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.

项目摘要/摘要 线粒体热休克蛋白(HSP)60分子伴侣在蛋白质平衡和应激中起关键作用 响应，催化线粒体蛋白质依赖于三磷酸腺苷水解的折叠。而精准的折叠 机制尚不清楚，Hsp60被认为具有与细菌直系同源基因GroEL相似的功能，在GroEL中展开了“客户”。 蛋白质被封闭在它的中央腔中，部分由它的辅助伴侣Hsp10形成，使它们能够折叠 不受其他蜂窝组件的干扰。Hsp60基因突变导致严重的神经退行性变 被称为遗传性痉挛截瘫的疾病，Hsp60在一组癌症中表达上调， 使这个伴侣成为一个重要的治疗目标。然而，HSP60上的高分辨率结构信息 是有限的，留下了关于齐聚物组织、变构调节和相互作用的关键问题 在其整个反应周期中与客户蛋白结合。这项建议的目标是确定机制 利用结构和化学生物学的综合方法对Hsp60的功能进行研究。在这里，我建议解决高- Hsp60-客户复合体在不同核苷酸水平上的分辨冷冻电子显微镜结构 结合状态和寡聚态，以确定对Hsp60辅助蛋白重要的客户相互作用表面 折叠(SA1)。在令人兴奋的初步数据中，我已经生成了高分辨率(2.5？)的低温电磁结构 结合ATP的Hsp60-Hsp10双环复合体。这种结构揭示了新颖的环间排列，表明 一个独特的伴侣作用机制，并为拟议的研究提供了良好的基础。至 作为这些研究的补充，我还将调查致病突变和小分子的影响 Hsp60结构和功能的抑制剂，以确定Hsp60功能的关键元件，并确定 这个复杂的大分子机器是如何被扰乱的(SA2)。这将通过使用 生化和生物物理分析，以及通过解决抑制物结合和突变体的低温EM结构 HSP60复合体。这些研究将极大地增加对HSP60机制的理解，以及 帮助开发了第一个经过充分验证的HSP60化学探针。这项工作，以及我的同伴 作为结构和化学生物学家的发展，将通过以下方式形成独特的培训环境 丹·索斯沃斯和杰森·格斯特维基的实验室，他们是使用低温EM和 分别是化学生物学。