Function and Expression of the Bile Acid Transport Protein m-Epoxide Hydrolase

胆汁酸转运蛋白间环氧化物水解酶的功能和表达

• 批准号: 7034097
• 负责人: DANIEL S LEVY
• 金额: $ 30.04万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-08-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7034097
• 关键词: biological transport; cholanate compound; clinical research; confocal scanning microscopy; enzyme induction /repression; enzyme structure; epoxide hydrolase; fluorescence microscopy; gene expression; gene mutation; genes; human genetic material tag; intracellular transport; liver cells; liver metabolism; membrane structure; membrane transport proteins; protein structure function; tissue /cell culture

DESCRIPTION (provided by applicant): Human microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays an important role in the sodium-dependent hepatic uptake of bile acids and in the metabolism of numerous xenobiotic carcinogens. Aberrant expression of mEH caused by polymorphisms in regulatory elements of the mEH gene (EPHX1) has been shown to result in defects in bile acid uptake resulting in hypercholanemia, and along with poly- morphisms in the coding region, in the altered metabolism of numerous carcinogens resulting in alterations in the susceptibility to various forms of cancer. Despite the critical role that mEH plays in bile acid transport and xenobiotic metabolism, the mechanisms that serve to regulate EPHX1 expression by endogenous and exogenous factors are now just beginning to be elucidated by studies from our laboratory. The long-term objectives of this research program are therefore to characterize the regulation of EPHX1 expression, further elucidate the role of mEH in mediating bile acid transport and to identify and functionally characterize human EPHX1 polymorphisms that affect transport function in hypercholanemic subjects. The specific aims of this proposal are A) to elucidate the transcriptional mechanisms involved in the bile acid induction of EPHX1 expression mediated by HNF-3beta, PPARalpha and PXR/CAR; B) to characterize mEH mediated transport of bile acids into hepatocytes and into the lumen of the endoplasmic reticulum where bile acid detoxification may take place via glucuronidation and C) to characterize the effects of human EPHX1 polymorphisms at regulatory sites or in the coding region on the transcription of EPHX1 and/or functional expression of mEH as related to the etiology of hypercholanemia. EPHX1 expression studies will use promoter activity assays, Northern and Western blot analyses, transcription factor expression vectors, footprinting, site-directed mutagenesis, electrophoretic mobility shift assays and chromatin immunoprecipitation. Transports studies will be done on wild type and mEH knockout mice. Polymorphism studies will utilize sequencing, genotyping and functional expression procedures. These studies should substantially increase our knowledge of this important transport/metabolic protein that plays a critical role in numerous physiological processes and in the etiology of several diseases including cholestatic liver disease.

描述（由申请方提供）：人微粒体环氧化物水解酶（mEH）是一种双功能蛋白质，在胆汁酸的钠依赖性肝摄取和多种外源性致癌物的代谢中发挥重要作用。由mEH基因（EPHX 1）的调节元件中的多态性引起的mEH的异常表达已显示导致胆汁酸摄取缺陷，从而导致高胆血症，并且沿着编码区中的多态性，导致许多致癌物的代谢改变，从而导致对各种形式癌症的易感性改变。尽管mEH在胆汁酸转运和外源性物质代谢中起着关键作用，但我们实验室的研究刚刚开始阐明通过内源性和外源性因素调节EPHX 1表达的机制。因此，该研究计划的长期目标是表征EPHX 1表达的调节，进一步阐明mEH在介导胆汁酸转运中的作用，并鉴定和功能表征影响高胆固醇血症受试者转运功能的人EPHX 1多态性。本发明的具体目的是：A）阐明胆汁酸诱导由HNF-3 β、PPARalpha和PXR/CAR介导的EPHX 1表达的转录机制; B）表征mEH介导的胆汁酸转运至肝细胞和内质网腔中，在内质网腔中胆汁酸解毒可能通过葡萄糖醛酸化发生，以及C）表征人EPHX 1在调控位点或编码区的多态性对EPHX 1转录和/或mEH功能表达的影响，其与高胆贫血的病因学相关。EPHX 1表达研究将使用启动子活性测定、北方和Western印迹分析、转录因子表达载体、足迹法、定点诱变、电泳迁移率变动测定和染色质免疫沉淀。将在野生型和mEH敲除小鼠中进行转运研究。多态性研究将利用测序、基因分型和功能表达程序。这些研究应该大大增加我们对这种重要的转运/代谢蛋白的认识，这种蛋白在许多生理过程和包括胆汁淤积性肝病在内的几种疾病的病因学中起着关键作用。