La mort cellulaire par apoptose est un processus biologique fondamental, nécessitant des interactions fines avec le système immunitaire pour une reconnaissance et une élimination efficace des cellules mortes. C'est ainsi que C1q, une molécule du complément, essentielle dans le système immunitaire inné, a été mise en évidence comme fortement impliquée dans le mécanisme de reconnaissance et d'élimination des cellules apoptotiques, notamment via sa région globulaire (C1qGR). Récemment, la translocation de la calréticuline (CRT) au niveau externe de la membrane plasmique des cellules cancéreuses a été identifiée comme un signal « eat-me » pouvant être immunogène. Initialement, l'interaction entre la CRT et C1q a été caractérisée à la surface des phagocytes et la CRT a été considérée comme un récepteur pour les queues collagènes de C1q (C1qCLF). L'ensemble de ces observations est en faveur d'un double rôle de la CRT, à la surface des phagocytes et des cellules apoptotiques. Dans un premier temps, l'élaboration d'une stratégie de FRET a permis de détecter une interaction directe entre la CRT et C1qGR à la surface de la cellule HeLa en apoptose précoce. Dans un second temps, la mise en place de tests de phagocytose a permis de montrer que la calréticuline exposée à la surface des cellules apoptotiques peut moduler la phagocytose : des effets opposés ont été observés selon le modèle cellulaire utilisé (HeLa traitées par des ARNi ou MEF CRT-/-) et dans certaines conditions, une modulation combinée de la calréticuline et de C1q a été observée sur la réponse inflammatoire (production de cytokines). / Cell death by apoptosis is a fundamental biological process, requiring fine interactions with the immune system for the effective recognition and removal of the apoptotic cells. C1q, a complement molecule, essential in the innate immune system, has been shown to be strongly involved in the mechanism of recognition and elimination of apoptotic cells, mainly through its globular region (C1qGR). Recently, the translocation of calreticulin (CRT) at the surface of cancer cells has been identified as an eat-me signal, which can be immunogenic. Initially, the interaction between CRT and C1q was characterized on phagocytes surface and CRT was described as a receptor for the collagenous tails of C1q (C1qCLF). All these observations support a dual role of CRT at the surface of phagocytes and their targets. At first, using a FRET strategy we achieve to detect a direct interaction between CRT and C1qGR at the surface of early apoptotic HeLa cells. Second, the establishment of phagocytosis assays showed that calreticulin exposed at the surface of apoptotic cells could modulate phagocytosis: opposite effects were observed depending on the cellular model used (RNAi treated HeLa or MEF CRT-/-) and under certain conditions, a combined modulation of calreticulin and C1q was observed on the inflammatory response (cytokine production).

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Class I major histocompatibility complex molecules present antigenic peptides to cytotoxic T lymphocytes, which leads to the elimination of virus infected cells. Class I molecules are heterotrimers consisting of a heavy chain, a light chain termed beta2-microglobulin, and a peptide ligand. Assembly of class I molecules begins in the endoplasmic reticulum where the heavy chain associates with beta2-microglobulin, and the heavy chain-beta2-microglobulin heterodimers enter a peptide loading complex where class I molecules acquire peptides. During the biogenesis of class I molecules, ERp57, a thiol oxidoreductase, associates with free class I heavy chains and, at a later stage, with the peptide loading complex. In this thesis, I show for the first time that ERp57 participates in oxidative folding of the heavy chain. Depletion of ERp57 by RNAi delayed heavy chain disulfide bond formation and slowed folding of the heavy chain alpha3 domain. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no such effect. Since ERp57 associates with the lectin-chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, I examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells, suggesting that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin-chaperones. ERp57 also forms a mixed disulfide with tapasin within the peptide loading complex and I found that the formation of this mixed disulfide is independent of its interaction with calnexin and calreticulin. I also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide-binding site in substrate recognition. Finally, by inactivating the redox active sites of ERp57, I demonstrate that its enzymatic activity is dispensable in stabilizing the loading complex and in supporting efficient peptide loading. Thus, ERp57 plays a structural rather than catalytic role within the peptide loading complex.

Class I major histocompatibility complex molecules present antigenic peptides to cytotoxic T lymphocytes, which leads to the elimination of virus infected cells. Class I molecules are heterotrimers consisting of a heavy chain, a light chain termed beta2-microglobulin, and a peptide ligand. Assembly of class I molecules begins in the endoplasmic reticulum where the heavy chain associates with beta2-microglobulin, and the heavy chain-beta2-microglobulin heterodimers enter a peptide loading complex where class I molecules acquire peptides. During the biogenesis of class I molecules, ERp57, a thiol oxidoreductase, associates with free class I heavy chains and, at a later stage, with the peptide loading complex. In this thesis, I show for the first time that ERp57 participates in oxidative folding of the heavy chain. Depletion of ERp57 by RNAi delayed heavy chain disulfide bond formation and slowed folding of the heavy chain alpha3 domain. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no such effect. Since ERp57 associates with the lectin-chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, I examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells, suggesting that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin-chaperones. ERp57 also forms a mixed disulfide with tapasin within the peptide loading complex and I found that the formation of this mixed disulfide is independent of its interaction with calnexin and calreticulin. I also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide-binding site in substrate recognition. Finally, by inactivating the redox active sites of ERp57, I demonstrate that its enzymatic activity is dispensable in stabilizing the loading complex and in supporting efficient peptide loading. Thus, ERp57 plays a structural rather than catalytic role within the peptide loading complex.

La mort cellulaire par apoptose est un processus biologique fondamental, nécessitant des interactions fines avec le système immunitaire pour une reconnaissance et une élimination efficace des cellules mortes. C'est ainsi que C1q, une molécule du complément, essentielle dans le système immunitaire inné, a été mise en évidence comme fortement impliquée dans le mécanisme de reconnaissance et d'élimination des cellules apoptotiques, notamment via sa région globulaire (C1qGR). Récemment, la translocation de la calréticuline (CRT) au niveau externe de la membrane plasmique des cellules cancéreuses a été identifiée comme un signal " eat-me " pouvant être immunogène. Initialement, l'interaction entre la CRT et C1q a été caractérisée à la surface des phagocytes et la CRT a été considérée comme un récepteur pour les queues collagènes de C1q (C1qCLF). L'ensemble de ces observations est en faveur d'un double rôle de la CRT, à la surface des phagocytes et des cellules apoptotiques. Dans un premier temps, l'élaboration d'une stratégie de FRET a permis de détecter une interaction directe entre la CRT et C1qGR à la surface de la cellule HeLa en apoptose précoce. Dans un second temps, la mise en place de tests de phagocytose a permis de montrer que la calréticuline exposée à la surface des cellules apoptotiques peut moduler la phagocytose : des effets opposés ont été observés selon le modèle cellulaire utilisé (HeLa traitées par des ARNi ou MEF CRT-/-) et dans certaines conditions, une modulation combinée de la calréticuline et de C1q a été observée sur la réponse inflammatoire (production de cytokines).

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.

Ras is a central player in signal transduction that mediates cellular proliferation and differentiation. Recent evidence has shown that lipid and non-lipid modified domains participate in Ras traffic and that plasma membrane association is mediated by vectorial vesicular transport from the endomembrane system. ERp57, an ER chaperone, has been shown to specifically bind farnesylated Hras but not non-farnesylated Hras. The objective of this study was to determine if ERp57 participates in Ras trafficking and function. First, the effect of ERp57 knock down by siRNA technology on Hras function was studied; there was a reduction in ERp57 cellular levels that led to a decrease of active ras. Second, specific anti-ERp57 antibodies were delivered into 3T3 cells expressing GFP-ras chimeras to observe the effect on intracellular trafficking. Anti-ERp57 antibodies blocked Hras plasma membrane localization but not Kras suggesting that ERp57 may be involved in Hras intracellular trafficking and function.