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

项目概述和摘要：肝受体同源物-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。这推动了 需要开发有效和具体的化学工具来调节受体的活性，并扩大我们的 了解其在健康和疾病途径中的作用。与其他NR类似，LRH-1的活性与 通过多种途径调节，包括配体结合、DNA结合、细胞定位、后 翻译修饰（PTM）和辅调节因子相互作用。然而，与许多其他NR不同，LRH-1结合 DNA作为一个单体和一个特定的候选内源性配体尚未确定。相反，LRH-1 已经显示出结合各种磷脂，以差异地控制下游信号传导 途径。因此，额外的互补或冲突的调节途径的相互作用是至关重要的， 严格控制LRH-1活性。这些调节途径的变化导致受体活性异常 有助于疾病的病理生理学。虽然使用化学方法探测代谢中的NR， 多年来，化学蛋白质组学在该领域的应用有所增加，但仍然有限。NR的蛋白质组学分析 由于NR的丰度相对较低并且与染色质紧密结合，因此仍然存在问题。因此，有必要 用于捕获内源性LRH-1和LRH-1转录复合物的工具，以更好地了解LRH-1的能力 来控制正常生理或驱动病理过程。我提出以下具体目标开发新的 方法和扩展LRH-1调节机制的理解。在目标1中，我将建立三个 LRH-1特异性探针以更有效地探测和/或捕获LRH-1和LRH-1转录复合物。 这些包括生物素化的LRH-1应答元件寡核苷酸探针、修饰的DLPC生物素探针和SR 1848， LRH-1小分子抑制剂。在目的2中，我将用SR1848或Cpd3探针LRH-1以抑制受体活性 和DLPC或RJW 101来驱动受体活性，以观察差异调节的PTM和辅调节因子 了解他们如何沟通，以调节LRH-1贩运和随后的信号。

Project Summary and 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 American 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. This propels the need to develop effective and specific chemical tools to modulate the receptor’s activity and expand our knowledge of its role in both healthy and disease pathways. Similarly to other NRs, LRH-1’s activity is tightly regulated via a multitude of pathways including ligand binding, DNA binding, cellular localization, post- translational modifications (PTMs), and coregulator interactions. However, unlike many other NRs, LRH-1 binds DNA as a monomer and a specific candidate endogenous ligand has yet to be identified. Rather, LRH-1 has been shown to bind an assortment of phospholipids in order to differentially control downstream signaling pathways. Hence, the interplay of additional complementary or conflicting regulatory pathways are essential for tight control of LRH-1 activity. Changes in these regulatory pathways leads to aberrant receptor activity contributing to disease pathophysiology. 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 NRs remains problematic as NRs are relatively low abundant and tightly bound to chromatin. As such, there is a need for tools to capture endogenous LRH-1 and LRH-1 transcriptional complexes to better understand LRH-1’s ability to control normal physiology or drive pathological processes. I propose the following specific aims develop novel methodologies and extend the understanding of LRH-1 regulatory mechanisms. In Aim 1, I will establish three LRH-1 specific probes to more efficiently probe and/or capture LRH-1 and LRH-1 transcriptional complexes. These include a biotinylated LRH-1 response element oligo probe, a modified DLPC biotin probe, and SR1848, small molecule inhibitor of LRH-1. In Aim 2, I will probe LRH-1 with SR1848 or Cpd3 to repress receptor activity and DLPC or RJW101 to drive receptor activity to observe differentially regulated PTMs and coregulators as well as understand how they communicate to regulate LRH-1 trafficking and subsequent signaling.