No description available.

No description available.

N-glycosylated proteins that traverse the endoplasmic reticulum (ER) can make use of the calnexin cycle to attain their correct fold. The calnexin cycle modifies the N-glycan structure and allows for association of the glycoprotein with the ER lectins calnexin and calreticulin, which in turn recruit further chaperones that assist folding. Most enveloped viruses encode glycoproteins, which, upon infection of a host cell, crucially depend on the calnexin cycle to aid their folding. This includes diverse families such as Flaviviridae, Retroviridae and Orthomyxoviridae. We studied the calnexin cycle components with the ultimate aim of developing broad-spectrum antivirals. X-ray crystallography was used to structurally characterise the murine ER α-glucosidase I, which controls entry into the calnexin cycle, with a number of inhibitory antiviral iminosugars. These data reveal flexibility in the ligands' alkyl tails and may act as a basis for the discovery of enzyme specific inhibitors. UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the quality control checkpoint of the calnexin cycle whose full-length structure from the thermophilic fungus Chaetomium thermophilum was recently determined. Presented here are a higher resolution structure in addition to SAXS studies of UGGT's interaction with Sep15, a protein that enhances UGGT activity. UGGT's reaction releases into the ER lumen UDP, which is the only known small molecule inhibitor of UGGT. An ER-resident UDPase, ENTPD5, breaks down UDP into UMP. Enzymatic characterisation of ENTPD5 reveals its substrate specificities; in addition we show a paralog, ENTPD6, possesses similar activities. Presented here is work towards crystallisation of these two proteins and a test of the anti-Zika activity of ENTPD5 inhibitors. Finally, CRISPR/Cas9 knock-out cells were generated to test, in principle, whether modulation of the activity of proteins involved in the calnexin cycle could be antiviral. The data confirm that the ER glucosidases are likely the best targets of those studied.

Major Histocompatibility Complex (MHC) class I receptors are glycoproteins which play a critical role in anti-viral immunity by displaying foreign peptides to cytotoxic T cell lymphocytes. The loading of high affinity peptides into the MHC class I receptor in mammals is coordinated by a multiple proteins that are collectively referred to as the peptide loading complex (PLC). To date, the composition of the peptide loading complex in fish is unknown and therefore the characterization of the molecules which may exist in this putative complex was pursued. This thesis includes the cloning and functional characterization of ERp57 and calnexin in rainbow trout which, in mammals, are known to interact with the MHC class I receptor either during its early biogenesis or later in the assembly of the PLC. Trout ERp57 and calnexin cDNA sequences are ubiquitously expressed in trout tissues and both the ERp57 and calnexin genes appear in at least two copies each in the trout genome. Interestingly, despite their high sequence identity with their mammalian homologues, some structural discrepancies were identified. ERp57 does not contain an endoplasmic reticulum (ER) retention signal or a nuclear localization signal, while one of the two isolated cDNA clones for calnexin does not contain an ER (endoplasmic reticulum) retention signal and lacks a conserved C-terminal serine phosphorylation site. These findings suggest that in trout, there may be unique versions of these proteins that have acquired different cellular functions. Through the production of polyclonal antibodies against trout ERp57, the conserved protein induction of ERp57 during ER stress was demonstrated concurrently with calnexin. In addition, this study shows for the first time that ERp57 can be induced transcriptionally by phytohemagglutinin and synthetic double stranded RNA, which implies its possible regulatory role during viral infection and the activation of the immune response. Furthermore, the functional characterization of the MHC class I specific chaperone tapasin, a key element in the PLC of mammals was pursued. Tissue and cell line distribution revealed that tapasin is expressed in high levels in immune system organs and in the rainbow trout macrophage cell line RTS11, at a relative molecular weight of 48 kDa with an additional 20 kDa band detected by the tapasin antibody. Tapasin protein was significantly up regulated upon exposure to synthetic double stranded RNA and during infection with two fish viruses: chum salmon virus and viral hemorrhagic septicemia virus genotype IVa, whereas the expression of the 20 kDa band was not affected by these stimuli. This study also examined the regulation of the MH class I heavy chain,β2 microglobulin and their associated machinery upon exposure to viral hemorrhagic septicemia virus genotype IVa at permissive and non-permissive temperatures. β2 microglobulin secretion into the cell media, a marker of MH class I receptor turnover, was detected in the conditioned media of RTS11 cells under normal conditions and was shown to be significantly enhanced during viral hemorrhagic septicemia virus genotype IVa infection. Furthermore, when RTS11 cells were maintained at cold temperatures, the secretion of β2 microglobulin was significantly reduced in both infected and non-infected cultures, while the cellular levels of β2 microglobulin remained unchanged. These results suggest that cold temperature can alter the expression of the MH class I molecule on the cell surface and therefore may be contributing to host susceptibility to viral hemorrhagic septicemia virus genotype IVa during the winter. Lastly, Co-immunoprecipitation demonstrated the interaction of the lectin chaperones: calnexin and calreticulin with the glycosylated MH class I receptor supporting their conserved role during MH class I receptor folding in fish. Concurrently, tapasin's interaction with transporter associated with antigen processing (TAP) and with the glycosylated form of the MH class I was revealed for the first time in fish, which supports their role in antigen presentation as in mammals. This study demonstrated that ERp57 and tapasin form a conserved disulfide linked heterodimer of 110 kDa, however unlike mammals, an additional 75 kDa heterodimer was detected which suggests a possible novel interaction of ERp57 with a 20 kDa tapasin version alternately regulating antigen presentation in fish. Overall, this study suggest that the interactions involved in antigen presentation in mammals are conserved in fish, however the presence of different protein versions of calnexin, ERp57 and tapasin might dictate a different mode of regulation for MH class I assembly in fish, as opposed to mammals. Elucidating these interactions during various viral infections in fish can help to uncover possible viral strategies to manipulate the host immune response and will provide information needed to assist in designing novel tools to prevent fish viral diseases.

Le repliement des protéines est un processus cellulaire crucial impliquant plusieurs protéines dont la calnexine, une chaperone du réticulum endoplasmique. Notre laboratoire et un autre groupe avons démontré que la calnexine est essentielle à la viabilité de la levure Schizosaccharomyces pombe. Dans le cadre d’études structure-fonction portant sur cette protéine, nous avons découvert un phénomène permettant la viabilité des cellules en absence de la calnexine. Cet état, nommé Cin pour calnexine independence, est induit par un mutant de la calnexine dépourvu du domaine central hautement conservé (Δhcd_Cnx1p). La caractérisation de l’état Cin a révélé plusieurs caractéristiques particulières telle la dominance, sa transmission de façon non-Mendélienne à la progéniture méïotique et sa transmission par des extraits protéiques dépourvus d’acides nucléiques. Toutes ces propriétés suggèrent donc que l’état Cin est médié via un élément de type prion. Le gène cif1+, pour calnexin independence factor, a été isolé lors de criblages visant à identifier des gènes impliqués dans l’état Cin. Il encode pour une protéine orpheline dont la surexpression induit de façon stable un état de viabilité en l’absence de la calnexine. Cet état diffère génétiquement et phénotypiquement de l’état Cin induit par le mutant Δhcd_Cnx1p préalablement caractérisé, ce qui suggère deux voies parallèles de signalisation du phénomène Cin. Une caractérisation exhaustive de Cif1p a permis de démontrer qu’il ne s’agissait pas du prion responsable de l’état Cin, malgré que cette protéine possède certaines propriétés typiques des prions in vitro. Finalement, Cif1p est une protéine nucléolaire dont la bonne localisation est essentielle à sa capacité à induire l’état Cin. Ceci suggère une interaction entre la fonction essentielle de la calnexine et une fonction exécutée dans le nucléole. Lors d’études visant à élucider la fonction cellulaire de Cif1p, il a été établi qu’elle interagissait avec certaines protéines de la grosse sous-unité du ribosome telle la protéine L3. Cependant, Cif1p ne co-sédimente pas avec des sous-unités ribosomales assemblées, des ribosomes ou des polysomes. De plus, des cellules contenant une délétion génomique de cif1 voient leur contenu en ribosomes perturbé lors de la phase stationnaire. Il semble donc que Cif1p joue un rôle dans la biosynthèse des ribosomes lors de la phase stationnaire. Ce rôle spécifique à cette phase de croissance coincide avec un clivage de la portion N-terminale de Cif1p, clivage qui a lieu lors de l’entrée des cellules en phase stationnaire. De plus, des études effectuées récemment dans notre laboratoire proposent que la calnexine joue un rôle important dans la signalisation de l’apoptose, et ce particulièrement en phase stationnaire. Ainsi, une voie impliquant Cif1p, sa fonction nucléolaire dans la biosynthèse des ribosomes en phase stationnaire, la calnexine et la médiation de l’apoptose semble se dessiner. D’autres travaux, notamment sur la fonction exacte de Cif1p, le rôle de son clivage et les autres composantes impliquées dans le phénomène Cin nous permettront de dessiner un portrait plus complet de cette voie cellulaire inédite. / Protein folding is a vital process that involves many proteins of the cell. One of them is calnexin, a chaperone of the endoplasmic reticulum. In the fission yeast Schizosaccharomyces pombe, calnexin is essential for survival of the cells. During structure-function studies on calnexin, our laboratory discovered a phenomenon allowing the viability of cells without this chaperone. This state, designated Cin for Calnexin INdependence, is induced by a calnexin mutant devoid of the highly conserved central domain (Δhcd_Cnx1p). Characterization of the Cin cells showed several exceptional properties such as dominance, non-Mendelian transmission and transmission via cell extracts devoid of nucleic acids of the Cin state. All these observations suggested that the Cin phenomenon is mediated via a prionic element. To identify genes implicated in the Cin state, genetic screens were performed. They led to the identification of the cif1+ gene, for calnexin independence factor. This gene encodes an orphan protein, the overexpression of which stably induces a state of viability in the absence of calnexin. Notably, this state is genetically and phenotypically distinct from the previously isolated Cin state arising from Δhcd_Cnx1p expression. This suggests the presence of two parallel pathways both able to signal the induction of the Cin phenomenon. The exhaustive characterization of Cif1p showed that it is not the prion solely responsible for the Cin state, although it displays prion-like properties in vitro. Finally, nucleolar localization of Cif1p is required to induce the Cincif1 state, thus suggesting an unexpected interaction between the vital cellular role of calnexin and a function of the nucleolus. While investigating Cif1p function in the cell, we observed that it interacts with ribosomal proteins of the large subunit, notably L3, but it does not sediment with assembled ribosomal subunits or whole ribosomes. However, cells containing a genomic deletion of cif1 also have a disrupted ribosome content during stationary phase. Altogether, these results suggest that Cif1p has a role in ribosomal biogenesis during stationary phase. This growth-phase specific role correlates with the occurence during stationary phase of a cleavage in the N-terminal part of Cif1p. Recent studies from our laboratory proposed that calnexin plays an important role in apoptosis signaling, especially in stationary phase. Thus, a pathway implicating Cif1p, its nucleolar function in ribosome biosynthesis in stationary phase, calnexin and apoptosis signaling is starting to emerge. However more studies, notably on the exact function of Cif1p, the role of its cleavage and the other proteins implicated in the Cin state will be necessary to draw the complete scheme of this unprecedented cellular pathway.

No description available.

No description available.

No description available.

Iminosugars are a class of small molecules defined by substitution of a sugar’s ring oxygen with nitrogen. Various chemical modifications of these basic structures (e.g. alkyl chain addition off of the ring nitrogen) have been developed during the last several decades. These molecules have been considered as therapeutics for a number of pathologies including viral infection, congenital disorders of glycosylation (of both glycoproteins and glycolipids), and diabetes. This thesis focuses on the application of a small subset of iminosugars, known as deoxynojirimycin derivatives, as therapeutics against dengue virus induced pathology. Dengue virus infection predominates in tropical climates, but autochthonous infection has recently emerged in areas of both southern Europe and the southern United States. With 390 million people infected annually, dengue is the most prevalent arthropod-borne viral infection worldwide, and the possibility of severe pathology including haemorrhage, shock, and/or death, necessitates development of effective antiviral therapies. Although the molecular mechanisms responsible for progression to severe dengue disease are not completely understood, there is considerable evidence for the role of both the innate and the adaptive immune responses in development of life-threatening complications. Excessive activation of the innate immune response, a phenomenon known as cytokine storm, has been hypothesised to explain development of symptoms related to vascular permeability, whereas the adaptive immune response has been implicated in severe disease through two hypotheses – the antibody dependent enhancement and original antigenic sin hypotheses. The evidence regarding each of these potential mechanisms of severe pathology is discussed throughout this thesis principally with respect to how iminosugar treatment could alter any detrimental effects of the immune response to dengue virus infection. The principal aim of this thesis is to consider the potential of deoxynojirimycin iminosugars as antiviral therapeutics in dengue infection with a focus on how these molecules exert their antiviral effects in primary human cells. I first consider the contributions of glycoprotein inhibition and glycolipid inhibition on production of infectious dengue virus. These experiments suggest that inhibition of glycoprotein folding is responsible for inhibition of infectious dengue virus production. I next consider the impact of treatment of a promising clinical candidate iminosugar, N9-methoxynonyl-deoxynojirimycin (MON-DNJ), on the primary human macrophage transcriptome. In uninfected macrophages as well as macrophages infected with dengue virus or treated with lipopolysaccharide to model bacterial sepsis, iminosugar treatment results in activation of the unfolded protein response and inhibition of several elements of the inflammatory response including signalling by the cytokines IFN-γ and TNF-α, and the inflammatory cascade mediated by NF-κB. Activation of the unfolded protein response as a result of treatment with MON-DNJ can be confirmed by analysis of phosphorylated (activated) NFE2L2, a transcription factor that functions principally to control oxidative stress in response to ER stress signals. Modulation of the inflammatory response of macrophages to dengue infection and bacterial sepsis is confirmed by analysis of secreted cytokines. As predicted by my transcriptomic experiments, levels of TNF-α and IFN-γ produced in response to dengue or lipopolysaccharide are reduced by treatment with MON-DNJ. Finally, I attempted to extend these observations to an animal model of dengue infection with a particular focus on TNF receptor and ligand superfamily members. Unfortunately, heterogeneity of cells types from tissue samples as well as limitations of the animal model complicate interpretation of these findings. Nevertheless, this thesis demonstrates that MON-DNJ is an effective dengue antiviral therapeutic and that this therapeutic activity may be related to both reduction of infectious virus as a consequence of inhibition of glycoprotein processing and as a result of changes to the host’s response to the pathogen. These results have been used in part to justify recently initiated clinical trials of MON-DNJ as a dengue antiviral therapy.

La mort cellulaire programmée (PCD pour Programmed Cell Death) est un processus essentiel aux cellules. Le PCD a d’abord été caractérisé dans le développement cellulaire et peut être divisé en plusieurs groupes selon les caractéristiques observées. L’apoptose, un sous-groupe du PCD, est caractérisé par plusieurs distinctions morphologiques et signalétiques attribué tout d’abord aux organismes complexes pour son rôle dans le développement et dans le maintien de l’intégrité tissulaire. Depuis la dernière décennie, de nombreuses études font état de l’existence d’un programme apoptotique dans des organismes unicellulaires comme les levures. Ce programme apoptotique a surtout été étudié chez les levures Saccharomyces cerevisiae et Schizosaccharomyces pombe et partage certaines caractéristiques avec l’apoptose des mammifères. Par contre, l’apoptose associé aux levures est distinct à certains égards entre autre par l’absence de certains homologues présents chez les mammifères. L’intérêt au niveau de l’étude du phénomène apoptotique chez les levures est sans cesse grandissant par la facilité avec laquelle les levures peuvent être utilisées comme système modèle. L’apoptose peut être induit dans les cellules de différentes façons en réponse à des stimuli internes ou externes. L’accumulation de protéines mal repliées au niveau du réticulum endoplasmique (RE) causant un stress est un inducteur bien caractérisé de la voie apoptotique. La signalisation de l’apoptose dans un cas de stress au RE fait appel aux transducteurs des signaux de la voie du UPR ( Unfolded Protein Response). Récemment, il a été montré que la calnexine, une chaperone transmembranaire du RE connue et caractérisée surtout pour ses fonctions d’aide au repliement des protéines et au contrôle de qualité, joue un rôle dans la transduction du signal apoptotique en réponse au stress du RE chez mammifères. Le rôle de la calnexine dans ce cas consiste principalement en l’échafaudage pour le clivage par la caspase 8 de la protéine apoptotique Bap31. Nous avons tout d’abord démontré que le stress du RE et que la déficience en inositol, un précurseur essentiel de nombreuses molécules signalétiques, sont deux inducteurs de l’apoptose chez la levure S. pombe. Ces deux voies semblent induire l’apoptose par deux voies distinctes puisque seule la voie de la déficience en inositol induit l’apoptose de façon dépendante à la métacaspase Pca1p. La calnexine, essentielle à la viabilité chez la levure S. pombe, est impliquée dans ces deux phénomènes apoptotiques. L’apoptose induit par le stress du RE nécessite une version de la calnexine ancrée à la membrane du RE pour être optimal. De façon opposée, l’apoptose induit par une déficience en inositol nécessite la présence de la queue cytosolique ancrée à la membrane de la calnexine pour être retardé. Ces deux actions différentes imputables à une même protéine laisse croire à une double fonction pro et anti-apoptotique de celle-ci. Suite à la découverte de l’existence d’un clivage endogène de la calnexine en situation normale de croissance, un modèle a été élaboré expliquant les rôles distincts de la calnexine dans ces deux voies apoptotiques. Ce modèle fait état d’un rôle associé au clivage de la calnexine dans l’apoptose. / Programmed Cell Death (PCD) is an essential process to the cells. PCD was first characterized in cell development and can be separated in sub-groups depending of cell death characteristics observed. Apoptosis is one of the PCD sub-groups that was first associated to complex organisms for its roles in cell development and in maintenance of tissues integrity. The apoptotic pathway is characterized by specific morphological and signalization characteristics. In the last ten years, numerous studies demonstrated the existence of apoptosis in unicellular organisms such as yeast. This apoptotic program was extensively studied in the two yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe and share characteristics with the mammalian one. However, yeast apoptosis is distinctive at many points as yeast do not encodes all the mammalian homologues of the apoptotic pathway. Although yeast and mammalian apoptosis seems to differs, the interest about yeast apoptosis is growing given that yeast is an excellent and easily tractable model system. External and internal stimuli can induce apoptosis by different ways. Accumulation of unfolded or incompletely folded proteins in the endoplasmic reticulum (ER) causing ER stress is a well-known inducer of the apoptotic pathway. Signalization of ER-stress induced apoptosis involves the same transducers than the UPR (Unfolded Protein Response) pathway. It was recently shown that calnexin, a transmembrane chaperone of the ER, is implicated in ER-stress apoptosis in mammalian cells. In this particular case, it was demonstrated that calnexin acts as a scaffold in the cleavage of the apoptotic protein Bap31 by caspase 8. We demonstrated that ER stress and inositol deficiency, a precursor of many important signalization molecules, are two situations leading to apoptosis in the yeast S. pombe. These two pathways leading to apoptosis seems to differ as only inositol deficiency is dependant of the yeast metacaspase Pca1p. We also demonstrated that S. pombe calnexin, essential for cell viability of this yeast, takes part in these two apoptotic process. ER stress induced apoptosis needs a calnexin anchors to the ER membrane to be efficient. However, apoptosis induced by inositol starvation needs the calnexin C-terminal tail with the transmembrane domain to be delayed. These two opposite actions from the same protein lead to the hypothesis that calnexin encodes both pro and anti-apoptotic functions. By the discovery that calnexin is cleaved under normal culture conditions, a model was elaborated explaining the distinctive roles of calnexin in these two apoptotic pathways. This model proposed a role of calnexin cleavage to apoptosis.