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.
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