Expressing proteins with high propensity to from amyloid, such as disease-associated huntingtin (Htt) fragments with 103 glutamines (HttQ103), is toxic in yeast containing either the PIN+ prion, which is the amyloid form of Rnq1, or PSI+ prion, which is the amyloid form of Sup35. In an effort to understand this toxicity we examined in detail the effects of expressing various Htt fragments in cells propagating different prion variants. HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in yeast with PSI+ than PIN+. This toxicity observed in cells with a strong PSI+ variant was not seen in a weak PSI+ variant, which has more soluble Sup35 than the strong variant. Additionally, expression of Sup35p MC region, which remains soluble in PSI+ yeast because it lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity. Overexpressing the essential Sup45p, which binds to and functions with Sup35p, did not restore viability. Therefore, the toxicity of HttQP103 in yeast containing the PSI+ prion is primarily due to sequestration of the essential Sup35 protein. We are also continuing studies of how alterations in primary structure of prion proteins can affect chaperone interactions. We have been isolating mutants of prion proteins that fall into two major classes, those that interfere with propagation of prions formed of wild type proteins and those that don't. Both types of mutants contain subclasses that, in the absence of wild type protein, either form prions with weakened phenotypes or are unable to form prions. We are using genetic and biochemical methods to determine if the observed effects on prion phenotype are due to differences in how the prion proteins self-associate or interact with various chaperones. Our preliminary work suggests that the prion protein mutations can influence the propagation of amyloid directly, or the interaction of chaperones that in turn affect how efficiently the prions propagate.

Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2012
Total Cost
$74,537
Indirect Cost
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State
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Reidy, Michael; Sharma, Ruchika; Roberts, Brittany-Lee et al. (2016) Human J-protein DnaJB6b Cures a Subset of Saccharomyces cerevisiae Prions and Selectively Blocks Assembly of Structurally Related Amyloids. J Biol Chem 291:4035-47
Zhao, Xiaohong; Park, Yang-Nim; Todor, Horia et al. (2012) Sequestration of Sup35 by aggregates of huntingtin fragments causes toxicity of [PSI+] yeast. J Biol Chem 287:23346-55