Developing chemoproteomic approaches to decipher the regulatory network of LRH-1, a nuclear receptor implicated in hepatic metabolism

开发化学蛋白质组学方法来破译 LRH-1（一种与肝脏代谢有关的核受体）的调控网络

• 批准号: 10249186
• 负责人: Valentine Virginie Courouble
• 金额: $ 2.09万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-04-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10249186
• 关键词: Acetylation; Affect; Affinity; Antibodies; Area; Atherosclerosis; Automobile Driving; Binding; Biology; Biotin; Cancer Etiology; Cardiovascular Diseases; Cell Nucleus; Cell physiology; Cessation of life; Chemicals; Cholesterol Homeostasis; Chromatin; Colon Carcinoma; Complex; Conflict (Psychology); DNA Binding; Diabetes Mellitus; Diagnosis; Disease; Disease Pathway; Epitopes; Fostering; Functional disorder; Gene Expression; Genetic Transcription; Growth; Health; Histone Deacetylase; Immunoprecipitation; Individual; Investigation; Knowledge; Ligand Binding; Ligands; Lipids; Liver; Malignant Neoplasms; Malignant neoplasm of liver; Mass Spectrum Analysis; Metabolic; Metabolic Diseases; Metabolic syndrome; Metabolism; Methodology; Molecular; Molecular Chaperones; Molecular Target; NR5A2 gene; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Nuclear Export; Nuclear Localization Signal; Nuclear Receptors; Oligonucleotides; Output; Ovary; Pancreas; Pathway interactions; Phospholipids; Physiological; Physiology; Play; Population; Post-Translational Protein Processing; Primary carcinoma of the liver cells; Process; Protein Isoforms; Proteins; Proteomics; Receptor Signaling; Regulatory Pathway; Reporting; Response Elements; Risk; Risk Factors; Role; Signal Pathway; Signal Transduction; Specificity; Steroid biosynthesis; Streptavidin; Structure-Activity Relationship; Tissues; Traction; Zinc Fingers; base; bile acid metabolism; chemoproteomics; design; interest; liver development; liver metabolism; malignant stomach neoplasm; non-alcoholic fatty liver disease; novel; overexpression; polypeptide; prevent; receptor; receptor function; scaffold; small molecule; small molecule inhibitor; success; therapeutic target; tool; tool development; trafficking; transcription factor

Project Summary/Abstract: Liver receptor homolog-1 (LRH-1; NR5A2) is a phospholipid-sensing nuclear receptor (NR) expressed predominantly in the liver, pancreas, and ovaries that plays an important role in metabolic physiologies and pathophysiologies. Specifically, it has been characterized to regulate bile acid metabolism, cholesterol homeostasis, and steroidogenesis, and in turn is involved in various disease states such as type 2 diabetes, atherosclerosis, nonalcoholic fatty liver disease as well as a multitude of cancers. These diseases are all risk factors for metabolic syndrome that affects nearly a third of the US population. This represents a significant health concern as individuals diagnosed with metabolic syndrome have increased risk for developing cardiovascular diseases as well as hepatocellular, gastric, and colon cancers; cancers that have been associated with overexpression of LRH-1. This suggests LRH-1 to be a promising therapeutic target for such metabolic diseases and cancers. However, a lack of a wholistic understanding of the receptors regulatory mechanism presents a significant limitation to the success of LRH-1 as a therapeutic target. Similar to other nuclear receptors, LRH-1's activity is tightly regulated via a multitude of pathways including cellular localization, ligand binding, DNA binding, post-translational modifications, and coregulator interactions. Cellular localization is central to these pathways as it determines the receptors milieu and thus available interacting partners. While, multiple nuclear localization signals and nuclear export signals have been identified to facilitate receptor shuttling in and out of the nucleus, there is no information regarding LRH-1 intranuclear trafficking, a key process that directly modulates DNA binding and transcriptional output. This propels the need to develop effective and specific tools to modulate LRH-1 localization and activity. While the use of chemical approaches to probe NRs in metabolism has increased over the years, the use of chemoproteomics in this area remains limited. Proteomic analysis of nuclear receptors remains problematic as they are relatively low abundant and tightly bound to chromatin. As such, there is a need for tools to probe and capture endogenous LRH-1 and LRH-1 transcriptional complexes. I propose the following specific aims to develop and employ LRH-1 specific chemoproteomic tools to elucidate the role of acetylation and a novel coregulator lamina associated polypeptide 2 (LAP2) in regulating LRH-1 intranuclear trafficking and signaling. In Aim 1, I will establish two LRH-1 specific probes to more efficiently modulate and/or capture LRH-1 and LRH-1 transcriptional complexes. These include a biotinylated LRH-1 response element oligo probe and SR1848, small molecule inhibitor of LRH-1 signaling. In Aim 2, I will identify LRH-1 acetylated residues and validate LAP2 isoforms as novel LRH-1 coregulators that coordinate to fine tune LRH-1 intranuclear trafficking and subsequent signaling. Elucidation of this regulatory pathway will directly enhance understanding of LRH-1 signaling driving hepatic cancer and other metabolic diseases.

项目摘要/摘要：肝受体同源物-1（LRH-1；NR5A2）是一种磷脂感应核 受体（NR）主要在肝脏、胰腺和卵巢中表达，在 代谢生理学和病理生理学。具体来说，它的特点是调节胆汁酸 代谢、胆固醇稳态和类固醇生成，进而参与各种疾病状态，例如 如 2 型糖尿病、动脉粥样硬化、非酒精性脂肪肝以及多种癌症。这些 这些疾病都是影响近三分之一美国人口的代谢综合征的危险因素。这 代表了一个重大的健康问题，因为被诊断患有代谢综合征的人风险增加 导致心血管疾病以及肝细胞癌、胃癌和结肠癌；癌症有 与 LRH-1 的过度表达有关。这表明 LRH-1 是一个有前途的治疗靶点 诸如代谢疾病和癌症。然而，人们对受体调节缺乏全面的了解。 机制对 LRH-1 作为治疗靶点的成功提出了重大限制。与其他类似 LRH-1 的活性通过多种途径受到严格调节，包括细胞定位、 配体结合、DNA 结合、翻译后修饰和共调节剂相互作用。细胞定位 它是这些途径的核心，因为它决定了受体环境，从而决定了可用的相互作用伙伴。尽管， 已鉴定出多个核定位信号和核输出信号以促进受体穿梭 在细胞核内外，没有关于 LRH-1 核内贩运的信息，这是一个关键过程， 直接调节 DNA 结合和转录输出。这推动了开发有效且具体的需求 调节 LRH-1 定位和活性的工具。虽然使用化学方法来探测 NR 多年来，新陈代谢有所增加，但化学蛋白质组学在该领域的应用仍然有限。蛋白质组学 核受体的分析仍然存在问题，因为它们的丰度相对较低并且与 染色质。因此，需要一种工具来探测和捕获内源性 LRH-1 和 LRH-1 转录 复合物。我提出以下具体目标来开发和使用 LRH-1 特异性化学蛋白质组学工具 阐明乙酰化和新型共调节子层相关多肽 2 (LAP2) 在调节中的作用 LRH-1 核内运输和信号传导。在目标1中，我将建立两个LRH-1特异性探针来更多 有效调节和/或捕获 LRH-1 和 LRH-1 转录复合物。这些包括生物素化的 LRH-1 反应元件寡核苷酸探针和 SR1848（LRH-1 信号传导小分子抑制剂）。在目标 2 中，我将 识别 LRH-1 乙酰化残基并验证 LAP2 亚型作为新型 LRH-1 辅助调节剂，可协调 微调 LRH-1 核内运输和后续信号传导。阐明这一监管途径将 直接增强对驱动肝癌和其他代谢疾病的 LRH-1 信号传导的了解。