HUMAN HEPATIC & INTESTINAL UDP GLUCURONOSYLTRANSFERASES

人肝UDP-葡萄糖醛酸基转移酶(UGT)对内源和外源化合物的生物转化是公认的。肠道的作用，不断暴露在潜在有毒的饮食成分，药物和胆汁中分泌的代谢物，还没有得到很好的研究。这项建议的长期目标是鉴定和表征2B家族的肠道UGT，并将它们与肝脏UGT2B酶进行比较，重点是类固醇导向的同工酶。需要检验的中心假设是，人体肠道参与内源和外来化合物的生物转化，并作为整体解毒机制的一部分发挥作用。具体地说，肠组织被认为是类固醇激素、某些胆汁酸(BA)和维甲酸生物转化的活跃部位。将使用序列特异性探针对肠道中的特定mRNA进行筛选。将进行平行实验来鉴定UGT2B的酶活性和蛋白质。将对人重组UGT2B4、2B7和新的2B亚型在类固醇激素、BA和维甲酸的葡萄糖醛酸化反应中的功能进行比较。这项资助还建议为肝脏和肠道的UGT2B亚型定义底物结合部位的结构特征，这些结构特征赋予底物专一性的独特差异。需要检验的假设是，UGT底物的选择性由蛋白质可变N-末端结构域中的氨基酸残基的子集决定。将使用的技术包括酶分析、光亲和标记、基因克隆、重组蛋白的表达和纯化、活性部位的蛋白水解图和诱变。为了解决这些问题，我们提出了两个具体的目标：1.鉴定和鉴定人类肠道UGT2B亚型，并将其与人肝脏UGT进行比较。2.定位UGT2B底物结合位点，并鉴定决定其独特底物特异性的残基。预计这些研究将导致关于肠道UGT表达对药物和饮食成分的解毒作用及其对肝脏和肠道疾病的影响的新概念。底物专一性的分子基础知识对于预测生物转化途径、抑制剂设计以及内源和外源化合物代谢的个体差异具有重要意义。

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.