Characterization of chaperone-prion specificity in Saccharomyces cerevisiae

描述（由申请人提供）：淀粉样疾病如阿尔茨海默病和帕金森病是快速增长的公共健康威胁。关于蛋白质单体形成淀粉样蛋白的许多已知知识来自于对依赖分子伴侣蛋白的酵母朊病毒的研究，但伴侣蛋白和朊病毒之间特异性的起源尚不清楚。该研究的长期目标是探索朊病毒-伴侣蛋白特异性的分子基础，这可能潜在地用于治疗淀粉样蛋白疾病。本研究以酿酒酵母（Saccharomyces cerevisiae）的[RNQ+]朊病毒为模型系统，试图通过遗传和生化相结合的方法确定伴侣朊病毒特异性的起源。将使用三个相关但独立的研究线，重点关注在[RNQ+]朊病毒维持中形成特定调控相互作用的三种蛋白质：伴侣蛋白Sis1和Ssa1，以及朊病毒蛋白Rnq1。具体目标是：1。通过分离了解SSa1在[RNQ+]维持中的特异性。利用遗传选择方法研究功能丧失突变体和抑制突变体，以及随后对这些突变体进行生化表征，以将表型变异与生化特性联系起来。2. 通过类似的抑制突变体的分离和表征来表征J-蛋白Sis1的特定区域在朊病毒维持中的作用，其中一些可能用作新的蛋白质NMR靶点。3. 通过体外合理地创建截断和嵌合体蛋白，确定Sis1依赖性朊病毒繁殖所需的Rnq1区域。遗传和生化测试将用于确定是否某些氨基酸序列是必要的特定相互作用的朊病毒和伴侣使用这种新的模型系统。调查是一个很好的训练机会，因为它将利用各种遗传和生化方法，并要求结合体外和体内观察。这项研究与美国国立卫生研究院的使命一致：对朊病毒和伴侣蛋白之间特异性的分子理解可能会导致在追求淀粉样蛋白和朊病毒疾病的治疗中操纵伴侣蛋白的能力。相关性：神经退行性疾病，如帕金森氏症和阿尔茨海默氏症是美国公众健康日益关注的问题。这项研究旨在促进对这些疾病和相关疾病原因的理解，同时也通过使用分子伴侣蛋白研究新的预防或治疗方法的潜力。

DESCRIPTION (provided by applicant): 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.