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（DME）的表达。I相和II相代谢的DME积极参与外源性物质和内源性化合物的解毒，并且是药物失活和干扰的主要来源。因此，了解开始与环境化合物和药物的摄入，并导致DME上调的分子事件是预测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基因的表达在其底物的存在下被激活，但对这种诱导机制的了解最近才得到阐明。在过去的几年中，核受体（NR）PXR已被牵连作为一个关键的介导的外源性诱导DME基因的表达在一些脊椎动物物种。最近完成的C.线虫测序项目揭示了超过260种线虫NR的存在，包括与PXR密切相关的几种。在这些PXR相关的NR中，NHR-8是C.线虫需要野生型对异生物质的抗性。NHR-8在异生物质抗性中的作用表明，与其脊椎动物同源物一样，NHR-8通过上调解毒网络的表达以去除有害化合物来响应异生物质毒性化合物的存在。本研究旨在通过以下方法验证这一预测：1）描述线虫解毒网络的成员在C。elegans基因组，2）确定由NHR-8上调的DME基因座，3）发现NHR-8表达的调节，和4）建立基于细胞培养的NR信号传导测定以研究NHR-8配体结合。梭将从线虫基因组序列中挖掘DME编码开放阅读框。将使用各种系统发育和比较基因组分析来表征所得数据，以确定线虫和脊椎动物蛋白之间的关系。本文对C. elegans解毒网络将允许生产DME特异性DNA微阵列，这将有助于发现NHR-8调控的DME。NR结构域的模块化性质将被利用来产生携带嵌合NR的转基因动物，所述嵌合NR含有限定的DNA结合结构域和NHR的配体结合结构域，以探测NHR-8响应于外源性物质的转录激活。

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.