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

项目摘要/摘要：肝受体同系物-1（LRH-1; NR 5A 2）是一种磷脂敏感核 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，并反过来参与各种疾病状态， 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 这些疾病都是代谢综合征的危险因素，影响了近三分之一的美国人口。这 这是一个重大的健康问题，因为被诊断患有代谢综合征的个体 发展心血管疾病以及肝细胞癌，胃癌和结肠癌;癌症， 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， 比如代谢疾病和癌症。然而，缺乏对受体调节的整体理解， 这一机制对LRH-1作为治疗靶点的成功提出了重大限制。类似于其他 作为核受体，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、翻译后修饰和辅调节因子相互作用。细胞定位 是这些途径的核心，因为它决定了受体的环境，从而决定了可用的相互作用伴侣。同时， 已经鉴定了多个核定位信号和核输出信号以促进受体穿梭 在核内外，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。这就需要制定有效和具体的 调节LRH-1定位和活性的工具。虽然使用化学方法来探测NRs， 尽管近年来蛋白质代谢增加，但化学蛋白质组学在这一领域的应用仍然有限。蛋白组 对核受体的分析仍然是有问题的，因为它们相对低丰度并且紧密结合到 染色质因此，需要探针和捕获内源性LRH-1和LRH-1转录因子的工具。 配合物我提出了以下具体目标，以开发和使用LRH-1特异性化学蛋白质组学工具 阐明乙酰化和一种新的辅助调节因子纤层相关多肽2（LAP 2）在调节 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， 有效地调节和/或捕获LRH-1和LRH-1转录复合物。其中包括生物素化的 LRH-1应答元件寡核苷酸探针和SR 1848，LRH-1信号传导的小分子抑制剂。在目标2中，我将 鉴定LRH-1乙酰化残基并验证LAP 2同种型作为新的LRH-1辅助调节剂， 微调LRH-1核内运输和随后的信号传导。阐明这一调控途径将 直接增强对LRH-1信号传导驱动肝癌和其他代谢疾病的理解。

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.