Structural Studies of Clp/Hsp 100 Molecular Chaperones

描述(申请人提供)：蛋白质必须正确折叠才能实现生物功能。同时，蛋白质聚集和错误折叠是许多毁灭性人类疾病的关键因素，如阿尔茨海默病、Pron介导性感染、II型糖尿病和囊性纤维化。虽然“常规”分子伴侣通过促进“向前”折叠或防止蛋白质聚集来帮助蛋白质折叠，但它们不能促进已经聚集的蛋白质解聚，如淀粉样蛋白，这与某些人类疾病有关。 细菌CIpB及其真核同源物Hsp104是热休克反应的必需蛋白质，形成大的环状结构，属于与多种细胞活动相关的Hsp100家族(AAA+)。与任何其他伴侣不同，包括Hsp100家族的其他成员，CIpB/Hsp104具有促进已经聚集的应激损伤蛋白质解聚的非凡能力。由于缺乏高分辨率的结构信息，其潜在机制目前尚不清楚。 长期目标是了解CIpB/Hsp104家族成员促进应激损伤蛋白解聚的分子机制。本研究的目标将通过以下具体目标进行：(1)用X射线结晶学解决CIpB/Hsp104的高分辨晶体结构；(2)用冷冻电子显微镜阐明0.6-MDA CIpB/Hsp104组装的三维结构；(3)确定CIpB/Hsp104识别和结合模型底物的结构基础。这些研究将得到突变和生化实验的补充，以检验从这些结构中推断的假说。这些方法的结合将提供对CIpB/Hsp104结构-功能关系的详细机制理解，并可能启发新技术的设计，从而可能导致潜在的人类Pron和淀粉样蛋白疾病的治疗。

DESCRIPTION (provided by applicant): Proteins must fold correctly in order to attain biological function. Concurrently, protein aggregation and misfolding are key contributors to many devastating human diseases such as Alzheimer's disease, prion-mediated infections, type II diabetes, and cystic fibrosis. While "conventional" molecular chaperones assist protein folding by promoting the "forward" folding or preventing protein aggregation, they are unable to promote the disaggregation of already aggregated proteins such as amyloids, which are associated with certain human diseases. Bacterial CIpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, form large ring-like structures, and belong to the Hsp100 family of ATPases associated with diverse cellular activities (AAA+). Unlike any other chaperone, including other members of the Hsp100 family, CIpB/Hsp104 has the remarkable ability to promote the disaggregation of already aggregated, stress-damaged proteins. The underlying mechanism is currently unknown due to the lack of high-resolution structural information. The long-term objective is to understand the molecular mechanism by which members of the CIpB/Hsp104 family promote the disaggregation of stress-damaged proteins. The goals of this research will be pursued through the following specific aims: (1) to solve the high-resolution crystal structure of CIpB/Hsp104 using X-ray crystallography, (2) to elucidate the three-dimensional structure of the 0.6-MDa CIpB/Hsp104 assembly by cryo-electron microscopy, and (3) to determine the structural basis by which CIpB/Hsp104 recognizes and binds model substrates. These studies will be complemented by mutational and biochemical experiments to test the hypotheses inferred from these structures. The combination of these approaches will provide a detailed mechanistic understanding of the structure-function relationship of CIpB/Hsp104, and may inspire the design of novel technology that could lead to a potential cure for human prion and amyloid diseases.