Structure-Function Relationship of CAR

描述（由申请人提供）：本提案的长期目标是了解组成型雄烷受体（CAR）的结构机制。CAR属于蛋白质的核受体超家族。这些都是基因调控的转录因子，几乎指导人类生理学的各个方面。CAR在不存在配体的情况下具有组成型活性。已经鉴定了调节CAR活性的小分子。甾体分子雄甾烷醇作为反向激动剂发挥作用并抑制CAR的组成性活性。另一种分子TCPOBOP从雄甾烷醇介导的抑制中拯救CAR，并可以增强CAR活性。我们假设CAR结构采用了独特的，但微妙的，来自经典核受体结构折叠的修饰，导致这种独特的行为。我们假设CAR的三种状态，即非活性的雄甾烷醇结合状态，活性的载脂蛋白和TCPOBOP结合的高活性状态，通过变构机制联系在一起。在具体目标1中，我们建议比较载脂蛋白CAR与雄甾烯醇结合CAR的结构。在具体目标2中，我们将比较CAR：TCPOBOP与载脂蛋白和雄甾烷醇结合的CAR结构。在具体目标3中，我们将通过对这些突变体进行基于结构的点突变以及结合和活性测定来研究CAR变构的机制。 CAR已显示指导属于细胞色素P50类酶的CYP 2和CYP 3基因产物的转录。这些酶负责异生物质如药物的代谢。CAR直接参与镇痛药、对乙酰氨基酚（商业命名：泰诺）等药物的代谢。了解CAR分子机制在核受体生物学中具有重要意义，在这些蛋白质的药理学中具有广泛的意义。由于其参与治疗剂的代谢，CAR本身是设计可以增强或减弱其转录活性的小分子配体的有吸引力的靶标。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the structural mechanism of the constitutive androstane receptor (CAR). CAR belongs to the nuclear receptor superfamily of proteins. These are hormone-modulated transcription factors that direct almost every aspect of human physiology. CAR is constitutively active in the absence of ligand. Small molecules have been identified that modulate CAR activity. The steroidal molecule, androstanol, functions as an inverse agonist and inhibits the constitutive activity of CAR. Another molecule, TCPOBOP, rescues CAR from the androstanol-mediated repression and can enhance CAR activity. We hypothesize that the CAR structure employs unique, but subtle, modifications from the canonical nuclear receptor structural fold that results in this distinctive behavior. We hypothesize that the three states of CAR, the inactive, androstanol-bound, the active, apo, and the TCPOBOP-bound highly active states are linked by an allosteric mechanism. In Specific Aim 1 we propose to compare the structures of apo CAR with androstenol-bound CAR. In Specific Aim 2, we will compare the CAR:TCPOBOP to apo and androstanol-bound CAR structures. In Specific Aim 3, we will study the mechanism of CAR allosterism by performing structure-based point mutations and binding and activity assays on these mutants. CAR has been shown to direct the transcription of the CYP2 and CYP3 gene products, which belong to the cytochrome P50 class of enzymes. These enzymes are responsible for the metabolism of xenobiotics such as pharmaceutical drugs. CAR has been directly implicated in the metabolism of the analgesic, acetaminophen (commercial nomenclature: Tylenol) other pharmaceutical products. Understanding the CAR molecular mechanism is of great significance in nuclear receptor biology with broad implications in the pharmacology of these proteins. Because of its involvement in metabolism of therapeutics, CAR is itself an attractive target for the design of small molecule ligands that can both enhance or diminish its transcriptional activity.