Nuclear Receptor Regulation of Detoxification Networks

描述(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(DME)的表达。I、II相代谢的DME积极参与外源物质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，了解从环境化合物和药物的摄入开始并导致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。虽然人们很早就认识到DME基因的表达是在底物存在的情况下被激活的，但对这种诱导机制的深入了解直到最近才被阐明。在过去的几年里，核受体(NR)PXR被认为是异源诱导DME基因在几种脊椎动物中表达的关键媒介。最近完成的线虫测序项目揭示了260多个线虫NRs的存在，其中包括几个与PXR密切相关的NRs。在这些与PXR相关的NRs中有NHR-8，线虫需要NHR-8来实现野生型对外源物质的抗性。NHR-8在异种抗药性中的作用表明，像它的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，从而对异生有毒化合物的存在做出反应。这项研究的目的是通过1)描述线虫基因组中线虫解毒网络的成员，2)确定受NHR-8上调的DME基因，3)发现NHR-8表达的调节，以及4)建立基于细胞培养的NR信号分析来研究NHR-8的配基结合来验证这一预测。线虫基因组序列将被挖掘为DME编码的开放阅读框。结果数据将使用各种系统发育和比较基因组分析来表征，以确定线虫和脊椎动物蛋白质之间的关系。线虫解毒网络的描述将允许生产特定于DME的DNA微阵列，这将有助于发现受NHR-8调控的DME。NHR结构域的模块化性质将被利用来产生携带嵌合NRs的转基因动物，该NRs包含定义的DNA结合域和NHR的配体结合结构域，以探测NHR-8对外源生物的转录激活。

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.