Protein misfolding is implicated in many neurodegenerative diseases such as Alzheimer’s,Creutzfeldt-Jakob and Bovine Spongiform Encephalopathy (BSE) disease, more commonly known as Mad Cow Disease. These misfolded proteins are known as prions and are capable of self-perpetuating changes in conformation and function that can serve as genetic elements. In Saccharomyces cereviseiae, the genetic element [PSI+] is believed to transmit phenotypes through the same type of self-perpetuating changes in conformation. However in yeast these changes in conformation are not lethal to the cell, but produce cytoplasmically transmitted heritable changes in phenotype. Current studies on the Amyloid Project in our lab on mutant peptides of Aß Amyloid protein, responsible for Alzheimer’s disease, have shown sequence specificity plays a critical role in self-assembly of these mutant peptides in vitro. This poses the question: Can we design an in vivo genetic assay to study the assembly of such proteins in vivo? If so, is it also possible to control the assembly process? The yeast prions, in particular Sup35 of S. cereviseiae, are ideal candidates for studying the mechanism of self-assembly because of their score able phenotype. [PSI+]’s protein determinant is Sup35, a translation termination factor. In [psi-] cells, Sup35 is soluble and functional while in [PSI+] cells most Sup35 is insoluble and non-functional which results in translation infidelity. There are three distinct regions (Figure 1) within Sup35: the NH2-terminus (1-123aa) N domain which plays a critical role in the proteins self-perpetuating change in state containing six imperfect oligopeptide repeats, the M domain (124-253aa) providing solubility and space function and the COOH-terminus, C domain (254-685aa), providing translation termination activity.
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Figure 1. (A) Schematic of Sup35 protein domains. (B) Sequence of prion domain 41-97aa. (C) Sequence of AB1-42 Amyloid protein
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Teresa Welch has mutaginized the N domain from 40-123aa to incorporate “cassettes” of repeats of (PGLVFFAS)2 and (PGLVFFAT)2. These specific “cassettes” show conversion to [PSI+]. In both cases the [PSI+] phenotype is not curable by 5 mM GuHCl, a known curing agent. The current results suggest that these cassettes have a higher propensity for self-assembly than wild type. Studies of the NM domain fused to GFP (Figure 2) allow us to observe aggregation of the protein in vivo. These cassettes are not dependent on the prion, Rnq1 ([Pin+]) or Hsp104. Studies are in progress to purify the protein with these cassettes and study the macromolecular assembly in vitro. We are also trying to determine if there is a dependence on number and cassette sequence for self-assembly. |
