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: 9764391
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9764391
• 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; Testing; Triage; Ubiquitination; misfolded protein; programs; protein misfolding; protein protein interaction; 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.

项目摘要 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理，包括神经退行性疾病、共济失调和癌症 可由E3泛素连接酶相互作用形成的蛋白质质量控制系统的缺陷引起 CHIP（Hsp 70相互作用蛋白的C末端）和ATP依赖性伴侣Hsp 70（70千道尔顿热 休克蛋白）。尽管CHIP/Hsp 70蛋白质质量控制复合物的重要性， CHIP/Hsp 70复合物用于靶向错误折叠的蛋白质进行Hsp 70介导的重折叠的机制 或泛素蛋白酶体介导的降解是未知的。在分流机制内， 仍然未被探索。这些包括：（1）CHIP/Hsp 70复合体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Hsp 70，然后是CHIP？(2)扮演什么角色 Hsp 70的核苷酸占有率以及在整个催化过程中Hsp 70的构象变化如何 周期决定了CHIP遍在错误折叠的客户端的能力？(3)什么是决定因素 一个错误折叠的客户端是变成泛素化的，还是被重新折叠的？该研究计划将研究 这些问题使用核磁共振（NMR），小角X射线散射（SAXS），电子 顺磁共振（EPR）和一套生物物理和生物化学技术。NMR数据将提供 CHIP/Hsp 70/客户端复合体内动态的高分辨率信息，并能够详细解剖 动力学在决定CHIP/Hsp 70分诊机制中的作用。将使用SAXS和EPR 在混合方法中一起确定动态CHIP/Hsp 70复合物的结构系综 并将动态构象变化与CHIP介导的Hsp 70泛素化的改变联系起来， 被误导的客户提出了一种SAXS/EPR混合方法， 确定将产生一种方法，将适用于许多蛋白质/蛋白质相互作用的调节， 动态构象变化。NMR、SAXS和EPR的结构数据将得到补充， 生物物理和生物化学方法，能够实时监测泛素化和直接测定 CHIP介导的泛素化和Hsp 70介导的蛋白质重折叠之间的竞争。这些测定 将允许快速测试从NMR和SAXS/EPR结构产生的机理假设 问题研究拟议的研究计划是建立发展，扩大到检查的作用，客户端发挥 在分流机制中。这些努力将扩展到可溶性蛋白质客户端， 能力，或控制的数量或位置的泛素化位点。