Retinoic acid (RA) has been recognized as a chemotherapeutic agent for various malignances such as lung, skin as well as cervical cancers. It binds to retinoid receptors heterodimers and consequently activates several RA-responsive genes which are involved in many biological processes including vertebrate development, bone growth, vision, haematopoiesis, cell growth, differentiation and apoptosis. These genes are under the control of numerous regulators to ensure their timely ordered activities. Among these regulators, we focused here on the 26S proteasome and ubiquitination. It has been reported that the activity of the ubiquitin/proteasome system (UPS) plays a fundamental role in retinoic acid receptor (RAR)-regulated transactivation. The mechanisms underlying this role, however, remain to be established. Chromatin immunoprecipitation (ChIP) assays in our study demonstrated that the 26S proteasome activity is important for preserving the occupancy of a TATA box-containing RA-responsive promoters by liganded retinoid receptors and thus by their coactivators. Additionally, by using coimmunoprecipitation assays and by measuring the half-life of retinoid receptors, we found that the non-proteolytic function of the proteasome is required for ligand-dependent association between DNA-free RAR-α and both DNA-free RXR-α and coactivators. Moreover, using immunofluorescent staining and in vivo ubiquitination assays, a proteasome inhibition-dependent cytoplasmic localization of RAR-α as well as ligand-enhanced ubiquitination and stabilization of RAR-α were shown. Our findings therefore, define novel mechanisms by which the UPS controls RAR-regulated genes. Furthermore, we shed new light on the regulators of retinoid receptors ubiquitination and subcellular localization.

SH3 domains are excellent models for probing folding and protein interactions. This thesis describes NMR studies of several SH3 domains, including the N-terminal SH3 domain of the Drosophila adaptor protein Drk (drkN SH3 domain), the SH3 domain of the proto-oncogene tyrosine-kinase Fyn, and the SH3 domains of the human adaptor protein CIN85, involved in Cbl-mediated downregulation of epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs). The drkN SH3 domain is an ideal system for studying disordered states. The unique quality of this isolated domain is that it exists in an approximately 50/50 equilibrium between its folded and unfolded states under non-denaturating buffer conditions. Interestingly, the single T22G mutation dramatically stabilizes the domain. Here the NMR structures of the drkN SH3 domain and its T22G mutant are determined and compared in order to illuminate the causes of the marginal stability of the domain. Solvent exposure of the folded and the unfolded drkN SH3 domains are probed and compared with a novel NMR technique using molecular oxygen dissolved in solution as a paramagnetic probe. The changes in partial molar volume along the folding trajectories of the drkN SH3 and Fyn SH3 domains are also studied and analyzed here in terms of changes in protein hydration and packing accompanying folding. Finally, the interactions between the SH3 domains of CIN85 and ubiquitin are discussed. All three are shown to bind ubiquitin. The structure of the SH3-C domain in complex with ubiquitin is presented and the effect of disruption of ubiquitin binding on ubiquitination of CIN85 and EGFR in vivo is discussed. SH3 domains are easily amendable to a wide range of NMR approaches and provide a good system for development and testing of novel methods. Through the use of these approaches significant insights into details of SH3 domain structure, stability, mechanisms of folding and cellular function have been gained.

A autofagia é o processo de degradação de estruturas celulares através do seu direcionamento ao lisossomo. As proteínas TRIMs reconhecem as -cargas? autofágicas e reúnem o complexo de nucleação do fagóforo, contudo se desconhece a função de cada domínio e a importância da atividade de E3 ligase para a sua atividade. A proteína TRIM49 clonada e expressa em E. coli ou em células humanas HEK293T não apresentou atividade de E3 ubiquitina ligase in vitro e reduziu os níveis totais de ubiquitinação in vivo, indicando que não é um E3 ubiquitina ligase. Células desafiadas com Htt74Q apresentaram menores níveis de citotoxicidade quando co-transfectadas com TRIM49 selvagem, mas não com os mutantes do domínio RING ou SPRY, indicando os dois domínios são necessários para sua atividade celular. A proteína selvagem se colocaliza com o marcador autofágico LC3, após o bloqueio da autofagia com bafilomicina A1. Os resultados indicam que a TRIM49 pode atuar na degradação intracelular de proteínas, por um mecanismo não dependente de atividade de E3 ligase. / Autophagy is the process of degradation of intracellular proteins through their directioning to the lysosome. TRIM proteins can directely recognize autophagic cargo and also act as a hub for the phagophore nucleation complex, however the function of each domain and the role of the E3 ligase activity in this process is unknown. The TRIM49 protein cloned and expressed in E. coli or in human cells HEK23T showed no ubiquitin E3 ligase activity in vitro and cells transfected with the wild type protein showed lower levels of polyubiquitinated proteins, indicating that TRIM49 is not a bona fide E3 ubiquitin ligase. Cells challenged with Htt74Q presented lower cytotoxicity levels when cotransfected with wild type TRIM49, when compared with the RING domain mutant or with the truncated protein lacking the SPRY domain, indicating that both domains are required for its cellular activity. The wild type protein colocalizes with the autophagic marker LC3 after treatment with the autophagy inhibitor bafilomycin A1. Taken together, these results indicate that the TRIM49 protein plays a role in protein degradation independently of a E3 ligase activity.

Ubiquitin (Ub) ligases are important regulatory and signalling molecules, which are involved in majority of cellular processes such as differentiation, DNA repair, and regulation of energetic metabolism or immune response. E3 Ubiquitin ligases are also responsible for pathophysiological changes in the organism and their activity is associated with many human diseases including cancers. This makes E3 Ubiquitin ligases to be new diagnostic markers and interesting pharmaceutical targets. Based on previous studies, these enzymes evince very specific expression in the level of tissues or cell populations. Determination of this specific expression is important for a better understanding of their biological function. In this diploma thesis we systematically screened presence of 370 genes of E3-Ub ligases in gastrointestinal tract under physiological conditions and during acute inflammatory damage of distal colon. Obtained data allowed us to select genes, which can play important role in homeostasis as well as pathophysiology and regeneration of gastrointestinal tract. The screening was based on the expression profiling using qPCR, followed by in situ hybridization to determine the exact localization of the gene expression within tissues. From qPCR analysis was predicted hundred thirty seven candidates for...

Retinoic acid (RA) has been recognized as a chemotherapeutic agent for various malignances such as lung, skin as well as cervical cancers. It binds to retinoid receptors heterodimers and consequently activates several RA-responsive genes which are involved in many biological processes including vertebrate development, bone growth, vision, haematopoiesis, cell growth, differentiation and apoptosis. These genes are under the control of numerous regulators to ensure their timely ordered activities. Among these regulators, we focused here on the 26S proteasome and ubiquitination. It has been reported that the activity of the ubiquitin/proteasome system (UPS) plays a fundamental role in retinoic acid receptor (RAR)-regulated transactivation. The mechanisms underlying this role, however, remain to be established. Chromatin immunoprecipitation (ChIP) assays in our study demonstrated that the 26S proteasome activity is important for preserving the occupancy of a TATA box-containing RA-responsive promoters by liganded retinoid receptors and thus by their coactivators. Additionally, by using coimmunoprecipitation assays and by measuring the half-life of retinoid receptors, we found that the non-proteolytic function of the proteasome is required for ligand-dependent association between DNA-free RAR-α and both DNA-free RXR-α and coactivators. Moreover, using immunofluorescent staining and in vivo ubiquitination assays, a proteasome inhibition-dependent cytoplasmic localization of RAR-α as well as ligand-enhanced ubiquitination and stabilization of RAR-α were shown. Our findings therefore, define novel mechanisms by which the UPS controls RAR-regulated genes. Furthermore, we shed new light on the regulators of retinoid receptors ubiquitination and subcellular localization.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

La mucoviscidose (CF, Cystic Fibrosis) est la maladie génétique la plus fréquente dans les populations d’origine caucasienne. Les malades présentent une symptomatologie variée, dominée par une bronchopneumopathie chronique obstructive due à des sécrétions de mucus abondantes et anormalement épaisses et une réponse inflammatoire chronique excessive. La mucoviscidose résulte de mutations dans le gène codant la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator), dont la plus fréquente est la délétion d’une phénylalanine en position 508 (F508del) qui est à l’origine d’un adressage défectueux et d’une fonction altérée de la protéine. Afin d’identifier différents partenaires moléculaires de CFTR qui participent à son processus de maturation, à son trafic à la membrane plasmique ou à sa fonction, un criblage double hybride de levure a été effectué en utilisant la troisième boucle intra-cytoplasmique de CFTR (ICL3) comme appât. A l’issue de ce criblage, 14 clones indépendants ont pu être identifiés dont la protéine COMMD1 qui a initialement été décrite comme un régulateur de l’homéostasie du cuivre, de l’absorption sodique et de la voie de signalisation NF-?B. L’objectif principal de ce travail a été de déterminer quel pouvait être l’impact de la protéine COMMD1 sur la maturation et le trafic intracellulaire de CFTR afin de déterminer le rôle de cette protéine dans la physiopathologie de la mucoviscidose. Nous avons montré que la protéine COMMD1 est un nouveau partenaire cytoplasmique du canal CFTR, qui régule le trafic intracellulaire de ce canal par inhibition de l’ubiquitinylation probablement au niveau des endosomes d’endocytose et de recyclage. Notre étude permet de proposer une nouvelle voie de trafic de CFTR via un modèle d’ubiquitinylation régulé par COMMD1. Au cours de cette étude, nous avons également identifié le récepteur 1 de la transferrine (TFR1) comme un nouveau partenaire de COMMD1 dont le mécanisme de régulation semble similaire à celui proposé pour CFTR. Dans un second temps, nous nous sommes intéressés aux propriétés inhibitrices de COMMD1 dans la réaction inflammatoire. COMMD1 a été décrit comme le prototype d’une nouvelle famille de protéines jouant un rôle dans l’inhibition de la voie de signalisation NF-kB. Nous avons observé que la distribution subcellulaire de COMMD1 est différente dans les cellules CF et non-CF. Nous avons mis en évidence que la surexpression de COMMD1 dans les cellules épithéliales bronchiques CF, qui présentent une inflammation excessive, pouvait restaurer un niveau d’inflammation comparable aux cellules non-CF. COMMD1 est impliquée dans plusieurs processus cellulaires altérés dans la physiopathologie de la mucoviscidose, affectant le trafic du canal CFTR, l’absorption de sodium et la réponse inflammatoire. Comprendre comment moduler d’une part le processus d’absorption/sécrétion des ions par le trafic de canaux ioniques et d’autre part, l’inflammation des cellules CF par rapport aux non-CF, devrait permettre d’identifier de nouvelles pistes thérapeutiques. Ces traitements permettraient à la fois de réduire la réaction inflammatoire exacerbée, sans nuire à l’activité essentielle de défense contre les pathogènes, et d’améliorer la sécrétion de fluide chez les patients atteints de mucoviscidose / Cystic fibrosis is mainly caused by mutations interfering with the biosynthetic folding of the CFTR protein. The aim of this study was to find proteins able to interact with CFTR and modify its processing. We have identified COMMD1 as a new CFTR partner. COMMD1 is a regulator of copper homeostasis and sodium uptake through interaction with ENaC, as well as the prototype of a new protein family that plays a role in inhibiting NF-?B signalling Co-immunoprecipitation experiments showed that COMMD1 associates with endogenous CFTR in HT29 cells and with F508del-CFTR in heterologously expressing epithelial cells. COMMD1 sub-cellular distribution is both nuclear and cytoplasmic, and more precisely in vesicular cytoplasmic compartments, as assessed by immunocytochemical microscopy. Further studies showed COMMD1 partial codistribution with an early endosomal compartments (TfR). COMMD1 is not involved in CFTR processing (C band) but wt-CFTR cell surface expression was halfreduced when COMMD1 expression was silenced. Unlike F508del-CFTR in temperature rescue, COMMD1 over-expression increased 15% wt-CFTR cell surface expression. Assessment of CFTR ubiquitination showed that COMMD1 over-expression strongly decreased CFTR ubiquitination therefore increasing CFTR cell surface expression. Finally, these data indicate that COMMD1 vesicular compartment is involved in CFTR trafficking through inhibition of CFTR ubiquitination. Understanding how COMMD1 modulation modifies transepithelial transport and inflammation in CF versus non CF cells should give new therapeutic clues to reduce exacerbated inflammation and improve fluid secretion in CF patients

>Magister Scientiae - MSc / Retinoblastoma Binding Protein 6 (RBBP6) is a 200 kDa RING finger-containing protein that plays a role in 3'-end poly-adenylation of mRNA transcripts as well as acting as an E3 ubiquitin ligase against a number of proteins involved in tumourigenesis, including p53. Since the human protein is too large and poorly structured for heterologous expression in bacteria, it would be advantageous to identify smaller fragments suitable for expression in bacteria. Many E3 ubiquitin ligases form homo-dimers and dimerisation is important for their activity; structural studies of the isolated RING finger of RBBP6 showed that it forms a weak homo-dimer. This poses the question of whether the complete RBBP6 protein forms homo-dimers in vivo, and, if so, whether a fragment of RBBP6 containing the RING finger could be identified which would be suitable for structural as well as functional studies. Such a construct would allow detailed investigation of the homo-dimeric state of the fragment, the relationship between dimerisation and ubiquitination activity, and the role of domains such as the DWNN domain and zinc finger in ubiquitination. A fragment consisting of the first 335 residues of RBBP6, dubbed R3 because it contained the first three domains of the protein, was expressed, along with three variants expressing mutations known to disrupt the dimerisation of the isolated RING finger. Size exclusion chromatography showed that R3 forms a strong homo-dimer that was not disrupted by the mutations, suggesting that additional parts of R3 outside of the isolated RING finger form part of the interface. To identify whether this included the DWNN domain or the zinc finger, a shorter fragment dubbed R2, excluding the N-terminal DWNN domain, was cloned and expressed. This was also found to form a strong homo-dimer, suggesting that the DWNN domain may not form an essential part of the dimer interface. Availability of the RING finger samples and monomerising mutations allowed investigation of whether the RING finger from RBBP6 was able to auto-ubiquitinate itself. Using a fully in vitro ubiquitination assay supplemented with intact proteasomes purified from human cell lysates, we found that wild type RING auto-ubiquitinates itself very efficiently, catalysing its own destruction in the proteasome. This provides an answer to the question of why RBBP6 is so difficult to detect in mammalian cells. Surprisingly, monomeric mutant RING fingers were also able to auto-ubiquitinate and catalyse their own destruction, although perhaps not as efficiently as wild type. This result would appear to rule out the hypothesis that dimerisation of RBBP6 is required for ubiquitination activity. Finally, samples of the RING finger from human MDM2 were expressed in bacteria and used to investigate whether the RING fingers of RBBP6 and MDM2 interact directly with each other. If so, this may provide a mechanism whereby RBBP6 and MDM2 cooperate in ubiquitination of p53. The results of a GST pull down assay using GST-MDM2-RING as ''bait'' and RBBP6-RING as ''prey'' provides evidence that such an interaction between the RING does exist. This work lays the foundation for future structural studies of the RING-RING hetero-dimer using protein Nuclear Magnetic Resonance Spectroscopy.

This paper was published in Nature 2009 Aug 13;460(7257):904-8. doi: 10.1038/nature08240. http://www.nature.com/nature/journal/v460/n7257/full/nature08240.html / 京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12855号 / 論医博第2085号 / 新制||医||1006(附属図書館) / 31535 / (主査)教授 髙折 晃史, 教授 羽賀 博典, 教授 岩井 一宏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le facteur de transcription NF-KB joue un rôle essentiel dans le développement, l’homéostasie, la survie du système immunitaire, mais également dans la propagation de certains lymphomes. L’activation optimale de NF-ΚB en réponse à l’engagement de nombreux immunorécepteurs repose sur la mise en place de larges signalosomes dans lesquels des adaptateurs spécifiques sont recrutés et poly-Ubiquitinylés de façon non-Dégradative. En réponse à des cytokines pro-Inflammatoires ou à l’activation des récepteurs antigéniques, ces adaptateurs ubiquitinylés s’accumulent sur la face cytoplasmique du réticulum endoplasmique (RE) via la protéine du RE metadherine (MTDH) qui assure la propagation du signal NF-KB. Toutefois, la nature des E3 ligases en charge de relayer NF-KB au niveau des organites intracellulaires reste méconnue. C’est pourquoi j’ai réalisé le crible par bioluminescence d’une librairie de siRNA dirigée contre les 46 E3 ubiquitine ligases humaines pourvues d’un domaine transmembranaire qui les ancrent au niveau de différents compartiments cellulaires afin d’étudier leur impact sur l’activation de NF-KB en réponse à une stimulation antigénique dans un modèle de lymphocytes T immortalisés Jurkat. Nous avons identifié la protéine du RE TRIM13 comme un régulateur positif de la signalisation NF-ΚB. Nos données suggèrent un modèle dans lequel TRIM13 régule l’activation de NF-KB en modulant indépendamment l’activation de deux membres clés de la famille NF-KB au cours de l’activation lymphocytaire, RelA (p65) et c-Rel.Lors de cette thèse, j’ai également participé au crible d’une librairie de siRNA ciblant les 96 déubiquitinases (DUBs) codées par le génome humain afin d’identifier celles en charge de ramener les cellules vers leur état basal. Ceci a permis la caractérisation de la protéase spécifique de l’ubiquitine USP34 (Ubiquitin specific protease 34). La réduction des niveaux endogènes de USP34 potentialise l’activation de NF-KB en réponse à l’engagement du récepteur antigénique T ou du récepteur au TNFa et la liaison de NF-KB à l’ADN est accrue. Collectivement, ces résultats suggèrent que USP34 est un nouvel acteur impliqué dans la régulation négative de NF-KB.Ces résultats illustrent l’importance des processus d’ubiquitination réversibles dans la régulation de la signalisation NF-ΚB et introduisent les cribles génétiques comme un outil efficace pour l’identification de régulateurs de processus biologiques divers. / The transcription factor NF-KappaB plays a critical role in the development, homeostasis, the survival of the immune system, but also in the propagation of certain lymphomas. The optimal activation of NF-KappaB in response to the engagement of many immunoreceptors rely on the implementation of large signalosomes where specific adaptors are recruited and poly-Ubiquitinylated in a non-Degradative manner. In response to proinflammatory cytokines or activation of antigen receptors, these Ubiquitinylated adaptors accumulate on the cytoplasmic leaflet of the endoplasmic reticulum (ER) via the ER protein metadherin (MTDH) providing NF-KappaB signal propagation . However, the nature of the E3 ligases responsible for relaying NF-KappaB in intracellular organelles remains unknown. This is why I made the screen ingby bioluminescence of a library of siRNAs targeting the 46 human ubiquitin E3 ligases provided with a transmembrane domain that anchor them at different cellular compartments to study their impact on the NF-KappaB activation in response to antigenic stimulation in immortalized T lymphocytes Jurkat. We identified the ER-Protein TRIM13 as a positive regulator of NF-KappaB signaling. Our data suggest a model in which TRIM13 regulates the activation of NF-KappaB activation by modulating independently two key members of the NF-KappaB family during lymphocyte activation, RelA (p65) and c-Rel. In this thesis, I also participated in the screening of a library of siRNAs targeting the 98 deubiquitinases (DUBs) encoded by the human genome to identify those in charge of the reset of the system to basal state. This screen allowed the characterization of the ubiquitin-Specific protease USP34 (ubiquitin specific protease 34). The reduction of endogenous levels of USP34 potentiates the activation of NF-KappaB in response to engagement of the antigen receptor or T receptor antagonists and enhances NF-KappaB DNA binding. Collectively, these results suggest that USP34 is a new player involved in the negative regulation of NF-KappaB. These results illustrate the importance of reversible ubiquitination process in the regulation of the NF-KappaB signaling and introduce genetic screens as an effective tool to identify regulators of diverse biological processes.