MOLECULAR DETERMINANTS OF UGT FUNCTION

UGT 功能的分子决定因素

• 批准号: 6387238
• 负责人: Michael H Court
• 金额: $ 20.88万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387238
• 关键词: acetaminophen; animal tissue; biotransformation; drug metabolism; enzyme mechanism; enzyme substrate; genetic polymorphism; glucuronosyltransferase; human tissue; isozymes; protein protein interaction; uridine diphosphate; yeast two hybrid system

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.

描述(改编自申请者摘要)：葡萄糖醛酸化反应，由 UDP-葡萄糖醛酸基转移酶(UGT)是一种重要的代谢酶 参与多种药物失活和排泄的途径， 毒素、潜在致癌物和内生菌。这个项目的长期目标是 研究是为了阐明个体变异性的分子决定因素 在UGT1A6函数中。在这样做的过程中，可能会发现 人群中可能存在药物不良反应高风险的个人 反应和相互作用，对环境毒素的敏感性和 致癌物，以及那些内源性新陈代谢先天缺陷的人。 UGT1A6优先葡萄糖醛酸化平面酚类化合物和 对扑热息痛的生物转化有很大贡献。 人类中对乙酰氨基酚的葡醛酸化反应似乎是异质性的，而 这种现象的分子基础目前尚不清楚。有证据表明 人类UGT1A6基因功能相关的多态，可能影响 底物亲和力或酶含量。此外，最近的研究 提示UGT异构体可以形成异二聚体，可以调节 UTG1A6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UTG1A6介导的葡萄糖醛酸化反应，将通过比较证实 用目前可用的方法测定活性和酶的动力学 表达的UGT异构体，并通过异构体特异性免疫抑制 人肝微粒体中对乙酰氨基酚的葡醛酸化反应：(2) UGT1A6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突变体UGT1A6的活性， 并通过使用人肝微粒体的表型-基因分析和(3) 研究蛋白质-蛋白质相互作用在调节中的潜在作用 UGT1A6通过鉴定与UGT1A6相互作用的蛋白介导的葡萄糖醛酸化 酵母双杂交表达系统的构建及功能验证 通过共表达研究这些相互作用的意义。