Folding and Activation of Client Proteins by the Hsp90 Molecular Chaperone Machine

分子伴侣调节细胞蛋白质的折叠，从合成到正确折叠或降解。大多数分子伴侣帮助蛋白质折叠，这些蛋白质大多是未折叠的。在分子伴侣中，Hsp90(热休克蛋白，90 kDa)是独一无二的，因为它优先与几乎完全折叠的蛋白质相互作用，并且是其活性所必需的。需要Hsp90才能发挥功能的客户蛋白几乎没有序列或结构同源性，而且人们对Hsp90如何促进一组不同的细胞客户蛋白的激活知之甚少。该项目的目标是确定Hsp90和两种截然不同的客户蛋白之间的相互作用位点。一组突变形式的Hsp90将被用作工具来识别和描述Hsp90与蛋白激酶和转录因子相互作用所需的关键特征。一旦确定了破坏体内两种不同客户蛋白相互作用的突变，进一步的研究将确定这些突变是否破坏了使用细胞裂解产物和纯化成分的Hsp90客户蛋白复合体的体外组装。进一步的研究将集中在确定特定的Hsp90突变是否通过阻止Hsp90与调节的辅助伴侣蛋白的相互作用间接干扰客户蛋白质的相互作用。这一研究项目将有助于澄清Hsp90和客户蛋白之间的接触部位，并更好地理解Hsp90和辅助伴侣如何合作促进不同客户蛋白的激活。该项目将对理解Hsp90如何与不同的客户蛋白相互作用产生广泛的影响。该项目将使每年一名研究生和两名本科生能够进行独立的研究项目。所有学生都将有机会在实验室会议、本科生研究海报论坛和/或其他科学会议上发展组织和展示数据的技能。这个实验室之外的其他研究生和本科生将通过微生物学、分子生物学和生物化学系以及生殖生物学中心(与华盛顿州立大学的联合项目)的教职员工之间的科学交流和研讨会来接触到这项工作。通过NIH-INBRE计划和多州WWAMI医学院计划的UDOC计划，来自代表性不足和少数群体的更多本科生和高中生也将接触到实验室的研究。

Molecular chaperones regulate the folding of cellular proteins from synthesis until they are properly folded or degraded. The majority of molecular chaperones assist the folding of proteins that are mostly unfolded. The abundant, essential molecular chaperone Hsp90 (heat shock protein, 90 kDa) is unique among chaperones because it preferentially interacts with and is required for the activity of proteins that are almost completely folded. The client proteins that require Hsp90 for function have little or no sequence or structural homology, and little is known about how Hsp90 promotes activation of a select group of diverse cellular client proteins. The goal of this project is to identify the sites of interaction between Hsp90 and two very different client proteins. A panel of mutant forms of Hsp90 will be used as tools to identify and describe critical features of Hsp90 required for interaction with a protein kinase and a transcription factor. Once mutations that disrupt the interaction with two distinct client proteins in vivo are identified, further research will determine whether the mutations disrupt the in vitro assembly of Hsp90-client protein complexes using cell lysates and purified components. Additional research will focus on establishing whether select Hsp90 mutations indirectly disrupt client protein interaction by preventing interaction of Hsp90 with regulatory co-chaperone proteins. This research project will lead to clarification of the contact sites between Hsp90 and client proteins and a greater understanding of how Hsp90 and co-chaperones cooperate to promote activation of a diverse group of client proteins. This project will have broad impacts on understanding how Hsp90 interacts with diverse client proteins. This project will enable one graduate student and two undergraduate students per year to conduct independent research projects. All students will have the opportunity to develop skills in organizing and presenting data during laboratory meetings, undergraduate research poster forums and/or additional scientific conferences. Additional graduate and undergraduate students outside of this lab will be exposed to this work through scientific exchanges and seminars among faculty members within the Microbiology, Molecular Biology and Biochemistry department, and the Center for Reproductive Biology, which is a joint program with Washington State University. Through the NIH-INBRE program and the UDOC program that is part of the multi-state WWAMI medical school program, additional undergraduate students and high school students from underrepresented and minority groups will also be exposed to research in the laboratory.