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/文摘