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

项目摘要/摘要：肝脏受体同源蛋白-1(LRH-1；NR5A2)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。具体地说，它的特征是调节胆汁酸。 新陈代谢、胆固醇稳态和类固醇生成，进而参与各种疾病状态，如 如2型糖尿病、动脉粥样硬化、非酒精性脂肪性肝病以及多种癌症。这些 疾病都是代谢综合征的风险因素，影响着近三分之一的美国人口。这 代表着一个重大的健康问题，因为被诊断为代谢综合征的人风险增加 罹患心血管疾病以及肝细胞癌、胃癌和结肠癌； 与LRH-1的过度表达有关。这表明LRH-1是一种有希望的治疗靶点。 这样的代谢性疾病和癌症。然而，缺乏对受体调控的整体了解 这一机制对LRH-1作为治疗靶点的成功提供了很大的限制。与其他类似 作为核受体，LRH-1‘S的活性受到多种途径的严格调控，包括细胞定位， 配基结合、DNA结合、翻译后修饰和协同调控相互作用。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伙伴。而与此同时， 多个核定位信号和核输出信号已被确定为促进受体穿梭 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。这推动了制定有效和具体的 调节LRH-1定位和活性的工具。虽然使用化学方法来探测NRS在 多年来，新陈代谢有所增加，但化学蛋白质组学在这一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，因为它们相对较低，含量较高，并与 染色质。因此，需要工具来探测和捕获内源性LRH-1和LRH-1转录 复合体。我提出了以下具体目标来开发和使用LRH-1特异性化学蛋白质组工具 阐明乙酰化和一种新的协调制层相关多肽2在调节中的作用 LRH-1的核内转运和信号转导。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转录复合体。其中包括生物素化的 LRH-1反应元件寡核苷酸探针和LRH-1信号小分子抑制物SR1848。在《目标2》中，我会 鉴定LRH-1乙酰化残基并验证LAP2亚型为新的LRH-1协调控子 微调LRH-1的核内转运和后续信号传递。阐明这一调控途径将 直接提高对LRH-1信号驱动肝癌和其他代谢性疾病的了解。

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.