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Author: Chandramowlishwaran, Pavithra × End date: 2019 × Start date: 2000 × 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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Polyglutamine toxicity is controlled by prion composition and gene dosage in yeast

2012-04-19 , Gong, He , Romanova, Nina V. , Allen, Kim D. , Chandramowlishwaran, Pavithra , Gokhale, Kavita , Newnam, Gary P. , Mieczkowski, Piotr , Sherman, Michael Y. , Chernoff, Yury O.

Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington’s disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45- bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines.

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