HUMAN HEPATIC & INTESTINAL UDP GLUCURONOSYLTRANSFERASES

内源性和外源性化合物的人肝UDP-葡萄糖醛酸基转移酶（UGT）生物转化已得到充分确立。 肠道持续暴露于胆汁中分泌的潜在有毒饮食成分、药物和代谢物，其作用尚未得到充分研究。 该提案的长期目标是鉴定和表征2B家族的肠道UGT，并将其与肝脏UGT 2B酶进行比较，主要强调类固醇定向同工酶。 待检验的中心假设是，人类肠道参与内源性和外源性化合物的生物转化，并作为总解毒机制的一部分发挥作用。 具体而言，假定肠组织是类固醇激素、某些胆汁酸（BA）和类维生素A生物转化的活性部位。将使用序列特异性探针筛选肠道中的特异性mRNA。 将进行平行实验以鉴定UGT 2B酶活性和蛋白质。 将对人重组UGT 2B 4、2B 7和新型2B亚型在类固醇激素、BA和类维生素A葡萄糖醛酸化方面的功能进行比较。 该授权还建议定义肝和肠UGT 2B亚型的底物结合位点的结构特征，这些结构特征赋予底物特异性的独特差异。 待检验的假设是UGT底物选择性由蛋白质可变N-末端结构域中的氨基酸残基子集决定。 将使用的技术包括酶测定、光亲和标记、cDNA克隆、重组蛋白的表达和纯化、活性位点的蛋白水解作图和诱变。 为了解决这些问题，提出了两个具体的目标：1。鉴定和表征人肠道UGT 2B亚型，并将其与人肝脏UGT进行比较。 2.定位UGT 2B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解毒的影响及其对肝脏和肠道疾病的影响的新概念。 底物特异性的分子基础的知识应该有重要的影响，预测的生物转化途径，抑制剂的设计和内源性和外源性化合物的代谢的个体间差异。

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.