Les protéines à bromodomaines (BCPs) sont notamment impliquées dans la régulation de la transcription de gènes et la signalisation cellulaire. Leur dérégulation conduit au développement pathologies, telles que les maladies inflammatoires, cardiovasculaires et plus particulièrement les cancers. Les BCPs sont capables de reconnaitre les lysines acétylées de protéines histones via leur module BromoDomaine(s) (BDs). Parmi les huit familles de BCPs, mon projet de thèse s’intéresse à la famille « BET ». Celle-ci, comprend quatre protéines constituées de deux BDs en tandem, formant deux sous-familles BD1 et BD2. L’architecture de la cavité centrale des BDs, qualifiée de « druggable », a permis l’émergence de ces protéines en tant que nouvelles cibles épigénétiques prometteuses. À ce jour, une vingtaine d’essais cliniques ont été initiés pour des molécules « pan-BET », inhibant l’ensemble des membres de cette famille. Cependant, l'inhibition « pan-BET » est problématique au niveau clinique puisqu’elle impacte de nombreuses voies de transcription et engendre l’apparition de cellules résistantes. Mon projet de thèse s’intègre au challenge actuel qui est de développer des inhibiteurs plus sélectifs, par exemple envers l’une des sous-familles BD1 ou BD2 ou plus idéalement envers un seul BD de la famille BET. Le développement de « sondes épigénétiques sélectives » ciblant des BDs de la famille BET, devrait permettre de décrypter leur rôle et leur mécanisme d’action dans les divers processus biologiques. L’identification de « candidat médicament » devrait aboutir à de nouvelles thérapies ciblées et de pallier les résistances liées à l’utilisation de molécules pan-BET inhibitrices. / Bromodomain-containing proteins (BCPs) are especially involved in the regulation of gene transcription and cell signalling. Their dysregulation lead to the development of pathologies, such as inflammatory, cardiovascular diseases, and more particularly cancers. BCPs involved in the recognition of acetylated lysine of the histone tails, through their BromoDomain(s) module(s) (BDs). Among the eight families of BCPs, my thesis project focuses on the “BET” family. This family comprises four proteins which are composed of a tandem of two BDs each belonging to the BD1 or the BD2 subfamily. The architecture of the central cavity of the BDs, qualified as "druggable", allows the emergence of these proteins as new promising epigenetic targets. To date, about twenty clinical trials targeting different types of cancer have been initiated for "pan-BET" molecules that target all the members of this family. However, "pan-BET" inhibition is clinically problematic because it impacts many transcriptional pathways and causes the appearance of resistant cells. My thesis project is part of the current challenge is to develop more selective inhibitors, for example towards the BET-BD1 subfamily or the BET-BD2 subfamily or ideally towards one single BD inside the BET family. The development of such "selective epigenetic probes" targeting BET family BDs should allow deciphering their role and mechanism of action in various biological processes. Identifying "drug candidates" should lead to new targeted therapies and overcome the resistances related to the use of pan-BET molecules.

La protéine chaperonne CDC48 (Cell division cycle 48) est un acteur important du contrôle qualité des protéines chez les eucaryotes et est associée à divers processus physio(patho)logiques chez les mammifères. En revanche, son rôle au sein du règne végétal a été peu appréhendé. Ce travail de thèse s’inscrit dans l’étude des fonctions de CDC48 chez les plantes et concerne plus particulièrement son implication dans la réponse immunité induite chez le tabac par cryptogéine produite par l’oomycète phytophthora cryptogea.Trois stratégies ont été adoptées. Premièrement, la dynamique d’accumulation de la protéine CDC48 ainsi que les événements intracellulaires sous-jacents à la réponse immunitaire ont été étudiés à la fois dans des cellules de tabac sauvages et des cellules sur-exprimant la protéine CDC48 (lignée CDC48-TAP). Deuxièmement, une liste de protéines interagissant avec CDC48 a été établie suite à des expériences d’immuno-précipitation de CDC48 suivit d’analyses de spectrométrie de masse. Parmi celles-ci, la forme cytosolique de l’ascorbate peroxydase (cAPX), une enzyme impliquée dans la détoxication du H2O2 intracellulaire, a fait l’objet d’une étude ciblée. Enfin, ces travaux ont été complétés par une analyse bio-informatique de l’ensemble des partenaires de CDC48 identifiés chez le tabac et d’établissement du réseau d’interaction protéique de CDC48 chez Arabidopsis thaliana.Les principaux résultats obtenus ont montré que l’activation de la réponse immunitaire s’accompagne de l’induction d’une accumulation des transcrits et la protéine CDC48. De plus, une mort cellulaire précoce a été observée chez les cellules CDC48-TAP, suggérant un rôle de cette dernière dans la régulation de la réponse hypersensible. L’interaction physique entre CDC48 et cAPX a été confirmée par différentes approches. De façon intéressante, il s’est avéré que l’activité et la dynamique d’accumulation de cAPX sont fortement impactées par la surexpression de CDC48. En accord avec ses résultats, le statut rédox s’est également révélé altéré dans la lignée surexpresseur. Enfin, l’analyse bio-informatique du réseau d’interaction protéique de CDC48 a permis de dégager de nouvelles protéines cibles, en particulier celles impliquées dans le métabolisme de la S-adenosylméthionine, une molécule substrat des réactions de trans-méthylation et précurseur de l’éthylène et de la nicotianamine. De plus, cette analyse a confirmé son rôle dans du système de dégradation Ubiquitine/protéasome.Pour conclure, ce travail de thèse apporte de nouvelles informations quant au rôle de CDC48 dans la biologie des plantes. Il indique que celle-ci est mobilisée dans les cellules végétales exprimant une réponse immunitaire et impacte le statut rédox via la régulation du turnover de cAPX. De nouvelles pistes de recherche ont été dégagées, en particulier un rôle probable de CDC48 dans la régulation de la synthèse de la S-adenosylméthionine et de la réponse hypersensible suivant des mécanismes restant à déterminer. / The chaperone protein CDC48 (Cell division cycle 48) is a major regulator of the quality control of proteins and is involved in various cellular processes in animals and yeast. In contrast, the role of CDC48 in plants is poorly known. In the present work, we investigated the function of CDC48 in plant immunity thanks to the cryptogein/tobacco biological model, cryptogein being produced by the oomycete phytophthora cryptogea.Three strategies were carried out. First, the dynamic of accumulation CDC48 together with intracellular events inherent to the immune response were analyzed in both wild-type and CDC48 overexpressing tobacco cells (CDC48-TAP line). Second, a list if CDC48 partners was established based on immunoprecipitation assays followed by mass spectroscopy analysis. Among those partners the cytosolic form of acorbate peroxidase (cAPX), a central enzyme of the regulation of the redox status regulation, has been specifically studied. Finally, a computational analysis of the partner list of CDC48 and the subsequent generation of the protein-protein interaction (PPI) network of CDC48 in Arabidopsis thaliana were undertook.Our data indicated that the activation of the immune response is accompanied by an induction of the accumulation of both CDC48 transcript and protein. In addition, an early and exacerbated cell death was observed in the CDC48-TAP line, suggesting a role for CDC48 in the hypersensitive response. The interaction between CDC48 and cAPX was confirmed by different approaches. Interestingly, the activity of CDC48 and its dynamic of accumulation were strongly impacted in the CDC48 overexpressing line. Accordingly, a dysregulation of the redox status also occurred in this line. Finally, the computational analysis of the CDC48 PPI network highlighted new potential target proteins including proteins involved in the metabolism of S-adenosylmethionine, a substrate molecule of trans-methylation reactions and precursor of ethylene and nicotianamine.To summarize, this work provides new information about CDC48 in plant biology. It indicates that CDC48 is mobilized by plant cells undergoing an immune response and impacts the redox status through the regulation of the cAPX turnover. New research avenues emerged from our study, notably a putative role of CDC48 in the regulation of S-adenosylmethionine biosynthesis and in the establishment of hypersensitive response through process which remain to be investigated

Abstract Acute myocardial infarction is a life-threatening condition that occurs as a result of reduced blood flow in the cardiac muscle, eventually leading to tissue damage. In infarcted areas, cardiomyocytes have insufficient ability to proliferate and replace the injured cells, which is associated with a deficient pumping capacity. A strictly regulated combinatorial interplay of transcription factors, e.g., GATA4, NKX2-5, TBX5, and MEF2C, orchestrates cardiac type gene expression during the cardiomyocyte differentiation and maturation processes. The aim of the present study was to (i) characterize the protein-protein interaction of the cardiac transcription factors GATA4-NKX2-5, (ii) evaluate the chemical agents that modify the synergy of GATA4-NKX2-5 in vitro, (iii) examine the capacity of the lead compound to promote myocardial repair in vivo after myocardial infarction and other cardiac injuries and (iv) study the structural features of the compound important for metabolism and cytotoxicity. Integration of the experimental mutagenic data with computational modeling suggests that the structural architecture of the GATA4-NKX2-5 interaction resembles the protein structure of the conserved DNA binding domain of nuclear receptors. Fragment-based screening, reporter gene-based optimization and pharmacophore searching were utilized to identify the most potent lead compound targeting the GATA4-NKX2-5 interaction: N-[4-(diethylamino)phenyl]-5-methyl-3-phenylisoxazole-4-carboxamide. This compound presented anti-hypertrophic effects in vitro and cardioprotective effects in vivo. In addition, structural analysis of the lead compound revealed the signature molecular features for metabolism and cytotoxicity. Current drug treatments are able to delay, but not prevent the progress of the heart failure; therefore, modulators of protein-protein interactions of key transcription factors may represent a novel class of pharmaceuticals for cardiac remodeling and repair. / Tiivistelmä Sydäninfarkti on henkeä uhkaava verenkierron häiriö, joka syntyy veren virtauksen äkillisen vähentymisen seurauksena sydänlihaksessa aiheuttaen kudosvaurion. Vaurioituneen sydänlihaskudoksen kyky uusiutua tai korvata kuolleet sydänlihassolut uusilla on puutteellinen, ja tämän seurauksena sydämen pumppauskyky heikkenee. Transkriptiotekijöiden GATA4, NKX2-5, TBX5 ja MEF2C muodostamat ja koordinoimat proteiinikompleksit säätelevät sydänsolujen geenien ilmenemistä solujen elinkaaren aikana. Väitöskirjatyön tavoitteena oli (i) karakterisoida geeninsäätelytekijöiden GATA4-NKX2-5 molekyylirakenteet ja niiden keskinäinen vuorovaikutus, (ii) seuloa kemiallisia yhdisteitä, jotka muokkaavat GATA4-NKX2-5 proteiinikompleksin aikaansaamaa geeniaktivaatiota, (iii) tutkia johtoyhdisteen vaikutuksia in vivo sydäninfarktia ja painekuormitusta kuvaavissa eläinmalleissa, ja (iv) tutkia johtoyhdisteen molekyylirakenteen yhteyttä yhdisteen metaboliaan ja sytotoksisuuteen. Väitöskirjatyö osoittaa molekyylimallinuksen ja kokeellisten tulosten perusteella, että geeninsäätelytekijöiden GATA4-NKX2-5 proteiinikompleksin orientaatio matkii tumareseptoriperheen DNA domeenin tertiäärirakennetta. Molekyylifragmenttien, lusiferaasi-reportterikokeen ja farmakoforimallin avulla seulottiin ja optimoitiin sitoutumisvoimakkuudeltaan lupaavin GATA4-NKX2-5 proteiinikompleksin toimintaan vaikuttava johtoyhdiste: N-[4-(dietyyliamino)fenyyli]-5-metyyli-3-fenyyli-isoksatsoli-4-karboksamidi. Johtoyhdisteellä havaittiin solu- ja eläinmalleissa hypertrofiaa estäviä vaikutuksia in vitro ja sydäntä suojaavia vaikutuksia in vivo. Väitöskirjatyö osoitti lisäksi aktiivisten molekyylien rakenneominaisuuksia, jotka keskeisesti vaikuttavat yhdisteiden metaboliaan ja sytotoksisuuteen. Nykyinen lääkehoito hidastaa, mutta ei pysäytä sydänlihasvaurioon liittyvän kroonisen sydämen vajaatoiminnan etenemistä. Lääkevaikutuksen kohdentaminen sydämen keskeisten transkriptiotekijöiden yhteisvaikutukseen avaa uuden mahdollisen tutkimuslinjan sydänlihasvaurion estossa ja korjauksessa.

Parkinson’s Disease (PD) is the second most common neurodegenerative disease in the Western world. It shows a high degree of genetic and phenotypic complexity with many implicated factors, various disease manifestations but few clear causal links. Ongoing research has identified a growing number of molecular alterations linked to the disease. Dopaminergic neurons in the substantia nigra, specifically their synapses, are the key-affected region in PD. Therefore, this work focuses on understanding the disease effects on the synapse, aiming to identify potential genetic triggers and synaptic PD associated mechanisms. Currently, one of the main challenges in this area is data quality and accessibility. In order to study PD, publicly available data were systematically retrieved and analysed. 418 PD associated genes could be identified, based on mutations and curated annotations. I curated an up-to-date and complete synaptic proteome map containing a total of 6,706 proteins. Region specific datasets describing the presynapse, postsynapse and synaptosome were also delimited. These datasets were analysed, investigating similarities and differences, including reproducibility and functional interpretations. The use of Protein-Protein-Interaction Network (PPIN) analysis was chosen to gain deeper knowledge regarding specific effects of PD on the synapse. Thus I generated a customised, filtered, human specific Protein-Protein Interaction (PPI) dataset, containing 211,824 direct interactions, from four public databases. Proteomics data and PPI information allowed the construction of PPINs. These were analysed and a set of low level statistics, including modularity, clustering coefficient and node degree, explaining the network’s topology from a mathematical point of view were obtained. Apart from low-level network statistics, high-level topology of the PPINs was studied. To identify functional network subgroups, different clustering algorithms were investigated. In the context of biological networks, the underlying hypothesis is that proteins in a structural community are more likely to share common functions. Therefore I attempted to identify PD enriched communities of synaptic proteins. Once identified, they were compared amongst each other. Three community clusters could be identified as containing largely overlapping gene sets. These contain 24 PD associated genes. Apart from the known disease associated genes in these communities, a total of 322 genes was identified. Each of the three clusters is specifically enriched for specific biological processes and cellular components, which include neurotransmitter secretion, positive regulation of synapse assembly, pre- and post-synaptic membrane, scaffolding proteins, neuromuscular junction development and complement activation (classical pathway) amongst others. The presented approach combined a curated set of PD associated genes, filtered PPI information and synaptic proteomes. Various small- and large-scale analytical approaches, including PPIN topology analysis, clustering algorithms and enrichment studies identified highly PD affected synaptic proteins and subregions. Specific disease associated functions confirmed known research insights and allowed me to propose a new list of so far unknown potential disease associated genes. Due to the open design, this approach can be used to answer similar research questions regarding other complex diseases amongst others.

Cdc42 is a member of the Rho family of small GTPases, which, together with its homologues RhoA and Rac1, controls a multitude of cellular functions via the actin cytoskeleton. Cdc42 exerts its effects on the cytoskeleton via effector proteins of the Wiskott-Aldrich Syndrome (WASP) family and the Transducer of Cdc42-dependent Actin assembly (TOCA) family. The WASP family and their activation by Cdc42 have been thoroughly studied in vitro and are well understood. Conversely, understanding of the TOCA family remains limited by a lack of biochemical, biophysical and structural insight. An investigation of the TOCA1-Cdc42 interaction is described here, revealing a relatively low affinity interaction with a dissociation constant in the micromolar range. This is 10-100x weaker than other Rho-effector interactions and suggests that TOCA1 must first be co-localised with Cdc42 to achieve stable binding in vivo. The solution NMR structure of the Cdc42 binding HR1 domain of TOCA1 provides the first structural data on this protein and reveals some interesting structural features that may relate to binding affinity and specificity. A structural model of the Cdc42-HR1 complex provides further insight into differential specificities and affinities of GTPase-effector interactions. NMR and actin polymerisation assays provide insight into the pathway of Cdc42/TOCA1/WASP-dependent actin assembly, suggesting unidirectional displacement of TOCA1 by N-WASP. A comparison of the Cdc42- TOCA1 model with an NMR structure of Cdc42 in complex with the GTPase binding domain of WASP reveals a possible mechanism by which an ‘effector handover’ from TOCA1 to N-WASP could take place. Small GTPases such as Cdc42 are lipid modified and membrane anchored via their C- termini in vivo, so in vitro studies using truncated, unmodified GTPases are limited in their biological interpretation. This project also aimed to develop methods to study full length and membrane-anchored GTPases in vitro. Lipid modified protein was produced, which showed a weak affinity for liposomes, and so structural studies of membrane anchored protein are within reach. Further method development is now required to achieve stable membrane anchoring of lipid modified GTPases for detailed NMR studies.

Vivant à des températures supérieures à 80°C, les archées hyperthermophiles ont démontré une capacité étonnante à se remettre de dommages dans leur ADN, suggérant la présence de gardiens du génome particulièrement efficaces. Ces gardiens, des protéines relativement similaires entre archées et eucaryotes, agissent et interagissent dans un ballet savamment orchestré par la cellule. Chez les archées, plusieurs protéines impliquées dans des voies essentielles à la réparation de I'ADN manquent à I'appel. Précédemment au laboratoire un réseau impliquant les protéines de la maintenance génomique de Pyrococcus abyssi a dévoilé de nouvelles interactions protéine-protéine. Décrire l'interaction pour mieux comprendre sa fonction, voici la démarche suivie et présentée dans ce manuscrit pour les duos: PCNA/Mrell-Rad50 et MCM/XPD. Pour la première fois chez Pyrococcus furiostts une interaction physique et fonctionnelle a été démontrée entre PCNA, le maestro de la réplication, et Mrell-Rad50, le complexe de la recombinaison. Pour la seconde étude, la caractérisation de l'hélicase réplicative MCM de P. abyssi a été menée via une approche biophysique basée sur des techniques de fluorescence. Les difficultés rencontrées durant la production de son partenaire potentiel XPD, n'ont toutefois pu permettre la caractérisation de leur interaction. Plus généralement ces interactions s'inscrivent dans un contexte où le couplage de la réplication avec des processus de réparation trouve son importance particulièrement chez les archées de l'extrême, archées qui se révèlent être de passionnants modèles pour l'étude des mécanismes de la maintenance génomique. / Living at temperatures above 80°C, hyperthermophilic Archaea demonstrated amazing capacity to recover from DNA damages, suggesting they arguably have efficient genome guardians. These guardians, proteins which are relatively similar between Archaea and eukaryotes, act and interact like a ballet orchestrated by the cell. Several proteins involved in essential repair pathway in eukaryotes are missing in Archaea. To gain insights into archaeal genome maintenance processes, a previous work proposed a protein-protein interaction network based on Pyrococcus abyssi proteins. Through this network, new interactions involving proteins from DNA replication and proteins from DNA repair were highlighted. To describe interactions for a better understanding of their functions, was the aim of the work presented here for two protein interactions: PCNA/Mrell-Rad50 and MCM/XPD. For the first time in Pyrococcusfuriosus, we demonstrated both physical and functional interplay between PCNA, the replication maestro, and Mrell-Rad50, a complex involved in recombination process. For the second studied interaction, we used a biophysics approach based on fluorescent technics to characterise helicase activity of P.abyssi MCM. As several problems were encountered for XPD production, we did not characterise the helicase interaction. These two interactions are part of a more general context, where combined DNA replication and DNA repair processes could be important, especially for extremophile Archaea, Archaea which are amazing study models for understanding molecular processes ensuring genome integrity.

Using computational methods to identify protein-protein interactions (PPIs) supports experimental techniques by using less time and less resources. Identifying PPIs can be made through a template-based approach that describes how unstudied proteins interact by aligning a common structural template that exists in both interacting proteins. A pipeline that uses this is InterPred, that combines homology modelling and massive template comparison to construct coarse interaction models. These models are reviewed by a machine learning classifier that classifies models that shows traits of being true, which can be further refined with a docking technique. However, InterPred is dependent on using complex structural information, that might not be available from unstudied proteins, while it is suggested that PPIs are dependent of the shape and interface of proteins. A method that aligns structures based on the interface attributes is InterComp, which uses topological and sequence-order independent structural comparison. Implementing this method into InterPred will lead to restricting structural information to the interface of proteins, which could lead to discovery of undetected PPI models. The result showed that the modified pipeline was not comparable based on the receiver operating characteristic (ROC) performance. However, the modified pipeline could identify new potential PPIs that were undetected by InterPred.

A divisão celular bacteriana é orquestrada por FtsZ, uma proteína homóloga à tubulina eucariótica que possui a capacidade de polimerizar e gerar uma estrutura chamada de anel Z. O local onde esta estrutura citoesquelética contrátil é formada determina o futuro sítio de divisão. O complexo MinCD é um dos principais reguladores da posição da divisão, favorecendo a montagem do anel Z precisamente na região medial da bactéria. MinCD age como um inibidor sítio específico da polimerização de FtsZ, atuando preferencialmente nos polos celulares. MinC é a proteína do complexo que atua diretamente sobre FtsZ e inibe sua polimerização. Essa tese elucida a interação entre FtsZ e MinC e sugere o mecanismo exercido por MinC em Bacillus subtilis. Foi triada uma biblioteca de mutantes randômicos de FtsZ para identificação de mutantes resistentes à ação de MinC. Dentre estes, as substituições K243R e D287V, quando caracterizados usando espalhamento de luz e espectroscopia de fluorescência impediram a interação com MinC. Como as mutações estavam localizados em torno das hélices H-9 e H-10 no domínio C-terminal de FtsZ, concluímos que esta região representa o sítio de interação com MinC desta proteína. Como complemento ao mapeamento do sitio de ligação de MinC em FtsZ, identificamos a região de MinC que interage com FtsZ. Para tanto, escolhemos resíduos de MinC para mutagênese e caracterização. A escolha priorizou os resíduos conservados entre espécies Gram-positivas, experimentos de RMN, carga e exposição ao solvente dos mesmos. Dentre os resíduos de MinC mutados que afetaram sua capacidade de inibir a polimerização de FtsZ in vitro foram: Y8 e K12 (β-1), K15 (alça-2), H55 (β-3) , H84 (β-4) e K149 (C-terminal). Sendo assim, podemos concluir que a face de interação para FtsZ em MinC de B. subtilis é a única folha β do domínio N-terminal desta proteína. Com base nos sítios mapeados das duas proteínas experimentalmente, criamos um modelo in silico do complexo MinC-FtsZ por docking molecular. De acordo com o modelo gerado, MinC interage com a porção lateral de polímeros de FtsZ. Isto sugere que MinC atue na inibição da formação de feixes de filamentos de FtsZ, impedindo assim a formação de anéis Z funcionais. Esse mecanismo de ação do sistema Min é diferente do proposto para E. coli, no qual MinC interage com a face de polimerização FtsZ-FtsZ e impede a formação de protofilamentos de FtsZ. / Bacterial cell division is orchestrated by FtsZ, a protein homologous to eukaryotic tubulin that has the ability to polymerize and generate a cytoplasmic structure called the Z ring. The subcellular location where this cytoskeletal structure is formed determines the future division site. The MinCD complex is one of the main regulators of the position of cell division, driving the assembly of Z-ring precisely at the medial region of the cell. MinCD acts as a site-specific inhibitor of FtsZ polymerization, blocking Z ring formation at the cell poles. MinC is the protein of the complex that acts directly on FtsZ and inhibits its polymerization. This thesis elucidates the interaction between FtsZ and MinC and suggests the MinC mechanism in Bacillus subtilis. An ftsZ randomly mutagenized library was screened to identify mutants that are resistant to MinC action. Using right-angle light scattering and fluorescence spectroscopy we showed that substitutions K243R and D287V lost the interaction to MinC. These substituted residues clustered around the H-9 and H-10 helices in the C-terminal domain of FtsZ, thus, we conclude that this region is the binding site for MinC. In addition to mapping the MinC binding site on FtsZ, we also identified the FtsZ binding site in MinC. Based on residue conservation, NMR experiments and exposure to solvent, we chose residues of MinC for mutagenesis and characterization. The substituted residues that di srupted MinC ability to inhibit FtsZ polymerization in vitro were: Y8 and K12 (β-1), K15 (turn-2) , H55 (β-3), H84 (β-4) and K149 (C-terminal). Thus, we conclude that the binding site of MinC for FtsZ is located on the β only sheet at the N-terminal domain of MinC from B. subtilis. Finally, based on the binding sites of the two proteins mapped experimentally, we created a model of the complex between MinC and FtsZ by molecular docking. According to the generated model, MinC interacts with the lateral portion of FtsZ polymers. This indicates that MinC should inhibit assembly of higher order FtsZ polymers, thereby preventing the formation of a functional Z-ring. This mechanism of Min is different from that proposed in E. coli, in which MinC interacts with FtsZ polymerization interface and inhibits FtsZ protofilament formation.

Oxidative stress is an important factor in carcinogenesis of oncohematological diseases. However its role in the pathogenesis of myelodysplastic syndromes (MDS) remains unclear. In this study, we have determined the oxidative status and evaluated proteomic changes in plasma of MDS patients as a consequence of oxidative dysbalance (oxidative modifications, protein-protein interaction and complex forming). We measured the levels of total cysteine, homocysteine, cysteinyglycine, glutathione, nitrites and nitrates in the plasma from 61 MDS patients and 23 healthy donors using high performance liquid chromatography. Glutathione and nitrites levels reduced significantly while other aminothiols levels increased significantly in plasma of MDS patients. This association with oxidative stress did not correlate with iron overload. We also found enhanced levels of asymmetric dimethylarginine in serums of middle aged patients with MDS that correlate to posttranslational modifications of proteins arginyl residues. Furthermore, carbonylated proteins level was significantly elevated in MDS patients compared to healthy donors. Using mass spectrometry, 5 S-nitrosylated blood platelets proteins were identified in plasma and blood platelets of MDS patients and set of 16 plasma proteins with high probability of...

Les RCPG sont exprimés dans tous types de tissus et sont impliqués dans la régulation de nombreux processus biologiques et ont pour rôle de capter un vaste panel de stimuli extracellulaires qu’ils transmettent à l’intérieur de la cellule. Récemment, le laboratoire a identifié une nouvelle famille de dix protéines, les GASP, qui interagissent avec les RCPG et moduleraient leur trafic intracellulaire. Alors que GASP-1 est le membre de cette famille le mieux caractérisé et que son interaction avec de nombreux RCPG soit documentée, peu d’informations sont disponibles sur les modalités d’interaction de cette protéineavec les RCPG au niveau moléculaire. La première partie de ce projet de thèse a consisté à étudier les modalités d’interaction entre les GASPs et les RCPG au niveau moléculaire. Nous avons ainsi pu montrer à l’aide de techniques biochimiques et biophysiques, l’importance d’un motif répété et conservé de 15 acides aminés pour l’interaction de GASP-1 avec divers RCPG. Par la suite, les résultats obtenus ont été exploités pour mettre en place un essai de criblage qui nous a permis d’identifier des petites molécules capables de perturber l’interaction entre GASP-1 et le récepteur beta-2 adrénergique. Enfin, l’absence de données structurales sur les protéines de la famille GASP nous a ensuite poussé à la réalisation d’études structurales de ces protéines à la fois par cristallographie et par RMN. Bien que les résultats obtenus ne nous aient pas encore permis d’obtenir la structure de ces protéines, des expériences préliminaires de RMN ont permis deconfirmer l’implication des acides aminés tryptophanes présents au sein des motifs GASP dans l’interaction avec les RCPG. / GPCRs represent one of the most diversified protein families in humans. They translate extracellular stimuli into intracellular signals to modulate a large panel of physiological processes making them unrivalled targets for development of new therapeutic agents. Recently, we identified the GASP family of proteins that interact with GPCRs and modulate the postendocyticfate of agonist activated receptors. GASP-1 is the well-characterized protein of this family and has been shown to be involved in the sorting of receptors that are quickly degraded following agonistpromoted internalization. Although GASP-1 was found to interact with numerous GPCR both in vitro and in vivo and that helix 8 of GPCRs is critically involved in this interaction, little is known about which region within GASP-1 is required for its interaction with GPCRs. In this work, we first present a detailed analysis of the molecular interaction between GASPs and GPCRs. By using biochemical and biophysical experiments we shown that the central domain of GASP-1 is critical for the interaction with GPCRs and that a conserved and repeated sequence of 15 amino acids plays a critical role in this interaction. In a second step, we developed an HTC assay allowing us to identify small molecules able to disrupt the interaction between GASP-1 and the beta-2 adrenergic receptor. Finally, preliminary NMR experiments have confirmed the importance of amino acid tryptophan for the interaction with GPCRs and a first crystallization trials were performed with a fragment of GASP -1.