La capacidad de Mycobacterium tuberculosis para sobrevivir dentro del macrófago contribuye grandemente a su patogenicidad, latencia y persistencia durante la infección. Este bacilo induce alteraciones en el ambiente intrafagosomal e inhibe la maduración del fagosoma, favoreciendo su supervivencia intracelular. M. tuberculosis PknG secuestra al macrófago precisamente al evitar la fusión fagosoma-lisosoma. En este sentido, PknG representa una familia de dianas novedosas para enfrentar la necesidad de nuevos antimicrobianos para la tuberculosis latente. Aquí, apuntamos a: (i) elucidar la base estructural-molecular del ATP y Mg2+ como cofactores de PknG; (ii) caracterizar los parámetros cinéticos que gobiernan la formación del complejo PknG:ATP; e, (iii) identificar péptidos capaces de unirse a PknG para investigar experimentalmente su interactoma usando enfoques combinatorios como “Phage Display”. Nuestros resultados confirman que PknG se une exclusivamente al ATP con una constante de disociación (KD) de 108.8  22.9 µM. El Mg2+ estabiliza térmicamente a PknG de forma ATP-dependiente. Análisis de estado pre-estacionario muestran que la unión y disociación del ATP es rápida en el complejo PknG:ATP. Usando PknGN-Ext, TPR resolvimos la estructura cristalina en el estado unido al ADP mientras que demostramos que el ATP imposibilita la cristalización. Los análisis bioinformáticos de las librerías enriquecidas por Phage Display identificaron 57 potenciales peptidos que interactuarían con PknG. Una comparación cercana con el proteoma de M. tuberculosis proporcionó un subconjunto de 20 proteínas que podrían interactuar con PknG. Nuestros resultados confirmaron cinco proteínas asociadas a PknG previamente reportadas: PknG, DnaK chaperona, transportador ABC Rv1747, Proteína Ribosomal L23 y Factor de Elongación Tu, resaltando la validez de nuestra plataforma para descubrir el interactoma de PknG. Así, nuestros resultados revelan interacciones proteína-proteína putativas que podrían participar en la supervivencia micobacteriana, mientras que también proporcionan bases sólidas para desarrollar drogas antituberculosas al interrumpir estas interacciones o explotar estos peptidos tipo compuesto líder. / The ability of Mycobacterium tuberculosis to survive inside the macrophage greatly contributes to its pathogenicity, latency and persistence during infection. This bacillus induces alterations in the intraphagosomal environment and inhibits phagosome maturation, thus promoting mycobacterial survival. M. tuberculosis PknG hijacks the macrophage precisely by avoiding phagosome-lysosome fusion. In this sense, PknG represents a family of novel targets to cope with the need for new antimicrobials for latent tuberculosis. Here, we aimed to: (i) elucidate the structural-molecular basis of ATP and Mg2+ as PknG cofactors; (ii) characterize the kinetic parameters governing PknG:ATP complex formation; and, (iii) identify PknG-binding peptides to experimentally query PknG’s interactome using combinatorial approach such as Phage Display. Our results confirm that PknG exclusively binds to ATP with a dissociation constant (KD) of 108.8  22.9 µM. Mg2+ thermally stabilizes PknG in an ATP-dependent manner. Pre-steady-state analyses show that ATP binding and dissociation are rapid in the PknG:ATP complex. Using PknGN-Ext, TPR we solved the ADP-state crystal structure while showing that ATP precludes crystallization. Phage Display and bioinformatic analyses identified 57 potential PknG binders. A close comparison to the M. tuberculosis proteome provided a subset of 20 proteins that may interact with PknG. Our results confirmed five previously reported PknG-associated proteins: PknG, DnaK chaperone, ABC transporter Rv1747, Ribosomal Protein L23 and Elongation Factor Tu, highlighting our platform’s validity to uncover the PknG interactome. Altogether, our results reveal putative protein-protein interactions that may play a role in mycobacterial survival, while also providing solid bases for the development of anti-tuberculosis drugs by disrupting these interactions or exploiting these lead-like peptide molecules. / Tesis

Das CCAAT/Enhancer-Binding-Protein α (C/EBPα) ist ein Transkriptionsfaktor, der das Zellschicksal des hämatopoetischen Systems bestimmt. C/EBPα reguliert die Selbsterneuerung hämatopoetischer Stammzellen und bestimmt die myelomonozytäre Zelldifferenzierung. C/EBPα besitzt zudem die Eigenschaft lymphoide Zellen in myeloischen Zellen zu transdifferenzieren. Die von C/EBPα induzierte lymphoid-myeloische Transdifferenzierung kann als Modellsystem dienen, um die Vorgänge der Linienfestlegung, der zellulären Plastizität sowie der Funktionen von C/EBPα in der Zelldifferenzierung und Leukämogenese zu untersuchen. C/EBPα ist ein unstrukturiertes Protein, das seine Funktionen durch wechselnde Proteininteraktionen ausübt. Intrinsisch unstrukturierte Proteine, wie C/EBPα, sind prädestiniert an schwachen, multivalenten und hochdynamischen Proteininteraktionen teilzunehmen. Solche Inter-aktionen werden hauptsächlich durch kurze lineare Motive der Protein-Primärstruktur vermittelt. Motiv-basierte Proteininteraktionen sind mit den herkömmlichen Methoden der Interaktom-Analyse schwer zu analysieren. Die Anwendung der neuartigen Biotin-Ligase basierten TurboID Methode mit schneller Enzym-Kinetik ermöglichte nun die Analyse eines dynamischen C/EBPα Interaktoms während der lymphoid-myeloiden Transdifferenzierung. Es wurde festgestellt, dass sich die Proteinexpression der meisten C/EBPα interagierenden Proteine während der Transdifferenzierung kaum änderte, trotz erheblicher Änderungen des C/EBPα Interaktoms, was eine Regulation durch alternative Mechanismen nahelegt. Es wurden mehrere epigenetische Komplexe gefunden, einschließlich Mediator, SWI/SNF und CAF-1, die als mögliche Verbindung zwischen Interaktom, Transkriptom, Chromatinstruktur und Phänotyp in Betracht gezogen werden können. / The CCAAT/enhancer binding protein α (C/EBPα) is a key lineage-instructive transcription factor in the haematopoietic system. C/EBPα regulates the self-renewal of haematopoietic stem cells and is one of the main determinants of myeloid commitment. C/EBPα induces transdifferentiation of B cells into myeloid cells. C/EBPα-induced lymphoid-myeloid transdifferentiation may serve as a model system to study lineage commitment and cellular plasticity as well as address open questions related to functions of C/EBPα in differentiation and leukaemogenesis. C/EBPα is an intrinsically disordered protein that exerts its functions through protein interactions. Intrinsically disordered proteins (IDPs) engage in highly dynamic, weak, and multivalent protein interactions, which are mediated by short linear motifs (SLiMs). SLiMs-based protein interactions are difficult to analyse by conventional methods of interactome analysis. However, to understand the connection between the C/EBPα interactome and its functions, analysis in the cellular context is required. The application of the novel biotin ligase TurboID with faster kinetics enabled the analysis of the dynamic interactome of C/EBPα in the process of lymphoid-myeloid transdifferentiation. For most of the interacting proteins, the protein level did not change, yet variable interactions were found, indicating regulated interactions. TurboID identified changes in the composition of the SWI/SNF complex during transdifferentiation, including the exchange of subunits specific for BAF and PBAF subcomplexes, Brg1 and Brm ATPases, and cell type-specific subunits. The analysis also identified the interaction pattern of the histone demethylase Kdm6b and functional assays confirmed its role in transdifferentiation. TurboID enabled the comparative analysis of the interactomes of C/EBPα isoforms and mutants, that alter the balance between differentiation and proliferation and its oncogenic functions.

Les Wolbachia sont des alpha-proteobactéries présentes chez de nombreux arthropodes et nématodes filaires. Ces bactéries héritées maternellement induisent chez leurs hôtes des phénotypes allant du parasitisme au mutualisme, avec le long de ce continuum des phénotypes tels que la féminisation (F), l'incompatibilité cytoplasmique (IC) ou la mort des mâles. Wolbachia est ainsi un modèle particulièrement intéressant pour étudier les différents types de relations symbiotiques.Chez Brugia malayi, comme pour les autres nématodes filaires, Wolbachia vit en symbiose obligatoire avec son hôte. L'élimination de la bactérie par des traitements antibiotiques entraîne une perte de fertilité voire la mort du nématode. Chez l'isopode terrestre Armadillidium vulgare, Wolbachia induit la féminisation des mâles génétiques en femelles fonctionnelles entraînant des biais de sex-ratio vers les femelles dans la descendance.Pour comprendre les mécanismes impliqués dans ces deux symbioses, nous avons mis au point une nouvelle méthode de capture pour isoler l'ADN de Wolbachia et séquencer 8 souches de Wolbachia d'isopodes (F et IC). Une étude de génomique comparative a permis d'établir un premier pan-génome des bactéries du genre Wolbachia et d'identifier 2, 5 et 3 gènes présents seulement chez les souches mutualistes, féminisantes ou induisant la mort des mâles. L'expression des gènes potentiellement impliqués dans la féminisation ou le mutualisme a été étudiée au cours du développement de l'hôte. L'étude de l'interactome protéique bactérie-hôte a ensuite été initiée en utilisant comme appât des protéines bactériennes à domaines eucaryotes en vue d'identifier les cibles de Wolbachia chez l'hôte. / Bacteria of the genus Wolbachia are gram-negative alpha-proteobacteria present in many arthropods and filarial nematodes. These obligate intracellular bacteria are maternally inherited and induce a large number of phenotypes across the symbiosis continuum from mutualism to parasitism, including feminization (F), cytoplasmic incompatibility (CI) or male killing. Studying Wolbachia symbioses is therefore of particular interest in the investigation of symbiotic relationships.In Brugia malayi and other filarial nematodes, they are obligate leading to a loss of worm fertility, and eventual death upon their depletion with antibiotic. In arthropods, they rather are parasitic. In the isopod crustacean Armadillidium vulgare they cause feminization when present: genetic males develop as functional female leading to female biased sex-ratio progenies.In order to understand the molecular mechanisms of these two symbioses, we set up a new capture procedure to catch Wolbachia DNA and performed whole-genome sequencing on 8 Wolbachia strains, symbionts of isopods (F & CI). Comparative genomics led to the establishment of the Wolbachia pan-genome as well as the identification of phenotype related gene patterns. We identified 2, 5 and 3 genes that are only found in mutualist, feminizing and male killing strains, respectively. Expression of genes potentially involved in feminization and mutualism were also analyzed throughout host post-embryonic development. Host-symbiont interactome approach was then initiated by protein-protein interaction studies using bacterial proteins with eukaryote like motifs as bait in order to identify Wolbachia host targets involved in symbiosis.

NUR77 est un récepteur nucléaire (RN) orphelin impliqué dans la régulation de processus biologiques dont la mort cellulaire, notamment dans la maladie de Parkinson (MP), découlant de la perte de neurones dopaminergiques, et dans le cancer du sein, résultant de la prolifération de cellules mammaires. NUR77 est impliqué dans le déclenchement et la protection de la mort cellulaire et son activité serait indépendante de la liaison d’un ligand. Nous avons émis l’hypothèse que l’activité de NUR77 est influencée par ses modifications post-traductionnelles (MPTs) et ses partenaires d’interactions. L’objectif général de cette thèse était de caractériser les MPTs et les partenaires d’interaction modulant l’activité de NUR77, dans des modèles de cellules en culture, afin de mieux comprendre ses fonctions biologiques - notamment dans la mort cellulaire. Le premier objectif de ce doctorat était de caractériser le rôle de la SUMOylation, une modification modulant l’activité des RN, chez NUR77, par des essais rapporteurs dans les cellules Human Embryonic Kidney 293 (HEK293). La surexpression de la E3 SUMO ligase PIASγ et/ou de l’isoforme 2 de la SUMO, protéines importantes dans la régulation de la SUMOylation chez les RN, a engendré un effet répresseur sur l’activité transcriptionnelle de NUR77. L’effet de PIASγ sur l’activité de NUR77 est modulé par la Sentrin SUMO-specific protease 1, qui hydrolyse la liaison des SUMO. Les mutations des résidus lysine dans des sites consensus de SUMOylation, de NUR77 (K102 et K577), empêchant cette MPT, ont causé des effets opposés sur son activité transcriptionnelle, suggérant le recrutement différent de corégulateurs de la transcription. Ces résultats combinés indiquent que la SUMOylation et les PIASγ et SUMO2 sont, respectivement, une MPT et des corégulateurs importants dans l’activité de NUR77. Le deuxième objectif de cette thèse était de caractériser l’interactome de NUR77 dans des HEK293 vivantes afin d’identifier les interacteurs pouvant moduler son activité, à l’aide d’une méthode de marquage des protéines proximales avec la biotine basée sur la peroxydase APEX2, combinée à la spectrométrie de masse. Ce procédé a identifié 336 potentiels interacteurs de NUR77, dont plusieurs connus. Des essais de coimmunoprécipitation et de coimmunofluorescence menés dans les HEK293 et dans les cellules du cancer du sein MCF-7 ont montré, respectivement, que la protéine régulatrice de l’apoptose Apoptosis Inhibitor 5 (API5), interagissait et colocalisait avec NUR77. La privation de sérum dans le milieu de culture des cellules et la diminution de l’expression de API5 a conduit à une augmentation des niveaux protéiques et de l’activité de NUR77 et à une diminution de la survie cellulaire. Ces données suggèrent que API5 constitue un régulateur de NUR77 dans les voies de signalisation associées à la mort cellulaire et que cette interaction pourrait constituer une cible pour moduler l’apoptose. Elles valident également l’approche d’identification d’interacteurs de NUR77. Les travaux de cette thèse ont donc permis de générer des outils pour caractériser l’activité de NUR77 et ont révélé des corégulateurs de cette activité. La poursuite de ces projets pourrait révéler le caractère opportun de cibler NUR77 comme modulateur de la mort cellulaire, notamment dans la MP et le cancer du sein. / NUR77 is an orphan nuclear receptor (NR) involved in the regulation of multiple cell biology processes including cell death, in particular in Parkinson's disease (PD), which results of the loss of dopaminergic neurons, and in breast cancer (BC), which is caused by the proliferation of mammary epithelial cells. NUR77 is involved in triggering and inhibiting cell death and its activity is believed to be independent of a ligand binding. We hypothesized that the regulation of NUR77 activity does not occur through a ligand, but through the influence of its post-translational modifications (PTMs) and its interaction partners. The general objective of this PhD project was to characterize the PTMs and the interacting partners that modulate the activity of NUR77 in cultured cell models, to better understand its physiological roles, in particular in the regulation of cell death. The first objective of this thesis was to characterize the role of SUMOylation, a modification that regulates NR activity, in regulating NUR77 transcriptional activity in reporter assays in Human Embryonic Kidney (HEK293) cells. Overexpression of the E3 SUMO ligase PIASγ or/and the isoform 2 of SUMO, both important regulators in SUMOylation of the NUR77 homolog NURR1, produced a repressive effect on the transcriptional activity of NUR77. The effect of PIASγ on the activity of NUR77 was shown to be modulated by the Sentrin SUMO-specific protease 1 protein, which removes SUMO tags on target proteins. In addition, mutations of lysine residues in SUMO consensus sites in NUR77 (K102 and K577) had opposite effects on its transcriptional activity, suggesting different recruitment of coregulators of transcription in the regions. The combination of these results indicates that SUMOylation is an important PTM for the regulation of NUR77 activity and that PIASγ and SUMO2 proteins are important transcriptional coregulators of NUR77. The second objective of this thesis was to evaluate NUR77 interactome in HEK293 living cells to identify the interactors that can modulate its activity, using a biotin-labelling method for proximal proteins based on the APEX2 peroxidase combined with mass spectrometry. This approach identified 336 potential interactors of NUR77, some that are consistent with the literature. Coimmunoprecipitation and coimmunofluorescence assays carried out in HEK293 cells and in MCF-7 breast cancer cell line have shown that the regulator of apoptosis Apoptosis Inhibitor 5 vi (API5), interacted and colocalized with NUR77. By depriving cells of serum and decreasing API5 expression, increased protein levels and activity of NUR77 was observed, as well as a decrease in cell viability. These data support that API5 is a regulator of NUR77 in its involvement in signalling pathways associated with cell death and that this interaction could be a target for modulating apoptosis. More generally, they validate the APEX2 tool which can be used to identify novel NUR77 interactors. In conclusion, the work of this thesis resulted in the generation of tools to better understand the activity of NUR77 and revealed important coregulators in this activity. The continued characterization of these interactors may provide opportunities to target NUR77 as a regulator of cell death, particularly in PD and in breast cancer.

Proteins and their intricate network of interactions are fundamental to many molecular processes that govern life. Mass spectrometry-based structural proteomics represents a powerful set of techniques for characterizing protein structures and interactions. The last decade has witnessed a large-scale adoption in the application of these techniques toward solving a variety of biological questions. Addressing these questions has often been coincident with the further development of these techniques. Insight into the structures of individual proteins and their interactions with other proteins in a proteome-wide context has been made possible by recent developments in the relatively new field of chemical crosslinking combined with mass spectrometry. In these experiments crosslinking reagents are used to capture protein-protein interactions by forming covalent linkages between proximal amino acid residues. The crosslinked proteins are then enzymatically digested into peptides, and the covalently-coupled crosslinked peptides are identified by mass spectrometry. These identified crosslinked peptides thus provide evidence of interacting regions within or between proteins. In this dissertation the development of tools and methods that facilitate this powerful technique are described. The primary arc of this work follows the development and application of mass spectrometry-based approaches for the identification of protein crosslinks ranging from those which exist endogenously to those which are introduced synthetically. Firstly, the development of a novel strategy for comprehensive determination of naturally occurring protein crosslinks in the form of disulfide bonds is described. Secondly, the application of crosslinking reagents to create synthetic crosslinks in proteins coupled with molecular dynamics simulations is explored in order to structurally characterize the intrinsically disordered tau protein. Thirdly, improvements to a crosslinking-mass spectrometry method for defining a protein-protein interactome in a complex sample is developed. Altogether, these described approaches represent a toolset to allow researchers to access information about protein structure and interactions. / Graduate

Le polyomavirus à cellules de Merkel est aujourd’hui reconnu comme l’agent étiologique du carcinome à cellules de Merkel (CCM). Le cycle viral et les mécanismes de l’oncogenèse viro-induite sont peu connus et les connaissances se basent essentiellement sur les études menées notamment sur le polyomavirus SV40. L’objectif des travaux de thèse était d’identifier les interactions entre les protéines virales et les protéines cellulaires lors de l’infection ou dans le contexte du carcinome à cellules de Merkel (CCM). Pour identifier ces interactions, nous avons réalisé des cribles double hybride en levures sur les oncogènes du MCPyV et du BKPyV. Afin valider les interactions obtenues en levures, nous avons utilisé une méthode orthogonale de validation par complémentation en cellules de mammifères reposant sur la restauration de la luciférase de Gaussia princeps. La combinaison de ses deux techniques nous a permis de valider des interactions avec des partenaires cellulaires impliqués dans la régulation du cycle cellulaire ou encore de la voie Akt-mTOR. Les précédents travaux du laboratoire, qui portaient sur l’interactome des protéines mineures de capsides VP2/VP3, avaient également permis d’identifier des interactions avec des protéines de la voie NF-kB. Nous avons alors testé les interactions entre les oncogènes et la protéine mineure de capside VP2 avec des protéines cellulaires impliquées dans cette voie. Ces travaux nous ont conduits à tester l’activation de la voie, l’expression des gènes sous le contrôle de NF-kB et la régulation de l’apoptose. Les résultats obtenus montrent une action de la protéine VP2 sur l’activation de la voie NF-kB et une induction de l’apoptose. / The Merkel cell polyomavirus is now recognized as the etiologic agent of Merkel cell carcinoma (MCC). The viral cycle and viro-induced oncogenesis mechanisms are not fully understood and the knowledge is mainly based on the studies carried out particularly on the SV40 polyomavirus. The aim of our work is to identify interactions between viral proteins and cellular proteins during productive infection or in MCC context. To identify these interactions, we performed yeast two hybrid screens on MCPyV and BKPyV oncogenes, as control. To validate the interactions obtained in yeasts, we used an orthogonal method of validation by complementation in mammalian cells based on the restoration of Gaussia princeps luciferase. The combination of these two orthogonal techniques allowed us to validate interactions with cellular partners involved in cell cycle regulation or Akt-mTOR pathway. Previous lab work on VP2/VP3 minor capsid proteins allowed the identification of interactions with NF-kB pathway involved proteins. We examined the interactions between oncogenes, VP2, with the cellular proteins involved in this pathway. This work led us to evaluate pathway activation, genes expression under the control of NF-kB and apoptosis regulation. These results evidenced an action of the VP2 protein on the activation of NF-kB pathway and an induction of apoptosis.

Kürzlich wurde mithilfe von Ribo-seq Experimenten die Translation hunderter Mikroproteine in menschlichen Herzen entdeckt. Diese blieben zuvor aufgrund ihrer geringen Größe (< 100 Aminosäuren) unentdeckt, und ihre physiologische Rolle ist noch weitgehend unbekannt. Ziel dieser Promotionsarbeit ist es, potentielle Funktionen dieser neuartigen Mikroproteine zu entschlüsseln. Dabei sollen insbesondere die Aufklärung ihrer evolutionären Konservierungssignatur, subzellulären Lokalisierung und ihres Proteininteraktoms helfen. Die Konservierungsanalyse ergab, dass fast 90% der Mikroproteine nur in Primaten konserviert ist. Weiterhin konnte ich die Produktion von Mikroproteine in vitro und in vivo nachweisen, die subzelluläre Lokalisierung von 92 Mikroproteinen definieren, und Interaktionspartner für 60 Mikroproteine identifizieren. Dutzende dieser Mikroproteine lokalisieren in Mitochondrien. Dazu gehörte ein im Herzen angereichertes Mikroprotein, das aufgrund der Interaktions- und Lokalisationsdaten einen neuartigen Modulator der mitochondrialen Proteintranslation darstellen könnte. Der Interaktom-Screen zeigte außerdem, dass evolutionär junge Mikroproteine ähnliche Interaktionsfähigkeiten wie konservierte Kandidaten haben. Schließlich wurden kurze Sequenzmotive identifiziert, die Mikroprotein-Protein-Wechselwirkungen vermitteln, wodurch junge Mikroproteine mit zellulären Prozessen – wie z.B. Endozytose und Spleißen – in Verbindung gebracht werden konnten. Zusammenfassend wurde die Produktion vieler kleiner Proteine im menschlichen Herzen bestätigt, von denen die meisten lediglich in Primaten konserviert sind. Zusätzlich verknüpften umfangreiche Lokalisierungs- und Interaktionsdaten mehrere Mikroproteine mit Prozessen wie Spleißen, Endozytose und mitochondrialer Translation. Weitere Untersuchungen dieses zuvor verborgenen Teils des Herzproteoms werden zu einem besseren Verständnis von evolutionär jungen Proteinen und kardiologischen Prozessen beitragen. / Recently, the active translation of hundreds of previously unknown microproteins was detected using ribosome profiling on tissues of human hearts. They had remained undetected due to their small size (< 100 amino acids), and their physiological roles are still largely unknown. This dissertation aims to investigate these novel microproteins and validate their translation by independent methods. Particularly, elucidating their conservation signature, subcellular localization, and protein interactome shall aid in deciphering their potential biological role. Conservation analysis revealed that sequence conservation of almost 90% of microproteins was restricted to primates. I next confirmed microprotein production in vitro and in vivo by in vitro translation assays and mass spectrometry-based approaches, defined the subcellular localization of 92 microproteins, and identified significant interaction partners for 60 candidates. Dozens of these microproteins localized to the mitochondrion. These included a novel cardiac-enriched microprotein that may present a novel modulator of mitochondrial protein translation based on its interaction profile and subcellular localization. The interactome screen further revealed that evolutionarily young microproteins have similar interaction capacities to conserved candidates. Finally, it allowed identifying short linear motifs that may mediate microprotein-protein interactions and implicated several young microproteins in distinct cellular processes such as endocytosis and splicing. I conclude that many novel small proteins are produced in the human heart, most of which exhibit poor sequence conservation. I provide a substantial resource of microprotein localization and interaction data that links several to cellular processes such as splicing, endocytosis, and mitochondrial translation. Further investigation into this hidden part of the cardiac proteome will contribute to our understanding of recently evolved proteins and heart biology.