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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK056226-03
Application #
6381608
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Serrano, Jose
Project Start
1999-09-01
Project End
2003-08-31
Budget Start
2001-09-01
Budget End
2002-08-31
Support Year
3
Fiscal Year
2001
Total Cost
$247,871
Indirect Cost
Name
University of Arkansas for Medical Sciences
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Little Rock
State
AR
Country
United States
Zip Code
72205
Xiong, Yan; Patana, Anne-Sisko; Miley, Michael J et al. (2008) The first aspartic acid of the DQxD motif for human UDP-glucuronosyltransferase 1A10 interacts with UDP-glucuronic acid during catalysis. Drug Metab Dispos 36:517-22
Zielinska, Agnieszka; Lichti, Cheryl F; Bratton, Stacie et al. (2008) Glucuronidation of monohydroxylated warfarin metabolites by human liver microsomes and human recombinant UDP-glucuronosyltransferases. J Pharmacol Exp Ther 324:139-48
Miley, Michael J; Zielinska, Agnieszka K; Keenan, Jeffrey E et al. (2007) Crystal structure of the cofactor-binding domain of the human phase II drug-metabolism enzyme UDP-glucuronosyltransferase 2B7. J Mol Biol 369:498-511
Starlard-Davenport, Athena; Xiong, Yan; Bratton, Stacie et al. (2007) Phenylalanine(90) and phenylalanine(93) are crucial amino acids within the estrogen binding site of the human UDP-glucuronosyltransferase 1A10. Steroids 72:85-94
Sabolovic, Nicole; Humbert, Anne-Claude; Radominska-Pandya, Anna et al. (2006) Resveratrol is efficiently glucuronidated by UDP-glucuronosyltransferases in the human gastrointestinal tract and in Caco-2 cells. Biopharm Drug Dispos 27:181-9
Radominska-Pandya, Anna; Bratton, Stacie; Little, Joanna M (2005) A historical overview of the heterologous expression of mammalian UDP-glucuronosyltransferase isoforms over the past twenty years. Curr Drug Metab 6:141-60
Radominska-Pandya, Anna; Ouzzine, Mohamed; Fournel-Gigleux, Sylvie et al. (2005) Structure of UDP-glucuronosyltransferases in membranes. Methods Enzymol 400:116-47
Lu, Yuan; Heydel, Jean-Marie; Li, Xin et al. (2005) Lithocholic acid decreases expression of UGT2B7 in Caco-2 cells: a potential role for a negative farnesoid X receptor response element. Drug Metab Dispos 33:937-46
Sabolovic, Nicole; Heydel, Jean-Marie; Li, Xin et al. (2004) Carboxyl nonsteroidal anti-inflammatory drugs are efficiently glucuronidated by microsomes of the human gastrointestinal tract. Biochim Biophys Acta 1675:120-9
Little, Joanna M; Kurkela, Mika; Sonka, Julia et al. (2004) Glucuronidation of oxidized fatty acids and prostaglandins B1 and E2 by human hepatic and recombinant UDP-glucuronosyltransferases. J Lipid Res 45:1694-703

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