Members of the 2-oxoglutarate (2OG)- and Fe(II)-dependent oxygenase (2OG oxygenase) superfamily catalyse a wide range of oxidative reactions in biology. 2OG oxygenases require Fe(II) and atmospheric oxygen for their activity, and couple substrate oxidation with the decarboxylation of 2OG into succinate and carbon dioxide. There are more than sixty known 2OG oxygenases in the human genome; they modify small molecules, nucleic acids and proteins implicated in diverse biological processes. Importantly, the seemingly disparate functions of 2OG oxygenases often converge to regulate gene expression. 2OG oxygenases have been shown to affect epigenetic reprogramming, chromatin remodelling, transcription factor activity and mRNA splicing. Emerging evidence indicates that 2OG oxygenases are also involved in the translational control of gene expression. Oxygenases TYW5, ALKBH8, ALKBH5 and FTO were found to catalyse modifications of tRNA and mRNA. The work in this thesis extends these observations by demonstrating that 2OG oxygenase-catalysed protein hydroxylations also play an important role in protein synthesis. The catalytic activities of two oxygenases belonging to the JmjC-only family, NO66 and JMJD4, are described. NO66 catalyses the histidinyl hydroxylation of 60S ribosomal subunit protein L8. NO66 is part of a conserved group of ribosomal protein hydroxylases that can be traced back to prokaryotes. JMJD4 is a lysyl hydroxylase of eRF1, the eukaryotic release factor responsible for translation termination. The hydroxylation of eRF1 takes place on a conserved NIKS motif important for release factor activity, and promotes effcient translational termination. JMJD4 is further implicated in cell growth and cancer, though the link between its activity and tumourigenesis remains to be determined. These results highlight the potential of 2OG oxygenases as regulators of protein synthesis, and further extend the scope of 2OG oxygenase function. The small molecule inhibition of 2OG oxygenases presents a novel therapeutic possibility targeting translational control in cancer and other diseases.

Master of Science / Biochemistry / John Tomich / One of the important keys to characterize the biological function of a protein is the study of post-translational modification (PTM). Formation of disulfide bond linkages between cysteine residues within a protein is a common PTM which not only contributes to folding and stabilizing the protein structure, but also to accomplishing its native function. Therefore, the study and discovery of structural-functional relationships of expressed proteins using an isolated proteomics approach has been one of the biggest advances within the field of structural biology in recent years. In this study, rapid disulfide bond mapping of freshly obtained equine serum albumin (ESA) was performed using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Highly sensitive MALDI-TOF MS is commonly used for the investigation of disulfide bond linkages in the proteomics field. However, it has also been known that the presence of disulfide bond linkages absorbs the energy which is created by the cysteine-cysteine kinetic vibration, resulting in a decrease of the instrumental sensitivity. To overcome this problem, the disulfide bond mapping method was optimized by applying a combination of chemical labeling, proteolytic enzymes, and matrices. With the optimized method, we were also able to achieve high protein sequence coverage. Obtaining higher sequence coverage of a protein provides more information about a protein which helps to identify the protein by peptide mass fingerprint (PMF) technique. These analyses eventually contribute to the estimation of the possible PTM sites.

L'autophagie est un processus d'autodigestion qui se produit dans toutes les cellules eucaryotes et conduit à la dégradation d'éléments du cytoplasme (organites, macromolécules) par le lysosome. Ce mécanisme, qui se produit de manière basale, permet le renouvellement du contenu cytoplasmique mais également la survie cellulaire lorsqu'il est induit par différents stress (carence nutritionnelle, hypoxie…). L'autophagie est alors impliquée dans diverses pathologies comme les maladies neurodégénératives et le cancer car sa dérégulation peut grandement perturber l'homéostasie cellulaire. Le but de ma thèse est de déterminer le rôle de la protéine nucléaire et cytoplasmique BAT3 dans l'autophagie et d'étudier son mécanisme de régulation. Cette protéine de 150 kDa, également appelée BAG6 ou Scythe, est composée de nombreux domaines protéiques (UBL, Prolin-rich, NLS, BAG) qui lui permettent d'interagir avec de multiples partenaires. Sa fonction majeure réside dans le contrôle qualité du cytoplasme mais BAT3 est aussi impliquée dans l'immunité ou l'apoptose. Ce travail identifie la protéine BAT3 comme essentiel pour l'autophagie basale et induite. Nous montrons que son mécanisme d'action passe par la régulation de la localisation de l'acétyltransférase p300 et l'acétylation de ces substrats : p53 et une protéine de la machinerie de l'autophagie : ATG7. En effet, BAT3 (i) limite la présence de p300 dans le cytosol en (ii) maintenant un faible et régulable niveau d'acétylation d'ATG7 et (iii) permet l'acétylation de p53 dans le noyau au cours de la carence nutritionnelle, événement indispensable à l'induction de l'autophagie. / Autophagy, literally meaning self-eating, is a highly evolutionary conserved process in eukaryotes in which parts of the cytoplasm (organelles, macromolecules) are degraded by lysosomes. Basal autophagy is a quality control mechanism allowing the renewal of the cytoplasm but autophagy is also induced by cellular stress (starvation, hypoxia…) to improve cell survival. Autophagy has been implicated in several physiopathologies such as cancer or neurodegenerative diseases. Deregulations of autophagy may profoundly affect homeostasis.The purpose of my thesis is to explore the role of the nucleo-cytoplasmic shuttling protein BAT3 in autophagy and the mechanism of BAT3-dependent autophagy.Also known as BAG6 or Scythe, this 150 kDa protein is composed of various domain (UBL, Prolin-Rich, NLS, BAG) by which BAT3 interacts with multiple partners. The major of role BAT3 seems to be the protein quality control but BAT3 is also implicated in immunity and apoptosis. Our work demonstrates that the protein BAT3 is essential for basal and starvation-induced autophagy. We show that BAT3 regulation of autophagy is mediated by the modulation of p300 acetyltransferase intracellular localization and acetylation of two subtrates: p53 and the autophagy-related protein ATG7. Indeed, Bat3 allows: (i) the limitation of p300 into cytosol resulting in (ii) the maintenance of a low level of ATG7 acetylation and (iii) the increase of the starvation-induced p53 autophagy leading to the induction of autophagy.

La polarité cellulaire est un processus fondamental qui contrôle les spécificités fonctionnelle et physiologique de la plupart des cellules eucaryotes. Cette asymétrie intracellulaire repose sur l’existence de compartiments membranaires distincts, à la fois dans leur composition en protéines mais également en phosphatidyl-inositols (PIs). Ainsi, la mise en place et le maintien de la localisation asymétrique de modules multi-protéiques associés notamment aux protéines PAR sont essentiels pour l’élaboration des domaines de polarité cellulaire. Durant ma thèse, j’ai étudié les relations entre les protéines de polarité et les PIs dans le contrôle de la polarité cellulaire. Plus particulièrement, en utilisant la chambre ovarienne de Drosophile, j’ai cherché à caractériser la suite d’évènements qui en amont régule l’activité de la PIP5K, Skittles (SKTL), qui produit le PI(4,5)P2 et à caractériser les mécanismes moléculaires qui lient le PI(4,5)P2, SKTL et les protéines PAR dans le contrôle et le maintien de la polarité cellulaire. J’ai contribué à caractériser l’importance de PI(4,5)P2 majoritairement produit par SKTL, dans le maintien de la polarité apico-basale et lors de la morphogenèse des cellules folliculaires de la chambre ovarienne. Le PI(4,5)P2 assure la localisation apicale de PAR3 et le maintien des jonctions adhérentes, sans affecter la localisation de PAR1. Par une méthode de quantification précise, j’ai ensuite démontré dans l’ovocyte que SKTL et le PI(4,5)P2, probablement grâce au trafic vésiculaire, étaient requis pour à la fois l’accumulation à l’antérieur de PAR3 et son exclusion au postérieur qui se fait à partir du stade 9B. L’accumulation antérieure de PAR3 est également dépendante d’un transport Dynéine dépendant et de la kinase IKKε tandis que son exclusion postérieure dépendant des phosphorylations par PAR1. Enfin, j’ai également étudié les modifications post traductionnelles de SKTL et leur importance dans la polarité cellulaire. J’ai identifié la présence de palmitoylation et de phosphorylations dont certaines impliquent la kinase PAR1 et la phosphatase PP1. Ces phosphorylations pourraient avoir un lien avec le rôle de SKTL dans le trafic vésiculaire. Ces résultats permettent donc d’élucider certains mécanismes cellulaires qui contrôlent la mise en place et le maintien de la polarité des cellules en liant les PIs et les protéines PAR / Cell polarity is a fundamental process that controls cell’s functional and physiological specificities. This process relies on membranous compartments differently composed both on proteins and on phosphatidyl-inositols (PIs). Indeed, through their asymmetric localization, polarity proteins, such as the PAR proteins, are essentials to establish and maintain polarity of the cells. During my PhD, I studied the interplay between the polarity proteins and the PIs. Using the Drosophila egg chamber, as a model, I aimed to characterized the upstream events that regulate the PI(4,5)P2 producing kinase (PIP5K), Skittles (SKTL), activity and localization. I also studied the downstream molecular process that link the PI(4,5)P2, SKTL and the PAR proteins in cell polarity. I contributed to the characterization of the importance of PI(4,5)P2, mainly produced by SKTL in maintaining the apical-basal polarity and during the morphogenesis of the follicle cells. The PI(4,5)P2 is ensuring PAR3 and adherens junctions but not PAR1 proper localizations. Next, through a precise quantification method, I showed that SKTL and the PI(4,5)P2, probably via vesicular traffic, were also ensuring PAR3 proper localizations (anterior accumulation and stage 9B posterior exclusion) in the oocyte. PAR3 accumulation also relies on a Dynein mediated transport and the IKKε kinase while its posterior exclusion relies on PAR1 phosphorylation. Finally, I studied SKTL post translational modifications and their relevance on cell polarity. I identified palmitoylation and phosphorylations that are regulated by the kinase PAR1 and the phosphatase PP1. SKTL phosphorylations seem to be related to its role on the vesicular traffic. Altogether these results clarify some mechanisms involving both PIs and PAR proteins in cell polarity maintaining and establishment

The important role of the central intermediary metabolite acetyl-coenzyme A (AcCoA)for several anabolic and catabolic pathways is well characterised. However, the role of AcCoA as the only known donor of acetyl groups for protein acetylation in regulation of enzyme activities, protein complex stability as well as epigenetic status off chromatin, is only recently emerging. Among multiple other pathways, the autophagy pathway has now been shown to be directly regulated by protein acetylation and deacetylation. Therefore, it was reasoned that the availability of AcCoA, via the modulation of AcCoA generating enzymes, may regulate autophagy. This study has focussed on the role of the acetate-mediated route to nuclear-cytosolic AcCoA synthesis, catalysed by AcCoA synthetase 2 (ACSS2), in the regulation of autophagy.

The dynamic S-palmitoylation regulates many intracellular events, including protein trafficking, anchoring, targeting, and protein-protein interactions. Direct detection of S-palmitoylation by conventional liquid chromatography-mass spectrometry (LC-MS) methods is challenging because of the tendency of palmitoyl loss during sample preparation and gas phase fragmentation. Additionally, the high hydrophobicity of the palmitoyl group can prevent proper elution of palmitoyl peptides from the commonly used C18 column. Here, we developed a comprehensive strategy tailored for S-palmitoyl detection using three palmitoyl peptide standards. We found that S-palmitoylation was largely preserved in neutral Tris buffer with tris(2-carboxyethyl)phosphine as the reducing agent and that various fragmentation methods provided complementary information for palmitoyl localization. Moreover, S-palmitoyl peptides were efficiently analyzed using a C4 column and the derivatization of free cysteine with a hydrophobic tag allowed relative quantification of palmitoyl peptides and their unmodified counterparts. We further discovered potential complications to S-palmitoylation analysis caused by the use of ProteaseMAXTM, an MS-compatible detergent. The hydrophobic degradation products of ProteaseMAXTM reacted with the free cysteine thiols, generating artifacts that mimic S-acylation and hydroxyfarnesylation. Another MS-compatible detergent, RapiGestTM, did not produce such artifacts, and showed the ability to stabilize S-palmitoylation by preventing thioester hydrolysis and dithiothreitol-induced thioester cleavage. Moreover, we found that the palmitoyl peptide GCpalmLGNAK could undergo intermolecular palmitoyl migration from the cysteine to the peptide N-terminus or the lysine side chain during sample preparation, and this could lead to false discovery of N-palmitoylation. RapiGestTM inhibited such migration, and is thus recommended for S-palmitoyl sample preparation. We then applied the established method to analyze the regulator of G-protein signaling 4 (RGS4) which had been reported to undergo S-palmitoylation by radioactive labeling. It had also been reported that the S-palmitoylation state of RGS4 affects its GTPase activity. With LC-MS/MS analysis, we found that the addition of palmitate to the cell culture medium in metabolic labeling experiments could boost the level of S-palmitoylation, leading to false discovery of new S-palmitoylation site(s). We also noted discrepancies between the S-palmitoylation sites identified by radioactive labeling and by LC-MS/MS analysis. Further studies are needed to evaluate the reliability of S-palmitoyl detection by these two methods.

Les modifications post-traductionnelles (MPTs) d’histones sont apparues comme un acteur majeur de la régulation de l’expression des gènes. Cependant peu de choses sont connues sur le réel impact des MPTs sur la chromatine. Il a été suggéré que les MPTs d’histones (H2A, H2B, H3 et H4) ont le potentiel de moduler la fonction chromatinienne selon un « codehistone » en recrutant des protéines spécifiques de liaison. L’objectif de mon projet est d’approfondir la fonction de l’acétylation du domaine globulaire de l’histone H3 et de comparer cette modification avec celles des queues N-terminale in vivo sur une lignée ES cellulaire. Pour étudier l’impact de ces MPTs in vivo, toutes les copies endogènes du gène H3 sauvage (WT) doivent être remplacées par des copies mutées. Ainsi la première étape de mon projet est d’établir une lignée cellulaire exprimant seulement H3 mutée (e.g reproduisant une acétylation permanente) afin d’étudier les effets des modifications sur le domaine globulaire de H3 sur (a) l’expression génique, (b) l’architecture chromatinienne mais également pour étudier (c) les effets réciproques et synergiques entre les différentes modifications du domaine globulaire et (d) comparer ces effets avec les modifications sur la queue N-terminale dans un système in vivo. / Post-translational modifications (PTMs) of histones have emerged as key players in the regulation of gene expression. However, little is known to what extent PTMs can directly impact chromatin. It has been suggested that PTMs of core histones (H2A, H2B, H3 and H4) have the potential to govern chromatin function according to the so called ‘‘histone code’’ hypothesis by recruiting specific binding proteins. The goal of my project is to gain insight in the function acetylation within the globular domain of H3 and to compare these modifications with histone tail modifications, in vivo by using the CRISPR in mouse embryonic stem cells (ES). To study the impact of PTMs in vivo, all endogenous wild type (WT) H3 gene copies have to be replaced with mutant copies. Hence, the primary focus of my project is to establish cell lines that exclusively express mutated H3 (e.g. mimicking acetylation) in order to study effects of H3 globular domain modifications on (a) gene expression (b) chromatin architecture as well as to study (c) cross talks and synergisms between globular domain modifications and (d) compare the effects with tail modifications in an vivo system.

The tumour suppressor p53 is often disrupted by missense mutations that can result in p53 protein accumulation and acquisition of novel oncogenic activities. Various studies have demonstrated that DNA-damaging drugs currently used in the clinic aimed at activating wild type p53, can also stabilise and activate mutant p53 oncogenic functions and thereby paradoxically enhance tumour progression, resulting in poor response to the treatment. In this study we aimed to investigate whether, like in wt p53, post-translational modifications (PTMs) drive such drug-induced mutant p53 accumulation and activation. For this purpose, we generated plasmids expressing non-phosphorylatable and phospho-mimic versions of R175H mutant p53 and tested them in different cell line models. We demonstrated that in response to DNA damage mutant p53 is accumulated and phosphorylated and these phenomena appeared to be mediated by ATM and ATR kinases. DNA-damage induced acetylation was also observed and occurred in a S15 phosphorylation-dependent manner. This suggested a role of the HAT p300, which is recruited by phosphorylated S15. Of note, other works have shown that p300 is required to trigger some oncogenic functions of mutant p53. We then aimed at developing systems to explore mutant p53 functions and their dependence on PTMs. Although we showed that cell growth is compromised upon endogenous mutant p53 depletion, exogenous expression of mutant p53 or its phosphorylation-site forms did not result in a successful rescue in our experimental conditions, thus we were unable to use this strategy to test the effect of PTMs. Ectopic expression of R175H mutant p53 or its phosphorylayion-site versions did not interfere with the growth rate and response to chemotherapy of the p53-null cell line H1299. We also found that mutant p53 phosphorylation does not affect subcellular localisation of mutant p53 and mutant p53-mediated inhibition of p63. Interestingly, ectopically expressed mutant p53 enhanced cell migration in H1299 cells. Notably, our results suggested an apparent threshold effect of mutant p53 levels required to induce migration. Due to the difficulty of obtaining cell lines expressing similar levels of the different phosphorylation-site mutants, the determination of the role of phosphorylation in mutant p53-induced migration was not conclusive. Remarkably, we found that, while S15 and S20 phosphorylation decreased MDM2-dependent degradation, only phosphorylated S20 interfered with CHIP-induced turnover in H1299 cells. Overall our data suggest that, despite exhibiting opposite biological effects, mutant and wt p53 can share upstream regulatory mechanisms and thus present phosphorylation as a promising target to prevent mutant p53 stabilisation and activation and improve response to therapy. Our results also highlight the challenge of developing a good system for determining the effects of the mutant p53 protein and its regulation by PTMs.

Les cils sont des organites très conservés au cours de l’évolution des eucaryotes et présents à la surface de presque tous les types cellulaires. Ils sont constitués d’une structure microtubulaire, l’axonème, entourée d’une membrane en continuité avec la membrane plasmique. Ils sont nucléés par un corps basal, centriole ancré à la surface cellulaire. Grâce aux nombreux récepteurs qu’ils concentrent à leur membrane, tous les cils sont des senseurs de leur environnement. Ils peuvent aussi être motiles et assurer, par leur battement coordonné, le déplacement relatif de la cellule et du fluide environnant. Tandis que cil et structure centriolaire, hérités du premier eucaryote, ont été perdus par certains champignons et par les plantes supérieures, certains gènes codant des protéines ciliaires et centriolaires sont pourtant retrouvés dans le génome de ces espèces. Cette conservation de protéines sans l’organite suggère soit que ces protéines interviennent dans un même processus moléculaire utilisé dans plusieurs organites, soit qu’elles jouent des rôles dans des processus moléculaires distincts via leur interaction avec différents types de partenaires.J’ai choisi d’étudier l’une de ces protéines ciliaires et centriolaires, Bug22p, hautement conservée en séquence protéique entre l'homme et la paramécie, mais également présente chez les plantes supérieures. J’ai mené cette étude principalement sur la paramécie, système modèle pour la biogénèse des corps basaux et des cils, mais aussi sur des cellules de mammifère et de végétaux supérieurs. Si Bug22p est impliquée dans la détermination du battement ciliaire chez la paramécie, elle se localise également dans des cils immotiles de cellules de mammifère suggérant que son activité ciliaire n’est pas réduite à cette seule fonction. Des expériences d’inactivation génique suggèrent par ailleurs un lien entre l’activité de Bug22p et la polyglycylation. Sa surexpression dans les cellules de mammifère en culture entraîne l’apparition d’extensions cellulaires et une augmentation des réseaux de tubulines acétylées probablement associées à une stabilisation des microtubules. L'ensemble de mes résultats suggère donc un rôle de Bug22p dans la régulation de modifications post-traductionnelles. En plus d’être présente dans les structures ciliaires, Bug22p se localise aussi bien dans les noyaux de la paramécie que dans ceux des cellules humaines et des plantes supérieures Arabidopsis et Nicotiana. Ces observations ouvrent un nouveau champ d’études. En effet, si l’on sait que les tubulines ciliaires sont soumises à différentes modifications post-traductionnelles telles que polyglycylation ou acétylation, ce type de modifications touchent également des protéines nucléaires régulant ainsi le trafic de protéines nucléaires ou l’expression génique. Nous pouvons donc avancer l’hypothèse selon laquelle Bug22p agirait sur la régulation de ces modifications dans le cil et dans le noyau. Il serait donc intéressant de caractériser les modifications post-traductionnelles chez les plantes supérieures afin de vérifier une possible implication de Bug22p dans leur régulation et donc comprendre les raisons de sa conservation chez les végétaux. / Cilia, organelles that have been conserved throughout the evolution of eukaryotes, are found at the surface of most cell types. They are composed of a microtubular structure, the axoneme, surrounded by a membrane continuous to the plasma membrane. Cilia are nucleated by basal body, which is a centriole anchored to the cell surface. Cilia are environmental sensors concentrated in the ciliary membrane. Cilia can be motile and ensure the relative movement of the cell with respect to the surrounding fluid by their coordinated beating. While cilia and centriolar structures inherited from the first eukaryote have been lost by certain fungi and higher plants, certain genes encoding ciliary and centriolar proteins are found in the genomes of organisms lacking these structures. The conservation of these proteins without organelle suggests that these proteins are involved in the same molecular process into different organelles or proteins are involved into different processes through some interactions with different partners.I chose to study the ciliary and centriolar protein, Bug22p, highly conserved between human and Paramecium proteins sequences, and also present in higher plants. My work addressed this study primarily on Paramecium, a model system for biogenesis of basal bodies and cilia, and I also pursued investigation of mammalian cells and higher plants. I was able to show that Bug22p is necessary for efficient Paramecium ciliary beating, but I also localized in the immotile cilia of mammalian cells suggesting that Bug22p is not only restricted to the motile ciliary function. By knockdown experiments in Paramecium, I obtained evidence that Bug22p is involved in polyglycylation. Bug22p overexpression in mammalian cells led to the appearance of cell extensions and increased acetylated tubulin networks consistent with microtubule stabilization. My results suggest that Bug22p may regulate post-translational proteins modifications.Bug22p is also localized in the nuclei of Paramecium, human and higher plants such as Arabidopsis and Nicotiana. These observations open a new field of study. The axoneme microtubules are highly modified by post-translational modifications such as acetylation and polyglycylation; we know that in the nucleus, theses modifications are involved in the control of nuclear trafficking of some proteins and the regulation of gene expression. We can therefore speculate that Bug22p acts on the regulation of these changes in the cilium and in the nucleus. Finally, it would be interesting to characterize the post-translational modifications in higher plants to verify the possible involvement of Bug22p in their regulation and thus understand the meaning of its conservation in higher plants lacking cilia.

Many mutations in superoxide dismutase 1 (SOD1) cause destabilization and misfolding of the protein and are implicated in amyotrophic lateral sclerosis. Likewise, a few post-translational modifications (PTMs) on SOD1 have been shown to cause the same phenotype. However, relatively few PTMs on SOD1 have been studied in depth and, in particular, very few studies have demonstrated how these PTMs affect SOD1's various biological roles. SOD1 is traditionally known for its role in reactive oxygen species (ROS)-scavenging but has also been found to have a few other biological roles, including transcription factor activity to promote genomic stability, preservation of cytoskeletal activity, maintaining zinc and copper homeostasis, and suppressing respiration. We have used the computational analysis tool, SAPH-ire, to find PTM 'hotspots' on SOD1 that have a high likelihood of affecting its biological functions. Interestingly, the top seven ranked PTM 'hotspots' were found in a small region of SOD1, between S98-K128. We focused our studies on one of the PTM 'hotspots' found in this region, lysine-122 (K122). K122 is found in the electrostatic loop of SOD1, a loop that is important for shuttling in superoxide radicals to be neutralized. According to our data, and other studies, this lysine is both succinylated and acetylated. We found that acetyl and succinyl-mimetics (K122Q and K122E, respectively) of this site do not affect its ROS scavenging activity but do prevent SOD1 from suppressing respiration and decrease its localization to the mitochondria. Further, when cells are depleted of SIRT5 (the desuccinylase for K122), SOD1 can no longer suppress respiration. Additionally, we found that SOD1 appears to suppress respiration at complex I, whether directly or through an indirect pathway is unknown. When HCT116 colon cancer cells were depleted of endogenous SOD1, the overexpressed succinyl K122-mimetic (K122E) could not recover growth as well as overexpressed WT SOD1. The K122E SOD1 expressing cells also exhibited increased mitochondrial ROS and unhealthier mitochondria. We propose a mechanism whereby SOD1 suppression of respiration acts as an additional regulator of oxidative stress: SOD1 suppresses the electron transport chain to decrease reactive oxygen species leakage and to promote healthier mitochondria and growth.