Probing the mechanism of the Hsp90 chaperone using systematic dominant negative mutational analyses

 描述(申请人提供)：HSP90是一种高度保守的伴侣蛋白，因为它在调节信号转导中发挥着独特的作用，所以引起了极大的兴趣。HSP90促进多种信号蛋白的成熟，包括大量的激酶、类固醇激素受体和转录因子，使其处于激活状态。重要的是，许多突变的激酶和转录调节因子是癌症的关键媒介，它们依赖于Hsp90进行适当的折叠，导致癌细胞对Hsp90功能的依赖增加。出于这个原因，Hsp90是许多不同癌症类型的生长抑制剂的一个有吸引力和有前途的靶点。尽管Hsp90具有巨大的临床重要性，但其激活客户的机制仍然知之甚少。研究Hsp90的机制是具有挑战性的，因为它是一种大的、动态的蛋白质，在ATP和许多瞬时相互作用的辅助伴侣的介导下经历了戏剧性的构象变化。本研究项目的目的是利用显性负性Hsp90突变体作为工具来探索伴侣蛋白促进客户折叠的机制。历史上，对显性负性蛋白质的分析通过提供动态过程的快照，为复杂蛋白质提供了有价值的机制信息。在批量竞争实验中，通过测量所有Hsp90点突变在野生型蛋白存在下的适合度影响，将系统地鉴定显性负Hsp90突变体。显性负突变对Hsp90结构和构象动力学的影响将通过一系列生物物理测试来评估，如荧光共振、能量转移和小角X射线散射。此外，还将确定显性负突变对Hsp90成熟模型客户能力的影响。对显性负性Hsp90突变体的检测将有助于确定客户折叠路径上的中间体，为了解 Hsp90激活客户端的机制。

 DESCRIPTION (provided by applicant): Hsp90 is a highly conserved chaperone of great interest because of the unique role it plays in modulating signal transduction. Hsp90 facilitates the maturation of many signaling proteins including a large number of kinases, steroid hormone receptors, and transcription factors to their active states. Importantly, many of the mutated kinases and transcription regulators that are key mediators of cancer depend on Hsp90 for proper folding, resulting in an increased dependence of cancer cells on Hsp90 function. For this reason, Hsp90 is an attractive and promising target for inhibitors of growth of many diverse cancer types. Despite its tremendous clinical importance, the mechanism by which Hsp90 activates its clients remains poorly understood. Investigating the mechanism of Hsp90 is challenging because it is a large, dynamic protein that undergoes dramatic conformational changes mediated by ATP and many transiently interacting co-chaperones. The goal of this research project is to use dominant negative Hsp90 mutants as tools to probe the mechanism used by the chaperone to facilitate client folding. Analysis of dominant negative proteins has historically provided valuable mechanistic information of complex proteins by providing snapshots of dynamic processes. Dominant negative Hsp90 mutants will be systematically identified by measuring the fitness effects of all Hsp90 point mutations in the presence of the wild-type protein in bulk competition experiments. The impact of the dominant negative mutations on Hsp90 structure and conformational dynamics will be assessed using a series of biophysical assays such as fluorescence resonance energy transfer and small-angle X-ray scattering. In addition, the effects of the dominant negative mutations on the ability of Hsp90 to mature model clients will be ascertained. Examination of the dominant negative Hsp90 mutants will help identify intermediates along the client folding pathway, providing novel insight into the mechanism of client activation by Hsp90.