Amyloid diseases such as Alzheimer and Parkinson diseases are rapidly growing threats to public health. Much of what is known about amyloid formation from protein monomers has arisen from studies of the yeast prions which are dependent on molecular chaperone proteins, but the origin of specificity between chaperones and prions is not understood. The long term goal of the investigation is to explore the molecular basis for prion-chaperone specificity which may potentially be exploited for the treatment of amyloid diseases. This study seeks to determine the origins of chaperone-prion specificity through a combined genetic and biochemical approach using the [RNQ+] prion of Saccharomyces cerevisiae as a model system. Three related but independent lines of inquiry will be used, focusing on the three proteins which form a specific regulatory interaction in [RNQ+] prion maintenance: chaperones Sis1 and Ssa1, and the prion protein Rnq1.
The specific aims are: 1. To understand the specificity of SSa1 in [RNQ+] maintenance through the isolation.of loss-of-function mutants and suppressors using genetic selection methods, and the subsequent biochemical characterization of these mutants to correlate phenotypic variations to biochemical properties. 2. To characterize the role of specific regions of the J- protein Sis1 in prion maintenance by similar isolation and characterization of suppressor mutants, some of which may be used as novel protein NMR targets. 3. To determine regions of Rnq1 necessary for Sis1- dependent prion propagation through rational in vitro creation of truncations and chimera proteins. Genetic and biochemical tests will be used to determine if certain amino acid sequences are necessary for the specific interaction of a prion and chaperone using this novel model system. The investigation is an excellent training opportunity because it will utilize a variety of genetic and biochemical methods and require the integration of both in vitro and in vivo observations. This investigation aligns with the mission of the NIH: a molecular understanding of the specificity between prions and chaperones may lead to the ability to manipulate chaperone proteins in the pursuit of treatments for amyloid and prion diseases. Relevance: Neurodegenerative diseases such as Parkinson's and Alzheimer's are of a growing concern to public health in the U.S. This investigation seeks to advance the understanding of the causes of these and related diseases, while also investigating the potential for new preventative or therapeutic treatments through the use of molecular chaperone proteins.