Triage mechanisms for directing protein refolding and degradation

项目总结 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 细胞功能正常。多种病理疾病，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中的缺陷造成的 ChIP(Hsp70相互作用蛋白的C末端)和依赖于ATP的伴侣Hsp70(70kodalton热) 休克蛋白)。尽管CHIP/Hsp70蛋白质质量控制复合体很重要，但分类 CHIP/Hsp70复合体用于靶向错误折叠的蛋白质进行Hsp70介导的重折叠的机制 或者泛素蛋白酶体介导的降解是未知的。在分类机制内，有几个关键领域 仍未被开发。这些包括：(1)芯片/Hsp70复合体如何执行观察到的分诊 首先泛化错误折叠的客户端，然后是Hsp70，然后是芯片的机制？(2) Hsp70的核苷酸占有率和整个催化过程中Hsp70的构象变化 周期决定芯片使错误折叠的客户端无处不在的能力？(3)控制的决定因素是什么 错误折叠的客户是变得无处不在，还是重新折叠？拟议的研究计划将审查 这些问题使用核磁共振(核磁共振)、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。核磁共振数据将提供 关于芯片/Hsp70/客户复合体内部动力学的高分辨率信息，并支持详细剖析 动力学在决定CHIP/HSP70分流机制中所起的作用。将使用SAXS和EPR 在混合方法中一起确定动态CHIP/HSP70复合体的结构系综 并将动态构象变化与芯片介导的Hsp70泛素化和 弄错了客户。SAXS/EPR混合方法在结构分析中的应用 确定将产生一种适用于许多蛋白质/蛋白质相互作用的方法 动态构象变化。来自核磁共振、SAXS和EPR的结构数据将得到补充 生物物理和生化方法，能够实时监测泛素化和直接化验 芯片介导的泛素化和Hsp70介导的蛋白质复性之间的竞争。这些化验 将允许快速测试从核磁共振和SAXS/EPR结构生成的机械假设 学习。建议的研究计划旨在通过扩展以检查客户所扮演的角色来进行发展 在分类机制中。这些努力将扩展到可溶性蛋白质客户，无论是受到干扰的折叠 泛素化能力，或泛素化位点的受控数量或位置。

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.