Helicobacter pylori (H. pylori) causes peptic ulcer or gastric cancer through different virulence factors including lipopolysaccharides (LPS), the cytotoxin-associated gene A product (CagA), and vacuolating cytotoxin A (VacA) etc. It stimulated mitogen-activated protein (MAP) kinase signaling cascades. Small ubiquitin-related modifier (SUMO) is a member of ubiquitin-related protein modifiers. However, the mechanisms of the involvement of SUMO-1 on H. pylori induced apoptosis were not clear. Our previous study showed that the expression of RFP-SUMO-1 and apoptosis were increased significantly by fluorescence microscopy assays on RFP-SUMO-1 transfectants during H. pylori infection. In addition, the cytoplasmic SUMO-1 was increased during infection and positively associated with apoptosis. Here, how SUMO-1 was involved in the apoptotic signaling enhancement during H. pylori infection was studied. Results showed that H. pylori infection enhanced MAP kinase activation and the effects were stronger on the SUMO-1 overexpressed cells. However, it was not affected by the secretion of CagA or VacA toxins of H. pylori. To investigate the possible role of SUMO-1 on MAPKs mediated signaling pathways, three selective MAPKs inhibitors were used on RFP-SUMO-1 overexpressed cells. Only p38 inhibitor decreased the levels of apoptosis during H. pylori infection and the expression of p53 was increased on RFP-SUMO-1 1 overexpressed cells. Thus, p38 and p53 pathways were suggested to be involved in SUMO-1 enhanced apoptosis during H. pylori infection. In addition, the nuclear localization of NF-£eB and expression of COX-2 were enhanced on RFP-SUMO-1 overexpressed cells. Moreover, more nuclear NF-£eB and cytoplasmic as well as nuclear RFP-SUMO-1 were observed during H. pylori infection. Our data suggest that H. pylori infection enhances SUMO-1 expression which activates MAPKs on both the pro-apoptotic p38-p53 pathway and the anti-apoptotic ERK-NF-£eB-COX2 pathway. The detail mechanisms on how cells making the final decision on the survival or apoptosis were still not clear and deserving to investigate.

Nuclear protein KLIP1 cooperates with myeloid leukemia factor 1 (MLF1) to inhibit the programmed cell death resulting in tumor formation. It also inhibits the activity of thymidine kinase promoter of Kaposi¡¦s sarcoma-associated Herpes Virus. KLIP1 functions as a centromere protein, hence acquires its name as CENP-U or CENP-50, to regulate the separation of sister-chromatids during mitosis. These results indicate that KLIP1 plays important roles in regulation of transcription and cell cycle. In this study, six potential SUMO modification sites, K33, K63, K126, K127, K185 and K210, were identified bioinformatically using SUMOplot. Many reports address that SUMO modification alters the transcriptional activity, protein-protein interaction, the subcellular localization and stability of its target protein. Recent data suggest that SUMO is required for centromere binding protein to mediate proper mitotic spindle attachment to the kinetochore, and previous research suggest that there has a SUMO-interaction motif (SIM) in KLIP1 protein sequence. To reveal the interaction between KLIP1 and SUMO-1, and study its effects on KLIP1 function, we co-express GFP-KLIP1 and His-tagged SUMO-1 in HEK 293 cells. After affinity purification of SUMOylated proteins from transfected cells using nickel conjugated beads and subsequent western blotted with anti-GFP. The results indicated the interaction between KLIP1 and SUMO-1 in co-transfected cells. Our confocal microscopy imaging also found colocalization of GFP-KLIP1 with RFP-SUMO-1 nuclear foci. In addition, we failed to detect the interaction between SUMO-1 and mutant KLIP1-M6 ,whose six potential SUMO modified lysine residues were mutated to arginine. Furthermore, we found a distinct nuclear localization of GFP-KLIP1-M6 as compared to the image of wildtype GFP-KLIP1, which show a significant higher frequency of colocalization with RFP-SUMO-1 foci. Taken together, our data suggest the interaction between KLIP1 and SUMO-1 may be related to these six potential lysine residues, which upon mutation blocks its colocalization with SUMO-1 in nuclear foci. The biological significance of their interaction are awaits for further investigation.

Abstract Human ubiquitin-like SUMO-1/2/3 proteins have been identified. The 3-D structure of the SUMO-1 has been shown to be very similar to that of ubiquitin, although their sequences share only 18 % identity. Unlike ubiquitination targets proteins for degradation, sumoylation appears to regulate a number of cellular processes such as protein-protein interaction, subcellular localization, protein stability, apoptosis, cell cycle and so on . Our laboratory has cloned cDNAs encoding human SUMO-2, mouse SUMO-2 and SUMO-3, as well as a single SUMO gene from nematode and Drosophila. Recently (Su & Li, Gene 296:65-73,2002), Su & Li have performed data-mining on current human genomic sequence and found the presence of only three SUMO-1/2/3 functional genes located at chromosome no. 2q33, 17q25.1 and 21q22.3, respectively, as well as eight SUMO-1 pseudogenes and 23 SUMO-2 pseudogenes. The protein-coding sequence of SUMO-1 gene is interrupted by four introns , while those of SUMO-2/3 genes are interrupted by only three introns. In this study , most of SUMO-1/2/3 proteins were show to be localized on nuclear membrane, nuclear bodies and cytoplasm, respectively. The N-terminus-deleted SUMO-1 proteins was further shown to be localized on nuclear membrane and in cytosol, while the mutant SUMO-2/3 proteins were localized only in the cytosol. The inactive precursor form of SUMO-3 was exclusively localized in the cytosol. The activation of SUMO-3 in HeLa cells was triggered by actinomycin D and its location was shifted from cytosol to nucleus. Further, the inactive precursor of SUMO-3 was reduced in HeLa cells treated with nocodazole and arsenic trioxide.

Orientador: Jörg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T01:34:02Z (GMT). No. of bitstreams: 1 Santos_MarcosTadeudos_D.pdf: 15943486 bytes, checksum: 39810fdf0ace76e5e8963354bdc460ca (MD5) Previous issue date: 2011 / Resumo: O objetivo deste projeto foi estudar genes ativados em células do estroma da medula óssea, induzidos pela co-cultura com blastos leucêmicos, na tentativa de uma melhor compreensão sobre o crostalk entre estas células no microambiente tumoral. Nós identificamos Stanniocalcina-1 (STC1) como um potencial marcador molecular do microambiente tumoral, uma vez que sua expressão foi aumentada cerca de 7 vezes em células do estroma co-cultivadas com blastos leucêmicos primários. STC1 humana é uma glicoproteína secretada e tem sido descrita participando em diferentes processos fisiológicos, incluindo a angiogênese, hipóxia e principalmente, a carcinogênese. Nós produzimos a proteína recombinante STC1 no sistema baculovírus e também anticorpos monoclonais, usados em um ensaio ELISA, que agora será testado como um novo kit de diagnóstico de leucemia por uma empresa brasileira. Além disso, identificamos novos parceiros de interação para STC1 através do sistema de duplo hibrido em levedura sendo que algumas destas interações foram confirmadas por GST-pull down. A região Nterminal foi identificada como sendo a região responsável pela interação de STC1 com seus parceiros. Estudos de localização sub-celular por microscopia, revelaram uma deposição ubíqua citoplasmática e puntiforme nuclear, lembrando corpúsculos nucleares relacionados a SUMOilação. Embora STC1 interaja com a proteína SUMO1 e tenha uma predição de alta probabilidade para ser SUMOilada, ensaios in vitro e in vivo não conseguiram detectar STC1 SUMOilada. No entanto, observamos que STC1 regula a SUMOilação de forma significativa em três outras proteínas. Essas descobertas sugerem um novo papel para STC1 no ciclo de SUMOilação, agindo como uma SUMO E3 ligase. Observamos também que STC1 possui um receptor na membrana plasmática em linhagem de células leucêmicas K562 e que a incubação de STC1 com outras células leucêmicas parece favorecer a proliferação destas células ao passo que estimula uma maior produção da própria STC1 intracelular em células do estroma. Juntos, todos estes resultados abrem novas pistas promissoras a serem exploradas no futuro, uma vez que todos os resultados mostram ligações interessantes com estudos funcionais anteriores em STC1 / Abstract: The aims of this project is to study upregulated genes on bone marrow stromal cells, induced by the co-culture with leukemic blasts, trying to have a better understand about the crosstalk between these cells in the tumor microenvironment. We identified Stanniocalcin-1 (STC1) as a putative molecular marker for the leukemic microenvironment, once its expression was increased around 7 times in stromal cells co-cultivated with primary leukemic blasts. Human STC1 is a secreted glycoprotein that has been implicated in different physiological process, including angiogenesis, hypoxia and mainly in carcinogenesis. We produced the recombinant protein STC1 in baculovirus system and monoclonal antibodies for an ELISA assay that now will be tested as a new leukemia diagnostic kit by a Brazilian company. Moreover, we identified new interacting protein partners for STC1 by yeast two hybrid system and some of these interactions were confirmed by GST-pull down assays. The N-terminal region was mapped to be the region that mediates the interaction between STC1 and its partners. Microscopic subcellular localization, revealed an ubiquitous cytoplasmic and dot-like nuclear deposition, resembling SUMOylation related nuclear bodies. Although STC1 interacts with SUMO-1 and has a high theoretical prediction score for a SUMOylation site, in vitro and in vivo assays could not detect STC1 SUMOylation. However, we found that STC1 significantly regulates the SUMOylation of three other proteins. These ??ndings suggest a new role for STC1 in SUMOylation cycle, acting as a SUMO E3 ligase. We either observe that STC1 has a plasmatic membrane receptor in K562 leukemic cell lines and the incubation of STC1 with other leukemic cells suggest a increase of proliferation of these cells and stimulates the production of more intracellular STC1 at stromal cells. Together, all of these findings open promising new avenues to be explored in future detailed studies, since they all show interesting connections with previous functional studies on STC1 / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Activated islet specific T cells are central to the destructive autoimmune response observed in type 1 diabetes (T1D). Not surprisingly, intense focus is placed on understanding how autoreactive T cell responses arise and contribute to disease pathology in the hope of using this information to develop novel therapeutic strategies for treatment of T1D. Here we investigate the mechanisms underlying defective c-Maf binding to the IL-4 promoter in T cells from diabetes prone mice and identify the mechanisms responsible for suppression of T cells by the inhibitory receptor Ly49A. It is not clear why development of protective Th2 cells is poor in T1D. c-Maf transactivates the IL-4 gene promoting Th2 cell development; therefore abnormalities in c-Maf may contribute to reduced IL-4 production by CD4 cells from nonobese diabetic (NOD) mice. Here we demonstrate that, despite normal expression, c-Maf binds poorly to the IL-4 promoter (IL-4p) in NOD CD4 cells. Immunoblots demonstrate that c-Maf can be modified at lysine 33 by small ubiquitin-like modifier-1 (SUMO-1). Sumoylation is facilitated by direct interaction with the E2 conjugating enzyme Ubc9 and increases following T cell stimulation. In addition, c-Maf physically interacts with p65/RelA. This interaction is dependent on the DNA binding domain of c-Maf and phosphorylation of p65 at serine 536. In transfected cells, overexpression of SUMO-1 or p65 decreases c-Maf transactivation of IL-4p-driven luciferase reporter activity, reduces c-Maf binding to the IL-4p in chromatin immunoprecipitation (ChIP) assays and enhances c-Maf localization into promyelocytic leukemia nuclear bodies (PML-NBs) or nucleoli, respectively. Sumoylation of c-Maf and phosphorylation of p65 are increased in NOD CD4 cells compared to CD4 cells from diabetes-resistant B10.D2 mice, suggesting that increased c-Maf sumoylation and interaction with p65 contribute to immune deviation in T1D by reducing c-Maf access to and transactivation of the IL-4 gene. Islet specific CD4 cells expressing inhibitory receptors may be a useful therapeutic tool for treating T1D. Engagement of transgenic Ly49A inhibits CD4 cell activation and delays onset of T1D in mice. However, in vitro studies suggest the inhibitory effect of Ly49A is incomplete. Here we report that following simultaneous T cell receptor (TCR) and Ly49A engagement, phosphorylation of Zap70, Erk1/2 and c-Jun were significantly diminished. Kinetic studies indicated that Ly49A did not simply delay activation but had a long-lasting effect. In contrast, when only costimulatory signals were provided through CD28, Ly49A engagement did not block p38 MapK or Akt phosphorylation. Likewise, expression of the downstream targets Bcl-xl and Baff were unaffected. Together these data suggest that engagement of Ly49A selectively inhibits signals downstream of the TCR but spares those unique to CD28. These results suggest that when considering its use as an immunotherapy, the potency of inhibitory receptors such as Ly49A may be further improved by pairing them with costimulatory blockade. Take together, these studies suggest that abnormal post-translational regulation of c-Maf function is a novel marker of altered T cell function in T1D and use of inhibitory receptors such as Ly49A may be optimized combining this approach with other complementary therapies.

The SUMO protein was named Small Ubiquitin-like MOdifier because its 3-D structure was similar to Ubiquitin. In human, three SUMO proteins were discovered, namely, SUMO-1/2/3. The recombinant ¡µ1-8, 94-95 SUMO-2 protein with 10 histidine residues at its N-terminus was expressed using E. coli. BL-21(DE3), purified at 4 oC and crystallized at room temperature. The surface properties of human SUMO-1/2/3 proteins and 3-D structure of ¡µ1-8, 94-95 SUMO-2 protein were analyzed using computer modeling and X-ray diffraction technology respectively. The two-step purification by immobilized metal ion affinity chromatography(IMAC) was developed to yield ¡µ1-8, 94-95 SUMO-2 protein that reached 60 mg/ml for crystallization. On protein expression, 120 mg protein was obtained from 6 L bacterial growth broth. Crystals of ¡µ1-8, 94-95 SUMO-2 were obtained by the hanging-drop vapor diffusion method and many different crystal forms were observed. One of single crystal with triangular plate polyhedron form diffracted to 1.6 Å resolution, the other one with rectangular polyhedron form diffracted to 1.2 Å. Analysis of the diffraction pattern suggests the crystals belong to R3 space group, the former one owned unit cell parameters a= b=75.3 Å, c=29.2 Å, £\=90¢X, £]=90¢X,£^=120¢X, and the later one owned unit cell parameters a= b=74.9 Å, c=33.2 Å and the same angles respectively. The R factor and Rfree of refinement are 0.133 and 0.190 with highly precise phase on 3-D structure of SUMO-2 protein. Comparison of crystal structure between human SUMO-2 and yeast SMT3 showed that the r.m.s. deviation of C£\ coordinate is 1.054 Å. In addition, comparison of SUMO-1 NMR structure and SMT3 crystal structure showed that the r.m.s. deviation of C£\ coordinates is 2.736 Å. Hence, the structures of SUMO-2 and SMT3 are more similar each other than those of SUMO-1and SMT3.

La protéine Rev du HIV-1 joue un rôle essentiel dans le cycle viral en permettant l'export vers le cytoplasme des ARNm viraux non ou partiellement épissés. Dans la perspective d'inhiber cette fonction, nous avons mis au point des peptides capables de reconnaître Rev et d'inhiber sa fonction, appelés SHPR (Sumo Heptapeptide Protein transduction domain for binding Rev). Nous avons établi que ces peptides sont capables de diffuser à travers les membranes plasmiques pour atteindre ensuite le noyau, où ils induisent la dégradation de la protéine virale, principalement par la voie du protéasome. Des expériences de mutagenèse ont précisé la contribution des différents domaines des SHPR dans l'action antivirale, et ont démontré que peu d'améliorations sont possibles pour optimiser leurs propriétés. Enfin, nous avons montré que Rev n'est pas régulée par sumoylation mais qu'elle peut être modifiée par addition d'une chaîne de polyubiquitine, qui stabilise la protéine mais module son activité

Les estrogènes jouent un rôle primordial dans le développement et le fonctionnement des tissus reproducteurs par leurs interactions avec les récepteurs des estrogènes ERα et ERβ. Ces récepteurs nucléaires agissent comme facteurs de transcription et contrôlent l’expression des gènes de façon hormono-dépendante et indépendante grâce à leurs deux domaines d’activation (AF-1 et AF-2). Une dérégulation de leur activité transcriptionnelle est souvent à l’origine de pathologies telles que le cancer du sein, de l’endomètre et des ovaires. Alors que ERα est utilisé comme facteur pronostic pour l’utilisation d’agents thérapeutiques, l’importance de la valeur clinique de ERβ est encore controversée. Toutefois, des évidences récentes lui associent un pouvoir anti-tumorigénique en démontrant que sa présence favorise l’inhibition de la progression de ces cancers ainsi que l’efficacité des traitements. En combinaisons avec d’autres études, ces observations démontrent que bien que les deux isoformes partagent une certaine similitude d’action, les ERs sont en mesure d’exercer des fonctions distinctes. Ces différences sont fortement attribuables au faible degré d’homologie observé entre certains domaines structuraux des ERs, comme le domaine AF-1, ce qui fait en sorte que les différents sites de modifications post-traductionnelles (MPTs) présents sur les ERs sont très peu conservés entre les isoformes. Or, l’activité transcriptionnelle ligand-dépendante et indépendante des ERs est hautement régulée par les MPTs. Elles sont impliquées à tous les niveaux de l’activation des ERs incluant la liaison et la sensibilité au ligand, la localisation cellulaire, la dimérisation, l’interaction avec l’ADN, le recrutement de corégulateurs transcriptionnels, la stabilité et l’arrêt de la transcription. Ainsi, de par leur dissimilitude, les ERs seront différemment régulés par la signalisation cellulaire. Comme un débalancement de plusieurs voies de signalisation ont été associées à la progression de tumeurs ER-positives ainsi qu’au développement d’une résistance, une meilleure compréhension de l’impact des MPTs sur la régulation spécifique des ERs s’avère essentielle en vue de proposer et/ou développer des traitements adéquats pour les cancers gynécologiques. Les résultats présentés dans cette thèse ont pour objectif de mieux comprendre les rôles des MPTs sur l’activité transcriptionnelle de ERβ qui sont, contrairement à ERα, très peu connus. Nous démontrons une régulation dynamique de ERβ par la phosphorylation, l’ubiquitination et la sumoylation. De plus, toutes les MPTs nouvellement découvertes par mes recherches se situent dans l’AF-1 de ERβ et permettent de mieux comprendre le rôle capital joué par ce domaine dans la régulation de l’activité ligand-dépendante et indépendante du récepteur. Dans la première étude, nous observons qu’en réponse aux MAPK, l’AF-1 de ERβ est phosphorylé au niveau de sérines spécifiques et qu’elles jouent un rôle important dans la régulation de l’activité ligand-indépendante de ERβ par la voie ubiquitine-protéasome. En effet, la phosphorylation de ces sérines régule le cycle d’activation-dégradation de ERβ en modulant son ubiquitination, sa mobilité nucléaire et sa stabilité en favorisant le recrutement de l’ubiquitine ligase E6-AP. De plus, ce mécanisme d’action semble être derrière la régulation différentielle de l’activité de ERα et ERβ observée lors de l’inhibition du protéasome. Dans le second papier, nous démontrons que l’activité et la stabilité de ERβ en présence d’estrogène sont étroitement régulées par la sumoylation phosphorylation-dépendante de l’AF-1, processus hautement favorisé par l’action de la kinase GSK-3. La sumoylation de ERβ par SUMO-1 prévient la dégradation du récepteur en entrant en compétition avec l’ubiquitination au niveau du même site accepteur. De plus, contrairement à ERα, SUMO-1 réprime l’activité de ERβ en altérant son interaction avec l’ADN et l’expression de ses gènes cibles dans les cellules de cancers du sein. Également, ces recherches ont permis d’identifier un motif de sumoylation dépendant de la phosphorylation (pSuM) jusqu’à lors inconnu de la communauté scientifique, offrant ainsi un outil supplémentaire à la prédiction de nouveau substrat de la sumoylation. En plus de permettre une meilleure compréhension du rôle des signaux intracellulaires dans la régulation de l’activité transcriptionnelle de ERβ, nos résultats soulignent l’importance des MPTs dans l’induction des différences fonctionnelles observées entre ERα et ERβ et apportent des pistes supplémentaires à la compréhension de leurs rôles physiopathologiques respectifs. / Estrogens play a pivotal role in reproductive physiology through direct interaction with the estrogen receptors ERα and ERβ, which belong to the nuclear hormone receptor family of ligand-activated transcription factors. Harbouring two activation domains (AF-1 and AF-2), gene expression can be controlled by ERs either in a hormone-dependent and/or independent manner. Disruption of ER transcriptional regulation is associated with pathological events such as breast and endometrial cancers. While ERα is considered a strong predictive factor in endocrine therapy of reproductive cancers, the clinical value of ERβ is still debated, although greater expression of ERβ has been associated with a favourable outcome since recent evidence has associated ERβ with anti-tumorigenic properties and a better response to anti-estrogenic compounds. Along with others studies, those individual outcomes indicate that even though the two receptors can exert similar roles by sharing resemblances in terms of structure and general response to hormone, they can also carry out distinct functions. These variations can be attributed to the fact that most of the structural domains shared by ERs exhibit a low level of homology, especially at the AF-1 domain. Consequently, the majority of the post-translational modifications sites (PTMs) on ERs are not shared between both isoforms. In fact, ligand-induced and ligand-independent activities of ERs are critically influenced by PTMs. PTMs controls the multiple aspects of ER-dependent activation by modulating ERs ligand binding, specificity, cellular localization, dimerization, interaction with their cognate DNA response element, combinatory recruitment of transcriptional coregulators, stability and transcriptional arrest. Hence, by their discrepancies, ERs will be differently influenced by the cellular environment. Furthermore, as the deregulation of different signalling pathway in cancers is associated with ER-dependant tumour progression and in the acquisition of a therapeutic resistant phenotype, it is crucial to understand the how PTMs affect ERs transactivation in order to eventually propose and/or develop adequate treatment. The results presented in this thesis were carried out with the objective of gaining a better understanding of PTM’s roles on ERβ transcriptional control which, as opposed to ERα, remain unclear. We demonstrate here a dynamic regulation of ERβ by phosphorylation, ubiquitination and sumoylation. Furthermore, as all the newly identified PTM are located within de AF-1 domain of ERβ, our results highlight the key role of this domain in the regulation of ligand-dependent and independent transcriptional properties of this receptor. The first study shows that in response to MAPK, specific serine residues in the AF-1 of ERβ are phosphorylated and play an important role in the regulation of ERβ ligand-independent activity by the ubiquitin-proteasome pathway. In fact, the activation-degradation cycle of ERβ induced by MAPK is regulated upon phosphorylation of these serines coordinating ERβ ubiquitination, subnuclear mobility and stability by promoting the recruitment of the ubiquitin ligase E6-AP. Moreover, this molecular process plays part in the differential regulation of ERα and ERβ activity upon proteasome inhibition. In the second paper, we demonstrate that ERβ activity and stability in presence of estrogen is closely regulated by the phosphorylation-dependent sumoylation of the AF-1 domain, amplified by GSK-3 action. SUMO-1 attachment prevents ERβ degradation by competing with ubiquitin at the same acceptor site and dictates ERβ transcriptional inhibition, as opposed to ERα, by altering estrogen-responsive target promoter occupancy and gene expression in breast cancer cells. Furthermore, these findings uncover a novel phosphorylated sumoylation motif (pSuM) and offer a valuable tool to predict novel SUMO substrates under protein kinase regulation. In combination to our better understanding on how intracellular signals controls ERβ transcriptional activity, our results highlight the significant role of PTMs in ERs isoforms discrepancies and allows supplementary comprehension of their respective physiopathologicals roles.

Les estrogènes jouent un rôle primordial dans le développement et le fonctionnement des tissus reproducteurs par leurs interactions avec les récepteurs des estrogènes ERα et ERβ. Ces récepteurs nucléaires agissent comme facteurs de transcription et contrôlent l’expression des gènes de façon hormono-dépendante et indépendante grâce à leurs deux domaines d’activation (AF-1 et AF-2). Une dérégulation de leur activité transcriptionnelle est souvent à l’origine de pathologies telles que le cancer du sein, de l’endomètre et des ovaires. Alors que ERα est utilisé comme facteur pronostic pour l’utilisation d’agents thérapeutiques, l’importance de la valeur clinique de ERβ est encore controversée. Toutefois, des évidences récentes lui associent un pouvoir anti-tumorigénique en démontrant que sa présence favorise l’inhibition de la progression de ces cancers ainsi que l’efficacité des traitements. En combinaisons avec d’autres études, ces observations démontrent que bien que les deux isoformes partagent une certaine similitude d’action, les ERs sont en mesure d’exercer des fonctions distinctes. Ces différences sont fortement attribuables au faible degré d’homologie observé entre certains domaines structuraux des ERs, comme le domaine AF-1, ce qui fait en sorte que les différents sites de modifications post-traductionnelles (MPTs) présents sur les ERs sont très peu conservés entre les isoformes. Or, l’activité transcriptionnelle ligand-dépendante et indépendante des ERs est hautement régulée par les MPTs. Elles sont impliquées à tous les niveaux de l’activation des ERs incluant la liaison et la sensibilité au ligand, la localisation cellulaire, la dimérisation, l’interaction avec l’ADN, le recrutement de corégulateurs transcriptionnels, la stabilité et l’arrêt de la transcription. Ainsi, de par leur dissimilitude, les ERs seront différemment régulés par la signalisation cellulaire. Comme un débalancement de plusieurs voies de signalisation ont été associées à la progression de tumeurs ER-positives ainsi qu’au développement d’une résistance, une meilleure compréhension de l’impact des MPTs sur la régulation spécifique des ERs s’avère essentielle en vue de proposer et/ou développer des traitements adéquats pour les cancers gynécologiques. Les résultats présentés dans cette thèse ont pour objectif de mieux comprendre les rôles des MPTs sur l’activité transcriptionnelle de ERβ qui sont, contrairement à ERα, très peu connus. Nous démontrons une régulation dynamique de ERβ par la phosphorylation, l’ubiquitination et la sumoylation. De plus, toutes les MPTs nouvellement découvertes par mes recherches se situent dans l’AF-1 de ERβ et permettent de mieux comprendre le rôle capital joué par ce domaine dans la régulation de l’activité ligand-dépendante et indépendante du récepteur. Dans la première étude, nous observons qu’en réponse aux MAPK, l’AF-1 de ERβ est phosphorylé au niveau de sérines spécifiques et qu’elles jouent un rôle important dans la régulation de l’activité ligand-indépendante de ERβ par la voie ubiquitine-protéasome. En effet, la phosphorylation de ces sérines régule le cycle d’activation-dégradation de ERβ en modulant son ubiquitination, sa mobilité nucléaire et sa stabilité en favorisant le recrutement de l’ubiquitine ligase E6-AP. De plus, ce mécanisme d’action semble être derrière la régulation différentielle de l’activité de ERα et ERβ observée lors de l’inhibition du protéasome. Dans le second papier, nous démontrons que l’activité et la stabilité de ERβ en présence d’estrogène sont étroitement régulées par la sumoylation phosphorylation-dépendante de l’AF-1, processus hautement favorisé par l’action de la kinase GSK-3. La sumoylation de ERβ par SUMO-1 prévient la dégradation du récepteur en entrant en compétition avec l’ubiquitination au niveau du même site accepteur. De plus, contrairement à ERα, SUMO-1 réprime l’activité de ERβ en altérant son interaction avec l’ADN et l’expression de ses gènes cibles dans les cellules de cancers du sein. Également, ces recherches ont permis d’identifier un motif de sumoylation dépendant de la phosphorylation (pSuM) jusqu’à lors inconnu de la communauté scientifique, offrant ainsi un outil supplémentaire à la prédiction de nouveau substrat de la sumoylation. En plus de permettre une meilleure compréhension du rôle des signaux intracellulaires dans la régulation de l’activité transcriptionnelle de ERβ, nos résultats soulignent l’importance des MPTs dans l’induction des différences fonctionnelles observées entre ERα et ERβ et apportent des pistes supplémentaires à la compréhension de leurs rôles physiopathologiques respectifs. / Estrogens play a pivotal role in reproductive physiology through direct interaction with the estrogen receptors ERα and ERβ, which belong to the nuclear hormone receptor family of ligand-activated transcription factors. Harbouring two activation domains (AF-1 and AF-2), gene expression can be controlled by ERs either in a hormone-dependent and/or independent manner. Disruption of ER transcriptional regulation is associated with pathological events such as breast and endometrial cancers. While ERα is considered a strong predictive factor in endocrine therapy of reproductive cancers, the clinical value of ERβ is still debated, although greater expression of ERβ has been associated with a favourable outcome since recent evidence has associated ERβ with anti-tumorigenic properties and a better response to anti-estrogenic compounds. Along with others studies, those individual outcomes indicate that even though the two receptors can exert similar roles by sharing resemblances in terms of structure and general response to hormone, they can also carry out distinct functions. These variations can be attributed to the fact that most of the structural domains shared by ERs exhibit a low level of homology, especially at the AF-1 domain. Consequently, the majority of the post-translational modifications sites (PTMs) on ERs are not shared between both isoforms. In fact, ligand-induced and ligand-independent activities of ERs are critically influenced by PTMs. PTMs controls the multiple aspects of ER-dependent activation by modulating ERs ligand binding, specificity, cellular localization, dimerization, interaction with their cognate DNA response element, combinatory recruitment of transcriptional coregulators, stability and transcriptional arrest. Hence, by their discrepancies, ERs will be differently influenced by the cellular environment. Furthermore, as the deregulation of different signalling pathway in cancers is associated with ER-dependant tumour progression and in the acquisition of a therapeutic resistant phenotype, it is crucial to understand the how PTMs affect ERs transactivation in order to eventually propose and/or develop adequate treatment. The results presented in this thesis were carried out with the objective of gaining a better understanding of PTM’s roles on ERβ transcriptional control which, as opposed to ERα, remain unclear. We demonstrate here a dynamic regulation of ERβ by phosphorylation, ubiquitination and sumoylation. Furthermore, as all the newly identified PTM are located within de AF-1 domain of ERβ, our results highlight the key role of this domain in the regulation of ligand-dependent and independent transcriptional properties of this receptor. The first study shows that in response to MAPK, specific serine residues in the AF-1 of ERβ are phosphorylated and play an important role in the regulation of ERβ ligand-independent activity by the ubiquitin-proteasome pathway. In fact, the activation-degradation cycle of ERβ induced by MAPK is regulated upon phosphorylation of these serines coordinating ERβ ubiquitination, subnuclear mobility and stability by promoting the recruitment of the ubiquitin ligase E6-AP. Moreover, this molecular process plays part in the differential regulation of ERα and ERβ activity upon proteasome inhibition. In the second paper, we demonstrate that ERβ activity and stability in presence of estrogen is closely regulated by the phosphorylation-dependent sumoylation of the AF-1 domain, amplified by GSK-3 action. SUMO-1 attachment prevents ERβ degradation by competing with ubiquitin at the same acceptor site and dictates ERβ transcriptional inhibition, as opposed to ERα, by altering estrogen-responsive target promoter occupancy and gene expression in breast cancer cells. Furthermore, these findings uncover a novel phosphorylated sumoylation motif (pSuM) and offer a valuable tool to predict novel SUMO substrates under protein kinase regulation. In combination to our better understanding on how intracellular signals controls ERβ transcriptional activity, our results highlight the significant role of PTMs in ERs isoforms discrepancies and allows supplementary comprehension of their respective physiopathologicals roles.

No / The surface expression and regulated endocytosis of kainate (KA) receptors (KARs) plays a critical role in neuronal function. PKC can modulate KAR trafficking, but the sites of action and molecular consequences have not been fully characterized. Small ubiquitin-like modifier (SUMO) modification of the KAR subunit GluK2 mediates agonist-evoked internalization, but how KAR activation leads to GluK2 SUMOylation is unclear. Here we show that KA stimulation causes rapid phosphorylation of GluK2 by PKC, and that PKC activation increases GluK2 SUMOylation both in vitro and in neurons. The intracellular C-terminal domain of GluK2 contains two predicted PKC phosphorylation sites, S846 and S868, both of which are phosphorylated in response to KA. Phosphomimetic mutagenesis of S868 increased GluK2 SUMOylation, and mutation of S868 to a nonphosphorylatable alanine prevented KA-induced SUMOylation and endocytosis in neurons. Infusion of SUMO-1 dramatically reduced KAR-mediated currents in HEK293 cells expressing WT GluK2 or nonphosphorylatable S846A mutant, but had no effect on currents mediated by the S868A mutant. These data demonstrate that agonist activation of GluK2 promotes PKC-dependent phosphorylation of S846 and S868, but that only S868 phosphorylation is required to enhance GluK2 SUMOylation and promote endocytosis. Thus, direct phosphorylation by PKC and GluK2 SUMOylation are intimately linked in regulating the surface expression and function of GluK2-containing KARs.