Characterization of chaperone-prion specificity in Saccharomyces cerevisiae

酿酒酵母伴侣蛋白-朊病毒特异性的表征

• 批准号: 7802817
• 负责人: Justin Keith Hines
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7802817
• 关键词: ATP phosphohydrolase; Affect; Alzheimer' s Disease; Amino Acid Sequence; Amino Acids; Amyloid; Amyloidosis; Binding; Biochemical; Biological Models; C-terminal; Cells; Chimera organism; Chimeric Proteins; Disease; Elements; Fiber; Genetic; Glycine; Goals; In Vitro; Individual; Investigation; Lead; Maintenance; Membrane; Methods; Mission; Molecular; Molecular Chaperones; Neurodegenerative Disorders; Parkinson Disease; Phenylalanine; Population; Prion Diseases; Prions; Property; Proteins; Public Health; Reaction; Reporting; Role; Saccharomyces cerevisiae; Specificity; Suppressor Mutations; System; Testing; Therapeutic; Training; United States National Institutes of Health; Variant; Work; amyloid formation; base; design; genetic element; genetic selection; in vivo; loss of function; monomer; mutant; novel; prion-based; sup35; yeast prion

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.

描述（由申请人提供）：淀粉样蛋白疾病，例如阿尔茨海默病和帕金森病，对公共健康的威胁正在迅速增长。关于蛋白质单体形成淀粉样蛋白的大部分知识源自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。该研究的长期目标是探索朊病毒伴侣特异性的分子基础，该特异性可能可用于治疗淀粉样蛋白疾病。本研究旨在使用酿酒酵母的 [RNQ+] 朊病毒作为模型系统，通过遗传和生化相结合的方法来确定伴侣蛋白 - 朊病毒特异性的起源。将使用三个相关但独立的研究线索，重点关注在 [RNQ+] 朊病毒维持中形成特定调节相互作用的三种蛋白质：伴侣 Sis1 和 Ssa1，以及朊病毒蛋白 Rnq1。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，以及随后对这些突变体进行生化表征，将表型变异与生化特性关联起来，了解 SSa1 在 [RNQ+] 维持中的特异性。 2.通过类似的抑制突变体分离和表征来表征J-蛋白Sis1的特定区域在朊病毒维持中的作用，其中一些突变体可用作新的蛋白质NMR靶标。 3.通过合理的体外截短和嵌合蛋白的产生来确定Sis1依赖性朊病毒繁殖所必需的Rnq1区域。使用这种新颖的模型系统，将使用遗传和生化测试来确定某些氨基酸序列对于朊病毒和伴侣蛋白的特定相互作用是否是必需的。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化方法，并需要整合体外和体内观察。这项研究与 NIH 的使命相一致：对朊病毒和伴侣蛋白之间特异性的分子理解可能会导致能够操纵伴侣蛋白来寻求淀粉样蛋白和朊病毒疾病的治疗方法。相关性：帕金森氏症和阿尔茨海默氏症等神经退行性疾病日益引起美国公众健康的关注。这项研究旨在增进对这些疾病及相关疾病病因的了解，同时还研究通过使用分子伴侣蛋白进行新的预防或治疗的潜力。