One ongoing study focuses on how alterations in structures of prion proteins can affect prion phenotypes, possibly related to effects on chaperone interactions. As an approach to identify sites on prion proteins that might interact with chaperones, we have been isolating mutants of prion proteins that have alterations outside the amyloid-forming region that interfere with ability of the proteins to propagate as stable prions. They fall into two major classes, those that interfere with propagation of endogenous prions formed of wild type proteins and those that don't. 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 some mutations can influence the ability of the proteins to form amyloid, while others affect interactions with chaperones, that in turn affect how efficiently the prions propagate. Sis1 is a major yeast J-protein of the Hsp40 family that is essential for growth and has important roles in prion propagation. Our earlier work implicates Sis1 as possessing a function that protects cells from lethal effects of PSI prions and we are investigating the basis of this toxicity. Preliminary findings point to a specific chaperone activity that becomes necessary when the prions are present, rather than a specific cellular process that is being perturbed by defective chaperone function. Continued work aims to identify this activity and the mechanisms of how it provides protection. Expressing huntingtin fragments with disease-associated expansion of glutamines (HttQ103) is toxic in yeast containing either the PIN prion, which is an amyloid form of Rnq1, or PSI prion, which is an amyloid form of Sup35. In an effort to understand this toxicity we are examining in detail the effects of expressing various Htt fragments in cells propagating different prion variants. The human J-protein DnaJB6 has potent anti-amyloid activity on several different mammalian proteins that have propensity to form amyloid, and it protects cells from toxic effects of expressing HttQ103. We showed DnaJB6 also protects yeast from HttQ103 toxicity, but by affecting HttQ103 aggregation in a different way. We also found that DnaJB6 cures yeast of URE3 prions and weak, but not strong versions of PSI prions. We found the amyloid structures of the curable prions composed of different proteins were more related than those of the differentially sensitive prions composed of the same protein. These findings showed that DnaJB6 anti-amyloid activity extends to yeast proteins. They also defined different structural variants of amyloid, even when formed by the same protein, that are differentially sensitive to this activity. These findings have important implications for the growing attraction to DnaJB6 as a potential therapy for amyloid disorders, especially because prions with the resistant structures of amyloid are more highly infectious.
