Cells have evolved a sophisticated and essential machinery of proteins called molecular chaperones to ensure the proper folding of newly made polypeptides. The importance of correct protein folding is underscored by the fact that a number of diseases, including Alzheimer's, Parkinson's polyglutamine repeat disorders (e.g. Huntington's Disease), and those involving infectious proteins (prions), are intimately associated with protein misfolding. Such misfolding events often lead to formation of a specific type of protein aggregate termed amyloid fibers. Efforts to understand why some proteins undergo self-propagating pathogenic changes in conformation have been hampered until recently because of the lack of a facile genetic or biochemical system for studying their formation and prion-like propagation. This situation has improved greatly with the finding that the [URE3] and [PSI+] states of yeast result from the prion-like aggregation of endogenous proteins. My laboratory has taken advantage of the [PSI+] phenomenon to study the endogenous proteins. My laboratory has taken advantage of the [PSI] phenomenon to study the mechanism of prion formation and propagation. The present proposal aims to establish a general set of tools for studying prion-like, self-propagating changes in protein conformations in vivo. In particular, we will search for novel prions in an effort to determine how generally prion-like aggregates occur in biological processes. In addition, a combination of genetic analyses in yeast and in vitro biochemical studies will be employed to identify and characterize properties of a pathogenic peptide (PrP89-143), derived from the mammalian PrP protein, that allow it to adopt a beta-sheet rich in conformation. The relationship between formation of this beta-sheet rich form and pathogenicity will then be explored using model systems such as cultured neuroblastoma cells and mice. These efforts will be greatly facilitated by the collaborations made possible with this program project grant. For example, one the one human our mutational analysis will both be guided by and help guide the structural studies of Wemmer and Pine (Project 2) and the chemical and computational approaches of Cohen (Project 1). On the other hand, analysis of the physiological significance of our findings in yeast will depend heavily on the expertise of the Prusiner and DeArmond groups in the production and analysis of transgenic animals.
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