Prion diseases are fatal neurodegenerative disorders of humans and animals. The prion hypothesis states that PrPSc, a misfolded conformational isoform of the cellular prion protein (PrPC), is the sole component of the infectious particle. Many open questions exist in prion biology including the cellular role of PrPC, the potential involvement of auxiliary factors in prion replication, and the mechanism of PrPSc-induced toxicity in prion disease. The identification of novel prion-like proteins and authentic in vivo prion protein-interacting proteins would certainly assist the process of demystifying these unsolved mysteries. Accordingly, two newly-identified proteins with potential relevance to prion protein biology, Shadoo and DPPX, were selected for biochemical and functional characterization. Shadoo, a hypothetical prion-like protein, is revealed as being a glycoprotein which possesses many overlapping properties with PrPC including neuronal expression, C1-like endoproteolytic processing, and the ability to protect against apoptotic stimuli in cerebellar neurons. Shadoo loosely resembles the disordered N-terminal domain of PrPC and consistent with this notion, Shadoo appears to lack a well-defined structure. Remarkably, Shadoo levels in the brains of mice with clinical prion disease are significantly decreased suggesting that Shadoo may be inherently linked to prion replication or prion disease pathogenesis. These experiments define Shadoo as the third member of the prion protein family and, because of its functional similarities to PrPC, Shadoo may be a useful tool for deciphering the in vivo function of PrPC. DPPX, a neuronal type II transmembrane protein, is demonstrated to be the first protein capable of interacting with all three members of the prion protein family (PrPC, Doppel, and Shadoo) in vivo. Complex formation between prion proteins and DPPX appears to be mediated by multiple binding sites. When coupled with high levels of DPPX expression in cerebellar granular neurons, DPPX is a strong candidate for mediating phenotypic interactions between prion proteins in cerebellar cells. Thus, Shadoo and DPPX comprise two new entry points for studying prion proteins. Further investigation of the roles of Shadoo and DPPX in both the cell biology of prion proteins and prion disease may yield important clues to these enigmatic topics.

Prion diseases are fatal neurodegenerative disorders of humans and animals. The prion hypothesis states that PrPSc, a misfolded conformational isoform of the cellular prion protein (PrPC), is the sole component of the infectious particle. Many open questions exist in prion biology including the cellular role of PrPC, the potential involvement of auxiliary factors in prion replication, and the mechanism of PrPSc-induced toxicity in prion disease. The identification of novel prion-like proteins and authentic in vivo prion protein-interacting proteins would certainly assist the process of demystifying these unsolved mysteries. Accordingly, two newly-identified proteins with potential relevance to prion protein biology, Shadoo and DPPX, were selected for biochemical and functional characterization. Shadoo, a hypothetical prion-like protein, is revealed as being a glycoprotein which possesses many overlapping properties with PrPC including neuronal expression, C1-like endoproteolytic processing, and the ability to protect against apoptotic stimuli in cerebellar neurons. Shadoo loosely resembles the disordered N-terminal domain of PrPC and consistent with this notion, Shadoo appears to lack a well-defined structure. Remarkably, Shadoo levels in the brains of mice with clinical prion disease are significantly decreased suggesting that Shadoo may be inherently linked to prion replication or prion disease pathogenesis. These experiments define Shadoo as the third member of the prion protein family and, because of its functional similarities to PrPC, Shadoo may be a useful tool for deciphering the in vivo function of PrPC. DPPX, a neuronal type II transmembrane protein, is demonstrated to be the first protein capable of interacting with all three members of the prion protein family (PrPC, Doppel, and Shadoo) in vivo. Complex formation between prion proteins and DPPX appears to be mediated by multiple binding sites. When coupled with high levels of DPPX expression in cerebellar granular neurons, DPPX is a strong candidate for mediating phenotypic interactions between prion proteins in cerebellar cells. Thus, Shadoo and DPPX comprise two new entry points for studying prion proteins. Further investigation of the roles of Shadoo and DPPX in both the cell biology of prion proteins and prion disease may yield important clues to these enigmatic topics.

Protein aggregation and physical stability are perpetual concerns in medicine and industry. Misfolded protein can form ordered protein aggregates, amyloids, which are associated with a host of neurodegenerative diseases in mammals and control heritable traits in fungi and yeast. Industrially, amorphous aggregates reduce the efficacy of protein-based therapeutics and activity of enzymes during production and storage. This work studies ion-specific and solvent-based effects on protein physical stability. We show that ion-specificity significantly affects amyloid formation kinetics, aggregate morphology, thermostability, frangibility, and, most intriguingly, prion infectivity in vivo. Forming amyloid in chaotropic or kosmotropic solutions generates predominately weak or strong prion variants, respectively. Ion-specific effects also influenced amorphous aggregation of model proteins and antibodies. To quantify protein - protein stability/affinity, we developed a rapid and reliable diffusion-based technique. Our technique was able to resolve relative differences in colloidal stability between various saline and saccharide solutions. In all, this dissertation expands our understanding of ion-specific and water-mediated interactions with prion proteins and protein dispersions.

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.

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Un défi majeur de l’ère post-génomique est de définir l’ensemble des protéines encodées par le génome : le protéome. Un ARNm mature est en général associé à un seul cadre ouvert de lecture (ORF, open reading frame en anglais) de référence (RefORF) codant pour une protéine. Des ORFs alternatifs (AltORFs) sont cependant présents dans les régions non-traduites (UTRs), ou chevauchant le RefORF dans les cadres de lecture alternatifs +2 et +3. Les AltORFs offrnt le potentiel d’augmenter la diversité protéique, mais leur réelle contribution au protéome est peu caractérisée. Par des techniques de biologie moléculaire, de biochimie, et de biologie cellulaire, j'ai tout d’abord mis en évidence chez plusieurs mammifères supérieurs, l’expression endogène d'une protéine alternative appelée AltPrP à partir du gène PRNP. La découverte d'AltPrP devrait améliorer notre compréhension des rôles pathologiques et physiologiques de ce gène. Suite à la découverte d'AltPrP, et basé sur ce modèle d’AltORF chevauchant un RefORF, j'ai entrepris d’investiguer l’étendue de l’utilisation de ces AltORFs comme source de diversité protéique, chez l’humain en particulier. Par des méthodes computationnelles, j'ai participé à la création d'une base de données d'AltORFs prédits dans les ARNm humains (HA1tORF, pour Human Alternative ORFs). HA1tORF est consultable et interrogeable en ligne. Elle facilitera et accélérera la découverte et l’étude des AltORFs. J'ai ensuite mis au point une approche protéomique afin d'apporter des preuves expérimentales de l’utilisation à grande échelle des AltORFs. Une base de données d’AltORFs, mise à jour pour inclure ceux chevauchant en tout ou partie les UTRs, a été créée et utilisée pour déterminer par spectrométrie de masse la contribution des protéines alternatives au protéome humain. J'ai validé l’expression de 1259 protéines alternatives prédites à travers différents échantillons. J'ai aussi démontré que l’expression d'AltORFs impliquait d’importants biais dans les dessins expérimentaux (transfections ou criblage de banques d’ADNc par exemple). Enfin, un grand nombre de protéines alternatives semblent conservées à travers l’évolution, suggérant leur importance fonctionnelle. En conclusion, mes travaux de doctorat ont permis de mettre en évidence que les AltORFs conduisent à l’expression de nouvelles protéines jusqu’alors ignorées. Ces résultats redéfinissent notre vision du protéome, remettent en question notre compréhension de la structure et de la fonction des gènes eucaryotes, et ouvrent la voie vers l’étude fonctionnelle des protéines alternatives.

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Orientador: Profa. Dra. Giselle Cerchiaro / Dissertação (mestrado) - Universidade Federal do ABC. Programa de Pós-Graduação em Ciência e Tecnologia/Química, 2014. / A proteina prion celular (PrPC) e expressa em varios tipos celulares, especialmente no tecido nervoso. Sua funcao fisiologica completa ainda nao e bem conhecida, no entanto,sua influencia no desenvolvimento de doencas neurodegenerativas foi bem descrito. Essas doencas, mais tarde denominadas encefalopatias espongiformes, resultam de uma mudanca conformacional da PrPC (rica em ¿¿ helices) para a forma Scrapie (PrPSC, rica em folhas ¿À). Diversos estudos mostraram a associacao da PrPC, o metabolismo do cobre e a atividade antioxidante da superoxido dismutase (SOD). Contudo, mecanismos de atuacao da PrPC, neste contexto, permanecem indefinidos. Considerando o papel protetor da PrPC amplamente reportado na literatura, foram comparados os comportamentos de celulas de astrocitos de camundongo do tipo selvagem (WWT), e nocaute para a PrPC (WKO), sob condicoes de estresse oxidativo induzido por peroxido de hidrogenio. A ativacao de caspases 3 e 8 foi avaliada, observando-se que a linhagem WKO sofre apoptose de maneira mais lenta que a linhagem WWT; e que a ativacao da apoptose na linhagem nocaute ocorre pela via extrinseca, enquanto que na linhagem selvagem ocorre pela via intrinseca. Os niveis de atividade e expressao da enzima SOD1 tambem foram acompanhados, mostrando que a PrPC modula a ativacao de SOD1 a curto prazo, mas tambem induz o aumento da expressao a medio prazo. No caso da WKO, os niveis de atividade e expressao de SOD1 se mantiveram constantes. Testes de viabilidade celular confirmaram que a linhagem WKO morre mais lentamente, e os parametros bioquimicos de oxidacao (TBARS e carbonilacao de proteinas) elevados mostraram que o delay observado e potencialmente prejudicial a cultura. Os niveis de expressao de APP bem como de GFAP foram monitorados, mostrando que a linhagem selvagem possui menor pre-disposicao a formacao de agregados proteicos e maior capacidade de recuperacao pos-estresse. / The cellular prion protein (PrPC) is expressed in many cell types, especially in nervous tissue. Its physiological function is still unclear, however, his influence on the development of neurodegenerative diseases has been well described. These diseases, later called spongiform encephalopathies, are the result of a conformational change of PrPC (rich in á-helix) to the scrapie form (PrPSC rich in â-sheet). Several studies have demonstrated the association between PrPC, copper metabolism and antioxidant activity of Cu,Zn-Superoxide Dismutase (SOD1). However, action mechanisms of PrPC in this context remain undefined. Considering the protective role of the PrPC widely reported in the literature, the behavior of mouse astrocytes cells were compared to wild type (WWT), and knockout to PrPC (WKO), under conditions of oxidative stress induced by hydrogen peroxide. The activation of caspase 3 and 8 and has been evaluated, and it was observed that the knockout strain undergoes apoptosis more slowly than the wild type strain. The activation of apoptosis in the knockout strain occurs by the extrinsic route, whereas the wild type strain occurs by the intrinsic pathway. Activity levels and expression of SOD1 were also followed, showing that PrPC modulates short-term activation SOD1, and also induces increased expression in the medium term. The WKO cell type, keep activity levels and SOD1 expression constant. Cell viability tests confirmed that the strain WKO die more slowly, and biochemical parameters oxidation (TBARS and protein carbonylation) showed that the higher observed delay is potentially harmful to the cell culture. APP and GFAP expression levels were monitored, showing that the wild type has a lower predisposition to the formation of protein aggregates and higher capacity to post-stress recovery.

The conformational conversion of the cellular prion protein (PrPc) to the misfolded isoform (PrPsc) is the central pathogenic event in the transmissible neurodegenerative prion diseases. The recently shown transmissibility of variant Creutzfeldt-Jakob disease by blood transfusion emphasizes the need for better understanding of the PrPc in blood. In the current thesis, we focused on blood platelet PrPc, which has not been very well described so far. In the first part of the thesis, platelet PrPc was characterized as glycosylphosphatidylinositol- anchored glycoprotein with dominant diglycosylated form. Platelet PrPc was shown to be sensitive to cleavage with proteinase K, which is a feature discriminating between cellular and pathological prion protein. We have confirmed that platelet PrPc binds copper ions by its N- terminal octapeptide repeat region. Regarding quantity of PrPc molecules expressed on blood elements we have proved that both platelets and red blood cells express considerable amount of PrPc and thus can not be neglected in the problematic of prions transmission by blood transfusion. The detailed study regarding PrPc localization in blood platelets is presented in the second part of the thesis. PrPc was shown to be expressed in -granules as well as on the cytoplasmic membrane of...