INHERITED DISORDERS OF HEPATIC BILIRUBIN GLUCORONIDATION

性状（由申请方提供）：胆红素是血红素分解的有毒终产物。胆红素的有效胆汁排泄需要由尿苷二磷酸葡萄糖醛酸葡萄糖醛酸转移酶-1A1（UGT 1A 1）介导的葡萄糖醛酸化，其催化位点位于肝细胞内质网（ER）腔内。遗传性UGT 1A 1缺乏导致未结合胆红素的积累，导致潜在致命的Crigler-Najjar综合征1型。UGT 1A 1还能解毒雌激素和几种药物和致癌物质。具体目的1是表征UGT 1A 1的结构-功能关系。我们已经证明UGT 1A 1形成二聚体，并将检验以下假设：通过生理性UGT激活剂UDP-N-乙酰葡糖胺（UDP-gluc-Nac）刺激UDP-葡萄糖醛酸（UDPGA）进入ER腔，激活酶需要二聚体。我们将测试二聚化是否解释了UGT 1A 1的某些突变形式的显性负功能。另一个有待检验的假设是UGT 1A 1活性受磷酸化调节。ER定位在UGT 1A 1功能中是重要的。因此，我们将描绘膜掺入和特定的ER定位所需的图案。由于几种药物通过抑制UGT 1A 1活性引起高胆红素血症，我们将描述与底物、UDPGA和胆红素结合相关的结构域。具体目的2是表征UGT 1A 1基因表达。我们假设UGT 1A 1上游调控区顺式作用元件与肝细胞反式激活因子的相互作用决定了UGT 1A 1表达的组织特异性、苯巴比妥和氯贝丁酯对其的诱导以及甲状腺激素对其的下调。我们将在分化的人肝癌细胞和永生化的人肝细胞中测试这种DNA酶足迹，电泳迁移率变动分析和启动子-报告基因构建体的表达。这项研究的成功完成将阐明UGT 1A 1在健康和遗传性疾病中的作用机制。了解UGT 1A 1的调节机制有助于确定酶诱导药物，以改善新生儿高胆红素血症和不完全UGT 1A 1缺乏症（Crigler-Najjar综合征2型）的治疗。

DESCRIPTION (provided by applicant): Bilirubin is the toxic end product of heme breakdown. Efficient biliary excretion of bilirubin requires glucuronidation mediated by uridinediphosphoglucuronate glucuronosyltransferase-1A1 (UGT1A1), the catalytic site of which is located inside the hepatocyte endoplasmic reticulum (ER) lumen. Inherited UGT1A1 deficiency leads to the accumulation of unconjugated bilirubin, causing the potentially lethal Crigler-Najjar syndrome type 1. UGT1A1 also detoxifies estrogen and several drugs and carcinogens. Specific Aim 1 is to characterize the structure-function relationship of UGT1A1. We have shown that UGT1A1 forms dimers, and will test the hypothesis that dimerization is required for activation of the enzyme by stimulation of the import of UDP-glucuronic acid (UDPGA) into the ER lumen by the physiological UGT activator, UDP-N-acetyl glucosamine (UDP-gluc-Nac). We will test whether dimerization explains the dominant negative function of some mutant forms of UGT1A1. Another hypothesis to be tested is that UGT1A1 activity is regulated by phosphorylation. ER localization is important in UGT1A1 function. Therefore, we will delineate the motifs required for membrane incorporation and specific ER localization. Since several drugs cause hyperbilirubinemia by inhibiting UGT1A1 activity, we will delineate domains involved in binding the substrtes, UDPGA and bilirubin. Specific Aim 2 is to characterize UGT1A1 gene expression. We hypothesize that the interaction of cis-acting elements within the regulatory upstream region of UGT1A1 with hepatocellular transactivating factors determine the tissue-specificity of UGT1A1 expression, its induction by Phenobarbital and clofibrate, and its down-regulation by thryroid hormone. We will test this DNase foot-printing, electrophoretic mobility shift assays and expression of promoter-reporter constructs in differentiated human hepatoma cells and immortalized human hepatocytes. Successful completion of this study will elucidate the mechanism of UGT1A1 function in health and inherited disorders. Understanding the regulatory mechanisms of UGT1A1 should assist in identifying enzyme-inducing drugs for improved treatment of neonatal hyperbilirubinemia mad incomplete UGT1A1 deficiency (Crigler-Najjar syndrome type 2).