HUMAN HEPATIC & INTESTINAL UDP GLUCURONOSYLTRANSFERASES

人肝

• 批准号: 6381608
• 负责人: Anna Radominska-Pandya
• 金额: $ 24.79万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6381608
• 关键词: active sites; affinity labeling; binding sites; biotransformation; chemical kinetics; detoxification; drug metabolism; enzyme activity; enzyme substrate; expression cloning; gender difference; glucuronosyltransferase; human tissue; intestines; isozymes; liver metabolism; protein purification; recombinant proteins

Human hepatic UDP-glucuronosyltransferase (UGT) biotransformations of endogenous and exogenous compounds are well established. The role of the intestine, continually exposed to potentially toxic dietary components, drugs and metabolites secreted in bile, has not been as well studied. The long term goals of this proposal are to identify and characterize intestinal UGTs of the 2B family and to compare them with hepatic UGT2B enzymes, with primary emphasis on steroid-directed isoenzymes. The central hypothesis to be tested is that the human intestine is involved in the biotransformation of endogenous and xenobiotic compounds and functions as part of the total detoxification mechanism. Specifically, it is postulated that intestinal tissue is an active site of biotransformation of steroid hormones, some bile acid (BA) and retinoids. The intestine will be screened for specific mRNA using sequence-specific probes. Parallel experiments will be carried out to identify UGT2B enzymatic activities and proteins. A functional comparison of human recombinant UGT2B4, 2B7 and novel 2B isoforms will be carried out in relation to glucuronidation of steroid hormones, BA and retinoids. This grant also proposes to define, for both hepatic and intestinal UGT2B isoforms, the structural characteristics of the substrate binding sites which confer unique differences in substrate specificity. The hypothesis to be tested is that UGT substrate selectivity is dictated by a subset of amino acid residues in the variable N-terminal domain of the protein. Enzymatic assays, photoaffinity labeling, cDNA cloning, expression and purification of recombinant protein, proteolytic mapping of active sites and mutagenesis are among the techniques that will be used. To approach these problems, two specific aims are proposed: 1. Identify and characterize human intestinal UGT2B isoforms and compare them with human hepatic UGTs. 2. Localize the UGT2B substrate binding sites and identify the residues that determine their unique substrate specificities. It is anticipated that these studies will lead to novel concepts regarding the effect of intestinal UGT expression on detoxification of drugs and dietary constituents and its impact on hepatic and intestinal diseases. Knowledge of the molecular basis of substrate specificity should have important implications for prediction of biotransformation pathways, inhibitor design and inter-individual differences in metabolism of endogenous and xenobiotic compounds.

人肝UDP葡萄糖基转移酶（UGT）的内源性和外源性化合物的生物转化已很好地确定。 肠道不断暴露于胆汁中分泌的潜在有毒饮食成分，药物和代谢产物的作用。 该提案的长期目标是识别和表征2B家族的肠道UGT，并将其与肝的UGT2B酶进行比较，主要强调了类固醇指导的同工酶。 要测试的中心假设是，人类肠与内源性和异种生物化合物的生物转化有关，作为总排毒机制的一部分。 具体而言，据推测，肠道组织是类固醇激素，一些胆汁酸（BA）和类维生素类动物的生物转化的活性部位。肠将使用序列特异性探针筛选特定的mRNA。 将进行平行实验以鉴定UGT2B酶活性和蛋白质。 将与类固醇激素，BA和类视网膜类似的葡萄糖醛酸化有关，对人重组UGT2B4，2B7和新型2B同工型进行功能比较。 该赠款还建议为肝和肠道UGT2B同工型定义，这是底物结合位点的结构特性，这些特性赋予了底物特异性的独特差异。 要检验的假设是UGT底物选择性取决于蛋白质可变的N末端结构域中的氨基酸残基的子集。 酶促测定，光亲和力标记，cDNA克隆，重组蛋白的表达和纯化，活性位点的蛋白水解映射和诱变是将使用的技术之一。 为了解决这些问题，提出了两个具体目标：1。识别和表征人类肠道UGT2B同工型，并将其与人类肝UGT进行比较。 2。定位UGT2B底物结合位点，并确定确定其独特底物特异性的残基。可以预料，这些研究将导致有关肠道UGT表达对药物和饮食成分排毒及其对肝和肠道疾病的影响的新概念。 了解底物特异性的分子基础的知识对预测生物转化途径，抑制剂设计以及内源性和异种生物化合物代谢的个体间差异的预测具有重要意义。