Yeast Prion Variants as Models of the Phenotypic and Pathological Consequences of Amyloid Polymorphism

Protein aggregation is the hallmark of protein conformational disorders such as Alzheimer's disease and prion diseases. Prions are infectious proteins that propagate a self- templating amyloid structure, and have become a model for studying these diseases. Interestingly, a single protein can form a variety of distinct amyloid structures, a phenomenon referred to as amyloid polymorphism. In prion diseases, these different structures, called prion strains, dictate variation in pathology. However, the underlying basis for how structural variation modulates pathology remains unclear. Yeast prions have been a valuable model for studying protein conformational disorders. Prion proteins endogenous to yeast similarly misfold and form different self-propagating prion strains (called variants) that modulate cellular phenotypes. Additionally, in both humans and yeast, molecular chaperones act to process misfolded substrates. Here, I explore the interplay between molecular chaperones and prion variants and reveal novel determinants for how distinct aggregate structures can dictate phenotype. Studies of the [PSI+] prion have served as the foundation for the biophysical analysis of prion strains for several years. I applied this knowledge to prion variants of another prion, [RNQ+]. I found a surprising diversity in the sequence elements that are required to maintain different [RNQ+] variants. Interestingly, I also found evidence to suggest that the prion conformation dictates the availability of interaction sites for chaperones. Moreover, different domains of the Hsp40 Sis1 are important for maintaining particular prion variants. In fact, Sis1 and its human homolog have distinct prion conformer selectivity, suggesting that the selectivity of Hsp40s has changed throughout evolution. I also apply the concept of amyloid polymorphism to examine mutations in the human Hsp40 DNAJB6 that cause limb-girdle muscular dystrophy type 1D (LGMD1D). Using a chimeric protein of DNAJB6 and Sis1, I found that LGMD1D mutations impaired the propagation of prion conformers in a manner that depended on both the conformation and mutation. Additionally, while other functions of Sis1 were unaffected, over-expression of these mutants caused Hsp70-dependent cellular toxicity. These data show that impairing chaperone- mediated processing of particular substrate conformers may be one mechanism involved in the development of chaperonopathies. Taken together, this dissertation highlights the complexity underlying the impact of amyloid polymorphism on dictating phenotypic diversity, and shows how amyloid conformation is an important variable when studying the pathogenesis of protein conformational disorders.