Characterization of chaperone-prion specificity in Saccharomyces cerevisiae

描述（由申请人提供）：淀粉样蛋白疾病，如阿尔茨海默病和帕金森病是对公共卫生的快速增长的威胁。许多关于蛋白单体形成淀粉样蛋白的知识来自于对依赖于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。这项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，这可能被用于治疗淀粉样疾病。本研究旨在确定伴侣蛋白-朊病毒特异性的起源，通过遗传和生物化学相结合的方法，使用[RNQ+]朊病毒的酿酒酵母作为模型系统。三个相关但独立的调查线将被使用，集中在三种蛋白质，形成一个特定的监管相互作用[RNQ+]朊病毒的维护：伴侣Sis 1和Ssa 1，和朊病毒蛋白Rnq 1。具体目标是：1.通过使用遗传选择方法分离功能丧失突变体和抑制子，并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相关联，了解SSa 1在[RNQ+]维持中的特异性。2.通过类似的抑制突变体的分离和表征来表征J-蛋白Sis 1的特定区域在朊病毒维持中的作用，其中一些可以用作新的蛋白质NMR靶标。3.通过合理的体外截短和嵌合蛋白的产生，确定Sis 1依赖性朊病毒繁殖所需的Rnq 1区域。遗传和生物化学测试将用于确定某些氨基酸序列是否是使用这种新的模型系统的朊病毒和伴侣蛋白的特异性相互作用所必需的。该研究是一个极好的培训机会，因为它将利用各种遗传和生物化学方法，并需要整合体外和体内观察。这项研究与NIH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导致在寻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一个日益关注的公共卫生在美国这项调查旨在促进这些和相关疾病的原因的理解，同时也调查了新的预防性或治疗性治疗的潜力，通过使用分子伴侣蛋白。

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.