La O-GlcNAcylation est une modification post-traductionnelle réversible qui consiste en l'addition de N-acétylglucosamine (GlcNAc) sur des résidus de sérine et de thréonine de protéines cytoplasmiques, nucléaires et mitochondriales. Elle est contrôlée par deux enzymes : la O-GlcNAc transférase (OGT), qui ajoute le résidu de GlcNAc, et la O-GlcNAcase (OGA), qui l'hydrolyse. La O-GlcNAcylation a été décrite chez Toxoplasma gondii, parasite apicomplexe responsable de la toxoplasmose. D'autre part, les plantes expriment deux OGTs potentielles, SPINDLY (SPY) et SECRET AGENT (SEC), interférant avec divers processus biologiques. In fine, peu de choses avaient été décrites concernant la O-GlcNAcylation et l’OGT chez ces organismes: le but de ma thèse a ainsi consisté à approfondir nos connaissances en ce domaine en nous focalisant sur les parasites, T. gondii et P. falciparum, la plante supérieure A. thaliana et l'algue verte unicellulaire Chlamydomonas reinhardtii. En utilisant différentes approches biochimiques et protéomiques nous avons identifié tout un panel de protéines O-GlcNAcylées chez T. gondii et P. falciparum. En ce qui concerne les modèles végétaux, nous avons identifié une OGT potentielle chez C. reinhardtii mais avons été surpris de constater que les niveaux de O-GlcNAcylation chez cet organisme ainsi que chez la plante A. thaliana étaient très faibles. SPY a récemment été reclassée comme une O-Fucosyltransférase, perdant son statut d’OGT. Ces résultats remettent en question l’existence de la O-GlcNAcylation chez les organismes végétaux et questionnent sur l’apparition et la diversification des OGTs au cours de l’évolution des espèces. / O-GlcNAcylation is a dynamic post-translational modification which consists in the addition of N-acetylglucosamine (GlcNAc) onto serine and threonine residues of proteins confined within the cytoplasm, the nucleus and the mitochondrion. O-GlcNAcylation is managed by two enzymes: the O-GlcNAc transferase (OGT) that adds the sugar donor UDP-GlcNAc, and the O-GlcNAcase (OGA) that hydrolyzes it. Occurrence of O-GlcNAcylation was described in Toxoplasma gondii, an apicomplexan parasite causing toxoplasmosis. In the other hand, plants express two distinct OGT, SPINDLY (SPY) and SECRET AGENT (SEC), capable to interfere with a variety of fundamental processes. In fine, relatively few things have been described regarding O-GlcNAcylation and OGT in these organisms: therefore, the aim of my thesis was to go further into our knowledges in this field by focusing on the parasites T. gondii and P. falciparum, the higher plant model Arabidopsis thaliana and the unicellular algae Chlamydomonas reinhardtii. By using a panel of biochemical and proteomics approaches we identified a set of O-GlcNAcylated proteins in T. gondii and P. falciparum, outlining for the very first time the O-GlcNAcome of these parasites. Concerning the plant models, we identified a putative OGT in C. reinhardtii but we were surprised to note that O-GlcNAcylation levels in this organism as for the plant A. thaliana were very low. Recently, it was revealed that SPY is in fact an O-Fucosyltransferase, losing its OGT status. The results question again the occurrence of O-GlcNAcylation in plants and about the apparition and the diversification of OGTs along species evolution.

Currently there is no curative therapy for Chronic Myelogenous Leukemia (CML), and patients must remain on the current prescribed treatment, tyrosine kinase inhibitors (TKI), indefinitely. Although many patients can survive in the chronic phase of the disease under TKI treatment, some patients do progress to the terminal blast crisis phase of the disease. Patients in this terminal phase do not respond to TKI treatment. We evaluated the therapeutic benefit of targeting the oncogene c-MYC in CML, using the CML cell line K562. This was achieved by inhibiting the enzyme O-linked β-N-acetylglucosamine Transferase (OGT), using two indirect inhibitors 2-deoxyglucose and Azaserine, and the direct inhibitor ST078925. Treatment with these inhibitors resulted in decreased half-life of c-MYC protein in K562, reduced c-MYC protein in K562 cells, and reduced K562 cell growth. Together these results suggest that targeting c-MYC through OGT may be a potential therapeutic option for patients with CML.

La O-GlcNAc est une glycosylation dynamique, résidente du cytosol et du noyau, participant à la régulation de processus biologiques tels que le cycle cellulaire et l'embryogenèse. Nos travaux ont porté dans un premier temps sur le contrôle par la O-GlcNAc de la reprise méiotique de l'ovocyte deXenopus laevis, processus analogue à la transition G2/M du cycle cellulaire. Cette transition G2/M est caractérisée par l'activation simultanée du M-phase Promoting Factor, facteur universel d'entrée en phase M et de la voie MAPK-Erk2, et par une augmentation du niveau de O-GlcNAc. Nous avons démontré que cette augmentation de O-GlcNAc était primordiale pour la reprise méiotique ovocytaire puisque l'inhibition de l'OGT, l'enzyme transférant le résidu de GlcNAc, empêche la transition G2/M de l'ovocyte alors que sa surexpression accélère ce phénomène. Nous avons identifié 24 protéines dont le niveau de O-GlcNAc augmente au cours de la reprise méiotique dont des protéines du cytosquelette, la kinase erk2, la phosphatase PP2A, des enzymes de la glycolyse et des protéines ribosomales. Nous avons également entrepris l'étude des variations de O-GlcNAc, d'OGT et d'UDP-GlcNAc au cours de l'ovogenèse et de l'embryogenèse précoce chez Xenopus laevis et avons montré que la dynamique de la O-GlcNAc était complexe tout au long de ces deux processus. Notamment, nous avons observé une diminution drastique et transitoire de la O-GlcNAc au début de la gastrulation suggérant une implication de la glycosylation dans les phénomènes de migration cellulaire caractéristiques de cette étape du développement mettant en place les trois feuillets embryonnaires à l'origine de tous les tissus de l'adulte. / O-GlcNAc is a dynamic and reversible post-translational modification found within the cytosol and the nucleus that take part in the regulation of many cellular processes among which cell cycle and embryogenesis. First, our works have focused on the study of O-GlcNAc implication in the control of Xenopus laevis oocyte meiotic resumption, a process analogous to the G2/M transition of the cell cycle. This G2/M transition is characterized by the simultaneous activation of the M-phase Promoting Factor, the universal regulator of the M-Phase entry and of the MAPK-Erk2 pathway but also by a sudden increase in the oocyte O-GlcNAc content. We have demonstrated that this O-GlcNAc increase was essential for meiotic resumption since the inhibition of OGT, the enzyme transferring the O-GlcNAc, prevents the oocyte G2/M transition whereas OGT overexpression accelerates this process. We identified 24 proteins that O-GlcNAc modification increases during meiotic resumption among which cytoskeletal proteins, the kinase erk2, the phosphatase PP2A, several glycolysis enzymes and sorne ribosomal proteins. Second, we have undertaken the study of O-GlcNAc, OGT and UDP-GlcNAc variations during the oogenesis and the early development of Xenopus laevis and we showed that the O-GlcNAc dynamism is intricate from the Xenopus oogenesis to embryogenesis. ln particular, we observed a drastic and transitory O-GlcNAc decrease at the onset of gastrulation, suggesting a role for O-GlcNAc in the regulation of cell migration characteristic of this stage of development since it permits the generation of the three germ layers, precursors ofthe whole adult tissues.

The O-linked N-acetylglucosamine post-translational modification (O-GlcNAcylation) is the dynamic and reversible attachment of N-acetylglucosamine to serine and threonine residues of target proteins. It is abundant in metazoa, involving hundreds of proteins linked to a plethora of biological functions with implications in human diseases. The process is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), that add and remove the sugar moiety, respectively. Ogt gene knock-out is embryonic lethal in a range of animal models, hampering the study of the biological role of O-GlcNAc. O-GlcNAcylation of nuclear and cytoplasmic proteins has been extensively studied, however little is known about the role of O-GlcNAc in mitochondria. A previous report suggested the presence of a mitochondrial OGT isoform (mOGT) in human cell lines in addition to the well-characterised nucleocytoplasmic one (ncOGT). Since this report more than one decade ago, this putative mOGT has not been studied further. Similarly, hundreds of O-GlcNAcylated nucleocytoplasmic proteins have been identified by high-throughput proteomic screens, whereas only a few mitochondrial proteins have been detected. Nevertheless, several studies suggest that altered O-GlcNAc signalling affects mitochondrial function and morphology, with potential clinical implications. The aim of this thesis work was to study and characterise the biological role of mOGT and determine the mitochondrial O-GlcNAc proteome. Firstly, the presence of mOGT in human cell lines and mouse tissues was investigated. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed at protein level in most of the species analysed, except human and some primates. In fact, the putative mOGT cDNA in most of the genomes analysed contains a stop codon that excludes the presence of such isoform. In addition, mOGT was not detected at protein level in a wide range of human cell lines. Knock-down experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, overexpression of ncOGT in HEK 293 suspension cells led to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the mitochondrial O-GlcNAc proteome. These data point to a model where O-GlcNAc cycling of mitochondrial proteins occurs in the cytosol, followed by their import into mitochondria. Alternatively, ncOGT itself might be transported into mitochondria where it can take part to O-GlcNAc cycling inside the organelle. In parallel, some advance in determining the O-GlcNAc mitochondrial proteome has been undertaken. Different mitochondrial fractionation protocols, combined with O-GlcNAc enrichment methods have been explored in order to map novel glycosylation sites on mitochondrial proteins. A novel technique established in our research group, employing a bacterial OGA orthologue as a bait to trap O-GlcNAcylated proteins, has been applied to crude mitochondrial fractions allowing the identification of several hits, although site mapping has not been yet achieved. The second chapter describes the work that has been done to improve and optimise novel O-GlcNAc inhibitors previously designed in the laboratory, called goblins. The original objective was to make these molecules cell-permeable and possibly target them to mitochondria in order to inhibit mOGT. Several strategies were explored to deliver the compounds into living cells, including the use of transfection reagents and covalent linkage to linear cell-penetrant peptides. The above methods did not achieve cellular uptake, although recently designed cyclic cell-penetrant peptides, linked to fluorescein, were internalised by HeLa cells with immediate diffuse nucleocytoplasmic staining. These molecules will be linked to goblins aiming to use the inhibitors for cell biology studies. A different approach, based on the permeabilisation of Drosophila embryos, enabled the penetration of goblins into the organisms with consequent reduction of global O-GlcNAc levels. This method allowed the use of these novel bisubstrate inhibitors in vivo for the first time, with potential applications in studying the role of O-GlcNAc in Drosophila development and possibly for future therapeutic purposes after further development of the scaffold.

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Les modifications post-traductionnelles telles que la phosphorylation, l’OGlcNAcylation et l’ubiquitination jouent des rôles critiques dans la coordination des fonctions protéiques et par conséquent influencent grandement de nombreux processus cellulaires. Il est à noter que ces modifications sont hautement dynamiques et finement regulées. Par exemple, l’ubiquitination peut être réversible via l’action des déubiquitinases comme le suppresseur de tumeurs BAP1. Parmis les gènes codant pour les déubiquitinases, BAP1 est la plus souvent mutée dans le cancer. Des études récentes ont démontré l’importance des dynamiques de modifications post-traductionnelles dans la régulation du complexe BAP1. En plus, BAP1 forme un complexe multi-protéiques contenant plusieurs régulateurs transcriptionnels comme la protéine polycomb OGT et les facteurs de transcription FOXK1 et FOXK2. OGT est une enzyme unique qui catalyze l’ajout d’un groupement O-GlcNAc sur ses substrats afin d’en moduler l’activité enzymatique, les interactions protéines-protéines et leur localisation cellulaire. Cette modification est aussi liée au métabolisme puisque son substrat donneur, l’UDP-GlcNAc, est dérivé de la voie biosynthétique des hexosamines. Parallèlement, FOXK1/2 ont aussi été démontrés comme étant critiques à des processus métaboliques telles que la myogenèse et l’autophagie. Lors de nos études, nous avons identifié FOXK1 comme un nouveau substrat d’OGT. De plus, les niveaux d’O-GlcNAcylation de FOXK1 fluctuent lors de l’entrée/sortie du cycle cellulaire. En outre, nous avons identifié l’importance de FOXK1 dans l’adipogenèse et observé que l’interaction FOXK1/BAP1 est affectée par le métabolisme cellulaire. En résumé, nos études ont révélé l’importance d’OGT dans la régulation de certaines composantes du complexe BAP1, ce qui aidera à la compréhension de l’effet suppresseur de tumeur de BAP1 ainsi que son mécanisme d'action dans différents processus tel que le remodelage de la chromatine. / Post-translational modifications such as phosphorylation, O-GlcNAcylation and ubiquitination play critical roles in coordinating protein function and are therefore involved in diverse cellular processes. Of relevance here, ubiquitination may be removed by deubiquitinases such as the tumour suppressor BAP1, which represents the most mutated deubiquitinase gene in the human genome. Recent studies have revealed that important and dynamic post-translational modifications regulate several functions of the BAP1 complex. Indeed, BAP1 has been shown to form a multi-protein complex with several transcriptional regulators including the polycomb group protein OGT and the transcription factors FOXK1 and FOXK2. OGT is a unique enzyme that catalyzes the addition of an O-GlcNAc moiety to target proteins, which impacts protein function including enzymatic activity, protein-protein interactions and subcellular localization. This modification is also highly linked to cellular metabolism, as the donor substrate for the reaction, UDP-GlcNAc, is derived from the hexosamine biosynthesis pathway. Similarly, FOXK1 and FOXK2 have been shown to be implicated in metabolic processes such as myogenesis and autophagy. During our studies, we identified FOXK1 but not FOXK2 as a novel substrate of OGT. Further, we found that this OGlcNAcylation is modulated during the entry/exit of cell cycle. We also found that FOXK1 is critical for adipogenesis and that the interaction between FOXK1/BAP1 is compromised during nutrient starvation. Thus, our studies have revealed that OGT selectively modulates and regulates components of the BAP1 complex which may impact different cellular processes, notably chromatin remodelling and could help understanding how BAP1 acts as a tumor suppressor.

L’O-GlcNAcylation est une modification post-traductionnelle qui consiste en l’ajout covalent du N-acetylglucosamine au groupement hydroxyle des sérines et thréonines des protéines nucléaires et cytoplasmiques. Ce type de glycosylation atypique est régulé de manière très dynamique par l’action de l’O-GlcNAc transférase (OGT) et de l’O-GlcNAcase (OGA) qui catalysent et hydrolysent cette modification respectivement. Aujourd’hui, OGT émerge comme un régulateur transcriptionnel et senseur critique du métabolisme où les protéines ciblées par l’O-GlcNAcylation couvrent la presque totalité des voies de signalisation cellulaire. Récemment, des études ont aussi proposé qu’OGT soit impliquée dans la régulation épigénétique par l’O-GlcNAcylation des histones. Dans le but de caractériser le rôle fonctionnel d’OGT dans la régulation épigénétique, nous avons revisité le concept d’O-GlcNAcylation des histones et, de manière surprenante, n’avons pu confirmer cette observation. En fait, nos données indiquent que les outils disponibles pour détecter l’O-GlcNAcylation des histones génèrent des artéfacts. De ce fait, nos travaux supportent plutôt un modèle où la régulation épigénétique médiée par OGT se fait par l’O-GlcNAcylation de régulateurs transcriptionnels recrutés à la chromatine. Parmi ceux-ci, OGT s’associe au complexe suppresseur de tumeurs BAP1. En étudiant le rôle d’OGT dans ce complexe, nous avons identifié le facteur de transcription FOXK1 comme un nouveau substrat d’OGT et démontrons qu’il est régulé par O-GlcNAcylation durant la prolifération cellulaire. Enfin, nous démontrons que FOXK1 est aussi requis pour l’adipogenèse. Ensemble, nos travaux suggèrent un rôle important d’OGT dans la régulation du complexe BAP1. / O-GlcNAcylation is a post-translational modification which consists in the covalent addition of an N-acetylglucosamine sugar to the hydroxyl group of serine and threonine residues of nuclear and cytoplasmic substrates. This atypical glycosylation is regulated in a very dynamic manner through the action of the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA) that catalyze and hydrolyze this modification respectively. OGT has emerged as a critical transcriptional regulator and sensor of metabolism whereby proteins targeted by O-GlcNAcylation cover several cell signaling pathways. Recently, studies have also suggested that OGT may be involved in epigenetic regulation through the O-GlcNAcylation of histones. For the purpose of characterizing the functional role of OGT in epigenetic regulation, our group revisited the concept of histone O-GlcNAcylation and surprisingly, our work could not confirm this observation. In fact, our data indicate that the available tools for histone O-GlcNAcylation detection generate artifacts. Consequently, our work rather supports a model whereby OGT-mediated epigenetic regulation is indirectly achieved through O-GlcNAcylation of chromatin-associated transcriptional regulators. Among these, OGT strongly associates with the BAP1 tumor suppressor complex. Thus, by focusing on the role of OGT in this complex, we identified the transcription factor FOXK1 as a novel substrate of OGT and demonstrate that it is regulated throught O-GlcNAcylation during cell proliferation. Finally, we demonstrate that FOXK1 is also required for adipogenesis. Taken together, these data suggest an important role of OGT in regulating the BAP1 complex.

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