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.

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.

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.

Though mass spectrometry has earned a central role in the field of proteomics due to its versatility in a wide range of experiments, challenges and complications are still encountered when using mass spectrometry to characterize protein structures, post-translational modifications (PTMs), and abundances. In this dissertation, analytical methods utilizing mass spectrometry have been developed to address challenges associated with both qualitative and quantitative protein characterization. The effectiveness of using multiple pepsin-like proteases, both separately and in mixtures, combined with online proteolysis using a special triaxial probe has been demonstrated on an amyloid beta peptide related to the onset of Alzheimer’s disease. These findings have broad implications in protein structural characterization studies using hydrogen-deuterium exchange mass spectrometry. A wider range of proteases (Lys-C, Glu-C, and trypsin) and multiple fragmentation methods (collisionally activated dissociation, electron transfer dissociation, and decision tree) have been utilized in the discovery-based PTM characterization of extracellular cellulosome proteins of the bioenergy-relevent organism Clostridium thermocellum, resulting in the identification of 85 previously unknown modification sites in 28 cellulosome proteins. These modifications may contribute to the structure and/or function of the cellulosome protein complex. By using peptide internal standards and a triple quadrupole mass spectrometer operating in selected reaction monitoring mode, a method has been developed for the absolute quantitation of the Clostridium thermocellum cellulosome protein machine in samples ranging in complexity from purified cellulosome samples to whole cell lysates as an alternative to a previously-developed enzyme-linked immunosorbent assay method of cellulosome quantitation. The precision of the cellulosome mass concentration in technical replicates is better than 5% relative standard deviation for all samples, indicating high precision of cellulosome mass concentration for this method. Though methods and results presented in this dissertation have implications in the study of Alzheimer’s disease and bioenergy research, more broadly this dissertation focuses on development of methods to contend with some of the more complex challenges associated with protein characterization currently presented to the field of proteomics.

H2A.Z is an essential histone variant that has multiple chromosomal functions. One such role is transcriptional regulation. However, its role in this process is complex since it has been reported to function both as a repressor and activator. Earlier work in our lab showed that H2A.Z can be post-translationally modified with monoubiquitin (H2A.Zub1) and this form of H2A.Z is linked to transcriptional silencing. We further predicted that changes in the H2A.Z ubiquitylation status directly modulated its function in transcription. Furthermore, H2A.Z-containing nucleosomes possess a unique set of post-translational modifications (PTMs), compared to H2A nucleosomes, many of which are linked to transcriptional activation. The central aim of this thesis was to characterize the role of PTMs on H2A.Z nucleosomes in transcriptional regulation. To this end, I have provided the first evidence linking H2A.Z deubiquitylation to transcriptional activation. I demonstrated that ubiquitin specific protease 10 (USP10) is a deubiquitylase that targets H2A.Z in vitro and in vivo. Moreover, I found that both H2A.Z and USP10 are required for activation of androgen-receptor (AR)-regulated genes, and that USP10 regulates the levels of H2A.Zub1 at these genes. To understand how H2A.Z engages downstream effector proteins, in the nucleosome context, we used a mass spectrometry approach to identify H2A.Z-nucleosome-interacting proteins. Many of the identified proteins contained conserved structural motifs that bind post-translationally modified histones. For example, we found that Brd2 contains tandem bromodomains that engage H2A.Z nucleosomes through acetylated H4 residues. To investigate the biological relevance of this interaction, I present evidence that Brd2 is recruited to AR-regulated genes in a manner dependent on H2A.Z and the bromodomains of Brd2. Consistent with this observation, chemical inhibition of Brd2 recruitment greatly inhibited AR-regulated gene expression. Collectively, these studies have defined how H2A.Z mediates transcriptional regulation through multiple mechanisms and pathways.

An array of analytical methods including density gradient ultracentrifugation, capillary electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), were utilized to analyze serum high density lipoprotein (HDL) subfractions from two cohorts of normolipidemic individuals, which included subjects with diagnosed coronary artery disease (CAD), and angiographically proven non-CAD controls. These methods collectively provided characteristic information about the two populations of individuals including composition, electrophoretic mobilities, molecular weights, isoforms, and post-translational modifications of HDL apolipoproteins. This information proved useful in identifying potential biomarkers for CAD risk, and understanding the biological functions of a novel atherogenic HDL phenotype in individuals with CAD. Through the implementation of the aforementioned methodologies, new isoforms of apoC-I were identified. MALDI-MS, detected a shifting of approximately 90 Da in the mass to charge ratios corresponding to apoC-I peaks in the serum subfractions from all CAD cohort patients. This shifting was not observed in the non-CAD cohort, which displayed apoC-I peaks in accordance with the known mass of this protein. In addition to the shifting observed in the CAD cohort, some CAD patients showed further modifications of apoC-I that were indicative of oxidative processes. Interestingly, one patient, who has not been diagnosed with CAD, and has a family history of the disease, contained the apoC-I isoforms. This feature could underlie this subject’s known family history of CAD, and serve as an initial screening that could indicate the future development of CAD in this individual. Through collaborative work with Johns Hopkins University, it was initially observed that apoC-I enriched HDL induced apoptosis of aortic smooth muscle cells. Conversely, apoC-I depleted HDL induced minimal to no apoptosis, which led to the hypothesis that apoC-I is a contributor to atherogenic HDL and is a potential risk factor for CAD. Further collaborative work with Johns Hopkins assessed the apoptosis levels induced by HDL from both cohorts of patients. A distinct difference in apoptosis was identified between the two cohorts. High density lipoprotein subfractions from subjects in the CAD cohort, all of which contained the apoC-I isoforms, induced marked apoptosis compared to the non-CAD controls. These results further supported the hypothesis that apoC-I compromises the functionality of HDL and showed that through the induction of apoptosis, apoC-I can contribute to the destabilization of atherosclerotic plaque and the acceleration of CAD.

There are many tools available for the study of post-translational modifications. The majority of these tools is specific towards the individual modification and involves separation of modified proteins from non-modified ones. The drawback of using a modification specific method is that there is a lack of flexibility in its usage for other modifications. The goal of these studies was to investigate the possibility of obtaining a similar separation effect by fractionating post-translationally modified proteins based on the physical properties of proteins. The post-translational modification chosen to be the basis of this study was the O-GlcNAc modification. Using the C2C12 mouse myoblast cell line, it was determined that the optimal conditions for producing lysates containing increased yields of O-GlcNAc modified proteins was to treat differentiated C2C12 cells with 10nM insulin, 12g/L glucose and 2mM of the O-GlcNAcase inhibitor Streptozotocin for 24 hours. Using the optimized lysis buffer, it was shown that protein separation by surface charge using standard anion exchange separation did not provide enough resolution or material to obtain any identifications of modified proteins. However, when a chromatofocusing method which separates proteins on the basis of their isoelectric points was used, a separation scheme with larger capacity and higher resolution was possible. Using this separation method followed by gel electrophoresis of individual fractions, proteins which are potentially O-GlcNAc modified were identified by mass spectrometry. It was evident from the number of protein bands observed per fraction on the Coomassie stained gels and the number of proteins identified per protein band by mass spectrometry that further reduction in sample complexity was required to assist in the positive identification of O-GlcNAc modified proteins. Among the identified proteins, 32 percent were metabolic proteins, 21 percent were protein processing proteins, 16 percent were structural proteins and the remainder a mix of other proteins. Unfortunately, it was not possible to validate the presence or absence of the O-GlcNAc modification on these proteins using available methodologies such as immunoprecipitation. As such, further work is required to optimize the separation strategy and to verify the usefulness of this separation strategy in identifying O-GlcNAc/post-translationally modified proteins.

The recognition of modified amino acids by reader proteins is governed by the competing interplay of weak, attractive, intermolecular forces and solvation effects. For the recognition of hydrophobic cations like methyl-lysines and methyl-arginines, native reader proteins utilize structural cages always containing multiple aromatic amino acids and sometimes an occasional acidic residue. Through the highly ordered arrangement of multiple aromatic surfaces, reader proteins can invoke the attractive forces of electrostatic, cation-pi, and in the case of arginine, pi-pi interactions. The hydrophobic effect can also significantly affect these binding events in aqueous environments. In this thesis, a number of small molecule, synthetic cages containing significant aromatic surface area have been synthesized. Variation in both total host hydrophobicity and degree of flexibility were explored to determine what effect they have on the overall binding of methylated amino acids in water. Significant flexibility in the first generation of highly aromatic hosts was shown to be detrimental to binding. However, strong binding was observed for guests with significant hydrophobic character despite this flexibility. The cause of the strong affinities in this family of synthetic cages was shown to be due to the hydrophobic effect, rather than any attraction due to cation-pi interactions. Synthetic efforts towards hosts with more rigid structures led to the use of Tröger’s base as a structural building block. Hosts incorporating Tröger’s bases into well-defined aromatic cavities were found to exhibit strong binding to both methyl-lysine and methyl-arginine derivatives in pure water. Differences in guest selectivity were due to the rigid altered host geometry introduced by the Tröger’s base cleft. / Graduate