Structure and function of mitochondrial Hsp60

线粒体 Hsp60 的结构和功能

• 批准号: 10631061
• 负责人: Julian Braxton
• 金额: $ 1.2万
• 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 2023-06-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10631061
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Adopted; Affinity; Allosteric Regulation; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Biology; Biophysics; Chaperonin 60; Chemicals; Client; Complement; Complex; Cryoelectron Microscopy; Data; Development; Disease; Elements; Environment; Future; Genetic; Genetic Diseases; Hereditary Spastic Paraplegia; Human; Investigation; Macromolecular Complexes; Malate Dehydrogenase; Malignant Neoplasms; Malignant neoplasm of prostate; Measures; Mentorship; Mitochondria; Mitochondrial Matrix; Mitochondrial Proteins; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Machines; Mutation; Mutation Analysis; Neurodegenerative Disorders; Nucleotides; Orthologous Gene; Play; Protein Dynamics; Protein Isoforms; Proteins; Publishing; Quality Control; Reaction; Resolution; Role; Sampling; Site; Structure; Structure-Activity Relationship; Surface; System; Techniques; Therapeutic; Training; Work; biological adaptation to stress; biophysical analysis; chaperonin; disease-causing mutation; in vitro Assay; inhibitor; member; misfolded protein; molecular dynamics; mutant; novel; protein complex; protein folding; proteostasis; response; 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.

项目概要/摘要 线粒体热休克蛋白 (Hsp) 60 分子伴侣对于蛋白质稳态和应激至关重要 响应，催化线粒体蛋白质的 ATP 水解依赖性折叠，同时进行精确折叠。 机制尚不清楚，Hsp60 的功能类似于细菌直系同源物 GroEL，其中展开了“客户” 蛋白质被封闭在其中央室中，部分由其共伴侣 Hsp10 形成，使它们能够折叠 不受其他细胞成分的干扰，Hsp60 突变会导致严重的神经退行性疾病。 称为遗传性痉挛性截瘫的疾病，并且 Hsp60 表达在一部分癌症中上调， 然而，Hsp60 的高分辨率结构信息使该伴侣成为重要的治疗靶点。 是有限的，留下了关于低聚物组织、变构调节和相互作用的关键问题 该提案的目的是确定其机制。 在此，我建议使用集成的结构和化学生物学方法来解决 Hsp60 功能的问题。 不同核苷酸下 Hsp60-客户复合物的分辨率冷冻电子显微镜 (cryo-EM) 结构 结合状态和寡聚状态，以确定对 Hsp60 辅助蛋白重要的客户相互作用表面 在令人兴奋的初步数据中，我生成了高分辨率 (2.5 Å) 的冷冻电镜结构。 ATP 结合的 Hsp60-Hsp10 双环复合物揭示了新的环间排列。 独特的伴侣蛋白作用机制，并为拟议的研究提供了良好的基础。 作为这些研究的补充，我还将研究致病突变和小分子的影响 Hsp60 结构和功能的抑制剂，以确定对 Hsp60 功能至关重要的元素，并确定 如何扰动这个复杂的大分子机器（SA2）这将通过使用来完成。 生物化学和生物物理测定，以及通过解决抑制剂结合和突变体的冷冻电镜结构 这些研究将极大地增进对 Hsp60 机制以及 Hsp60 机制的理解。 为第一个经过充分验证的 Hsp60 化学探针的开发做出了贡献。 作为结构和化学生物学家的发展，将通过独特的培训环境来实现 Dan Southworth 和 Jason Gestwicki 的实验室是使用冷冻电镜研究分子伴侣的专家 分别是化学生物学。