Quantifying and modeling ligand-dependent control of RORγ dynamics via structural proteomics

通过结构蛋白质组学对 RORγ 动力学的配体依赖性控制进行量化和建模

• 批准号: 10704173
• 负责人: Patrick Robert Griffin
• 金额: $ 59.28万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-13 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10704173
• 关键词: Affinity; Agonist; Allosteric Regulation; Binding; Biological Assay; Bone Growth; CD4 Positive T Lymphocytes; Cells; Circadian Rhythms; Communication; Complex; Crosslinker; DNA; DNA Sequence; Data; Development; Genes; Hepatocyte; Immunity; Immunosuppression; Length; Ligand Binding; Ligands; Liver; Modeling; Mutation; Mutation Analysis; N-terminal; NCOA2 gene; NCOA3 gene; NR1 gene; Names; Nuclear Receptors; Oligonucleotides; Orphan; Pathogenicity; Peptides; Pharmacology; Physiological; Physiology; Play; Process; Protein Isoforms; Proteins; Proteomics; Receptor Activation; Regulation; Reporter; Reporting; Response Elements; Retinoic Acid Receptor; Role; Source; Steroid Receptors; Sterols; Structural Models; Structure; T-Lymphocyte; Testing; Transactivation; Transcription Coactivator; Validation; antagonist; blood glucose regulation; circadian pacemaker; crosslink; design; genetic analysis; glucose metabolism; human disease; immune function; insight; lipid metabolism; monomer; mutant; novel therapeutic intervention; overexpression; programs; promoter; protein purification; receptor; recruit; response; small molecule; therapeutic development; therapeutic target; tool; transcription factor; transcriptome; tumor

The nuclear receptor (NR) superfamily of ligand regulated transcription factors has proven to be a rich source of targets for the development of therapeutics for a wide range of human diseases. Endogenous small molecule regulation of these allosteric proteins control processes central to most aspects of mammalian physiology. Our lab has focused on synthetic ligand development and structure-function analysis of the NR1F subfamily of NRs known as the retinoic acid receptor-related orphan receptors or the RORs. This subfamily contains three genes that are involved in but not limited to regulation of glucose and lipid metabolism, bone growth, and immune functions. In this proposal we seek to expand our understanding of ligand-dependent regulation of NR1F3 (RORγ; gene name RORC), in the context of the intact full-length receptor. There are two isoforms of RORγ, RORγ1 and RORγ2, that differ in only their N-terminal sequence. RORγ1 is broadly expressed, and in the liver it plays an important role in circadian rhythms and glucose and lipid metabolism. The expression of RORγ2 or RORγt, is T cell specific and has been shown to be the key lineage-defining transcription factor to initiate the differentiation program of TH17 cells making RORγt an essential regulator for TH17 and Tc17 differentiation. Importantly, these cells that have demonstrated anti-tumor efficacy and RORγt controls gene programs that enhance immunity and decrease immune suppression. We have reported sterols and oxygenated sterols as high affinity endogenous ligands and others have confirmed these findings and provided key evidence that they are indeed physiological RORγ ligands. Although in certain experimental paradigms RORγ can recruit coactivators without addition of exogenous ligand, suggesting the receptor may be constitutively active. Recent evidence clearly demonstrates that RORγ is dependent on ligand binding for activation. While extensive structural studies on isolated domains of the receptor have provided important insight into high affinity ligand binding for both agonists and antagonists, there is a lack of information on how the modular domains of RORγ act together in the context of the intact full-length receptor. Given the importance of RORγ as a therapeutic target, it is surprising that we have an incomplete understanding of how small molecules modulate its activity. The mechanism for “turning off” RORγ activity appears straightforward; however, we have an incomplete understanding on how the receptor is “turned on.” We hypothesize that ligand-dependent structural perturbations manipulate the localization and PTM status of the receptor influencing its coregulator and DNA interactions to modulate of the RORγ transcriptome. To provide the groundwork to test this hypothesis, we propose to develop and validate an integrated structural model of intact full-length RORγ/DNA complex to expand our understanding of ligand-dependent regulation of RORγ. Illuminating RORγ activation mechanisms will help develop better tools to study its pharmacology and may lead to new therapeutic strategies by designing functionally selective ligands.

配体调节的转录因子的核受体（NR）超家族已被证明是一个丰富的来源， 用于开发用于广泛的人类疾病的治疗剂的靶点。内源性小分子 这些变构蛋白的调节控制对哺乳动物生理学的大多数方面至关重要的过程。我们 实验室专注于NR的NR 1F亚家族的合成配体开发和结构-功能分析 称为视黄酸受体相关孤儿受体或ROR。这个亚家族包含三个基因 参与但不限于调节葡萄糖和脂质代谢、骨生长和免疫功能， 功能协调发展的在这个提议中，我们试图扩大我们对NR 1F 3（RORγ; 基因名称RORC），在完整全长受体的情况下。RORγ有两种亚型，RORγ1和 RORγ2，仅N-末端序列不同。RORγ1广泛表达，在肝脏中起着重要作用。 在昼夜节律和糖脂代谢中起重要作用。RORγ2或RORγt的表达是T 细胞特异性，并已被证明是启动分化的关键谱系定义转录因子 TH 17细胞的程序使RORγt成为TH 17和Tc 17分化的重要调节因子。重要的是这些 已证明具有抗肿瘤功效的细胞，RORγt控制增强免疫力的基因程序， 减少免疫抑制。我们已经报道了甾醇和氧化甾醇作为高亲和力内源性 配体和其他人已经证实了这些发现，并提供了关键证据，证明它们确实是生理性的。 RORγ配体。尽管在某些实验范例中，RORγ可以募集辅激活因子而不添加 外源性配体，表明受体可能是组成型活性的。最近的证据清楚地表明 RORγ的激活依赖于配体结合。 虽然对受体的分离结构域的广泛结构研究提供了重要的洞察力， 尽管对于激动剂和拮抗剂两者的亲和配体结合，缺乏关于模块化配体如何结合的信息。 RORγ的结构域在完整全长受体的情况下一起起作用。鉴于RORγ的重要性， 作为治疗靶点，令人惊讶的是，我们对小分子如何调节 其活动。“关闭”RORγ活性的机制似乎很简单;然而，我们有一个 对受体如何“开启”的理解不完整。我们假设配体依赖性结构 扰动操纵受体的定位和PTM状态，影响其辅助调节因子和DNA R 0 R γ转录组的调节。为了提供测试这一假设的基础，我们 建议开发和验证完整全长RORγ/DNA复合物的整合结构模型， 扩大我们对RORγ的配体依赖性调节的理解。阐明RORγ激活机制 将有助于开发更好的工具来研究其药理学，并可能通过设计新的治疗策略， 功能选择性配体。