Human Glutathione Peroxidase Gene Research Articles

The characterization and purification of a human transcription factor modulating the glutathione peroxidase gene in response to oxygen tension.

An oxygen responsive transcription factor regulating human glutathione peroxidase gene (GPx) through two oxygen responsive elements (ORE I and ORE2) has been purified and characterized by sequence-specific DNA affinity chromatography. The DNA binding activity, termed Oxygen Responsive Element Binding Protein (OREBP), was partially represented by a 77 kD polypeptide (p70) possessing a blocked N-terminus. The p70 subunit co-eluted with an 86 kD subunit (p80) from affinity columns. N-terminal sequencing analysis of the 86 kD component revealed that this protein represented the larger member of the Ku antigen complex. The identity of the purified 77 kD subunit was determined by Western blot analysis using an antibody directed against the p70 protein. In addition to binding the GPx-ORE, the OREBP was itself regulated by oxygen tension. It was found that the abundance of the ORE binding activity was decreased in cells maintained at low oxygen tension (40 mm Hg). Anti-Ku-antibodies specifically supershifted the OREBP-ORE DNA complex. These observations further add to the numerous nuclear roles of the Ku-transcription factor.

Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing.

Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPX1) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template-preparation steps is discussed.

Attenuation of 6-OHDA-induced neurotoxicity in glutathione peroxidase transgenic mice.

Normal cellular metabolism produces oxidants which are neutralized within cells by antioxidant enzymes and other antioxidants. An imbalance between oxidants and antioxidants has been postulated to lead to the degeneration of specific populations of neurons in neurodegenerative diseases, e.g. Parkinson's disease. The present study investigates whether overexpression of glutathione peroxidase, the enzyme which metabolizes hydrogen peroxide to water, can prevent or slow down neuronal injury in an animal model of Parkinson's disease. Transgenic mice overexpressing the human glutathione peroxidase gene under the control of the mouse hydroxymethylglutaryl-coenzyme A promoter and genetically matched control mice were injected intracerebroventricularly with the dopaminergic neurotoxin 6-hydroxydopamine. Seven days after injection, the number of tyrosine hydroxylase-positive nigral dopaminergic neurons was decreased by 52.4% and 20.5% in 6-hydroxydopamine-injected control and glutathione peroxidase transgenic mice, respectively. Similarly, 3 days after injection of the neurotoxin, striatal dopamine was decreased by 71.2% and 56.5%, respectively. Overexpression of glutathione peroxidase therefore partially protects dopaminergic neurons against 6-hydroxydopamine-induced toxicity.

Identification of oxygen responsive elements in the 5'-flanking region of the human glutathione peroxidase gene.

The -1389 to +588 region of the human genomic glutathione peroxidase gene (hgpx1) was amplified using the polymerase chain reaction. This DNA fragment was cloned and sequenced, and various deletion constructs derived from the hgpx1 5'-flanking region were fused to the chloramphenicol acetyltransferase gene. These reporter genes were analyzed in transient transfection assays using primary cultured human ventricular cardiomyocytes obtained from patients with tetralogy of Fallot. Two distinct regions upstream from the transcription start site, which was determined using S1 nuclease analysis, were identified to be responsive to the oxygen tension in culture (pO2 values of 150 or 40 mm Hg). Methylation interference footprinting assays revealed proteins closely apposed to two sequences located at -1232 to -1213 and -282 to -275. We have designated these oxygen responsive elements ORE1 and ORE2, respectively. Gel mobility shift assays using double-stranded oligonucleotides corresponding to each site have demonstrated the formation of specific complexes using both cultured human cardiomyocyte and HeLa nuclear extracts. ORE1 and ORE2 bind disparate proteins with equal precision as bound complexes could be competed away with identical sequences but not with either the other ORE or an unrelated sequence. Insertion of these oxygen responsive elements into a reporter gene governed by a SV40 promoter similarly regulated chloramphenicol acetyltransferase activity according to the oxygen tension in culture.

The 5′ untranslated region of the human cellular glutathione peroxidase gene is indispensable for its expression in COS-7 cells

We studied the expression of the human cellular glutathione peroxidase (GPx) gene, from which a key enzyme containing selenocysteine (Scy) at the active site is produced. Expression of some human GPx gene mutants in COS-7 cells revealed that the 5′ untranslated region (utr) was necessary for expression of the GPx gene, since mutant genes having 10 base pairs (bps) at the 5′utr (the complete had 311 bps) expressed GPx at very low levels. The genes with 311 or 408 bps at the 5′utr were better expressed than those having 257 bps. The GPx gene having 133 bps at the 3′utr (80 bps shorter than the entire length) was highly expressed. This deletion did not influence expression. We constructed some mutants in which 3 bases were altered at the upstream region of the Scy UGA codon in the frame of the GPx gene, by site-directed mutagenesis. GPx expression decreased but the expression was restored. Therefore, the upstream region of the in-frame Scy codon was not essential in the Scy decoding mechanisms. Finally, the 5′utr was essential for the expression of GPx gene. However, the deletion of a part of the 3′utr and the site-directed mutation upstream of the Scy codon did not show drastic effects on the expression.

Isolation and chromosomal localization of the human glutathione peroxidase gene

We have isolated cDNA clones for the gene, termed GPX1, encoding the major human selenoprotein, glutathione peroxidase. Sequence analysis confirmed previous findings that the unusual amino acid selenocysteine is encoded by the opal terminator codon UGA. Southern blot analysis of human genomic DNA with the GPX1 cDNA showed that restriction endonucleases without sites in the probe sequence produced three hybridizing bands at standard stringency, diminishing to one strongly and one weakly hybridizing band at high stringency. In situ hybridization localized the human GPX1 gene to a single site on chromosome 3, at region 3q11–13.1. Thus, three genomic sites bear sequence homology to the GPX1 cDNA, and the one most homologous maps to 3q11–13.1.

Post-transcriptional regulation of glutathione peroxidase gene expression by selenium in the HL-60 human myeloid cell line

We have used a cloned cDNA for the major human selenoprotein, glutathione peroxidase (GPx), to assess the mode of regulation of human GPx gene (GPX-1) expression by selenium. When the HL-60 human myeloid cell line is grown in a selenium-deficient medium, GPx enzymatic activity decreases 30-fold compared with selenium-replete cells. Upon return to a medium containing selenium in the form of selenite, GPx activity in the cells starts to increase within 48 hours and reaches maximal (selenium-replete) levels at 7 days. Steady-state immunoreactive protein levels correlate with enzymatic activity. Cycloheximide inhibits the rise in GPx activity that accompanies selenium replenishment, indicating that protein synthesis is required for the increase. However, GPx mRNA levels and the rate of transcription of the human GPx gene change very little and thus appear to be independent of the selenium supply. Thus the human GPx gene appears to be regulated post-transcriptionally, probably cotranslationally, in response to selenium availability.