Functional Analysis of Nuclear Receptor Variants

描述(申请人提供)：构成雄烷受体(CAR，NR1I3)是核受体超家族的成员，主要在肝脏表达。CAR在调节生物转化的所有三个阶段中发挥着关键作用，这些途径决定了新陈代谢的命运，以及与药物、其他外源物质和内源性物质(如胆汁酸、甲状腺激素、血红素和类固醇)的接触有关的毒性。由于这一受体系统作为人类异种处理的决定因素的重要性，以及CAR作为生理调节因子的作用日益得到认可，充分描述由人类CAR蛋白质组指导的生物学过程是势在必行的。先前研究计划的进展包括发现新形式的CAR、CAR2和CAR3；通过使用替代剪接机制产生受体，该机制导致在受体的配体结合域中分别插入4-氨基酸和5-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本身不同的是，CAR变体是配体激活的受体，表现出选择性的化学激活特征，并似乎差异地调节靶基因的表达。我们假设，选择性剪接增强了CAR分子的功能多样性，导致作为异源传感器的重叠但有区别的角色，推动肝脏基因表达网络的相互作用，进而提供关键的整合信号，指导人类对化学环境的生理反应。为了验证我们的假设，我们将展开三个特定目标的进展，包括：1)结晶CAR蛋白的结构分析；2)使用原代人类肝细胞培养模型来实现受体驱动的转录图谱、靶基因相互作用和评估受体细胞内定位的动力学；以及3)人源化转基因小鼠的研究，以分析体内受体调控的转录调控，以及一系列血清标志物中受体表达的表型影响。我们预测，基因组程序的各自调控和由此产生的信号电路对人类健康具有关键的功能影响，决定了药物-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平衡的调节等结果。这项拟议的研究结果将有助于对CAR核受体作为动态基因调控网络的直接调节器的作用的重要新进展和见解，动态基因调控网络决定了对外来物质和内源物质的毒理学和生理反应。 与公共健康相关：在这项研究计划中，我们的研究将表征一系列新型受体蛋白的作用，这些蛋白与药物和环境化学品相互作用，并发挥调节肝脏代谢物质的能力的功能。这些受体被称为结构性雄烷受体，是存在于肝细胞中的一系列传感蛋白的一部分，这些蛋白有助于处理细胞化学环境的性质，以便它能够更好地调整其解毒能力，使我们接触到的化学物质。

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.