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

 描述（由申请人提供）：Hsp90是一种高度保守的伴侣蛋白，由于其在调节信号转导中发挥着独特的作用，因此引起了人们的极大兴趣。热休克蛋白90促进许多信号蛋白的成熟，包括大量的激酶，类固醇激素受体和转录因子的活性状态。重要的是，许多突变的激酶和转录调节因子是癌症的关键介质，它们依赖于Hsp90进行正确折叠，导致癌细胞对Hsp90功能的依赖性增加。由于这个原因，Hsp90是许多不同癌症类型生长抑制剂的有吸引力和有前途的靶点。尽管其巨大的临床重要性，热休克蛋白90激活其客户端的机制仍然知之甚少。研究Hsp90的机制是具有挑战性的，因为它是一个大的，动态的蛋白质，经历了由ATP和许多瞬时相互作用的辅分子介导的巨大的构象变化。本研究的目的是利用显性负性热休克蛋白90突变体作为工具来探测分子伴侣促进客户端折叠的机制。显性负性蛋白质的分析历史上提供了复杂蛋白质的动态过程的快照提供有价值的机制信息。显性负性Hsp90突变体将通过在批量竞争实验中测量野生型蛋白存在下所有Hsp90点突变的适应性效应来系统地鉴定。显性失活突变对Hsp90结构和构象动力学的影响将使用一系列生物物理测定如荧光共振能量转移和小角X射线散射进行评估。此外，显性负性突变对Hsp90成熟模型客户的能力的影响将被确定。显性负性Hsp90突变体的检测将有助于鉴定沿着客户端折叠途径的中间体，从而为研究Hsp90蛋白的表达提供新的视角。 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.