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Platelets are anucleated cell fragments derived from megakaryocytes. They play a fundamental role in hemostasis, but there is rising evidence that they are also involved in immunological processes. Despite absence of a nucleus, human platelets are capable of de novo protein synthesis and contain a fully functional proteasome system, which is, in nucleated cells, involved in processes like cell cycle progression or apoptosis by its ability of protein degradation. The physiological significance of the proteasome system in human platelets is not yet fully understood and subject of ongoing research.
Therefore, this study was conducted with the intention to outline the role of the proteasome system for functional characteristics of human platelets. For experimentation, citrated whole blood from healthy donors was obtained and preincubated with proteasome inhibitors. In addition to the commonly used bortezomib, the potent and selective proteasome inhibitor carfilzomib was selected as a second inhibitor to rule out agent-specific effects and to confirm that observed changes are related to proteasome inhibition.
Irreversibly induced platelet activation and aggregation were not affected by proteasome blockade with bortezomib up to 24 hours. Conversely, proteasome inhibition led to enhanced threshold aggregation and agglutination up to 25 %, accompanied by partial alleviation of induced VASP phosphorylation of approximately 10-15 %. Expression of different receptors were almost unaffected. Instead, a significant increase of PP2A activity was observable in platelets after proteasome blockade, accompanied by facilitated platelet adhesion to coated surfaces in static experiments or flow chamber experiments.
Carfilzomib, used for the first time in functional experimentation with human platelets in vitro, led to a dose-dependent decrease of proteasome activity with accumulation of poly ubiquitylated proteins. Like bortezomib, carfilzomib treatment resulted in enhanced threshold aggregation with attenuated VASP phosphorylation.
As the main conclusion of this thesis, proteasome inhibition enhances the responsiveness of human platelets, provided by an alleviation of platelet inhibitory pathways and by an additional increase of PP2A activity, resulting in facilitated platelet adhesion under static and flow conditions. The proteasome system appears to be involved in the promotion of inhibitory counterregulation in platelets. The potential of proteasome inhibitors for triggering thromboembolic adverse events in patients must be clarified in further studies, in addition to their possible use for targeting platelet function to improve the hemostatic reactivity of platelets.
Eukaryotic cells react to various stress conditions with the rapid formation of membrane-less organelles called stress granules (SGs). SGs form by multivalent interactions between RNAs and RNA-binding proteins and are believed to protect stalled translation initiation complexes from stress-induced degradation. SGs contain hundreds of different mRNAs and proteins, and their assembly and disassembly are tightly controlled by post-translational modifications. The ubiquitin system, which mediates the covalent modification of target proteins with the small protein ubiquitin (‘ubiquitylation’), has been implicated in different aspects of SG metabolism, but specific functions in SG turnover have only recently emerged. Here, we summarize the evidence for the presence of ubiquitylated proteins at SGs, review the functions of different components of the ubiquitin system in SG formation and clearance, and discuss the link between perturbed SG clearance and the pathogenesis of neurodegenerative disorders. We conclude that the ubiquitin system plays an important, medically relevant role in SG biology.
This decade saw the development of new high-end light microscopy approaches. These technologies are increasingly used to expand our understanding of cellular function and the molecular mechanisms of life and disease. The precision of state-of-the-art super resolution microscopy is limited by the properties of the applied fluorescent label. Here I describe the synthesis and evaluation of new functional fluorescent probes that specifically stain gephyrin, universal marker of the neuronal inhibitory post-synapse. Selected probe precursor peptides were synthesised using solid phase peptide synthesis and conjugated with selected super resolution capable fluorescent dyes. Identity and purity were defined using chromatography and mass spectrometric methods. To probe the target specificity of the resulting probe variants in cellular context, a high-throughput assay was established. The established semi-automated and parallel workflow was used for the evaluation of three selected probes by defining their co-localization with the expressed fluorescent target protein. My work provided NN1Dc and established the probe as a visualisation tool for essentially background-free visualisation of the synaptic marker protein gephyrin in a cellular context. Furthermore, NN1DA became part of a toolbox for studying the inhibitory synapse ultrastructure and brain connectivity and turned out useful for the development of a label-free, high-throughput protein interaction quantification assay.
Im Zellkern eukaryotischer Zellen werden Gene in mRNAs transkribiert, welche umfangreich prozessiert und aus dem Zellkern exportiert werden. Im Zytoplasma erfolgt die Translation der mRNAs in Proteine, ein Prozess, welcher viel Energie benötigt und daher mittels vielfältiger Mechanismen streng reguliert wird. Ein Beispiel hierfür stellt die Klasse der TOP-mRNAs dar, eine RNA-Spezies, welche hauptsächlich Transkripte von Genen umfasst, die selbst in die Translation involviert sind. Die prominentesten Vertreter dieser Klasse sind die Proteine der kleinen und großen ribosomalen Untereinheiten. TOP-mRNAs zeichnen sich durch ein gemeinsames Sequenz-Motiv am Anfang Ihrer 5’-UTR aus, welches aus einem Pyrimidinstrang besteht und unmittelbar nach dem Cap mit einem Cytosin beginnt. Dieses allen TOP-RNAs gemeinsame Motiv ermöglicht die zeitgleiche Translationskontrolle dieser RNA-Klasse. So kann die Translation der TOP-mRNAs unter Stressbedingungen wie z.B. Nährstoffmangel koordiniert inhibiert werden, wodurch Energie eingespart wird.
Bereits lange wird nach einem Regulator gesucht, der an dieses TOP-Motiv bindet und die koordinierte Regulation ermöglicht. Man kann sich hier einen Inhibitor oder auch einen Aktivator vorstellen. Verschiedene Proteine wurden bereits in Erwägung gezogen. In dieser Arbeit wurde das Protein TIAR mittels Massenspektrometrie als TOP-interagierender Faktor identifiziert und dessen Bindungseigenschaften mit dem TOP-Motiv durch Shift Assays untersucht. Hierbei konnten Minimalkonstrukte verschiedener Organismen sowie RNA-TOP – Sequenzen identifiziert werden, welche sich für Strukturanalysen eignen würden. Als weiterer TOP-interagierender Faktor wurde über verschiedene sequenzielle Reinigungsschritte das Protein 14-3-3ε identifiziert.
Weiterhin wurden die TOP-Motiv-bindenden Proteine LARP1 und LARP7 auf Ihre Bindungseigenschaften mit Ihren Zielsequenzen untersucht. Während gezeigt werden konnte, dass LARP1 einen inhibierenden Einfluss auf TOP-RNAs hat, wurde in weiteren Shift-Assays die Bindungseigenschaften von LARP7 mit 7SK untersucht, wobei ebenfalls ein minimales LARP7–Konstrukt sowie 7SK-Konstrukte für Strukturanalysen identifiziert werden konnten. Weiterhin konnte gezeigt werden, dass verschiedene Substanzen wie tRNA und Arginin einen starken Einfluss auf die LARP7-7SK – Interaktion ausüben, welcher in weiteren Studien berücksichtigt werden sollte.
Antikörper, die gegen eine klinisch relevante Gruppe von Rezeptoren innerhalb der Tumornekrosefaktor-Rezeptor-Superfamilie (TNFRSF) gerichtet sind, darunter CD40 und CD95 (Fas/Apo-1), benötigen ebenfalls eine Bindung an Fc-Gamma-Rezeptoren (FcγRs), um eine starke agonistische Wirkung zu entfalten. Diese FcγR-Abhängigkeit beruht weitgehend auf der bloßen zellulären Verankerung durch die Fc-Domäne des Antikörpers und benötigt dabei kein FcγR-Signalling. Ziel dieser Doktorarbeit war es, das agonistische Potenzial von αCD40- und αCD95-Antikörpern unabhängig von der Bindung an FcγRs durch die Verankerung an Myelomzellen zu entfalten. Zu diesem Zweck wurden verschiedene Antikörpervarianten (IgG1, IgG1-N297A, Fab2) gegen die TNFRSF-Mitglieder CD40 und CD95 genetisch mit einem einzelkettig kodierten B-Zell-aktivierenden Faktor (scBaff) Trimer als C-terminale myelom-spezifische Verankerungsdomäne fusioniert, welche die Fc-Domäne-vermittelte FcγR-Bindung ersetzt. Diese bispezifischen Antikörper-scBaff-Fusionsproteine wurden in Bindungsstudien und funktionellen Assays mit Tumorzelllinien untersucht, die einen oder mehrere der drei Baff-Rezeptoren exprimieren: BaffR, Transmembran-Aktivator und CAML-Interaktor (TACI) und B-Zell-Reifungsantigen (BCMA). Zelluläre Bindungsstudien zeigten, dass die Bindungseigenschaften der verschiedenen Domänen innerhalb der Antikörper-scBaff-Fusionen gegenüber der Zielantigene vollständig intakt blieben. In Ko-Kulturversuchen von CD40- und CD95-responsiven Zellen mit BaffR-, BCMA- oder TACI-exprimierenden Verankerungszellen zeigten die Antikörper-Fusionsproteine einen starken Agonismus, während in Ko-Kulturen mit Zellen ohne Expression von Baff-interagierenden Rezeptoren nur eine geringe Rezeptorstimulation beobachtet wurde. Die hier vorgestellten αCD40- und αCD95-Antikörper-scBaff-Fusionsproteine zeigen also Myelom-spezifische Aktivität und versprechen im Vergleich zu herkömmlichen CD40- und CD95-Agonisten geringere systemische Nebenwirkungen.
Under physiological conditions, protein synthesis controls cell growth and survival and is strictly regulated. Deregulation of protein synthesis is a frequent event in cancer. The majority of mutations found in colorectal cancer (CRC), including alterations in the WNT pathway as well as activation of RAS/MAPK and PI3K/AKT and, subsequently, mTOR signaling, lead to deregulation of the translational machinery. Besides mutations in upstream signaling pathways, deregulation of global protein synthesis occurs through additional mechanisms including altered expression or activity of initiation and elongation factors (e.g., eIF4F, eIF2α/eIF2B, eEF2) as well as upregulation of components involved in ribosome biogenesis and factors that control the adaptation of translation in response to stress (e.g., GCN2). Therefore, influencing mechanisms that control mRNA translation may open a therapeutic window for CRC. Over the last decade, several potential therapeutic strategies targeting these alterations have been investigated and have shown promising results in cell lines, intestinal organoids, and mouse models. Despite these encouraging in vitro results, patients have not clinically benefited from those advances so far. In this review, we outline the mechanisms that lead to deregulated mRNA translation in CRC and highlight recent progress that has been made in developing therapeutic strategies that target these mechanisms for tumor therapy.
∆Np63 is a master regulator of squamous cell identity and regulates several signaling pathways that crucially
contribute to the development of squamous cell carcinoma (SCC) tumors. Its contribution to coordinating the
expression of genes involved in oncogenesis, epithelial identity, DNA repair, and genome stability has been
extensively studied and characterized. For SCC, the expression of ∆Np63 is an essential requirement to
maintain the malignant phenotype. Additionally, ∆Np63 functionally contributes to the development of cancer
resistance toward therapies inducing DNA damage.
SCC patients are currently treated with the same conventional Cisplatin therapy as they would have been
treated 30 years ago. In contrast to patients with other tumor entities, the survival of SCC patients is limited,
and the efficacy of the current therapies is rather low. Considering the rising incidences of these tumor entities,
the development of novel SCC therapies is urgently required. Targeting ∆Np63, the transcription factor, is a
potential alternative to improve the therapeutic response and clinical outcomes of SCC patients.
However, ∆Np63 is considered “undruggable.” As is commonly observed in transcription factors, ∆Np63 does
not provide any suitable domains for the binding of small molecule inhibitors. ∆Np63 regulates a plethora of
different pathways and cellular processes, making it difficult to counteract its function by targeting
downstream effectors. As ∆Np63 is strongly regulated by the ubiquitin–proteasome system (UPS), the
development of deubiquitinating enzyme inhibitors has emerged as a promising therapeutic strategy to target
∆Np63 in SCC treatment.
This work involved identifying the first deubiquitinating enzyme that regulates ∆Np63 protein stability. Stateof-the-art SCC models were used to prove that USP28 deubiquitinates ∆Np63, regulates its protein stability,
and affects squamous transcriptional profiles in vivo and ex vivo. Accordingly, SCC depends on USP28 to
maintain essential levels of ∆Np63 protein abundance in tumor formation and maintenance. For the first time,
∆Np63, the transcription factor, was targeted in vivo using a small molecule inhibitor targeting the activity of
USP28. The pharmacological inhibition of USP28 was sufficient to hinder the growth of SCC tumors in
preclinical mouse models.
Finally, this work demonstrated that the combination of Cisplatin with USP28 inhibitors as a novel therapeutic
alternative could expand the limited available portfolio of SCC therapeutics. Collectively, the data presented
within this dissertation demonstrates that the inhibition of USP28 in SCC decreases ∆Np63 protein abundance,
thus downregulating the Fanconi anemia (FA) pathway and recombinational DNA repair. Accordingly, USP28
inhibition reduces the DNA damage response, thereby sensitizing SCC tumors to DNA damage therapies, such
as Cisplatin.
The macromolecular SMN complex facilitates the formation of Sm-class ribonucleoproteins involved in mRNA processing (UsnRNPs). While biochemical studies have revealed key activities of the SMN complex, its structural investigation is lagging behind. Here we report on the identification and structural determination of the SMN complex from the lower eukaryote Schizosaccharomyces pombe, consisting of SMN, Gemin2, 6, 7, 8 and Sm proteins. The core of the SMN complex is formed by several copies of SMN tethered through its C-terminal alpha-helices arranged with alternating polarity. This creates a central platform onto which Gemin8 binds and recruits Gemins 6 and 7. The N-terminal parts of the SMN molecules extrude via flexible linkers from the core and enable binding of Gemin2 and Sm proteins. Our data identify the SMN complex as a multivalent hub where Sm proteins are collected in its periphery to allow their joining with UsnRNA.
Bacteria thrive and survive in many different environments, and as a result, they have developed robust mechanisms to adapt rapidly to alterations in their surroundings. The protection against osmotic forces is provided by mechanosensitive channels: their primary function is to maintain the integrity of the cell upon a hypoosmotic shock. The mechanosensitive channel of small conductance (MscS) is not only the smallest common structural unit of a diverse family that allows for a tailored response in osmoregulation; it is also the most intensively studied homologue. Mechanosensitive channels directly sense elevated membrane tension levels generated by increased pressure within the cell and open transiently. Escherichia coli has six paralogues that differ in their gating properties and the number of additional transmembrane (TM) helices. These TM helices, termed sensor paddles, are essential for sensing, as they directly contact the surrounding membrane; however, the role of the additional TM helices is still unclear. Furthermore, lipids occupy hydrophobic pockets far away from the membrane plane. A recent gating model for MscS states that increased membrane tension triggers the expulsion of lipids out of those pockets, modulating different conformational states of MscS. This model focuses on bound lipids, but it is still unclear to what extent the direct interaction with the membrane influences sensing and how relevant it is for the larger paralogues.
In the herein described work, structural studies on two larger paralogues, the medium-sized channel YnaI and the large channel YbiO were realised using electron cryomicroscopy (cryo-EM). Lipids were identified in YnaI in the pockets in a similar position and orientation as in MscS, suggesting a conserved sensing mechanism. Moreover, the copolymer diisobutylene/maleic acid (DIBMA) allowed the extraction of artificially activated YnaI from plasma membranes, leading to an open-like form of this channel. This novel conformation indicated that the pore helices bend at a GGxGG motif during gating, which is unique among the Escherichia coli paralogues, concomitant with a structural reorganisation of the sensor paddles. Thus, despite a high similarity of their closed states, the gating mechanisms of MscS and YnaI are surprisingly different. Furthermore, the comparison of MscS, YnaI, and YbiO accentuates variations and similarities between the differently sized family members, implying fine-tuning of channel properties in the pore regions and the cytosolic lateral entry sides into the channel. Structural analyses of MscS reconstituted into different systems showed the advantages and disadvantages of certain polymers and detergents. The novel DIBMA copolymer and the more conventional amphiphilic polymers, so-called Amphipols, perturb contacting transmembrane helices or lead to their denaturation. Due to this observation, the obtained structures of YnaI must also be cautiously considered. The structures obtained in detergents resulted in unaffected channels; however, the applicability of detergents for MscS-like channels is limited by the increased required sample concentration.
The role of lipids for gating MscS in the absence of a membrane was examined by deliberately removing coordinated lipid molecules from MscS using different amounts and kinds of detergent. The effects on the channel were inspected by cryo-EM. These experiments showed that closed MscS adopts the open conformation when it is enough delipidated by incubation with the detergent n-dodecyl-β-D-maltoside, and adding lipids to the open channel reverses this process. The results agree with the state-of-the-art model that the amount of lipid molecules in the pockets and grooves is responsible for the conformational state of MscS. Furthermore, incubation with the detergent lauryl maltose neopentyl glycol, which has stabilising and delipidating characteristics, resulted in a high-resolution structure of open MscS exhibiting an intricate network of ligands. Based on this structure, an updated gating model is proposed, which states that upon opening, lipids from the pockets migrate into the cytosolic membrane leaflet, while lipids from the periplasmic leaflet enter the grooves that arise between the sensor paddles.
Stress granules (SGs) are cytoplasmic condensates containing untranslated mRNP complexes. They are induced by various proteotoxic conditions such as heat, oxidative, and osmotic stress. SGs are believed to protect mRNPs from degradation and to enable cells to rapidly resume translation when stress conditions subside. SG dynamics are controlled by various posttranslationalmodifications, but the role of the ubiquitin system has remained controversial. Here, we present a comparative analysis addressing the involvement of the ubiquitin system in SG clearance. Using high-resolution immuno-fluorescence microscopy, we found that ubiquitin associated to varying extent with SGs induced by heat, arsenite, H2O2, sorbitol, or combined puromycin and Hsp70 inhibitor treatment. SG-associated ubiquitin species included K48- and K63-linked conjugates, whereas free ubiquitin was not significantly enriched. Inhibition of the ubiquitin activating enzyme, deubiquitylating enzymes, the 26S proteasome and p97/VCP impaired the clearance of arsenite- and heat-induced SGs, whereas SGs induced by other stress conditions were little affected. Our data underline the differential involvement of the ubiquitin system in SG clearance, a process important to prevent the formation of disease-linked aberrant SGs.