The trascriptional activation of the human copper/zinc superoxide dismutase gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin through two different regulator sites,the anitoxidant responsive element and xenobiotic responsive element

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced during biological oxidations and environmental stress. The most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces SOD1 in human liver cells. Deletion analyses showed that the promoter region between -400 and -239 was responsible for the induction, in which two different characteristic regulatory elements, the antioxidant responsive element (ARE) and xenobiotic responsive element (XRE), are located. When the cells transfected with the plasmid containing those two cis-elements, the transactivation of SOD1 promoter was about 4-fold by TCDD, whereas mutation either on the ARE or XRE elevated the promoter activity by about 2-fold. Functional analyses of these two elements by deletion, mutation in the natural context, heterologous promoter assay, and gel mobility shift assay supported the notion that the activation of the SOD1 promoter was induced by TCDD through these two regulatory elements ARE and XRE. These results alongside our previous data indicate that the induction of SOD1 in response to TCDD is mediated by either Nrf2 protein or Ah receptor protein through ARE and XRE, respectively. These results also imply that the SOD1 can be induced by dioxin either in combination with or independently of these two regulatory elements to effectively defend cells from oxidative stress.     

The amino-acid sequence of copper/zinc superoxide dismutase from swordfish liver. Comparison of copper/zinc superoxide dismutase sequences

The amino acid sequence of copper/zinc superoxide dismutase from swordfish (Xiphias gladius) liver has been determined by alignment of the tryptic peptides according to the known sequence of bovine erythrocyte copper/zinc superoxide dismutase. This alignment has resulted in the ligands to the copper (His-47, 49, 76 and 94) and the zinc (His-76, 85, 134 and Asp-97) being conserved in all the copper/zinc superoxide dismutases sequenced so far. Also conserved in the sequences are the cysteines forming the intrachain disulphide bridge (Cys-58 and 160) and the essential arginine (Arg-157). Comparison of the amino acid sequence of swordfish liver copper/zinc superoxide dismutase with the bovine, human, horse, yeast and Photobacterium leiognathi indicates that the swordfish enzyme has a high homology with the other eukaryotic enzymes. Low homology is, however, observed with the P. leiognathi enzyme.     

Xenobiotic-responsive element for the transcriptional activation of the rat Cu/Zn superoxide dismutase gene

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced from biological oxidation and environmental stresses. A number of xenobiotics are toxic because they generate free radicals, such as superoxide and hydroxyl radicals, through a redox cycle. The xenobiotic responsive element (XRE) was located between the nt -268 and -262 region of the 5'-flanking sequence of the SOD1 gene. Functional analyses of this element by deletion, mutations, and heterologous promoter systems confirmed that the expression of the SOD1 gene was induced by a xenobiotic through the XRE. Gel mobility shift assays showed the xenobiotic inducible binding of the receptor-ligand complex to XRE. The cytoplasmic fraction from nontreated HepG2 cells also contains the factor as a cryptic form and prominently reveals its DNA-binding activity by incubation with betaNF in vitro. These results suggest that the XRE participates in the induction of the rat SOD1 gene by xenobiotics.     

Transcriptional activation of the human Cu/Zn superoxide dismutase gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin through the xenobiotic-responsive element

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced during biological oxidations and environmental stress. Here we have investigated the effect of the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the promoter of the Cu/Zn superoxide dismutase (SOD1) gene in HepG2 and HeLa cells using the chloramphenicol acetyltransferase gene as a reporter. The SOD1 promoter was activated 4- to 5-fold by TCDD treatment, in a concentration-dependent manner. In addition, the level of SOD1 mRNA and the enzymatic activity of the SOD1 protein were also enhanced on exposure of the cells to TCDD. Functional analysis of the regulatory region of the SOD1 gene by deletion and point mutation, and the use of a heterologous promoter system, showed that the SOD1 gene was transactivated by TCDD via the xenobiotic-responsive element (XRE). Gel mobility shift assays also confirmed the induction and the inducible binding of a receptor-ligand complex to XRE. Yeast cells that overexpress hSOD1 appeared to be more resistant to TCDD than the wild type. These results demonstrate that SOD1 is induced by TCDD via the XRE. The induced SOD1 may accelerate the neutralization of the superoxide anion and thus reduce the oxidative damage associated with dioxin toxicity.     

Heavy metal-mediated activation of the rat Cu/Zn superoxide dismutase gene via a metal-responsive element

The Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced in the course of biological oxidations. When placed under the control of the rat SOD1 gene promoter and transfected into human HepG2 hepatoma cells, the activity of a chloramphenicol acetyltransferase reporter gene was found to increase three- to four-fold in the presence of heavy metals (cadmium, zinc and copper). Functional analysis of mutant derivatives of the SOD1 gene promoter and the use of a heterologous promoter system confirmed that the induction of the SOD1 gene by metal ions requires a metal-responsive element (MRE) located between positions −273 and −267 (GCGCGCA). It was also shown by gel mobility shift assays that an MRE binding protein is induced by the exposure of the human liver cell line HepG2 to heavy metals. These results suggest that the MRE participates in the induction of the SOD1 gene by heavy metals. Received: 5 February 1999 / Accepted: 21 May 1999     

Molecular cloning and characterization of a copper chaperone for copper/zinc superoxide dismutase from the rat

Copper chaperone is an essential cytosolic factor that maintains copper homeostasis in living cells. Cytosolic metallochaperones have been recently identified in plant, yeast, rodents, and human cells. During our investigation, we found a new member of the copper chaperone family for copper/zinc superoxide dismutase, which was cloned from rats. The new copper chaperone was named rCCS (rat Copper Chaperone for Superoxide dismutase). The cDNA of rCCS was found to have a length of 1094 bp, and the protein analyzed from the cDNA was deduced to contain 274 amino acids. The amino acid sequence of rCCS consists of three domains: A metal binding domain, which has a MXCXXC motif in domain I, a homolog of the Cu/Zn SOD in domain II, and a CXC motif in domain III. The binding of rCCS to Cu/Zn SOD was analyzed by GST column binding assay, and the domain II of rCCS was found to be essential for binding to Cu/Zn SOD, which in turn activates Cu/Zn SOD.     

Characterization of the Bacillus stearothermophilus manganese superoxide dismutase gene and its ability to complement copper/zinc superoxide dismutase deficiency in Saccharomyces cerevisiae.

Recombinant clones containing the manganese superoxide dismutase (MnSOD) gene of Bacillus stearothermophilus were isolated with an oligonucleotide probe designed to match a part of the previously determined amino acid sequence. Complementation analyses, performed by introducing each plasmid into a superoxide dismutase-deficient mutant of Escherichia coli, allowed us to define the region of DNA which encodes the MnSOD structural gene and to identify a promoter region immediately upstream from the gene. These data were subsequently confirmed by DNA sequencing. Since MnSOD is normally restricted to the mitochondria in eucaryotes, we were interested (i) in determining whether B. stearothermophilus MnSOD could function in eucaryotic cytosol and (ii) in determining whether MnSOD could replace the structurally unrelated copper/zinc superoxide dismutase (Cu/ZnSOD) which is normally found there. To test this, the sequence encoding bacterial MnSOD was cloned into a yeast expression vector and subsequently introduced into a Cu/ZnSOD-deficient mutant of the yeast Saccharomyces cerevisiae. Functional expression of the protein was demonstrated, and complementation tests revealed that the protein was able to provide tolerance at wild-type levels to conditions which are normally restrictive for this mutant. Thus, in spite of the evolutionary unrelatedness of these two enzymes, Cu/ZnSOD can be functionally replaced by MnSOD in yeast cytosol.     

Rat copper/zinc superoxide dismutase gene: isolation, characterization, and species comparison.

A 13 kb rat Cu/ZnSOD genomic clone has been purified from a rat liver genomic library and completely characterized by restriction mapping, detailed sequencing and Southern blot analysis. This gene spans approximately 6 kb and contains five exons and four introns. Comparison of rat, mouse, and human Cu/ZnSOD genes reveals a high conservation in genomic organization and exon-intron junctions, including an unusual 5'GC donor sequence at the first intron. The gene contains a TATA box as well as an inverted CCAAT box, a feature common to both the mouse and human genes. Furthermore, several repeats were identified in the 5' promoter region of this gene, and these regulatory elements are also strikingly conserved in these three species.     

Mechanisms of biosynthesis of mammalian copper/zinc superoxide dismutase

Copper/zinc superoxide dismutase (SOD1) is an abundant intracellular enzyme with an essential role in antioxidant defense. The activity of SOD1 is dependent upon the presence of a bound copper ion incorporated by the copper chaperone for superoxide dismutase, CCS. To elucidate the cell biological mechanisms of this process, SOD1 synthesis and turnover were examined following 64Cu metabolic labeling of fibroblasts derived from CCS+/+ and CCS-/- embryos. The data indicate that copper is rapidly incorporated into both newly synthesized SOD1 and preformed SOD1 apoprotein, that each process is dependent upon CCS and that once incorporated, copper is unavailable for cellular exchange. The abundance of apoSOD1 is inversely proportional to the intracellular copper content and immunoblot and gel filtration analysis indicate that this apoprotein exists as a homodimer that is distinguishable from SOD1. Despite these distinct differences, the abundance and half-life of SOD1 is equivalent in CCS+/+ and CCS-/- fibroblasts, indicating that neither CCS nor copper incorporation has any essential role in the stability or turnover of SOD1 in vivo. Taken together, these data provide a cell biological model of SOD1 biosynthesis that is consistent with the concept of limited intracellular copper availability and indicate that the metallochaperone CCS is a critical determinant of SOD1 activity in mammalian cells. These kinetic and biochemical findings also provide an important framework for understanding the role of mutant SOD1 in the pathogenesis of familial amyotrophic lateral sclerosis.     

Elevation of superoxide dismutase in Halobacterium halobium by heat shock.

Exposure of Halobacterium halobium to 50 degrees C for 2.5 h in an aerobic environment resulted in a greater than twofold increase in the activity of the manganese-containing superoxide dismutase. Nondenaturing polyacrylamide gels stained for enzymatic activity did not reveal any additional isozymes of superoxide dismutase induced by the heat shock. The maximal effect was observed at 50 degrees C, and the elevated levels of activity remained constant during 5 h of recovery at 40 degrees C. The induction of enzymatic activity was sensitive to protein synthesis inhibitors. The results are discussed relative to heat shock and stress-related proteins as well as alterations in metabolism brought about by elevated temperatures.     

A copper chaperone for superoxide dismutase that confers three types of copper/zinc superoxide dismutase activity in Arabidopsis

The copper chaperone for superoxide dismutase (CCS) has been identified as a key factor integrating copper into copper/zinc superoxide dismutase (CuZnSOD) in yeast (Saccharomyces cerevisiae) and mammals. In Arabidopsis (Arabidopsis thaliana), only one putative CCS gene (AtCCS, At1g12520) has been identified. The predicted AtCCS polypeptide contains three distinct domains: a central domain, flanked by an ATX1-like domain, and a C-terminal domain. The ATX1-like and C-terminal domains contain putative copper-binding motifs. We have investigated the function of this putative AtCCS gene and shown that a cDNA encoding the open reading frame predicted by The Arabidopsis Information Resource complemented only the cytosolic and peroxisomal CuZnSOD activities in the Atccs knockout mutant, which has lost all CuZnSOD activities. However, a longer AtCCS cDNA, as predicted by the Munich Information Centre for Protein Sequences and encoding an extra 66 amino acids at the N terminus, could restore all three, including the chloroplastic CuZnSOD activities in the Atccs mutant. The extra 66 amino acids were shown to direct the import of AtCCS into chloroplasts. Our results indicated that one AtCCS gene was responsible for the activation of all three types of CuZnSOD activity. In addition, a truncated AtCCS, containing only the central and C-terminal domains without the ATX1-like domain failed to restore any CuZnSOD activity in the Atccs mutant. This result indicates that the ATX1-like domain is essential for the copper chaperone function of AtCCS in planta.     

Overexpression of copper/zinc superoxide dismutase decreases ischemia-like astrocyte injury

Overexpression of copper/zinc superoxide dismutase (SOD1) in transgenic mice protects from transient focal cerebral ischemia in adult animals, but increases oxidative injury in perinatal mice. The effect of SOD1 overexpression on astrocytes subjected to ischemia-like insults has not yet been determined. Overexpression of human SOD1 in astrocytes resulted in a 3-fold increase in SOD1 activity without coupled up-regulation of catalase or glutathione peroxidase activities. Cells subjected to oxygen-glucose deprivation (OGD) or glucose deprivation to mimic ischemic injury were protected by SOD1 overexpression. OGD injury was reduced 47.6+/-9.3%, assessed by release of lactate dehydrogenase. OGD also caused a significant increase in catalase activity which was moderated by SOD1 overexpression. The level of glutathione in astrocytes overexpressing SOD1 was maintained at higher levels following 5 h OGD compared to control cultures under the same conditions. Reduction of glutathione prior to OGD significantly increased cell death of SOD1-overexpressing astrocytes as well as controls, but SOD1 still provided significant protection, suggesting that both GSH-dependent scavenging and GSH-independent scavenging are relevant to SOD1 protection in astrocytes.     

Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.