Structure and function of mitochondrial Hsp60

线粒体 Hsp60 的结构和功能

• 批准号: 10406155
• 负责人: Julian Braxton
• 金额: $ 3.93万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10406155
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Adopted; Affinity; Allosteric Regulation; Binding Sites; Biochemical; Biological Assay; Biology; Biophysics; Chaperonin 60; Chemicals; Client; Complement; Complex; Cryoelectron Microscopy; Data; Development; Disease; Elements; Environment; Future; Genetic; Hereditary Spastic Paraplegia; Human; Investigation; Macromolecular Complexes; Malates; Malignant Neoplasms; Malignant neoplasm of prostate; Measures; Mentorship; Mitochondria; Mitochondrial Matrix; Mitochondrial Proteins; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Machines; Mutation; Mutation Analysis; Neurodegenerative Disorders; Nucleotides; Orthologous Gene; Play; Protein Isoforms; Proteins; Publishing; Quality Control; Reaction; Resolution; Role; Sampling; Site; Structure; Structure-Activity Relationship; Surface; System; Techniques; Therapeutic; Time; Training; Work; base; biological adaptation to stress; biophysical analysis; chaperonin; chemical genetics; disease-causing mutation; in vitro Assay; inhibitor; member; mutant; novel; protein complex; protein folding; proteostasis; small molecule inhibitor; structural biology; therapeutic target; tool

PROJECT SUMMARY/ABSTRACT The mitochondrial heat shock protein (Hsp) 60 molecular chaperone is critical in proteostasis and stress response, catalyzing ATP hydrolysis-dependent folding of mitochondrial proteins. While the precise folding mechanism is unknown, Hsp60 is proposed to function like the bacterial ortholog GroEL in which unfolded `client' proteins are enclosed in its central chamber, formed in part by its co-chaperone Hsp10, allowing them to fold without interference from other cellular components. Mutations in Hsp60 cause severe neurodegenerative diseases termed hereditary spastic paraplegias, and Hsp60 expression is upregulated in a subset of cancers, making this chaperone a significant therapeutic target. However, high-resolution structural information on Hsp60 is limited, leaving unresolved critical questions about oligomer organization, allosteric regulation, and interactions with client proteins throughout its reaction cycle. The objective of this proposal is to determine the mechanism of Hsp60 function using an integrated structural and chemical biology approach. Herein, I propose to solve high- resolution cryo-electron microscopy (cryo-EM) structures of Hsp60-client complexes at different nucleotide- bound and oligomeric states, in order to identify client interaction surfaces important for Hsp60-assisted protein folding (SA1). In exciting preliminary data, I have generated high-resolution (2.5 Å) cryo-EM structures of the ATP-bound Hsp60-Hsp10 double ring complex. This structure reveals novel inter-ring arrangements, indicating a distinct chaperonin mechanism of action and providing an excellent basis for the proposed studies. To complement these studies, I will also investigate the effects of disease-causing mutations and small molecule inhibitors on Hsp60 structure and function, in order to identify elements critical for Hsp60 function, and determine how this complex macromolecular machine can be perturbed (SA2). This will be accomplished by using biochemical and biophysical assays, as well as by solving cryo-EM structures of inhibitor-bound and mutant Hsp60 complexes. These studies will dramatically increase understanding of Hsp60 mechanism, as well as contribute to the development of the first well-validated Hsp60 chemical probes. This work, and my concomitant development as a structural and chemical biologist, will be enabled by a unique training environment formed by the labs of Dan Southworth and Jason Gestwicki, experts in studying molecular chaperones using cryo-EM and chemical biology, respectively.

项目总结/摘要 线粒体热休克蛋白（Hsp）60分子伴侣在蛋白质稳态和应激中起着关键作用 反应，催化线粒体蛋白的ATP水解依赖性折叠。虽然精确的折叠 机制尚不清楚，Hsp 60被认为与细菌直系同源物GroEL一样起作用，其中未折叠的“客户端” 蛋白质被包裹在其中央腔室中，部分由其辅助分子Hsp 10形成，使它们能够折叠 而不受其他蜂窝组件的干扰。Hsp 60突变导致严重的神经退行性变 被称为遗传性痉挛性截瘫的疾病，并且Hsp 60表达在一个癌症亚组中上调， 使该分子伴侣成为重要的治疗靶点。然而，Hsp 60的高分辨率结构信息 是有限的，留下未解决的关键问题，寡聚体组织，变构调节，和相互作用 在整个反应周期中与客户蛋白质结合。本提案的目的是确定机制 Hsp 60的功能，使用综合结构和化学生物学方法。在此，我建议解决高- 热休克蛋白60-客户端复合物在不同核苷酸的分辨率冷冻电镜（cryo-EM）结构， 结合态和寡聚态，以鉴定对Hsp 60辅助蛋白重要的客户相互作用表面 折叠（SA 1）。在令人兴奋的初步数据中，我已经生成了高分辨率（2.5 μ m）的冷冻EM结构， ATP结合的Hsp 60-Hsp 10双环复合物。这种结构揭示了新颖的环间排列，表明 一个独特的伴侣蛋白的作用机制，并提供了一个很好的基础上提出的研究。到 作为对这些研究的补充，我还将研究致病突变和小分子 抑制剂对Hsp 60结构和功能的影响，以确定Hsp 60功能的关键因素，并确定 这台复杂的大分子机器是如何被扰乱的（SA 2）。这将通过使用 生物化学和生物物理测定，以及通过解决冷冻EM结构的走廊结合和突变 Hsp 60复合物。这些研究将大大增加对Hsp 60机制的理解， 有助于开发第一个经过充分验证的Hsp 60化学探针。这项工作，和我的伴随 作为一个结构和化学生物学家的发展，将通过一个独特的培训环境， Dan Southworth和Jason Gestwicki的实验室，他们是使用冷冻EM研究分子伴侣的专家， 化学生物学，分别。