Cloning and Characterization of the Promoter Region of the Wilson Disease Gene

Oh, Won Zin; Kim, Min Jung; Park, Kee Don; Hahn, Si Houn; Yoo, Ook Joon

doi:10.1006/bbrc.1999.0732

Cited by 18 publications

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“…Human WD promoter-reporter gene constructs A 1.6-kb construction of the WD promoter, named pPWDLuc, was described previously [7]. Trinucleotide mutations in each of the four MREs of the WD gene promoter were constructed using the PCR-based quick change site-directed mutagenesis method (Stratagene).…”

Section: Cell Culturementioning

confidence: 99%

“…HepG2 cells were cultured at 40-60% confluence in 6-well dishes and transfected with 1 lg of DNA mixture containing various Ku cDNAs, WD promoter report construct (pPWD-Luc) [7], and pSVb-gal which served as an internal control for transfection efficiency. The transfected cells were cultured for a further 24 or 48 h, and the expression levels of luciferase and b-galactosidase were determined.…”

Section: Transfection and Luciferase Assaysmentioning

confidence: 99%

“…Hepatic cirrhosis and neuronal degeneration are the most debilitating symptoms of WD and are caused by the impairment of biliary copper excretion and the accumulation of toxic concentration of copper. The WD gene product shares a high degree of sequence similarity with the cation-transporting P-type ATPases [1][2][3][4][5] and functions in the binding and translocation of copper [6].Interestingly, multiple copies of metal-response elements (MREs) are located in the 1.3-kb promoter of the WD gene [7]. Five or more nonidentical MREs are present in the 5¢-flanking region of the vertebrate metallothionein (MT) gene [8,9] and are believed to mediate the transcriptional activation of the MT gene by heavy metals [8,[10][11][12] and oxidative stress [13,14].…”

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confidence: 99%

“…Interestingly, multiple copies of metal-response elements (MREs) are located in the 1.3-kb promoter of the WD gene [7]. Five or more nonidentical MREs are present in the 5¢-flanking region of the vertebrate metallothionein (MT) gene [8,9] and are believed to mediate the transcriptional activation of the MT gene by heavy metals [8,[10][11][12] and oxidative stress [13,14].…”

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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

Kim

et al. 2002

European Journal of Biochemistry

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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 1.3-kb 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 (DKu-80) mutant inhibited WD promoter activity in HepG2 cells in a dominant-negative manner. We also found that WD promoter activity was decreased in Xrs5 cells, which, unlike the CHO-K1 cells, are defective in the Ku-80 protein.When Ku-80 cDNA was transfected into Xrs5 and CHO cells, WD promoter activity was recovered only in Xrs5 cells. Taken together, our findings suggest that the Ku-80 subunit is required for constitutive expression of the WD gene.Keywords: ATP7B gene; Ku antoantigen; RNA interference; site-directed mutagenesis.Wilson disease (WD) is an autosomal recessive disorder characterized by defect in copper transport. Hepatic cirrhosis and neuronal degeneration are the most debilitating symptoms of WD and are caused by the impairment of biliary copper excretion and the accumulation of toxic concentration of copper. The WD gene product shares a high degree of sequence similarity with the cation-transporting P-type ATPases [1][2][3][4][5] and functions in the binding and translocation of copper [6].Interestingly, multiple copies of metal-response elements (MREs) are located in the 1.3-kb promoter of the WD gene [7]. Five or more nonidentical MREs are present in the 5¢-flanking region of the vertebrate metallothionein (MT) gene [8,9] and are believed to mediate the transcriptional activation of the MT gene by heavy metals [8,[10][11][12] and oxidative stress [13,14]. The MREs consist of 12-base pair sequences that contain a seven-nucleotide core sequence (TGCRCNC) surrounded by less well-conserved flanking nucleotides [15]. MTs are small (6-7 kDa), cysteine-rich, metal-binding proteins that funct...

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Section: Cell Culturementioning

confidence: 99%

Section: Transfection and Luciferase Assaysmentioning

confidence: 99%

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confidence: 99%

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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

Kim

et al. 2002

European Journal of Biochemistry

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“…Barada et al (2010) Mutações sinônimas -às vezes chamadas mutações "silenciosas"-são amplamente reconhecidas como sendo capazes de provocar alterações na expressão, conformação e função proteica (Sauna & Kimchi-Sarfaty 2011 . Oh et al (1999) Filhos de pais afetados com DW só podem ser afetados se o outro progenitor é ao menos heterozigoto. Considerando-se que a prevalência da DW é estimada para ser da ordem de 30 por um milhão, com uma frequência de portadores de cerca de 1:90, a probabilidade de ocorrência da doença em descendentes é estimada em 0,56% nos EUA e Europa (Bull et al 1993;Petrukhin et al 1993).…”

Section: Diversidade Genotípica E Fenotípica Em Uma Mesma Famíliaunclassified

Importância da detecção de mutações do gene ATP7B para o diagnóstico da doença de Wilson

Araújo¹

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Quantitative relationship between mutated amino-acid sequence of human copper-transporting ATPases and their related diseases

Yan

Wu²

2008

Mol Divers

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Copper-transporting ATPase 1 and 2 (ATP7A and ATP7B) are two highly homologous P-type copper ATPase exporters. Mutations in ATP7A can lead to Menkes disease which is an X-linked disorder of copper deficiency. Mutations in ATP7B can cause Wilson disease which is an autosomal recessive disorder of copper toxicity. In this study, we attempt to build a quantitative relationship between mutated ATPase and Menkes/Wilson disease. First, we use the amino-acid distribution probability as a measure to quantify the difference in ATPase before and after mutation. Second, we use the cross-impact analysis to define the quantitative relationship between mutant ATPase protein and Menkes/Wilson disease, and compute various probabilities. Finally, we use the Bayesian equation to determine the probability that Menkes/Wilson disease is diagnosed under a mutation. The results show (i) the vast majority of mutations lead to the amino-acid distribution probability increase in mutant ATP7As and decrease in ATP7Bs, and (ii) the probability that a mutation causes Menkes/Wilson disease is about nine tenth. Thus we provide a way to use the descriptively probabilistic method to couple the mutation with its clinical outcome after quantifying mutations in proteins.

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Cloning and Characterization of the Promoter Region of the Wilson Disease Gene

Cited by 18 publications

References 19 publications

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

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

Importância da detecção de mutações do gene ATP7B para o diagnóstico da doença de Wilson

Quantitative relationship between mutated amino-acid sequence of human copper-transporting ATPases and their related diseases

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