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).

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK039137-14A2
Application #
6613241
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
Serrano, Jose
Project Start
1987-07-01
Project End
2007-06-30
Budget Start
2003-09-01
Budget End
2004-06-30
Support Year
14
Fiscal Year
2003
Total Cost
$365,313
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
071036636
City
Bronx
State
NY
Country
United States
Zip Code
10461
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