Triage mechanisms for directing protein refolding and degradation

指导蛋白质重折叠和降解的分类机制

• 批准号: 10462587
• 负责人: Richard C Page
• 金额: $ 35.93万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462587
• 关键词: Area; Ataxia; Biochemical; Biological Assay; Biophysics; Cell physiology; Cerebellar Ataxia; Client; Complement; Complex; Data; Defect; Degradation Pathway; Development; Dissection; Electron Spin Resonance Spectroscopy; Future; Heat Shock 70kD Protein Binding Protein; Heat shock proteins; Human; Hybrids; Inherited; Knowledge; Lead; Location; Malignant Neoplasms; Mediating; Methods; Molecular Chaperones; Molecular Conformation; Neurodegenerative Disorders; Nuclear Magnetic Resonance; Nucleotides; Pathology; Pathway interactions; Physiology; Play; Proteins; Quality Control; Reperfusion Injury; Research; Resolution; Roentgen Rays; Role; Site; Structure; System; Techniques; Triage; Ubiquitination; misfolded protein; programs; protein misfolding; protein protein interaction; rapid testing; real time monitoring; response; therapeutic target; ubiquitin mediated proteasome degradation; ubiquitin-protein ligase

PROJECT SUMMARY Cellular protein quality control systems enable the robust response to protein misfolding that is essential for normal cellular function. Numerous pathologies including neurodegenerative disorders, ataxias, and cancers can result from defects in the protein quality control system formed by the interaction of the E3 ubiquitin ligase CHIP (C-terminus of Hsp70 Interacting Protein) and the ATP-dependent chaperone Hsp70 (70 kilodalton heat shock protein). Despite the importance of the CHIP/Hsp70 protein quality control complex, the triage mechanism used by the CHIP/Hsp70 complex to target misfolded proteins for either Hsp70-mediated refolding or ubiquitin proteasome-mediated degradation is unknown. Within the triage mechanism, several key areas remain unexplored. These include: (1) How does the CHIP/Hsp70 complex execute the observed triage mechanism that ubiquitinates misfolded clients first, then Hsp70, then CHIP? (2) What is the role played by nucleotide occupancy of Hsp70 and how do the conformational changes in Hsp70 throughout the catalytic cycle dictate the ability of CHIP to ubiquitinate misfolded clients? (3) What are the determinants that control whether a misfolded client becomes ubiquitinated, or is refolded? The proposed research program will examine these issues using nuclear magnetic resonance (NMR), small angle X-ray scattering (SAXS), electron paramagnetic resonance (EPR), and a suite of biophysical and biochemical techniques. NMR data will provide high-resolution information on dynamics within CHIP/Hsp70/client complexes and enable a detailed dissection of the role that dynamics play in dictating the CHIP/Hsp70 triage mechanism. SAXS and EPR will be used together in a hybrid methods approach to determine structural ensembles for dynamic CHIP/Hsp70 complexes and connect dynamic conformational changes to alterations in CHIP-mediated ubiquitination of Hsp70 and misfolded clients. The proposed development of a SAXS/EPR hybrid methods approach to structure determination will produce an approach that will be applicable to many protein/protein interactions regulated by dynamic conformational changes. Structural data from NMR, SAXS, and EPR will be complemented with biophysical and biochemical approaches that enable real-time monitoring of ubiquitination and directly assay the competition between CHIP-mediated ubiquitination and Hsp70-mediated protein refolding. These assays will allow for rapid testing of mechanistic hypotheses generated from the NMR and SAXS/EPR structural studies. The proposed research program is set up to evolve by expanding to examine the role that clients play in the triage mechanism. These efforts will be extended to soluble protein clients with either perturbed refolding capabilities, or controlled numbers or locations of ubiquitination sites.

项目概要 细胞蛋白质质量控​​制系统能够对蛋白质错误折叠做出强有力的反应，这对于 正常的细胞功能。多种病症，包括神经退行性疾病、共济失调和癌症 可能是由 E3 泛素连接酶相互作用形成的蛋白质质量控​​制系统缺陷造成的 CHIP（Hsp70 相互作用蛋白的 C 末端）和 ATP 依赖性伴侣 Hsp70（70 千道尔顿热量） 休克蛋白）。尽管 CHIP/Hsp70 蛋白质质量控​​制复合物很重要，但分类 CHIP/Hsp70 复合物使用的机制来靶向错误折叠的蛋白质以进行 Hsp70 介导的重折叠 或泛素蛋白酶体介导的降解尚不清楚。在分流机制内，几个关键领域 仍未被探索。其中包括： (1) CHIP/Hsp70 复合物如何执行观察到的分类 首先泛素化错误折叠的客户端，然后是 Hsp70，然后是 CHIP 的机制？ (2) 发挥什么作用 Hsp70 的核苷酸占据以及 Hsp70 在整个催化过程中的构象变化如何 周期决定了 CHIP 泛素化错误折叠客户的能力吗？ (3) 影响因素有哪些？ 错误折叠的客户是否会被泛素化，或者被重新折叠？拟议的研究计划将审查 使用核磁共振 (NMR)、小角 X 射线散射 (SAXS)、电子 顺磁共振（EPR）以及一套生物物理和生化技术。 NMR 数据将提供 有关 CHIP/Hsp70/客户复合物内动态的高分辨率信息，并可进行详细解剖 动力学在决定 CHIP/Hsp70 分类机制中所起的作用。将使用 SAXS 和 EPR 采用混合方法确定动态 CHIP/Hsp70 复合物的结构整体 并将动态构象变化与 CHIP 介导的 Hsp70 泛素化变化联系起来 错误折叠的客户。建议开发 SAXS/EPR 混合方法来构建结构 确定将产生一种方法，该方法将适用于受调节的许多蛋白质/蛋白质相互作用 动态构象变化。来自 NMR、SAXS 和 EPR 的结构数据将得到补充 生物物理和生化方法，可实时监测泛素化并直接进行测定 CHIP 介导的泛素化和 Hsp70 介导的蛋白质重折叠之间的竞争。这些化验 将允许快速测试由 NMR 和 SAXS/EPR 结构生成的机械假设 研究。拟议的研究计划旨在通过扩大研究客户所扮演的角色来发展 在分流机制中。这些努力将扩展到具有扰动重折叠的可溶性蛋白客户 能力，或受控的泛素化位点数量或位置。