The Rho GTPases form a family of enzymes that control numerous cellular processes including cell migration and proliferation through effects on the cytoskeleton, membrane trafficking and cell adhesion. The activity of these molecular switches is modulated by GTPase-activating proteins (GAPs), a group of negative-regulators which includes Cdc42-GTPase-Activating Protein (CdGAP). This protein specifically negatively-regulates the Rho GTPases Cdc42 and Rac1. In this study, we show that CdGAP is regulated by lipid-, protein- and intramolecular-interactions. First, we demonstrate that a polybasic region (PBR) of CdGAP preceding the GAP domain and found in numerous Rho family GAPs is required for CdGAP specific association with phosphatidilinositol-3,4,5-trisphosphate (PI(3,4,5)P3). We show that the binding of PI(3,4,5)P3 is required for CdGAP-mediated GAP activity in vitro, and that an intact PBR is required for its CdGAP-mediated GAP activity in vivo. Second, we characterize the binding site for the negative-regulator of CdGAP Intersectin-1 located in the Basic-Rich (BR) domain of CdGAP. We present evidence that this interaction mediated by the SH3D domain of Intersectin requires one to three lysine residues located in the Basic-Rich (BR) domain of CdGAP. Thirdly, we show that CdGAP is negatively-regulated by its C-terminal domain. This observation is part of a study that links two human CdGAP gene mutations to a syndrome which presents a combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). In this syndrome, the deletion-mutant gene products which lack the residual amino-acid of CdGAP at its C-terminus have an increased activity compared to wild-type proteins. We show that this C-terminus can bind to the GAP domain of CdGAP, providing a model to explain how the absence of the C-terminus induces this syndrome. In summary, this work provides novel insight into understanding the mechanisms of regulation of CdGAP, a protein involved in cell migration and adhesion with unexpected roles related to human diseases. / Les Rho GTPases forment une famille d'enzymes qui contrôlent de nombreux processus cellulaires, tels que la migration cellulaire et la prolifération, grâce à leurs effets sur le cytosquelette, le trafic membranaire et l'adhésion cellulaire. L'activité de ces interrupteurs moléculaires est modulée par les protéines activatrices de GTPases (GAPs), un groupe de régulateurs négatifs qui inclu CdGAP (Cdc42-GTPase activating protein). Cette protéine régule négativement les Rho GTPases Cdc42 et Rac1 de façon spécifique. Dans la présente étude, nous montrons que CdGAP est régulée par des interactions lipidiques, protéiques et intramoléculaire. Premièrement, nous démontrons qu'une région polybasique (PBR), précédant le domaine GAP et retrouvée dans plusieurs GAP de la famille Rho, est requise pour l'association spécifique de CdGAP avec le phosphatidilinositol-3,4,5-trisphosphate (PI(3,4,5)P3). Nos résultats suggèrent que l'activation des GAP requiert la liaison du PI(3,4,5)P3 à CdGAP dans un contexte in vitro et un PBR intact pour que CdGAP provoque ses effets GAP-dépendants dans un contexte in vivo. Deuxièmement, nous caractérisons le site de liaison du régulateur négatif de CdGAP Intersectin-1. Ce site est localisé dans le domaine riche en résidus basiques (BR) de CdGAP. Nous suggérons que cette interaction, médiée par le domaine SH3D d'Intersectin, requiert de un à trois résidus lysine dans le domaine BR de CdGAP. Troisièmement, nous montrons que CdGAP est régulé de manière négative par son propre domaine C-terminal. Cette observation fait partie d'une étude qui associe deux mutations humaines du gène CdGAP à un syndrôme présentant une combinaison d'aplasie cutis congenita (ACC) et de malformation des doigts et des orteils (TTLD). Les gènes mutants produisent des protéines tronquées qui ont une activité GAP supérieure à la protéine de type sauvage. Nous montrons que ce C-terminal peut lier le domaine GAP de CdGAP, supportant un modèle expliquant comment l'absence du C-terminal induit ce syndrome. En bref, ce travail présente un nouvel aperçu des mécanismes de régulation de CdGAP, une protéine impliquée dans la migration cellulaire et dans l'adhésion des cellules en plus d'être directement impliquée dans une maladie humaine.

The recruitment of ribosomes to the 5' end of mRNAs in eukaryotic cells is generally thought to be the rate-limiting step of translation initiation and this process is mediated by the eukaryotic initiation factor 4F complex (eIF4F). This complex consists of three subunits: eIF4E, a mRNA-cap binding protein, eIF4A, a RNA helicase and eIF4G, a large molecular scaffold that mediates the binding of mRNA to ribosomes. Deregulation of translation initiation through eIF4F activation has been widely observed in human cancers. The eIF4F complex lies downstream of key signalling pathways involved in oncogenesis (such as c-Myc and PI3K/Akt/mTOR), thus targeting translation initiation provides an attractive therapeutic approach for cancer therapy. Here, we show that c-Myc stimulates protein synthesis by up-regulating the expression and activity of not only eIF4E, but that of the other two subunits of eIF4F. In turn, this elevated eIF4F levels result in increased synthesis and function of c-Myc, establishing a positive feedforward loop. We used the Eμ-myc lymphoma mouse model to show that expression of the three eIF4F subunits is also up-regulated by c-Myc in vivo. Most importantly, we demonstrate that loss of eIF4E function using inducible and reversible RNA interference (RNAi) greatly delays the rates of c-Myc-induced lymphoma development. These data suggest that targeting eIF4E in vivo is an effective therapeutic approach. Since the assembly of eIF4F complex is regulated by mTOR signaling, the coupling of c-Myc to eIF4F is under mTOR control. In the course of a screen of inhibitors of the PI3K/Akt/mTOR signalling pathway, we found two small molecules, silibinin and the anti-depressant sertraline, both of which show anti-proliferative effects on breast cancer cells. Silibinin and sertraline effectively target eIF4F complex function by downregulating mTOR signaling. Importantly, sertraline is able to enhance the chemosensitivity of PTEN (+/-)/Eμ-Myc lymphomas to the chemotherapeutic agent doxorubicin in vivo. Thus, targeting mTOR-dependent translation initiation shows anti-cancer activity in this pre-clinical setting. / Il est généralement admis que le recrutement des ribosomes à l'extrémité 5' des ARN messagers (ARNm) est l'étape limitante de l'initiation de la traduction chez les eucaryotes. Cette étape est dépendante de l'activité du complexe d'initiation eIF4F qui comprend trois sous-unités: eIF4E, une protéine liant la coiffe des ARNm, eIF4A, une hélicase d'ARN et eIF4G, une grande protéine d'échafaudage dont le rôle est de coordonner la liaison du ribosome à l'ARNm. Le dérèglement du contrôle de l'initiation de la traduction suite à l'activation d'eIF4F est observé fréquemment chez les cancers humains. L'activité de ce complexe est contrôlée par plusieurs voies de signalisation clés qui sont impliquées dans la formation des tumeurs (tels que c-Myc et PI3K/Akt/mTOR). Donc, cibler l'initiation de la traduction représente une avenue attrayante pour contrer le cancer. Nous démontrons ici que l'oncogène c-Myc peut stimuler la synthèse protéique en favorisant l'expression et l'activité de non-seulement eIF4E, mais aussi des deux autres sous-unités du complexe eIF4F. En réponse à cela, les niveaux supérieurs d'eIF4F permettent une augmentation de la synthèse et donc de l'activité de c-MYC, établissant alors une boucle auto-stimulante. Nous avons utilisé le modèle de souris Eμ-myc pour démontrer que l'expression de chacune des sous-unités d'eIF4F est stimulée par c-Myc in vivo. Plus important encore, nous avons démontré que la réduction des niveaux d'eIF4E en utilisant la technique d'interférence à ARN (ARNi) de manière inductible et réversible freine considérablement le développement de lymphomes par c-Myc. Ces données suggèrent que cibler eIF4E in vivo est une approche thérapeutique viable et efficace. De plus, puisque l'assemblage d'eIF4E est contrôlé de mTOR, il en résulte donc que le couplage de c-Myc et d'eIF4F est donc aussi sous contrôle de cette voie de signalisation. Suite à un cribblage de molécules inhibitrices de la voie PI3K/Akt/mTOR, nous avons identifié deux molécules, la silibinine et l'anti-dépresseur sertraline, qui ont la propriété de bloquer la prolifération de cellules du cancer du sein. La silibinin et la sertraline inhibent efficacement l'activité du complexe eIF4F en ciblant la voie de signalisation de mTOR. Par surcroît, la sertraline accentue fortement la sensibilité des lymphomes PTEN (+/-)/Eμ-Myc à l'agent chimiothérapeutique doxorubicin in vivo. En conclusion, il appert que cibler le contrôle de la traduction par mTOR peut contrer efficacement le cancer dans ce modèle de cancer préclinique.

The 45kDa isoform of TC-PTP is a ubiquitous, predominantly nuclear protein. Reports demonstrate that it can exist outside the nucleus; however, immunofluorescence and cellular fractionation techniques point to an almost exclusive residence in the nucleus, and even within the nucleolus. Though the conditions of nucleolar enrichment appear to be serum-dependent, and may involve S52 phosphorylation, the exact circumstances necessary for this localization remain unknown, though TC-PTP C216S mutant, and not an S52A/C216S double mutant had the ability to trap a phosphorylated substrate of ∼105kDa, thus encouraging further research into the identity of this phosphoprotein. There is also data implicating TC-PTP in the p53-dependent DNA damage response. TC-PTP WT pMEF's are more susceptible to apoptosis than KO pMEF's, thus suggesting a possible role as a tumor suppressor. Finally, in order to explore the physiological significance of possible non-hematopoietic roles of TC-PTP, a Cre-Lox targeting vector was constructed for the eventual generation of a conditional knockout.

Angiogenesis is a process of new blood vessel formation and is the culmination of both mitogenic and tissue remodeling events, resulting in neovascularization. It is a physiological process that is required for, amongst others, normal embryonic development, female reproductive function, and wound healing. Angiogenesis is a tightly regulated process which is balanced by both positive and negative factors. However, in many disease states, including diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, and several cancers, dysregulation of angiogenesis contributes to disease progression. Previous published reports have implicated the coordinated activities of at least two families of receptor tyrosine kinases (RTKs), the vascular endothelial growth factor receptor (VEGFR) and the Tie receptor families, in this process. / Tie2, an endothelial-specific receptor tyrosine kinase, plays an essential role in normal blood vessel maturation, remodeling, and stability. Tie2 expression is also upregulated in various cancers indicating a role in tumor angiogenesis. The human Tie2 mRNA transcript contains an unusually long (372 nucleotides) 5' untranslated region (UTR) with five upstream open reading frames (uORFs). In this thesis, we demonstrate that the Tie2 5' UTR promotes cap-independent translation, indicating the presence of functional internal ribosome entry site (IRES). In addition, we illustrate that Tie2 IRES activity is maintained, and even slightly stimulated, under hypoxic conditions when cap-dependent protein synthesis is attenuated. We further show that the Tie2 IRES is functional during quiescence, another condition known to compromise cap-dependent translation. These results present how Tie2 mRNA is translated despite a cumbersome structured 5' UTR and how its production is secured under unfavorable environmental conditions. / We define experimental conditions where the Tie2 IRES is not active, allowing us to assess the contribution of cap-dependent translation to Tie2 protein synthesis. We demonstrate evidence that Tie2 mRNA can be translated via both cap-dependent scanning mechanism and internal initiation. Moreover, we show that the presence of the uORFs within the 5' UTR is inhibitory to downstream translation initiation. Our results suggest that the uORFs serve to decrease the proportion of ribosomes competent for reinitiation as they traverse the mRNA 5' UTR and thus minimizing interference with the IRES and/or mediating inefficient translation of the potent protein under normal conditions. Like many other cellular IRESes, the entire Tie2 5' UTR appears to be required for maximum IRES activity. / Taken together, our results underscore the complex mechanisms to control gene expression at the level of translation initiation of the Tie2 mRNA.

During spermatogenesis histones must be degraded in late round and early elongating spermatids to permit chromatin condensation. Ubiquitin conjugation is activated and histones are ubiquitinated at this stage, suggesting that histone degradation may be mediated by ubiquitination. The activation of ubiquitin conjugation during spermatogenesis is dependent on the ubiquitin conjugating enzyme (E2) UBC4. We therefore studied whether histones are ubiquitinated by a UBC4 dependent ubiquitin protein ligase (E3) during spermatogenesis. E3Histone was identified by a biochemical screen and purified to near homogeneity. Mass spectrometry identified E3Histone as LASU1, a 482 kDa HECT domain protein and E3Histone conjugates ubiquitin to all core histories in vitro. UBC4-1 and UBC4-testis were the preferred E2s for E3Histone-dependent ubiquitination of histones. E3Histone was the major UBC4-1 dependent histone ubiquitinating E3 in testis. Anti-LASU1 antibody immunodepleted E3 Histone activity. Immunohistochemistry showed that E3Histone /LASU1 was predominantly expressed in nuclei from spermatogonia to mid-pachytene cells, but not detectable in spermatids. Histones are also ubiquitinated in spermatocytes. E3Histone/LASU1 was widely expressed in different mouse tissues. It was mainly expressed in the cytoplasm in most tissues, except in neurons of the brain and in early germ cells of the testis where it was expressed in the nucleus. In most tissues, E3Histone/LASU1 was expressed in epithelia. The wide expression of E3Histone/LASU1 suggests the existence of substrates of this E3 other than histones. Indeed, our assays showed that in vitro purified E3Histone stimulates polyubiquitination of Mcl-1, a BH3 region containing antiapoptotic protein. E3Histone may therefore regulate cell apoptosis by mediating degradation of Mcl-1. Since E3Histone/LASU1 was found previously to affect gene transcription and histone monoubiquitination is known to regulate gene transcription, we also evaluated the role of E3 Histone/LASU1 in histone ubiquitination in somatic cells. Depletion of E3Histone/LASU1 protein by siRNA did not affect the levels of free or ubiquitinated histories. In summary, E3Histone /LASU1 is a novel multi-functional protein that may mediate histone ubiquitination during meiosis and may be involved in apoptosis by triggering Mcl-1 degradation. Its wide expression and large non-catalytic region indicate that there are likely many other substrates of E3Histone/LASU1.

Human adenovirus type 5 (Ad5) has been engineered as therapeutic oncolytic viruses to selectively kill cancer cells. The earliest and most widely used is the Ad5 ONYX-015 virus which lacks the viral protein E1B55K. The adenoviral proteins E1B55K and E4orf6 were shown previously to be important for multiple functions during viral infection. Most of these functions are dependent on their formation of the E3 ligase complex with cellular proteins Cul5, Elongins B and C, and Rbx1. Therefore, in order to improve the current oncolytic adenovirus therapy, a better understanding on this E3 ligase complex is required. As the E4orf6/E1B55K E3 ligase contains components similar to other cullin E3 ligase complexes, the mechanism of the E3 ligase assembly was investigated. I showed that the E4orf6/E1B55K E3 ligase complex is formed in an unconventional way: E4orf6 uniquely contains three BC box motifs for its interaction with Elongin C unlike other cellular proteins, which contain only one BC box. In addition, the complex utilises neither of the two known mechanisms for recruiting Cul5. Ad5 is by far the best characterized of the more than fifty different adenovirus serotypes; however, it is unclear how representative its properties are with respect to all adenoviruses. Thus, the conservation of the E4orf6/E1B55K E3 ligase was studied systematically in members of other adenovirus subgroups. I demonstrated that the E4orf6 and E1B55K proteins from all serotypes can form an E3 ligase complex but with different cullin specificities: Ad4, Ad5, Ad9 and Ad34 recruit primarily Cul5, Ad12 and Ad40 recruit primarily Cul2, and Ad16 can recruit both. As for function, I showed that different serotypes degrade different ranges of substrates with the only common substrate to all being DNA ligase IV. I found clear evidence that E1B55K is the substrate binding component of the complex; however, I demonstrated that there is no correlation between binding and the capability to degrade specific substrates. These studies have shown clearly that considerable heterogeneity exists in the formation and function of the adenovirus E4orf6/E1B55K E3 ligase. / L'adénovirus humain de type 5 (Ad5) a été modifié génétiquement à des fins thérapeutiques afin d'éliminer sélectivement les cellules cancéreuses. Le premier virus décrit et le plus communément utilisé est le Ad5 virus ONYX-015 dans lequel la protéine virale E1B55K est absente. Il a été précédemment démontré que les protéines adénovirales E1B55K et E4orf6 sont importantes pour de multiples fonctions lors de l'infection virale. La plupart de ces fonctions dépendent de la formation du complexe E3 ligase avec les protéines cellulaires Cul5, Elongine B et C, ainsi que Rbx1. Ainsi, une meilleure compréhension de ce complexe E3 ligase est nécessaire à l'amélioration des thérapies oncolytiques adénovirales actuelles. Puisque le complexe E4orf6/E1B55K E3 ligase contient des composants similaires à d'autres complexes culline E3 ligase, nous avons étudié le mécanisme d'assemblage de l'E3 ligase. J'ai ainsi démontré que le complexe E4orf6/E1B55K E3 ligase est formé de façon non conventionnelle : E4orf6 contient uniquement trois motifs BC pour son interaction avec l'Elongine C contrairement aux protéines cellulaires qui n'en contiennent qu'un. De plus, le complexe n'utilise aucun des deux mécanismes connus pour recruter Cul5. Ad5 est de loin le mieux caractérisé des plus de cinquante sérotypes différents d'adénovirus, pourtant on ne sait pas exactement à quel point ses propriétés sont représentatives des autres sérotypes, d'où l'importance de l'étude systématique de la conservation du complexe E4orf6/E1B55K E3 ligase parmi les membres des autres sous-groupes d'adénovirus. J'ai démontré que les protéines E4orf6 et E1B55K peuvent former un complexe E3 ligase dans tous les sérotypes, mais avec des cullines spécifiques différentes : les sérotypes viraux Ad4, Ad5, Ad9 et Ad34 recrutent principalement Cul5, Ad12 et Ad40 recrute principalement Cul2 alors que Ad16 recrute à la fois Cul5 et Cul2. En ce qui concerne les fonctions, j'ai démontré que différents sérotypes sont capables de dégrader des substrats différents, le seul substrat commun à tous les sérotypes étant la ligase d'ADN IV. J'ai mis en évidence qu'E1B55K est le membre du complexe qui se lie au substrat, mais il n'y a pas de correspondance entre la capacité de lier un substrat et celle de le dégrader. Ces expériences ont clairement démontré la grande hétérogénéité existant entre la formation et les fonctions du complexe adénoviral E4orf6/E1B55K E3 ligase.

Members of the MAF (proto-) oncogene and CNC families of basic leucine zipper transcription factors (bZIP) play important roles in development, differentiation, mammalian gene expression and stress signalling. We analyzed the regulation of the human small MAF transcription factor family members, MAFF, MAFG and MAFK, as well as two CNC family members, NRF2 and NRF3, in human reproductive tissue. We found that MAFF expression was induced by the proinflammatory cytokines interleukin 1 beta (IL1B) and tumor necrosis factor (TNF) in PHM1-31 myometrial cells. It was particularly interesting that the proinflammatory cytokine Interleukin 6 not affect the expression of MAFF. Additionally, the transcript and protein levels of the highly homologous MAFG and MAFK genes were not modulated by these cytokines. Using electromobility shift assays (EMSA)s we showed that MAFF is capable of heterodimerzing with NRF3, and since they are both highly expressed in the placenta we investigated their role in more detail in this tissue. We found that MAFF and NRF3 are expressed in the anchoring villi from 12 weeks to term. Furthermore, we demonstrated that MAFF and NRF3 are highly expressed in primary placental cytotrophoblasts, but not in placental fibroblasts. This led us to examine whether NRF3 is also regulated by proinflammatory cytokines and we showed that NRF3, is also modulated by TNF in the human JAR placental cell line. / Parallel studies investigated the role NRF2 plays in the antioxidant response of placental cells due to exposure to arsenic. We provided evidence for the involvement of NRF2 by confirming the increase in binding of endogenous NRF2/small MAF heterodimers to Stress Response Element (StRE), and the upregulation of heme oxygenase-1 (HOI) expression, upon exposure of JAR cells to arsenic. Our results suggest a role for MAFF, NRF3 and NRF2 in proinflammatory cytokine control of myometrial and placental gene expression as well as arsenic mediated stress in placental cells.

In order to study the functions of SIV Nef in vivo, in a small animal model, transgenic (Tg) mice expressing the SIVmac239 nef gene, under the control of the human CD4 gene promoter (CD4C) were generated. The transgene was found to be expressed in the same cells targeted by the virus, in vivo. These CD4C/SHIV-nef SIV Tg mice develop a severe AIDS-like disease, including premature death, failure to thrive/weight loss, wasting, thymic atrophy, exhibit an especially low number of peripheral CD8+ T cells as well as low number of peripheral CD4+ T cells, diarrhea, splenomegaly, kidney (interstitial nephritis, segmental glomerulosclerosis), lung (lymphocytic interstitial pneumonitis) and heart disease. In addition, these Tg mice fail to mount a class-switched antibody response after immunization with ovalbumin, produce anti-DNA autoantibodies and some of them develop P. Carinii lung infection. These CD4C/SHIV-nefSIV Tg mice develop an AIDS-like disease very similar to that of CD4C/HIV Tg mice, except that the kidney and cardiac diseases were more severe, and that a thymic developmental defect was observed. Heart enlargement was very severe in CD4C/SIV Tg mice during early breeding on the C3H background. Histopathological lesions in the heart of these mice were also multifocal and were similar to those found in CD4C/HIV Tg mice. Data from echocardiography analysis are not yet available for these Tg mice. The low number of peripheral CD8+ and CD4 + T cells likely reflects a thymic defect and may be similar to the DiGeorge-like "thymic defect" immunophenotype described in a subgroup of HIV-1 infected children. Ontogeny studies show that the Tg mice were born with a smaller thymus and that this phenotype is not progressive in nature. As young as embryonic day 17, the thymic absolute cell numbers are lower in the Tg mice when compared to their non-Tg controls and there is a defect in thymocyte maturation in the transition between DN3 and DN4, with a failure to generate normal numbers of DP cells. Fetal liver transplantation studies have ruled out a significant impairment of the thymic epithelium and have suggested that this defect is likely a direct consequence of abnormal T cell progenitors in the thymus. / Therefore, it appears that SIV Nef alone expressed in mice, in appropriate cell types and at sufficient levels, can elicit many of the phenotypes of simian and human AIDS. These Tg mice should be instrumental in studying the pathogenesis of SIV Nef-induced phenotypes.

The innate immune system senses pathogen invasion through the recognition of distinct molecular patterns collectively known as PAMPs (Pathogen-Associated Molecular Patterns). The mitochondrial molecule MAVS a central adaptor linking sensing of incoming viruses by the cytoplasmic RNA helicases RIG-I/MDA5 to downstream activation of the classical IKK complex and IKK-related kinases IKKepsilon and TBK1, resulting in the activation of transcription factors NF-kappaB and IRF3/IRF7 respectively. NF-kappaB and IRFs co-operate in the induction of type I interferons, but also play specific roles in the induction of pro-inflammatory and antiviral genes, respectively. Strict regulation of the RIG-I pathway must exist to mount an effective immune response while protecting the host from excessive inflammation and tissue damage. The work presented here is a compilation of three studies that highlight both positive and negative aspects of RIG-I signaling that contributed to a better understanding of the molecular events surrounding MAVS, TRAF3, and IKKepsilon. In the first study, presented in Chapter II, we linked RIG-I signaling to the phosphorylation events mediated by TBK1 and IKKepsilon and established the minimal serine consensus motif targeted by the kinases TBK1 and IKKepsilon in the transcription factors IRF3 and IRF7. This study confirmed the role of TBK1 and IKKepsilon as positive regulators of the RIG-I pathway. In the study presented in Chapter III, we followed up on a precious finding that IKKepsilon, but not TBK1, was recruited to the mitochondria. Here, we mapped the interaction of MAVS and IKKepsilon to the C-terminal region of MAVS, and more specifically to Lysine 500. We showed that Lysine 500 is a K63-linked polyubiquitin acceptor site; thus demonstrating that MAVS is ubiquitinated following virus infection. Mutation of Lysine 500 lead to an upregulation of downstream genes involved in the inflammatory and antiviral responses; demonstrating for the first time a novel role for IKKepsilon in the negative regulation of the RIG-I pathway. Chapter IV demonstrated that recruitment of IKKepsilon to ubiquitinated Lysine 500 lead to a disruption of TRAF3-MAVS interaction; which was shown to be mediated by a novel and functional TRAF3 binding site in the C-terminus of MAVS. / La réponse immune innée est déclenchée suite à la détection d'une variété considérable de pathogènes. Ces derniers sont reconnus comme étrangers à l'hôte à travers des motifs spécifiques conservés communément appelés PAMPs (Pathogen-Associated Molecular Patterns). La protéine mitochondriale MAVS, suite à la reconnaissance d'ARNs viraux intra cytoplasmiques par les ARN hélicases RIG-I et MDA5, permet les activations cruciales du complexe classique des kinases IKK et des kinases IKKepsilon et TBK1 qui sont responsables in fine de l'activation des facteurs de transcription NF-kappaB et IRF3/IRF7. NF-kappaB et IRFs, coopèrent pour l'induction des interférons de type 1 et stimulent respectivement l'expression spécifique de gènes pro inflammatoires et antiviraux. Une régulation stricte de ces signalisations est essentielle pour la mise en place d'une réponse immune efficace tout en protégeant l'hôte d'une inflammation excessive. Le travail présenté ici dans cette thèse est une compilation de trois études qui, d'une part mettent en avant les propriétés régulatrices positives et négatives de la voie de signalisation RIG-I, d'autre part contribuent à une meilleure compréhension des mécanismes moléculaires impliquant MAVS, TRAF3 et IKKepsilon. Dans la première étude présentée dans le Chapitre II, nous avons approfondi nos précédentes études et avons établie une séquence spécifique minimal de phosphorylations des IRF3 et IRF7 par les kinases TBK1 et IKKepsilon et donc confirmé le rôle des kinases dans la régulation positive de la signalisation par RIG-I. Dans la seconde étude du Chapitre III, nous avons approfondi l'observation antérieure démontrant que IKKepsilon et non TBK1 est recruté à la mitochondrie suite à une infection virale. Ici, nous avons démontrés que IKKepsilon était recrutée par MAVS via la région C-terminale et plus spécifiquement à la Lysine 500. De plus, nous avons caractérisé la Lysine 500 de MAVS comme étant capable de lier une chaîne de polyubiquitine K63 et donc démontré que MAVS est ubiquitiné suite à une infection virale. Enfin, la mutation de la Lysine 500 aboutit à une stimulation des voies pro inflammatoires et antiviraux; ainsi, pour la première fois nous avons démontré un nouveau rôle pour IKKepsilon dans la régulation de la voie de signalisation dépendante de RIG-I. Chapitre IV met en évidence que le recrutement d'IKKepsilon modifiait l'interaction de TRAF3 à MAVS à un motif C-terminal nouvellement identifié dans cette étude et démontré essentiel pour l'induction de la voie de signalisation.

Antibiotic resistance is a serious concern in nosocomial bacterial infections. The extensive use of antibiotics has led to the emergence of multidrug-resistant bacteria such as Enterococci. Strains of E. faecium are frequently encountered that are resistant to a diverse range of antibiotics, including beta-lactams, glycopeptides, as well as aminoglycosides. The resistance profile of E. faecium to many aminoglycosides is attributed to an acetyltransferase. The enzyme, aminoglycoside 6'-N-acetyltransferase type-Ii (AAC(6')-Ii), uses acetyl coenzyme A (AcCoA) to modify all clinically relevant aminoglycosides that contain a 6'-amino sugar. Such modifications render aminoglycosides incapable of binding to the 30S bacterial ribosome, thereby preventing antibiotics from inducing protein mistranslation and thus abolishing their bactericidal properties.We aimed to explore different possibilities to restore aminoglycoside susceptibility by either protecting aminoglycosides from being modified by AAC(6')-Ii or inhibiting the enzyme so that it can no longer modify aminoglycosides. To this end, we pursued the structural description of the entire AAC(6')-Ii reaction cycle. AAC(6')-Ii employs an ordered sequential mechanism, where AcCoA binds prior to the substrate, and where the modified substrate leaves before coenzyme A (CoASH). Thus, the enzyme cycles through four distinct states: apo, AcCoA complex, ternary complex, and CoASH complex. Two out of the four states (AcCoA and CoASH complexed forms) had already been structurally characterized. In addition, diverse mutagenesis and kinetic studies have determined many aspects of the kinetic mechanism governing the enzyme. However, the most important states (apo and ternary state) remain uncharacterized and no conclusive AAC(6')-Ii reaction mechanism has been proposed.Here, we report structural studies for the two remaining states of AAC(6')-Ii. X-ray crystallographic studies of the apo and ternary complex permitted us to describe conformational changes between the different states, define the active site/binding pocket of the enzyme, and rationalize its broad substrate specificity. Complementary solution methods such as nuclear magnetic resonance (NMR), circular dichroism (CD) and small angle X-ray scattering (SAXS) allowed us to gain a better understanding of the dynamics of conformational changes between the apo and complexed states. Estimating quantitative binding energies between the enzyme and several aminoglycosides enabled us to postulate a complete reaction mechanism and extend this mechanism to other acetyltransferases. Ultimately, this detailed structural analysis of AAC(6')-Ii allows us to propose avenues to rejuvenate aminoglycoside therapy against multidrug-resistant pathogens such as E. faecium. / La résistance aux antibiotiques est un grave problème pour les infections nosocomiales bactériennes. L'utilisation excessive d'antibiotiques a conduit à l'émergence de bactéries multirésistantes telles que Enterococci. Plusieurs souches d'E. faecium sont résistantes à un large éventail d'antibiotiques, dont les bêtalactamines, les glycopeptides et les aminoglycosides. Le profil de résistance d'E. faecium à plusieurs aminoglycosides est attribué en majeure partie à une acétyltransférase. Cette enzyme, aminoglycoside 6'-N-acétyltransférase de type-Ii AAC(6')-Ii, se sert de l'acétyl-coenzyme A (AcCoA) pour modifier, en position 6', tous les aminoglycosides utilisés en milieux hospitaliers. Ces modifications rendent les aminoglycosides incapables de se lier à la sous-unité 30S du ribosome bactérien, empêchant ainsi l'antibiotique de modifier la traduction des protéines et entraînant donc la suppression de ses propriétés bactéricides.Le but premier de cette étude était de déterminer par quels moyens il serait possible de redonner aux aminoglycosides leur sensibilité, soit en les protégeant d'une modification par AAC(6')-Ii ou en inhibant directement l'enzyme, de sorte qu'elle ne puisse plus modifier les aminoglycosides. À cette fin, nous avons commencé par décrire, à partir de la structure d'AAC(6')-Ii, le cycle des réactions de l'enzyme menant à l'inactivation des aminoglycosides. AAC(6')-Ii utilise un mécanisme séquentiel ordonné, où AcCoA se lie à l'enzyme avant le substrat et où le substrat modifié quitte le site actif avant la coenzyme A (CoASH). Ainsi, la réaction enzymatique se décrit en quatre états distincts: apo, complexe AcCoA, complexe ternaire et complexe CoASH. Deux de ces états (AcCoA et CoASH complexés) ont déjà été caractérisés structurellement. De plus, diverses mutagenéses et études cinétiques ont minutieusement déterminé de nombreux aspects des mécanismes cinétiques régissant l'enzyme. Toutefois, les états les plus importants (apo et ternaire) sont toujours indéterminés et aucun mécanisme concluant n'a été proposé pour l'activité d'AAC(6')-Ii.Nous présentons ici les deux états enzymatiques manquants d'AAC(6')-Ii. Les cristallographies par rayons X des complexes apo et ternaire nous ont permis de décrire les changements de conformation entre les différents états, de définir le site de liaison et le site actif de l'enzyme, et de rationaliser son large spectre de substrats. Grâce à des méthodes complémentaires telles que la résonance magnétique nucléaire (RMN), le dichroïsme circulaire (CD) et la diffusion de rayons X à petit angle (SAXS) nous avons pu mieux comprendre la dynamique qui guide les changements de conformation entre les états apo et complexés. Des estimations quantitatives des énergies de liaison entre l'enzyme et les aminoglycosides nous ont ainsi permis de proposer un mécanisme réactionnel complet pour AAC(6')-Ii et d'étendre ce mécanisme à d'autres membres de la famille des acétyltransférases. Pour conclure, ces études détaillées de la structure d'AAC(6')-Ii nous permettent de proposer des pistes vers l'élaboration de solutions pour surmonter la résistance aux aminoglycosides de pathogènes tels que E. faecium.