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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

基本信息

批准号：
7760905
负责人：
DANIEL S LEVY
金额：
$ 28.33万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
1979
资助国家：
美国
起止时间：
1979-08-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7760905
关键词：
3&apos Untranslated Regions 5&apos Flanking Region Affect Agonist Amish Antibodies Bile Acids Binding Biological Assay Carcinogens Carrier Proteins Cells Code DNA Sequence Analysis Defect Disease Drug Metabolic Detoxication EPHX1 gene Electrons Electrophoretic Mobility Shift Assay Endogenous Factors Endoplasmic Reticulum Epoxide hydrolase Etiology Exogenous Factors Exons Fibrates Funding Genetic Polymorphism Genetic Transcription Genomics Genotype Glucuronides Hepatic Hepatocyte Human Hydrocarbons Hydrolase Gene Individual Introns Knockout Mice Knowledge Label Laboratories Liquid Chromatography Liver diseases Malignant Neoplasms Mediating Membrane Metabolic Metabolism Microsomal Epoxide Hydrolase Mutation Nuclear Receptors PPAR alpha Physiological Processes Plasma Play Poison Population Predisposition Procedures Proteins RNA Splicing Radiolabeled Regulation Regulatory Element Research Research Personnel Role Serum Site Site-Directed Mutagenesis Sodium Technology Transfection Translations Transmembrane Transport Uridine Diphosphate Glucuronic Acid Urine Western Blotting Xenobiotic Metabolism Xenobiotics chromatin immunoprecipitation deletion analysis expression vector genetic linkage analysis kindred programs promoter radiotracer tandem mass spectrometry transcription factor uptake

项目摘要

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.

人微粒体环氧化物水解酶（mEH）是一种双功能蛋白，在钠依赖性肝脏对胆汁酸的吸收以及多种外源致癌物的代谢。 mEH 基因 (EPHX1) 调控元件多态性引起的 mEH 异常表达已被证明会导致胆汁酸摄取缺陷，从而导致高胆碱血症，并且与多聚编码区的态射，导致多种致癌物代谢改变对各种形式的癌症的易感性。尽管 mEH 在胆汁酸转运中发挥着关键作用和外源代谢，通过内源性和外源性调节 EPHX1 表达的机制我们实验室的研究现在刚刚开始阐明外源因素。长期来看因此，该研究计划的目标是表征 EPHX1 表达的调节，进一步阐明 mEH 在介导胆汁酸转运中的作用，并鉴定和功能表征人类 EPHX1 多态性影响高胆碱血症受试者的转运功能。本次活动的具体目标建议是 A) 阐明参与 EPHX1 胆汁酸诱导的转录机制 HNF-3beta、PPARα 和 PXR/CAR 介导的表达； B) 表征 mEH 介导的转运胆汁酸进入肝细胞和内质网腔，在此进行胆汁酸解毒可能通过葡萄糖醛酸化发生，并且 C) 表征人类 EPHX1 多态性在 EPHX1 转录和/或 mEH 功能表达的调控位点或编码区，如与高胆碱血症的病因有关。 EPHX1 表达研究将使用启动子活性测定， Northern 和 Western blot 分析、转录因子表达载体、足迹、定点诱变、电泳迁移率变动分析和染色质免疫沉淀。运输研究将在野生型和 mEH 敲除小鼠上进行。多态性研究将利用测序、基因分型和功能表达程序。这些研究应该会大大增加我们对此的了解重要的转运/代谢蛋白，在许多生理过程和包括胆汁淤积性肝病在内的多种疾病的病因学。