Determining the structural basis for ligand-mediated RORγ activation

确定配体介导的 RORγ 激活的结构基础

• 批准号: 9468687
• 负责人: Timothy Silas Strutzenberg
• 金额: $ 3.1万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-11-15 至 2019-11-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9468687
• 关键词: Adipocytes; Affinity; Agonist; Apoptosis; Automobile Driving; Binding; Biochemical; Biological; Biological Assay; Bone Marrow; Cell Line; Cells; Chemicals; Co-Immunoprecipitations; Communities; Complement; Complex; Consensus; Coupled; Crystallography; Cultured Cells; Deposition; Deuterium; Duodenum; Exhibits; Fracture; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genetic Transcription; Goals; Hydrogen; Hypercalcemia; Immunologic Surveillance; Immunoprecipitation; Immunotherapy; In Vitro; Interleukin-17; Ligands; Lymphoma; Mass Spectrum Analysis; Measurement; Mediating; Mesenchymal Stem Cells; Messenger RNA; Methodology; Modeling; Molecular; Molecular Conformation; Molecular Profiling; Monitor; Mus; Nuclear Receptors; Obesity; Osteoclasts; Outcome; Output; PPAR gamma; Pharmacology; Phosphorylation; Physiological; Production; Property; Proteins; Proxy; Receptor Activation; Recruitment Activity; Regulation; Research Project Grants; Response Elements; Roentgen Rays; Role; Specificity; Structural Models; Structure; System; T-Cell Activation; T-Lymphocyte; Therapeutic; Toxic effect; Transcript; Tumor Immunity; Validation; Western Blotting; base; biological systems; biophysical properties; bone; chromatin immunoprecipitation; crosslink; drug development; experimental study; improved; insight; insulin sensitizing drugs; lipid biosynthesis; mRNA Expression; novel; orphan nuclear receptor ROR-gamma; prevent; programmed cell death ligand 1; programs; promoter; proteogenomics; receptor; screening; transcriptome sequencing

Project Summary/Abstract This proposal seeks to reveal the underlying structural mechanism of consensus hyperactivation of the nuclear receptor (NR) RORγ driving maximal transcriptional output of target genes. To achieve this goal, I will perform detailed mechanism of action studies on a range of novel synthetic agonists of RORγ to further our understanding the molecular mechanism driving their ‘functional selectivity’ and to evaluate the physiological role of RORγ. I hypothesize that ligand-dependent structural perturbations manipulate coregulator interactions and PTM status of the receptor to directly influence gene program regulation. I aim to correlate structure based measurements and proteogenomic studies with biochemical and biological activity of synthetic agonists for RORγ and contrasting this to results obtained using repressive antagonist/inverse agonist ligands. RORγ agonists have already been developed with various potencies, binding modes, and receptor activation potential in cell based assays7,8. To accomplish the goals of my research project, I propose the following specific aims. Aim 1: Develop a comprehensive structural model for ligand activation of RORγ. I will perform structure determination of agonist-LBD complexes via X-ray co-crystallography and will determine ligand-dependent perturbation in receptor plasticity with hydrogen/deuterium exchange mass spectrometry (HDX-MS). As a proxy for receptor conformation, a coregulator interaction screen will be developed to determine ligand effects on specificity and affinity for coregulator recruitment. Crosslinking coupled with mass spectrometry (XL-MS) and HDX-MS will be conducted to quantitate ligand-dependent effects on LBD conformation ensembles and coregulator interactions as well as determining specific NR box motifs receptor interactions. These comprehensive biophysical measurements will be used to cluster compounds with similar properties where the functional consequences of these structural perturbations will be determined in Aim 2. Aim 2: Validation of structural model in cultured cells in vitro and ex vivo for RORγ agonists. Agonist activity in cultured cells will be characterized in relevant systems by expression profiling of RORγ target genes and coregulator associations using rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME). The murine T lymphoma EL4 cell line will be used for initial compound screening by monitoring transcript modulation. This system will also be used to develop RIME methodology. Induced T helper 17 cells (iTH17) will serve as a more physiologically relevant system for agonist characterization. iTh17 target gene programs and coregulator perturbations will be determined by RNA-seq and RIME, respectively. Co-immunoprecipitation with western blotting and chromatin immunoprecipitation experiments will be conducted to confirm findings from RIME.

项目摘要/摘要 该提议旨在揭示共识过度激活的潜在结构机制 核接收器（NR）RORγ驱动目标基因的最大转录输出。为了实现这一目标，我将 对RORγ的一系列新型合成激动剂进行详细的作用研究机理，以进一步 了解推动其“功能选择性”的分子机制并评估生理 RORγ的作用。我假设配体依赖性结构扰动操纵了核心调节器的相互作用 接收器的PTM状态直接影响基因程序调节。我的目标是基于结构 合成激动剂的生化和生物学活性的测量和蛋白质研究 RORγ并将其与使用抑制性拮抗剂/反激动剂配体获得的结果进行对比。 激动剂已经以各种能力，结合模式和受体激活潜力开发 在基于细胞的测定中7,8。为了实现我的研究项目的目标，我提出了以下特定目标。 AIM 1：开发一种用于RORγ的配体激活的综合结构模型。我将执行结构 通过X射线共结晶确定激动剂-LBD复合物，并将确定配体依赖性 氢/氘交换质谱法（HDX-MS）的受体可塑性扰动。作为代理 对于受体构象，将开发一个核心调节器相互作用屏幕以确定配体对 对核心招聘的特异性和亲和力。交联与质谱（XL-MS）和 将进行HDX-MS，以量化配体依赖性对LBD构象合并的影响和 核心节相互作用以及确定特定的NR盒基序受体相互作用。这些 全面的生物物理测量将用于群集具有具有相似特性的化合物 这些结构扰动的功能后果将在AIM 2中确定。 AIM 2：在体外和体内对RORγ激动剂的培养细胞中结构模型的验证。激动剂 通过表达RORγ靶基因的表达分析，将在相关系统中表征培养细胞中的活性 使用内源性蛋白的快速免疫沉淀质谱法的核心结合 （rime）。鼠T淋巴瘤EL4细胞系将通过监测转录本来用于初始化合物筛选 调制。该系统还将用于开发Rime方法。诱导的T辅助17细胞（ITH17）将 作为激动剂表征的更加相关的系统。 ITH17目标基因程序和 核心扰动将分别由RNA-seq和Rime确定。与 将进行蛋白质印迹和染色质免疫沉淀实验，以确认从 rime。