In virtually all modern species, including man, the formation of amino acid conjugates (amidates) of bile acids (BA) is quantitatively the most important step in BA metabolism. These conjugates because of their low pKas are effective micellar detergents even in the acid milieu of the upper gut. BA amidation is catalyzed by two enzymes, bile acid CoA ligase and bile acid CoA:amino acid N-acyltransferase (BAT). The long-term goal of this project is to determine the effects of defects in bile acid amidation in human health and disease. This will be approached in several ways using various molecular biology reagents (cloned cDNAs and specific antibodies) that we have prepared in the previous grant periods. We propose to determine how BAT could be sensitive to specific genetic mutations and to post-translational modifications that may occur in liver disease. This involves (1) the identification of critical residues that (a) account for BAT enzyme activity, (b) for the marked differences in glycine/taurine substrate specificity amongst, humans, mice and rats, (c) the effect of mutation of the Cys-235 to Ser on the enzymatic properties of this protein, (d) the effect of the Met- 76 to Val mutation observed in children with cholestasis and other hepatobiliary disease symptoms in an Amish kindred, and (e) the effect of each mutation on BAT structure using H-D exchange/mass spectrometry; (2) the control of peroxisomal localization of BAT using biochemical and molecular biology approaches; and (3) the effect of reactive oxygen species and reactive nitrogen species generated in cholestatic and inflammatory liver disease on BAT activity (modeled in vitro, in hepatocytes and in animal models), in particular, on the cysteine sulfhydryl groups (Cys-235 and Cys372), but potentially other groups such as tyrosine and histidine residues. Overall, these experiments will utilize a combination of molecular biology, enzyme biochemistry, animal models, and mass spectrometry to identify protein modifications of BAT in order to determine the consequence of defects in BA amidation that occur genetically or epigenetically as a consequence of the inflammatory complications of hepatobiliary disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK046390-09
Application #
7101799
Study Section
Special Emphasis Panel (ZRG1-GMA-3 (01))
Program Officer
Serrano, Jose
Project Start
1994-08-15
Project End
2008-07-31
Budget Start
2006-08-01
Budget End
2008-07-31
Support Year
9
Fiscal Year
2006
Total Cost
$203,186
Indirect Cost
Name
University of Alabama Birmingham
Department
Pharmacology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
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Kang, Dae-Joong; Ridlon, Jason M; Moore 2nd, Doyle Ray et al. (2008) Clostridium scindens baiCD and baiH genes encode stereo-specific 7alpha/7beta-hydroxy-3-oxo-delta4-cholenoic acid oxidoreductases. Biochim Biophys Acta 1781:16-25
Shonsey, E M; Eliuk, S M; Johnson, M S et al. (2008) Inactivation of human liver bile acid CoA:amino acid N-acyltransferase by the electrophilic lipid, 4-hydroxynonenal. J Lipid Res 49:282-94
Styles, Nathan A; Falany, Josie L; Barnes, Stephen et al. (2007) Quantification and regulation of the subcellular distribution of bile acid coenzyme A:amino acid N-acyltransferase activity in rat liver. J Lipid Res 48:1305-15
Shonsey, Erin M; Wheeler, James; Johnson, Michelle et al. (2005) Synthesis of bile acid coenzyme a thioesters in the amino acid conjugation of bile acids. Methods Enzymol 400:360-73
Shonsey, Erin M; Sfakianos, Mindan; Johnson, Michelle et al. (2005) Bile acid coenzyme A: amino acid N-acyltransferase in the amino acid conjugation of bile acids. Methods Enzymol 400:374-94
He, Dongning; Barnes, Stephen; Falany, Charles N (2003) Rat liver bile acid CoA:amino acid N-acyltransferase: expression, characterization, and peroxisomal localization. J Lipid Res 44:2242-9
Sfakianos, Mindan K; Wilson, Landon; Sakalian, Michael et al. (2002) Conserved residues in the putative catalytic triad of human bile acid Coenzyme A:amino acid N-acyltransferase. J Biol Chem 277:47270-5
Falany, Charles N; Xie, Xiaowei; Wheeler, James B et al. (2002) Molecular cloning and expression of rat liver bile acid CoA ligase. J Lipid Res 43:2062-71
King 3rd, L; Barnes, S; Glufke, U et al. (2000) The enzymatic formation of novel bile acid primary amides. Arch Biochem Biophys 374:107-17

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