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Thorne, Christopher Mark Cornelius. "Characterisation of ubiquitin specific protease 33." Thesis, University of Liverpool, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548811.
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Groll, Michael. "Strukturelle und funktionelle Zusammenhänge und Unterschiede archaebakterieller und eukaryontischer 20S-Proteasome." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2005. http://dx.doi.org/10.18452/13957.
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In eukaryotes protein degradation is performed by the ubiquitin-proteasome system. The 26S proteasome, a 2.5MDa large multimeric molecular machine, consists of more than 30 subunits and represents the core component of this proteolytic pathway. The complex is assembled from a proteolytically active 20S proteasome and two 19S regulator cap complexes. So far crystal structure, topology and enzymatic mechanism have only been elucidated for the 20S proteasome core particle (CP). CPs are assembled from four stacked rings of seven subunits each, following an alpha7beta7beta7alpha7-stochiometry. The strict established order of the proteasomal assembly and maturation is essential to prevent uncontrolled and premature protein degradation in the cell. CPs belong to the class of Ntn-hydrolases. Peptide hydrolysis is performed inside a central cavity at the active sites of the beta-type subunits, with Ogam of the hydroxyl group of the N-terminal threonine acting as the nucleophile. Release of the proteolytically active threonine through N-O-Acetyl rearrangement is the last step of the proteasomal assembly. Compartmentalisation of CPs is an important way to regulate substrate access to the central cavity as well as release of the generated oligopeptides. The activity of eukaryotic CPs are controlled by an unique mechanism: docking of regulatory complexes, like Blm3, PA28 or 19S, causes a conformational change of the N-terminal residues of the latent alpha-subunits, resulting in an activation of the proteolytically active sites. Archaebacterial CPs lack such regulatory gating mechanism. The controlled degradation of proteins by the proteasome dominates a variety of biological essential processes, like metabolic adaptation, apoptosis, inflammation, immune and stress response, as well as cell proliferation and cell differentiation. Selective and specific natural and synthetic inhibitors of CPs might find their practical application in treatment of cancer or inflammatory diseases.
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Blanchette, Paola. "Functional analysis of Unp, a mammalian ubiquitin protease." Thesis, University of Ottawa (Canada), 2002. http://hdl.handle.net/10393/6281.
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The murine Unp gene encodes a ubiquitously expressed protein that fractionates with the nuclear fraction (hence its name ubiquitous nuclear protein) (Gupta, K., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and Gray, D. A. (1993). Oncogene 8, 2307-10). It possesses proprieties of an oncogene, such as the ability to promote tumours in a nude mouse assay (Gupta, K., Chevrette, M., and Gray, D. A. (1994). Oncogene 9, 1729--31), and its human homologue, USP4 (previously known as Unph), was shown to be over expressed in certain types of human lung tumours (Gray, D. A., Inazawa, J., Gupta, K., Wong, A., Ueda, R., and Takahashi, T. (1995). Oncogene 10, 2179--83). Although very little was known about this protein's normal function, even less on its mechanism of tumorigenicity, the predicted protein sequence gave some clues on its function. It possesses the two conserved domains present in all ubiquitin specific proteases, and the two motifs common to viral oncoproteins through which they interact with the retinoblastoma gene product pRb. In addition to these features it also possesses a region that resembles a nuclear localisation signal. A mutational approach was taken in combination with ubiquitin cleavage assays and binding assays to study Unp's function. With these, it was confirmed that Unp is a ubiquitin specific protease, its ability to cleave ubiquitin dependent on the conserved cysteine, and may be dependent on Unp phosphorylation status. Unp is a phosphoprotein, being phosphorylated on serine residue(s). It is capable of binding to pRb's hypophosphorylated as well as the hyperphosphorylated forms, a binding that is dependent on an intact conserved motif 2 (CR2). It is also capable of binding to the other pocket proteins, p107 and p130, although with different requirements of conserved regions that for pRb. With these and other results obtained, a model is proposed linking Unp's activity as a deubiquitinating enzyme and its interactions with the pocket proteins with its role as an oncogene. The data obtained also supports the newer view that ubiquitin specific proteases, have a role in the specific regulation of protein levels and not just as general ubiquitin recycling enzymes as previously believed.
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Tibbo, Emma. "Cell cycle aspects of the Unp ubiquitin protease." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/nq21019.pdf.
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Albrecht, Brian Keith. "A concise total synthesis of the TMC-95A and TMC-95B proteasome inhibitors." Access citation, abstract and download form; downloadable file 12.48 Mb, 2004. http://wwwlib.umi.com/dissertations/fullcit/3131652.
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MASSA, FILOMENA. "THE UBIQUITIN-SPECIFIC PROTEASE USP14 CONTROLS CILIOGENESIS AND THE HEDGEHOG PATHWAY." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/562686.
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Primary cilia are microtubule-based organelles on the apical surface of mammalian cells, and play a crucial role in vertebrate development and tissue homeostasis. Consequently, ciliary defects are associated with human disorders called ciliopathies. This organelle represents an organizing center for signaling pathways. In particular, in vertebrates, the Hedgehog (Hh) pathway controls embryonic development and adult homeostasis using the primary cilium to transduce its signal. Hh components localize to cilia and Kif7, a key player in cilia structure and length, controls the ciliary localization of Hh signaling molecules. Phenotypes associated to Hh signaling impairment are often observed in ciliopathies. Recent studies established a link between ciliary proteins and the Ubiquitin proteasome system (UPS) pathway, however much remains to be understood. The main role of the UPS is to mark proteins for degradation although it also functions in a wide variety of cellular processes. The aim of my PhD project was to investigate the association between cilioproteins and proteasomal functions, with particular emphasis on the cilia- associated OFD1 protein, which is responsible for the rare OFD type I syndrome. The results obtained demonstrate that OFD1 controls proteasomal complex composition through direct binding with proteasomal components (see Liu et al. in appendix). Our results also demonstrate a role for Usp14, a deubiquitinating enzyme, in the control of ciliogenesis, cilia length and proper activation of the Hh pathway. We propose a new mechanism by which cilia maintenance and the Hh pathway are regulated by Usp14 via modulation of Kif7 proteasomal degradation (manuscript in preparation). This mechanism may be relevant not only in ciliopathies but also in other pathological conditions associated to Hedgehog signaling defects. Overall our results provide new insight into the spectrum of action of the UPS and may provide novel opportunities for therapeutic intervention in pathological conditions associated to cilia dysfunction.
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Boehringer, Jonas. "Substrate recognition by the proteasome." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.669968.
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The ubiquitin proteasome system targets proteins to the proteasome where they are degraded. Substrate recognition and processing prior to degradation take place at the 19S regulatory particle of the proteasome. A polyubiquitin chain, linked through isopeptide bonds formed between the C-terminal G76 and K48, is the signal responsible for delivery to the proteasome. Because chains linked via any of the seven lysine residues of ubiquitin exist in vivo and encode signals unrelated to protein degradation it is crucial for cells to avoid crosstalk between these different pathways. Several ubiquitin receptors related to proteasomal degradation have been identified but the selectivity between the different ubiquitin chains has not been assessed quantitatively while avoiding artefacts attributed to GST-dimerisation. By employing isothermal titration calorimetry, analytical ultracentrifugation and nuclear magnetic resonance, discrimination between K48- and K63-linked diubiquitin was established for the S. pombe proteasomal receptor Rpn10 and the shuttle protein Rhp23. The same methods allowed us to propose a discriminatory model for Rpn10. The crystal structures of the 19S regulatory particle subunits Rpn101-193 and Rpn121-224 have been determined and possible protein-protein interaction sites were identified by surface conservation and electrostatics analysis. Rpn12 surface residues were identified that had a negative effect on Rpn10-binding. This interaction was studied by surface plasmon resonance, fluorescence anisotropy and nuclear magnetic resonance. These experiments revealed a binding site on Rpn10 that is exclusively occupied by either ubiquitin or Rpn12 and for the first time demonstrated the interaction of a ubiquitin interacting motif with a protein other than ubiquitin.
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Fischer, Susanne. "Untersuchungen zur Funktion der Ubiquitin spezifischen Protease nonstop im visuellen System von Drosophila melanogaster." [S.l. : s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=961831448.
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Sharif, Azar. "Structural characterization of the polycomb repressor complex 1 binding partner ubiquitin specific protease 11." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/39355.
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Ubiquitin Specific Protease 11 (USP11), USP4 and USP15 are highly conserved and are characterised by an N-terminal 'domain present in ubiquitin specific proteases' (DUSP) and 'ubiquitin-like' (UBL) domains. This DUSP-UBL (DU) domain is thought to be involved in substrate recognition. It was shown that USP11 co-purifies with human Polycomb Repressive Complex type 1 (PRC1) and regulates the stability of the E3 ligase component of PRC1 (Maertens et al, 2010). PRC1 repress transcription from the INK4a tumour suppressor locus. Hence knockdown of USP11 in primary human fibroblasts causes de-repression of INK4a, followed by a senescence-like proliferative arrest. In this project we aimed to map the interaction between USP11 and PRC1 components (BMI1, RING2, MEL18 and CBX8). We used two methods to investigate their interactions; yeast two-hybrid and in vitro pull down. Unexpectedly, we could not confirm a direct interaction between USP11 and any PRC1 component. We hypothesize that the lack of post-translation modifications, the presence of fusion tags and/or the need of a multi-subunit PRC1 complex might be needed to observe a high affinity interaction. We also aimed to map the interaction between three PRC1 components; RING2, BMI1 and RYBP, with the ultimate aim of solving the X-ray structure of the complex. The main obstacle in this project was to express, extract and purify these proteins at high levels in bacterial culture. Preliminary data suggests that RYBP and BMI1 do not interact directly. Here we report the 3.6 Å resolution X-ray structure of the human USP11 DU. The sequence linking the DUSP and UBL domains, the DU finger, could not be assigned in the electron density map due to low resolution. Comparison with the related USP4 DU crystal structure reveals that the structures are mostly conserved.
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Nunes, Gonçalo Pedro da Silva. "The role of Ubiquitin Specific Protease 7 on Latency Associated Nuclear Antigen DNA binding." Master's thesis, Universidade Nova de Lisboa. Instituto de Tecnologia Quimica e Biológica António Xavier, 2018. http://hdl.handle.net/10362/130062.
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Dissertation presented to obtain the Master degree in Biochemistry for HealthThe implications of USP7 (Ubiquitin Specific Protease 7, deubiquitinating enzyme involved in several crucial molecular pathways) interactions with LANA (Latency Associated Nuclear Antigen protein, that facilitates the tethering of viral γ-Herpesvirus episomes into the host’s DNA and promotes its replication) has multiple health consequences, since USP7 is a protein that has an essential role it the regulation of many cellular functions, like the p53-mdm2 pathway, that regulates cellular apoptosis and prevents cancer.
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Bromby, Heather Joanne. "The role and regulation of ubiquitin-specific protease 4 in nuclear factor kappa B signalling." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/2987.
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Deubiquitinating enzymes (DUBs) constitute a diverse family of regulators of ubiquitin signalling. Aberrations in this can underlie pathologies including osteoarthritis, cancers, inflammatory diseases, and even ageing. Understanding the biology of ubiquitin signalling components and how they regulate cell signalling pathways is therefore a widening area of research. The aim of this thesis was to further understanding on the function of the DUB, ubiquitin-specific protease 4 (USP4), and the mechanism of Usp4 regulation by its post-translational modification (PTM) at two serine residues, focusing on the nuclear factor of κB (NF-κB) pathway. Initial observations in Usp4 null (-/-) mice suggested a role for Usp4 in regulating circulating levels of specific inflammatory cytokines and, perhaps consequentially, a role in viral infection clearance. Thus, in vitro, the role of USP4 in IL-1- and TNFα-mediated NF-κB signalling was examined. Overexpression of Usp4 significantly increased NF-κB signalling, and using an RNAi approach to deplete USP4 resulted in suppressed NF-κB activity and significantly reduced expression of NF-κB-regulated genes (MMP13, IL-6 and IL-8). Depletion of USP4 also significantly reduced ubiquitination and degradation of IκBα, and phosphorylation of p65 and IKK. Together, data were indicative of USP4 as a positive regulator of NF-κB signalling by functioning upstream of the IKK complex. Importantly, aspects of these findings were confirmed in mouse embryonic fibroblasts (MEFs) isolated from the Usp4-/- mice. Further, identification of potential USP4 substrates were assessed. Reductions in K48- and K63-linked polyubiquitination were observed with depletion of USP4. To further understanding of the regulation of USP4, the PTM at serines 675 and 680 was examined. Lentiviral-mediated delivery of Usp4 phosphorylation variants revealed that a complex mechanism of phosphorylation/dephosphorylation of USP4 may regulate its function in NF-κB signalling. In conclusion, this thesis identifies USP4 as a positive regulator of IL-1-induced NF-κB signalling, potentially regulating itself through its PTM.
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Kathoria, Meeta. "An investigation of the properties and functions of the herpes associated ubiquitin-specific protease, Hausp." Thesis, University of Glasgow, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284737.
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Hennig, Thomas. "Function and transport of a herpesvirus encoded ubiquitin-specific protease in virus entry and assembly." Thesis, Imperial College London, 2015. http://hdl.handle.net/10044/1/55292.
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Herpes simplex virus type I (HSV-1), the prototype α-herpesvirus (HV), is a double stranded DNA virus that replicates in the nucleus of infected cells. The nuclear pore represents a gateway that must be engaged and navigated immediately after cell entry for successful infection by many classes of human viruses. For herpesviruses, capsid-tegument assemblies must be targeted to the pore where the viral genome exits and transport into the nucleus occurs. We currently have little mechanistic knowledge of this fundamental step of infection. A swath of evidence indicates that the conserved tegument protein VP1-2 is essential for early capsid transport and pore binding, and that it contains a conserved nuclear localisation signal (NLS) required for pore docking. In this thesis I undertook a detailed analysis to dissect functional determinants within the NLS from herpes simplex virus, to examine putative NLSs in VP1-2 homologues from representatives of all sub-families, to characterise protein interactions with VP1-2 and finally to construct a GFP expressing entry defective recombinant virus to study the consequences of infection. I show that the HSV NLS can function as a mono-or bipartite motif and has a particular organisation conserved in the a-herpesvirus homologues but distinct from those in the \beta- and \gamma-herpesviruses. The representatives of all 3 classes contain a functional NLS at approximately the same position. All bi-partite motifs were able to rescue the HSV VP1-2ΔNLS virus defect albeit to varying extent whereas the mono-partite HHV-8 motif did not. I constructed and purified chimeric recombinant viruses for the VZV, HCMV and EBV motifs and show distinct differences in their ability to replicate in non-complementing cells. In HSV, NLS function in the context of protein nuclear import or of viable virus replication, was dependent on lysine 428 and the integrity of the full bi-partite motif. Mutations which reduced NLS activity generally caused reduced fitness of recombinant viruses. For the analysis of interaction partners of VP1-2 and the NLS, I developed a one-step approach to analyse the capsid interactome during entry. Additionally, I constructed mammalian GST-VP1-2.NLS fusion proteins and cell lines which inducibly express the N-terminal region of VP1-2 for analysis of NLS interacting proteins. Using mass spectrometry (MS) I identified a number of VP1-2-interacting, cellular proteins including DTX3L, an important regulator of the DNA damage response.
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Gewies, Andreas. "Investigation of the ubiquitin-specific protease UBP41 and of the lysosomal cysteine proteases cathepsin-L and cathepsin-B as potential mediators of proapoptotic signalling." Diss., lmu, 2004. http://nbn-resolving.de/urn:nbn:de:bvb:19-16836.
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Reyskens, Kathleen Maria Simone Elise. "The maladaptive effects of HIV protease inhibitors (Lopinavir/Ritonavir) on the rat heart." Stellenbosch : Stellenbosch University, 2013. http://hdl.handle.net/10019.1/85782.
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Thesis (PhD)--Stellenbosch University, 2013.ENGLISH ABSTRACT: Although antiretroviral treatment decreases HIV-AIDS morbidity/mortality, long-term effects include onset of insulin resistance and cardiovascular diseases. Increased oxidative stress and dysregulation of the ubiquitin-proteasome system (UPS) are implicated in protease-inhibitor (PI)-mediated cardio-metabolic pathophysiology. We hypothesized that PI treatment (Lopinavir/Ritonavir) elevates myocardial oxidative stress and concomitantly inhibits the UPS, thereby attenuating cardiac function. Lopinavir/Ritonavir was dissolved in 1% ethanol (vehicle) and injected into mini-osmotic pumps that were surgically implanted into Wistar rats for eight weeks vs. vehicle and sham controls. Subsequently, we evaluated metabolic parameters and heart function (ex vivo and in vivo methods) at baseline and following ischemia-reperfusion. PI-treated rats exhibited weight gain, increased serum LDL-cholesterol, higher tissue triglycerides (heart, liver), but no evidence of insulin resistance. It also upregulated hepatic gene expression of acetyl-CoA carboxylase β and 3-hydroxy-3-methylglutaryl-CoA-reductase, key regulators of fatty acid oxidation and cholesterol synthesis, respectively. Further, PI-treated hearts displayed impaired UPS, increased superoxide dismutase (SOD) activity and unaltered superoxide levels, and elevated peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) peptide levels. Perfusion data revealed contractile dysfunction at baseline and following ischemia-reperfusion, while post-ischemic hearts exhibited decreased ATPase specific activity vs. matched controls. Early changes initiated by PI treatment resemble the metabolic syndrome and reflect a pre-atherogenic profile. Moreover, the effects of PIs on cardiac contractile function may in part be triggered by impaired UPS activity together with strain on the mitochondrial energetic system. Our study alerts to cardio-metabolic side effects of PI treatment and raises the question of the most appropriate co-therapies for patients on chronic antiretroviral treatment.
AFRIKAANSE OPSOMMING: Alhoewel anti-retrovirale behandeling MIV-VIGS morbiditeit/mortaliteit verlaag, bestaan daar langtermyn effekte soos die aanvang van insulienweerstandigheid en kardiovaskulêre siektes. Verhoogde oksidatiewe stres en wanregulering van die ubikwitien-proteosoomsisteem (UPS) word geïmpliseer met protease-inhibeerder (PI) gemediëerde kardio-metaboliese patofisiologie. Ons hipotetiseer dat PI behandeling (Lopinavir/Ritonavir) miokardiale oksidatiewe stres verhoog, en gevolglik die UPS inhibeer waardeur dit kardiale funksie verander. Lopinavir/Ritonavir is in 1% etanol (draer) opgelos en in ‘n mini-osmotiese pomp ingespuit wat chirurgies in Wistar rottes ingeplant is vir agt weke vs. draer en valskontroles. Gevolglik het ons die metabolise parameters en hartfunksie (ex vivo en in vivo metodes) op basislyn en na afloop van ischemie-reperfusie ondersoek. PI-behandelde rotte het ‘n toename in massa getoon asook verhoogde serum LDL-cholesterol, hoër weefseltrigliseriede (hart, lewer), maar geen bewys van insulienweerstandigheid nie. Dit het ook hepatiese asetielko-ensiem A karboksilase β en 3-hidrokise-3-metielglutariel KoA reduktase geenuidrukking opwaarts gereguleer, wat sleutel reguleerders van vetsuuroksidasie en cholesterolsintese onderskeidelik is. Verder, het PI-behandelde harte ingeperkte UPS, verhoogde SOD aktiwiteit en onveranderde superoksiedvlakke vertoon, asook verhoogde peroksisoomproliferator-geaktiveerde reseptor-γ ko-aktiveerder 1-α (PGC-1α) peptiedvlakke. Perfusie data toon kontraktiele wanfunskionering gedurende basislyn en na afloop van ischemie-reperfussie, terwyl post-ischemiese harte verlaagde ATPase spesifieke aktiwiteit vs gepaarde kontrole vertoon. Vroeë veranderinge wat deur PI behandeling veroorsaak word, kom ooreen met die metabolise sindroom en reflekteer op ‘n pre-aterogeniese profiel. Bowendien kan die effekte van PIs op kardiale kontraktiele funksie deels veroorsaak word deur die ingeperkte UPS aktiwiteit tesame met die las op die mitochondriale energie sisteem. Ons studie waarsku teen kardio-metaboliese newe effekte met PI behandeling en rig die vraag; wat die mees gepaste ko-behandeling vir pasiënte op chroniese anti-retrovirale behandeling is.
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Veiga, Inês Margarida Berenguer [Verfasser]. "The UL36-encoded ubiquitin-specific protease in Marek's disease virus replication and tumourigenesis / Inês Margarida Berenguer Veiga." Berlin : Freie Universität Berlin, 2013. http://d-nb.info/1034527819/34.
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Crespo-Yañez, Xènia. "Découverte de l'ubiquitination en tant que nouveau mécanisme de régulation de la protéine ESCRT-III CHMP1B." Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAV035/document.
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J’ai effectué ma thèse dans le groupe du Dr. Marie-Odile Fauvarque qui met en œuvre des stratégies de génétique moléculaire sur des modèles de cellules humaines et chez la mouche drosophile pour l'étude de la fonction des protéines dans la signalisation intracellulaire. Dans ce contexte, mes travaux visaient à produire des connaissances fondamentales sur le système ubiquitine dans le contrôle du trafic endocytaire, en particulier de récepteurs membranaires impliqués dans la réponse inflammatoire (TNFR, ILR) ou la différenciation et la croissance cellulaire (EGFR). Je me suis notamment intéressée au rôle du complexe formé par l’interaction entre une protéine de la voie endocytaire, CHMP1B, et la protéase d’ubiquitine UBPY (synonyme USP8). CHMP1B est un membre de la famille ESCRT-III qui, via des processus de changements de conformation et de polymérisation à la membrane, contrôle la biogenèse des vésicules intraluménales (ILVs) au niveau des endosomes tardifs pour former les corps multivésiculaires (MVBs). Ces derniers fusionnent avec les lysosomes, assurant ainsi la protéolyse des récepteurs internalisés et l‘arrêt de la signalisation intracellulaire. Alternativement, les récepteurs peuvent être renvoyés à la membrane plasmique à partir des endosomes précoces ou tardifs via des vésicules de recyclage. Le trafic intracellulaire et le tri des récepteurs dans ces différents compartiments subcellulaires jouent un rôle majeur dans l’activation, la durée et la terminaison des signaux intracellulaires. Or, la liaison covalente d’une ou plusieurs ubiquitine (un polypeptide très conservé de 76 aminoacides) au niveau des récepteurs est un signal majeur déclenchant leur internalisation. En hydrolysant cette ubiquitine, UBPY peut stopper l’internalisation des récepteurs au niveau de la membrane plasmique, ou bien, favoriser leur entrée dans le MVB. UBPY jouerait ainsi deux rôles opposés sur la stabilité des récepteurs selon son niveau d’action dans la cellule. L’interaction entre CHMP1B et UBPY avait été décrite dans la littérature chez la levure ou par co-immunoprécipitation à partir de lysat cellulaires. Cependant, les travaux de l’équipe montraient l’absence d’interaction forte entre les domaines d’interaction de ces deux protéines in vitro et par ailleurs, la fonction de cette interaction dans le processus d’endocytose n’avait été que partiellement élucidée. J’ai confirmé l’existence du complexe CHMP1B-UBPY in cellulo qui se localise essentiellement au niveau des endosomes tardifs. J’ai déterminé la région impliquée dans cette interaction et prouvé que l’existence de ce complexe permet de stabiliser les deux protéines dans les cellules. J’ai ensuite démontré l’existence de formes ubiquitinées monomériques et dimériques de CHMP1B dans lesquelles la liaison d’une molécule d’ubiquitine sur une des deux lysines d’une boucle flexible de la protéine induit un probable changement de conformation. De plus, UBPY hydrolyse cette ubiquitine et favorise l’accumulation d’oligomères de CHMP1B qui sont dépourvues d’ubiquitine. Finalement, le traitement des cellules par l’EGF, qui se lie à l’EGFR et provoque son internalisation, induit le recrutement transitoire des dimères ubiquitinés de CHMP1B aux membranes. L’analyse du trafic intracellulaire de l’EGFR et de la morphogenèse de l’aile de drosophile dans différents contextes génétiques a également prouvé que la forme ubiquitinée de CHMP1B est essentielle à sa fonction. L’ensemble de mes travaux m’autorisent à formuler une hypothèse complètement nouvelle dans laquelle l’ubiquitination de CHMP1B induit une conformation ouverte de la protéine incapable de polymériser qui est recrutée sous forme de dimères à la membrane des endosomes où la présence d’UBPY induit la deubiquitination et la polymérisation concomitante de CHMP1B, très probablement en hétéro-complexes avec d’autres membres de la famille ESCRT-III agissant de concert pour la déformation et la scission des membranesI did my thesis in the group of Dr. Marie-Odile Fauvarque who implements strategies of molecular genetics on human cell culture models and in the Drosophila fly for the identification and study of the function of proteins in intracellular signaling. In this context, my work aimed to produce fundamental knowledge about the ubiquitin system in the control of the endocytic trafficking, in particular of membrane receptors involved in the inflammatory response (TNFR, ILR) or cell differentiation and growth (EGFR). I was particularly interested in the role of the complex formed by the interaction between an endocytic protein, CHMP1B, and the ubiquitin protease UBPY (synonym USP8). CHMP1B is a member of the ESCRT-III family that controls the biogenesis of intraluminal vesicles (ILVs) at the late endosomes to form multivesicular bodies (MVBs) Conformational change and polymerization at lipidic membrane processes are needed for CHMP1B function. MVBs fuse with the lysosomes, thus ensuring the proteolysis of the internalized receptors and the stoppage of the intracellular signaling. Alternatively, the receptors may be returned to the plasma membrane from early or late endosomes via recycling vesicles. Intracellular trafficking and receptor sorting in these different subcellular compartments play a major role in the activation, duration and termination of intracellular signals. The covalent bond of one or more ubiquitin (a highly conserved polypeptide of 76 amino acids) at the receptors is a major signal triggering their internalization. By hydrolyzing this ubiquitin, UBPY can stop the internalization of receptors at the plasma membrane, or promote their entry into the MVB. UBPY would thus play two opposing roles on the stability of the receptors depending on its level of action in the cell. The interaction between the two proteins CHMP1B and UBPY had been described in the literature in the two-hybrid system in yeast or by co-immunoprecipitation from cell lysates. However, the team's work showed no strong interaction between the domains of interaction of these two proteins in vitro and the function of this interaction in the endocytosis process had only been partially elucidated.During my thesis, I confirmed the existence of the CHMP1B-UBPY in cellulo complex, which is located mainly at the level of late endosomes. I determined the region involved in this interaction and proved that the existence of this complex makes possible the stabilization of both proteins into the cells. I then demonstrated the existence of monomeric and dimeric ubiquitinated forms of CHMP1B in which the binding of a molecule of ubiquitin to one of the two lysines of a flexible loop of the protein likely induces and/or stabilize a conformational conformation. In addition, UBPY hydrolyses this ubiquitin and promotes the accumulation of CHMP1B oligomers which are devoid of ubiquitin. Finally, the treatment of cells by EGF, which binds to EGFR and causes its internalization, induces transient recruitment of ubiquitinated CHMP1B dimers to the membranes. Analysis of the intracellular trafficking of EGFR and the morphogenesis of Drosophila wing in different genetic contexts has also shown that the ubiquitination of CHMP1B is essential to its function. My work has allowed me to formulate a completely new hypothesis in which the ubiquitination of CHMP1B induces an open conformation of the protein incapable of polymerizing in this state which is recruited in the form of dimers to the membrane of the endosomes and there the presence of UBPY induces the deubiquitination and the concomitant polymerization of CHMP1B, most probably in hetero-complexes with other members of the ESCRT-III family acting in concert for deformation and scission of the membranes
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Rimsa, Vadim. "Targeting an E3 ubiquitin ligase Siah1 and a cysteine protease SENP1 using SPR and DSF-based fragment screening." Thesis, University of Dundee, 2013. https://discovery.dundee.ac.uk/en/studentTheses/0dfaa23f-8048-423a-b3c0-560ac40de2a4.
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This dissertation presents fragment screening studies against two human proteins Siah1 and SENP1, which function in post-translational modification pathways. Siah1 is an E3 ubiquitin ligase that functions as a scaffold to transfer ubiquitin bound to an E2 ubiquitin-conjugating enzyme to a substrate as part of the ubiquitination pathway. SENP1 is a cysteine protease that catalyses two essential reactions in the SUMO pathway. It processes pre-SUMO proteins to their mature form and removes SUMO from the target proteins. Siah1 interactions with other proteins involve large surface areas, while SENP1 has a small active site, making it hard to identify ligands for these proteins. The fragment-based approach has emerged as a complementary method to high-throughput screening of finding novel small molecules. The main aim of the study was to examine whether fragment screening would identify any ligands against these targets. Chapter 1 introduces post-translational modifications and presents fragment-based approach used in drug discovery. Chapter 2 describes the experimental methods used. The results from fragment screening against Siah1 using SPR and DSF are reported in chapter 3. The chapter also presents the structure of Siah1 refined to 1.95 Å that displays new parts of the structure, previously missing due to the absence of reliable electron density. Chapter 4 contains results from the fragment screens against SENP1 using DSF and NMR. The crystal structure of SENP1 was determined with a number of improvements made over earlier structures. Besides performing fragment screening, the binding between Siah1 interacting proteins reported in the literature and Siah1 was investigated. A number of Siah1 binding partners were successfully expressed and purified as described in chapter 5. One of those, SIP showed a clear interaction with Siah1, as observed by the shift on a size exclusion column of the complex relative to the individual protein species. Siah1 was reported to collaborate with PEG3 in the regulation of ß-catenin degradation. A SCAN domain, located at the N-terminus of PEG3, was tested for binding using gel filtration chromatography and NMR, but no interaction was observed. PEG3 was used in the crystallographic studies and a structure of its SCAN domain was solved using molecular replacement and refined to 1.95 Å. The structure of PEG3-SCAN domain revealed a stable homodimer with an extensive dimerization interface. The structure of a zinc-dependent cytosolic carboxypeptidase from Burkholderiacenocepacia was determined and is reported in chapter 6. This work was a side project assessing a new refinement strategy, which involved the use of the automated protocols embedded in the PDB_REDO server. The structure revealed that carboxypeptidase is a tetramer and provides details of its active site, whose spatial conformation of residues supports the notion that the protein might function as a deglutamylase.
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Reid, Jocelyn. "Fun[c]tions of the N-terminal extensions of the ubiquitin-specific processing protease-testis 1 and 2 isoforms." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=19440.
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Ubiquitin is a small, globular 8 kDa protein whose function is accomplished by its conjugation to targeted substrates. This conjugation is a multi-step process involving three different enzymes. Upon polyubiquitination of a target protein, the 26S proteasome is recruited, leading to the degradation of the substrate. The ubiquitination of proteins, and hence their degradation, can be prevented through the action of deubiquitinating enzymes (DUBs), cysteine proteases that cleave the isopeptide bond through which ubiquitin moieties are attached to their substrates. How DUBs recognize their substrates has been unclear. Two isoforms of a novel testis-specific deubiquitinating enzyme, UBP-tl and UBP-t2, which contain identical core regions but different N-termini, were identified 1. This thesis describes the further characterization of the function of the N-termini. Assessment of the kinetic parameters of UBP-tl, UBP-t2 and the core domain of the two enzymes alone (UBP-core) for the substrate ubiquitin-PEST revealed that the different N-termini increased the Km's of the enzymes, thereby diminishing their affinity towards the substrate. The Vmax of UBP-t2 and the core domain were similar, suggesting that the N-termini do not affect catalytic efficiency. As well, UBP-tl and UBP-t2 had lower activity than UBP-core against endogenously ubiquitinated testis proteins. Thus, the N-termini probably restrict the deubiquitinating activity of the core domain to specific substrates. The UBP-core deubiquitinating activity was not exclusive to testis proteins, as endogenously ubiquitinated proteins in liver, brain, and muscle extracts were also deubiquitinated, however at a slower rate than those in testis. UBP-testis appears to be specific for ubiquitinated proteins, as the enzymes could not remove Nedd8 (the ubiquitin-like protein most similar to ubiquitin) from endogenously neddylated proteins. Since the N-termini appeared to be involved in regulating substrate specificity, they were used as ligands in affinity columns to try to bind substrates and/or interacting proteins from testis extracts. AUFl, a protein regulating mRNA stability was found to be a possible interacting protein with the UBP-tl N-terminus.
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Czech-Sioli, Manja [Verfasser], and Nicole [Akademischer Betreuer] Fischer. "Einfluss der Ubiquitin spezifischen Protease 7 auf den Lebenszyklus des Merkel Zell Polyomavirus / Manja Czech-Sioli ; Betreuer: Nicole Fischer." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/1209676192/34.
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Engel, Elodie. "Identification des "ubiquitin specific proteases" impliquées dans la régulation des voies de l'immunité chez la drosophile." Phd thesis, Grenoble 1, 2009. http://www.theses.fr/2009GRE10110.
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La dérégulation des facteurs NF-kappaB, impliqués dans la survie cellulaire et l'inflammation, peut entraîne des pathologies inflammatoires chroniques et des cancers. Dans ce contexte, l'objectif de ma thèse était d'identifier de régulateurs négatifs des voies NF-KB conservées au cours de l'évolution, Tollet Imd, chez la drosophile. De nombre investigation pour rechercher des régulateurs de ces voies. J'ai réalisé le crible d'une collectio d'ARN interférents permettant l'inactivation des 21 USPs de drosophile en cellules S2. Ce crible a mis en évidence trois regulateurs négatifs de la voie Imd, dont un montre également une activité sur la voie Toll. Parmi ces candidats, dUSP36 un homologue de la protéine humaine USP36, avait été préalablement sélectionné par un crible génétique au laboratoire. Des études de l'équipe auxquelles j'ai contribué, montrent son rôle in vivo dans la régulation négative de la protéine adaptatrice Imd via son activité catalytique. Afin de caractériser la fonction des deux autres USPs, j'ai mené de! expériences de transgénèse chez la drosophile qui prouvent que ces deux USPs répriment la voie Imd en cas d'infectioI et qu'elles sont requises pour maintenir l'état inactif de la voie Imd en l'absence d'infection. J'ai également entrepris de caractériser l'activité catalytique des deux USPs in vitro. L'originalité de mon travail a consisté à limiter le crible à une famille de gènes, ce qui a permis de détecter de nouveaux régulateurs qui n'avaient pas été mis en évidence dans des cribles antérieursThe deregulation of NF-KB signalling pathways, involved in cell survival and inflammation, leads t< chronic inflammation and cancers. The aim of this thesis was to identify negative regulators of the conserved NF-KE pathways, Toll and Imd, in Drosophila melanogaster. The stability or activity of several compounds of NF-kappaB pathway is regulated by ubiquitination. Consequently, the ubiquitine specific proteases (USPs) constitute a new area to look fo regulators ofthese pathways. To do this, 1 constructed a collection of interfering RNA able to inactivate the 21 USPs 0 drosophila in S2 cells. The screening of this collection identified three negative regulators of the Imd pathway, one 0 them having also an effect on the Toll pathway. The target specificity of USPs could explain the small number 0 candidates. Among these candidates, dUSP36, a homologue of human USP36, was previously detected in the lab in : genetic screening. Team studies, in which 1 participated, show its in vivo effect on the adaptative protein Imd through it: catalytic activity. Ln order to characterise the two other candidates 1 performed trangenesis experiments in drosophila These studies show that the two USPs are able to prevent activation of the Imd pathway in case of infection and that the: are required to maintain the inactivated state of the Imd pathway in absencê of infection. 1 also started to characterise th4catalytic activity of both candidates in vitro. My work's novelty was to limit the screening to only one gene family which allowed the detection of new regulating genes which had not been revealed in previous screenings performed on ; large part or the entire genome
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Grazette, Affif. "Molecular characterisation of squamous cell carcinoma antigen recognised by T-cells 3, an adaptor protein of ubiquitin specific protease 15." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33081/.
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The deubiquitinating enzyme USP15, a member of the USP family, reverses the process of ubiquitination thereby altering the fate of a plethora of substrates. As such, USP15 has been implicated in a numerous important cellular pathways including cell cycle progression, transcriptional modification and DNA damage repair. The spliceosomal subunit recycling protein SART3 has been shown to bind to USP15 enhancing its deubiquitination of histone H2B thereby providing histone dimers for reassembly during subsequent rounds of transcription and splicing. Furthermore SART3 has also been shown to bind to USP4, a close homologue of USP15. In addition to this histone chaperone activity, SART3 primarily functions by mediating the re-annealing of the U4 and U6 snRNPs to facilitate consequent rounds of splicing in addition to its implication in several disease states including numerous cancer types and HIV through interactions with various cellular proteins. In this thesis ITC, ESI-MS, analytical size exclusion, X-ray crystallography, SAXS and pull-down assays are used to characterise the interaction between USP15 and SART3, solve the structure of the N-terminus of SART3 and identify novel binding partners of the USP15-SART3 complex. The structure of SART3’s N-terminus (residues 96-574) has been solved to 3.04Å, revealing that it is a homodimer comprised of a series of anti-parallel α-helices that form a shape reminiscent of a bowtie. This dimeric arrangement is retained in solution and can bind two molecules of USP15DU. The DU-finger of USP15 plays a pivotal role in co-ordinating the binding interaction with SART3, as small changes to this region can affect the nanomolar affinity interaction between USP15 and SART3. In addition the USP15-SART3 complex appears to interact with several cellular proteins which could have significant impact in several disease states.
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Engel, Elodie. "Identification des "Ubiquitin Specific proteases" impliquées dans la régulation des voies de l'immunité chez la drosophile." Phd thesis, Université Joseph Fourier (Grenoble), 2009. http://tel.archives-ouvertes.fr/tel-00403268.
Full textAbstract:
La dérégulation des facteurs NF-κB, impliqués dans la survie cellulaire et l'inflammation, peut entraîner des pathologies inflammatoires chroniques et des cancers. Dans ce contexte, l'objectif de ma thèse était d'identifier des régulateurs négatifs des voies NF-κB conservées au cours de l'évolution, Toll et Imd, chez la drosophile. De nombreux éléments de ces voies sont régulés par ubiquitination. Les « Ubiquitine Specific Proteases » (USPs) constituent ainsi un nouveau champ d'investigation pour rechercher des régulateurs de ces voies. J'ai réalisé le crible d'une collection d'ARN interférents permettant l'inactivation des 21 USPs de drosophile en cellules S2. Ce crible a mis en évidence trois régulateurs négatifs de la voie Imd, dont un montre également une activité sur la voie Toll. Parmi ces candidats, dUSP36, un homologue de la protéine humaine USP36, avait été préalablement sélectionné par un crible génétique au laboratoire. Des études de l'équipe auxquelles j'ai contribué, montrent son rôle in vivo dans la régulation négative de la protéine adaptatrice Imd via son activité catalytique. Afin de caractériser la fonction des deux autres USPs, j'ai mené des expériences de transgénèse chez la drosophile qui prouvent que ces deux USPs répriment la voie Imd en cas d'infection et qu'elles sont requises pour maintenir l'état inactif de la voie Imd en l'absence d'infection. J'ai également entrepris de caractériser l'activité catalytique des deux USPs in vitro. L'originalité de mon travail a consisté à limiter le crible à une famille de gènes, ce qui a permis de détecter de nouveaux régulateurs qui n'avaient pas été mis en évidence dans des cribles antérieurs.
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Kondrajew, Jana [Verfasser], and Thomas [Akademischer Betreuer] Dobner. "Analysen zur Rolle der Ubiquitin-spezifischen Protease 7 im Verlauf der produktiven Infektion mit dem Adenovirus Typ 5 / Jana Kondrajew ; Betreuer: Thomas Dobner." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/1216629374/34.
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Kasherman, Maria A. "USP9X in Cortical Development and Behaviour." Thesis, Griffith University, 2020. http://hdl.handle.net/10072/394719.
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Autism Spectrum Disorder (ASD) and Intellectual Disability (ID) are two of the most common neurodevelopmental disorders classified by the Diagnostic and Statistical Manual of Mental disorders 5th Edition. Both are often comorbid, meaning that they often exist in the same patient. Due to this, it has been hypothesised that there may be a common cause for ID and ASD, a common disruption in the same neurodevelopmental pathway. A gene that has been implicated in both the pathogenesis of ID and ASD is the Ubiquitin-Specific Protease 9 XLinked (USP9X). Targets of USP9X and USP9X itself have been found to be part of mTOR, TGFb, and WNT signalling pathways, all equally important pathways in the developing brain. In addition, deletion of Usp9x in mice has been found to have immense effects on the postnatal development of the hippocampus. Recently, a study reported that Usp9x-null mice had learning and memory deficits, similar to that seen in ID patients with USP9X mutations. However, to date, there has been no extensive study into how the deletion of Usp9x in mice affects cortical development nor investigated if any ASD-related behaviour manifested from the deletion. Therefore, this study set out to address those questions by deleting Usp9x from the dorsal telencephalon of mice and used them as a model system to investigate the role of Usp9x in cortical development and behaviour.
Adult male mice where Usp9x has been deleted (Usp9x-/y) and their control littermates (Usp9x+/y) were used in this project. Firstly, the adult neocortex was characterised using haematoxylin staining, where measurements were made in several areas of the neocortex. Results indicate that the neocortex were minimally affected with the deletion of Usp9x; the thickness of most areas remained not significantly different to controls, barring the retrosplenial cortex being thicker in Usp9x-/y mice compared to Usp9x+/y mice. However, the deletion of Usp9x lead to a significantly smaller corpus callosum compared to controls. Following that, the axonal initiation ability of neurons lacking Usp9x was investigated by culturing neurons from embryonic mouse cortices. Usp9x-/y cortical neurons were found to grow axons normally, comparable to the controls.
To characterise any changes in behaviour as a result of deleting Usp9x from the dorsal telencephalon, Usp9x-/y mice were subjected to a battery of behavioural assays. The first set of assays measured anxiety-related behaviour, including open-field test, elevated plus maze (EPM), light/dark box, and long-term readings using the Phenomaster. The results showed that Usp9x-/y mice displayed hyperactivity and low anxiety behaviour in short-term open field, EPM, and light/dark tests. However, Usp9x-/y mice also showed high anxiety-like traits in the home-cage Phenomaster readings, indicating that Usp9x-/y mice may have chronic, long-term anxiety. Next, Usp9x-/y mice were subjected to several tests that measured the three diagnostic traits of ASD, impaired communication, deficits in social interaction, and repetitive behaviour. To test for communication, Usp9x-/y pups were recorded for their ultrasonic vocalisations (USVs). Usp9x-/y pups were found to have changes in communication after being separated from the dams. Next, Usp9x-/y mice were tested for any impairments in their socialisation skills. The mice were subjected to the standard three-chambered sociability test and a five-day social habituation/dishabituation test. Results from both tests indicated that Usp9x-/y mice have low social interest, confirming one of the most indicative traits of ASD is present in Usp9x-/y mice. However, Usp9x-/y mice did not display any repetitive behaviour when tested using the Y-maze. Despite that, based on these results, the morphological and behavioural phenotypes seen in the Usp9x-/y mouse model correspond to the symptoms seen in the human patients. This study shows that Usp9x-/y mouse model has the potential to be used as a model organism to explore therapeutic avenues, such as drug or compound screening.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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Lionnard, Loïc. "Régulation de la stabilité de la protéine anti-apoptotique BCL2A1." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT003/document.
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L’apoptose ou mort cellulaire programmée joue un rôle prépondérant dans l’homéostasie cellulaire. Ce processus est très finement régulé par les protéines de la famille BCL-2 qui contrôlent la perméabilité de membrane mitochondriale externe et la libération du cytochrome c, deux événements majeurs précédant la mort cellulaire. Les protéines anti-apoptotiques de la famille BCL-2 contribuent à la tumorigenèse et sont impliquées dans la résistance des cancers aux molécules chimiothérapeutiques ; à ce titre, elles représentent des cibles importantes pour le développement de nouvelles thérapies. BCL2A1 est un membre anti-apoptotique de la famille BCL-2 impliqué dans la chimiorésistance de nombreuses tumeurs. La protéine BCL2A1 a pour caractéristique d’avoir une demi-vie courte due à sa dégradation constitutive par le système ubiquitine-protéasome. Ceci régule la stabilité et la fonction anti-apoptotique de BCL2A1 et représente un mécanisme suppresseur de tumeur majeur. Cependant, les enzymes qui contrôlent les modifications post-traductionnelles impliquées dans l’ubiquitination et la dégradation de BCL2A1 demeurent, à ce jour, inconnues. Dans la présente thèse, nous donnons un aperçu des acteurs et des mécanismes impliqués dans la régulation de l’ubiquitination de BCL2A1. Nous présentons des preuves que TRIM28 est une E3 ubiquitine-ligase pour BCL2A1. En effet, les protéines TRIM28 et BCL2A1 endogènes interagissent ensemble au niveau des mitochondries et la déplétion de TRIM28 diminue l’ubiquitination de BCL2A1. Nous montrons aussi que TRIM17 stabilise BCL2A1 en empêchant son interaction avec TRIM28 et son ubiquitination médiée par TRIM28, et que l’activité de GSK3 est impliquée dans l’inhibition de la dégradation de BCL2A1. Ainsi, BCL2A1 et son proche homologue MCL-1 sont régulés par des facteurs communs mais de façon opposé. Finalement, la surexpression de TRIM28 ou l’inactivation de TRIM17 diminue le niveau protéique de BCL2A1 et restaure la sensibilité des cellules de mélanomes aux thérapies utilisant des inhibiteurs de la kinase BRAF. Globalement, nos résultats décrivent un rhéostat moléculaire au sein duquel deux protéines de la famille TRIM régulent de façon antagoniste la stabilité de BCL2A1 et modulent ainsi la mort cellulaireApoptosis or programmed cell death plays a crucial role in tissue homeostasis and is regulated by the Bcl-2 proteins, which control mitochondria membrane permeability and cytochrome c release, two events that precede cell demise. Anti-apoptotic Bcl-2 family members can contribute to tumorigenesis and cause resistance to anti-cancer regimens, therefore representing important targets for novel therapeutics. BCL2A1 is an anti-apoptotic member of the BCL-2 family that contributes to chemoresistance in a subset of tumors. BCL2A1 has a short half-life due to its constitutive processing by the ubiquitin-proteasome system. This constitutes a major tumor-suppressor mechanism regulating BCL2A1 function. However, the enzymes involved in the regulation of BCL2A1 protein stability are currently unknown. Here we provide the first insight into the regulation of BCL2A1 ubiquitination. We present evidence that TRIM28 is an E3 ubiquitin-ligase for BCL2A1. Indeed, endogenous TRIM28 and BCL2A1 bind to each other at the mitochondria and TRIM28 knock-down decreases BCL2A1 ubiquitination. We also show that TRIM17 stabilizes BCL2A1 by blocking TRIM28 from binding and ubiquitinating BCL2A1, and that GSK3 is involved in the phosphorylation-mediated inhibition of BCL2A1 degradation. BCL2A1 and its close relative MCL1 are thus regulated by common factors but with opposite outcome. Finally, overexpression of TRIM28 or knock-out of TRIM17 reduced BCLA1 protein levels and restored sensitivity of melanoma cells to BRAF-targeted therapy. Therefore, our data describe a molecular rheostat in which two proteins of the TRIM family antagonistically regulate BCL2A1 stability and modulate cell death.Sommaire
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Andreazza, Simonetta. "Analysis of new genes controlling Drosophila melanogaster rest-activity rhythms." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA112314.
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Les mécanismes moléculaires contrôlant les rythmes circadiens sont conservés parmi les organismes des différents règnes (plantes, animaux et champignons). Ils se composent de boucles de rétroaction où un complexe d’activation transcriptionnelle, l’hétérodimère CLK/CYC chez la drosophile, entraîne l'expression des répresseurs de son activité, les gènes et protéines PER et TIM chez la mouche. De manière importante, la période de l'oscillateur dépend en grande partie par des mécanismes post-transcriptionnels qui régulent l’accumulation et l'activité des composantes positifs et négatifs de la boucle. Bien que de nombreux partenaires d'interaction modifiant les composants d'horloge de base ont déjà pu être isolés, le schéma reste encore incomplet. Dans le cadre de la recherche de nouveaux composants de cette horloge, nous avons réalisé un crible comportemental basé sur l'expression ciblée de transgènes ARNi dirigés contre la moitié du génome de Drosophila melanogaster. Cinquante-quatre nouveaux gènes putatifs ont pu être identifiés. Au cours de ce travail, j'ai étudié le rôle de deux d’entre eux, sélectionnés pour les forts défauts comportementaux de l'expression de leur transgène ARNi. Le gène CG12082 de la drosophile est l’orthologue de l’Ubiquitin-specific protéase 5 (USP5) chez l’homme. La dérégulation d’Usp5 retarde les oscillations de la protéine PER dans les neurones d'horloge et allonge la période d'activité locomotrice des mouches. Chez les mouches ARNi Usp5, des formes à haut poids moléculaire des protéines PER et TIM s'accumulent pendant le matin, alors qu’elles sont normalement dégradées chez les contrôles. On a pu montrer que Usp5 participe directement à la dégradation de la protéine PER, indépendamment de TIM. En accord avec le rôle décrit pour l’orthologue humaine, Usp5 serait susceptible de contrôler la dégradation des protéines par son activité de démontage des chaînes libres de polyubiquitine présents dans la cellule, qui peuvent entrer en compétition avec les protéines ubiquitinylées pour la reconnaissance au niveau du protéasome, bloquant leur dégradation. La majorité des travaux ont porté sur un gène isolé au cours de notre crible, Strip, dont les fonctions étaient encore inconnues. Strip interagit avec Cka, une nouvelle sous-unité régulatrice de l’enzyme phosphatase PP2A. La dérégulation à la fois de Strip et/ou de Cka amène à des phénotypes comportementaux de période longue. D’un point de vue moléculaire, des formes hyper-phosphorylées de la protéine CLK s’accumulent dans la matinée quand Cka et/ou Strip sont perturbées. La dérégulation des activités générales de PP2A produit également une hyper-phosphorylation de CLK le matin, indiquant que, grâce à Cka/Strip, les complexes PP2A contrôlent la déphosphorylation de CLK à la fin du cycle. Il est connu que les formes hyper-phosphorylés de CLK sont transcriptionnellement inactives. En effet, la transcription des gènes tim et vrille, cibles de CLK, est fortement réduite dans les mouches ARNi Cka. En plus de PP2A/Cka, des complexes PP2A contenant une autre sous-unité régulatrice, Wdb, ont été montré pour déstabiliser CLK en culture des cellules (Kim et Edery, 2006). Nous montrons que la dérégulation de Wdb affecte la stabilité du CLK également dans la mouche adulte, sans toutefois induire aucun effet apparent sur sa phosphorylation. En conclusion, deux complexes PP2A différents agissent sur la protéine CLK : le complexe PP2A/Cka/Strip contrôle la déphosphorylation de CLK et sa réactivation, tandis que PP2A/Wdb affecte la stabilité de CLK indépendamment ou après PP2A/Cka. Ces résultats enrichissent l’étude de la régulation post-traductionnelle de la protéine CLK, qui était largement mal connue.Pour conclure, cette étude a permis de décrire deux nouveaux composants de la boucle moléculaire qui contrôle les rythmes circadiens chez la mouche du vinaigre, Drosophila melanogasterThe molecular mechanism underlying circadian rhythms is conserved among organisms and consists of feedback loops where a transcriptional activating complex (the CLOCK (CLK)/CYCLE (CYC) heterodimer in Drosophila) drives the expression of the repressors of its activity (the period (per) and timeless (tim) genes and proteins in Drosophila). Importantly, the pace of the oscillator largely depends on post-transcriptional mechanisms that regulate the accumulation and activity of both the positive and negative components of the loop. A number of interacting partners that modify core clock components have already been isolated, but more are expected. Looking for new clock components, we set up a behavioral screen based on targeted expression of RNAi transgenes directed to half of the Drosophila genome. 54 putative new clock genes have been identified. Among them, some were independently reported to function within the fruit fly molecular clock, thus validating the screen. In this work, I investigated the circadian role of additional “positive” genes, selected for the strong behavioral defect induced by the expression of the corresponding RNAi. The CG12082 gene codes for the fruit fly ortholog of the human Ubiquitin-specific protease 5 (USP5). Downregulation of USP5 in clock cells lengthens the period of locomotor activity of flies as well as PER protein oscillations in clock neurons. High molecular weight forms of PER and TIM proteins accumulate during the morning after USP5 knockdown, while these forms are degraded in controls. In addition, TIM is not stabilized in the absence of PER, while PER still accumulate in the absence of TIM. Therefore, USP5 directly participates in the degradation of the PER protein and, later, of the TIM protein at the end of the cycle. Being a deubiquitinylase enzyme, USP5 may directly deubiquitinate PER. However, accordingly to the role described for the human ortholog, USP5 likely controls protein degradation through the disassembling of the unanchored polyubiquitin chains present in the cell that could compete with ubiquitinated-PER for proteasome recognition and subsequent breakdown.The majority of the work has focused on an unknown gene isolated in the screen, that, accordingly to the human homolog, we named STRIP. We show that STRIP interacts with Connector of Kinase to AP-1 (CKA), a novel regulatory subunit for the PP2A phosphatase holoenzyme, both in insect S2 cells and in fly head extracts. Downregulation of both STRIP and/or CKA causes long-period behavioral phenotypes and high molecular weight forms of the CLK protein to accumulate in the morning. Perturbation of general PP2A activities also produces hyper-phosphorylated CLK in the morning indicating that, through CKA/STRIP, PP2A complexes controls CLK dephosphorylation at the end of the cycle. Hyper-phosphorylated CLK forms are transcriptionally inactive. Accordingly, transcription of the tim and vrille (vri) CLK targets is strongly reduced in Cka-RNAi fly head extracts. PP2A complexes containing the Widerborst (WDB) regulatory subunits were already shown to affect CLK stability in insect S2 cells (Kim and Edery, 2006). We show that WDB downregulation also affects the stability of CLK in fly head extracts, but has no apparent effects on CLK phosphorylation. Therefore, we could describe two different PP2A complexes acting on the CLK protein: PP2A/CKA/STRIP complex controls CLK dephosphorylation and reactivation, while PP2A/WDB affects CLK stability independently or after PP2A/CKA functions. Moreover, STRIP, but not CKA, downregulation affects the stability of PER, indicating that STRIP possesses some functions unrelated to CKA. In conclusion, this work has allowed the isolation of new components of the Drosophila molecular clock. In particular, we give evidence for a double role for the PP2A phosphatase in modulating the activity and stability of the CLK protein, the regulation of which is not well understood yet
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Maghames, Chantal. "Regulation of proteotoxicity through atypical NEDDylation." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT041.
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Les cellules sont constamment exposées à des stress « protéotoxiques » qui altèrent leurs protéines. Si les protéines endommagées ne sont pas réparées ou éliminées, elles peuvent former des agrégats toxiques pouvant conduire à l’émergence de plusieurs maladies, telle que les maladies neurodégénératives et le cancer. Pour éviter cette toxicité, les cellules ont développé plusieurs stratégies qui collaborent et communiquent afin d'assurer le contrôle de qualité des protéines et maintenir l’intégrité du protéome cellulaire. L’ensemble de ces stratégies forment le réseau de l’homéostasie protéique ou « protéostasie ». Ce réseau inclus les chaperonnes moléculaires, les systèmes protéolytiques (lysosomes, protéasomes) et des systèmes de séquestration des protéines endommagées. L’Ubiquitine et les protéines apparentées à l’Ubiquitine telle que SUMO et NEDD8, sont des effecteurs essentiels de ce réseau. Ces molécules modifient leurs substrats de façon covalente, grâce à l’action d’une cascade d’enzymes E1, E2 et E3. En principe, on considérait que chacune de ces voies employait sa propre cascade enzymatique pour la modification post-traductionnelle de ses substrats. L’Ubiquitination joue un rôle essentiel dans la réponse au stress cellulaire, surtout en assurant la dégradation protéasomique des protéines mal repliées. Récemment, notre laboratoire a trouvé que plusieurs stress protéotoxiques telle que l’inhibition du protéasome, un choc thermique et un stress oxydatif, causent une augmentation de NEDDylation. De manière remarquable, cette augmentation ne dépend pas de l’enzyme d’activation de NEDD8 NAE, mais plutôt de celle de l’Ubiquitine Ube1. De plus, elle se caractérise par la formation des chaînes poly-NEDD8 et des chaînes mixtes entre NEDD8 et Ubiquitine. Ce processus est réversible et une restauration cellulaire est obtenue une fois le stress atténué. Le but de notre projet est de caractériser la réponse de NEDD8 au stress cellulaire ou ce qu’on appelle « la NEDDylation atypique » en vue de comprendre son effet biologique pendant ces conditions. Nos résultats montrent que la NEDDylation atypique dépend des protéines de stress Hsp70/90 et qu’elle cible principalement les protéines nouvellement synthétisées et mal repliées. On montre que, suite à leur modification par NEDD8/Ubiquitin, ces protéines sont transloquées du cytosol au noyau, où elles sont dégradées par le protéasome. Cependant, des conditions de stress prolongé causent une atténuation de l’activité nucléaire des protéasomes 26S, ce qui provoque alors l’accumulation des protéines endommagées sous forme d’inclusions nucléaires. Ces dernières sont réversibles et peuvent être éliminées par le protéasome une fois le stress atténué. Afin d’identifier les cibles de NEDD8 dans des conditions de stress, nous avons développé une approche protéomique basée sur une stratégie de mutation ponctuelle (NEDD8R74K). Cette stratégie permet l’identification des sites spécifiques de NEDDylation au sein des protéines cibles. Cette approche en combinaison avec le SILAC a permis l’identification de NEDD8, Ubiquitine, SUMO-2 et les protéines ribosomiques en tant que principales cibles de NEDD8 en réponse au stress. Ce qui était plus intéressant est que, en appliquant l’étude protéomique SILAC, on a pu constater que le rôle essentiel de la NEDDylation atypique est d’induire l’agrégation/séquestration d’un ensemble spécifique de protéines au sein des inclusions nucléaires. De plus, nous avons montré que l’agrégation induite par NEDD8 protège les protéasomes nucléaires d’une sévère déficience et permet une meilleure survie cellulaire pendant le stress. Notre étude présente NEDD8 comme un nouvel effecteur dans le réseau de protéostasie, elle identifie une nouvelle inclusion nucléaire cytoprotectrice et montre que la NEDDylation atypique est essentielle pour la réponse cellulaire au stressCells are continuously endangered by a variety of proteotoxic stresses that cause protein misfolding and accumulation. Defects in repair or elimination of protein damage can lead to the formation of toxic aggregates that have been associated with diseases, such as neurodegenerative disorders and cancer. To prevent this toxicity, cells have evolved multiple quality control processes that interact and cooperate to maintain protein homeostasis leading to cellular fitness. These processes form “the proteostasis network”, and include molecular chaperones, proteolytic machineries (lysosomes, proteasomes) and pathways for protein damage sequestration. One of the main effectors of this network is the Ubiquitin and the Ubiquitin-like molecules, such as SUMO and NEDD8. These molecules covalently modify proteins through the action of E1, E2 and E3 enzymes. Historically, it was believed that each pathway employed its own and unique set of enzymes to post-translationally modify its substrates. Ubiquitination is essential for the cellular response to stress, especially by targeting misfolded proteins for proteasomal degradation. However, we recently discovered that proteotoxic stresses including proteasome inhibition, heat shock and oxidative stress induce a global increase in protein NEDDylation. Surprisingly, this increase does not depend on the NEDD8 activating enzyme NAE, but rather on the Ubiquitin activating enzyme Ube1, and is characterized by the formation of poly-NEDD8 chains and mixed chains between NEDD8 and Ubiquitin. Importantly, this process is reversible and cell recovery is accomplished once stress is alleviated. In this study, we focused on characterizing the NEDD8 response to stress or “atypical NEDDylation” in order to understand its biological relevance under these conditions.Our results showed that atypical NEDDylation depends on Hsp70/90 and targets mainly newly synthesized damaged proteins. We showed that, after their NEDDylation/Ubiquitination, misfolded proteins are progressively translocated from the cytosol into the nucleus for proteasomal degradation. However, upon prolonged stress conditions, the activity of nuclear 26S proteasome is compromised, resulting in the accumulation of these conjugates into nuclear inclusions. These inclusions are reversible and eliminated by nuclear proteasomes once stress is alleviated. In order to identify NEDD8 targets upon these conditions, we developed a proteomic approach based on a point mutation strategy (NEDD8R74K) that enables a site-specific analysis of NEDDylated proteins. This approach in combination with SILAC allowed the identification of NEDD8, Ubiquitin, SUMO-2, and ribosomal proteins as the major NEDD8 targets upon stress. Interestingly, by SILAC proteomics we found that the main function of atypical NEDDylation is to induce the aggregation/sequestration of a specific subset of proteins within the nuclear inclusions. We showed that this NEDD8-induced aggregation protects nuclear proteasomes from a severe impairment and allows a better cell survival upon proteotoxic stress.Our study defines NEDD8 as a new effector in the proteostasis network, identifies a new cytoprotective nuclear inclusion and shows that atypical NEDDylation is essential for the cellular response to stress
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Rückrich, Thomas. "Einsatz synthetischer Aktivitätssonden im Ubiquitin-Proteasom-System : Analyse deubiquitinierender Proteasen und Charakterisierung Proteasominhibitor-adaptierter Zellen /." Tübingen, 2008. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000253092.
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Depaux, Arnaud. "Régulation des complexes d'ubiquitinylation et de sumoylation par la ligase E3 hSIAH2." Paris 7, 2006. http://www.theses.fr/2006PA077094.
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Les modifications post-traductionnelles des protéines (phosphorylation, l'acétylation ou l'ubiquitinylation) permettent de réguler leur activité, stabilité, localisation ou interactions avec d'autres facteurs. Les complexes permettant la modification par l'ubiquitine ou Sumo bien que d'organisation similaire sont composés de protéines différentes : une ligase El qui active le résidu, une ligase E2 permettant le transfert de l'ubiquitine sur le substrat et une ligase E3 qui assure la spécificité de reconnaissance du substrat. Plusieurs familles de ligases E3 ont été décrites mais seule la famille de protéines à domaine RING Finger présente des membres impliqués dans les complexes de la sumoylation et de l'ubiquitinylation. Afin de caractériser de nouveaux partenaires des ligases à domaine RING Finger hSIAHl et hSIAH2 (human Seven In Absentia homolog), nous avons développé une expérience de double-hybride chez la levure en utilisant hSIAH2 pour appât. La caractérisation des partenaires ainsi isolés a fait l'objet de mon projet de thèse. J'ai mis en évidence des protéines impliquées dans l'ubiquitinylation (Ubiquitine, Ubc5 ou hSIAH) et la sumoylation (PIAS, SUMO et Ubc9). J'ai ainsi démontré que hSIAH2 est capable de former des homodimères et des hétérodimères avec hSIAH et que cette dimérisation permet de réguler la propre stabilité des deux protéines. D'autre part, j'ai montré que hSIAH2 catalyse l'ubiquitinylation de PIAS et sa dégradation par le protéasome. L'ensemble de ce travail a mis en évidence le rôle spécifique de hSIAH2 dans la régulation de la stabilité d'intermédiaires essentiels, à la fois, aux complexes d'ubiquitinylation et de sumoylationAfter synthesis, proteins are targeted to post-translational modifications such as acetylation, phosphorylation or ubiquitination. These mechanisms regulate their function, stability, localization or interaction with partners. Modification process by ubiquitin or sumo named ubiquitination or sumoylation respectively involve complexes with similar organization but compose of different enzymes. Their organization relies on Sumo or ubiquitin activating El enzyme, transferring E2-ligase and E3-ligase or sub-complex conferring the substrate specific récognition. El-ligase is unique for each complex, whereas E2 and E3-ligases are multiple. Among E3-ligase families, RING Finger protein family only has been involved in both modifications complexes. Two human homologs of Drosophila Seven In Absentia (hSIAHl et hSIAH2), belong to RING Finger E3-ligase family. In a yeast two hybrid assay, we have identified new SIAH interacting proteins. Their characterization has been the purpose of my PhD project. We have characterized partners implicated in both ubiquitination (ubiquitin, Ubc5 or hSIAH) and sumoylation (Sumo, Ubc9 and PIAS) pathways. In a first attempt, I have demonstrated that hSIAH proteins can form homo- or hetero-dimers. Dimerization régulates their stability via a proteasome dependent degradation. I have also demonstrated that hSIAH2 catalyzes the proteasome dependent degradation of PIAS1, a sumo E3-ligase. Altogether this study evidences an important rôle for hSIAH2 in the regulation of the stability of ubiquitination and sumolation complexes
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Boehringer, Jonas. "Ubiquitin recognition by the proteasome." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:036151d9-5062-4ffe-a1ef-a58e1b020680.
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The ubiquitin proteasome system targets proteins to the proteasome where they are degraded. Substrate recognition and processing prior to degradation take place at the 19S regulatory particle of the proteasome. A polyubiquitin chain, linked through isopeptide bonds formed between the C-terminal G76 and K48, is the signal responsible for delivery to the proteasome. Because chains linked via any of the seven lysine residues of ubiquitin exist in vivo and encode signals unrelated to protein degradation it is crucial for cells to avoid crosstalk between these different pathways. Several ubiquitin receptors related to proteasomal degradation have been identified but the selectivity between the different ubiquitin chains has not been assessed quantitatively while avoiding artefacts attributed to GST-dimerisation. By employing isothermal titration calorimetry, analytical ultracentrifugation and nuclear magnetic resonance, discrimination between K48- and K63-linked diubiquitin was established for the S. pombe proteasomal receptor Rpn10 and the shuttle protein Rhp23. The same methods allowed us to propose a discriminatory model for Rpn10. The crystal structures of the 19S regulatory particle subunits Rpn101-193 and Rpn121-224 have been determined and possible protein-protein interaction sites were identified by surface conservation and electrostatics analysis. Rpn12 surface residues were identified that had a negative effect on Rpn10-binding. This interaction was studied by surface plasmon resonance, fluorescence anisotropy and nuclear magnetic resonance. These experiments revealed a binding site on Rpn10 that is exclusively occupied by either ubiquitin or Rpn12 and for the first time demonstrated the interaction of a ubiquitin interacting motif with a protein other than ubiquitin.
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Shi, Yuan. "Ubiquitin Recognition by the Proteasome." Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13070024.
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Ubiquitin proteasome pathway is an important cellular pathway that affects the fate of almost all intracellular proteins. Misregulation of this pathway has been found to be associated with a broad range of human diseases, such as cancer, neurodegenerative diseases, as well as viral infections. Ubiquitin recognition by the proteasome is of central importance to this pathway. So far, two proteasome subunits, Rpn10 and Rpn13, have been identified as ubiquitin receptors. An alternative pathway is mediated by shuttling factors. In yeast, three shuttling factors, known as UBL-UBA proteins, have been found. A UBL receptor activity of the proteasome has been attributed to Rpn1. However, yeast cell mutated all five proteasomal ubiquitin receptors is still viable.
To identify the additional proteasomal ubiquitin receptor in cells, I first obtained and characterized a new Rpn13 mutant allele. This Rpn13 mutant completely abolished its ubiquitin binding activity, and functionally resembles a null allele. Rpn13 substrate pool has also been sought in this mutant cells.
In the second part of this dissertation, I reported a novel ubiquitin binding site on proteasomal subunit Rpn1. With the help of NMR analysis, Rpn1's ubiquitin and UBL binding surfaces were resolved at high resolution and found to substantially overlap. A specific Rpn1 mutation that disrupts both ubiquitin and UBL binding while not compromising the folding of Rpn1 was obtained. This mutant allele shows a pleiotropic proteasomal defect in vivo. Moreover, I found that the dual ubiquitin/UBL binding activity is not unique in Rpn1, but a common feature in all three proteasomal ubiquitin receptors. In summary, the proteasome adopts a multilayer ubiquitin/UBL binding surface to ensure flexible substrate recognition.
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Viargues, Perrine. "L'ubiquitination et le trafic endocytaire régulent la réponse immunitaire de la drosophile." Thesis, Grenoble, 2013. http://www.theses.fr/2013GRENV040/document.
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Le système immunitaire inné repose sur la détection de motifs microbiens et l'activation de réponses adaptées, parmi lesquelles les voies de signalisation dépendantes des facteurs NF-κB jouent un rôle primordial. Ces voies sont finement régulées afin d'éviter une réponse immunitaire excessive et soutenue dans le temps qui peut causer de nombreuses pathologies, comme les maladies auto-immunes et pro-inflammatoires. Au cours de ma thèse, j'ai élucidé certains mécanismes de régulation des voies de signalisation NF-κB, Toll et IMD, chez la drosophile, qui reposent sur l'ubiquitination de protéines et leur dégradation par la voie endocytaire ou le protéasome. L'ubiquitination réversible des protéines est une modification post-traductionnelle qui permet de réguler leur activité, leur stabilité et leur localisation subcellulaire. En particulier, l'ubiquitination des récepteurs membranaires peut servir de signal d'endocytose et de dégradation lysosomale. Chez la drosophile, le récepteur PGRP-LC reconnaît spécifiquement le peptidoglycane (PGN) bactérien de type acide diaminopimélique et induit la voie de signalisation IMD. J'ai montré que PGRP-LC est ubiquitiné, internalisé et dégradé par la voie endocytaire. Dans ce processus, j'ai identifié le rôle majeur de la déubiquitinase USP8 qui contrôle la dégradation de PGRP-LC ubiquitiné. J'ai aussi mis en évidence que la stimulation de la voie IMD par les PGN augmente l'internalisation et la dégradation de PGRP-LC, assurant l'élimination des récepteurs après que la voie IMD ait été activée. En outre, j'ai participé à des études visant à comprendre le rôle des déubiquitinases USP2, USP34 et USP36, préalablement sélectionnées par l'équipe comme des régulateurs négatifs des voies IMD et/ou Toll. Mes résultats ont notamment contribué à montrer que USP2 agit principalement au niveau de la protéine adaptatrice Imd, en permettant l'hydrolyse de ses chaînes d'ubiquitine K48 et sa dégradation par le protéasome. Finalement, j'ai observé que USP2 interagit également avec PGRP-LC et favorise l'hydrolyse des chaînes K48 associées à ce récepteur, bien que dans ce cas, la dégradation des formes poly-ubiquitinées K48 de PGRP-LC ne dépende pas du protéasome, mais des protéines de la voie endocytaire Hrs, Rab5 et de la déubiquitinase USP8The innate immune system relies on the recognition of “non-self” and on the activation of adapted responses, among which NF-κB signaling pathways play a crucial role. These pathways are tightly regulated, in order to prevent an excessive and sustained immune response, responsible for several pathologies, such as autoimmune and pro-inflammatory diseases. During my PhD thesis, I elucidated some Drosophila regulatory mechanisms of NF-κB pathways, Toll and IMD, which rely on protein ubiquitination and their subsequent degradation by the endocytic pathway or proteasome. Reversible ubiquitination of proteins is a post-translational modification, regulating their activity, their stability and the subcellular localization. In particular, ubiquitination of membrane receptors could trigger their internalization and their subsequent lysosomal degradation. In Drosophila, the PGRP-LC receptor specifically recognizes diaminopimelic acid containing peptidoglycan (PGN) and induces the IMD signaling pathway. I proved that PGRP-LC receptor is ubiquitinated, internalized and degraded by the endocytic pathway. In this process, I identified the major role of the USP8 deubiquitinating enzyme, which controls the degradation of ubiquitinated PGRP-LC. Besides, I showed that the IMD stimulation by PGN enhances the PGRP-LC internalization and its degradation, ensuring receptors elimination once the IMD pathway has been activated. Moreover, I took part to studies, aiming to understand the role of USP2, USP34 and USP36, previously selected by the team as negative regulators of the IMD and/or Toll pathways. In particular, my results showed that USP2 principally acts at the Imd level, allowing for the hydrolysis of its K48 poly-ubiquitin chains and its proteasomal degradation. Finally, I observed that USP2 also interacts with PGRP-LC and favors the hydrolysis of PGRP-LC associated K48 chains, whereas the degradation of K48 poly-ubiquitinated PGRP-LC is independent from the proteasome, but rather depends on the Hrs and Rab5 endocytic proteins and on the USP8 deubiquitinating enzyme
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Ligr, Martin. "Apoptosis in the yeast Saccharomyces cerevisiae a novel cell death process regulated by the Ubiquitin-Proteasome system /." [S.l. : s.n.], 2001. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB9203728.
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Regelmann, Jochen. "Katabolitinaktivierung der Fructose-1,6-bisphosphatase: Identifizierung und Charakterisierung neuer, für ihren Ubiquitin-Proteasom-katalysierten Abbau benötigter Proteine." [S.l. : s.n.], 2005. http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-25854.
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Gupta, Nilaksh. "UBIQUITIN-PROTEASOME SYSTEM MODULATES PLATELET FUNCTION." Cleveland State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=csu1408896695.
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Bingol, Baris Zinn Kai George. "Ubiquitin-proteasome system at the synapse /." Diss., Pasadena, Calif. : Caltech, 2006. http://resolver.caltech.edu/CaltechETD:etd-05272006-184911.
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Dreger, Henryk. "Suppression der Hypertrophie kardialer Myozyten durch Inhibition des Ubiquitin-Proteasom-Systems." Doctoral thesis, [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969239076.
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Rieger, Melanie. "Strukturelle und funktionelle Anpassung des Ubiquitin-Proteasomsystems an IFN-gamma." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15888.
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Das Ubiquitin-Proteasom-System ist an der Degradation cytosolischer Proteine und der Generierung von Antigenen beteiligt, die über MHC Klasse I Moleküle CD8+ T Zellen präsentiert werden. Die Antigenprozessierung wird durch Typ I und II Interferone beeinflusst, welche die Formierung des Immunoproteasoms und des Proteasomen-Aktivators PA28 induzieren und so die katalytische Aktivität des Ubiquitin-Proteasom-Systems qualitativ verändern. In der vorliegenden Arbeit wurde im Zellkulturmodell unter dem Einfluss von IFN gamma die zunehmende Inkorporation der Immunountereinheiten in de novo assemblierende 20S Proteasomen und die daraus resultierende Veränderung der proteolytische Aktivität untersucht. Die Inkorporation der Immunountereinheiten wurde mittels 2D Gelelektrophorese und Western Blots von 20S Proteasomen untersucht, die nach unterschiedlicher Stimulationsdauer mit IFN gamma aus HeLa Zellen isoliert wurden. Es konnte gezeigt werden, dass innerhalb der ersten 24h einer IFN gamma Stimulation die strukturelle Heterogenität des zellulären Proteasomenpools zunimmt, indem sowohl intermediäre als auch Immunoproteasomen assemblieren. In der Nativ-PAGE von Lysaten IFN gamma stimulierter Zellen wurde eine Zunahme des 20S Proteasoms als freier Komplex und in Assoziation mit PA28 beobachtet, während die Menge des zum ATP-abhängigen Abbau von polyubiquitinierten Proteinen notwendigen 26S Proteasoms unverändert blieb. Die Stimulation mit IFN gamma hatte eine Steigerung der gesamtproteasomalen Aktivität zur Folge, die unter Inhibition der Interaktion zwischen 20S Proteasom und PA28 verzögert erfolgte. Die katalytischen Eigenschaften isolierter Proteasomen wurden anhand der Generierung eines immunrelevanten Hepatitis C CTL Epitops des viralen Core Proteins in vitro untersucht. Im Verlauf der IFN gamma Stimulation de novo assemblierte Proteasomen wiesen jeweils unterschiedliche Präferenzen für die Generierung des untersuchten CTL Epitops auf. Eine weitere, proteasomen-spezifische Änderung der katalytischen Aktivität bewirkte die Assoziation des Proteasomen-Aktivators. Innerhalb der ersten zwölf Stunden einer IFN gamma Stimulation wurde das Epitop vermehrt mit der Unterstützung des Proteasomen-Aktivators generiert, nach 24 Stunden zunehmend durch freies 20S Proteasom. Die Ergebnisse der vorgestellten Arbeit zeigen, dass Strukturvarianten des Proteasoms zusammen mit PA28 redundant funktionieren und eine hohe proteolytische Plastizität des UPS gewährleisten.The ubiquitin proteasome system is responsible for the degradation of cytosolic proteins and the processing of MHC class I restricted antigens. The generation of these antigens is influenced by type I and II interferons which induce the expression of immunoproteasomes and the proteasome activator PA28; and thereby impact the quality of peptides processed by the proteasome system. The adoption of the proteasome system to a proinflammatory environment has been investigated in a cell culture model by isolating proteasomes after different stages of IFN gamma stimulation. The composition of isolated proteasomes was analysed by 2D PAGE and western blot approach. The presented work shows that within 24h of IFN gamma stimulation an increasing heterogeneity of the cellular proteasome pool is observed, resulting from the assembly of both intermediate type proteasomes and immunoproteasomes at the early stage of IFN gamma stimulation. It could be shown by native PAGE of HeLa cell lysates that IFN gamma induces increasing amounts of 20S proteasomes and PA28 associated proteasomes without decreasing the amount of 26S proteasomes that are necessary for the ATP dependent degradation of ubiquitinated proteins; and resulting in an enhanced total proteasomal activity in vitro. This increase in activity was delayed when the interaction of 20S proteasomes and PA28 was inhibited. A comparative analysis of the ability of isolated 20S proteasomes to generate a known hepatitis C virus derived CTL epitope in vitro proved that during early IFN gamma stimulation de novo assembled proteasomes exhibited a structure specific preference to generate the HCV CTL epitope either alone or in combination with the proteasome activator PA28. Within the first 12h of IFN gamma stimulation the epitope was generated with higher efficiency by 20S proteasomes in association with PA28, whereas after 24h the impact of PA28 on the proteasome pool was less pronounced. The presented work shows that IFN gamma induces a heterogeneity of 20S proteasomes in the early stage of stimulation, acting in combination with the proteasome activator in a redundant manner; and provides a high proteolytic placticity of the proteasome system.
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Didier, Robin. "Implication des enzymes de déubiquitination associés au protéasome dans la pathogénie du mélanome." Thesis, Université Côte d'Azur (ComUE), 2018. http://www.theses.fr/2018AZUR4219/document.
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Le mélanome cutané est un cancer très agressif, responsable de 80% des décès liés aux cancers de la peau. Le mélanome métastatique (MM) est souvent résistant à la radiothérapie et aux chimiothérapies. Sa progression est majoritairement initiée par des mutations oncogéniques des gènes BRAF et NRAS activant la voie de prolifération MEK/ERK. Le MM est difficile à traiter malgré le succès de nouveaux traitements (thérapies ciblant l’oncogène BRAFV600E et immunothérapies), qui sont cependant limités à certains patients. De plus l'émergence de résistances ne permet pas d’obtenir une réponse durable, ce qui incite à rechercher de nouvelles cibles tumorales. Dans les cellules cancéreuses, l’accumulation d’altérations génétiques et le fort index prolifératif accroissent leur addiction aux mécanismes de contrôle de la qualité du protéome, comme le système ubiquitine-protéasome (UPS). L’UPS comprend une machinerie protéolytique (le protéasome 26S) et un réseau d’enzymes régulant l’ubiquitination de protéines cibles. La réaction enzymatique de retrait de l’ubiquitine est la déubiquitination, réalisés par de protéases spécifiques appelées DéUBiquitinases (DUBs). Malgré l’importance des DUBs dans de nombreuses situations pathologiques comme le cancer, leur implication dans la physiopathologie du mélanome est mal connue. Afin d’identifier des DUBs dont l’activité est modulée dans le mélanome, nous avons utilisé une méthode d’étiquettage biochimique in vitro des DUBs actives (‘’DUB trap assay’’) qui nous a permis d’identifier USP14 (Ubiquitin Specific Protease 14) dont l’activité est augmentée dans nos lignées de mélanome par rapport aux mélanocytes. USP14 est associée physiquement au protéasome, avec un rôle important sur la protéostasie cellulaire en général. L’analyse de données bioinformatiques publiques confirme l’importance de USP14 dans le mélanome en associant l’expression du gène USP14 à la progression du mélanome et à un mauvais pronostic. Nous avons ensuite montré que cibler USP14 par des approches génétique (siRNA) ou pharmacologique (inhibiteurs de l’activité) a un effet anti-mélanome in vitro et in vivo, associé à une accumulation de protéines polyubiquitinées, générant un stress du réticulum endoplasmique, la dépolarisation de la mitochondrie et une production de ROS, aboutissant à une mort indépendante des caspases. Cet effet cytotoxique est obtenu indépendamment du statut mutationnel des protéines oncogéniques (BRAFV600E, NRAS, NF1), des suppresseurs de tumeurs (TP53, PTEN), du niveau de résistance aux thérapies ciblées ou du statut phénotypique des mélanomes. Ces résultats indiquent que USP14 représente une nouvelle cible thérapeutique pertinente dans le mélanome. Dans la continuité de ces travaux, j’ai cherché à identifier d'autres DUBs pouvant jouer un rôle dans la prolifération et la survie des cellules de mélanome en réalisant le criblage d'une banque de siRNA ciblant 90 DUBs sur une lignée de cellules de mélanome. Outre le fait de confirmer l’implication de USP14 dans la prolifération du mélanome, ce criblage génétique révèle que la déplétion d’une autre DUB associée au protéasome a un puissant effet antiprolifératif sur les cellules de mélanome. Nos travaux préliminaires montrent que le ciblage de cette nouvelle DUB se traduit par un arrêt de prolifération suivi d’une mort cellulaire associée à des dommages à l’ADN in vitro et in vivo. Dans l’ensemble, mes travaux de thèse révèlent un rôle essentiel des DUBs associées au protéasome dans la prolifération et la survie du mélanome, et ouvrent la piste à de nouvelles stratégies thérapeutiques ciblant les mécanismes aberrants de la protéostasie tumorale de ce cancerNon communiqué
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Menéndez, Benito Victoria. "The ubiquitin-proteasome system during proteotoxic stress /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-706-5/.
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Ghannam, Khetam [Verfasser]. "Ubiquitin proteasome system and myopathies / Khetam Ghannam." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2015. http://d-nb.info/1075493374/34.
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Min, Mingwei. "Decoding the mitotic exit ubiquitin-proteasome system." Thesis, University of Cambridge, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708388.
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Jolly, R. S. "The ubiquitin proteasome system in Huntington's disease." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444454/.
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Huntington's disease (HD) is an autosomally dominant, progressive movement disorder, caused by an expansion in the polyglutamine tract of huntingtin protein. HD is pathologically characterised by the presence of insoluble, proteinaceous neuronal intranuclear inclusions (NHs) and dystrophic neurite inclusions (DNIs) in affected neurons that can be immunostained for ubiquitin and other proteins involved in the ubiquitin-proteasome system (UPS). The UPS is a highly conserved mechanism for degradation of both normal and misfolded proteins in eukaryotic cells. This has led to suggestions that the UPS is inhibited in HD. This study utilises microscopy, biochemistry and fluorometric assays to examine the molecular composition of aggregates and the potential dysfunction of the UPS in the R6/2 mouse line, an established model of HD. The ultrastructure of aggregates is shown to be predominantly amorphous and granular in appearance and likely to be formed through the process of transglutamination. Immunohistochemical data shows that certain chaperones, ubiquitin-like proteins (UBLs) and proteins involved in the UPS localise to Nils and DNIs differentially. Fluorometric assays demonstrate that the proteasome exhibits a differential profile in R6/2 mice where both chymotrypsin-like and PGPH-like activities are markedly increased whilst trypsin-like activity is decreased relative to litter-mate control mice. Furthermore, these activity changes may be explained by alterations in proteasome regulation, levels and maturation. These results suggest that, in the R6/2 line, the proteasome is not inhibited by the presence of mutant huntingtin, rather that there are alterations of the catalytic activities of the proteasome. It appears that Nil's act not only as focal points of proteolysis, but also of proteasome biogenesis. This is consistent with, and extends, the concept of clastosomes.
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Ulbricht, David, Jan Pippel, Stephan Schultz, René Meier, Norbert Sträter, and John T. Heiker. "A unique serpin P1′ glutamate and a conserved β-sheet C arginine are key residues for activity, protease recognition and stability of serpinA12 (vaspin)." Portland Press, 2015. https://ul.qucosa.de/id/qucosa%3A33439.
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SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1′) of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1′ glutamate (Glu379) with an arginine residue (Arg302) of the β-sheet C. A P1′ glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1′ residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin–protease interaction. Arg302 is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1′ Glu379, which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu379 and Arg302 influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin.
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Keller, Martin. "Die Modulation des Ubiquitin-Proteasom-Systems als Immunevasionsmechanismus des malignen Melanoms." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15960.
Full textAbstract:
Die effiziente Präsentation von Tumorepitopen stellt einen kritischen Faktor zur Eliminierung von Tumorzellen durch die CD8+ cytotoxische T-Lymphozyten (CTL) vermittelte Immunantwort dar. Eine wichtige Rolle spielt in diesem Zusammenhang die effiziente Generierung von Tumorepitopen durch das Ubiquitin-Proteasom-System (UPS). Veränderungen von Komponenten des UPS, die an der Degradation und Prozessierung von Antigenen beteiligt sind, können daher zur Immunevasion von Tumorzellen gegenüber CTL führen. In der vorliegenden Arbeit wurden zwei unterschiedliche UPS-assoziierte Immunevasionsmechanismen des malignen Melanoms identifiziert, die auf einer ineffizienten Präsentation des immundominanten Tumorepitops Melan-A26-35 basieren. Ein Mechanismus beruht auf den unterschiedlichen katalytischen Eigenschaften von Proteasomsubtypen, deren Expression durch INFgamma induziert wird. Proteasomen, die einerseits die Immunountereinheiten beta1i und/oder beta2i beinhalten, oder anderseits mit dem Proteasomaktivator 28 (PA28) assoziiert sind, führen zu einer drastisch reduzierten Generierung des Tumorepitops Melan-A26-35. In beiden Fällen ist dies auf eine ineffiziente Prozessierung des N-Terminus des Epitops zurückzuführen. Der andere Immunevasionsmechanismus steht im Zusammenhang mit der ER-assoziierten Degradation (ERAD), die den retrograden Transport von Proteinen aus dem Endoplasmatischen Retikulum (ER) ins Cytosol zur proteasomalen Degradation beinhaltet. Durch Immunselektion mittels Melan-A26-35-spezifischen CTL wurden cytolyseresistente Melanomzellen identifiziert, deren Resistenz auf eine defiziente ER-assoziierte Degradation zurückzuführen ist. Dieser Defekt beruht auf einer verminderten Expression von ERAD-Komponenten, deren Reduktion die Verfügbarkeit des Antigens Melan-A zur proteasomalen Degradation und Generierung des immundominanten Epitops Melan-A26-35 wesentlich limitiert.Efficient presentation of tumor epitopes by MHC class I molecules on the cell surface is a prerequisite for the elimination of tumor cells by cytotoxic CD8+ T lymphocytes. The generation of these epitopes requires the degradation and processing of proteins by the ubiquitin proteasome system (UPS). Therefore alterations of UPS components can lead to tumor escape from immune recognition as a result of decreased epitope generation. In the present thesis two different UPS connected immune escape mechanisms of melanoma cells were identified. Both are based on an impaired generation of the immunodominant epitope Melan-A26-35 derived from Melan-A/MART-1 tumor antigen. One mechanism is mediated by the expression of different INFgamma-inducible proteasome immunosubunits leading to the formation of intermediate proteasome subtypes, which differ in their cleavage site preferences. Purified proteasomes harboring the immunosubunits beta1i and/or beta2i show a dramatic decrease in the generation of the Melan-A26-35 epitope. In addition, the INFgamma induced association of proteasomes with the proteaosome activator 28 (PA28) results in a reduced epitope generation. Both mechanisms are induced by an inefficient processing of the epitope’s N-terminus. The second immune escape mechanism is caused by defects of the ER-associated degradation pathway (ERAD). ERAD mediates the transport of ER-proteins back to the cytosolic compartment for proteasomal degradation. Via immunselection of tumor cells with Melan-A26-35 specific CTL, cytolysis resistant cells were identified. Resistance to CTL mediated lysis was shown to be connected to a decreased expression of ERAD components. This defect of the ERAD pathway limits the availability of the Melan-A protein and as consequence the generation of the immunodominant Melan-A26-35 epitope by proteasomes.
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Sotzny, Franziska. "Regulation des Ubiquitin-Proteasom-Systems unter proteotoxischem Stress." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2016. http://dx.doi.org/10.18452/17599.
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Das Ubiquitin-Proteasom-System (UPS) stellt eines der wichtigsten zellulären Abbausysteme dar. Es vermittelt die Degradation fehlgefalteter, beschädigter sowie regulatorischer Proteine. Folglich ist es essentiell für die Proteinqualitätskontrolle und für eine Vielzahl zellulärer Prozesse. Eine Störung des UPS steht im engen Zusammenhang mit neurodegenerativen Erkrankungen und malignen Tumoren. Adaptive Mechanismen ermöglichen es der Zelle das UPS an den stetig schwankenden Bedarf proteolytischer Aktivität anzupassen. So wirkt eine erhöhte Expression proteasomaler Gene einem Abfall der proteasomalen Aktivität entgegen. Der Transkriptionsfaktor TCF11/Nrf1 wurde hierbei als Hauptregulator identifiziert. Unter physiologischen Bedingungen ist TCF11/Nrf1 in der ER-Membran lokalisiert und wird über das ER-assoziierte Degradationssystem (ERAD) abgebaut. In Antwort auf Proteasominhibition wird der Transkriptionsfaktor aktiviert und in den Nukleus transferiert. Hier vermittelt er durch Bindung der regulatorischen antioxidative response elements die Genexpression proteasomaler Untereinheiten. Die Ergebnisse dieser Arbeit zeigten, dass es sich bei diesem autoregulatorischen Rückkopplungsmechanismus um einen generellen adaptiven Regulationsmechanismus in Mammalia handelt. Zudem ergaben weitere Untersuchungen, dass der durch Proteasominhibition hervorgerufene oxidative Stress, die TCF11/Nrf1-vermittelte Aktivierung der Genexpression fördert. Die induzierende Wirkung von oxidativem Stress wurde ferner unter Verwendung des Pro-Oxidans Rotenon bekräftigt. Dieses Neurotoxin induziert die TCF11/Nrf1-abhängige Transkription proteasomaler Untereinheiten und folglich die Neubildung aktiver Proteasomkomplexe. Der Transkriptionsfaktor förderte ferner die Zellviabilität Rotenon-behandelter SH-SY5Y Zellen. Diese Ergebnisse demonstrieren, dass die TCF11/Nrf1-vermittelte Genexpression proteasomaler Untereinheiten bedeutend für die Aufrechterhaltung der Redox- sowie der Protein Homöostase ist.The ubiquitin proteasome system (UPS) represents a major protein degradation machinery. It facilitates the degradation of misfolded and damaged as well as regulatory proteins, thereby ensuring protein quality control and regulation of various cellular processes. Disturbances of the UPS are strongly associated with neurodegeneration and cancer. Adaptive mechanisms enable the cell to deal with changing demand in proteolytic activity. A rise in proteasomal gene expression compensates for decreased proteasomal activity. This adaption is mainly regulated by the transcription factor TCF11/Nrf1. Under unstressed conditions TCF11/Nrf1 resides in the ER-membrane where it is degraded via the ER-associated protein degradation system (ERAD). Proteasome inhibition causes the nuclear translocation of TCF11/Nrf1. In the nucleus, it mediates the gene expression of proteasomal subunits by interacting with their regulatory antioxidant response elements. Within this thesis, it was shown, that this autoregulatory feedback loop represents a general adaptive mechanism in mammalian cells. Moreover, experiments using antioxidative compounds revealed, that the oxidative stress induced by proteasomal inhibition promotes the TCF11/Nrf1-dependent proteasomal gene expression. The inducing effect of oxidative stress was verified using the pro-oxidant rotenone. This neurotoxin activates the transcription of the proteasomal genes resulting in the formation of newly synthesised, active proteasome complexes. Thus, TCF11/Nrf1 exerts a cytoprotective function in response to oxidative and proteotoxic stress in SH-SY5Y cells. In conclusion, this thesis revealed that TCF11/Nrf1-dependent induction of the proteasome expression promotes the maintenance of the redox as well as protein homeostasis.
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Steffen, Janos. "Regulation des Ubiquitin-Proteasom-Systems in Säugetierzellen durch den Transkriptionsfaktor TCF11." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2010. http://dx.doi.org/10.18452/16184.
Full textAbstract:
Das Ubiquitin-Proteasom-System (UPS) ist das wichtigste System für den Abbau von nicht mehr benötigten oder beschädigten Proteinen innerhalb der eukaryotischen Zelle und ist somit an der Aufrechterhaltung der zellulären Homöostase beteiligt. Ein Abfall der proteasomalen Aktivität führt zu intrazellulärem Stress. Die Zelle wirkt diesem Abfall entgegen, indem sie die proteasomalen Gene verstärkt exprimiert und dadurch die Neubildung von 26S Proteasomen bewirkt. Während in der Bäckerhefe Saccharomyces cerevisiae mit Rpn4 der Transkriptionsfaktor für die verstärkte Expression identifiziert wurde, war dieser in Säugetieren noch nicht bekannt. In der vorliegenden Arbeit konnte TCF11 (transcription factor 11) als der verantwortliche Transkriptionsfaktor identifiziert werden, der in der humanen Endothelzelllinie Ea.hy926 die Transkription der proteasomalen Gene nach Proteasominhibition induziert. Unter physiologischen Bedingungen ist TCF11 ein N-glykosyliertes ER-ständiges Membranprotein, welches durch die ER-assoziierte Protein Degradation, unter der Mitwirkung des E3-Enzyms HRD1 und der AAA-ATPase p97, schnell abgebaut wird. Nach der Proteasominhibition kommt es zur Akkumulation von oxidierten Proteinen, und TCF11 wird aktiviert und in den Zellkern transportiert. Im Zellkern bindet TCF11 an AREs (antioxidant response element) in den proteasomalen Promotoren und aktiviert dadurch die Transkription der proteasomalen Gene. Darüber hinaus reguliert TCF11 auch die Expression von zahlreichen Enzymen, die die Ubiquitinierung von Proteinen katalysieren. Dadurch wird die zelluläre Homöostase wiederhergestellt und TCF11 sehr wahrscheinlich durch die neu gebildeten Proteasomen abgebaut. Die Ergebnisse der vorliegenden Arbeit zeigen auf, dass die Integrität des UPS nach Proteasominhibition in der humanen Endolthelzelllinie Ea.hy926 über einen TCF11 abhängigen Rückkopplungsmechanismus aufrechterhalten wird.The ubiquitin-proteasome-system (UPS) is the most important system for regulated protein degradation in eukaryotes. Therefore it is involved in the regulation of cellular homeostasis. Reduced proteasome activity results in proteotoxic stress. To counteract for reduced proteasome activity, eukaryotic cells enhance proteasome gene expression, which results in formation of new 26S proteasomes and recovery of physiological conditions. While in bakers yeast Saccharomyces cerevisiae the transcription factor Rpn4 is responsible for enhanced proteasome gene expression in response to proteasome inhibition, in mammals the responsible transcription factor was unknown. In this thesis, transcription factor TCF11 (transcription factor 11) was identified as a key regulator for 26S-proteasome formation in the human cell line Ea.hy926 to compensate for reduced proteolytic activity. Under non-inducing conditions N-glycosylated TCF11 resides in the endoplasmic reticulum (ER) membrane, where TCF11 is targeted to ER-associated protein degradation system requiring the E3-ubiquitin ligase HRD1 and the AAA-ATPase p97. Proteasome inhibitors trigger the accumulation of oxidant-damaged proteins, and promote the nuclear translocation of TCF11 from the ER, permitting activation of proteasome gene expression by binding of TCF11 to antioxidant response elements (ARE) in their promoter regions. Furthermore TCF11 controlls the expression of additional UPS-related genes. Thus the transcriptional feedback loop regulating human proteasome dependent protein degradation to counteract proteotoxic stress caused by proteasome inhibition was uncovered.
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Loukil, Abdelhalim. "Etude de la cycline A2 : interactions, dégradation et mise en évidence du rôle de l'autophagie." Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20115.
Full textAbstract:
Le cycle cellulaire est finement régulé dans le temps et l'espace. Nous avons abordé les aspects dynamiques des interactions que la cycline A2 entretient avec ses partenaires Cdk1, Cdk2 et l'ubiquitine au cours du cycle cellulaire, dans des lignées cellulaires humaines. A cette fin, nous avons eu recours aux approches de FRET (Förster/fluorescence resonance energy transfer) et de FLIM (fluorescence lifetime imaging microscopy). Ceci nous a permis de montrer que les formes ubiquitinylées de la cycline A2 apparaissent principalement sous forme de foyers en prométaphase et se propagent ensuite à l'ensemble de la cellule. En outre, nous avons découvert que l'autophagie participe à la dégradation de cette cycline en mitose. Nous discutons les implications de ces observations quant à un rôle éventuel de la cycline A2 au moment de la formation de l'anneau de constriction, ainsi que de la participation de l'autophagie via cette cycline, dans la réponse aux dommages à l'ADN en mitoseThe cell cycle is finely regulated in time and space. We have studied the dynamical aspect of the interactions between cyclin A2 and its partners Cdk1, Cdk2 and ubiquitin during the cell cycle, in human cell lines. To this aim, we have used FRET (Förster/fluorescence resonance energy transfer) and FLIM (fluorescence lifetime imaging microscopy) techniques. We have thus shown that ubiquitylated forms of cyclin A2 are detected predominantly in foci in prometaphase, before spreading throughout the cell. Moreover, we have shown that autophagy contributes to cyclin A2 degradation in mitosis. We discuss the implications of these observations regarding a possible role of cyclin A2 when the cleavage furrow forms, and the participation of autophagy in DNA damage response in mitosis
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Vrij, Femke Maaike Sophia de. "Mutant ubiquitin and the proteasome in Alzheimer's disease." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2005. http://dare.uva.nl/document/79250.
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