MOLECULAR DETERMINANTS OF UGT FUNCTION

描述（改编自申请人摘要）：葡萄糖醛酸化，由 UDP-葡萄糖醛酸基转移酶（UGT）是一种重要的代谢酶， 参与多种药物的失活和排泄的途径， 毒素、潜在致癌物和内毒素。这一长期目标 研究的目的是阐明个体差异的分子决定因素 UGT 1A 6功能。这样，就有可能确定 人群中可能存在不良药物高风险的个体 反应和相互作用，对环境毒素的敏感性， 致癌物，以及那些与先天性缺陷的内源性代谢。 UGT 1A 6优先葡糖醛酸化平面酚类异生物质， 基本上有助于对乙酰氨基酚的生物转化。 人体中对乙酰氨基酚葡萄糖醛酸化似乎是异质性的， 这种现象的分子基础目前尚不清楚。有证据表明 人类UGT 1A 6基因的功能相关多态性，可能影响 底物亲和力或酶含量。此外，最近的研究 提示UGT同种型可以形成异二聚体， UTG 1A 6通过蛋白质-蛋白质相互作用介导的葡萄糖醛酸化。三 具体目标是：（1）利用对乙酰氨基酚作为探针底物 对于UTG 1A 6介导的葡萄糖醛酸化，将通过比较 活性和酶动力学测定， cDNA表达的UGT亚型，并通过亚型特异性免疫抑制 对乙酰氨基酚在人肝微粒体中的葡萄糖醛酸化：（2）研究 UGT 1A 6基因多态性对同工酶含量和特异性的影响 通过比较表达的野生型和变体UGT 1A 6确定活性， 以及通过使用人肝微粒体的表型-基因型分析和（3） 研究蛋白质-蛋白质相互作用在调节 UGT 1A 6介导的葡萄糖醛酸化，通过鉴定与 酵母双杂交表达系统，并证实了功能 通过共表达研究这些相互作用的意义。

DESCRIPTION (Adapted from Applicant's Abstract): Glucuronidation, catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes, is an important metabolic pathway involved in the inactivation and excretion of a multitude of drugs, toxins, potential carcinogens and endobiotics. The long-term objectives of this research are to elucidate the molecular determinates of individual variability in UGT1A6 function. In doing so, it may then be possible to identify individuals within a population that may be at high risk for adverse drug reactions and interactions, susceptibility to environmental toxins and carcinogens, as well as those with inborn errors of endogenous metabolism. UGT1A6 preferentially glucuronidates planar phenolic xenobiotics and substantially contributes to the biotransformation of acetaminophen. Acetaminophen glucuronidation in humans appears to be heterogenous and the molecular basis for this phenomenon is currently unknown. There is evidence for functionally relevant polymorphisms in the human UGT1A6 gene, which may affect either substrate affinity or enzyme content. Furthermore, recent studies suggest that UGT isoforms can form heterodimers which could modulate UTG1A6-mediated glucuronidation through protein-protein interactions. Three specific aims are proposed: (1) To utilize acetaminophen as a probe substrate for UTG1A6-mediated glucuronidation which will be substantiated by comparative activity and enzyme kinetic determinations using currently available cDNA-expressed UGT isoforms, and by isoform-specific immunoinhibition of acetaminophen glucuronidation in human liver microsomes: (2) To investigate the influence of polymorphisms in the UGT1A6 gene on isoenzyme content and specific activity ascertained by comparisons of expressed wild-type and variant UGT1A6, and by phenotypic-genotypic analyses using human liver microsomes and (3) To investigate the potential role for protein-protein interactions in modulating UGT1A6-mediated glucuronidation by identifying interacting proteins with the yeast two-hybrid expression system, and substantiating the functional significance of these interactions by coexpression studies.