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.     

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.     

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     

A Novel Superoxide Dismutase Gene Encoding Membrane-bound and Extracellular Isoforms by Alternative Splicing in Caenorhabditis elegans

We have identified a novel Cu/Zn superoxide dismutase gene (termedSOD-4) in Caenorhabditis elegans. Characterization of its complementaryDNA revealed that the gene encodes two isoforms by alternativesplicing, SOD4-1 and SOD4-2 which differ in their C-terminalexons. Their predicted amino acid sequences include a consensussignal peptide at their N-termini and are homologous to theextracellular-types of Cu/Zn superoxide dismutase in mammals.In addition, SOD4-2 possesses a putative transmembrane domainat the C-terminal region. When transiently expressed in Chinesehamster ovary cells, both types were found in the membranesand SOD4-1 also in the culture fluid. It is, therefore, indicatedthat SOD4-1 is an extracellular form and SOD4-2 a membrane-boundform, the latter representing a novel type of SOD. In C. elegans,SOD4-2 mRNA was found to be preferentially expressed in eggs.     

Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has altered redox activities and is toxic to motor neurons in vitro. Using bovine SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding activity six times faster than superoxide dismutase activity, whereas inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine. Although urate, xanthine, and ascorbate can protect superoxide dismutase activity of Cu,Zn SOD from inactivation, they were not effective at protecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydrogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible.     

Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli

Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.     

Aggregate formation in Cu,Zn superoxide dismutase-related proteins

Aggregation of Cu,Zn superoxide dismutase (SOD1) protein is a pathologic hallmark of familial amyotrophic lateral sclerosis linked to mutations in the SOD1 gene, although the structural motifs within mutant SOD1 that are responsible for its aggregation are unknown. Copper chaperone for SOD1 (CCS) and extracellular Cu,Zn superoxide dismutase (SOD3) have some sequence identity with SOD1, particularly in the regions of metal binding, but play no significant role in mutant SOD1-induced disease. We hypothesized that it would be possible to form CCS- or SOD3-positive aggregates by making these molecules resemble mutant SOD1 via the introduction of point mutations in codons homologous to a disease causing G85R SOD1 mutation. Using an in vitro assay system, we found that expression of wild type human CCS or a modified intracellular wild type SOD3 does not result in significant aggregate formation. In contrast, expression of G168R CCS or G146R SOD3 produced aggregates as evidenced by the presence of high molecular weight protein complexes on Western gels or inclusion bodies on immunofluorescence. CCS- and SOD3-positive inclusions appear to be ubiquitinated and localized to aggresomes. These results suggest that proteins sharing structural similarities to mutant SOD1 are also at risk for aggregate formation.     

Identification of copper/zinc superoxide dismutase as a novel nitric oxide-regulated gene in rat glomerular mesangial cells and kidneys of endotoxemic rats.

To define the mechanism of nitric oxide (NO) action in the glomerulus, we attempted to identify genes that are regulated by NO in rat glomerular mesangial cells. We identified a Cu/Zn superoxide dismutase (SOD) that was strongly induced in these cells by treatment with S-nitroso-glutathione as a NO-donating agent. Bacterial lipopolysaccharide (LPS) acutely decreased Cu/Zn SOD mRNA levels. The LPS-mediated decrease in Cu/Zn SOD is reversed by endogenously produced NO, as LPS also induced a delayed strong iNOS expression in these cells in vitro, which is accompanied by increased Cu/Zn SOD expression. NO dependency of Cu/Zn SOD mRNA recovery could be demonstrated by inhibition of this process by L-NG-monomethylarginine, an inhibitor of NOS enzymatic activity. To demonstrate the in vivo relevance of our observations, we have chosen LPS-treated rats as a model for induction of a systemic inflammatory response. In these animals, we demonstrate a direct coupling of Cu/Zn SOD expression levels to the presence of NO, as Cu/Zn SOD mRNA levels declined during acute inflammation in the presence of a selective inhibitor of iNOS. We propose that the up-regulation of Cu/Zn SOD by endogenous NO may serve as an adaptive, protective mechanism to prevent the formation of toxic quantities of peroxynitrite in conditions associated with iNOS induction during endotoxic shock.     

Functional role of AhR in the expression of toxic effects by TCDD

Cytochrome P450 1A1 (CYP1A1) is one of the xenobiotic metabolizing enzymes (XMEs), which is induced by polycyclic aromatic hydrocarbons (PAHs). The most potent inducer of CYP1A1 is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, TCDD induces a broad spectrum of biochemical and toxic effects, such as teratogenesis, immunosuppression and tumor promotion. Most, if not all, of the effects caused by TCDD and other PAHs are known to be mediated by AhR (aryl hydrocarbon receptor or dioxin receptor) which has a high binding affinity to TCDD. The liganded AhR translocates from cytoplasm to nuclei where it switches its partner molecule from Hsp90 to Arnt. Thus formed AhR/Arnt heterodimer binds a specific DNA sequence designated XRE in the promoter region of the target genes including CYP1A1, UDP-glucuronosyl transferase and others to enhance their expression. Although it remains to be studied how AhR is involved in the other TCDD-induced biological effects such as teratogenesis and immunosuppression than induction of XMEs, it is believed that these adverse TCDD effects are caused by untimely activation of gene expression by ligand-activated AhR in the biological process. We summarize the present knowledge about functional role of AhR in TCDD-induced biological effects.     

Superoxide Dismutase Concentration and Activity in Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal-dominant familial amyotrophic lateral sclerosis (FALS) have been associated with mutations in SOD1 , the gene that encodes Cu/Zn superoxide dismutase (Cu/Zn SOD). We determined the concentrations (µg of Cu/Zn SOD/mg of total protein), specific activities (U/µg of total protein), and apparent turnover numbers (U/µmol of Cu/Zn SOD) of Cu/Zn SOD in erythrocyte lysates from patients with known SOD1 mutations. We also measured the concentrations and activities of Cu/Zn SOD in FALS patients with no identifiable SOD1 mutations, sporadic ALS (SALS) patients, and patients with other neurologic disorders. The concentration and specific activity of Cu/Zn SOD were decreased in all patients with SOD1 mutations, with mean reductions of 51 and 46%, respectively, relative to controls. In contrast, the apparent turnover number of the enzyme was not altered in these patients. For the six mutations studied, there was no correlation between enzyme concentration or specific activity and disease severity, expressed as either duration of disease or age of onset. No significant alterations in the concentration, specific activity, or apparent turnover number of Cu/Zn SOD were detected in the FALS patients with no identifiable SOD1 mutations, SALS patients, or patients with other neurologic disorders. That Cu/Zn SOD concentration and specific activity are equivalently reduced in erythrocytes from patients with SOD1 mutations suggests that mutant Cu/Zn SOD is unstable in these cells. That concentration and specific activity do not correlate with disease severity suggests that an altered, novel function of the enzyme, rather than reduction of its dismutase activity, may be responsible for the pathogenesis of FALS.     

Evaluation of oxysterol levels of patients with silicosis by LC–MS/MS method

Molecular and Cellular Biochemistry - Aberrant structural formations of Cu/Zn superoxide dismutase enzyme (SOD1) are the probable mechanism by which circumscribed mutations in the SOD1 gene cause...     

Identification of a single-nucleotide insertion in the promoter region affecting the sodC promoter activity in Brucella neotomae

Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.     

人铜锌超氧化物歧化酶基因在乳酸乳球菌中的食品级表达

以lacF基因为食品级选择标记,构建了乳酸乳球菌食品级基因表达系统,并进而实现了人铜锌超氧化物歧化酶基因在乳酸乳球菌中的食品级表达。首先构建了含有lacF基因两侧同源DNA序列(0.5kb)的整合型质粒pUCEmDE,通过pUCEmDE与乳酸乳球菌MG5267染色体上单拷贝的乳糖操纵子之间的同源双交换,构建了lacF基因缺失突变的食品级受体菌WZ103 (Lac-),并经PCR及Lac表型检测所验证。然后构建了互补质粒pMG36eF,其lacF基因的表达受组成型的强启动子P32的控制。将其电转化导入WZ103后,Lac+表型得到恢复,表明WZ103中lacF基因的功能可被互补质粒pMG36eF上的lacF基因互补。随后,以互补质粒pMG36eF为基础,构建了不含任何抗生素抗性选择标记的人铜锌超氧化物歧化酶基因的食品级表达质粒pWZ104。通过非变性聚丙烯酰胺凝胶电泳和SOD活性凝胶染色分析,检测到WZ103(pWZ104)中Cu/Zn SOD的表达,并且具有生物活性。     

铜锌超氧化物歧化酶(SOD)研究进展——从基因到功能

超氧化物歧化酶(SOD,EC 1.15.1.1),己经在多种组织中发现,它能将O2.-催化生成H2O2及O2.迄今为止,已经从哺乳动物体内分离出三种SOD:CuZnSOD(SOD1)、MnSOD(SOD2)TLEC-SOD(胞外超氧化物歧化酶,SOD3),各自具有不同的生化及分子特性.CuZnSOD(SOD1),是一类含有Cu及Zn原子的二聚体,存在于特定细胞的基质内,约占SOD总量的90%.在胞质及周质中,SOD以二聚体形式存在,而在线粒体及质外,则以四聚体形式存在.在保护脑、肺及其它组织的氧化应激中,CuZnSOD被认为起着保护作用.运动神经元肌萎缩侧索硬化症(ALS),据称也与同源二聚体CuZnSOD的错误折叠有关,己经报导,有多个CuZnSOD基因位点突变与ALS有关.本文将从基因的结构、表达、调节及蛋白的结构与功能等方面,对CuZnSOD进行简要论述.     

Tumor necrosis factor-alpha down-regulates human Cu/Zn superoxide dismutase 1 promoter via JNK/AP-1 signaling pathway

Overexpression of Cu/Zn superoxide dismutase 1 (SOD1) in monocytes blocks reactive oxygen species-induced inhibition of cell growth and apoptosis and renders cells resistant to the toxic effect of tumor necrosis factor (TNF)-alpha, suggesting that TNF-alpha represses the SOD1 gene in these cells. We herein show that TNF-alpha decreases SOD1 mRNA, protein, and promoter activity in U937 cells. Electrophoretic mobility-shift assays (EMSA) show that TNF-alpha decreased binding of three different complexes. Ectopic Sp1 overexpression markedly increased SOD1-basal promoter activity and partially antagonized the TNF-alpha inhibitory effect. In contrast, ectopic c-Jun overexpression mimics TNF-alpha inhibitory effects and antagonizes Sp1 stimulatory effects. In agreement with these findings, EMSA shows a TNF-alpha-induced increase in AP-1 and a decrease in Sp1 DNA binding. Disruption of the C/EBP site decreases, whereas mutation in the Sp1/Egr-1 site completely abolishes DNA-binding and promoter activity. A JNK inhibitor antagonized the negative effects of TNF-alpha on SOD1 promoter activity, suggesting that JNK signaling through c-Jun protein activation is critical for the TNF-alpha-dependent SOD1 repression. A greater understanding of the mechanisms of TNF-alpha-induced SOD1 repression could facilitate the design and development of novel therapeutic drugs for inflammatory conditions.     

Role of superoxide dismutase in the oxidation of N-alkanes by yeasts.

Yeast microorganisms from Candida genus are investigated for their superoxide dismutase (SOD) and catalase activity during cultivation on N-alkanes. The later caused a considerable increase of Cu/Zn SOD activity of yeast cells in comparison with glucose. A correlation between SOD and catalase activity existed. It is further observed that cells of Candida lipolytica 68-72 which contain a high level of Cu/Zn SOD were more resistant to lethality of exogenous O2-. An over-production of Cu/Zn SOD during the assimilation of N-alkanes by yeasts is also connected to their considerable resistance to increased concentrations of Cu2+ and Zn2+ ions in the nutrient medium. The results are consistent with the assumption that the enhanced resistance of yeast cells to O2- and high concentrations of Cu2+ and Zn(2+)-ions are due to the increased activity of Cu/Zn SOD and that SOD is involved in the protection of some cellular components. Polyacrylamide gel electrophoresis of Candida lipolytica cell-free extracts revealed the same chromatic bands of SOD activity under growth on glucose and N-alkanes. The type of the carbon source used from yeast cells as a single source of carbon and energy had no influence on the SOD profile of the cell.