Functional Analysis of Nuclear Receptor Variants

描述（由申请人提供）：组成型雄烷受体（CAR，NR 1 I3）是主要在肝脏中表达的核受体超家族成员。CAR在调节生物转化的所有三个阶段中起关键作用，生物转化是决定与暴露于药物、其他外源性物质和内源性物质（如胆汁酸、甲状腺激素、血红素和类固醇）相关的代谢和毒性的命运的途径。由于该受体系统作为人类异生质处置的决定因素的重要性，以及CAR作为生理调节剂的作用日益被认可，因此必须充分表征由人CAR蛋白质组指导的生物过程。先前研究计划的进展包括发现新形式的CAR、CAR 2和CAR 3;通过使用选择性剪接机制产生的受体，该机制分别导致在受体的配体结合结构域内插入4个和5个氨基酸。这些受体在人类肝脏中与CAR同时表达，但具有高度独特的功能属性。与CAR本身不同，变体汽车是配体激活的受体，表现出选择性化学激活谱，并且似乎差异性地调节靶基因表达。我们假设选择性剪接增强了CAR分子的功能多样性，导致作为异生物质传感器的重叠但有区别的作用，驱动肝脏基因表达网络的相互作用，反过来，提供指导人类对化学环境的生理反应的关键整合信号。为了验证我们的假设，将部署三个具体目标的进展，包括：1）结晶CAR蛋白的结构分析; 2）使用原代人肝细胞培养模型以实现受体驱动的转录谱分析、靶基因相互作用和受体细胞内定位的动力学评估;和3）人源化转基因小鼠研究，以分析受体在体内调节的转录控制，以及受体表达在一系列血清标志物内的表型影响。我们预测，基因组程序的相应调节和由变体汽车控制的所得信号传导回路对人类健康具有关键的功能性影响，决定诸如药物-药物相互作用、药物和胆汁酸诱导的肝毒性、致癌物和类固醇代谢以及脂质和能量稳态的调节等结果。拟议研究的结果将有助于重要的新进展和见解，关于CAR核受体作为动态基因调控网络的直接调节剂的作用，这些动态基因调控网络决定了对外源性和内源性物质的毒理学和生理学反应。 公共卫生相关性：在这项研究计划中，我们的研究将描述一系列新的受体蛋白的作用，这些蛋白与药物和环境化学物质相互作用，并调节肝脏代谢物质的能力。这些受体被称为组成型雄烷受体，是与肝细胞一起存在的传感蛋白电池的一部分，以帮助处理细胞化学环境的性质，以便更好地调节其对我们暴露的化学物质进行解毒的能力。

DESCRIPTION (provided by applicant): The constitutive androstane receptor (CAR, NR1I3) is a member of the nuclear receptor superfamily that is expressed primarily in the liver. CAR plays a key role in regulating all three phases of biotransformation, pathways that determine the fates of metabolism and toxicity associated with exposures to drugs, other xenobiotics, and endogenous substances such as bile acids, thyroid hormone, heme, and steroids. Due to the importance of this receptor system as a determinant of xenobiotic disposition in man, together with CAR's increasingly recognized role as a physiological regulator, it is imperative to fully characterize the biological processes directed by the human CAR proteome. Progress in the previous research program included the discovery of novel forms of CAR, CAR2 and CAR3; receptors generated through the use of alternative splicing mechanisms that result in the insertion of 4- and 5- amino acids, respectively, within the receptors' ligand binding domain. These receptors are expressed simultaneously with CAR in human livers, but possess highly unique functional attributes. Unlike CAR itself, the variant CARs are ligand-activated receptors, exhibit selective chemical activation profiles, and appear to differentially modulate target gene expression. We hypothesize that alternative splicing enhances the functional diversity of CAR molecules, resulting in overlapping, yet discriminating roles as xenobiotic sensors, driving the interplay of hepatic gene expression networks that in turn, provide critical integration signals directing human physiological responses to the chemical milieu. To test our hypotheses, a progression of three specific aims will be deployed that include: 1) the structural analysis of crystallized CAR proteins; 2) use of primary human hepatocyte culture models to enable receptor-driven transcriptional profiling, target gene interactions, and assessment of the dynamics of receptor intracellular localization; and, 3) humanized transgenic mouse investigations to analyze the transcriptional controls modulated by the receptors in vivo, and the phenotypic impact of receptor expression within a series of serum markers. We predict that the respective modulation of genomic programs and the resulting signaling circuitry controlled by the variant CARs have critical functional implications for human health, determining outcomes such as drug-drug interactions, drug and bile acid induced hepatotoxicity, carcinogen and steroid metabolism, and the regulation of lipid and energy homeostasis. The results of the proposed research will contribute important new advances and insights regarding the role of the CAR nuclear receptors as direct modulators of the dynamic gene regulatory networks that determine toxicological and physiological responses to both xenobiotic and endogenous substances. PUBLIC HEALTH RELEVANCE: In this research program, our studies will characterize the roles of a novel series of receptor proteins that interact with pharmaceuticals and environmental chemicals and function to regulate the liver's capacity to metabolize substances. These receptors are termed constitutive androstane receptors and are part of a battery of sensing proteins that exist with liver cells to assist in processing the nature of the cell's chemical environment so that it can better tune its ability to detoxify chemicals we are exposed to.