SUMOylation is a post-translational modification in which the small ubiquitinlike protein SUMO is attached to lysine residues of the target protein. In plants, wide-spread SUMOylation is observed upon a variety of different stress cues. We tested Arabidopsis SUMOylation machinery knockout mutants for impaired disease resistance against Pseudomonas syringae pv. tomato (Pst) and identified sumo2-1, sae1a-1 and sae2-3 as showing moderate but statistically insignificant resistance. sumo2-1 also exhibited slightly reduced HR compared to the wild-type plants after PstDC3000(avrB) challenge. Change in the cellular redox status is an important outcome of attempted pathogen ingress. Therefore, we also looked at the redox regulation of SUMOylation both in vivo and in vitro. We found a significant increase in SUMO1/2 conjugation and free SUMO1/2 accumulation in atgsnor1-3 plants after PstDC3000(avrB) challenge which was reversed during the establishment of disease in the absence of an AvrB, suggesting an important role of S-nitrosylation in modulating plant SUMOylation. High basal level of high molecular weight (HMW) SUMO1/2 conjugates was also apparent in atgsnor1-3 plants even in the absence of pathogens. The changes in SUMO3 and SUMO5 remained less significant and their regulation was found to be independent of GSNOR. Biotin switch technique was employed to test further if SUMO enzymes are modified by NO. It was found that SCE1 and SAE1a are S-nitrosylated in vitro in a GSNO dose dependent fashion. MS analysis and site directed mutagenesis revealed Cys139 in SCE, and Cys93, Cys158 and Cys231 in SAE1a as the targets of S-nitrosylation. We established that GSNO treatment to SCE1 differentially regulates in vitro SUMOylation of the model substrate ScPCNA. Furthermore, the S-nitrosylation of Cys139 of SCE1 is important in fine-tuning protein SUMOylation under changing cellular redox tone. These data highlight the complexity of cross-communication between two different post-translational modifications (i.e. S-nitrosylation and SUMOylation) in the control of protein function.

SUMO (small ubiquitin-like modifier) protein, also known as Smt3 (suppressor of Mif2 protein 3) of Saccharomyces cerevisiae is an ubiquitin-like protein due to the similar post-transcriptional modifications to their substrates. There are three members of SUMO genes (SUMO-1, SUMO-2 & SUMO-3) in the vertebrate, while only one SUMO gene exists in the invertebrate. Covalent modification of cellular proteins by the SUMO regulates various cellular processions, such as nuclear transport, transcription repression and cellular apoptosis. To investigate the biological functions of SUMO-1 and SUMO-2, yeast two hybrid assays were applied. Results showed that N-terminus (Daxx1, 1-282 amino acids) and C-terminus (Daxx4, 607-740 amino acids) of Daxx were the SUMOs interacting fragments. For identification of the sumoylation site on Daxx1 and Daxx4, six mutants (K60R, K630A, K631A, K634A, K630, 631A and K630, 631, 634A) were constructed. In vitro sumoylation were applied in the Daxx1 fragment and mutated Daxx1 (K60R) as well as the Daxx4 fragment and mutated Daxx4 (K630A, K631A , K634A, K630, 631A and K630, 631, 634A) to identify the sumoylation sites of Daxx. Our results showed that Daxx1 K60 was one of the sumoylation sites, neverthless it was not a major sumoylation site. The major sumoylation sites were on the C-terminus of Daxx (Daxx4). The major sumoylation sites of SUMO-1 on Daxx4 seemed different from those of SUMO-2. Mutants (K631A and K634A) of Daxx4 decreased the yields of sumoylation complexes of SUMO-1 more than that of Daxx4 K630. However, mutants (K630A and K631A) of Daxx4 decreased the yields of sumoylation complexes of SUMO-2 more than that of Daxx4 K634. Thus we propose that the major sumoylation sites of SUMO-1 on Daxx are K631A and K634A and that of SUMO-2 are K630A and K631A. Daxx may have other sumoylation sites on the Daxx C-terminal 635-740 amino acids fragment, unless the sumoylation reactions of Daxx mutants were pseudo-positive reactions which might be caused by the improper folding of Daxx4 during in vitro sumoylation.

The 1£\, 25-dihydroxyvitamin D3 (1,25(OH)2D3) is involved in various physiological processes, including calcium/phosphorous homeostasis, cell growth, differentiation and apoptosis. 1,25(OH)2D3 induces the formation of VDR/RXR complex to up-regulate or down-regulate target gene expression. Recent studies find that VDR undergoes several post-translational modifications, such as phosphorylation and ubiquitination, which may regulate its transcriptional activity and/or stability. In this study, we identified VDR as a new target for small ubiquitin-related modifier (SUMO)-2 modification in vitro. In E. coli. SUMO-conjugation system, VDR is mainly sumoylated at Lys-103. SUMOylation of VDR enhanced VDR/RXR-mediated transcriptional activation as determined by promoter activity assay. In addition, 1,25(OH)2D3-induced expression of osteopontin was attenuated after mutation of VDR SUMOylation site. However, chromatin immunoprecipitation assay indicated that wild type and K103A mutant of VDR bound to the osteopontin promoter with similar affinity. Collectivity, our results suggest that SUMOylation of VDR may affect its transcriptional activity by modulating the interaction between VDR and co-activators.

No description available.

Le syndrome de l’X-fragile est la forme la plus fréquente de déficience intellectuelle héréditaire liée au chromosome X. Cette maladie résulte de la mutation du gène FMR1 localisé sur le chromosome X. La protéine correspondante, FMRP, est absente chez les patients atteints de la maladie. Il faut noter ici qu’il existe un modèle murin mimant la pathologie humaine. Ainsi dans ces animaux qui n’expriment pas la protéine FMRP, les neurones présentent des anomalies architecturales de la synapse entraînant d’importants dysfonctionnements dans la transmission et la plasticité synaptique qui sont à l’origine des déficits intellectuels observés chez les patients atteints du syndrome de l’X-fragile. FMRP joue donc un rôle majeur dans la genèse et la maturation des épines dendritiques. Une des fonctions de FMRP est de lier de nombreux ARNm, de les transporter et d’inhiber leur traduction jusqu’à la synapse. Pour accomplir ses fonctions, FMRP interagit avec de nombreux partenaires cellulaires et ses interactions sont finement régulées par différentes modifications post-traductionnelles. Nous avons montré in vivo que la protéine FMRP est un substrat d’une nouvelle modification, la sumoylation. Nous avons également montré que la sumoylation de FMRP est impliquée dans le maintien de l’architecture synaptique et participe à la régulation de la transmission synaptique. Et enfin, nous avons montré que la sumoylation de FMRP permet sa dissociation avec ses partenaires protéiques au sein des complexes ribonucléoprotéiques se trouvant à la base des épines dendritiques. Les ARNm réprimés par FMRP au sein de ces complexes sont ainsi libérés puis traduits. / Fragile X Syndrome is the most frequent inherited cause of intellectual disability in children and is caused by the lack of the mRNA-binding Fragile-X Mental Retardation Protein (FMRP) expression. FMRP plays a role in the activity-dependent targeting and translation of specific mRNAs in dendrites. The absence of FMRP expression in neurons leads to an abnormal neuronal morphology with increased spine length and density. FMRP is therefore playing key roles both in neuronal development and synaptic plasticity. However, the molecular mechanisms underlying the functional regulation of FMRP-mediated mRNA trafficking, translation and subsequent protein synthesis are still largely unknown. My host laboratory has recently discovered that FMRP is sumoylated in vivo. Sumoylation is a post-translational modification that consists in the covalent conjugation of the protein SUMO to specific lysine residues of target proteins. To start unraveling the functional consequences of FMRP sumoylation, I studied first the spine morphology of the WT and FMRP Knock Out mice that recapitulated the human disease. Morphological analysis of fmr1-KO neurons transfected with the WT form of FMRP restores the correct mature spine morphology whereas the non-sumoylatable protein failed to do so. Moreover the non-sumoylatable form of FMRP acts as a dominant negative on WT neurons so confirming the important role of FMRP sumoylation in its function. We report here that FMRP sumoylation is required for the control of spine morphology.

Loss of DNA damage-induced G2 checkpoint control is associated with genome instability, tumour formation and the therapeutic response of tumours to genotoxic agents. The large 3418 residue protein encoded by BRCA2 – heterozygous germline mutations in which predispose to cancer - has recently been implicated in G2 checkpoint maintenance. However, the mechanistic basis of BRCA2’s role in the G2 checkpoint remains unknown. The overall aim of my research is to understand the mechanism by which BRCA2 regulates the G2 checkpoint. Domain mapping studies, using overlapping fragments encoding the full-length BRCA2 protein, carried out in our laboratory suggest that BRCA2’s function at the G2 checkpoint is mediated through regions that span BRCA2 amino acids (aa) 1-454 and aa 2438-2824. My research has focused on understanding how these two regions contribute to G2 checkpoint function through the interrogation of two novel interactions of BRCA2 mediated at these regions. My experiments have identified that BRCA2 interacts with the serine/threonine kinase ATR (aa 2438-2824) and the deSUMOylase SENP1 (aa 1-454). ATR is known to play a key role at the G2 checkpoint and my results identify that loss of BRCA2 leads to a reduction in ATR activity at sites of damage. This leads to a downstream attenuation in the phosphorylation of Chk1 – an important effector protein of the G2 checkpoint. BRCA2 is known to function in DNA repair and is recruited to sites of DNA damage, where it displaces RPA bound to exposed single-strand DNA. RPA is required to localise and activate ATR during the G2 checkpoint and therefore I hypothesise that BRCA2’s role at the checkpoint is to substitute for RPA and mediate ATR activation. I have also shown that SENP1 interacts with BRCA2 at a region within the N-terminus (aa 290-454). SUMOylation has been increasingly recognised as an important post-translational modification (PTM) in the context of the DNA damage response, and SENP1 is involved in reversing this PTM. I have shown that BRCA2 is SUMOylated. Moreover, I have shown that loss of SENP1 prevents BRCA2 from being recruited to chromatin in a timely manner after DNA damage and that SENP1 depletion exacerbates the loss of G2 checkpoint maintenance seen in BRCA2 deficiency. Taken together, this work reveals novel insights into the mechanism by which BRCA2 maintains genomic stability through enforcement of the G2 checkpoint. This new knowledge has the potential to translate into a better understanding of how mutations in BRCA2 may lead to cancer.

La sumoylation représente, après l’ubiquitination, l’exemple le plus étudié de modification post-traductionnelle impliquant la liaison d’une protéine à une autre. Cependant, alors que l’ubiquitination est impliquée principalement dans la dégradation des protéines par le protéasome, la sumoylation semble réguler les propriétés biochimiques de ses substrats (localisation cellulaire, interaction protéique, activité, …). Pour venir lier une protéine appelée Sumo (Small Ubiquitin-like Modifier) sur un substrat, la sumoylation emprunte une voie enzymatique analogue à celle de l’ubiquitination mais utilise des enzymes différentes. A ce jour, bien que plusieurs centaines de substrats de la sumoylation aient été identifiés, seules 5 structures de protéines sumoylées ont été résolues. Elles ne sont vraisemblablement pas représentatives de l’ensemble des substrats de la sumoylation et mon travail de thèse vise à élargir les connaissances structurales sur la sumoylation pour permettre de dégager des concepts généraux. Les études sur la sumoylation se heurtent généralement à la difficulté d’obtenir les substrats sumoylés. Ce projet a donc nécessité, au niveau technique, la mise au point d’un système de sumoylation in vivo en bactérie permettant de modifier des quantités importantes de protéines et de les purifier efficacement. Des analyses bioinformatiques nous ont permis d’identifier des substrats de la sumoylation propices à une étude structurale de leur forme sumoylée. Au terme de ces analyses, nous avons retenu 3 protéines : DJ-1, PPARγ et IκBα. Bien que la complexité du sujet nous ait ensuite amené à écarter DJ-1 et PPARγ, nous sommes parvenus à purifier la forme sumoylée d’IκBα. Ce résultat nous a permis d’entreprendre une campagne de cristallogenèse d’IκBα complexé au facteur de transcription NF-κB. L’obtention d’IκBα sumoylé permettra également d’aborder des études fonctionnelles pour améliorer la compréhension du rôle de la sumoylation de ce substrat. Nos analyses bioinformatiques ont également révélé que dans plus de 60% des cas, les sites de sumoylation des substrats se trouvent dans des zones prédites intrinsèquement désordonnées. L’importance du désordre dans le processus de sumoylation était jusqu’alors largement sous-estimée. A titre d’exemple, nous avons étudié par diffusion des rayons X aux petits angles la structure du domaine transactivateur du facteur de transcription ERM sous forme non modifiée et sous forme sumoylée. Cette étude indique que la sumoylation d’ERM n’induit pas le repliement de ce domaine transactivateur. De même, il apparait peu probable, au vu de la flexibilité de cette région, que la sumoylation empêche des interactions avec certains partenaires cellulaires. Dans ce contexte, la sumoylation semble servir de plateforme de recrutement de partenaires, reconnaissant de manière spécifique le Sumo. Ce mécanisme pourrait se généraliser à l’ensemble des sites de sumoylation prédits dans des zones intrinsèquement désordonnées. Le système de sumoylation que nous avons développé permet de produire des protéines sumoylées pures en grande quantité et pourra également servir à identifier des protéines reconnaissant spécifiquement les substrats modifiés. Tous ces éléments devraient permettre de progresser dans la compréhension de cette modification post-traductionnelle impliquée dans de nombreux processus cellulaires fondamentaux.

Diverse extracellular stimuli, including ultraviolet light, irradiation, heat shock, proinflammatory cytokines, trigger activation of MAPK pathway through phosphorylation on a TGY motif within the kinase activation loop. Protein MAPK appears to play a major role in apoptosis. It has been causally implicated in sepsis and arthritis. The translational small ubiquitin related modifier (SUMO) modification of proteins has been shown to play multiple functional roles in several cellular processes, including signal transduction, protein targeting, stabilization, transcriptional activation and apoptosis. Our previous study demonstrated that the expression levels of SUMO-1 rnRNA and proteins were enhanced in Helicobacter pylori infected human gastric epithelial cells. The activation of MAPK pathway and cellular apoptosis of AGS cell lines were increased during Helicobacter pylori infection. It was hypothesized that Helicobacter pylori functioning as a biological stress that induced MAPK mediated apoptosis which may be regulated by sumoylation. Results showed that MAPK phosphorylation and cellular apoptosis were enhanced in RFP-SUMOs or GFP-MAPK expressing cells, especially during Helicobacter pylori infection. It was inhibited by pretreatment of MAPK inhibitor. The enhanced phosphorylation and apoptosis were observed during GFP-MAPK overexpression. It¡¦s suggested that MAPK is a target protein for SUMOs.

SUMOylation is a type of post-translational protein modification similar to ubiquitination and it involves the covalent attachment of a small ubiquitin-like modifier (SUMO) protein to the lysine residue of a target substrate. While there is strong evidence for the role of protein ubiquitination in the formation of fear-based memories, few studies have been conducted examining the role that SUMOylation plays in this same process. The amygdala is the main site of storage for emotional memories and there is strong evidence that protein ubiquitination is critical for fear memory formation in this region. However, it has not previously been studied whether protein SUMOylation in the amygdala is also involved in fear memory formation. Additionally, although there is evidence to support sex differences in ubiquitin signaling during fear memory formation in the amygdala, whether males and females differ in their need for protein SUMOylation during fear memory formation has not been investigated. We have found significant sex differences in protein SUMOylation in the amygdala both at baseline (rest) and during fear memory formation. Western blot analysis revealed higher resting levels of SUMOylated proteins in females when compared to males, though both sexes showed global increases following fear conditioning. A SUMOylation-specific proteomic analysis discovered that only females had increased protein targeting with SUMO following fear conditioning, with four proteins being identified that gained SUMOylation modifications, the main target being a heat shock protein. One heat shock protein in males was identified as having lower SUMOylation levels following fear conditioning. This suggests sex differences in the interaction and targeting of proteins by SUMOylation following fear conditioning. We also inhibited the function of the only E2 conjugase for SUMOylation, Ube2i, via siRNA in the amygdala and found impaired fear memory in males but enhanced fear memory in females, though the latter only occurred under high siRNA concentrations. Interestingly, western blot analysis revealed that knockdown of Ube2i caused an increase in protein SUMOylation levels in females but a decrease in males, indicating that compensation is likely occurring in females. This suggests that in females, protein SUMOylation may be critical for basal cellular functioning, which precludes us from directly determining its role in fear memory formation. Collectively, these data reveal a novel, sex-specific role for protein SUMOylation in the amygdala during fear memory formation and expand our understanding of how ubiquitin-like signaling regulates memory formation. / Master of Science / SUMOylation is a modification of protein which plays a key role in various biological processes and is similar to the protein modification process called ubiquitination, which has been implicated in the formation of fear-based memories for traumatic events. Despite this and the established role of SUMOylation in genomic stability, cell proliferation, and migration, less is known about its role in the process of memory formation. Importantly, ubiquitination and SUMOylation of proteins often work in tandem to regulate cell signaling and recent evidence suggests that SUMOylation may also be involved in fear memory formation. However, the role of protein SUMOylation in regulating fear memory formation in the amygdala, the primary site of storage for emotional memories, has never been directly examined. Additionally, there is also a significant gap in the literature regarding whether sex differences exist for the requirement of protein SUMOylation in fear memory formation. We have found that there are significant differences between the sexes regarding protein SUMOylation during fear memory formation in the amygdala. Western blot analysis showed that females have higher resting (baseline) levels of SUMOylated proteins in the amygdala compared to males, though both sexes showed global increases in protein SUMOylation following fear conditioning. In addition, a proteomic analysis revealed that four proteins in females gained a SUMOylation modification following fear conditioning. In contrast, one protein was identified in males which lost a SUMOylation modification, together suggesting unique targeting of proteins by SUMOylation across sexes during fear memory formation. Further, when the function of an essential enzyme for protein SUMOylation was inhibited in vivo, fear memory in males was impaired but enhanced in females. Collectively, these data reveal a novel, sex-specific role for protein SUMOylation in the amygdala during fear memory formation and expand our understanding of how ubiquitin-like signaling regulates memory formation.

Les fichiers qui accompagnent mon document sont des tableaux supplémentaires réalisés avec Excel (Microsoft Office), dans la version papier du mémoire ces fichiers sont sur un CD-ROM. / Une large gamme d’événements cellulaires est régulée par la SUMOylation des protéines. Cette modification post-traductionnelle est impliquée dans le cancer notamment dans la leucémie promyélocytaire aigue. À ce jour, peu d’études à grande échelle ont porté sur l’identification des sites de modification. Ce mémoire présente une approche protéomique quantitative unique qui combine une double purification par affinité au niveau des protéines cibles ainsi que des peptides modifiés. L’approche la plus répandue de purification des protéines SUMOylés implique l’utilisation d’une forme de SUMO modifié avec une étiquette (His6-SUMO). A ce jour, les approches permettant l’enrichissement au niveau peptidique nécessite une forme mutante de SUMO. Notre analyse consiste à premièrement enrichir en protéines SUMOylés dans les cellules humaines vierges ou sur exprimant His6-SUMO-1/3 en présence ou pas de trioxyde de diarsenic, un traitement de leucémie promyélocytaire aigue. Par la suite, les échantillons sont digérés et les peptides obtenus des protéines SUMOylés conservent un branchement caractéristique. Les peptides sont soit immunoprécipités avec un anticorps spécifique au branchement SUMO ou directement analysés par nano LC/LC-MS/MS par un spectromètre de masse LTQ-Orbitrap. Une analyse manuelle des données révèle des fragments caractéristiques correspondant à la chaîne latérale de SUMO. L’originalité de l’approche réside dans l’identification quantitative et sans ambigüité des sites de SUMOylation. Cette approche a permis l’identification de 17 et 3 sites de SUMO-3 et SUMO-1 respectivement dans les cellules HEK293. Finalement, la SUMOylation de PML est induite suite au traitement d’arsenic. / A wide range of cellular events are regulated by protein SUMOylation. This posttranslational modification was involved in APL (acute promyelocytic leukemia). Only a few large scale studies in mammalian cells have focused on identifying the conjugation sites. This thesis presents a unique quantitative proteomics approach that combines double affinity purification at the protein and peptide level. A common approach to purification of SUMOylated proteins involves the use of a tagged SUMO (His6-SUMO). To date, the SUMO peptide isolation is addressed using an engineered SUMO. In presence or absence of arsenic trioxide, a treatment of APL, mock and His6-SUMO1/3 expressing cells are lysed and the SUMOylated proteins are isolated under denaturing conditions. Subsequently, these samples are digested and the peptides bearing the modification site bear a specific SUMO stub. They are either immunoprecipitated with an anti SUMO stub antibody or directly analyzed by nano LC coupled to an LTQ-Orbitrap mass spectrometer. Manual analysis of the data reveals characteristic fragmentation corresponding to the side chain of SUMO. The originality of the approach lies in the quantitative and unambiguous identification of SUMOylation sites in vivo. This approach allowed the identification of 17 and 3 sites of SUMO-3 and SUMO-1, respectively, in HEK293 cells. Finally, PML was identified as the major SUMOylation target following arsenic treatment.