Mechanism of Hsp90-Dependent Glucocorticoid Receptor Activation

项目总结/摘要 维持蛋白质稳态是生物体生存的基础。这一进程不可或缺的是 分子伴侣，包括高度丰富和进化保守的热休克蛋白90（Hsp 90）， 其促进数百种“客户”蛋白质的折叠。Hsp 90客户富含信号分子， 例如激酶和转录因子，其调节细胞生长和存活。因此，许多HSP 90 客户是癌蛋白，使Hsp 90成为Hsp 90抑制剂治疗癌症的重要药物靶点 目前在临床肿瘤试验中。然而，对Hsp 90如何重塑客户蛋白的机制理解 缺乏并阻碍了Hsp 90靶向癌症治疗的进一步发展。一类临床上 Hsp 90的重要客户是类固醇激素受体（SHR），类固醇激活的转录因子， 控制细胞生长和发育，是癌症的有效治疗靶点。一个SHR，糖皮质激素 受体（GR），是一种模型Hsp 90客户端，它经历一个“伴侣循环”，其中GR结合Hsp 90，Hsp 70， 和多种辅助分子来维持其活性。GR功能的多个方面受Hsp 90调节， 包括配体结合、核转位和染色质结合，这些都是GR依赖性基因表达的重要步骤。 表达调控了解Hsp 90调节GR的机制将有助于阐明Hsp 90 影响了无数临床上重要的信号通路，推进了针对这一主调节因子的努力 用于癌症治疗。为了研究Hsp 90是如何重折叠和重激活GR的，我确定了冷冻电镜结构， 天然的，活性GR配体结合域（LBD）结合到Hsp 90，揭示了，第一次，机制 热休克蛋白90介导的客户端构象重塑。我将利用我的结构知识， 确定Hsp 90如何调节配体结合下游的GR功能。目标1将确定Hsp 90 在Hsp 90辅助分子伴侣FK 506结合蛋白51（FKBP 51）的帮助下调节GR核转位 和FKBP 52，其通过连接动力蛋白来调节GR核转位。使用冷冻EM以及体外 和体内生化分析，我将确定FKBP如何与GR-伴侣循环结合， 影响GR构象，使GR：Hsp 90与动力蛋白连接，进行核转位。目标2将调查 Hsp 90如何调节GR与DNA和染色质的结合。以前的研究表明Hsp 90是抑制性的， GR DNA和染色质结合，但抑制机制尚不清楚。使用第一个重组 纯化的多结构域GR，包含LBD和DNA结合结构域（DBD），我将确定GR如何 使用冷冻EM和体外生物化学方法，伴侣蛋白循环调节GR与DNA和染色质底物的结合 结合测定。该项目包括结构生物学、生物化学和基于细胞的测定， 蛋白质折叠和染色质领域。为了我成为首席研究员的目标，这个项目 为研究事业提供了坚实的基础，阐明了临床重要的潜在机制， 细胞通路

PROJECT SUMMARY/ABSTRACT Maintaining protein homeostasis is fundamental for organismal survival. Integral to this process are molecular chaperones, including the highly abundant and evolutionary conserved heat shock protein 90 (Hsp90), which facilitates the folding of hundreds of `client' proteins. Hsp90 clients are enriched in signaling molecules, such as kinases and transcription factors, which regulate cell growth and survival. Consequently, many Hsp90 clients are oncoproteins, making Hsp90 an important pharmaceutical target for cancer with Hsp90 inhibitors currently in clinical oncology trials. However, a mechanistic understanding of how Hsp90 remodels client proteins is lacking and precludes further advancements in Hsp90-targeted cancer therapies. One class of clinically important Hsp90 clients are the steroid hormone receptors (SHRs), steroid-activated transcription factors that control cell growth and development and are potent therapeutic targets for cancer. One SHR, the glucocorticoid receptor (GR), is a model Hsp90 client that goes through a `chaperone cycle', where GR binds to Hsp90, Hsp70, and a variety of co-chaperones to maintain its activity. Multiple aspects of GR function are regulated by Hsp90, including ligand binding, nuclear translocation, and chromatin binding—all essential steps in GR-dependent gene expression regulation. Understanding the mechanism by which Hsp90 regulates GR will elucidate how Hsp90 influences a myriad of clinically important signaling pathways, advancing efforts to target this master regulator for cancer therapies. To investigate how Hsp90 refolds and reactivates GR, I determined the cryo-EM structure of the native, active GR ligand binding domain (LBD) bound to Hsp90, revealing, for the first time, the mechanism of Hsp90-mediated conformational remodeling of a client. I will build on the knowledge from my structure to determine how Hsp90 regulates GR functions downstream of ligand binding. Aim 1 will determine how Hsp90 regulates GR nuclear translocation with the aid of the Hsp90 co-chaperones FK506 binding protein 51 (FKBP51) and FKBP52, which regulate GR nuclear translocation by connecting to dynein. Using cryo-EM as well as in vitro and in vivo biochemical assays, I will determine how the FKBPs incorporate with the GR-chaperone cycle, influence GR conformation, and connect GR:Hsp90 to dynein for nuclear translocation. Aim 2 will investigate how Hsp90 regulates GR binding to DNA and chromatin. Previous studies have suggested Hsp90 is inhibitory to GR DNA and chromatin binding, but the mechanism of inhibition is unknown. Using the first recombinantly purified multidomain GR, containing both the LBD and DNA binding domain (DBD), I will determine how the GR chaperone cycle modulates GR binding to DNA and chromatin substrates using cryo-EM and in vitro biochemical binding assays. This project encompasses structural biology, biochemistry, and cell-based assays, while bridging the protein folding and chromatin fields. Towards my goal of becoming a principal investigator, this project provides a strong foundation for a career in research, elucidating mechanisms underlying clinically important cellular pathways.