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.

项目总结/摘要 这个建议试图揭示共识超激活的潜在结构机制， 核受体（NR）RORγ驱动靶基因的最大转录输出。为了实现这一目标，我将 对一系列新型RORγ合成激动剂进行详细的作用机制研究，以进一步推动我们的研究 了解驱动其“功能选择性”的分子机制，并评估其生理功能。 RORγ的作用我假设配体依赖的结构扰动操纵辅调节因子的相互作用 和受体的PTM状态直接影响基因程序调控。我的目标是将结构 合成激动剂的生物化学和生物学活性的测量和蛋白质基因组学研究， RORγ并将其与使用抑制性拮抗剂/反向激动剂配体获得的结果进行对比。RORγ 已经开发出具有各种效力、结合模式和受体活化潜力的激动剂 在基于细胞的分析中7，8.为了实现我的研究项目的目标，我提出了以下具体目标。 目的1：建立RORγ配体活化的结构模型。我会表演结构 通过X-射线共晶体学测定激动剂-LBD复合物，并将测定配体依赖性 用氢/氘交换质谱法（HDX-MS）测定受体可塑性的扰动。作为代理 对于受体构象，将开发共调节因子相互作用筛选以确定配体对 辅助调节因子募集的特异性和亲和力。交联与质谱联用（XL-MS）和 将进行HDX-MS以定量对LBD构象系综的配体依赖性影响， 辅助调节因子相互作用以及确定特定NR盒基序受体相互作用。这些 将使用全面的生物物理测量来聚集具有相似性质的化合物，其中 这些结构扰动的功能后果将在目标2中确定。 目的2：验证RORγ激动剂在体外和离体培养细胞中的结构模型。激动剂 通过RORγ靶基因的表达谱，在相关系统中表征培养细胞中的活性 使用内源性蛋白质的快速免疫沉淀质谱分析法 （RIME）。鼠T淋巴瘤EL 4细胞系将用于通过监测转录物来进行初始化合物筛选。 调变该系统还将用于开发RIME方法。诱导的T辅助细胞17（iTH 17）将 作为激动剂表征的更生理相关的系统。iTh 17靶基因程序和 将分别通过RNA-seq和RIME确定共调节子扰动。免疫共沉淀 将进行蛋白质印迹和染色质免疫沉淀实验，以证实来自 RIME。