Characterization of chaperone-prion specificity in Saccharomyces cerevisiae

描述(申请人提供)：淀粉样疾病，如阿尔茨海默病和帕金森病，正在迅速增加对公共健康的威胁。关于蛋白质单体形成淀粉样蛋白的许多已知知识都源于对依赖于分子伴侣蛋白的酵母普里恩的研究，但伴侣蛋白和普鲁恩蛋白之间的特异性起源尚不清楚。这项研究的长期目标是探索蛋白-伴侣特异性的分子基础，这可能被用于淀粉样蛋白疾病的治疗。本研究试图以酿酒酵母的[RNQ+]Prion为模型系统，通过遗传和生化相结合的方法来确定伴侣-Prion专一性的起源。将使用三条相互关联但相互独立的研究路线，重点研究在[RNQ+]Prion维持过程中形成特定调控相互作用的三种蛋白质：伴侣Sis1和Ssa1，以及Prion蛋白Rnq1。其具体目的是：1.通过分离功能缺失的突变体和抑制子，以及随后这些突变体的生化特征，了解SSa1在[RNQ+]维持中的特异性，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鉴定来研究J-蛋白Sis1的特定区域在蛋白维持中的作用，其中一些突变体可能被用作新的蛋白质核磁共振靶标。3.通过在体外合理构建截短片段和嵌合体蛋白，确定依赖SIS1的普恩病毒繁殖所必需的Rnq1区域。使用这个新的模型系统，遗传和生化测试将被用来确定特定的氨基酸序列是否对于普里恩和伴侣的特定相互作用是必要的。这项研究是一个极好的培训机会，因为它将利用各种遗传和生化方法，并需要整合体外和体内观察。这项研究与美国国立卫生研究院的使命一致：从分子上了解普鲁恩和伴侣之间的特异性，可能会导致在寻求治疗淀粉样蛋白和普鲁恩疾病的过程中操纵伴侣蛋白的能力。相关性：帕金森氏症和阿尔茨海默氏症等神经退行性疾病在美国越来越受到公共卫生的关注。这项调查旨在促进对这些疾病和相关疾病的原因的了解，同时也调查通过使用分子伴侣蛋白进行新的预防或治疗方法的可能性。

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.