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Elucidating the Interaction between the Molecular Chaperone Hsp104 and the Yeast Prion Sup35Helsen, Christopher W 26 March 2012 (has links)
Hsp104 is a protein remodeling factor that is crucially important for induced thermotolerance and prion propagation in yeast. Recent work demonstrates that Hsp104 is able to directly recognize and interact with synthetic polypeptide substrates, and that this interaction is dependent on the amino acid composition or sequence (Lum et al., 2008). Here this concept is applied to the in vivo substrate Sup35. Sup35, a translation termination factor, also forms the yeast prion [PSI+]. The maintenance of the prion is critically dependent on the expression levels of Hsp104. Over-expression of Hsp104 leads to the loss of prions, as does inhibition of this protein remodeling factor. As part of this thesis, an in vitro assay was established in which spontaneous nucleation, the event preceding of fiber formation, was suppressed. Fibrilization itself then becomes strictly dependent on the chaperones Hsp104, huHsp70p and Ydj1. In line with in vivo observations, Hsp104 mutants that fail to propagate [PSI+] also fail to overcome nucleation inhibition in this assay. Following this, the next part of this work established that the middle (M) domain of Sup35 inhibited this process, while not affecting spontaneous fibrilization under non-inhibitory conditions. This finding was reproduced in vivo, as middle domain over-expression also led to curing of weak [PSI+]. This suggested that the M-domain contains an Hsp104 binding site. This hypothesis is supported by data presented in this thesis which show that a small segment 129-148 within the Middle domain has enhanced Hsp104 binding properties. Deletion of this 20-mer peptide also reduced the Hsp104 ability to interact with this prion substrate; it also results in the destabilization of the prion and enhanced curing by the prion curing agent guandidinium hydrochloride. This represents the first ever Hsp104 binding site identified within a natural substrate.
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Elucidating the Interaction between the Molecular Chaperone Hsp104 and the Yeast Prion Sup35Helsen, Christopher W. 26 March 2012 (has links)
Hsp104 is a protein remodeling factor that is crucially important for induced thermotolerance and prion propagation in yeast. Recent work demonstrates that Hsp104 is able to directly recognize and interact with synthetic polypeptide substrates, and that this interaction is dependent on the amino acid composition or sequence (Lum et al., 2008). Here this concept is applied to the in vivo substrate Sup35. Sup35, a translation termination factor, also forms the yeast prion [PSI+]. The maintenance of the prion is critically dependent on the expression levels of Hsp104. Over-expression of Hsp104 leads to the loss of prions, as does inhibition of this protein remodeling factor. As part of this thesis, an in vitro assay was established in which spontaneous nucleation, the event preceding of fiber formation, was suppressed. Fibrilization itself then becomes strictly dependent on the chaperones Hsp104, huHsp70p and Ydj1. In line with in vivo observations, Hsp104 mutants that fail to propagate [PSI+] also fail to overcome nucleation inhibition in this assay. Following this, the next part of this work established that the middle (M) domain of Sup35 inhibited this process, while not affecting spontaneous fibrilization under non-inhibitory conditions. This finding was reproduced in vivo, as middle domain over-expression also led to curing of weak [PSI+]. This suggested that the M-domain contains an Hsp104 binding site. This hypothesis is supported by data presented in this thesis which show that a small segment 129-148 within the Middle domain has enhanced Hsp104 binding properties. Deletion of this 20-mer peptide also reduced the Hsp104 ability to interact with this prion substrate; it also results in the destabilization of the prion and enhanced curing by the prion curing agent guandidinium hydrochloride. This represents the first ever Hsp104 binding site identified within a natural substrate.
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Determinants of Yeast Prion StabilityDavies, Linda Emily 24 February 2009 (has links)
S. cerevisiae Sup35p inhabits two metastable states: functional translation termination factor; and prion-like aggregate [PSI+], which propagates by converting soluble Sup35p to its own misfolded form. Once initiated, Sup35p polymerization in [PSI+] cells is spontaneous, but [PSI+] prion inheritance depends on the Hsp104p disaggregase. To identify Hsp104-interacting sequences, Sup35p was subjected to a systematic deletion screen. [PSI+] maintenance by mutant Sup35p was assessed in both presence and absence of plasmid-encoded WT Sup35p in haploid sup35 cells. Large deletions abolished [PSI+], implying perturbations of prion structure, while others imparted [PSI+]-dependent toxicity. Removal of a single 25aa segment destabilised [PSI+] inheritance, resulting in enhanced rates of prion loss. This is consistent with the expected prion propagation defect in response to reduced Hsp104p interaction. However, several mutants containing this 25aa segment share the destabilised prion phenotype, suggesting chaperone/prion interactions are strongly context-dependent, and no one Sup35p region is solely responsible for Hsp104p recognition.
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Determinants of Yeast Prion StabilityDavies, Linda Emily 24 February 2009 (has links)
S. cerevisiae Sup35p inhabits two metastable states: functional translation termination factor; and prion-like aggregate [PSI+], which propagates by converting soluble Sup35p to its own misfolded form. Once initiated, Sup35p polymerization in [PSI+] cells is spontaneous, but [PSI+] prion inheritance depends on the Hsp104p disaggregase. To identify Hsp104-interacting sequences, Sup35p was subjected to a systematic deletion screen. [PSI+] maintenance by mutant Sup35p was assessed in both presence and absence of plasmid-encoded WT Sup35p in haploid sup35 cells. Large deletions abolished [PSI+], implying perturbations of prion structure, while others imparted [PSI+]-dependent toxicity. Removal of a single 25aa segment destabilised [PSI+] inheritance, resulting in enhanced rates of prion loss. This is consistent with the expected prion propagation defect in response to reduced Hsp104p interaction. However, several mutants containing this 25aa segment share the destabilised prion phenotype, suggesting chaperone/prion interactions are strongly context-dependent, and no one Sup35p region is solely responsible for Hsp104p recognition.
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Implication de l'activité chaperon de protéines du ribosome (PFAR) dans les mécanismes de prionisation & identification de nouvelles molécules antiprion / .Nguyen, Phuhai 11 December 2013 (has links)
Les maladies à prion font partie des maladies neurodégénératives. L’agent responsable est la protéine prion PrPSc. La conversion de la forme cellulaire nativePrPC en forme pathologique PrPSc et son agrégation sous forme des fibres amyloïdeconstituent des éléments clés de la physiopathologie des maladies à prion. Pourtant,les mécanismes contrôlant/favorisant cette conversion sont très mal connus. Chez lalevure Saccharomyces cerevisiae, il n’existe pas d’homologue de la protéine PrP,mais des protéines se comportant comme des prions existent, telle que Sup35p quiest responsable du prion [PSI+] ou encore la protéine Ure2p qui est responsable duprion [URE3]. Lors d’études antérieures à cette thèse, le laboratoire a isolé la 6AP etle GA, des molécules actives contre les prions de levure [PSI+] et [URE3] et contre leprion de mammifère PrPSc dans des tests cellulaires ainsi que in vivo dans unmodèle murin pour les maladies à prion. Ces résultats démontrent au moins certainsdes mécanismes de prionisation sont conservés de la levure aux mammifères.L’équipe a ensuite montré que la 6AP et le GA étaient des inhibiteurs spécifiques etcompétitifs de l’activité chaperon de protéines du ribosome (ou PFAR pour ProteinFolding Activity of the Ribosome). Ces résultats suggéraient donc que l’activité PFARreprésente un nouveau mécanisme de prionisation conservé de la levure auxmammifères. Par ailleurs, la 6AP et le GA s’étant révélées actives dans des modèlespour d’autres maladies neurodégénératives à fibres amyloïdes, l’activité PFARpourrait également être un acteur physiopathologique majeur de ces protéinopathies.Ma thèse avait deux objets : tester l’implication de l’activité PFAR dans l’apparitionet/ou la propagation des prions et enfin identifier de nouvelles molécules antiprion etcomprendre leurs mécanismes d’action. Mes résultats montrent que l’activité PFARjoue bien un rôle dans la propagation des prions de levure. En effet, l’enrichissementen PFAR favorise l’apparition spontanée du prion [PSI+]. Il conduit également à uneinstabilité accrue de ce même prion. Ainsi, l’activité PFAR ressemble à celle duchaperon de protéine Hsp104p, une protéine indispensable au maintien et à lapropagation de tous les prions de levure, mais qui n’a pas d’homologue chez lesmammifères. Mes résultats suggèrent que les activités PFAR et Hsp104p sontpartiellement redondantes pour le maintien des prions chez la levure et que, chez lesmammifères, seule l’activité PFAR jouerait ce rôle. Parallèlement, nous avonsidentifié de nouvelles familles de molécules antiprion, actives tant contre les prionsde levure que de mammifères. Ces molécules inhibent toutes l’activité PFAR. Nosrésultats contribuent ainsi à une meilleure compréhension des mécanismes deprionisation. Ils indiquent également que l’activité PFAR est une cible thérapeutiqueprometteuse pour les maladies à prion, mais aussi probablement pour d’autresprotéinopathies beaucoup plus fréquentes. / Prion diseases are considered neurodegenerative diseases. The incriminated agentis the prion protein PrPSc. The conversion of PrP from its native conformation PrPC tothe pathologic form PrPSc is the major element of the pathogenesis of prion diseases.However, the mechanisms involved in this conversion are poorly understood. In theyeast Saccharomyces cerevisiae, there is no counterpart of the PrP protein. Howeverproteins acting as prion do exist in yeast, such as the Sup35 protein responsible forthe prion [PSI+], or the Ure2 protein responsible for the prion [URE3]. In previousstudies, our team isolated two compounds, 6AP and GA, which are active against theyeast prions [PSI+] and [URE3 ] and against the mammalian prion PrPSc in cellbasedassays as well as in vivo in a mouse model for prion diseases. These resultsdemonstrated that the prionisation mechanisms are at least partially conserved fromyeast to mammals. 6AP and GA specific and competitive inhibitors of the ProteinFolding Activity of the Ribosome (PFAR) thereby showing that the PFAR is oneconserved mechanism of the prionisation. Moreover, 6AP and GA have been provenactive against other amyloid diseases thus placing the PFAR as a key player in thepathophysiology of protein folding diseases. My thesis aims were to test theinvolvement of the PFAR in the initiation and / or propagation of prion, to identify newantiprion molecules and to understand their mechanisms of action. My results showthat the PFAR plays a central role in the yeast prion propagation. Indeed, PFARenrichment promotes the spontaneous appearance of the prion [PSI+] and at thesame time leads to an increased instability of the same prion. Thus, PFAR activityresembles the yeast Hsp104p chaperone protein activity in the maintenance andpropagation of all yeast prions. My results suggest that the PFAR and Hsp104pactivity are partially redundant and that only the PFAR should play this role inmammals. Meanwhile, we have identified new antiprion drugs that are active againstboth yeast and mammal’s prions. These compounds are all inhibitors of the PFAR.Our results contribute to a better understanding of the prionisation mechanisms andindicate that the PFAR is a promising therapeutic target for prion diseases andprobably also for common protein folding diseases.Keywords: prion, yeast, ribosome, protein chaperon, Hsp104
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Towards a Mechanistic Understanding of the Molecular Chaperone Hsp104Lum, Ronnie 18 February 2011 (has links)
The AAA+ chaperone Hsp104 mediates the reactivation of aggregated proteins in Saccharomyces cerevisiae and is crucial for cell survival after exposure to stress. Protein disaggregation depends on cooperation between Hsp104 and a cognate Hsp70 chaperone system. Hsp104 forms a hexameric ring with a narrow axial channel penetrating the centre of the complex. In Chapter 2, I show that conserved loops in each AAA+ module that line this channel are required for disaggregation and that the position of these loops is likely determined by the nucleotide bound state of Hsp104. This evidence supports a common protein remodeling mechanism among Hsp100 members in which proteins are unfolded and threaded along the axial channel. In Chapter 3, I use a peptide-based substrate mimetic to reveal other novel features of Hsp104’s disaggregation mechanism. An Hsp104-binding peptide selected from solid phase arrays recapitulated several properties of an authentic Hsp104 substrate. Inactivation of the pore loops in either AAA+ module prevented stable peptide or protein binding. However, when the loop in the first AAA+ was inactivated, stimulation of ATPase turnover in the second AAA+ module of this mutant was abolished. Drawing on these data, I propose a detailed mechanistic model of protein unfolding by Hsp104 in which an initial unstable interaction involving the loop in the first AAA+ module simultaneously promotes penetration of the substrate into the second axial channel binding site and activates ATP turnover in the second AAA+ module. In Chapter 4, I explore the recognition elements within a model Hsp104-binding peptide that are required for rapid binding to Hsp104. Removal of bulky hydrophobic residues and lysines abrogated the ability of this peptide to function as a peptide-based substrate mimetic for Hsp104. Furthermore, rapid binding of a model unfolded protein to Hsp104 required an intact N-terminal domain and ATP binding at the first AAA+ module. Taken together, I have defined numerous structural features within Hsp104 and its model substrates that are crucial for substrate binding and processing by Hsp104. This work provides a theoretical framework that will encourage research in other protein remodeling AAA+ ATPases.
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Caractérisation de la chaperone Hsp104 chez la levure Schizosaccharomyces pombe et étude de son rôle dans la propagation des prions de levureSénéchal, Patrick January 2007 (has links)
Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.
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Interaction of Hsp104 with Hsp70: Insight into the Mechanism of Protein DisaggregationMoradi, Shoeib 18 March 2013 (has links)
Hsp104 and ClpB are hexameric ATPases that resolubilize aggregated proteins in collaboration with the Hsp70 chaperone system. Hsp104/ClpB functionally interact only with their respective Hsp70 system and this specificity is mapped to the Hsp104/ClpB coiled-coil domain (CCD). We hypothesize that the interaction between Hsp70 and Hsp104/ClpB CCD stimulates nucleotide exchange and release of substrate from Hsp70. In the current study, the CCDs of E. coli ClpB and S. cerevisiae Hsp104 have been purified. Isolated domains are monomeric and well folded. They inhibit refolding of aggregated firefly luciferase in a species-specific manner. We found that the ATPase activity of E. coli DnaK is stimulated at low concentrations of the E. coli ClpB CCD but not by yeast Hsp104 CCD. However, in another bacterial system (Thermus thermophilus) we found that the ClpB CCD inhibits The ATPase activity of DnaK suggesting that the interaction may have different consequences in distinct chaperone networks.
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Caractérisation de la chaperone Hsp104 chez la levure Schizosaccharomyces pombe et étude de son rôle dans la propagation des prions de levureSénéchal, Patrick January 2007 (has links)
Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal
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Towards a Mechanistic Understanding of the Molecular Chaperone Hsp104Lum, Ronnie 18 February 2011 (has links)
The AAA+ chaperone Hsp104 mediates the reactivation of aggregated proteins in Saccharomyces cerevisiae and is crucial for cell survival after exposure to stress. Protein disaggregation depends on cooperation between Hsp104 and a cognate Hsp70 chaperone system. Hsp104 forms a hexameric ring with a narrow axial channel penetrating the centre of the complex. In Chapter 2, I show that conserved loops in each AAA+ module that line this channel are required for disaggregation and that the position of these loops is likely determined by the nucleotide bound state of Hsp104. This evidence supports a common protein remodeling mechanism among Hsp100 members in which proteins are unfolded and threaded along the axial channel. In Chapter 3, I use a peptide-based substrate mimetic to reveal other novel features of Hsp104’s disaggregation mechanism. An Hsp104-binding peptide selected from solid phase arrays recapitulated several properties of an authentic Hsp104 substrate. Inactivation of the pore loops in either AAA+ module prevented stable peptide or protein binding. However, when the loop in the first AAA+ was inactivated, stimulation of ATPase turnover in the second AAA+ module of this mutant was abolished. Drawing on these data, I propose a detailed mechanistic model of protein unfolding by Hsp104 in which an initial unstable interaction involving the loop in the first AAA+ module simultaneously promotes penetration of the substrate into the second axial channel binding site and activates ATP turnover in the second AAA+ module. In Chapter 4, I explore the recognition elements within a model Hsp104-binding peptide that are required for rapid binding to Hsp104. Removal of bulky hydrophobic residues and lysines abrogated the ability of this peptide to function as a peptide-based substrate mimetic for Hsp104. Furthermore, rapid binding of a model unfolded protein to Hsp104 required an intact N-terminal domain and ATP binding at the first AAA+ module. Taken together, I have defined numerous structural features within Hsp104 and its model substrates that are crucial for substrate binding and processing by Hsp104. This work provides a theoretical framework that will encourage research in other protein remodeling AAA+ ATPases.
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