Prions cause tremendous concern as the causative agents of lethal neurodegenerative disorders such as CJD and BSE. Comparative analysis of prions and other proteins linked to human amyloidosis, and the identification of prions among benign epigenetic modifiers of cellular functions in fungi suggest the existence of molecular mechanisms governing the formation and elimination of self-perpetuating protein conformations. Revealing these mechanisms is expected to facilitate the development of new strategies for preventing the onset and transmission of prion diseases. The long-term goal of PI's research is to understand the biogenesis and biological role of prions, whereas the objective of the current proposal is to elucidate the mechanism of initial establishment of prion state. Genetic analysis in yeast is the major approach. The first indication that the de novo formation of prions may not be truly spontaneous came from the finding that the [PSI+] prion appears only in the presence of a prion named [PIN+] (for [PSI+] inducibility). The demonstrations that various prions can substitute for [PIN+] during [PSI+] induction and that formation of prions other than [PSI+] can be similarly facilitated by heterologous prions suggest that there is a general mechanism through which pre-existing prion aggregates seed novel prions.
Aim 1 is to elucidate this mechanism. The working hypothesis postulates that the formation of [PSI+] is initiated by the formation of a short """"""""polar zipper"""""""" between Q/N-rich sequences in the prion domains of [PSI+] and [PIN+]-composing proteins, Sup35 and Rnq1. The initial deletion analysis of Rnq1 will determine, which of multiple Q/N-rich regions of Rnq1 are involved in the facilitation of the [PSI*] formation by [PIN*]. Then mutagenesis will be directed to these region(s). Screening for compensatory Sup35 mutations that restore disrupted seeding will be used validate the hypothesis. The conclusions of in vivo studies will be verified in vitro with bacterially expressed proteins, and atomic force microscopy will be used to monitor the seeding process. Also, the efficiency of interconversion of prions encompassing Sup35 prion domains from different Saccharomycetes species will be analyzed to test if the seeding model is applicable to prion transmission between species.
Aim 2 is to identify new prions. Strategy (A) will test whether the proteins that satisfy several previously established criteria for presumptive prions can indeed take on self-propagating conformations. Among them, Lsm4 and Yck1 play roles in mRNA processing and signaling, respectively, and their ability to become prions is expected to advance our understanding of splicing, mRNA stability and signal transduction. Lsm4 studies may also shed light on the mechanism of spinal muscular atrophy. Strategy (B) involves designing several libraries of peptides. Screening these peptides for the ability to form prions or promote prion formation, and subsequent computer analysis of data will allow finding an algorithm for aggregation-prone proteins. Such proteins may not cause detectable prion phenotypes themselves but may induce dangerous prions. ? ? ?
