A critical barrier to advancing the understanding of chaperone function in prion biology is that the fundamental chaperone requirements for most yeast prions remain unidentified. Existing knowledge is disjointed due to a lack of systematic evaluation that controls for variation in yeast strain background and prion structure. The continued existence of this barrier is an important problem because, until it is overcome, an understanding of how protein sequences give rise to amyloids with distinct patterns of chaperone interaction cannot be fully realized. The long- term goal is to utilize the highly tractable budding yeast, S. cerevisiae, to systematically decipher the complex relationships between amyloid-forming yeast prions and molecular chaperone proteins with a goal of better un- derstanding J-protein chaperone function and prion behavior. The objective of this particular application is to determine the functional elements involved in specific prion-chaperone interactions and to utilize two newly de- veloped genetic systems to broadly evaluate chaperone requirements in a tightly controlled yeast system. The central hypothesis is that differences in amyloid structure, arising primarily from amino acid composition, create distinct challenges for prion transmission which are overcome by specific J-protein functions that buffer prions against loss during mitosis. The hypothesis has been formulated on the basis of data produced in the applicant?s laboratory. The rationale for the proposed research is that unambiguous determinations of J-protein functional requirements are a necessary step toward understanding the mechanisms of J-protein function in amyloid biol- ogy. Using two distinct sets of chimeric prions, our own collection of well-studied naturally occurring prions, and the yeast cytosol as model systems, this hypothesis will be tested by pursuing two specific aims: 1) Identify prion sequence characteristics responsible for J-protein requirements, and 2) Determine the roles of J-proteins in Hsp104-mediated prion elimination. Proven yeast genetic manipulations, which have been established as feasi- ble in the applicant?s hands, will be the primary methods used to accomplish these aims. The approach is inno- vative because it represents a substantive departure from the status quo by placing emphasis on the ability to draw distinctions and make comparisons among multiple J-proteins and yeast prions as a way to broadly under- stand J-protein function. The contribution of the proposed research is expected to be the elucidation of the roles of several distinct J-proteins in prion propagation and the identification of new prion-chaperone requirements. This contribution is significant because it is the next step in a continuum of research which is expected to con- tribute to the understanding of the biochemical basis of J-protein?amyloid interactions. A molecular understand- ing of prion-chaperone interactions has the potential to inform the development of interventions for protein mis- folding disorders, including the increasing prevalent neurodegenerative disorders Alzheimer?s and Parkinson?s.
This proposal seeks to characterize the interactions between amyloid aggregates, which are associated with many neurodegenerative diseases that are a tremendous threat to public health, and yeast chaperones, which, unlike human chaperones, have evolved the ability to recognize, bind, and even resolubilize otherwise intractable protein aggregates. The proposed research is therefore relevant to public health because knowledge of the biochemical basis of chaperone?amyloid interactions may allow their manipulation pharmacologically as interventions for protein misfolding disorders. Thus, the project is relevant to both the specific mission of NIGMS and the mission of the NIH because it seeks to further understanding of poorly explored but fundamental life processes which will lay the groundwork for new treatments for increasingly prevalent neurodegenerative disorders.
