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.

项目摘要/摘要 维持蛋白质稳态是有机生存的基础。这个过程不可或缺的是 分子伴侣，包括高度丰富和进化保守的热休克蛋白90（HSP90）， 这有助于折叠数百种“客户”蛋白质。 HSP90客户端富含信号分子， 例如激酶和转录因子，这些因子调节细胞生长和存活。因此，许多HSP90 客户是癌蛋白，使HSP90成为HSP90抑制剂的癌症的重要药物靶标 目前正在临床肿瘤学试验中。但是，对HSP90如何重塑客户端蛋白的机械理解 缺乏和排除了针对HSP90靶向的癌症疗法的进一步进步。一类临床 重要的HSP90客户是类固醇骑马受体（SHR），类固醇激活的转录因子 控制细胞的生长和发育，是癌症的潜在治疗靶标。一个SHR，糖皮质激素 接收器（gr），是通过“伴侣周期”的模型HSP90客户端，其中GR与HSP90，HSP70， 以及各种伴侣维持其活性。 GR函数的多个方面受HSP90的调节， 包括配体结合，核易位和染色质结合 - GR依赖性基因的所有基本步骤 表达调节。了解HSP90调节GR的机制将阐明HSP90如何 影响无数临床上重要的信号通路，促进针对该主调节器的努力 用于癌症疗法。为了研究HSP90的反映和反应GR，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）的首次揭示 HSP90介导的会议重塑对客户的重塑。我将基于从我的结构到 确定HSP90如何调节配体结合下游的GR功能。 AIM 1将决定HSP90 借助于HSP90共伴侣FK506结合蛋白51（FKBP51）来调节GR核易位。 和FKBP52，它们通过连接与动力蛋白来调节GR核易位。使用冷冻EM以及体外 和体内生化测定，我将确定FKBP如何与GR-Chaperone循环合并， 影响GR构象，并将GR：HSP90连接到Dynein，以进行核易位。 AIM 2将调查 HSP90如何调节GR与DNA和染色质结合。先前的研究表明HSP90是抑制性的 GR DNA和染色质结合，但抑制机制尚不清楚。重组使用第一个 纯化的多域GR，包含LBD和DNA结合域（DBD），我将如何确定GR 伴侣循环使用冷冻EM和体外生化调节GR与DNA和染色质底物的结合 绑定测定。该项目包括结构生物学，生物化学和基于细胞的测定法 蛋白质折叠和染色质场。为了成为成为首席调查员的目标，这个项目 为研究职业，阐明临床重要的基础机制奠定了坚实的基础 细胞途径。