Structural Characterization of Metabolic Gene Regulators

描述(由申请人提供)：本提案侧重于肝细胞核因子4α(HNF4a)的结构和功能特征，HNF4a是代谢基因计划的主要转录调节因子。HNF4a对配体结合没有反应，而是似乎依赖于甲基化和磷酸化，以及调节其转录活性的辅活化子的诱导。我们最近对HNF4a的结构表征揭示了多个出乎意料的结构域-结构域相互作用。高度互连的结构域排列将亚单位二聚与DNA和共激活物结合联系起来。我们希望通过一系列生化和基于细胞的研究来验证这种对受体变构排列的理解，这些研究探索了这些结构域之间的联系。我们还发现了几个映射到关键的域-域连接的翻译后修饰。因此，第二个目标是了解共价修饰传递到受体的DNA结合域的变构机制。第三个目标是扩大我们对共激活剂如何与HNF4a相互作用的结构性和机械性理解。在这里，我们将检验一个新的假设，即一些辅助激活剂，包括PGC-1a，可以物理地驱动和稳定HNF4a在DNA上的生产性复合体。 公共卫生相关性：我们建议的研究集中在HNF4a上，HNF4a是肝脏和胰岛细胞基因的主要调节者。HNF4a调节葡萄糖和胰岛素的产生，并与一种形式的糖尿病有关。为了了解HNF4a的功能，我们将应用一些技术，使我们能够可视化其蛋白质结构，并检查其与其他蛋白质的相互作用。

DESCRIPTION (provided by applicant): This proposal focuses on the structural and functional characterization of the hepatocyte nuclear factor 4 alpha (HNF4a), a master transcriptional regulator of metabolic gene programs. HNF4a does not respond to ligand binding, but instead appears to rely on methylation and phosphorylation, and on the induction of coactivators for regulating its transcriptional activity. Our recent structural characterization of HNF4a reveals multiple domain-domain interactions that were unanticipated. A highly interconnected domain arrangement links subunit dimerization to both DNA and coactivator binding. We wish to validate this understanding of the receptor's allosteric arrangement using a series of biochemical and cell-based studies that probe these inter-domain connections. We also found several posttranslational modifications that map to key domain-domain junctions. Therefore, a second goal is to understand the allosteric mechanism by which covalent modifications are transmitted to the DNA binding domain of the receptor. The third goal is to expand our structural and mechanistic understanding of how coactivators interact with HNF4a. Here we will test a novel hypothesis that some coactivators, including PGC-1a, can physically drive and stabilize the productive complex of HNF4a on DNA. PUBLIC HEALTH RELEVANCE: The studies we propose are focused on HNF4a, a master regulator of genes in the liver and in pancreatic islet cells. HNF4a regulates the production of glucose and insulin, and has been linked to a form of diabetes. To understand the way in which HNF4a functions, we will apply techniques that allow us visualize its protein architecture, and examine its interactions with other proteins.