J-PROTEIN REGULATION OF YEAST PRION PROPAGATION

描述（由申请人提供）：促进对朊病毒生物学中伴侣功能的理解的一个关键障碍是大多数酵母朊病毒的基本伴侣需求仍未确定。由于缺乏控制酵母菌株背景和朊病毒结构变化的系统评估，现有的知识是脱节的。这一屏障的持续存在是一个重要的问题，因为在它被克服之前，对蛋白质序列如何产生具有不同伴侣相互作用模式的淀粉样蛋白的理解不能完全实现。长期目标是利用高度易处理的出芽酵母酿酒酵母，系统地解读淀粉样蛋白形成酵母朊病毒与分子伴侣蛋白之间的复杂关系，从而更好地理解j蛋白伴侣蛋白的功能和朊病毒的行为。这一特殊应用的目的是确定两种特定朊病毒-伴侣相互作用的功能元件，并利用新开发的遗传系统在严格控制的真核生物模型中评估伴侣的需求。主要的假设是淀粉样蛋白结构的差异，主要是由氨基酸组成引起的，这对蛋白质产生了不同的挑战

DESCRIPTION (provided by applicant): 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 understanding J-protein chaperone function and prion behavior. The objective of this particular application is to determine the functional elements involved in two specific prion-chaperone interactions and to utilize a newly developed genetic system to evaluate chaperone requirements in a tightly controlled eukaryotic model. 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 biology. Using two distinct yeast prions, [URE3] and [SWI+], and the yeast cytosol as model systems, this hypothesis will be tested by pursuing three specific aims: 1) Identify the functional role of the auxilin homolog Swa2 in [URE3] prion propagation; 2) Determine the structural elements responsible for the specificity of the J-protein Ydj1 toward the prion [SWI+]; and 3) Determine whether Asn-content is the primary determinant of secondary J-protein requirements among prion-forming proteins in yeast. Proven yeast genetic manipulations, which have been established as feasible in the applicant's hands, will be the primary methods used to accomplish these aims. The approach is innovative 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 understand J-protein function. The contribution of the proposed research is expected to be the elucidation of the roles of two distinct J-proteins in prion propagation and the identification of new prion-chaperone requirements. This contribution is significant because it is the first step in a continuum of research which is expected to contribute to the understanding of the biochemical basis of J-protein-amyloid interactions. A molecular understanding of prion-chaperone interactions has the potential to inform the development of interventions for protein misfolding disorders, including the increasing prevalent neurodegenerative disorders Alzheimer's and Parkinson's.