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Characterisation and manipulation of a plant proteasome subunit geneRahrami, Ahmad Reza January 1999 (has links)
No description available.
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Binding of SGTA to Rpn13 selectively modulates protein quality controlLeznicki, P., Korac-Prlic, J., Kliza, K., Husnjak, K., Nyathi, Yvonne, Dikic, I., High, S. 10 June 2020 (has links)
Yes / Rpn13 is an intrinsic ubiquitin receptor of the 26S proteasome regulatory subunit that facilitates substrate capture prior to degradation. Here we show that the C-terminal region of Rpn13 binds to the tetratricopeptide repeat (TPR) domain of SGTA, a cytosolic factor implicated in the quality control of mislocalised membrane proteins (MLPs). The overexpression of SGTA results in a substantial increase in steady-state MLP levels, consistent with an effect on proteasomal degradation. However, this effect is strongly dependent upon the interaction of SGTA with the proteasomal component Rpn13. Hence, overexpression of the SGTA-binding region of Rpn13 or point mutations within the SGTA TPR domain both inhibit SGTA binding to the proteasome and substantially reduce MLP levels. These findings suggest that SGTA can regulate the access of MLPs to the proteolytic core of the proteasome, implying that a protein quality control cycle that involves SGTA and the BAG6 complex can operate at the 19S regulatory particle. We speculate that the binding of SGTA to Rpn13 enables specific polypeptides to escape proteasomal degradation and/or selectively modulates substrate degradation. / BBSRC [grant number: BB/L006510/1] and the Wellcome Trust [grant number: 092107/Z/10/Z]. K.K. was supported by the UPStream network [EU, FP7, ITN project 290257]
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BPV Entry and Trafficking in EBTr CellsDudleenamjil, Enkhmart 19 November 2009 (has links) (PDF)
Bovine Parvovirus (BPV) belongs to the genus Bocavirus, family Parvoviridae. BPV is the leading etiologic agent among the pathogens that cause primary gastroenteritis of cattle. Many of the intracellular events associated with virus replication are unknown. In this research project, we investigated BPV internalization into the host cell and trafficking in the cytosol. Preliminarily, EBTr cells had abundant clathrin, virus attached to purified clathrin, and EM micrographs revealed virus in endocytic vacuoles. Assays detecting virus infectivity (i.e. viral protein synthesis), virus production (completion of the replication cycle), and quantitative PCR (qPCR) to detect viral transcripts were used to evaluate virus uptake and subsequent trafficking events in the presence of selective inhibitors. Cell toxicity mediated by the drugs was evaluated by the MTT test. Virucidal effects of the drugs were assessed. A control virus was used to verify the inhibitor technology. Immunofluoresceinated virus particles were found in clathrin-rich early endosomes. Clathrin-mediated endocytosis (CME) was examined by clathrin polymerization inhibiting agent (chloropromazine), lysosomotropic agents (ammonium chloride and chloroquine), a vacuolar ATPase inhibitor (bafilomycin A1), and a blocker of transition between endosomes (brefeldin A). Caveosome pathway inhibitors included phorbol 12-myristate 13-acetate (a suppressor of caveolae formation), nystatin and methyl-beta-cyclodextrin (lipid raft blockers), and genistein (a tyrosine kinase phosphorylation inhibitor). Trafficking of BPV was investigated using specific inhibitors of proteasomal activity, actin-myosin function, and microtubule-dynein function. The proteasomal protease suppressor (lactacystin), and a proteasomal chymotrypsin inhibitor (epoxomicin) were used. The role of actin was probed by cytochlasin D, latrunculin A, and ML-7. The microtubule inhibitors nocodazole, vanadate, and EHNA were used to probe microtubule function. The inhibitors of CME reduced virus production and reduced infectivity, a result confirmed by qPCR. The blockers of caveolin-mediated entry did not interfere with virus production nor virus infectivity. Proteasome activity blockage did not affect the virus replication. But the virus cycle was affected by actin blockage and by microtubule blockage detected by qPCR. Taken together these data indicate that BPV uptake is mediated by clathrin coated pits and is acid-dependent. Further processing of BPV in the cytosol does not require proteasomal enzymes. Actin-associated vesicular transport appears to be essential to virus replication and trafficking to the nucleus appears to be mediated by microtubules.
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Von Toleranz zur AutoimmunitätSteinhoff, Ulrich Johannes 05 November 2002 (has links)
Immunologische Toleranz ist eine elementare Eigenschaft des Immunsystems, die primär durch die klonale Deletion autoreaktiver T-Zellen im Thymus gewährleistet wird. Neben diesem als zentrale Toleranz bezeichneten Mechanismus, verfügt ein Organismus gleichzeitig über periphere Toleranzmechanismen wie Ignoranz, Anergie und regulatorische T-Zellen. Trotz dieser Kontrollmechanismen können in bestimmten Situationen autoreaktive CD4+ und CD8+ T-Zellen aktiviert werden und meistens zu örtlich und zeitlich begrenzten Autoimmunreaktionen führen. Ursache hierfür kann die hormonelle Regulation oder das gewebespezifische Vorkommen eines Selbsttantigens sein. Am Beispiel von HSP60-kreuzreaktiven CD8+ T-Zellen konnte gezeigt werden, dass der Transfer dieser T-Zellen in Tiere zu einer Entzündung des Dünndarms aber nicht des Dickdarms führt, obwohl das Selbstantigen im letzteren wesentlich stärker exprimiert wird. Die Gewebespezifität der Autoimmunpathologie konnte durch die in den Organen unterschiedliche, proteasomale Antigenprozessierung, erklärt werden. Proteinbiochemische und immunologische Analysen ergaben, dass sich die 20S Proteasomen verschiedener Organe strukturell und funktionell deutlich unterscheiden und somit jedes Gewebe ein individuelles Repertoire von MHC-Klasse I restringierten Peptiden präsentiert. Damit wurde ein weiterer Mechanismus entdeckt, durch den Reaktivität von protektiven und pathologischen CD8+ T-Zellen kontrolliert wird. / Immunological tolerance which is primarily mediated by the clonal deletion of autoreactive T cells in the thymus is a key feature of the immune system. Besides this central tolerance, several mechanisms act also in the periphery including ignorance, anergy and regulatory T cells. Despite all these checkpoints, autoreactive CD4+ and CD8+ T cells may still be activated causing local and time restricted autoimmune-reactions. This may refer primarily to self-antigens which are hormonally regulated or tissue-specifically expressed. Adoptive transfer of crossreactive, hsp60-specific CD8+ T cells into mice induced an local inflammation of the small intestine but not the colon despite elevated expression of hsp60 in the latter organ. The pathology could be explained by the finding that the proteasomal antigen processing varies between different organs. Biochemical and immunological analyses revealed that 20S proteasomes of different organs vary in their structural and functional properties indicating that every tissue displays an individual and distinct repertoire of MHC class I peptides. This represents a new mechanism by which the activity of protective and pathological CD8+ T cell responses may be controlled.
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Prozessierung des pp89 MCMV MHC Klasse I Epitops durch das ProteasomVoigt, Antje 20 April 2004 (has links)
Das Proteasom ist eine ATP- abhängige Protease, die sich aus vielen Untereinheiten zusammensetzt. Es ist für die Generierung der MHC Klasse I- restringierten Peptide verantwortlich, die im Folgenden auf der Zelloberfläche präsentiert werden. Nicht-funktionelle Proteine, die als so genannte defective ribosomal products (DRIP) bezeichnet werden, stellen eine wichtige Quelle für die Generierung von antigenen Peptiden, insbesondere jedoch von viralen Peptiden dar. Generell wird die Lehrmeinung vertreten, dass der Abbau von polyubiquitinierten Proteinen durch das 26S Proteasom zur Generierung von MHC Klasse I- Liganden führt. Allerdings ist weiterhin unklar, ob virale Proteine Ubiquitin- abhängig vom Proteasom abgebaut werden. Demnach sollte im Rahmen dieser Arbeit der Proteasom- abhängige Abbau des mCMV ie pp89 Proteins vor allem hinsichtlich einer Ubiquitinierung untersucht werden. Folglich wurden Konstrukte sowohl für ein rekombinantes pp89 (rek pp89) als auch für ein ODCpp89 Fusionsprotein entworfen. Somit konnten sowohl der in vitro Abbau dieser Proteine als auch die Prozessierung des spezifischen MHC Klasse I H2-Ld Epitops verfolgt werden. Experimente zum Nachweis von Ubiquitin- Protein- Konjugaten wurden in vivo mit stabil transfizierten Mausfibroblasten (B8 Zellen) durchgeführt. Die experimentellen Daten sprechen für einen schnellen in vitro Abbau des rek pp89 durch das 20S Proteasom. Das MHC Klasse I pp89 Epitop bzw. dessen 11mer Precursorpeptid wurden dabei mit hoher Präzision generiert. Spezifische CTL Assays weisen auf die Generierung des korrekten Epitops bzw. des Precursors hin. Nach Verdau des ODCpp89 Fusionsproteins durch 26S Proteasomen in Anwesenheit von Antizym konnten mit diesem Test ebenfalls das 9mer Epitop respektive das 11mer des pp89 nachgewiesen werden. Eine potentielle Ubiquitinierung des pp89 wurde in vivo in Zellkulturen untersucht. Nach Gabe von Proteasomeninhibitoren zu Mausfibroblasten konnte eine starke Akkumulierung von Ubiquitin- Konjugaten beobachtet werden. Allerdings konnte in den verschiedenen Versuchsansätzen kein Nachweis von pp89- Ubiquitin- Konjugaten erbracht werden. Demzufolge ist für die Generierung von viralen Epitopen ein Proteasom- abhängiger, aber Ubiquitin- unabhängiger Abbauweg denkbar. / The proteasome, an ATP-dependent, multisubunit protease, is responsible for the generation of most MHC class I restricted epitopes presented on the cell surface. Non-functional proteins, also known as defectice ribosomal products (DRiP), represent an important source for the generation of antigenic peptides in general and of viral epitopes in particular. It is widely accepted that the degradation of polyubiquitinated proteins by the 26S proteasome is a prerequisite for the generation of MHC class I ligands. However, the ubiquitin dependence for the proteasomal degradation of viral proteins is an issue so far unresolved. Therefore, the aim of this study was to analyze the proteasomal degradation of the mCMV ie pp89 in respect to an anticipated ubiquitinylation. Thus, a recombinant pp89 (recpp89) as well as an ODCpp89 fusion protein were generated. The in vitro processing of these proteins and the generation of a MHC class I H2-Ld epitope by proteasomes was further studied. Murine fibroblast cell lines (B8 cells) were used to analyze any in vivo evidence for potentially existing ubiquitin-protein conjugates. The experiments show that the recpp89 protein is rapidly degraded in vitro by the 20S proteasomes and that the correct MHC class I pp89 epitope or its 11mer precursor are generated with high fidelity. Furthermore, CTL assays, indicating the generation of the specific pp89 epitope or the 11mer precursor, also suggested a 26S proteasome-dependent degradation of the mCMV pp89-ODC fusion protein in the presence of antizyme. Treating cell cultures with proteasome inhibitors resulted in a significant accumulation of ubiquitin-conjugates in vivo. However, higher molecular weight pp89-ubiquitin conjugates were not detectable throughout the entire experimental set-up. Consequently, a proteasome-dependent, but ubiquitin-independent pathway can be postulated for the generation of viral epitopes.
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