L’incidence du mélanome a augmenté de façon considérable lors des trente dernières années avec un doublement tous les dix ans. Le mélanome ne représente que 5% des cancers cutanés mais entraîne 80% de décès, ce qui constitue un problème majeur de santé publique. En effet, cette tumeur est extrêmement agressive et possède un fort potentiel métastatique. Dès l’apparition de métastases, le pronostic vital devient fortement défavorable. Malgré des avancées thérapeutiques majeures, de nombreux patients sont encore réfractaires à ces nouveaux traitements. La compréhension des mécanismes impliqués dans le développement de cette tumeur reste donc un enjeu de premier ordre. Le séquençage d'exomes a conduit à l'identification d'une mutation dans le gène RAC1 (la mutation P29S) constituant une des mutations somatiques les plus fréquentes dans le mélanome (après les mutations BRAFV600, NRASQ61 et NF1). RAC1 est une petite GTPase qui fonctionne dans plusieurs processus cellulaires. Dans des conditions physiologiques, l'activité de RAC1 est principalement contrôlée par des protéines activatrices de l'activité GTPase (GAPs) et des facteurs d'échange Nucléotidique (GEF). GAPs et GEFs contrôlent le niveau de RAC1-GTP et régulent donc son activité. L'activité de RAC1 est aussi dépendante de son niveau d'expression protéique qui est contrôlé par des E3 ubiquitine ligases, parmi lesquelles HACE1. HACE1 est considérée comme un suppresseur de tumeur. De façon inattendue, les données obtenues montrent clairement que HACE1 favorise les propriétés migratoires et tumorigéniques des cellules de mélanome. / Melanoma incidence has considerably increased over the last thirty years, with a doubling every ten years. Melanoma accounts for only 5% of cutaneous cancers but causes more than 80% of deaths, which is a major public health problem. Indeed, this tumor is extremely aggressive and has a high metastatic potential. After the onset of metastases, the prognosis becomes highly unfavorable. Despite major therapeutic advances, many patients are still refractory to these new treatments. Understanding the mechanisms involved in the development of this tumor and the identification of new therapies remain a major issue. The sequencing of exomes led to the identification of a mutation in the RAC1 gene (P29S) constituting one of the most frequent somatic mutations in melanoma (after the BRAFV600, NRASQ61 and NF1 mutations). RAC1 is a small GTPase that is involved in several key cellular processes. Under physiological conditions, the activity of RAC1 is mainly controlled by GTPase activating proteins (GAPs) and Nucleotide Exchange (GEF) exchange factors. GAPs and GEFs control the level of RAC1- GTP and thus regulate its activity. The activity of RAC1 is also dependent on its protein level of expression which is controlled by E3 ubiquitin ligases, including HACE1. HACE1 is considered a tumor suppressor. Unexpectedly, our data clearly show that HACE1 promotes migratory and tumorigenic properties of melanoma cells. Indeed, inhibition of HACE1 alters migration of melanoma cells in vitro, as well as in vivo pulmonary colonization in mice. Transcriptomic analysis of 4 melanoma cell lines demonstrated that HACE1 suppression inhibits ITGAV and ITGB1 expression.

L'inflammation est un processus déclenché suite à la détection de pathogènes et de dommages tissulaires. Elle conduit à la sécrétion de cytokines pro-inflammatoires par les cellules immunitaires innées ainsi qu'au déclenchement de la pyroptose, mort cellulaire pro-inflammatoire. NLRP3 est une protéine senseur de stress cellulaire régulant le déclenchement de ces processus via la formation d'une plateforme multiprotéique appelée inflammasome. L'activation non contrôllée de NLRP3 conduit au développement d'une maladie auto-inflammatoire appelée CAPS (Cryopyrin associated periodic syndrome). De plus, l'inflammasome est impliqué dans le développement et la sévérité des symptômes de nombreuses maladies multifactorielles (diabète de type 2, athérosclérose, maladie de Parkinson et d'Alzheimer, sclérose en plaques, cancers...). Les mécanismes régulant l'activation de NLRP3 ne sont pas encore compris, mais les modifications post-traductionnelles de NLRP3 sont impliquées dans ce processus. Notre laboratoire a identifié différents sites d'ubiquitination et de phosphorylation sur NLRP3 par des approches biochimiques. Via la création de lignées cellulaires NLRP3 knock out reconstituées pour exprimer NLRP3 muté sur les résidus précédemment identifiés et de souris NLRP3 knock-in par la technique de CRISPR/Cas9, le travail de thèse a consisté en l'étude de l'impact fonctionnel de ces modifications. Ces résultats montrent que les substitutions de deux lysines identifiées comme étant ubiquitinées conduisent à une dérégulation de l’activation de l'inflammasome NLRP3 dans les cellules primaires. Un nouveau point de contrôle de l'activation de NLRP3 a ainsi pu être mis en lumière. / Inflammation is triggered after the sensing of pathogens or tissue damages. This process leads to the secretion of pro-inflammatory cytokines by innate immune cells and to the triggering of a pro-inflammatory form of cell death called pyroptosis. NLRP3 protein is a sensor of cellular stress and regulates the triggering of these events through the formation of a multiproteic platform called inflammasome. NLRP3 activation has to be tightly controlled as its deregulation leads to the development of an auto-inflammatory disease called CAPS (Cryopyrin associated periodic syndrome). Moreover, NLRP3 inflammasome is associated with the development and the severity of numerous multifactorial diseases (type 2 diabetes, atherosclerosis, Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, cancers…). The mechanisms involved in the regulation of NLRP3 activation are not fully understood. Recently, post-translational modifications of NLRP3 were shown to be important for the regulation of NLRP3 activation. Our lab has identified several phosphorylation and ubiquitination sites on this protein through biochemical studies. This phD work aims to identify the functional impact of these modifications. Thus, the generation of reconstituted cell lines expressing NLRP3 mutated on the previously identified residues and the generation of NLRP3 knock in mice via CRISPR/Cas9 technology were performed. The results show that substitution of two lysine residues previously identified as ubiquitinated leads to the deregulation of NLRP3 inflammasome activation in primary cells. This work highlights a new point of control in NLRP3 activation.

Les brassinostéroïdes (BR) sont des hormones stéroïdes végétales qui jouent un rôle dans la croissance et le développement des plantes. Ils sont perçus à la surface cellulaire par le récepteur kinase BRI1 (BR insensitive 1) situé au niveau de la membrane plasmique. La voie de signalisation des BRs implique la cis et trans-phosphorylation de BRI1 et de son corécepteur BAK1 (BRI1 Associated Receptor Kinase 1) et aboutit à la déphosphorylation des facteurs de transcription BZR1 (Brassinazole Resistant 1) et BES1 (bri1-EMS-Supressor 1) et l’activation des réponses génomiques aux BRs. Les mécanismes permettant la dé-activation du complexe de perception des BRs sont encore peu connus mais peuvent impliquer la déphosphorylation de BRI1, la fixation de régulateur négatif comme BKI1, et l’endocytose de BRI1. Mon travail de doctorat a consisté à étudier les mécanismes d’endocytoses et de dégradation de BRI1 et leurs régulations par les conditions environnementales.L’ubiquitination est une modification post-traductionnelle impliquant l’attachement d’un polypeptide d’ubiquitine (Ub) sur une ou plusieurs lysines (K) d’une protéine cible. L’ubiquitine elle-même peut être sujette à l’ubiquitination, créant ainsi des chaînes de poly-ubiquitine (polyUb) qui peuvent adopter des topologies différentes selon le résidu K de l’ubiquitine impliqué dans la formation de la chaîne. Parmi ces chaînes de polyUb, la chaîne impliquant la lysine-63 (K63) est connue pour être impliquée dans la dégradation des protéines par endocytose chez les levures et les mammifères. Néanmoins, peu de choses sont connues sur l’endocytose dépendante de l’ubiquitine chez les plantes. Durant la première partie de ma thèse, j’ai démontré que BRI1 est modifié in vivo par des chaînes de polyUb K63 et j’ai pu identifier des sites putatifs d’ubiquitination dans BRI1. En utilisant une forme artificiellement ubiquitinée de BRI1 ainsi qu’une forme non ubiquitinable de BRI1, j’ai montré que l’ubiquitination joue un rôle sur l’internalisation de BRI1 à la surface cellulaire et est essentielle pour son adressage à la vacuole. Par ailleurs, j’ai établi que la dynamique de la protéine BRI1 médiée par son ubiquitination joue un rôle important dans le contrôle des réponses des BR.La deuxième partie de ma thèse m’a permis de découvrir une connexion entre l’endocytose dépendante de l’ubiquitine de BRI1 et la réponse des plantes à une élévation de température. J’ai notamment montré que l’accumulation de la protéine BRI1 est réduite dans les racines lorsque les plantes sont cultivées à une température plus élevée (i.e. 26°C). Cependant, la forme non ubiquitinable de BRI1 ne répond pas à cette élévation de la température suggérant une implication de l’endocytose dépendant de l’ubiquitine dans de telles conditions. Cette déstabilisation de BRI1 observée à température plus élevée se traduit au niveau moléculaire par une inhibition de la voie de signalisation des BRs et une hypersensibilité à des traitements BRs exogènes. Les plantes répondent à une montée subite de la température par une augmentation de la longueur de l’hypocotyle, des pétioles et de la racine principale ; processus largement sous le contrôle de l’auxine. J’ai accumulé au cours de ma thèse des évidences génétiques et génomiques montrant que la déstabilisation du BRI1 et l’inhibition de la signalisation des BR à plus hautes températures contrôle l’élongation des racines indépendamment de l’auxine. En conclusion, les résultats obtenus indiquent que BRI1 intègre les informations de température et de signalisation des BRs pour ajuster la croissance des racines lors de variations des conditions environnementales. / Brassinosteroids (BRs) are plant steroid hormones that play important roles on plant growth and development, and that are perceived at the plasma membrane (PM) by the receptor kinase BRI1 (BR insensitive 1). The perception of BRs by BRI1 triggers the cis and trans-phosphorylation of BRI1 and its co-receptor BAK1 (BRI1 Associated Receptor Kinase 1) initiating this way the BR signaling cascade that culminates with the de-phosphorylation of the transcription factors BZR1 (Brassinazole Resistant 1) and BES1 (bri1-EMS-Supressor 1), allowing these transcription factors to activate the transcription of thousands of BR-responsive genes. The mechanisms allowing the deactivation of the BR receptor complex are still elusive but may involve dephosphorylation, binding to negative regulators such as BKI1 and endocytosis. My PhD work focused on the endocytosis and the degradation of BRI1, and its regulation by environmental conditions.Ubiquitination is a post-translational modification that consists of the attachment of ubiquitin (Ub) polypeptides to one or several lysine (K) of a target protein. Ubiquitin itself can undergo self-ubiquitination thereby creating polyubiquitin (polyUb) chains that can harbor different topologies depending on the lysine residue from ubiquitin involved in the chain formation. Among these, polyUb chains involving lysine-63 (K63) are known to be involved in the degradation of proteins by endocytosis in yeast and mammals, while very little is known in plants. On a first part of my thesis, I demonstrated that BRI1 is post-translationally modified by K63 poly-ubiquitin chains and I identified putative ubiquitination sites in BRI1. Using both artificial ubiquitination of BRI1 and the generation of an ubiquitination-defective BRI1, I showed that ubiquitination plays a role on the internalization of BRI1 from the cell-surface, and is essential for its vacuolar targeting. Furthermore, ubiquitin-mediated BRI1 protein dynamics was also shown to play an important role in the control of BR responses by stabilizing BRI1 at the PM.The second part of the PhD uncovered a connection between BRI1 ubiquitin-mediated endocytosis and plant responses to elevated ambient temperature. I showed that BRI1 protein accumulation is lower at elevated temperature (i.e. 26°C) in roots, while the ubiquitin-defective form of BRI1 failed to respond, indicating a role of the BRI1 ubiquitin-mediated endocytosis in temperature responses. This temperature-mediated destabilization of BRI1 correlates with a downregulation of BR signaling and BR responses. Plants respond sudden rise in temperature by increasing their primary root length, a process that is under the control of auxin. Importantly, I accumulated genetic and genomic evidence that BRI1 destabilization and downregulation of BR signaling controls root elongation upon elevated ambient growth temperature, with little or no contribution of auxin. Altogether, our results establish that BRI1 integrates temperature and BR signaling to regulate root growth upon long-term changes in environmental conditions.

No description available.

The significance of poxvirus manipulation of the host ubiquitin proteasome system has become increasingly apparent. Ubiquitin is post-translationally added to target proteins by a highly conserved enzymatic cascade, typically resulting in protein degradation via the 26S proteasome. The highly conserved poxvirus protein, p28, is a functional ubiquitin ligase and a critical virulence factor. Here, we investigate the relationship between p28 and ubiquitination. We observed that the KilA-N DNA binding domain in p28 targeted p28 to viral factories, where p28 co-localized with conjugated ubiquitin. Furthermore, we determined that p28 is highly regulated by ubiquitination and proteasomal degradation. Disruption of p28 ubiquitin ligase activity revealed that p28 is regulated through auto-ubiquitination and ubiquitination by an additional unknown ubiquitin ligase. Moreover, we observed Lysine-48 ubiquitin linkages, Lysine-63 ubiquitin linkages and a proteasomal subunit co-localizing with p28 at the viral factory, suggesting an intricate relationship between p28 and proteasomal degradation. / Virology

Accurate regulation of gene transcription is essential for organismal survival, and is orchestrated by myriad transcription factors and cofactors (TFs). Little is known about how the intrinsic activity of TFs is controlled. Recent work has indicated that the selective proteolysis of TFs provided by the ubiquitin-proteasome system (UPS) plays an important role in stimulating gene expression through a ‘destruction-activation’ mechanism, whereby the degradation of a ‘used’ TF is thought to stimulate further ‘fresh’ TF binding and reinitiate gene transcription. TFs are targeted to the proteasome via E3 ligases that mediate the addition of ubiquitin molecules to form a chain on the substrate TF. These polyubiquitin chains may be extended by E4 ligases, which recognize substrates with four or more ubiquitin molecules, amplifying substrate targeting to the proteasome. In plants the immune response to many pathogens is regulated by the hormone salicylic acid (SA), which operates through the transcriptional coactivator NPR1 to induce large scale changes in gene expression. Proteasome-mediated degradation of NPR1 appears to be required for the activation of its target genes. Mutation of the E3 ligase prevents ubiquitination of NPR1, leading its to stabilisation and suppression of transcription. Chapter 3 of this work identifies the first E4 ligase, UBE4, involved in NPR1 regulation. Mutation of UBE4 resulted in reduced capacity to polyubiquitinate substrates and stabilized NPR1. In contrast to E3 ligase mutants, however, mutant ube4 plants displayed increased NPR1 target gene expression. These results suggest that initial ubiquitination of NPR1 may stimulate its ability to initiate transcription and that subsequent ubiquitin chain elongation limits NPR1 activity by targeting it to the proteasome. Chapter 4 describes a ubiquitin-protein-ligase (UPL) which is both novel and crucial to the SA-mediated defence response. Mutation of this UPL leads a large reduction in total cellular polyubiquitinated proteins and was associated with strongly enhanced disease susceptibility. Gene expression profiling of upl mutants revealed an intimate connection between cellular polyubiquitination and appropriate activation of SA-responsive gene expression programmes. Destruction activation was first described in yeast and is required for the regulation of yeast amino acid synthesis TF GCN4. GCN4 requires proteasome-mediated degradation to induce genes involved in amino acid production. Chapter 5 investigates the role of two E4 ligases in GCN4 turnover. While one mutation had little effect of GCN4-mediated transcription a second increased basal transcriptional levels, suggesting that an E4 is required for the prevention of spurious GCN4-mediated transcription. In summary the work presented here describes cellular mechanisms by which global and substrate-specific polyubiqutination are vital to regulation of gene transcription.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25489号 / 医博第5089号 / 新制||医||1073(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 岩田 想, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Nrf2 (NF-E2-related factor 2) is a transcription factor that regulates a battery of downstream genes that contain an antioxidant response element (ARE) in their promoters, including intracellular redox-balancing proteins, phase II detoxifying enzymes, and transporters. These Nrf2-dependent proteins work in collaboration to protect against many diseases where oxidative stress plays an essential role in disease onset and progression. Consequently, it is imperative to understand the basic molecular mechanisms of how Nrf2 is regulated so we can target this pathway for disease prevention and treatment.Nrf2 is mainly regulated at the protein level by the ubiquitin proteasome system. Under basal conditions Nrf2 is constantly ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and subsequently degraded by the 26S proteasome. Currently, regulation of the Nrf2-Keap1 pathway by ubiquitination is largely understood. However, the mechanism responsible for removal of ubiquitin conjugated to Nrf2 or Keap1 remains unknown. In this dissertation, we identified two molecular mechanisms that are important in understanding how the Nrf2-Keap1 pathway is regulated: (i) USP15 negatively regulates the Nrf2-Keap1 pathway by deubiquitinating Keap1 and (ii) deubiquitinated-Keap1 binds in the Cul3-Keap1-E3 ligase complex more tightly than ubiquitinated-Keap1. Additionally, (iii) we demonstrated the importance of the Nrf2-Keap1 pathway in USP15-dependent paclitaxel-chemoresistance.Under oxidative stressed or induced conditions the ability of the E3-ligase to target Nrf2 for degradation becomes impaired. As a result, Nrf2 is stabilized and free Nrf2 translocates to the nucleus and initiates transcription of ARE-bearing genes. Activation of this pathway is advantageous for chemoprevention. In Chapters 4 and 5, we identified and characterized two activators of the Nrf2 cytoprotective pathway, oridonin and cinnamic aldehyde. These compounds inhibit Cul3-Keap1-dependent degradation of Nrf2, stabilize Nrf2 protein levels, and activate the antioxidant response. Furthermore, both compounds are able to protect against cytotoxic and genotoxic stress-induced cell death. Moreover, our study on USP15 has elucidated an additional mechanism that allows small molecules, such as oridonin, to activate Nrf2 by causing a switch in ubiquitination from Nrf2 to Keap1. Taken together, these findings further our understanding of how the Nrf2-Keap1 pathway is regulated, which is imperative in targeting this pathway for chemoprevention or chemotherapy.

Protein ubiquitination is a key regulator of both protein stability and activity, and is involved in the regulation of a vast variety of cellular pathways. The ubiquitination system therefore provides an exciting target for drug development aiming to regulate the function of specific proteins. Our understanding of ubiquitin signalling is far from complete; and if we are to exploit this system for the benefit of human health, it is important to gain a better understanding of this complex posttranslational modification system as well as the effect of ubiquitination on the target protein. The E3 ligases MDM2 and CHIP were implicated in the control of the two transcriptional activators (TAs) IRF-1 and p53, that normally function to maintain health at the cellular and organismal level. Research carried out as part of my PhD has focused on gaining a mechanistic understanding of the ubiquitination process in particular the relationship between the E3 ligase and its substrate. Broadly, the mechanisms of E3 ligase regulation have been linked to substrate specificity and then to the physiological outcome of site-specific ubiquitination of the DNA binding domain of the TAs IRF-1 and p53. More specifically I have; (i) identified a mechanism by which the E3 ligase activity of the CHIP U-box can be allosterically regulated by ligand binding to its TPR domain. (ii) Residues on IRF-1 that are targeted by MDM2 and CHIP have been mapped, revealing that both ligases modify sites exclusively in IRF-1's DNA binding domain (DBD). Furthermore, I showed that, in its DNA bound conformation, IRF-1 is neither bound nor ubiquitinated by the ligases, suggesting a mechanism by which IRF-1 ubiquitination and possibly degradation can be regulated through its DNA binding state. And lastly, (iii) I have shown that both IRF-1 and p53, which have ubiquitin acceptor lysines in their DBD, bind DNA more stably when ubiquitinated. Modelling suggests that interactions between a positively charged surface area of ubiquitin and the negatively charged DNA can stabilises the TA-ubiquitin complex. DBD ubiquitination sites are required for full transactivation potential of both TAs, supporting a role of ubiquitin in their activation. p53 is ubiquitinated in response to activation by IR or Nutlin-3 and these ubiquitinated forms of p53 are localised in the cell nucleus associated with chromatin and do not lead to protein degradation. Taken together, the data imply that p53 and IRF-1 DNA binding ability, and thereby activity, can be modulated by ubiquitin modification.

<p>The bacterial pathogen <i>Legionella pneumophila</i> modulates host immunity using effectors translocated by its Dot/Icm transporter to facilitate its intracellular replication. A number of these effectors employ diverse mechanisms to interfere with protein ubiquitination, a post-translational modification essential for immunity. Here, we have found that <i>L. pneumophila</i> induces monoubiquitination of the E2 enzyme UBE2N by its Dot/Icm substrate MavC(Lpg2147). Ubiquitination of UBE2N by MavC abolishes its activity in the formation of K63-linked polyubiquitin chains, which dampens NF-kB signaling in the initial phase of bacterial infection. The inhibition of UBE2N activity by MavC creates a conundrum because this E2 enzyme is important in multiple signaling pathways, including some that are important for intracellular <i>L. pneumophila</i> replication. Here we also show that the activity of UBE2N is restored by MvcA(Lpg2148), an ortholog of MavC. MvcA functions to deubiquitinate UBE2N-Ub using the same catalytic triad required for its deamidase activity. Structural analysis of the MvcA-UBE2N-Ub complex reveals a crucial role of the insertion domain in MvcA in substrate recognition. Our findings reveal that two remarkably similar proteins catalyze the forward and reverse reactions to impose temporal regulation of the activity of UBE2N during <i>L. pneumophila</i> infection.</p>