Mechanism of Hsp90-Dependent Glucocorticoid Receptor Activation

Hsp90 依赖性糖皮质激素受体激活机制

• 批准号: 10311692
• 负责人: Chari Noddings
• 金额: $ 4.14万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10311692
• 关键词: Affect; Affinity; Binding; Binding Sites; Biochemical; Biochemistry; Biological Assay; Cancer Cell Growth; Cell Nucleus; Cell Survival; Cells; Chromatin; Client; Clinical; Complex; Cryoelectron Microscopy; DCTN2 gene; DNA; DNA Binding; DNA Binding Domain; DNA receptor; Dimerization; Drug Targeting; Dynein ATPase; Foundations; Gene Expression Regulation; Genetic Transcription; Genome; Glucocorticoid Receptor; Goals; Growth and Development function; Heat-Shock Proteins 90; Hormone Receptor; In Vitro; Knowledge; Ligand Binding; Ligand Binding Domain; Ligands; Malignant Neoplasms; Mediating; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Nuclear Translocation; Nucleosomes; Oncogenic; Oncoproteins; Pathway interactions; Pharmacologic Substance; Phosphotransferases; Physiological; Principal Investigator; Process; Proteins; Proteome; Receptor Activation; Recombinants; Regulation; Reporting; Research; Response Elements; Role; Signal Pathway; Signaling Molecule; Steroids; Structure; Tacrolimus Binding Proteins; Testing; Time; Transcriptional Activation; Work; base; cancer cell; cancer clinical trial; cancer therapy; career; cell growth; dynactin; experimental study; glucocorticoid receptor alpha; in vivo; inhibitor/antagonist; protein folding; protein function; proteostasis; receptor binding; receptor function; reconstitution; steroid hormone receptor; structural biology; tacrolimus binding protein 4; targeted cancer therapy; therapeutic target; transcription factor

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.

项目概要/摘要 维持蛋白质稳态是有机体生存的基础。这个过程的组成部分是 分子伴侣，包括高度丰富且进化保守的热休克蛋白 90 (Hsp90)， 它促进了数百种“客户”蛋白质的折叠。 Hsp90 客户端富含信号分子， 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，使得 Hsp90 成为 Hsp90 抑制剂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 如何重塑客户蛋白的机械理解 缺乏并阻碍了 Hsp90 靶向癌症治疗的进一步进展。临床上一类 Hsp90 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。一种 SHR，糖皮质激素 受体 (GR) 是一种 Hsp90 客户端模型，会经历“伴侣循环”，其中 GR 与 Hsp90、Hsp70 结合， 以及各种辅助伴侣来维持其活性。 GR 功能的多个方面均受 Hsp90 调节， 包括配体结合、核易位和染色质结合——GR 依赖性基因的所有重要步骤 表达调节。了解 Hsp90 调节 GR 的机制将阐明 Hsp90 如何调节 GR 影响无数临床上重要的信号通路，推进针对这一主调节因子的努力 用于癌症治疗。为了研究 Hsp90 如何重折叠和重新激活 GR，我确定了冷冻电镜结构 结合 Hsp90 的天然活性 GR 配体结合域 (LBD)，首次揭示了该机制 Hsp90 介导的客户构象重塑。我将利用我的结构中的知识 确定 Hsp90 如何调节配体结合下游的 GR 功能。目标 1 将确定 Hsp90 如何 借助 Hsp90 共伴侣 FK506 结合蛋白 51 (FKBP51) 调节 GR 核易位 和 FKBP52，通过连接动力蛋白来调节 GR 核易位。使用冷冻电镜以及体外 和体内生化测定，我将确定 FKBP 如何与 GR 伴侣循环结合， 影响 GR 构象，并将 GR:Hsp90 连接至动力蛋白以进行核转位。目标 2 将进行调查 Hsp90 如何调节 GR 与 DNA 和染色质的结合。先前的研究表明 Hsp90 具有抑制作用 GR DNA 和染色质结合，但抑制机制尚不清楚。使用第一个重组 纯化的多域GR，包含LBD和DNA结合域（DBD），我将确定GR如何 伴侣循环利用冷冻电镜和体外生化调节 GR 与 DNA 和染色质底物的结合 结合测定。该项目涵盖结构生物学、生物化学和基于细胞的测定，同时桥接 蛋白质折叠和染色质区域。为了实现我成为首席研究员的目标，这个项目 为研究事业奠定坚实的基础，阐明临床重要的潜在机制 细胞途径。