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Sekiyama, Naotaka. "STRUCTURAL ANALYSIS OF UBIQUITIN AND UBIQUITIN-LIKE PROTEIN RECEPTORS." 京都大学 (Kyoto University), 2010. http://hdl.handle.net/2433/120884.
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Braxton, Courtney N. "The progress on mapping ubiquitin signaling using photocrosslinking mono and di-ubiquitin probes and other ubiquitin moieties." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5382.
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Ubiquitin (Ub) is a small, 76 amino acid, and post-translational modification (PTM) protein in eukaryotes. Modification of a substrate protein via the covalent attachment of the C-terminal glycine of Ub to the ε-amino group of lysine residues in a substrate is termed ubiquitination. Unlike, other PTM proteins, Ub can form polyUb chains at one or more of its seven lysine residues. (K6, K11, K27, K29, K33, K48, and K68). The consequence of these different polymerization sites is altered biological response with different polyUb linkages conferring different fates to target proteins. Unfortunately, the study of these chains have been limited by the inability to generate homogeneous polyUbs chains linked at known lysine residues. Furthermore, a three step enzymatic cascade consisting of activating-enzymes (E1s), conjugating enzymes (E2s), and ligase enzymes (E3s) tightly controls this modification. In response, our laboratory has developed a system that creates polyUb chains through bacterial expression and "synthetic" building blocks. Now, the main questions are what do these chains interact with in the cell and how do these interactions mediate biological responses?
In an attempt to answer these questions, this dissertation looks at different molecular techniques created to capture the transient interactions of monoUb and diUb probes with Ub substrates, such as, ubiquitin binding domains (UBDs) and conjugating E2 enzymes. One molecular technique focuses on the use of incorporating a genetically encoded, photo-crosslinker, p-Benzoyl-L-phenylalanine (pBpa) into diUb probes to capture their interaction with UBDs. This sets the foundation for understanding Ub’s cellular signaling recognition of UBDs. Another technique is creating diUb probes that contain lysine derivatives, Nε-L-Thiaprolyl-L-lysine (ThzK) or Nε-L-Cysteinyl-L-lysine (CysK), and can form a disulfide bonds with E2 enzymes to capture their complex, opening an opportunity to understand mechanistically the role E2 enzymes have with polyUb chain formation. Herein, these techniques are established to help unravel the complexity of Ub signaling.
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Haririnia, Aydin. "Molecular interactions of ubiquitin and polyubiquitin with ubiquitin binding domains." College Park, Md. : University of Maryland, 2007. http://hdl.handle.net/1903/7627.
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Thesis (Ph. D.)--University of Maryland, College Park, 2007.Thesis research directed by: Dept. of Chemistry and Biochemistry. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Lange, Anja. "Structural characterization of the interaction of the Stam2's ubiquitin binding domains with ubiquitin chains by NMR : Cooperativity or not, that is the question !" Thesis, Lyon 1, 2010. http://www.theses.fr/2010LYO10308.
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Résumé en anglais uniquementFrom the discovery of ubiquitin and its function as signal for proteasomal degradation over 20 years ago to this days, it became evident that ubiquitin is a universal signal in eukaryotic cells. Ubiquitin in its different forms is involved in many versatile cellular processes. Knowing that the ubiquitin signal is differently translated, depending on its occurrences as mono-ubiquitin or poly-ubiquitin, raises the question: how do cells distinguish between the different occurrences of ubiquitin and translate it into the proper response? Proteins interacting with ubiquitin contain so called ubiquitin binding domains (UBDs), whereas the affinities to ubiquitin vary from a few _M to mM. So far only three (K63, K48 and linear chains) out of the eight possible chain-linkages can be produced in sufficient amounts to characterize their interaction with UBDs. K48- and K63- linked ubiquitin chains regulate different cellular events and need to be recognized by different proteins. Thus, it is of prime importance to characterize the binding of different UBDs to these two kinds of ubiquitin chains, as it can give important clues related to the general mechanism of chain discrimination by ubiquitin adapter proteins. Some isolated UBDs exhibit a preference for one chain linkage type over the other, whereas others do not discriminate between mono-ubiquitin or K63- and K48-linked chains. Interestingly, many ubiquitin adapter proteins harbor more than one UBD. STAM2 is a ubiquitin adapter protein, that is involved in endosomal receptor sorting and supposed to preferentially bind mono-ubiquitin and K63- over K48-linked ubiquitin. STAM2 contains two UBDs (a VHS and UIM domain) that were shown to bind to ubiquitin . The current manuscript shows that STAM2’s SH3 domain binds ubiquitin as well. To understand the function of the sequential arrangement of three UBDs in one protein, first binding of the individual VHS and UIM domains to monoubiquitin as well as K48- and K63-linked di-ubiquitin was investigated. This work shows, that the VHS domain displays a different mode of binding for K63- and K48-linked diubiquitin. In spite of the fact, that the apparent Kd for both chains is the same, only one VHS domain can bind to K48-linked di-ubiquitin chains (with a preference for the distal domain), whereas K63-linked di-ubiquitin can accommodate two VHS domains at a time. Since no conclusion can be drawn with respect to the apparent Kds, the different binding modes might gain more impact in consideration of the ensemble of three UBDs. Results presented in this manuscript, based on a construct containing the VHS and UIM domain, show that binding to K63- but not K48-linked di-ubiquitin is cooperative
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Pirim, Ibrahim. "Ubiquitin and neurogenerative diseases." Thesis, University of Nottingham, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335277.
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Deschutter, Julie. "Identification de la monoubiquitination de la protéine SHIP2 et caractérisation des mécanismes régulateurs associés." Doctoral thesis, Universite Libre de Bruxelles, 2009. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/241308.
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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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Bazirgan, Omar Al-Kasim. "Functional analysis of the ubiquitin ligase Hrd1p with the ubiquitin-conjugating enzyme Ubc7p." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3246079.
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Thesis (Ph. D.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed March 9, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Rumsby, Ellen Louise. "Regulation of the cell division cycle by ubiquitin and ubiquitin-like modifications in yeast." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/2938.
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The ability of a cell to regulate its cell cycle in response to external stimuli, such as oxidative stress, is important to maintain viability by preventing damage and allowing time for repair. However, the underlying sensing and signalling mechanisms behind cell cycle regulation in response to oxidative stress remain largely unclear. Ubiquitin and ubiquitin-like (Ubl) proteins are a family of highly conserved protein modifiers with a role in many cellular processes including cell cycle regulation. The use of catalytic cysteine residues in the conjugation pathways of ubiquitin and Ubls suggest a mechanism by which these modifiers can be redox-regulated. Thus the aim of this project was to investigate the regulation of the cell division cycle by ubiquitin and Ubls in response to two conditions previously observed to lead to G1 phase cell cycle arrest in S. cerevisiae, treatment with the oxidising agent diamide and glutathione depletion. We find that in response to diamide the ubiquitin E2, Cdc34 is particularly sensitive to oxidation compared to the other E2s examined. Oxidation of Cdc34 was shown to lead to an increase in the stability of the Cdc34 substrate Sic1, coincident with G1 phase arrest. We also find that the Rub1 Ubl modifier is essential for regulation of the cell cycle in response to diamide. Interestingly, we find that Rub1 is also required to prevent budding in response to glutathione depletion. Importantly, here we reveal that SIC1 is essential to maintain viability by preventing replication-induced DNA damage following glutathione depletion. Our studies demonstrate that G1 phase cell cycle arrest in response to diamide and glutathione depletion is multifaceted, involving many of the same proteins but that these proteins are regulated differently in response to the two conditions.
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Souza, Letícia Martins Ignácio de 1987. "Sistema ubiquitina-proteassoma no hipotálamo : implicações para a gênese da obesidade." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/310374.
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Orientadores: Lício Augusto Velloso, Marciane Milanski FerreiraTese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-22T00:15:51Z (GMT). No. of bitstreams: 1 Souza_LeticiaMartinsIgnaciode_D.pdf: 4083691 bytes, checksum: 4627dc93519577a00d2747b36d1a406f (MD5) Previous issue date: 2013
Resumo: Dentre os fatores ambientais que contribuem para o desenvolvimento de obesidade, o consumo de dietas ricas em ácidos graxos saturados desempenha o papel mais importante. Estudos recentes realizados por vários grupos, inclusive o nosso, revelam que ácidos graxos saturados presentes na dieta levam ao desenvolvimento de resistência hipotalâmica à ação dos hormônios leptina e insulina, fenômeno este fundamental para que ocorra a quebra no equilíbrio entre ingestão e gasto calórico. Até o momento caracterizaram-se dois mecanismos moleculares potencialmente envolvidos na iniciação do processo que resulta na disfunção hipotalâmica na obesidade, a ativação de TLR4 e a indução de estresse de retículo endoplasmático, ambos levando a uma resposta inflamatória local e, eventualmente, a apoptose neuronal. Estudos recentes têm revelado que frente a situações que oferecem risco de dano celular, ativa-se um mecanismo de controle de tráfico e degradação protéica chamado sistema ubiquitina-proteassoma (UPS). O acúmulo de agregados protéicos positivos para ubiquitina pode gerar toxicidade celular e regular a plasticidade neuronal. Também a modulação de componentes do UPS pode gerar neurodegeneração hipotalâmica e fenótipo obeso em animais experimentais. Neste estudo aventamos a hipótese que durante períodos prolongados de obesidade a ativação anômala do UPS contribuiria para a perpetuação do quadro de obesidade. De fato, os resultados obtidos revelam que roedores com predisposição para a obesidade induzida por dieta mantém, a princípio, a capacidade de regular adequadamente a UPS no hipotálamo. Com o passar do tempo esta capacidade é perdida resultando numa maior dificuldade para perda de peso frente à redução do aporte calórico. Roedores com mutações que os protegem da inflamação, não apresentam distúrbio funcional do UPS quando expostos a dieta rica em ácidos graxos e, são também protegidos da obesidade. Portanto, o defeito funcional do UPS no hipotálamo no curso de obesidade prolongada, constitui-se num fator importante contribuindo para a refratariedade ao tratamento e perpetuação da doença
Abstract: The consumption of high-fat diets, especially those rich in saturated fatty acids, plays the most important role in the development of obesity. Recent studies by several groups, including ours, have shown that dietary long-chain saturated fatty acids lead to the development of hypothalamic resistance to leptin and insulin, an important condition contributing for breaking of the balance between caloric intake and energy expenditure. Two molecular mechanisms are currently known to play a triggering role in this process; activation of TLR4 and endoplasmic reticulum stress, both leading to local inflammation and eventually apoptosis of neurons. The ubiquitin-proteasome system (UPS) plays an important role in the control of protein recycling in the cell. The accumulation of ubiquitin-positive protein aggregates can cause cell toxicity and regulate neuronal plasticity. Also the modulation or differential activation of UPS can produce hypothalamic neurodegeneration and obese phenotype in experimental animals. Here, we hypothesized that under prolonged diet-induced obesity, a defect in the UPS in the hypothalamus could contribute for the defective control of energy homeostasis leading to the refractoriness of obesity to caloric restriction. In fact in an obesity-prone rodent strain, prolonged, but not short-term obesity was accompanied by functional abnormality of the UPS in the hypothalamus. In mutants protected from inflammation, resistance to diet-induced obesity was accompanied by stability of the UPS in the hypothalamus. Thus, defect of the UPS in the hypothalamus, during prolonged obesity is an important factor contributing the refractoriness of obesity to caloric restriction
Doutorado
Biologia Estrutural, Celular, Molecular e do Desenvolvimento
Doutora em Fisiopatologia Médica
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Schick, Martin Christopher. "High pressure NMR of ubiquitin /." Zürich, 1998. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=12586.
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Tsirigotis, Maria. "Mutational analysis of mammalian ubiquitin." Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/29267.
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Much of what is known about the ubiquitin/proteasome pathway has been deduced from mutational analysis performed in the yeast model system. From the high level of conservation between yeast and mammalian ubiquitin it would be expected that expression of analogous ubiquitin isoforms in higher eukaryotes would result in similar phenotypes. A site directed mutagenesis approach was employed to investigate the phenotypes of expression of mutant ubiquitin in higher eukaryotes and in the in vivo setting of novel ubiquitin transgenic mice. It was found that Ub-EGFP fusion proteins are efficiently recognized and processed by ubiquitin specific proteases both in mammalian cells and in transgenic mice; the transgene-derived ubiquitin moiety was found to substitute for endogenous ubiquitin in poly-Ub chain assembly and ubiquitinated conjugates were recovered using standard purification methodologies. The expression of chain-terminating ubiquitin derivatives (K48R and K63R) predisposed cells to the toxic effects of misfolded proteins and sensitized cells to DNA damaging agents. In transgenic mice, the expression of K48R mutant ubiquitin was found to confer protective effects and delay the deterioration of Purkinje neurons in a mouse model of SCA-1. The neuroprotective effect of K48R mutant ubiquitin may be mediated though stabilization of key transcription factors whose loss figured in the normal course of the SCA1 disease.
The expression of C-terminal variants in yeast has been proposed to have profound effects on ubiquitin metabolism. A mechanistically related mechanism has been proposed to contribute to the pathogenesis of Alzheimer's disease wherein transcriptional frameshifting of the ubiquitin B mRNA generates an aberrant ubiquitin protein (termed UBB+1) with an altered C-terminus. To investigate the constraints with regard to processing/conjugation and recycling of ubiquitin in higher eukaryotes a plethora of C-terminal ubiquitin variants were generated and introduced in mammalian cells as linear fusions with EGFP. Mutations that inactivate yeast ubiquitin did not abolish the function of ubiquitin in higher eukaryotes; C-terminal ubiquitin variants were processed by deubiquitinating enzymes and in some cases were found to conjugate to cellular proteins. The tolerance of mammalian cells to mutant ubiquitin may be attributable to loosened constraints that exist at the C-terminus due to mechanisms that couple deubiquitination, targeting and destruction of Ub-EGFP fusion proteins. Preliminary data suggest that prolonged exposure of cells of neuronal lineage to C-terminal ubiquitin variant as assessed in transgenic mice may result in perturbed ubiquitin homeostasis, a feature observed in the pathogenesis of Alzheimer's disease.
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Ligr, Martin. "Ubiquitin metabolism in Chlamydomonas reinhardtii." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1995. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/MQ33407.pdf.
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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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Ye, Yu. "Targeting ubiquitin chains with deubiquitinases." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610783.
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Wilken, Jill. "Synthesis and structure of ubiquitin." Thesis, University of Edinburgh, 1995. http://hdl.handle.net/1842/13236.
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The protein ubiquitin has been used as a model for folding studies with the synthesis of ubiquitin analogues including both single and double substitutions. Chemical synthesis has allowed for the facile incorporation of both unnatural amino acids and a novel synthetic fluorinated amino acid. Purification of synthetic ubiquitin by a variety of protocols has identified important steps in the folding pathway. We have investigated the placement of fluorine within the hydrophobic core of ubiquitin through the synthesis of three double substitutions at positions 43&67, 50&67, 56&67 and one single substitution at position 67 using (2S,4S)-5-fluoroleucine to replace leucine. Methyl transfer across the hydrophobic core has been considered using the double substitution analogue [3-Norleucine,43-norvaline]ubiquitin involving a modification in the distribution of alkyl groups. The importance of the chirality of the single histidine on the final strand of β-sheet has been examined in the synthesis of the analogue [68-DHistidine]ubiquitin. The contribution of secondary structural features in protein folding has been investigated by studying peptide fragments corresponding to the N and C-terminal halves of ubiquitin and three overlapping peptides on the final strand of β-sheet. The complementation of the N and C-terminal halves to regenerate the native fold has also been studied.
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Green, Jeremy. "Towards the synthesis of ubiquitin." Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/14941.
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Oughtred, Rose W. "Characterization of ubiquitin-protein ligases in the testis interacting with the UBC4UBC5 ubiquitin-conjugating enzymes." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36825.
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Ubiquitin is a highly conserved 8 kDa protein whose many cellular functions are mediated by its covalent ligation to other proteins. Conjugation of ubiquitin is a multistep process involving a ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (UBCs or E2s), and ubiquitin protein ligases (E3s). The multi-ubiquitination of substrates marks them for degradation by the 26S proteasome.Previously, rat UBC4 isoforms homologous to S. cerevisiae UBC4/UBC5 were cloned and characterized (Wing and Jain, 1995) (Wing et al., 1996). The UBC4-1 isoform is highly expressed in the testis, and the UBC4-testis isoform is induced in round spermatids. This thesis describes the identification and characterization of E3s interacting with these UBC4 isoforms expressed in the rat testis.
First, the isolation and characterization of a novel E3 from rat testis extracts, E3Histone, is described. E3Histone mediates conjugation of ubiquitin to histones in a UBC4-dependent manner. Interestingly, E3Histone was immunodepleted by antibodies against Cdc27, a subunit of the a&barbelow;naphase-p&barbelow;romoting c&barbelow;omplex (APC), an E3 which plays a critical role in the regulation of the cell cycle. However, E3Histone and the APC are distinct complexes. Gel filtration resolved the 600 kDa E3Histone from the 1500 kDa APC. E3Histone interacts preferentially with UBC4, whereas the APC interacts preferentially with UbcH10 and shows specificity for the substrate cyclin. E3Histone and the APC may be members of a newly-recognized family of combinatorial E3s that share some common core subunits, such as CDC27, yet possess distinct subunits that confer upon them their respective E2 and substrate specificities. In addition, E3Histone activity was detected in extracts from various purified germ cells. Induction of UBC4 may lead to the increased ubiquitination of histories and together with E3 Histone may play a role in the chromatin condensation that occurs during spermatid maturation.
Secondly, the characterization of a HECT domain E3, Rat100, is described. UBC4-1 and UBC4-testis were found to transfer ubiquitin to Rat100 in vitro. Immunoblotting showed that Rat100 has a molecular weight of 300 kDa, and that the developmental and cell-specific expression of Rat100 correlates with that of UBC4. The induction of Rat 100 may playa role in the activation of ubiquitin-dependent proteolysis during spermatogenesis.
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Kummer, Anne Verfasser], and Lothar [Akademischer Betreuer] [Jänsch. "Ubiquitin and ubiquitin-like host modifications in Listeria monocytogenes infection / Anne Kummer ; Betreuer: Lothar Jänsch." Braunschweig : Technische Universität Braunschweig, 2017. http://d-nb.info/1175817945/34.
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Matta, Camacho Edna. "Structural studies of Ubiquitin related proteins." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110562.
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Posttranslational attachment of ubiquitin (Ub) or poly-Ub chains marks proteins for various cellular fates and functions, including proteasomal degradation, endocytosis, endosomal sorting, and DNA repair. In general, three enzymes catalyze the ubiquitination reaction: activating enzyme (E1), conjugating enzyme (E2), specificity-determining Ub-protein ligase (E3). This thesis examines three distinct aspects of the ubiquitination of proteins.The recognition of substrates by UBR1 and UBR2 proteins: UBR proteins are E3-ligases that recognize certain N-terminal residues (N-degrons) as signals for protein degradation. For this reason, they are called N-recognins. N-recognition follows a turnover rule known as the N-end rule. Using X-ray crystallography, we determined the structure of the substrate-recognition domain of the UBR1 and UBR2 proteins with a bound ligand. These structures showed that the domain adopts a novel fold stabilized through the binding of three zinc ions and forms a binding pocket for N-degrons. Additionally, biochemical affinity measurements established the molecular principles for the recognition of positively charged N-degrons. The elucidation of the ubr-box structure in complex with a type-1 N-degron pioneered the understanding of N-end rule substrate recognition.Substrate binding and ubiquitination by UBR5 protein: UBR5 is an E3 Ub ligase containing a catalytic HECT domain and a peptide-binding domain. The peptide-binding MLLE domain recruits different regulatory proteins through a 12-residue peptide motif, termed PAM2. Using pull-down and ubiquitination assays, we demonstrated that the HECT domain interacts with the adjacent MLLE domain in a PAM2 dependent manner. This interaction modulates binding and ubiquitination of TopBP1, a substrate of UBR5. These results provided a new view on how different domains in UBR5 proteins work together for efficient substrate recognition and ubiquitination. The recognition of Ub or poly-Ub chains by the UBA domain of Swa2 protein: Ub-associated (UBA) domains are ∼40 amino acid motifs occurring in proteins associated with ubiquitination. Most UBA domains bind to mono-Ub as well as poly-Ub. A structural model of the Swa2p UBA domain in complex with Ub showed that Ub recognition occurs predominantly through an atypical interaction through helix α1 of the UBA. Mutagenesis and surface plasmon resonance revealed a second low-affinity Ub-binding site and preference of Swa2p UBA for poly-Ub binding. These results revealed a potential role of Swa2p UBA domain in binding poly-ubiquitinated proteins in vivo, and the UBA domains as communication elements in the Ub system.This thesis provides a better understanding of the mechanisms that the Ub pathway employs to assure selectivity and processivity at three different levels: substrate recognition by the N-end rule, substrate binding and ubiquitination by an E3 ligase, and the recognition of Ub species by UBA domains.Les modifications post-traductionnelles des protéines par une molécule d'ubiquitine (Ub) ou par des chaînes de poly-Ub déterminent leur destin pour diverses fonctions cellulaires notamment la dégradation à travers le protéosome, l'endocytose, le tri endosomique ainsi que la réparation de l'ADN. De façon générale, trois enzymes catalysent une réaction d'ubiquitination: une enzyme d'activation (Ub-activating enzyme E1), une enzyme de conjugaison (Ub-conjugating enzyme E2), et une enzyme qui détermine la spécificité pour le substrat (specificity-determining Ub-protein ligase E3). Cette thèse porte sur trois aspects distincts impliqués dans le processus d'ubiquitination des protéines:La reconnaissance de substrats par les protéines UBR1 et UBR2: les Ub-recognins (UBR) sont des ligases E3 qui reconnaissent des résidus N-terminaux (N-degrons) comme signaux pour la dégradation des protéines. Cette reconnaissance suit une règle de rotation du nombre d'entités ciblées nommée N-end rule (règle du N-terminus). A l'aide de données de cristallographie aux rayons X, nous avons déterminé la structure du domaine UBR-box impliqué dans la reconnaissance du substrat des protéines UBR1 et UBR2 en complexe avec un N-degron. Ces structures montrent que le domaine UBR-box adopte un nouveau repliement stabilisé par trois ions zinc et qu'il forme une poche pour accueillir les N-degrons. Aussi, des mesures biochimiques d'affinité ont permis d'établir des principes moléculaires pour la reconnaissance des N-degrons qui sont chargés positivement.La fixation du substrat et son ubiquitination par la protéine UBR5: UBR5 est une Ub ligase E3 contenant un domaine catalytique HECT et un domaine de liaison peptidique, MLLE. Le domaine MLLE recrute plusieurs protéines régulatrices grâce à leur motif PAM2 composé de douze résidus. En procédant à des expériences de pull-down et à des essais d'ubiquitination, nous avons démontré que le domaine HECT interagissait avec le domaine MLLE adjacent de manière dépendante du motif PAM2. Cette interaction conduit à des changements conformationnels qui modulent le repliement et l'ubiquitination de TopBP1, un substrat d'UBR5.La reconnaissance d'Ubiquitine et de chaînes poly-Ub par le domaine UBA de la protéine Swa2: les domaines UBA (Ub-associated) sont composés d'environ quarante acides aminés présents dans des protéines associées à l'ubiquitination. Les domaines UBA se lient à des mono-Ub in vitro, mais semblent avoir une affinité supérieure pour les chaînes de poly-Ub. Un modèle structurel du domaine UBA de la protéine Swa2p en complexe avec une Ub montre que la reconnaissance de l'Ub se produit principalement par une interaction atypique grâce à l'hélice α1 du domaine UBA. Des essais de résonance plasmonique de surface combinés à de la mutagenèse ont révélé une faible affinité secondaire pour le site de liaison à l'Ub et une préférence de l'UBA de Swa2p pour la liaison aux poly-Ub. Ces résultats ont révélé un rôle potentiel du domaine UBA de Swa2p pour lier les protéines poly-ubiquitinylées in vivo.À trois niveaux différents, la reconnaissance du substrat par le règle N-fin, fixation du substrat et l'ubiquitination par une ligase E3, et la reconnaissance des espèces de l'ubiquitine par domaines UBA, cette thèse a fournir une meilleure compréhension du mécanisme qui la voie Ub emploie pour assurer la sélectivité et la processivité de garder le bon environnement homéostatique dans la cellule.
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Meldrum, Jill Kathleen. "Engineering the Folding Pathway of Ubiquitin." Thesis, University of Nottingham, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.519401.
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Mesquita, Francisco Sarmento. "Interactions between salonella and ubiquitin pathways." Thesis, Imperial College London, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.534978.
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Crespo, Solans MariÌa Dolores. "Probing the folding pathway of ubiquitin." Thesis, Nottingham Trent University, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415685.
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Simpson, Emma Rhiannon. "Exploring the folding pathway of ubiquitin." Thesis, University of Nottingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437025.
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Canning, Mary. "Ubiquitin-mediated proteolysis and Drosophila embryogenesis." Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/13305.
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Ubiquitination provides a means of rapidly and irreversibly eliminating an unwanted protein from the cell, and is therefore a potentially effective tool for regulating cellular behaviour. Ubiquitin-mediated proteolysis is involved in such diverse physiological functions as growth control, cell signalling, differentiation and the immune response. The aim of this research has been to investigate its role in Drosophila embryogenesis. Protein ubiquitination is a stepwise process carried out by three classes of enzyme known as E1s, E2s and E3s. The E1 (ubiquitin-activating enzyme), generates a thiolester linkage with a ubiquitin cysteine residue. The activated ubiquitin is then transferred to an E2 (ubiquitin-conjugating enzyme) which, with the help of an E3 (ubiquitin-protein ligase), recruits the substrate protein which is to be degraded. I examined the embryonic expression patterns of several known and novel genes encoding each type of ubiquitinating enzyme. The E2 UbcD4 is transcribed during early to mid-embryogenesis in a variety of tissues, with specific germcell expression in stage 10 embryos. This suggested a possible role for UbcD4 in germ cell migration towards the somatic gonadal precursors. UbcD4 mRNA was also abundant in git and nervous system during germband retraction and dorsal closure. I screened for UbcD4 - interacting proteins using the yeast two-hybrid system, and identified several putative substrates for, as well as ancillary factors involved in, ubiquitination by UbcD4. These included a novel E3 of the Hect-domain family. In an attempt to examine the function of UbcD4 directly, I used RNA interference to disrupt UbcD4 function. The results suggest a post-germband retraction requirement for UbcD4.
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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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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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Schubert, Alexander Fabian. "Mechanism of PINK1-mediated ubiquitin phosphorylation." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/277271.
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Ubiquitin phosphorylation by PINK1 (PTEN-induced Putative Kinase 1) is crucial for mitochondrial quality control and loss or mutation of PINK1 can lead to autosomal recessive juvenile parkinsonism (AR-JP). PINK1 is an unusual kinase, as it is characterised by three unique insertions in its kinase N lobe and a C-terminal region after the kinase domain. Despite great effort, a structure of PINK1 could not be determined and the molecular mechanism of ubiquitin phosphorylation and the effect of the PINK1 AR-JP patient mutations remained elusive. The versatile modifier ubiquitin (Ub) is also an unusual kinase substrate, as its phosphorylation site (Ser65) is not exposed, but protected by the Ub fold. Hence, it was not clear how a kinase would be able to target Ser65 of Ub. This work shows that Ub needs to adopt a previously described conformation in order to be efficiently phosphorylated by PINK1. NMR experiments revealed that in a small population of Ub the last β-strand is retracted, resulting in a more accessible Ser65 loop. It could be shown that PINK1 binds the Ser65 loop in this C-terminally retracted conformation (Ub-CR), but not in the ‘common’ conformation. In addition, it could be shown that Ub trapped in the Ub-CR conformation by point mutations (Ub TVLN) is phosphorylated significantly faster than Ub wt, which only adopts the Ub-CR conformation at very low frequency. To further elucidate how PINK1 binds and phosphorylates Ub, the kinase domain of Pediculus humanus corporis (Ph)PINK1 was crystallised in complex with Ub TVLN stabilised by a nanobody. The structure revealed many peculiarities of PINK1, such as the architecture of the unique insertions and the C-terminal region. Together with NMR and mass spectrometry studies, the structure explains how PINK1 interacts with ubiquitin via insertion-3 and its activation segment, and how PINK1 utilises the Ub- CR conformation for efficient Ser65 phosphorylation. In addition, the structure shows that two autophosphorylation sites in the N lobe regulate PINK1, by stabilising the functionally important insertions. The structure helped our understanding of the molecular basis of over 40 AR-JP patient mutations and may guide the design of ARJP therapeutics in the future.
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Michel, Martin Alex. "Specific recognition of atypical ubiquitin modifications." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/277694.
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Protein ubiquitination regulates various cellular events, such as protein degradation, immune signalling and DNA repair. This functional versatility arises from the ability of ubiquitin (Ub) to form distinct polymers linked through one of its eight primary amines (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63 and the N terminus of Met1). Discrimination of these differently linked polyUb chains by linkage-specific ubiquitin-binding domains (UBDs) is crucial to ensure an appropriate cellular response to a particular stimulus. While Lys48- and Lys63-linked chains have been studied extensively and their functions are well-described, the remaining, so-called atypical Ub chains have largely resisted characterisation. The broad aim of this thesis was to better our understanding of atypical Ub modifications with a focus on Ub recognition by linkage-specific binders. In the first part, the UBD family of Npl4-like zinc fingers (NZFs) will be discussed, as this family has previously been shown to be capable of linkage-specific recognition. A candidate-based approach was used to screen UBDs for linkage-preference for atypical Ub chains. This revealed that TRABID NZF1 is specific for Lys29/Lys33 chains and is the first UBD to specifically recognise these linkages. Moreover, [a protein] was found to be specific for Lys6/Lys63 chains and is the first UBD with specificity for Lys6 chains. Biochemical and structural characterisation of these UBDs in complex with their preferred chain types uncovered the molecular mechanisms that confer linkage specificity. Using insights gained from studying NZF UBDs, further investigations into the versatility of the NZF scaffold were performed. Unexpectedly, this revealed that [a protein] is able to recognise substrate ubiquitination of [a protein] in a substrate- and site-specific manner and this interaction was structurally and biochemically characterised. In the second part, the UBDs of all selective autophagy receptors were screened for interesting interactions with Ub. None of the UBDs tested exhibited linkage-specific interaction with Ub, except for OPTN, which prefers Met1-linked chains. Moreover, because autophagy receptors can also act in mitophagy, these UBDs were tested for preferential interaction with Ub phosphorylated on Ser65. However, none of the UBDs tested prefer phosphorylated Ub. Two structures of these UBDs in complex with Ub are presented, which together with previously published structures, allowed us to rationalise the observed binding behaviours. In the final part, the generation of linkage-specific tools for Lys6- and Lys33-/Lys11-linked chain is described. In collaboration with Avacta, linkage-specific affimer binders against Lys6- and Lys33/Lys11-linked polyUb chains have been developed. Crystal structures of the affimers in complex with their cognate diUb linkages revealed the basis of linkage specificity and further allowed structure-guided improvements. These improved affimers prefer their cognate diUb >106-fold over other Ub linkages. Furthermore, affimers were validated in a number of applications: using the Lys6-affimer as a pull-down reagent allowed enrichment of Lys6 chains over 100-fold compared to other linkages. This led us to identify the HECT E3 ligase HUWE1 as a major source of cellular Lys6 chains. Furthermore, the Lys6-affimer was used in confocal imaging where it localised to depolarized mitochondria in a Parkin-dependent manner, consistent with Parkin assembling Ub chains, including Lys6 linkages, upon mitochondrial damage. Together, these affimers are the first linkage-specific tools for these atypical Ub chains and enable targeted investigations into the biological functions of specific atypical Ub linkages. Taken together, the research presented in this thesis identified novel, linkage-specific components of the Ub system. Moreover, the developed tools will lay the basis for further, mechanistic investigations into the functions of atypical Ub linkages in important signalling pathways which will hopefully shed light on the biological roles of these understudied chain types.
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Sokratous, Kleitos. "Probing the affinity, selectivity and inhibition of ubiquitin-ubiquitin binding domain complexes by electrospray ionization mass spectrometry." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/14368/.
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This thesis describes the development and application of a rapid and sensitive electrospray ionization-mass spectrometry (ESI-MS) method to study the weak hydrophobic interactions seen in many Ub-Ub-binding domain (UBD) complexes. A range of UBDs has been screened against mono-Ub, di-Ub (Ub2) and tetra-Ub (Ub4). Affinities in the 2-200 J.lM range were found to be in excellent agreement with data obtained from other biophysical techniques. Insights into the UBD's preference for poly-Ub chain linkage and length are also provided by this methodology. Detection of a ternary complex involving Ub interacting simultaneously with two different UBDs demonstrated the co-existence of multisite interactions. A simple, clean and effective method for reducing charge states observed in ESI-MS without the use of any solution additives or instrumental modifications is also reported; with the charge reduction method ultimately promoting the investigation ofthe Ub-UBD interactions. Moreover, the development and application of a top-down proteomics approach to characterize the topology of an unanchored Ub dimer purified from rat skeletal muscle is also described in this thesis. This study has identified the topology of the Ub2 to be Lys48-linked. In addition, ESI-MS of endogenous Ub2 species has revealed the presence of cyclic Lys48-linked Ub2 and demonstrates for the first time that cyclisation of poly-Ub can also occur in vivo. Further to these studies, the inhibitory activity of small peptides against the complex formed by Ub with the ZnF domain of isopeptidase T (IsoT) is also investigated. Finally, the unusual effects of cation adduction upon the gas-phase conformation of three-helix bundle UBDs are revealed by ESI-IMS-MS and reported in this thesis.
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Bebington, Catherine. "Ubiquitin and ubiquitin-like proteins in the uterus and placenta of the human and non-human primate." Thesis, University of Nottingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312211.
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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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Wu, George Tatung. "The role of anaphase-promoting complex in cellular differentiation and tumorigenesis /." Access full-text from WCMC, 2008. http://proquest.umi.com/pqdweb?did=1528351821&sid=7&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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BINET, MARIE-NOELLE. "Structure et expression de genes d'ubiquitine de tournesol (helianthus annuus)." Strasbourg 1, 1991. http://www.theses.fr/1991STR13102.
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Afin d'etudier la regulation differentielle des genes d'ubiquitine chez le tournesol, une banque de cdna de fleurs de tournesol a ete criblee d'une sonde d'ubiquitine humaine. Deux cdnas de polyubiquitine, appeles ubf et ubb1, ont ete sequences et correspondent a des transcrits de 1600 nucleotides, codant pour une hexaubiquitine. Les genes correspondants a ces cdna ont ete recherches dans une banque genomique de racines de tournesol afin d'etudier les sequences regulatrices controlant leur transcription. Deux genes homologues de polyubiquitine, ubb1 et ubb2, ont ete isoles a l'aide d'une sonde specifique du cdna ubb1. Les transcrits ubb1 et ubb2 sont exprimes a un niveau faible dans les tissus analyses alors que les transcrits ubf sont abondants dans les fleurs. Inversement, les genes ubb1 et ubb2 sont induits dans les hypocotyles soumis a un stress thermique alors que les transcrits ubf ne varient pas dans ces conditions. Les deux genes isoles presentent une region 5 de 250 nucleotides en amont de leur site d'initiation de la transcription, qui est extremement conservee. L'activite promotrice de cette region a ete analysee par des experiences d'expression transitoires avec le gene rapporteur de la beta-glucuronidase (gus) dans les protoplastes de tabac. En l'absence d'inducteurs externes, l'activite relative gus sous le controle de cette derniere region est 5 fois superieure a celle obtenue avec le gene gus place sous le controle du promoteur 35s du camv. Des deletions reduisant la taille du promoteur ubb1 ont permis d'identifier une region de 65 pb responsable de l'essentiel de l'activite du promoteur ubb1 dans les protoplastes de tabac. Ces resultats suggerent que le gene de polyubiquitine ubb1 est sous le controle de multiples elements regulateurs et soulignent l'importance de ce gene dans la reponse cellulaire au stress
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Ratti, Francesca. "Role of HDAC6 in Skeletal Muscle Atrophy." Thesis, Lyon, École normale supérieure, 2014. http://www.theses.fr/2014ENSL0887.
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HDAC6 est une histone déacétylase hautement conservée, principalement cytoplasmique. Contrairement à d'autres désacétylases, HDAC6 a une spécificité de substrat unique pour les protéines non - histones . Outre les domaines de désacétylation, HDAC6 contient également un domaine de liaison à l'ubiquitine , qui relie HDAC6 de la voie ubiquitine / protéasome .L’atrophie du muscle squelettique est une condition sévère de perte progressive de masse musculaire au cours de certaines maladies telles le cancer, le diabète, le SIDA ou également immobilizations prolongées. Le contrôle de la masse musculaire est sous la dépendance d’un équilibre entre les processus anaboliques et cataboliques. L’atrophie se caractérise par une augmentation substantielle de la dégradation des protéines par le système ubiquitine-protéasome, causée par l'expression d'une série de gènes spécifiques, les atrogenes . Un des atrogenes induits plus spectaculaire est le muscle spécifique de l'ubiquitine ligase E3 MAFbx/Atrogin-1, qui prend soin de la dégradation de MyoD et de eIF3 -f. La dégradation de ces deux protéines inhibe l'expression de gènes et la traduction myotrophiques empêchant le remplacement de protéines dégradées.Récemment, nous avons identifié l’Histone Deacetylase 6 (HDAC6) comme un nouvel atrogène. L’expression de HDAC6 augmente au cours de l’atrophie musculaire, à la fois chez la souris et l’homme, à travers un mécanisme FOXO3 -dépendante. La déplétion de cet enzyme in vivo (electroporation de l’shRNA contre HDAC6 dans des muscle squelettiques de souris ou analyse de souris invalidées pour ce gène) protège contre l’atrophie. De plus, l’inhibition de HDAC6 après déclenchement de l’atrophie peut aussi atténuer le phénotype. Lors de la caractérisation du mécanisme d’action de HDAC6, nous avons montré que HDAC6 intéragit avec MAFbx et que elle est nécessaire pour l’ubiquitination de MyoD par MAFbx. Nos résultats montrent que la surexpression d’un mutant MyoD resistant à la degradation par MAFbx protège contre l’atrophie provoqué par la denervation.. De plus, certaines données préliminaires indiquent une implication de HDAC6 dans la dégradation de eIF3-f et dans le processus de autophagy dans le tissu musculaire , révélant une double rôle de HDAC6 dans le muscle squelettique .Ces preuves suggèrent que HDAC6 représente potentiellement une cible utile pour des traitements curatifsHDAC6 is a highly conserved histone deacetylase, mostly cytoplasmic. Unlike other deacetylases, HDAC6 has unique substrate specificity for non-histone proteins. Besides the deacetylation domains, HDAC6 also contains an ubiquitin-binding domain, which links HDAC6 to the ubiquitin/proteasome pathway. Skeletal muscle atrophy is a severe condition of muscle mass loss occurring during aging or in many clinical disorders as cancer, diabetes and AIDS. The maintenance of muscle mass is subtly controlled by an equilibrium between catabolic and anabolic processes. Muscle atrophy results as a partial suppression of protein synthesis and a substantial increase of protein breakdown by the ubiquitin-proteasome system, caused by the expression of a series of specific genes, the atrogenes. One of the atrogenes induced more dramatically is the muscle specific E3 ubiquitin ligase MAFbx/Atrogin-1, which takes care of the degradation of MyoD and of eIF3-f. Degradation of those two proteins inhibits expression of myotrophic genes and translation preventing the replacement of degraded proteins.We identified HDAC6 as a new atrogene. HDAC6 expression is up regulated during muscle atrophy in mouse and human through a mechanism FoxO3-dependent. In vivo depletion of this enzyme by shRNA electroporation or homologous recombination gives protection against atrophy and its inhibition during atrophy can partially reverse the muscle wasting phenotype. HDAC6 can interact with MAFbx and is required for MAFbx-mediated degradation of MyoD. According to our results, forced expression of a MyoD mutant resistant to HDAC6 and MAFbx dependent degradation prevents muscle wasting induced by denervation. Furthermore, some preliminary data show an involvement of HDAC6 in the degradation of eIF3-f and in the autophagy process in muscle tissue, revealing a double role of HDAC6 in skeletal muscle.These evidences suggest that HDAC6 potentially represents a valuable target for curative treatments
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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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Cheng, Yen-Fu. "Role of the ubiquitin-proteasome pathway in the inner ear : identification of an E3 ubiquitin ligase for Atoh1." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/96458.
Full textAbstract:
Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 91-105).
Atoh1, the proneural basic-helix-loop-helix transcription factor, is critical for the differentiation of inner ear hair cells. Hair cells do not develop in mice that lack Atoh1, and overexpression of the transcription factor in embryonic ears induces differentiation of extra hair cells. The level of Atoh1 expression is under the control of a Wnt and Notch transcriptional regulatory network to keep the level of mRNA within a narrow range. Once the protein is made, it activates its own expression through an interaction with the Atoh1 enhancer, such that Atoh1 transcription is self-perpetuating. Because of this autoregulatory loop, halting transcription of the gene to maintain Atoh1 at an appropriate level would require that the amount of protein be decreased. Since the ubiquitin-proteasome pathway regulates catabolism of key regulatory proteins, we assessed its role in the degradation of Atoh1. E3 ubiquitin ligases confer substrate specificity to degradation of proteins by transferring a ubiquitin tag to a specific protein substrate. Using an immunoprecipitation/mass spectrometry screening approach, we identified Huwe1, a HECT domain E3 ubiquitin ligase, as an Atoh1 binding partner. We validated the binding between Atoh1 and Huwe1 through reciprocal co-immunoprecipitation and mass spectrometry. We found that Huwe1 promoted polyubiquitylation of Atoh1 through a lysine 48-linked polyubiquitin chain. Mutation at a catalytic cysteine within the HECT domain of Huwe1 reduced the polyubiquitylation. We also defined a motif in the C-terminus of Atoh1 responsible for interaction with Huwe1. Inhibition of proteasomal activity, as well as Huwe1 depletion, stabilized Atoh1 in the cochlea and resulted in generation of new hair cells in the newborn cochlea.
by Yen-Fu Cheng.
Ph. D.
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Crimmins, Stephen Lewis. "Characterization and functional analysis of Usp14." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2007p/crimmins.pdf.
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Parvatiyar, Kislay. "Ubiquitin Dependent Regulation of Innate Antiviral Signaling." Scholarly Repository, 2010. http://scholarlyrepository.miami.edu/oa_dissertations/651.
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Induction of type I interferons by the transcription factors IRF3 and IRF7 is essential in the initiation of antiviral innate immunity. Activation of IRF3/7 requires C-terminal phosphorylation by the upstream kinases TBK1/IKKi, where IRF3/7 phosphorylation promotes dimerization, and subsequent nuclear translocation to the IFN-beta promoter. Recent studies have described the ubiquitin-editing enzyme A20 as a negative regulator of IRF3 signaling by associating with TBK1/IKKi, however the regulatory mechanism of A20 inhibition remains unclear. Here we describe the adaptor protein, TAX1BP1, as a key regulator of A20 function in terminating signaling to IRF3. Murine embryonic fibroblasts (MEFs) deficient in TAX1BP1 displayed increased amounts of IFN-beta production upon viral challenge compared to WT MEFs. TAX1BP1 inhibited virus-mediated activation of IRF3 at the level of TBK1/IKKi. TAX1BP1 and A20 blocked antiviral signaling by disrupting K63-linked polyubiquitination of TBK1/IKKi independently of the A20 deubiquitination (DUB) domain. Furthermore, TAX1BP1 was required for A20 effector function as A20 was defective for the targeting and inactivation of TBK1 and IKKi in Tax1bp1/ MEFs. Additionally, we found the E3 ubiquitin ligase TRAF3 to play a critical role in promoting TBK1/IKKi ubiquitination. Collectively, our results demonstrate TBK1/IKKi to be novel substrates for A20 and further identifies a novel mechanism whereby A20 and TAX1BP1 restrict antiviral signaling by disrupting a TRAF3/TBK1/IKKi signaling complex. Several viruses utilize a number of strategies to evade the host innate immune response by inhibiting the production of type I interferons. The Human T-cell leukemia virus type 1 (HTLV-1) has been shown to block interferon signaling, however the mechanism of inhibition is poorly understood. We show here that the HTLV-1 encoded protein, Tax plays a critical role in blunting the activation of type I interferons. Tax expression rendered MEFs hyper-permissive in supporting virus replication. Correspondingly, Tax blocked the production of IFN-beta. Interestingly, Tax did not require NEMO interaction to inhibit antiviral signaling to IRF3/7. Instead, Tax targeted RIP1 and further blocked IRF7 K63-linked polyubiquitination. Altogether, we show that Tax inhibits IFN activation by disrupting the ubiquitin dependent activation of IRF7 mediated by RIP1.
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Dixon, James Edward. "Arkadia Family Ubiquitin-Ligases in TGFp Signalling." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486282.
Full textAbstract:
During vertebrate development and in human pathology the regulation of the level and duration of TGFp signalling is important in controlling cell plasticity, migration, proliferation and apoptosis. My work has focused on Arkadia (Akd) which is a nuclear RING-H2 domain E3 ubiquitin-ligase that enhances Smad2/3-mediated TGFp sign!llling and is essential in mammalian development. Akd has recently been shown to destroy inhibitory (I)-Smads, sensitising cells to TGFp ligands. Here I describe data that demonstrates a second more important mechanism by which Akd enhances and terminates Smad2/3 signalling in which phosphorylated(P)Smad2 and Smad3 are the targets for Akd. I have employed reporter' analyses to investigate the Akd protein domains required for ubiquitination, and highlight�· the requirement ofP-Smad2/3-interaction to activate Smad2/3-mediated signalling. This has lead to the hypothesis that the primary pathway of Akd is through ubiquitination of PSmad2/ 3s, which many co-operate with the function of Akd to destroy I-Smads. Furthermore, I have isolated a second Akd gene (Akd2) in mouse and human, and shown its function is complementary to enhance Smad2/3 signalling in reporter analyses. Moreover, I confirmed the importance of Akd in TGFp signalling by isolating a functional Akd homologue in the invertebrate drosophila melanogaster (termed dAkd). Akd and certain isoforms of Akd2 are ubiquitpusly expressed. I have employed reporter analyses to address the regulation of Akd genes post-transcriptionally and engineered tools to analyse these mechanisms in the�· embryo. My data strongly implicates post-translational regulation of Akd genes to fine-tune TGFp signalling in vivo. My study confirms the central role of the Akd family in regulation of TGFpsignals and highlights the importance of post-transcriptional control ofAkd genes. These data will be important in understanding how the alternate cellular interpretation of TGFp ligands is achieved in many important biological processes.
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Ptak, Christopher. "Ubiquitin conjugating enzymes, relating form to function." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq23058.pdf.
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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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Adlington, Jennifer. "Ubiquitin binding by the p62 UBA domain." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/14519/.
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Over 30 different mutations in p62 UBA have been identified in patients with Paget's disease of bone (PDB). The mechanisms which underlie PDB are poorly understood, although impaired ubiquitin binding has been identified as a mechanism in the onset of disease. However, the decrease in affinity is subtle for many PDB mutants. The p62 UBA is unique amongst UBAs since it exists as a highly stable dimer but binds to ubiquitin as a monomer. The dimerization interface partially occludes the ubiquitin binding interface resulting in competing equilibria. The factors which regulate the affinity of p62 UBA were examined in this thesis. In isolated p62 UBA the monomer-dimer equilibrium and the effects of phosphorylation were investigated. By mutating residues at the dimerization interface, weaker dimers which had a higher affinity for ubiquitin were produced. The weak dimers had an increased population of monomer at equilibrium. A phosphorylation site at Ser403 in p62 UBA was recently identified. Phosphomimetic mutants which showed subtle increases in affinity for ubiquitin were generated. The increase was attributed to the close proximity of Ser403 to the ubiquitin binding interface. Factors outside the UBA also have a role in regulating UBA affinity. Binding by p62 UBA was therefore probed in longer p62 constructs. A fragment of p62 encoding residues 300-440 was used to investigate p62 binding to multiple proteins. A ternary complex was formed, but an allosteric relationship was not observed by ubiquitin and MAP-Le3 in binding to the p62 fragment. A model of full length oligomeric p62 was generated to probe avid binding to polyubiquitin chains. The model showed higher affinity for linear diubiquitin than monoubiquitin suggesting avidity effects are influential in oligomeric p62 binding. Since the effects of PDB mutations and phosphorylation are subtle, they are likely to be amplified by avidity in vivo.
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Tinsley, Caroline L. "Multi-protein complexes, ubiquitin and muscle disease." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510248.
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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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Marsh, Sadie. "Ubiquitin-like proteins in the human uterus." Thesis, University of Nottingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272574.
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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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Nathan, James Alexander. "The RING-CH ubiquitin E3 ligase MARCH7." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612286.
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