HUMAN HEPATIC & INTESTINAL UDP GLUCURONOSYLTRANSFERASES

人肝脏udp -葡萄糖醛酸糖基转移酶（UGT）内源性和外源性化合物的生物转化已经得到了很好的证实。肠道的作用，持续暴露于潜在有毒的饮食成分，药物和胆汁分泌的代谢物，还没有得到很好的研究。本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UGT2B酶进行比较，主要侧重于类固醇导向的同工酶。待验证的中心假设是，人类肠道参与内源性和外源性化合物的生物转化，并作为整个解毒机制的一部分发挥作用。具体来说，肠道组织被认为是类固醇激素、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特定的mRNA。将进行平行实验以鉴定UGT2B酶活性和蛋白。将对重组人UGT2B4、2B7和新型2B亚型在糖醛酸化类固醇激素、BA和类维生素A方面的功能进行比较。该资助还建议定义，对于肝脏和肠道UGT2B亚型，底物结合位点的结构特征赋予底物特异性的独特差异。待验证的假设是，UGT底物选择性是由蛋白质可变n端结构域中的氨基酸残基子集决定的。酶分析、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定位和诱变是将使用的技术之一。为了解决这些问题，我们提出了两个具体目标：1。鉴定和表征人类肠道UGT2B亚型，并与人类肝脏UGT2B进行比较。2. 定位UGT2B底物结合位点，鉴定决定其独特底物特异性的残基。预计这些研究将导致肠道UGT表达对药物和膳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。了解底物特异性的分子基础对预测生物转化途径、抑制剂设计和内源性和外源性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.