Hsp110 protein chaperone function in yeast

Hsp110 蛋白伴侣在酵母中的功能

• 批准号: 8234723
• 负责人: KEVIN ANTHONY MORANO
• 金额: $ 29.95万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8234723
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affinity; Age; Alleles; Alzheimer' s Disease; Ataxia; Binding; Binding Sites; Biochemical; Biogenesis; Biological; Cell physiology; Cells; Complex; Conflict (Psychology); Cytosol; Data; Defect; Disease; Dissociation; Endoplasmic Reticulum; Escherichia coli; Eukaryota; Eukaryotic Cell; Family; Funding; Genetic; Genomics; Goals; Guanine Nucleotide Exchange Factors; Heat-Shock Proteins 70; Homeostasis; Homologous Gene; Human; Human Resources; Huntington Disease; In Vitro; Investigation; Lead; Locales; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Modeling; Modification; Molecular Chaperones; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Nucleotides; Outcome; Parkinson Disease; Physiological; Play; Process; Protein Family; Proteins; Publishing; Quality Control; Recruitment Activity; Regulation; Relative (related person); Reperfusion Injury; Research; Ribosomes; Role; Saccharomyces cerevisiae; Saccharomycetales; Specificity; Stress; Testing; Therapeutic Intervention; Translations; Work; Yeasts; base; biological systems; chemical genetics; cofactor; design; experience; genetic analysis; genome-wide; heat-shock proteins 110; human disease; in vivo; mutant; novel; polypeptide; protein folding; protein misfolding; repaired; research study; therapeutic target; tool

DESCRIPTION (provided by applicant): In humans, defects in protein folding can lead to neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's disease and debilitating forms of ataxia. The 70 kD heat-shock proteins (Hsp70) are a ubiquitous family of protein chaperones involved in all aspects of protein homeostasis including protein biogenesis, repair and degradation. Hsp70 activity is governed by two types of cofactors; J domain-containing proteins activate the ATP hydrolysis step leading to high affinity substrate binding, while nucleotide exchange factors (NEFs) promote ADP dissociation and polypeptide release. Eukaryotes from yeast to humans express three recently described families of structurally diverse cytosolic NEFs (Hsp110 (Sse1/2 in yeast), HspBP1 (Fes1), and Bag-domain (Snl1)) that perform essentially the same biochemical function. The distinct contributions these proteins make to Hsp70-dependent activities are unknown in any biological system. The primary goal of this proposal is to determine how these NEFs partner with cytosolic Hsp70 chaperones to mediate proteostasis in eukaryotic cells. We hypothesize that while the NEFs share a common ability to stimulate Hsp70 activity, they differentially interact with the Ssa and Ssb families of cytosolic Hsp70 to promote protein folding and repair. In addition, we hypothesize that the Bag homolog Snl1 simultaneously recruits Hsp70 and the ribosome to promote protein biogenesis at the endoplasmic reticulum (ER). Three lines of investigation are proposed to test these hypotheses. In the first aim, we will determine how the apparently principal NEF Hsp110 is integrated into the cytosolic NEF network to promote protein biogenesis and repair. A major component of this aim will be genetic and cell biological experiments to assess the contribution of the Hsp110 substrate binding domain to Hsp70- dependent protein folding in vivo. In contrast to published results that Fes1 binds Ssa and Ssb in vitro, we have obtained preliminary evidence that Fes1 interacts solely with Ssa in vivo. In Aim 2 we will resolve these conflicting findings and determine the mechanism of Fes1-Hsp70 specificity. Lastly, we have discovered that the ER membrane-associated Snl1 binds the ribosome in addition to Hsp70. Aim 3 will be focused on determining the physiological significance of membrane recruitment of the translation machinery by an Hsp70 NEF. These studies represent the first comprehensive analysis of cytosolic Hsp70 NEFs by genetic, biochemical, and genomic approaches. Due to the high degree of conservation of these and other components of the Hsp70 chaperone network in eukaryotes, the results will be directly applicable to understanding the roles of Hsp70 NEFs in protein biogenesis and quality control in human cells. PUBLIC HEALTH RELEVANCE: In humans, defects in protein folding can lead to neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's disease and debilitating forms of ataxia. Dedicated cellular machines called "molecular chaperones" assist newly made proteins to fold and help repair proteins damaged by stress or age. This proposal seeks to understand how the Hsp70 molecular chaperone performs this task inside cells. More specifically, we will focus on helper proteins called "exchange factors" that control how fast the Hsp70 machine works. These studies will be carried out using yeast cells, a convenient, tractable a tested model for understanding how human cells function. The outcomes of this project should allow us to more accurately predict the disease consequences of genetic defects in the protein repair machinery, and facilitate therapeutic intervention to ameliorate protein misfolding disorders.

描述(申请人提供)：在人类中，蛋白质折叠缺陷可能导致神经退行性疾病，如阿尔茨海默氏症、帕金森氏症和亨廷顿病以及衰弱形式的共济失调。70kD热休克蛋白(Hsp70)是一个普遍存在的蛋白质伴侣家族，参与蛋白质生物发生、修复和降解等蛋白质动态平衡的各个方面。HSP70的活性受两种辅因子控制：含J结构域的蛋白质激活ATP水解步骤，导致高亲和力底物结合，而核苷酸交换因子(NEF)促进ADP解离和多肽释放。从酵母到人类，真核生物表达三个结构不同的胞质NEF家族(Hsp110(酵母中的Sse1/2)、HspBP1(Fes1)和Bag结构域(Snl1))，它们执行基本相同的生化功能。这些蛋白质对Hsp70依赖的活性的不同贡献在任何生物系统中都是未知的。这项建议的主要目标是确定这些NEF如何与胞浆HSP70伴侣蛋白在真核细胞中介导蛋白平衡。我们假设，虽然NEF具有共同的刺激Hsp70活性的能力，但它们与胞质Hsp70的SSA和SSB家族有不同的相互作用，以促进蛋白质折叠和修复。此外，我们假设BAG同源物Snl1同时招募Hsp70和核糖体来促进内质网(ER)的蛋白质生物发生。为了检验这些假说，本文提出了三条研究路线。在第一个目标中，我们将确定明显主要的NEF Hsp110如何整合到胞质NEF网络中，以促进蛋白质的生物发生和修复。这一目标的一个主要组成部分将是遗传和细胞生物学实验，以评估Hsp110底物结合结构域在体内对Hsp70依赖的蛋白质折叠的贡献。与已发表的Fes1在体外结合SSA和SSB的结果相反，我们获得了Fes1在体内仅与SSA相互作用的初步证据。在目标2中，我们将解决这些相互矛盾的发现，并确定Fes1-Hsp70特异性的机制。最后，我们发现内质网膜相关的Snl1除了与Hsp70结合外，还与核糖体结合。目标3将集中于确定Hsp70 NEF对翻译机器的膜招募的生理意义。这些研究代表了首次通过遗传、生化和基因组方法对胞质HSP70 NEF进行全面分析。由于Hsp70分子伴侣网络在真核生物中的高度保守性，这些结果将直接适用于理解Hsp70分子伴侣网络在人类细胞中蛋白质生物发生和质量控制中的作用。 与公共卫生相关：在人类中，蛋白质折叠缺陷可能导致神经退行性疾病，如阿尔茨海默氏症、帕金森氏症和亨廷顿病以及衰弱形式的共济失调。被称为“分子伴侣”的专用细胞机器帮助新合成的蛋白质折叠，并帮助修复因压力或年龄而受损的蛋白质。这项提议试图了解Hsp70分子伴侣如何在细胞内执行这一任务。更具体地说，我们将关注被称为“交换因子”的辅助蛋白，它控制着Hsp70机器工作的速度。这些研究将使用酵母细胞进行，酵母细胞是一种方便、易处理的测试模型，用于了解人类细胞的功能。这个项目的结果应该使我们能够更准确地预测蛋白质修复机制中遗传缺陷的疾病后果，并促进治疗干预以改善蛋白质错误折叠障碍。