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 这一建议旨在揭示共识过度激活的潜在结构性机制 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RoRγ的一系列新型合成激动剂进行详细的作用机理研究，以促进我们的 了解驱动其功能选择性的分子机制，并评估其生理活性 RoRγ的角色。我假设配体依赖的结构扰动操纵协同调节因子的相互作用。 而受体的PTM状态直接影响基因程序的调控。我的目标是将结构基于 合成激动剂的生化和生物活性测定及蛋白质组学研究 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 已经开发出具有不同效力、结合模式和受体激活潜力的激动剂 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：建立一个完整的RORγ配体激活结构模型。我会表演结构 激动剂-LBD络合物的X射线共结晶学测定及其配体依赖性 氢/氢交换质谱仪(HDX-MS)在受体可塑性中的扰动。作为代理人 对于受体构象，将开发一个辅助调节相互作用屏幕来确定配体对 协同调节因子招募的特异性和亲和力。交联质谱(XL-MS)和 HDX-MS将被用来定量LBD构象系综上的配体依赖效应和 协同调节相互作用以及确定特定的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类似性质的化合物，其中 这些结构扰动的功能后果将在目标2中确定。 目的2：在体外和体外培养细胞中验证RoRγ激动剂的结构模型。激动剂 培养细胞的活性将通过RoRγ靶基因的表达谱在相关系统中进行表征 利用内源蛋白的快速免疫沉淀质谱学研究 (Rime)。小鼠T淋巴瘤EL4细胞株将用于通过监测转录本进行初步化合物筛选 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 作为一种更具生理学意义的激动剂表征系统。ITh17靶基因程序和 共同调节因子的扰动将分别由RNA-seq和RIME确定。免疫共沉淀 将进行Western blotting和染色质免疫沉淀实验，以确认 里姆。