INHERITED DISORDERS OF HEPATIC BILIRUBIN GLUCORONIDATION

描述（由申请人提供）：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 （UGT1A1）介导的糖醛酸化，其催化位点位于肝细胞内质网（ER）管腔内。遗传性UGT1A1缺乏会导致未结合胆红素的积累，从而导致潜在致命的Crigler-Najjar综合征1型。UGT1A1还能解毒雌激素、几种药物和致癌物。具体目的1是表征UGT1A1的结构-功能关系。我们已经证明UGT1A1形成二聚体，并将验证二聚体是激活酶所必需的假设，即通过刺激udp -葡糖苷酸（UDPGA）通过生理UGT激活剂udp - n -乙酰氨基葡萄糖（udp -葡糖苷- nac）进入内质网腔。我们将测试二聚化是否解释了一些UGT1A1突变形式的主要负功能。另一个有待验证的假设是，UGT1A1的活性受磷酸化调节。内质网定位在UGT1A1功能中很重要。因此，我们将描述膜结合和特异性内质网定位所需的基序。由于几种药物通过抑制UGT1A1活性导致高胆红素血症，我们将描述与底物、UDPGA和胆红素结合有关的结构域。特异性目的2是表征UGT1A1基因的表达。我们推测，UGT1A1上游调控区域内的顺式作用元件与肝细胞反活化因子的相互作用决定了UGT1A1表达的组织特异性、苯巴比妥和氯贝特诱导UGT1A1表达以及甲状腺激素下调UGT1A1表达。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酶印迹、电泳迁移率转移试验和启动子报告基因构建的表达。本研究的成功完成将阐明UGT1A1在健康和遗传疾病中的作用机制。了解UGT1A1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UGT1A1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).