Amino acid conjugation of bile acids

描述（由申请人提供）：在几乎所有现代物种中，包括人类，胆汁酸（BA）的氨基酸偶联物（氨基酸）的形成是BA代谢中最重要的定量步骤。这些偶联物由于其低pKas是有效的胶束清洁剂，即使在上肠的酸性环境。BA酰胺化由两种酶催化，胆汁酸辅酶a连接酶和胆汁酸辅酶a：氨基酸n -酰基转移酶（BAT）。该项目的长期目标是确定胆汁酸酰胺化缺陷对人类健康和疾病的影响。这将通过几种方式来实现，使用我们在之前的拨款期间准备的各种分子生物学试剂（克隆cdna和特异性抗体）。我们建议确定BAT如何对肝脏疾病中可能发生的特定基因突变和翻译后修饰敏感。这涉及(1)鉴定关键残基，(a)解释BAT酶活性，(b)人类、小鼠和大鼠之间甘氨酸/牛磺酸底物特异性的显著差异，(c) Cys-235对Ser的突变对该蛋白质酶特性的影响，(d)在阿米什族患有胆汁潴存和其他肝胆疾病症状的儿童中观察到的Met- 76对Val突变的影响。(e) H-D交换/质谱法分析各突变对BAT结构的影响；(2)利用生物化学和分子生物学方法控制BAT过氧化物酶体的定位；(3)胆汁淤积症和炎症性肝病中产生的活性氧和活性氮对BAT活性的影响（在体外、肝细胞和动物模型中建模），特别是对半胱氨酸巯基（Cys-235和Cys372）的影响，但也可能对酪氨酸和组氨酸残基等其他基团的影响。总的来说，这些实验将结合分子生物学、酶生物化学、动物模型和质谱来鉴定BAT的蛋白质修饰，以确定由于肝胆疾病的炎症并发症而发生的遗传或表观遗传的BA酰胺化缺陷的后果。

DESCRIPTION (provided by applicant): 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.