Title:
Prion nucleation and propagation by mammalian amyloidogenic proteins in yeast
Prion nucleation and propagation by mammalian amyloidogenic proteins in yeast
Author(s)
Chandramowlishwaran, Pavithra
Advisor(s)
Chernoff, Yury O.
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Abstract
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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Date Issued
2018-04-17
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Dissertation