 kDa. Thus, to the best of my knowledge no protein exceeding 100 kDa has been used in amber codon suppression so far.
In this thesis a low-complex, well-plate based reporter assay is presented, based on an ACP-GFP fusion protein for fast and efficient screening of ncAA incorporation. Reliability and applicability of the reporter assay is demonstrated by successful upscaling to larger protein constructs and increased expression scale.
As outlined in this thesis, we have carefully set up methods for the modification of murine FAS and made several achievements:
(i) We have created our own toolbox with a multitude of suppressor plasmids and various orthogonal pairs. pACU and pACE plasmids are compatible for fast exchange of cassettes, and cloning procedures are optimized for modification of synthetases by site-directed mutagenesis. (ii) We have organic synthesis of several ncAAs stably running in the lab and synthesis of other ncAAs can be established when required. Therefore, extensive screening at moderate costs is possible. (iii) We have established a reporter assay for screening our own library of vectors for amber codon suppression and for optimizing incorporation of ncAAs. (iv) We successfully incorporated ncAAs into subconstructs and full-length murine FAS, and collected initial promising results for the application of these proteins in spectroscopic methods. Thus, laying the foundation for future studies to address fundamental questions of the ACP-mediated substrate shuttling and other conformational dynamics of these enzymes.
Paramyxo- and pneumoviruses include many pathogens with great relevance for human and animal health. To identify common host factors involved in the Paramyxo- and Pneumoviridae life cycle as a basis for new insights in the biology of these viruses and the development of rationally designed therapeutics, genome scale siRNA screens with wild-type measles, mumps, and respiratory syncytial viruses in A549 cells, a human lung adenocarcinoma cell line, were performed. A comparative bioinformatics analysis yielded different members of the coatomer complex I, the translation factors ABCE1 and eIF3A, and several RNA binding proteins as cellular proteins with proviral activity for all three viruses. The strongest common hit, ABCE1, an ATP-binding cassette transporter member, was chosen for further study. We found that ABCE1 supports replication of all three viruses, confirming its importance for both virus families. While viral protein kinetics showed that ABCE1 knockdown resulted in a drastic decrease of MeV protein expression, viral mRNA kinetics are not directly affected by a reduction of ABCE1.
The impact of ABCE1 on viral and global cellular translation was investigated using both 35S metabolic labelling and non radioactive fluorescent protein labelling. ABCE1 knockdown strongly inhibited the production of MeV proteins, while only modestly affecting global cellular protein synthesis and showed that ABCE1 is specifically required for efficient viral, but not general cellular, protein synthesis, indicating that paramyxoand pneumoviral mRNAs may exploit specific translation mechanisms.
In a second approach the efficacy of the small-molecule polymerase inhibitor ERDRP-0519 against MeV was assessed in squirrel monkeys. Animals treated with the drug experienced less severe clinical disease compared to untreated controls, and this effect correlated with the onset of drug treatment.
We observed a reduction of levels of PBMC-associated viremia and virus release in the upper airways, illustrating effective inhibition of virus replication by the drug treatment. ERDRP-0519 drug treatment also alleviated MeV-induced immunosuppression. In addition to providing proof-of-concept for the support of MeV eradication efforts by preventing disease and transmission with a small-molecule polymerase inhibitor, this dissertation provides a novel perspective on cellular proteins that impact the replication of MeV, MuV and HRSV and highlights the role of ABCE1 as host factor that is required for efficient paramyxo- and pneumovirus translation.
ATP-binding cassette (ABC) transporters constitute an omnipresent superfamily of integral membrane proteins, which catalyze the translocation of a multitude of chemically diverse substrates across biological membranes. In humans, ABC transporters typically act as highly promiscuous exporters, responsible for many physiological processes, multi-drug resistance, and severe diseases, such as hypercholesterolemia, lipid trafficking disorders, and immune deficiency. In all ABC transporters, ATP-driven movements within two highly conserved nucleotide-binding domains (NBDs) are coupled to conformational changes of two transmembrane domains (TMDs), which provide a framework for substrate binding and release on the opposite side of the membrane and enable the transporter to cycle between inward-facing and outward-facing orientations. Several structures of ABC transporters determined either by X-ray crystallography or single-particle electron cryo-microscopy (cryo-EM) have been reported, mostly exhibiting a variation of the inward-facing state, which highlights their dynamic behavior. However, for a complete understanding of the conformational dynamics, further structural information on intermediates is needed – especially for heterodimeric ABC transporters, which are predominant in humans and for which only limited structural information is available.
One prime example of such human heterodimeric ABC transport complexes is the transporter associated with antigen processing (TAP). TAP is a key player of the adaptive immune response, because it translocates proteasomal degradation products into the ER lumen for loading of MHC I molecules. Many functional aspects of TAP have been disclosed in recent years. However, structural information is lacking far behind and a major challenge in the field of medical relevant transporters. Recently, the heterodimeric ABC export system TmrAB (Thermus thermophilus multidrug resistance proteins A and B) was identified as an ortholog of TAP, by sharing structural homology with TAP and, intriguingly, being able to restore antigen presentation in human TAP-deficient cells. Thus, TmrAB is a biochemically well-characterized ABC exporter that can be regarded as a functional ortholog of TAP and serves as a model system for (heterodimeric) ABC export systems in general.
Thus, to illuminate the molecular basis of substrate translocation by single-particle cryo-EM, one of the main objectives of this work was the generation of stabilizing chaperones (synthetic antibodies, nanobodies, cyclic peptides) to reduce the conformational heterogeneity of TAP and TmrAB. Selected antibodies were analyzed with respect to stable complex formation, conformational trapping, and the ability to serve as alignment tools for structural studies by single-particle cryo-EM. Both antibody types were shown to form sufficiently stable complexes to serve as a rigid body for EM analyses. However, all selected antibodies bound to the inward-facing state exclusively.
Hence, for EM studies, various ligands were added to elucidate the full spectrum of conformational states during the catalytic cycle. For TAP, first attempts by negative-stain EM revealed a homogenous distribution of particles on the grid. Surprisingly, no transporter-like features were observed although various attempts were applied to increase the overall sample quality.
For TmrAB, in contrast, the complete conformational space in a native-like lipid environment under turnover conditions was mapped. Cryo-EM analysis of TmrAB incubated with ATP-Mg2+ and substrate revealed two distinct inward-facing conformations (IFwide and IFnarrow) as well as two asymmetric conformations with dimerized NBDs, which were markedly different from all previously reported structures. Here, the catalytically active site was slightly wider and contained ADP, while ATP was still bound at the catalytically-inactive site within the NBDs, demonstrating an asymmetric post-hydrolysis state. Intriguingly for the inward-facing conformations, a weak additional density close to residues M139TmrB and W297TmrB was observed in the inward-facing conformation, which displayed a higher degree of cytosolic gate opening (IFwide) indicating the presence of substrate. To verify that this density corresponds to substrate, single alanine mutations of M139TmrB and W297TmrB were introduced, leading to a strong reduction in substrate binding and transport. Since substrate release requires the opening of the extracellular gate, the absence of an outward-facing open conformation indicated that the opening must be highly transient. In order to explore the outward-facing open conformation, a cryo-EM analysis of the catalytically-inactive TmrAE523QB mutant upon incubation with ATP-Mg2+ was performed. Remarkably, within the same dataset, two different outward-facing conformations (occluded and open) were resolved, both in an ATP-bound state, which indicated that binding of ATP is sufficient to drive the large-scale conformational transition from inward-facing to outward-facing open. To explore the effect of nucleotide hydrolysis, TmrAB was trapped by vanadate. Again, two populations were observed, representing the outward-facing open and outward-facing occluded conformation.
Based on several structures of key intermediates, determined under turnover conditions or trapped in the pre-hydrolysis and hydrolysis transition state, for the first time the complete description of the ATP hydrolysis and translocation cycle of a heterodimeric ABC transport complex was elucidated in one single study. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates, fundamental and so-far hidden steps of the substrate translocation cycle of asymmetric ABC transporters were resolved and a general template for (heterodimeric) ABC exporter-catalyzed substrate translocation was provided.
Proteostasis stressors that destabilize the cellular proteome, like heat shock, trigger transcription and translational reactions leading to the accumulation of heat shock proteins, also called molecular chaperones. During stress, induction of stress response genes is prioritized so that molecular chaperones and other stress response proteins are synthesized to cope with proteome misfolding and aggregation. In order to promote the selective translation of stress-specific genes, translation of others genes that are nonessential for cell survival has to stop. Nonessential protein-coding mRNAs accumulate in the cytosol with the associated proteins to form granular structures called stress granules (SG). These membrane-less organelles are thought to be involved in cell survival, mRNA stabilization and mRNA triage. They were proposed to form via the liquid-liquid phase separation which can be triggered by the high local concentration of RNA-binding proteins. mRNAs were long thought to simply play a scaffolding role by bringing RNA-binding proteins together and allowing their concentration and local aggregation. Recently, the active role of mRNAs in the SG assembly became apparent, too. For example, the spontaneous assembly of total yeast RNA into granules was observed, and these RNA granules showed a large overlap with SG transcriptome. Furthermore, cytosolic mRNAs can be released from polyribosomes under stress and be exposed to the cytosolic contents as free mRNAs. It has been suggested that this massive increase of free mRNA in the cytosol might overload the capacities of RNA-stabilizing proteins. The remaining free mRNA molecules would then become exposed to misfolded and aggregation-prone proteins and trigger granulation.
We investigated the role of free mRNAs in different stress conditions during the early and chronic phases of stress response and explored their involvement in SGs assembly and amlyoidogenesis. We identified and studied the interactome of a free mRNA probe incubated with heat shocked cell lysate by means of quantitative mass spectrometry. Proteomics analysis allowed us to identify 79 interactors of free mRNA. Among these interactors, we focused on the translation initiation factor eIF2α and on the RNA methyltransferase TRMT6/61A. Both interactions were verified biochemically, which confirmed that the association is enhanced in heat shocked lysate. In vitro reconstitution showed that free mRNA and TRMT6 interact directly. Ex vivo pulldowns revealed that eIF2α and TRMT6/61A interact under stress conditions and that this interaction is RNA-dependent.
TRMT6/61A is a tRNA methytransferase responsible for the methylation of the adenosine 58 at the position 1 producing m1A. However, also mRNAs have been recently found to be methylated by TRMT6/61A. Our bioinformatics analyses revealed that significantly more mRNAs enriched in SG contain the motif for methylation than SG-depleted mRNAs. We hypothesized that m1A methylation of mRNAs could constitute a tag for the mRNAs targeting to SGs. TRMT61A knock-down (KD) cell lines were generated using the CRISPR-Cas9 technique. In TRMT61A KD cells, m1A was significantly reduced on mRNAs, which correlated with an increased sensitivity of the cells to proteostasis stress. KD cells also showed defects in SG assembly. In heat shocked cells, an m1A motif-containing mRNA recovered better after returning to normal temperature than a control mRNA with mutated motif. In addition, we could isolate SGs and analyze their m1A and m6A content by mass spectrometry. While m6A content in SG mRNAs was very similar to cytosolic mRNAs, m1A was almost 8 times enriched in SGs. Thus, we could confirm experimentally the results of the bioinformatics analysis and directly support the hypothesis that m1A is a tag to direct mRNAs for sequestration. Finally, we compared amyloidogenesis in wild-type and TRMT61A KD cell lines. Cells with reduced levels of TRMT61A demonstrated an increased accumulation of transfected Aβ and an impaired aggregate clearance. Various assays led us to conclude that the lack of m1A deposition on mRNAs enhanced RNA co-aggregation with amyloids.
Based on our results, we propose a model explaining the fate of free mRNA during proteostasis stress. Upon polysome disassembly, free mRNA is released and becomes free to interact with other proteins, including the methyltransferase TRMT6/61A. TRMT6/61A methylates the freed mRNAs containing the cognate motif. The m1A tag then targets mRNAs to SGs promoting sequestration. Upon stress release, SGs disassemble, thus releasing rescued mRNAs which could now reenter translation and support cell recovery. On the other hand, non-sequestered mRNAs increasingly co-aggregate with aggregating proteins. Thus, deficiency of the N1-adenine methylation of mRNAs due to the lack of TRMT6/61A increases the amount of unpacked mRNAs. The deposition of m1A on mRNAs could then be a way to protect them during exposure to stress, to limit their co-aggregation with misfolded proteins and to allow a faster recovery upon stress release.
Enolase is a glycolytic enzyme, which catalyzes the inter-conversion of 2-phosphoglycerate to phosphoenolpyruvate. Altered expression of this enzyme is frequently observed in cancer and accounts for the Warburg effect, an adaptive response of tumor cells to hypoxia. In addition to its catalytic function, ENO-1 exhibits other activities, which strongly depend on its cellular and extracellular localization. For example, the association of ENO-1 with mitochondria membrane was found to be important for the stability of the mitochondrial membrane, and ENO-1 sequestration on the cell surface was crucial for plasmin-mediated pericellular proteolysis. The latter activity of ENO-1 enables many pathogens but also immune and cancer cells to invade the tissue, leading further to infection, inflammation or metastasis formation. The ability of ENO-1 to conduct so many diverse processes is reflected by its contribution to a high number of pathologies, including type 2 diabetes, cardiovascular hypertrophy, fungal and bacterial infections, cancer, systemic lupus erythematosus, hepatic fibrosis, Alzheimer’s disease, rheumatoid arthritis, and systemic sclerosis. These unexpected non-catalytic functions of ENO-1 and their contributions to diseases are the subjects of this review.
Die Lebensfunktion der Zelle beruht unter anderem auf der Funktion und Wechselwirkung der Nukleinsäuren DNA (2’-Desoxyribonukleinsäure) und RNA (Ribonukleinsäure). Mit Hilfe von PDS (engl. ’pulsed dipolare spectroscopy’)-Techniken, basierend auf der EPR (engl. ’electron paramagnetic resonance’)-Spektroskopie, können Abstände in einem Bereich von 2-10 nm zwischen zwei markierten Positionen einer Nukleinsäure bestimmt werden. Daneben kann mit der Abstandsverteilung auf die Flexibilität des Moleküls geschlossen werden. Durch PDS-Messungen eröffnet sich die Möglichkeit, Bewegungen und Zustandsänderungen zu untersuchen. Die Messungen beruhen auf der dipolaren Kopplung von Radikalen (Spinlabel). Da die gemessenen dipolaren Kopplungen eine anisotrope Wechselwirkung sind, können an starren Systemen neben den Abstandsinformationen auch die Orientierungen der beiden Spinlabel zueinander bestimmt werden. Diese zusätzliche Information ermöglicht es, mittels orientierungsselektiver PDS-Messungen noch genauer die Geometrie und Flexibilität des Systems zu untersuchen. Klassischerweise werden alle Messungen mit der Doppelfrequenztechnik PELDOR (engl. ’pulsed electron-electron double resonance’) durchgeführt. Einzelfrequenzmethoden basieren dagegen auf Breitbandanregung, die mit den technischen Gegebenheiten l nge nicht möglich war. Eine solche Sequenz ist 2D-SIFTER.ImmRahmen dieser Arbeit von PELDOR ausgehende, weiterentwickelte Simulationsprozedur etabliert. Eine große Herausforderung ist die eindeutige Interpretation der sensitiven orientierungsselektiven PELDOR-Messungen. Sie mittels MD (Moleküldynamik)-Simulationen zu beschreiben war bisher nur qualitativ möglich. Allerdings wurden mehrere neue Kraftfelder publiziert. Mit einem quantitativen Vergleich mit orientierungsselektiven PELDOR-Daten kann sichergestellt werden, dass die Flexibilität des Systems durch Kraftfelder richtig beschrieben ist. PELDOR-Zeitspuren, gemessen bei Raumtemperatur und 50 K, unterscheiden sich besonders in ihrer Dämpfung. Der physikalische Unterschied beider Messungen konnte durch MD-Simulationen qualitativ nachvollzogen worden. Eine Schwierigkeit für speziell orientierungsselektive PELDOR-Messungen ist die aufwendige Synthese von mit dem starren Ç-Label markierten Nukleinsäuren. Als Alternative wurde in der Sigurdsson-Gruppe das halbstarre IMU-Label entwickelt. Die Analyse der orientierungsselektiven Daten ergab ein klares Bild der Dynamik dieses Labels. Ein weiterer interessanter Spinlabel ist der `G. Dieser Label ist nicht kovalent gebunden, sondern interkaliert in eine Stelle der Nukleinsäure, in der eine Guanin- Base fehlt. MD-Simulationen im quantitativen Vergleich mit orientierungsselektiven PELDOR-Messungen an verschiedenen Magnetfeldern haben eine hohe Übereinstimmung. Dabei konnte gezeigt werden, dass der Label, interkaliert in eine dsDNA, flippen kann, was zu einer Ausmittelung der Anisotropie führt, allerdings zu keiner Verbreiterung der Abstandsverteilung. Dagegen wird in der dsRNA dieses Flippen um die Einfachbindung sterisch gehindert, so dass neben dem Abstand auch die Orientierung des Labels bestimmt werden kann. Kurze dsRNA-Bausteine tendieren dazu, Oligomere zu bilden, was zu Multispineffekten führte. Zusätzlich beeinflusst diese Aggregation die Dynamik der einzelnen RNAs. Daher musste dieses ’end-to-end’-Stacking verhindert werden. Eine Nukleobasean einem Ende der dsRNA führt zu einer Dimerisierung, während eine Nukleobase an beiden Seiten dieses Stacking vollständig verhindert. Messungen mit unterschiedlichen Salzkonzentrationen konnten zusätzlich zeigen, dass die Interaktion zweier dsRNAs bei höheren Salzkonzentrationen zunimmt.
The three major autoimmune diseases (ADs) of the liver are primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and autoimmune hepatitis (AIH). All of those diseases show an aggressive immune reaction resulting in the destruction of liver tissue and finally to the development of hepatic fibrosis.
PSC is an autoimmune mediated disease of unknown etiology. It is characterized by inflammation of intra- and extrahepatic bile ducts. The progressive destruction of the bile ducts can lead to liver cirrhosis and finally to liver failure. Clinical signs for PSC are increased alkaline phosphatase (AP) and gamma glutamyltransferase (GGT) levels, presence of perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) and bile ducts with characteristic strictures and dilations of the biliary tree as well as onion skin fibrosis surrounding the damaged bile ducts. Currently, there is no established treatment for PSC patients. The administration of ursodeoxycholic acid (UDCA) is being use as a therapy. However, it merely serves a symptomatic treatment to reduce serum AP and GGT as well as the formation of gallstones. In the advanced stage of PSC, liver transplantation is the last therapeutic option. Mdr2-/- mice are an excepted mouse model for human PSC. Such mice show lymphocytes infiltration into the liver, bile duct lesions, as well as the presence of the typical onion skin-like pericholangitis and periductal fibrosis.
AIH is a rare chronic autoimmune disease of the liver that results from the loss of self-tolerance to hepatocytes and leads to destruction of the hepatic parenchyma with the onset of cirrhosis. Clinical signs for AIH are elevated alanine aminotransferase (ALT) and aspartate transaminase (AST) levels, hypergammaglobulinemia and different types of autoantibodies. In addition, interphase hepatitis with lymphocytic and plasmacellular infiltrates in the periportal field are characteristic for AIH. Two different subtypes of AIH exist and depending on their autoantibody profile they can be distinguished into AIH type 1 which is characterized by the presence of anti-nuclear (ANA) and/or anti-smooth muscular (SMA) autoantibodies, and AIH type 2 showing liver/kidney microsomal autoantibodies (LKM-1). LKM-1 recognizes the major autoantigen, the 2D6 isoform of the cytochrome P450 enzyme family (CYP2D6). One mouse model for AIH is the CYP2D6 model in which the injection of Ad-2D6 leads to a breakdown of the immune tolerance by the destruction of hepatocytes.
There are some patients with autoimmune diseases of the liver who have both cholestatic and hepatic liver enzymes and histological features suggestive of two different liver diseases. These patients are diagnosed with an overlap syndrome (OS).
In my thesis I generated an animal model with characteristics of both diseases, which would mimic features of human PSC-AIH OS. Mdr2-/- mice which spontaneously develop PSC were infected with Ad-2D6 to trigger the autoimmune-driven hepatic injury. Pathogenesis of PSC-AIH OS mice was compared to mice with solitary PSC or AIH. Naïve FVB wild type mice have been used as healthy controls. The characterization of the PSC-AIH OS model was done by analyzing serological parameters like ALT, AP, different antibodies like pANCA, LKM-1 like CYP2D6 and total IgG. Additionally, fibrosis and cholangitis were analyzed by immunohistochemistry and Western blotting. Moreover, cellular infiltrations of CD4+ and CD8+ T cells, dendritic cells (DCs), monocytes/macrophages and neutrophils were determined with immunohistochemistry. Finally, the overall immune balance in the liver and the frequency of CYP specific T cells were analyzed via flow cytometry. Our new mouse model indeed represents the characteristics of both PSC and AIH and mimics features of the human PSC-AIH OS. It allows studying the development of a PSC-AIH OS and how the two overlapping diseases are influencing one another. In a second approach I wanted to induce CYP2D6-specific tolerance in AIH mice. Therefore, I tried four different approaches, namely intranasal peptide administration, injection of tolerogenic DCs, antigen-coupled splenocytes, and Ag-coupled nanoparticles (NP) and evaluated their potential to induce CYP2D6 specific Treg with the capacity to prevent AIH in mice. Unfortunately, the intranasal peptide administration and also the injection of tolerogenic DCs did not increase the amount of CYP2D6 specific Treg which would lead to a reduction of the frequency of inflammatory T cells. Surprisingly, the injection of antigen-coupled splenocytes showed the opposite effect characterized by a very strong cytokine secretion in the tolerized mice. The use of NPs led to an increase in CYP2D6 specific Treg as well as in decrease in the frequency of inflammatory T cells and finally has the potential for a therapeutic approach.
In summary, the generated PSC-AIH OS model represents many clinical signs which can also be observed in PSC-AIH OS patients. This model can be used to study the etiology of this overlap syndrome and further to test potential therapeutic approaches. The different immune tolerance induction pathways which I tried in the AIH model show that NPs have to potential to induce immune tolerance but this approach has to be refined and the outcome has to be characterized in more detail.
Die CXCR4-CXCL12-Signalachse gilt als eines der bislang am besten studierten Signalsysteme in der Hämatopoese. Allerdings stammt unser Wissen über diesen kritischen Signalweg maßgeblich aus subtraktiven Studien, wie z.B. knock-out Modellen oder pharmakologischer Inaktivierung. Zwar können aus diesen Modellen wichtige Erkenntnisse über die physiologische Rolle dieses Signalwegs abgeleitet werden, aber dennoch bleiben einige Phänomene ungeklärt. So konnte gezeigt werden, dass es sowohl bei CXCR4-Defizienz als auch bei Patienten mit dem WHIM-Syndrom (ausgelöst durch eine überaktive CXCR4-Mutante) zu einer ausgeprägten B-Zellaplasie kommt. Dies scheint intuitiv nicht vereinbar. Daher wurde in der vorliegenden Arbeit ein Modell mit einer überaktiven CXCR4- Mutante (CXCR41013/1013) hinsichtlich der (un)reifen Hämatopoese systematisch untersucht.
Zunächst wurden hämatopoetische Stamm- und Vorläuferzellen (HSPC) hinsichtlich der aberranten CXCR4-Signalweiterleitung ex vivo analysiert. Die CXCR4-Überaktivierung konnte sowohl in frühen Effekten nach Aktivierung des Rezeptors (F-Aktinpolymerisierung, Aktivierung des MAPK- Signalweges), als auch in späten, zellfunktionellen Effekten (Migrationsassay) nachgewiesen werden. Die veränderte CXCR4 Signalintegration hatte auch bereits in der Homöostase organismische Konsequenzen im Mausmodell. So konnte eine massiv vergrößerte HSPC-Population in der Milz von CXCR41013/1013-Tieren detektiert werden, im Sinne einer extramedullären Hämatopoese. Knochenmarks-HSPC aus CXCR41013/1013-Tiere zeigten ein massiv eingeschränktes (serielles) Repopulationspotenzial. Kombiniert mit der oben genannten ausgeprägten extramedullären Hämatopoese in diesen Tiere interpretieren wir diese Beobachtung als starken Hinweis auf eine dysfunktionelle Interaktion der Stammzellen mit der hämatopoetischen Stammzellnische im Knochenmark. In diesem Zusammenhang besonders interessant ist die Tatsache, dass auch ein Kompetitorknochenmark das Überleben einer Sekundärtransplantation nicht sichert. Dabei ist zu diskutieren, ob dieser Effekt durch eine effizientere Besetzung von Stammzellnischen durch CXCR41013/1013-Zellen, eine Akkumulation von CXCL12 in der Knochenmarkflüssigkeit (siehe unten) oder eventuell sogar ein vesikelabhänginger Transport von mutiertem CXCR4 in Kompetitorzellen ausgelöst wird. Ein weiteres Merkmal dieser Dysfunktion könnte ebenfalls die gezeigte Akkumulation von CXCL12 in der Knochenmarkflüssigkeit von CXCR41013/1013-Tiere darstellen. Diese Akkumulation könnte die Suppression co-transplantierter wildtypischer Hämatopoese sowie die verminderte Effizienz der G-CSF-induzierten Stammzellmobilisierung funktionell erklären. Zusätzlich konnte gezeigt werden, dass die Mobilisierung von Stammzellen aus dem Knochenmark durch einen CXCR4- Inhibitor in CXCR41013/1013-Tieren ebenfalls erheblich hinter der in Wildtypmäusen zurückbleibt.
Analog zu Patienten mit WHIM-Syndrom zeichnen sich CXCR41013/1013-Mäuse weiterhin durch eine ausgeprägte Leukopenie, insbesondere durch einen schweren B-Zell-Mangel, aus. Aus diesem Grund wurde die B-Lymphopoese und humorale Immunfunktion genauer analysiert. Eine grundsätzliche humorale Immunkompetenz von CXCR41013/1013-Tieren konnte nachgewiesen werden, jedoch ist die B-Memory-Funktion erheblich eingeschränkt. Durchflusszytometrisch und funktionell konnte eine reduzierte preB/pro-B Population im Knochenmark bei einer gleichzeitig vergrößerten preB/pro-B Population in der Milz (vgl. extramedulläre Hämatopoese) nachgewiesen werden. Ebenfalls konnten wir in dieser Zellpopulation eine stark erhöhte CXCR4-Oberflächenexpression im Vergleich zu wildtypischen Zellen nachweisen. Da diese unreifen B-Zellen keine verstärke Apoptoserate aufweisen, gehen wir derzeit davon aus, dass der Differenzierungsstopp nicht durch selektiven Zelltod, sondern durch aberrante Retention der preB/proB-Zellen in einer primitiven B-Vorläufer- Nische im Knochenmark zustande kommt, beziehungsweise durch eine gestörte Migration in differenzierende Nischen im Knochenmark. Alternativ könnte die Überdosis CXCR4-Signal differenzierenden Signalen entgegenstehen. Beide Hypothesen können das eingangs erwähnte Paradoxon bezüglich einer B-Zellaplasie in CXCR4-defizienten und CXCR4-überaktiven Zellen hinreichend erklären.
As central component of the peptide loading complex, the ABC transporter TAP is a key player in the adaptive immune response. By recognizing and translocating antigenic peptides derived from proteasomal degradation into the ER lumen it connects the processing of harmful intruders and the marking of an infected cell for elimination. This work focused mainly on the interaction between TAP and one of its viral inhibitors. Of the five known TAP inhibitors, ICP47 is the only one that is not anchored in the ER membrane and has a nonomolar affinity to TAP. These properties and its specific architecture make it an interesting protein engineering tool that can be used in a variety of ways to generate functionally arrested TAP complexes. Different lengths of ICP47 were chosen to map the optimal distance between the binding pocket and the N-terminal elbow helix of either TAP1 or TAP2. I demonstrated that the interaction of fused ICP47 with coreTAP inhibits antigen presentation via MHC I. Interestingly, the loss of MHC I surface expression only depended on the presence of the active domain and not on the length of the fused ICP47 fragments. Summarizing it can be said that TAP complexes containing an intact active domain of ICP47 successfully suppressed MHC I surface expression. Considering the MHC I surface expression in the use of free ICP47 fragments it was revealed that the active domain may not be sufficient. All free constructs, except the one that contains exclusively the active domain (1-35), were able to fully arrest peptide translocation, while the fragment 1-35 partially restored MHC I surface expression. This was the first evidence suggesting that more residues might be present in the ICP47 sequence that contribute to the interaction with TAP.
Further characterization of the ICP47-coreTAP fusion complexes comprised the determination of their thermostability and melting temperatures. The ICP47-coreTAP fusion complexes revealed a preferred orientation for ICP47. The ICP47(1-65) fragment led to a stable complex only if fused to TAP2, highlighting an interesting asymmetry at the TAP1/TAP2 interface, which suggests a shorter distance of the C-terminus of the stabilizing region to the elbow helix of TAP2 than of TAP1. The shorter fragments 1-35 and 1-50, and the ICP47 linker fragments, which inhibited, but did not trigger any thermostabilizing effects on TAP, revealed a second hint for the presence of other residues important for the ICP47/TAP interaction. To define the thermostability in more detail, the melting temperature of complexes with fused or freely bound ICP47 fragments was determined. Short fused fragments of ICP47 (residues 1-35 or 1-50) did not fully stabilize the TAP complex. Only ICP47 fragments longer than residues 1-50 raised the melting temperature to the full extent and led to a completely stabilized complex, suggesting that the critical melting temperature, which determines whether a complex is fully stabilized or not, is about 44-45°C. By comparing different ICP47 proteins from the herpesviral clade, I further noticed that the 21 residues following the active domain are highly conserved. The residues in this region were exchanged by glycines and alanines to study their impact on the thermostabilization of TAP. I demonstrated that several charged residues, an alanine rich, and a proline rich sequence were mainly responsible for the preservation of high melting temperatures. In summary, these findings reveal a dual inhibition mechanism of ICP47. While the active domain of ICP47 is wedged at the TAP1/2 interface and arrests the complex in an open-inward facing conformation, the highly conserved C-terminal region stabilizes the ICP47/TAP interaction and generates a thermostabilized TAP complex.
The second part of this thesis deals with two alternative expression and stabilization strategies for coreTAP, designed to provide a 1:1 ratio of TAP subunits during protein biosynthesis. Different glycine-serine (GS) linkers and a self cleaving 2A site were im- plemented into the TAP sequence and used for comparison with the classical coreTAP. Despite their functionality in antigen translocation, the utilization of GS linkers proved to be unsuitable due to low expression and scarce purification efficiency caused by the unfeasible orthogonal purification. In contrast, the use of a 2A site allowed orthogonal His10- and SBP-tag purification and yielded comparable amounts to the classical coreTAP. However, the ICP47/coreTAP interaction appeared to be hampered by the modified N-terminus of ICP47, due to the cleavage process.
The third and last part of this work deals with the Thermus thermophilus ABC trans- porter TmrAB, which was identified to be part of the same ABC subfamily as TAP. The structure of TmrAB is similar to that of coreTAP and includes a TMD and an NBD for each subunit. In comparison to TAP, TmrAB has a broader substrate range, but it can transport peptides, which are also transported by TAP. Since the natural substrate, and thus the actual function, of TmrAB has not yet been identified, it is counted among the multidrug resistance ABC transporters, from where it also takes its name. In this work, the question was investigated whether TmrAB can be utilized as a TAP substitute. To compare the function of TmrAB and TAP in a natural cell environment, the N-terminal domains of the TAP subunits called TMD0s were fused to the TmrAB subunits and subsequently expressed as different combinations. I found that especially the hybrid complexes containing a TMD0 of TAP2 were functional in terms of MHC I surface expression. Furthermore, TmrAB with TMD0 co-localized prevalently with the ER marker PDI while complexes without TMD0 did not co-localize. Interestingly, the analysis of the interaction with components of the PLC revealed that interaction with tapasin could only occur when a TMD0 was present. In turn, calreticulin, MHC I, and ERp57 were bound, regardless of the presence of a TMD0. It is remarkable that a bacterial protein, sharing only 27-30% sequence identity with human TAP is able to take over a key function of our adaptive immune system. Yet, TmrAB originates from a hyperthermophilic bacterium and may have assembly and folding difficulties that the human cell seeks to overcome by recruiting chaperones like calreticulin and ERp57. Although further experiments will be necessary to analyze the interaction of TmrAB with the PLC components in more detail, TmrAB appears to be homologous to coreTAP, not only in terms of sequence and structure, but also in terms of function.
Supersaturating formulations are widely used to improve the oral bioavailability of poorly soluble drugs. However, supersaturated solutions are thermodynamically unstable and such formulations often must include a precipitation inhibitor (PI) to sustain the increased concentrations to ensure that sufficient absorption will take place from the gastrointestinal tract. Recent advances in understanding the importance of drug-polymer interaction for successful precipitation inhibition have been encouraging. However, there still exists a gap in how this newfound understanding can be applied to improve the efficiency of PI screening and selection, which is still largely carried out with trial and error-based approaches. The aim of this study was to demonstrate how drug-polymer mixing enthalpy, calculated with the Conductor like Screening Model for Real Solvents (COSMO-RS), can be used as a parameter to select the most efficient precipitation inhibitors, and thus realise the most successful supersaturating formulations. This approach was tested for three different Biopharmaceutical Classification System (BCS) II compounds: dipyridamole, fenofibrate and glibenclamide, formulated with the supersaturating formulation, mesoporous silica. For all three compounds, precipitation was evident in mesoporous silica formulations without a precipitation inhibitor. Of the nine precipitation inhibitors studied, there was a strong positive correlation between the drug-polymer mixing enthalpy and the overall formulation performance, as measured by the area under the concentration-time curve in in vitro dissolution experiments. The data suggest that a rank-order based approach using calculated drug-polymer mixing enthalpy can be reliably used to select precipitation inhibitors for a more focused screening. Such an approach improves efficiency of precipitation inhibitor selection, whilst also improving the likelihood that the most optimal formulation will be realised.
Objectives: The objective of this review is to provide an overview of PK/PD models, focusing on drug-specific PK/PD models and highlighting their value-added in drug development and regulatory decision-making.
Key findings: Many PK/PD models, with varying degrees of complexity and physiological understanding, have been developed to evaluate the safety and efficacy of drug products. In special populations (e.g. pediatrics), in cases where there is genetic polymorphism and in other instances where therapeutic outcomes are not well described solely by PK metrics, the implementation of PK/PD models is crucial to assure the desired clinical outcome. Since dissociation between the pharmacokinetic and pharmacodynamic profiles is often observed, it is proposed that physiologically-based pharmacokinetic (PBPK) and PK/PD models be given more weight by regulatory authorities when assessing the therapeutic equivalence of drug products.
Summary: Modeling and simulation approaches already play an important role in drug development. While slowly moving away from “one-size fits all” PK methodologies to assess therapeutic outcomes, further work is required to increase confidence in PK/PD models in translatability and prediction of various clinical scenarios to encourage more widespread implementation in regulatory decision-making.
Die Steuerung biochemischer Prozesse oder die Verbesserung von Materialien erfordert zunächst ein tiefgründiges Verständnis über die zugrundeliegenden Systeme. Zur Untersuchung eignet sich Licht als ideales Werkzeug, da hiermit nützliche Informationen über die chemische Struktur, ihre Eigenschaften sowie den zusammenhängenden, schnellen Reaktionsabläufen erhalten werden können. Um die Aufklärung zu erleichtern können kleine, chemische Verbindungen eingeführt werden, welche beispielsweise ein Fluoreszenzmarker, eine photolabile Schutzgruppe oder eine photoschaltbare Verbindung sein können. Von jeweils einem Vertreter dieser Moleküle wurden unterschiedliche Studien durchgeführt, dessen Ergebnisse in dieser Arbeit in insgesamt drei Projekten zusammengefasst werden.
Zunächst wurde die Funktionalität der Helikase RhlB untersucht, die der Familie der DEAD-Box Proteine zugeordnet wird, und RNA-Duplexe in ihre Einzelstränge entwindet. Als RNA-Modellduplex diente JM2h, an dem ein RNA-Einzelstrang fluoreszenzmarkiert war (M2AP6). Die Einführung dieses Markers ermöglichte die Durchführung von statischen Fluoreszenzmessungen sowie von Mischexperimenten, die mit Hilfe der stopped-flow-Technik durchgeführt wurden. In den einleitenden Studien wurde die Helikase weggelassen, wodurch der Fokus auf den Fluoreszenzeigenschaften der RNA gelegt wurde. Die Ergebnisse hierzu zeigten, dass die Fluoreszenzintensität des Einzelstrangs durch Zugabe des komplementären Strangs deutlich abnimmt, wobei das Minimum bei einem äquimolaren Verhältnis erreicht wird. Die dazugehörigen stopped-flow-Messungen zeigten eine Beschleunigung der Hybridisierungsreaktion, wenn höhere Konzentrationen des Gegenstrangs in der Lösung vorhanden waren. Nach anschließender Zugabe der Helikase zur Lösung wurde ein Anstieg der Fluoreszenzintensität erwartet, der vom separierten Einzelstrang M2AP6 herrühren sollte. Dieser Anstieg wurde jedoch erst nach weiterer Zugabe von ATP beobachtet, der auf eine ATP-Abhängigkeit der Entwindungsreaktion von RhlB hindeutet. Diese Abhängigkeit wurde auch bereits für andere Helikasen der DEAD-Box Familie entdeckt. Die korrekte Funktionalität sowie die ATP-Abhängigkeit wurden in stopped-flow-Messungen verfiziert, bei denen der Fluoreszenzanstieg auch zeitaufgelöst betrachtet werden konnte. Für die spektralen Korrekturen der Fluoreszenzspektren wurde ein selbstgeschriebenes MATLAB-Programm namens FluCY verwendet (engl.: Fluorescence Correction & Quantum yield), welches eine schnelle und fehlerfreie Verarbeitung des Datensatzes ermöglichte.
Die zwei im folgenden beschriebenen Projekte handeln von photoaktivierbaren Molekülen. Zum einen photolabile Verbindungen, welche die Funktion z.B. eines Biomoleküls durch eine chemische Modifikation deaktivieren können. Durch eine lichtinduzierte Reaktion kommt es zur Abspaltung der Modifikation und die Funktion ist wiederhergestellt. In dieser Arbeit wurden verschiedene photolabile Schutzgruppen untersucht, die denselben Chromophor BIST (BIsStyryl-Thiophen) tragen. Durch die Einführung dieses Chromophors absorbierten sämtliche untersuchte Verbindungen sehr effizient sichtbares Licht (epsilon(445)=55.700 M^(-1) cm^(-1)), wodurch der photoinduzierte Bindungsbruch mit Wellenlängen durchgeführt werden, die bei einer biologischen Anwendungen keinen Schaden an der Zelle anrichten würden. Hieraufhin wurden in statischen und zeitaufgelösten Absorptionsmessungen Teilschritte der Freisetzungsreaktion untersucht, indem nach Photoanregung die Absorptionsänderungen auf verschiedenen Zeitskalen analysiert wurden. Die ultraschnelle Dynamik im Piko- bis Nanosekundenbereich (10^(-12)-10^(-9) s) wird durch eine spektral breite, positive Absorptionsänderng dominiert. Diese impliziert, dass die Deaktivierung über den Triplettpfad abläuft, der die vergleichsweise niedrigen Freisetzungsausbeuten erklärt (phi(u) < 5). Aufgrund des hohen Extinktionskoeffizienten reichen dennoch bereits niedrige Strahlungsdosen aus, um eine Freisetzung zu initiieren. Der geschwindigkeitsbestimmende Schritt dieser Reaktion ist dem Zerfall des aci-nitro Intermediats zugeordnet. Für ein sekundäres Amin, welches mit BIST geschützt wurde, ist eine Lebensdauer des Intermediats von 71 µs gefunden worden.
In einigen Fällen ist es erwünscht, eine vorliegende Aktivität nicht nur ein-, sondern auch ausschalten zu können, wofür photochrome Verbindungen (oder Photoschalter) verwendet werden. Die in dieser Arbeit untersuchte Verbindung ceCAM ist ein Alken-Photoschalter und vollführt bei Bestrahlung mit Licht eine cis/trans-Isomerisierung. ceCAM ist das Cyanoester-Derivat (ce) von Cumarin-substituierten Allylidenmalonat, von denen beide Konformere sehr effizient sichtbares Licht absorbieren trans: epsilon(489)=50.300 M^(-1) cm^(-1); cis: epsilon(437)=18.600 M^(-1) cm^(-1)). Andere photophysikalische Eigenschaften umfassen u.a. hohe thermische und photochemische Stabilität. Letztere wurde über ein Experiment nachgewiesen, bei dem die lichtinduzierte Isomerisierung alternierend durchgeführt wurde und selbst bei über 250 Zyklen keine signifikate Abnahme der Absorption beobachtet werden konnte. Des Weiteren konnte die Reaktion mit Quantenausbeuten von 39% (trans) und 42% (cis) induziert werden, wobei im photostationären Gleichgewicht auch hohe Isomerenverhältnisse mit bis zu 80% (trans) und 96% (cis) akkumuliert werden konnten. Die Geschwindigkeit der Reaktion wurde mit Hilfe der Ultakurzzeit-Spektroskopie untersucht. Die Dynamik im Zeitbereich von ps-ns zeigte, dass die trans/cis-Isomerisierung unterhalb von 0,5 ns und die umgekehrte Reaktion noch viel schneller (wenige ps) abgeschlossen ist. Durch die Untersuchungen in dieser Arbeit an den BIST-Verbindungen und ceCAM sind viele vorteilhafte, photophysikalische Eigenschaften charakterisiert worden, wodurch sie als verbesserte Alternative zu den bisher bekannten photolabilen Schutzgruppen oder Photoschaltern anzusehen sind.