MOLECULAR DETERMINANTS OF UGT FUNCTION

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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.