FUNCTIONAL ANALYSIS OF NUCLEAR RECEPTOR VARIANTS

描述(申请人提供)：基因的激活，以响应细胞外刺激，病原体，药物管理，或环境因素，需要一个高度集成的信号转导过程，指导转录机制，以调节适当的基因网络。一系列细胞色素P450(CYP)基因以及其他生物转化功能是这些信号的靶标。与Cyps一样，核激素受体(NHR)是由一个超家族基因编码的，主要协调由化学和激素效应器引发的反应。这项研究提案将特别关注人类结构性雄烷受体(CAR)和RXRpha受体(RXRA)，这两种NHR最近被鉴定为介导包括各种药物和异生药物在内的“苯巴比妥类”诱导化合物的转录活性。该计划将检验的主要假设是，CAR和RXRA核受体在人类群体中具有遗传多态和结构变异。此外，我们将测试假设，各个不同的受体决定不同的生物反应。提出了一系列3个实验目标，以促进研究计划的进展，从最初的发现阶段到体外和体内方法，以实现CAR和RXRA结构变体的生物学特性。研究将包括部署新的基因敲除小鼠模型，使人类NHR及其结构变体能够在原代肝细胞和原本具有零受体背景的肝组织中表达。“人源化”的小鼠将被分析与受体亚型表达相关的改变的生物学活动，包括对药物暴露的反应的干扰，使用扫描荧光显微镜的异常受体相互作用，以及使用DNA微阵列杂交的基因表达谱的变化。人群中NHR受体变异的存在可能决定了化学暴露引起的药物反应和毒性的个体间差异。此外，结构变异受体的存在可能会导致人类疾病风险的个体差异，包括某些癌症。从这些调查中获得的数据将产生关于NHR超家族重要成员的结构多样性的重要的新的遗传和生物学信息。

DESCRIPTION (provided by applicant): Activation of genes in response to extracellular stimuli, pathogens, pharmaceutical administration, or environmental agents, requires a highly integrated signal transduction process that directs the transcriptional machinery to modulate the appropriate network of genes. An array of cytochrome P450 (CYP) genes, as well as other biotransformation functions, are targets of these signals. Like the CYPs, the nuclear hormone receptors (NHRs) are encoded by a superfamily of genes and largely orchestrate the responses triggered by chemical and hormonal effectors. This research proposal will focus in particular on the human constitutive androstane receptor (CAR) and the RXRalpha receptor (RXRa), NHRs that have been characterized recently to mediate the transcriptional activity of 'phenobarbital-like' inducer compounds that include a variety of pharmaceuticals and xenobiotic agents. The primary hypothesis that will be tested with this program is that the CAR and RXRa nuclear receptors are genetically polymorphic and structurally variant in human populations. Furthermore, we will test the hypothesis that the respective variant receptors dictate differential biological responses. A series of 3 experimental aims are advanced to facilitate the progression of the research program through initial discovery phases to in vitro and in vivo approaches to enable biological characterization of CAR and RXRa structural variants. Investigations will include the deployment of novel knockout mouse models that enable expression of the human NHRs, and structural variants thereof, in primary hepatocytes and in liver tissues that otherwise possess null receptor backgrounds. The "humanized" mice will be analyzed for altered biological activities associated with expression of the receptor isoforms, including perturbations in response to drug exposures, aberrant receptor interactions using scanning fluorescence microscopy, and changes in the repertoire of gene expression using DNA microarray hybridizations. The presence of variant NHR receptor variants in the population likely dictates substantial interindividual differences in drug response and toxicities resulting from chemical exposures. In addition, the existence of structurally variant receptors may contribute to individual differences in the risk for human diseases, including certain cancers. The data obtained from these investigations will generate important new genetic and biological information regarding the structural diversity of prominent members of the NHR superfamily.