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.

配体调节转录因子的核受体 (NR) 超家族已被证明是丰富的来源 开发针对多种人类疾病的治疗方法的目标。 这些变构蛋白的调节控制过程是哺乳动物生理学大多数方面的核心。 实验室专注于 NR NR1F 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 该亚家族包含三个基因。 参与但不限于葡萄糖和脂质代谢、骨骼生长和免疫的调节 在本提案中，我们寻求扩大我们对 NR1F3 (RORγ；配体依赖性调节) 的理解。 基因名称 RORC），在完整的全长受体的背景下，有两种 RORγ 亚型，RORγ1 和 RORγ1。 RORγ2 仅在 N 末端序列上有所不同，RORγ1 广泛表达，并且在肝脏中发挥着重要作用。 RORγ2或RORγt的表达是T，在昼夜节律和糖脂代谢中发挥重要作用。 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 TH17 细胞的程序使 RORγt 成为 TH17 和 Tc17 分化的重要调节因子。 已证明具有抗肿瘤功效的细胞和 RORγt 控制增强免疫力和 我们已报道甾醇和含氧甾醇是高亲和力的内源性物质。 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学的 尽管在某些实验范例中，RORγ 可以招募共激活剂而无需添加 外源配体，表明受体可能具有组成型活性。 RORγ 的激活依赖于配体结合。 虽然对受体分离域的广泛结构研究为高 亲和配体结合激动剂和拮抗剂，缺乏关于模块化如何结合的信息 鉴于 RORγ 的重要性，RORγ 结构域在完整的全长受体中共同作用。 作为治疗靶标，令人惊讶的是我们对小分子如何调节的了解不完全 “关闭”RORγ 活性的机制看似简单，但我们有一个方法。 我们对受体如何“开启”的了解不完全。 扰动操纵受体的定位和 PTM 状态，影响其辅助调节子和 DNA 为了提供检验这一假设的基础，我们进行了 RORγ 转录组的相互作用调节。 建议开发并验证完整全长 RORγ/DNA 复合物的集成结构模型 扩大我们对 RORγ 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，并可能通过设计产生新的治疗策略 功能选择性配体。