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search.filters.applied.f.advisor: Chernoff, Yury O. × End date: 2018 × Start date: 2018 × search.filters.applied.f.isOrgUnitOfPublicationTitle: College of Sciences ×

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Prion nucleation and propagation by mammalian amyloidogenic proteins in yeast

2018-04-17 , Chandramowlishwaran, Pavithra

Cross-β fibrous protein polymers or “amyloids” are associated with a variety of human and animal diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) and are suspected to possess transmissible (prion) properties. However, the molecular mechanisms of amyloid formation and propagation are difficult to investigate in vivo due to complexity of the human organism. While evolutionarily distant from humans, yeast cells carry transmissible amyloids (yeast prions) that can be detected phenotypically. The objectives of the work presented in this dissertation were to understand the molecular mechanisms of initial prion nucleation and propagation by mammalian proteins in yeast. Our model employed chimeric constructs, containing the mammalian amyloidogenic proteins (or domains) fused to various fragments of the yeast prion protein Sup35. Phenotypic and biochemical detection assays, previously developed for the Sup35 prion, enabled us to detect prion nucleation and propagation by mammalian proteins. We have demonstrated that several non-Q/N rich, mammalian amyloidogenic proteins, nucleated a prion in yeast in the absence of pre-existing prions. Sequence alterations antagonizing or enhancing amyloidogenicity of human Aβ (associated with AD) and mouse PrP (associated with prion diseases) respectively antagonized or enhanced nucleation of a yeast prion by these proteins. Mutational dissection of Aβ identified sequences and chemicals that influence initial amyloid nucleation. We have also shown that Aβ and microtubule-associated binding protein tau that is also associated with AD, could propagate a prion state on their own or after transfection with in vitro generated amyloid seeds, in yeast. Aβ- and tau-based chimeric constructs formed distinct variants (“strains”) in the yeast cell. Our data show that prion properties of mammalian proteins detected in the yeast assays correspond with those found in mammals or in vitro, thus making yeast a powerful model for deciphering molecular foundations of amyloid/prion diseases.

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Role of asymmetric segregation and the ribosome associated complex in prion formation and propagation

2018-04-06 , Howie, Rebecca Leigh

Self-perpetuating transmissible protein aggregates—prions—are implicated in mammalian diseases and control phenotypically detectable traits in yeast. Yeast heat shock-induced chaperone proteins counteract stress-induced aggregation but also control prion propagation. Heat-damaged proteins that are not disaggregated by chaperones are cleared from daughter cells via mother-specific asymmetric segregation in cell divisions following heat shock. Heat shock-mediated destabilization of [PSI+], a prion isoform of the yeast translation termination factor Sup35, was previously shown to coincide with the imbalance between the Hsp104 and Ssa chaperones. The ribosome associated complex chaperone Ssb has previously been shown to antagonize the function of Ssa in prion propagation. The objective of this work was to better understand prion curing and formation in yeast, and specifically to understand the roles of asymmetric segregation and the ribosome associated complex. We show that cells lacking Sir2, which is responsible for asymmetric segregation of heat-damaged proteins, are impaired in the heat shock-mediated destabilization of [PSI+], and that Sup35 aggregates co-localize with aggregates of heat-damaged proteins. These results support the role of asymmetric segregation in prion destabilization. We then show that depletion of Ssb decreases heat shock-mediated destabilization of [PSI+], while disruption of a co-chaperone complex mediating the binding of Ssb to the ribosome increases prion loss. Ssb is shown to relocate from the ribosome to the cytosol during heat stress. These data support the role of Ssb, a stress non-inducible protein, in prion curing during stress and further implicate chaperone imbalance in prion curing. Lastly, we demonstrate that increased aggregation due to disruption of Ssb or the ribosome-associated complex increases formation of various prions, especially during stress, establishing these as anti-prion components that are necessary for both curing and protection against prion formation.

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