TAILIEUCHUNG - Báo cáo Y học: Nuclear proteins that bind to metal response element a (MREa) in the Wilson disease gene promoter are Ku autoantigens and the Ku-80 subunit is necessary for basal transcription of the WD gene

Wilson disease (WD), an inherited disorder affecting copper metabolism, is characterized by hepatic cirrhosis and neuronal degeneration, which result from toxic levels of copper that accumulate in the liver and brain, respectively. We reported previously that the promoter of the WD gene contains four metal response elements (MREs). Among the four MREs, MREa plays the most important role in the transcriptional activation of the WD promoter. Electrophoretic mobility shift assays (EMSAs) using synthetic MREa and an oligonucleotide containing the binding site for transcription factor Sp1 revealed the presence of nuclear factors that bind specifically to MREa. Two MREa-binding. | Eur. J. Biochem. 269 2151-2161 2002 FEBS 2002 doi Nuclear proteins that bind to metal response element a MREa in the Wilson disease gene promoter are Ku autoantigens and the Ku-80 subunit is necessary for basal transcription of the WD gene Won Jun Oh1 Eun Kyung Kim1 Jung Ho Ko1 Seung Hee Yoo1 Si Houn Hahn2 and Ook-Joon Yoo1 1 Department of Biological Sciences Korea Advanced Institute of Science and Technology KAIST Taejon Korea department of Pediatrics Ajou University School of Medicine Suwon Korea Wilson disease WD an inherited disorder affecting copper metabolism is characterized by hepatic cirrhosis and neuronal degeneration which result from toxic levels of copper that accumulate in the liver and brain respectively. We reported previously that the w promoter of the WD gene contains four metal response elements MREs . Among the four MREs MREa plays the most important role in the transcriptional activation of the WD promoter. Electrophoretic mobility shift assays EMSAs using synthetic MREa and an oligonucleotide containing the binding site for transcription factor Sp1 revealed the presence of nuclear factors that bind specifically to MREa. Two MREa-binding proteins of 70 and 82 kDa were purified using avidin-biotin affinity chromatography. Amino acid sequences of peptides from each protein were found to be highly homologous to the Ku proteins. Immunoblot analysis and EMSAs showed that the MREa-binding proteins are immunologically related to the Ku proteins. To study further the functional significance of these Ku-related proteins in transcriptional regulation of the WD gene we performed RNA interference RNAi assays using a Ku-80 inverted-repeat gene to inhibit expression of the Ku-80 gene in vivo. Results of the RNAi assays showed that expression of the Ku-80 protein was suppressed in transfected cells which in turn led to the suppression of the WD gene. In addition a truncated Ku-80 AKu-80 mutant inhibited WD promoter .

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