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Com a idéia de identificar proteínas envolvidas no processo de enovelamento das proteínas sintetizadas na glândula salivar de Rhynchosciara americana, no início deste projeto adotou-se como estratégia o seqüenciamento de uma biblioteca de cDNA. Esta biblioteca foi construída utilizando-se glândulas salivares de Rhynchosciara americana do período de seu desenvolvimento onde tem início a síntese de seu casulo. Mensagens de proteínas envolvidas no processo de enovelamento, transporte e proteólise foram isoladas, alguns exemplos são hsc70, hsp83, hip, hop, dnaJ, trap1 e prolil isomerase, sec61α/β, sec23, peptidase de sinal, rab7, partícula reconhecedora de sinal (srp), enzima conjugadora de ubiquitina e complexo regulatório proteassomo 26, cop 1 e ubiquitina ligase. A identificação destes genes permitiu o isolamento de clones genômicos através de triagem em banco de fagos e caracterização dos genes hsc70 e hsp83 para verificação de sua organização em Rhynchosciara americana. A expressão dos seus respectivos mRNAs foi avaliada em vários períodos do último estágio larval. A localização por hibridização in situ mostrou que estes genes estão localizados em regiões dos cromossomos politênicos próximas a dois pufes de DNA, C3 e C8. O estudo dos níveis de expressão das proteínas codificadas pelos genes hsc70 e hsp83 mostrou a diferença de comportamento destes genes sob condições de estresse térmico e que a expressão destas proteínas deve ser regulada pelo período de desenvolvimento das larvas de Rhynchosciara americana. Quando evidenciada por imunofluorescência a proteína Hsc70 mostra localização predominantemente no citoplasma. / With the idea of identify some of these proteins involved in the folding process of the proteins synthesized on the Rhynchosciara salivary gland, this project started adopting the shotgun cDNA sequencing strategy. This cDNA library was constructed utilizing salivary glands of Rhynchosciara americana at a developmental period where the cocoon construction begins. Messengers of important proteins involved in the folding, transport and proteolysis process were isolated, some examples are hsc70, hsp83, hip, hop, sec61 α/β, sec23, signal peptidase, rab7, signal recognition particle (srp), ubiquitin conjugating enzyme e 26 proteasome regulatory complex, cop 1 and ubiquitin ligase. Identification of these genes allowed the screening of genomic clones from a phage library; hsc70 and hsp83 characterization was carried out to verify the arrangement of these genes on genome of Rhynchosciara americana. The study of these genes will contribute with phylogenetic information about the specie. The mRNA expression of these genes was analyzed during several periods of the last larval developmental stage. In situ localization showed that these genes are located in polytene chromosomes regions near two DNAs puffs, C3 and C8. The expression levels of the proteins codified by genes hsc70 and hsp83 showed different behaviors of these genes under heat stress conditions and mainly, that the regulation of the proteins Hsc70 and Hsp83 can be related to the period of development of the larvae of Rhynchosciara americana. When revealed by immunofluorescence, Hsc70 protein shows localization predominantly on the cytoplasm.

Com a idéia de identificar proteínas envolvidas no processo de enovelamento das proteínas sintetizadas na glândula salivar de Rhynchosciara americana, no início deste projeto adotou-se como estratégia o seqüenciamento de uma biblioteca de cDNA. Esta biblioteca foi construída utilizando-se glândulas salivares de Rhynchosciara americana do período de seu desenvolvimento onde tem início a síntese de seu casulo. Mensagens de proteínas envolvidas no processo de enovelamento, transporte e proteólise foram isoladas, alguns exemplos são hsc70, hsp83, hip, hop, dnaJ, trap1 e prolil isomerase, sec61α/β, sec23, peptidase de sinal, rab7, partícula reconhecedora de sinal (srp), enzima conjugadora de ubiquitina e complexo regulatório proteassomo 26, cop 1 e ubiquitina ligase. A identificação destes genes permitiu o isolamento de clones genômicos através de triagem em banco de fagos e caracterização dos genes hsc70 e hsp83 para verificação de sua organização em Rhynchosciara americana. A expressão dos seus respectivos mRNAs foi avaliada em vários períodos do último estágio larval. A localização por hibridização in situ mostrou que estes genes estão localizados em regiões dos cromossomos politênicos próximas a dois pufes de DNA, C3 e C8. O estudo dos níveis de expressão das proteínas codificadas pelos genes hsc70 e hsp83 mostrou a diferença de comportamento destes genes sob condições de estresse térmico e que a expressão destas proteínas deve ser regulada pelo período de desenvolvimento das larvas de Rhynchosciara americana. Quando evidenciada por imunofluorescência a proteína Hsc70 mostra localização predominantemente no citoplasma. / With the idea of identify some of these proteins involved in the folding process of the proteins synthesized on the Rhynchosciara salivary gland, this project started adopting the shotgun cDNA sequencing strategy. This cDNA library was constructed utilizing salivary glands of Rhynchosciara americana at a developmental period where the cocoon construction begins. Messengers of important proteins involved in the folding, transport and proteolysis process were isolated, some examples are hsc70, hsp83, hip, hop, sec61 α/β, sec23, signal peptidase, rab7, signal recognition particle (srp), ubiquitin conjugating enzyme e 26 proteasome regulatory complex, cop 1 and ubiquitin ligase. Identification of these genes allowed the screening of genomic clones from a phage library; hsc70 and hsp83 characterization was carried out to verify the arrangement of these genes on genome of Rhynchosciara americana. The study of these genes will contribute with phylogenetic information about the specie. The mRNA expression of these genes was analyzed during several periods of the last larval developmental stage. In situ localization showed that these genes are located in polytene chromosomes regions near two DNAs puffs, C3 and C8. The expression levels of the proteins codified by genes hsc70 and hsp83 showed different behaviors of these genes under heat stress conditions and mainly, that the regulation of the proteins Hsc70 and Hsp83 can be related to the period of development of the larvae of Rhynchosciara americana. When revealed by immunofluorescence, Hsc70 protein shows localization predominantly on the cytoplasm.

Le lupus érythémateux disséminé est une maladie autoimmune systémique provoquant des lésions tissulaires graves. Notre laboratoire a découvert un peptide phosphorylé, appelé P140, présentant des propriétés thérapeutiques pour le traitement du lupus. Le mode d’action du peptide P140 dans le traitement du lupus repose sur son interaction avec la protéine de choc thermique HSPA8/HSC70 et l’objectif de mon projet de thèse a été de consolider et compléter ce mécanisme. Nous avons démontré qu’après internalisation par endocytose dépendante des clathrines, le peptide P140 se localise rapidement au sein du lysosome des lymphocytes B. Dans cet organelle, il réduit l’import de substrat cytosolique par autophagie dépendante des chaperonnes en ciblant et réduisant l’activité de HSPA8 intralysosomale. Nous avons également entrepris une analyse comparative du répertoire des lymphocytes T et B des souris lupiques par rapport aux souris saines. Nos résultats révèlent un changement dans la fréquence de certains réarrangements du TCR entre les souris lupiques et les souris saines et un effet bénéfique du peptide P140 sur certains réarrangements associés au lupus. / Systemic lupus erythematosus is a multi-organ autoimmune disease provoking tissue damages. Our laboratory has discovered a phosphorylated peptide, named P140, with therapeutic activities in lupus. The mode of action used by P140 peptide relies on its interaction with the heat shock protein HSPA8/HSC70. The aim of my thesis project was to consolidate and complete this HSPA8-dependent mechanism. We have demonstrated that, upon internalization by clathrin-mediated endocytosis, P140 peptide homes rapidly into B cell lysosome. In this organelle, the peptide reduces chaperone-mediated autophagy by interacting and inhibiting intralysosomal HSPA8 activity. We also performed a comparative analysis of T cell and B cell repertoire on lupic mice compared to healthy mice Our results show a modification in the frequency of certain TCR rearrangements between lupus-prone mice and healthy mice and a beneficial effect of P140 peptide on certain lupus-associated rearrangements.

La formation et le dépôt de fibres d'α-Synucléine dans le cerveau humain sont à l‟origine de la maladie de Parkinson. Cette thèse documente le rôle de deux chaperons moléculaires dans l‟assemblage en fibres de l'α-Syn : Hsc70 (protéine de choc thermique constitutivement exprimée chez l‟Homme) et Ssa1p (son équivalent chez la levure). Le but était d'élargir le catalogue d'effets connus des chaperons moléculaires sur α-Syn, pour éventuellement ouvrir la voie à des applications thérapeutiques. Nous avons montré que Hsc70 inhibe l'assemblage de l'α-Syn en fibres, en se liant avec une forte affinité à la forme soluble de l'α-Syn. Hsc70 se lie préférentiellement aux fibres de l'α-Syn, et cette liaison a un effet cytoprotecteur puisqu'elle rend les fibres moins toxiques pour les cellules de mammifères en culture. Pareillement à Hsc70, Ssa1p inhibe l'assemblage de l'α-Syn en fibres, et a une plus forte affinité pour les fibres que pour la forme soluble de l'α-Syn. En revanche, la liaison de Ssa1p aux fibres de l'α-Syn n'a pas d'effet cytoprotecteur, sûrement due aux différences entre les séquences du site de liaison aux peptides des deux chaperons moléculaires, qui fait que Ssa1p a une affinité plus faible que Hsc70 pour les fibres d'α-Syn. Nous avons fixé le complexe entre Ssa1p et α-Syn avec des agents pontants, pour ensuite établir une carte du site d'interaction entre les deux protéines en utilisant la spectrométrie de masse. Ceci est indispensable si un « mini » Ssa1p, constitué des éléments nécessaires et suffisants sera utilisé comme agent thérapeutique pour réduire la toxicité des fibres d'α-Syn. / The formation and deposition of α-Synuclein fibrils in the human brain is at the origin of Parkinson’s disease. The objective of my thesis was to document the role of two molecular chaperones on the assembly of α-Syn into fibrils: Hsc70, a constitutively expressed human heat shock protein, and Ssa1p, its yeast equivalent. The aim was to expand the catalogue of known effects of molecular chaperones on the PD implicated protein, which could have therapeutic significance. We showed that Hsc70 inhibits the assembly of α-Syn into fibrils, by binding with high affinity to the soluble form of α-Syn. We documented that Hsc70 binds preferentially to α-Syn fibrils and that this binding has a cytoprotective effect, as it renders the fibrils less toxic to cultured mammalian cells. Similarly to Hsc70, Ssa1p inhibits the assembly of α-Syn into fibrils, and has a higher affinity for fibrils than for the soluble form of α-Syn. On the other hand, binding of Ssa1p to α-Syn fibrils does not have a cytoprotective effect, almost certainly due to differences in the amino acid sequences of the peptide binding sites of the two molecular chaperones, which mean that Ssa1p has a lower affinity than Hsc70 for α-Syn fibrils. We stabilized the complex between Ssa1p and α-Syn using chemical cross-linkers, to then map the interaction site between the two proteins. This is indispensable if a “mini” Ssa1p, comprised of only what is necessary and sufficient of Ssa1p, is to be used as a therapeutic agent to decrease the toxicity of α-Syn fibrils. A therapeutic agent based on exogenous protein Ssa1p is less likely to trigger an autoimmune response than for example the endogenous protein Hsc70.

Notch signaling is important for cell-cell signaling during development. Notch signaling is highly conserved across all metazoans and failure in Notch signaling is causative in many human diseases. In the Drosophila eye, activation of the Notch pathway requires Lqf (Drosophila Epsin)-dependent and Clathrin-dependent internalization of the Notch receptor ligands, Delta or Serrate, by the signal-sending cells. However, it is unclear why ligand must be internalized into the signal-sending cells to activate Notch signaling in the signal-receiving cells. Evidence suggests that in addition to Clathrin and Epsin, Auxilin is essential for signaling and is indirectly required for internalization of the Notch receptor ligand Delta. Auxilin functions in uncoating Clathrin-coated vesicles to maintain a pool of free Clathrin and Epsin in the cell. auxilin mutants were used as an entryway to identify previously unknown components of the Notch signaling pathway. An F1, FLP/FRT, EMS screen was performed and enhancers of an auxilin mutant rough eye defect were isolated. The enhancers ultimately formed one complementation group on the 2nd chromosome and fourteen complementation groups on the 3rd chromosome. Three of the 3rd chromosome complementation groups were each identified as Delta, lqf, or hsc70. A single allele was identified as faf. Delta and Epsin have known roles in signaling cells to activate Notch as described above. Hsc70 is an ATPase that functions with Auxilin to uncoat Clathrin-coated vesicles and Faf is a deubiquitinating enzyme that maintains levels of active Epsin in the cell. These results suggest I have isolated mutations in genes closely tied to Notch signaling or functioning directly with Auxilin. Mutations in two genes previously undescribed in Notch signaling in the developing Drosophila eye were also isolated from the screen and identified. The second chromosome complementation group was identified as α-adaptin. α-Adaptin is a subunit of the heterotetrameric Clathrin adaptor protein AP-2. One of the third chromosome complementation groups was identified as crumbs. Crumbs is an integral membrane protein that functions at adherens junctions and in establishing apical/basal polarity in cells. Characterizing roles for α-Adaptin and Crumbs during Notch signaling may elucidate the purpose for Delta internalization to activate Notch signaling. / text

Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Le mépliement et l'agrégation des protéines sont à l'origine de nombreuses maladies neurodégénératives, dont la maladie de Huntington (HD) et la maladie de Parkinson (PD). Même si l’agrégation de différentes protéines liées à des maladies est bien documentée, on en sait peu sur l'interaction entre les protéines mal repliées et les cellules neuronales, qui leur permettent de se propager et affecter différentes régions du cerveau. L'objectif de ma thèse était de générer des modèles cellulaires rapporteurs de la huntingtine et l’α-synucléine, protéines dont le mauvais repliement et l'agrégation sont à l'origine de HD et PD respectivement, et utiliser ces modèles cellulaires pour étudier les interactions entre les agrégats et des lignées cellulaires de mammifères. Notre but c’était de documenter les propriétés de liaison et d’absorption de ces agrégats par les cellules rapporteuses, et les conséquences de leur internalisation pour les cellules. Deux modèles cellulaires de neuroblastome (SH-SY5Y et Neuro2A) et un modèle de cellules d’ostéoblastome (U2OS) exprimant la protéine fluorescente ChFP ont été générés pour HD. Pour simuler ce qui se passe au sein de neurones réels, des cellules de neuroblastome ont été induites à se différencier. Des différences de fixation, internalisation, nucléation de la protéine endogène et localisation finale des agrégats de polyglutamine internalisés ont été observées entre les cellules différenciées et non différenciées. Des cellules rapporteuses U2OS ont été utilisées pour déterminer les différences d’infectiosité entre des fibres de HttExon1 assemblés en présence ou en l’absence de la protéine de choc thermique constitutivement exprimée chez l'Homme Hsc70. Hsc70 a un effet protecteur car il rend les fibres moins infectieuses pour les cellules de mammifères en culture. Enfin, un modèle cellulaire de neuroblastome (Neuro2A) rapporteur pour PD exprimant l’α-synucléine fusionnée à la protéine ChFP a été utilisé pour déterminer des différences de liaison, pénétration, absorption, nucléation de la protéine endogène et persistance entre deux polymorphismes d’α-synucléine générés par notre équipe. L'hétérogénéité observée dans différents patients souffrant de synucléinopaties pourrait s'expliquer par différents polymorphes d’assemblages protéiques d’α-synucléine présents dans les cerveaux des malades, ce qui doit être pris en compte pour les développements thérapeutiques futurs.Ces modèles cellulaires rapporteurs pour différentes maladies sont un système valable pour l'étude de différents processus cellulaires liés à l'interaction entre les protéines agrégées exogènes et des cellules de mammifères en culture. Nos résultats indiquent un mécanisme commun par lequel les différentes protéines agrégées peuvent interagir avec des cellules en culture: les protéines mal repliées exogènes sont capables de se lier à des membranes cellulaires, les pénétrer, entrer dans l'espace intracellulaire et recruter des protéines endogènes solubles. Même si cela semble être un mécanisme générique pour des protéines infectieuses telles que la α-synucléine ou la huntingtine, des lignées cellulaires avec différents phénotypes montrent différences de vulnérabilité à la présence de protéines agrégées. Ceci suggère la présence de récepteurs spécifiques à la surface de la cellule capables de reconnaître des structures de type amyloïde. D'autres études sont nécessaires pour déterminer la nature de ces récepteurs et si sa modulation pourrait être utile pour contrôler la propagation des ces maladies dans le cerveau. / Protein misfolding and aggregation are at the origin of many neurodegenerative diseases, including Huntington’s disease (HD) and Parkinson’s disease (PD). Even if the aggregation of different disease-related proteins is well documented, little is known about the interaction between those misfolded proteins and neuronal cells that allow them to spread and affect several regions of the brain. The objective of my thesis was to generate reporter cellular models of huntingtin and α-synuclein, proteins whose misfolding and aggregation are at the origin of HD and PD respectively, and use these cell models for studying the interactions between misfolded protein aggregates and mammalian cell lines. We aimed to document the binding and uptake properties of those aggregates by reporter cells and the consequences of their internalization for the cells. Two neuroblastoma cell models (SH-SY5Y and Neuro2A) and an osteoblastoma cell model (U2OS) expressing the fluorescent protein ChFP were generated as mammalian reporter cell lines for HD. To mimic what happens in real neurons, neuroblastoma reporter cells were induced to differentiate. Differences in binding, internalization, nucleation of the endogenous protein and final localization of the internalized polyglutamine aggregates were observed between differentiated and undifferentiated cells. U2OS reporter cells were used for determining differences in the infectivity of HttExon1 fibrils assembled in the presence or in the absence of the constitutively expressed heat shock protein Hsc70, suggesting a protective effect of Hsc70, since it renders the fibrils less infectious to mammalian cells. Finally, a neuroblastoma reporter cell model (Neuro2A) of PD expressing α-synuclein fused to the fluorescent and reporter protein ChFP was used to determine the different binding, penetration, uptake, nucleation of the endogenous protein and persistence properties of two α-synuclein polymorphs generated by our team. The heterogeneity observed in different patients suffering from synucleinopathies could be explained due to different α-synuclein assemblies present in diseased brains, what needs to be taken into account for future therapeutic developments. These reporter cellular models for different diseases are a valid system for the study of different cellular processes related with the interaction between exogenous aggregated proteins and mammalian cells in culture. Our results indicate a common mechanism by which different aggregated proteins can interact with cells in culture: exogenous misfolded proteins are able to bind cell membranes, penetrate them, enter the intracellular space and recruit endogenous soluble proteins. Even if this seems to be a generic mechanism for infectious proteins such as α-synuclein or huntingtin, different cell lines or cell phenotypes show distinct vulnerability to the presence of aggregated proteins. This strongly suggests the presence of specific receptors at the surface of the cell able to recognize amyloid-like structures. Further investigations are needed to determine the nature of these receptors and whether their modulation might be helpful for controlling the spread of these diseases within the brain.

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 are also unknown. Here, we show by isotope-edited Fourier transform infrared (FTIR) spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and its refolding of CTA1 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance (SPR) experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allowed disordered CTA1 to attain an active state and did not prevent further stimulation of toxin activity by ADP-ribosylation factor 6, a host cofactor for CTA1. This activity is consistent with its role as a chaperone that refolds endogenous cytosolic proteins as part of a foldosome complex consisting of Hsp90, Hop, Hsp40, p23, and Hsc70. A role for Hsc70 in CT intoxication has not yet been established. Here, biophysical, biochemical, and cell-based assays demonstrate Hsp90 and Hsc70 play overlapping roles in the processing of CTA1. Using SPR we determined that Hsp90 and Hsc70 could bind independently to CTA1 at distinct locations with high affinity, even in the absence of the Hop linker. Studies using isotope-edited FTIR spectroscopy found that, like Hsp90, Hsc70 induces a gain-of-structure in unfolded CTA1. The interaction between CTA1 and Hsc70 is essential for intoxication, as an RNAi-induced loss of the Hsc70 protein generates a toxin-resistant phenotype. Further analysis using isotope-edited FTIR spectroscopy demonstrated that the addition of both Hsc70 and Hsp90 to unfolded CTA1 produced a gain-of-structure above that of the individual chaperones. Our data suggest that CTA1 translocation involves a ratchet mechanism which couples the Hsp90-mediated refolding of CTA1 with extraction from the ER. The subsequent binding of Hsc70 further refolds CTA1 in a manner not previously observed in foldosome complex formation. The interaction of CTA1 with these chaperones is essential to intoxication and this work elucidates details of the intoxication process not previously known.

Thesis (Ph.D.). / Written for the Dept. of Plant Science. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.