Nuclear Receptor Regulation of Detoxification Networks

描述（由申请人提供）：本研究的长期目标是了解环境化学信号如何影响药物代谢酶（DMEs）的表达。I期和II期代谢的DMEs积极参与外源和内源性化合物的解毒，是药物失活和干扰的主要来源。因此，了解从摄取环境化合物和药物开始并导致DME上调的分子事件是预测药物-药物和药物-环境相互作用的关键组成部分。虽然人们早就认识到DME基因表达在它们的底物存在下被激活，但对这种诱导机制的见解直到最近才被阐明。在过去的几年里，核受体（NR） PXR被认为是几种脊椎动物中外源诱导二甲醚基因表达的关键介质。最近完成的秀丽隐杆线虫测序项目揭示了260多个线虫NRs的存在，其中一些与PXR密切相关。在这些与pxr相关的核糖核酸中，NHR-8是秀丽隐杆线虫对外源药物产生野生型抗性所需的核糖核酸。NHR-8在外源耐药中的作用表明，与其脊椎动物同源物一样，NHR-8通过上调解毒网络的表达来清除有害化合物，从而对外源有毒化合物的存在做出反应。本文的研究旨在验证这一预测：1)描述线虫解毒网络在线虫基因组中的成员，2)定义NHR-8上调的DME位点，3)发现NHR-8表达的调控，4)建立基于细胞培养的NR信号分析来研究NHR-8配体结合。秀丽隐杆线虫基因组序列将被挖掘为dme编码开放阅读框。由此产生的数据将使用各种系统发育和比较基因组分析来确定线虫和脊椎动物蛋白质之间的关系。秀丽隐杆线虫解毒网络的描述将允许生产dme特异性DNA微阵列，这将有助于发现由NHR-8调节的dme。NR结构域的模块化特性将被利用来生产带有嵌合NR的转基因动物，嵌合NR包含定义的DNA结合结构域和NHR的配体结合结构域，以探测NHR-8在响应外源药物时的转录激活。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand how environmental chemical signals impact the expression of drug metabolism enzymes (DMEs). DMEs of Phase I and II metabolism are actively involved in detoxification of xenobiotics and endogenous compounds and are a major source of drug inactivation and interference. Therefore, understanding the molecular events that begin with the ingestion of environmental compounds and drugs and result in DME upregulation is a key component of predicting drug-drug and drug environment interactions. Although it has long been recognized that DME gene expression is activated in the presence of their substrates, insights into the mechanisms of this induction have only recently been elucidated. In the past several years, the nuclear receptor (NR) PXR has been implicated as a key mediator of xenobiotic induction of DME gene expression in several vertebrate species. The recent completion of the C. elegans sequencing project revealed the existence of more than 260 nematode NRs including several that are closely related to PXR. Among these PXR-related NRs is NHR-8, which C. elegans requires for wild type resistance to xenobiotics. The role of NHR-8 in xenobiotic resistance suggests that like its vertebrate homolog, NHR-8 responds to the presence of xenobiotic toxic compounds by upregulating the expression of a detoxification network to remove the offending compounds. The research proposed here is designed to test this prediction by 1) describing the members of the nematode detoxification network within the C. elegans genome, 2) defining the DME loci that are upregulated by NHR-8, 3) discovering the regulation of NHR-8 expression, and 4) establishing a cell culture based NR signaling assay to investigate NHR-8 ligand binding. The C. elegans genomic sequence will be mined for DME-coding open reading frames. The resultant data will be characterized using a variety of phylogenetic and comparative genomic analyses to identify the relationships between the nematode and vertebrate proteins. The description of the C. elegans detoxification network will allow the production of a DME-specific DNA microarray that will facilitate the discovery of DMEs regulated by NHR-8. The modular nature of NR domains will be harnessed to produce transgenic animals bearing chimeric NRs containing defined DNA binding domains and NHR's ligand binding domain to probe NHR-8 for transcriptional activation in response to xenobiotics.