Molecular mechanism of PIN1-mediated regulation of the nuclear receptor PPARy

PIN1介导的核受体PPARγ调节的分子机制

• 批准号: 10607310
• 负责人: Christopher Conrad Williams
• 金额: $ 3.47万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607310
• 关键词: 3-Dimensional; Adipocytes; Affect; Affinity; Agonist; Automobile Driving; Binding; Binding Proteins; Binding Sites; Biological Assay; Biology; C-terminal; Cell physiology; Cells; Clinical; Complement; Complex; Cryoelectron Microscopy; DNA Binding Domain; Data; Data Correlations; Diabetes Mellitus; Disease; Enzymes; Epitopes; FDA approved; Gene Expression Profiling; Genes; Genetic Transcription; Glean; Human; In Vitro; Insulin; Interferometry; Isomerase; Isomerism; Isotope Labeling; Kinetics; Label; Length; Ligand Binding; Ligand Binding Domain; Ligands; Lipolysis; MAPK1 gene; Mass Spectrum Analysis; Measures; Mediating; Mediator; Metabolic syndrome; Methods; Modeling; Molecular; Molecular Conformation; Monitor; Mutagenesis; N-terminal; NIMA; NMR Spectroscopy; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Magnetic Resonance; Nuclear Protein; Nuclear Receptors; PPAR gamma; Pattern; Peptides; Peptidylprolyl Isomerase; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Plasmids; Population; Post-Translational Protein Processing; Proline; Protein Conformation; Protein Region; Proteins; Proteomics; Rationalization; Regulation; Reporter; Role; Site; Structure; Tertiary Protein Structure; Therapeutic; Vertebral column; Visualization; X-Ray Crystallography; adipocyte differentiation; antagonist; biophysical techniques; crosslink; experimental study; flexibility; genetic corepressor; insight; insulin sensitivity; knock-down; lipid biosynthesis; overexpression; programs; recruit; small hairpin RNA; structural biology; tool; transcription factor

Project Summary/Abstract Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand sensitive nuclear receptor and master regulator of adipogenesis. There exist FDA approved drugs that bind the ligand binding domain (LBD) of PPARγ to induce a well characterized conformational change, which alters the activity of this transcription factor. However, the functionally critical N-terminal intrinsically disordered AF1 domain remains poorly understood from a structural standpoint due to the limited number of biophysical methods that can provide atomic level detail of binding interactions and AF1 conformational states. Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1) is a known protein binding partner of PPARγ that exerts its catalytic activity specifically at pSer/pThr-Pro motifs, facilitating the cis/trans isomerization of proline bonds along the peptide backbone. Preliminary data from our lab and others suggest PIN1 binds the AF1 region of PPARγ with a much greater affinity than the LBD, an observation which presents the opportunity to better understand the role of AF1 function in PPARγ biology. This binding interaction suggests a cascade of post translational modifications (PTMs), including kinase-mediated phosphorylation and PIN1 enzyme-catalyzed proline cist/trans isomerization of the AF1 region, may be responsible for tuning the transcriptional activity of PPARγ and maintaining activation of adipogenic gene programs. In this project I will use nuclear magnetic resonance (NMR) spectroscopy to monitor PTM (phosphorylation and isomerization) induced changes in AF1 conformational states and correlate this data with cellular function associated with PPARγ-mediated gene programs that influence adipogenesis and insulin sensitization. Aim 1 of this project outlines the use of NMR methods to monitor and describe the phosphorylation- dependent and isomerization-dependent conformational changes the AF1 domain undergoes. These NMR studies will be complemented by kinetic and structural insight gleaned from biolayer interferometry (BLI) experiments to measure binding affinity of AF1 peptides to PIN1, X-ray crystallography studies of PIN1 in complex with AF1 peptides, and cross-linking mass spectrometry (XL-MS) using full length PPARγ and PIN1 to identify intermolecular protein contacts which may not be exclusive to the AF1 domain. To correlate the structural and molecular mechanisms described by Aim 1 with functional effects observed in cells, Aim 2 of this project uses structure-guided mutagenesis, transcriptional reporter assays, and knockdown experiments in pre- adipocytes to understand the role of PIN1 as a mediator of PPARγ function during adipogenesis. Gene expression analysis and assays that measure cellular endpoints associated with PPARγ function will further assess the roles of these PTMs within the AF1 domain in promoting adipogenic function.

项目概要/摘要 过氧化物酶体增殖物激活受体 γ (PPARγ) 是一种配体敏感的核受体， 脂肪生成的主要调节剂存在 FDA 批准的与配体结合域 (LBD) 结合的药物。 PPARγ 可诱导特征明确的构象变化，从而改变该转录因子的活性。 然而，功能关键的 N 端本质上无序的 AF1 结构域仍然知之甚少。 由于生物物理方法数量有限，可以提供原子级细节的结构结构 结合相互作用和 AF1 构象状态 NIMA 相互作用 1。 (PIN1) 是 PPARγ 的已知蛋白结合伴侣，特异性在 pSer/pThr-Pro 上发挥其催化活性 基序，促进脯氨酸键沿肽主链的顺式/反式异构化。 我们的实验室和其他人表明 PIN1 与 PPARγ 的 AF1 区域结合的亲和力比 LBD 大得多，LBD 是一种 观察提供了更好地了解 AF1 功能在 PPARγ 生物学中的作用的机会。 结合相互作用表明一系列翻译后修饰 (PTM)，包括激酶介导的 AF1 区域的磷酸化和 PIN1 酶催化的脯氨酸顺式/反式异构化可能是 负责调节 PPARγ 的转录活性并维持脂肪形成基因的激活 在这个项目中，我将使用核磁共振 (NMR) 光谱来监测 PTM。 （磷酸化和异构化）诱导 AF1 构象状态的变化，并将该数据与 与影响脂肪生成和胰岛素的 PPARγ 介导的基因程序相关的细胞功能 该项目的目标 1 概述了使用 NMR 方法来监测和描述磷酸化。 AF1 结构域经历依赖和异构化依赖的构象变化。 研究将得到从生物层干涉测量 (BLI) 中收集的动力学和结构见解的补充 测量 AF1 肽与 PIN1 结合亲和力的实验，PIN1 的 X 射线晶体学研究 与 AF1 肽形成复合物，并使用全长 PPARγ 和 PIN1 进行交联质谱 (XL-MS) 识别可能不属于 AF1 结构域的分子间蛋白质接触以关联结构。 以及目标 1 描述的分子机制以及在细胞中观察到的功能效应，该项目的目标 2 使用结构引导诱变、转录报告分析和敲低实验 脂肪细胞了解 PIN1 在脂肪生成过程中作为 PPARγ 功能调节剂的作用。 表达分析和测定与 PPARγ 功能相关的细胞终点将进一步 评估 AF1 结构域内这些 PTM 在促进脂肪生成功能中的作用。