Mechanism of Hsp90-Dependent Glucocorticoid Receptor Activation

项目摘要/摘要 维持蛋白质的动态平衡是生物体生存的基础。这一过程中不可或缺的是 分子伴侣，包括高度丰富和进化保守的热休克蛋白90(Hsp90)， 这有助于数百种“客户”蛋白质的折叠。HSP90客户富含信号分子， 例如，调节细胞生长和存活的激酶和转录因子。因此，许多Hsp90 客户是癌蛋白，使Hsp90成为治疗癌症的重要药物靶点 目前正在进行临床肿瘤学试验。然而，对Hsp90如何重塑客户蛋白质的机械性理解 缺乏Hsp90靶向癌症治疗的进一步进展。一类临床上的 重要的Hsp90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。一种是自发性高血压，糖皮质激素 受体(GR)，是一种典型的Hsp90客户，经历一个‘伴侣循环’，其中GR结合到Hsp90，Hsp70， 以及各种辅助伴侣来维持它的活动。GR功能的多个方面受HSP90调节， 包括配体结合、核转位和染色质结合--所有这些都是GR依赖基因的基本步骤 表达调控。了解Hsp90调节GR的机制将有助于阐明Hsp90是如何 影响了无数临床上重要的信号通路，推动了针对这一主要调控因子的努力 用于癌症治疗。为了研究Hsp90是如何折叠和重新激活GR的，我确定了冷冻EM结构 与Hsp90结合的天然活性GR配体结合域(LBD)的研究，首次揭示了Hsp90的作用机制 Hsp90介导的客户构象重塑。我将以我的结构中的知识为基础 确定Hsp90如何调节配体结合下游的GR功能。目标1将决定Hsp90如何 借助Hsp90辅伴蛋白FK506结合蛋白51(FKBP51)调节GR核转位 和FKBP52，它通过连接动力蛋白来调节GR核转位。使用冷冻-EM以及在体外 在活体生化分析中，我将确定FKBP如何与GR-伴侣循环结合， 影响GR构象，并将GR：HSP90连接到动力蛋白上进行核转位。AIM 2将调查 Hsp90如何调节GR与DNA和染色质的结合。此前的研究表明，Hsp90具有抑制性 能与GR DNA和染色质结合，但抑制机制尚不清楚。重新组合使用第一个 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 利用低温EM和体外生化技术研究伴侣循环调节GR与DNA和染色质底物的结合 有约束力的分析。该项目包括结构生物学、生物化学和基于细胞的分析，同时桥接 蛋白质折叠和染色质领域。对于我成为首席调查员的目标，这个项目 为从事研究奠定了坚实的基础，阐明了临床上重要的潜在机制 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.