In eukaryotic organisms, polyubiquitination is the modification of a protein with polymerized ubiquitin (Ub) chain. This process is well known for its function in targeting proteins for degradation by the 26S proteasome. However, a polyUb chain assembled through the lysine 63 residue of the Ub moiety (Lys63-linked polyubiquitination) has been shown to play a signaling role rather than targeting proteins for degradation. In plants, the functions of Lys63-linked polyubiquitination are currently not well understood. Ub-protein ligase (E3) catalyzes the last step in the ubiquitination reactions, and to a large extent it also determines the substrate specificity of protein ubiquitination. In order to study the roles of Lys63-linked polyubiquitination in plants, two E3s of Arabidopsis thaliana, proteins encoded by AtCHIP and At1g74370 (tentatively named E3-A1), were chosen for functional studies, since they interacted with AtUbc13A protein. Sequence analysis showed that AtCHIP is the only member in the family. A T-DNA insertion mutant line (Atchip-1) was obtained to study the AtCHIP gene knock-out effect. The mutant line was grown in normal conditions and further tested in a variety of conditions: hormonal treatments, osmotic stress, seed deterioration, high temperature stress, high-intensity light stress, oxidative stress and DNA damaging stress. However, no clear difference was observed between the mutant and wild type plants based on the several parameters measured. Sequence analysis of E3-A1 indicated two closely related proteins, tentatively named E3-A2 and E3-A3. As E3-A1 and E3-A2 appeared to share more sequence similarity, RNA interference (RNAi) transformants, with the level of transcripts for either of the two E3-A genes reduced by over 90% were generated. Selected RNAi mutant lines for E3-A1 and E3-A2 were crossed with each other, and double RNAi mutants were obtained. However, no distinct phenotype was detected under normal, high-sucrose or hormonal conditions for either single or double RNAi lines. Although various assays did not reveal a significant phenotype in the mutants in this study, the materials generated and the assays used will benefit a wider range of phenotypic survey in the future.

Les évidences scientifiques révèlent l’implication des actions proinflammatoires de l’angiotensine II (Ang II) dans le développement de l’athérosclérose. Cependant, la caractérisation des bases moléculaires de l’Ang II sur le tissu vasculaire n’est pas totalement élucidée. La majorité des actions de l’Ang II implique l’activation d’une variété de cascades de signalisation dont les voies mitogen-activated protein kinases (MAPKs) ; c-Jun N-terminal kinases (JNKs), p38 kinases et extracellular signal-regulated kinases (ERK) et l’activation du facteur de transcription NF-κB via le complexe IKK. Récemment, une nouvelle modification post-traductionnelle dans les actions de l’Ang II, soit la polyubiquitination de la sous-unité NF-κB essential modulator (NEMO) du complexe IKK, a été révélée. L’objectif de mon projet de recherche est de vérifier l’importance de la polyubiquitination en K63 tout en caractérisant les protéines impliquées dans la modification de NEMO dans des cellules musculaires lisses vasculaires (CMLV) exposées à l’Ang II. Notre étude suggère, selon une approche siARN combinant Ubc7 et Ubc13, la diminution de la phosphorylation du complexe IKK, de Akt et des MAPKs. De plus, nos résultats illustrent l’implication de TRAF6 dans la signalisation cellulaire de l’Ang II. Finalement, notre étude révèle la présence de la polyubiquitination en K63 dans la signalisation cellulaire de l’Ang II par chromatographie d’affinité. Cette étude met en évidence l’implication de la polyubiquitination en K63 dans la signalisation de l’Ang II dans des CMLV et implique Ubc13 et Ubc7 dans le remodelage vasculaire et l’inflammation dépendante de l’Ang II dans des CMLV. / Several studies have demonstrated that the proinflammatory and growth promoting actions of Angiotensin II (Ang II) are implicated in cardiovascular disease. However, the underlying molecular mechanisms involved in Ang II actions are still not completely elucidated. Most of the known biological effects of Ang II are through the activation of several cell signaling pathways, such as the mitogen-activated protein kinase pathways (MAPKs; c-Jun N-terminal kinases (JNKs), p38 kinases and extracellular signal-regulated kinases (ERK)), and nuclear factor kappaB transcription factor (NF-κB) pathway by IKK complex activation. Recently, another post-translational modification, polyubiquitination of the IKK complex sub-unit NF-κB essential modulator (NEMO) has been demonstrated to be implicated in Ang II signaling. The objective of my research project was to illustrate the importance of K63-linked polyubiquitination, and characterizing the proteins involved in the modification of NEMO in vascular smooth muscle cells (VSMC) exposed to Ang II. Using siRNA, we show that Ubc7 and Ubc13 together are involved in MAPK, IKK and Akt phosphorylation in VSMC exposed to Ang II. Moreover, our results show that TRAF6, a ubiquitin ligase, is involved in Ang II signaling. Finally, our study reveals involvement of K63-linked polyubiquitination in Ang II signaling by chromatography affinity. Here, we report K63-linked polyubiquitination involvement in Ang II signaling and also identify Ubc7 and Ubc13 as a ubiquitin conjugating enzymes involved in MAPKs, ERK and NF-kB signalling pathway suggesting a role of these proteins in Ang II-dependent vascular remodelling and proinflammatory effects in VSMC.

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