FXR interactions with ligands and coregulators

描述（由申请人提供）：胆汁酸是肝脏胆固醇的终端代谢物，有助于肠道脂质和脂溶性维生素的溶解。此外，通过与核受体FXR结合，某些胆汁酸可以调节负责其生物合成和运输的基因的表达。FXR作为肝脏胆汁酸池的传感器，优先结合鹅脱氧胆酸（CDCA），并转录调节负责胆固醇和胆汁酸稳态的基因。该建议依赖于结构，生化和基于细胞的转录报告分析来解读FXR与其配体的相互作用，并表征负责辅激活因子和辅抑制因子结合的受体构象。在前四个目标中，我们依赖于FXR的分离配体结合域（LBD），并提出了一组实验来提供关于FXR如何识别甾体和非甾体配体，以及配体结合如何改变受体对含有基序的辅激活子衍生的LXXLL和辅抑制子衍生的（I/L）XXII基序的亲和力的立体化学信息。我们还建议利用FXR在关键功能位点的突变，以及与胆汁酸相关的新化学实体，进一步扩展和测试我们关于FXR如何形成其分子相互作用的概念。在第五个目标中，我们建议确定FXR-RXR异源二聚体在其同源DNA响应元件上的晶体结构，以了解RXR和DNA结合如何进一步影响FXR的功能表面。

DESCRIPTION (provided by applicant): Bile acids are the terminal metabolites of hepatic cholesterol and assist in the solubilization of intestinal lipids and fat-soluble vitamins. In addition, through their binding to the nuclear receptor FXR, certain bile acids can regulate the expression of genes responsible for their biosynthesis and transport. FXR acts as the liver's sensor of bile acid pools, binding preferentially to chenodeoxycholic acid (CDCA), and transcriptionally regulating genes responsible for cholesterol and bile acid homeostasis. This proposal relies on structural, biochemical and cell-based transcriptional reporter assays to decipher the interactions of FXR with its ligands, and to characterize the receptor conformations responsible for both coactivator and corepressor binding. In the first four aims, we rely on the isolated ligand binding domain (LBD) of FXR and propose a set of experiments to provide stereochemical information about how FXR recognizes both steroidal and nonsteroidal ligands, and how ligand binding alters the receptor's affinities for coactivator derived LXXLL containing motifs and corepressor-derived (I/L)XXII motifs. We also propose to use mutations of FXR at critical functional sites, as well as novel chemical entities related to bile acids, to further expand and test our notions of how FXR forms its molecular interactions. In the fifth aim, we propose to determine the crystal structure of the FXR-RXR heterodimer on its cognate DNA response element, in order to understand how RXR and DNA binding further impact FXR's functional surfaces.