Genetic and biochemical characterization of Cu,Zn superoxide dismutase mutants in Saccharomyces cerevisiae

The allele scd 1 is a recessive chromosomal mutation in Saccharomyces cerevisiae that eliminates Cu,Zn superoxide dismutase (SOD-1) activity. SOD-1- strains are unable to grow in 100% O2 in rich medium and are methionine and lysine auxotrophic when grown in air (Bilinski, T., Krawiec, Z., Liczmanski, A., and Litwinska, J. (1985) Biochem. Biophys. Res. Commun. 130, 533-539). In this report, scd1 was genetically mapped to the right arm of chromosome X, 11 centimorgans proximal to cdc11. The gene for SOD-1 (SOD1) was physically mapped by Southern blot to restriction fragments containing CDC11. scd1 failed to complement a complete deletion of SOD1. Thus, scd1 maps to the SOD1 locus and is designated sod1-1. The molecular basis for the lack of SOD-1 activity in sodl-1 carrying strains has also been established. The size and amount of SOD-1 mRNA in the mutant were essentially the same as in wild type cells. Western blot analysis showed that the SOD-1 dimer and 16-kilodalton subunit that co-migrated electrophoretically with wild type yeast SOD-1 were abundant in mutant cell extracts. However, two additional SOD-1 immunoreactive polypeptides were detected in these extracts in both denaturing and nondenaturing gels. None of the SOD-1 immunoreactive species in the mutant extracts exhibited superoxide dismutase activity. Transformants of the mutant strain carrying episomal, wild type SOD1 expressed wild type, active SOD-1 protein, indicating that the mutant allele had no discernible effect on the correct synthesis and activation of apoSOD-1. Size exclusion chromatography of soluble cell extracts derived from wild type and SOD1 deletion strains identified a copper binding peak that corresponded to SOD-1. This copper-binding fraction was absent in cell extracts from the sod1-1-containing strain although Western blot analysis of the corresponding chromatographic fractions showed that SOD-1 polypeptide was present in these fractions. Sequence data derived from the cloned genes showed that sod1-1 differed from SOD1 only in the adjacent 5'-noncoding region. The biochemical data indicate that this genetic alteration results in the synthesis of a collection of SOD-1 polypeptides that fail to bind copper and may also fail to completely self-associate. Both phenotypes could be due to the inability of these polypeptides to adopt the native SOD-1 conformation.     

Null mutants of Saccharomyces cerevisiae Cu,Zn superoxide dismutase: characterization and spontaneous mutation rates.

Deletion-replacement mutations of the Saccharomyces cerevisiae Cu,Zn superoxide dismutase gene were constructed. They were exquisitely sensitive to redox cycling drugs and showed slight sensitivity to other agents. The aerobic spontaneous mutation rate was three- to fourfold higher in sod1 delta 1 mutants, while the anaerobic rate was similar to that of the wild type.     

Tolerance of spermatogonia to oxidative stress is due to high levels of Zn and Cu/Zn superoxide dismutase

Background

Spermatogonia are highly tolerant to reactive oxygen species (ROS) attack while advanced-stage germ cells such as spermatozoa are much more susceptible, but the precise reason for this variation in ROS tolerance remains unknown.

Methodology/Principal Findings

Using the Japanese eel testicular culture system that enables a complete spermatogenesis in vitro, we report that advanced-stage germ cells undergo intense apoptosis and exhibit strong signal for 8-hydroxy-2′-deoxyguanosine, an oxidative DNA damage marker, upon exposure to hypoxanthine-generated ROS while spermatogonia remain unaltered. Activity assay of antioxidant enzyme, superoxide dismutase (SOD) and Western blot analysis using an anti-Copper/Zinc (Cu/Zn) SOD antibody showed a high SOD activity and Cu/Zn SOD protein concentration during early spermatogenesis. Immunohistochemistry showed a strong expression for Cu/Zn SOD in spermatogonia but weak expression in advanced-stage germ cells. Zn deficiency reduced activity of the recombinant eel Cu/Zn SOD protein. Cu/Zn SOD siRNA decreased Cu/Zn SOD expression in spermatogonia and led to increased oxidative damage.

Conclusions/Significance

These data indicate that the presence of high levels of Cu/Zn SOD and Zn render spermatogonia resistant to ROS, and consequently protected from oxidative stress. These findings provide the biochemical basis for the high tolerance of spermatogonia to oxidative stress.     

Intracellular Mn (II)-associated superoxide scavenging activity protects Cu,Zn superoxide dismutase-deficient Saccharomyces cerevisiae against dioxygen stress

Three Cu,Zn superoxide dismutase (SOD-1)-deficient Saccharomyces cerevisiae mutants do not grow in 100% O2 in rich medium and require Met and Lys when grown in air (Bilinski, T., Krawiec, Z., Liczmanski, A., and Litwinska, J. (1985) Biochem. Biophys. Res. Commun. 130, 533-539). We show herein that medium manganese (II) accumulated by the mutants rescues these O2-sensitive phenotypes; 2 mM medium Mn2+ represented the threshold required for cell growth. The accumulation of Mn2+ was not oxygen-inducible since mutants grown aerobically and anaerobically accumulated the same amount of Mn2+. Mn2+ accumulation is not unique to these mutants since wild type accumulated almost twice as much Mn2+ as did mutant. ESR spectra of the cell extracts and whole cells loaded with Mn2+ were typical of free Mn(II) ion. These spectra could not account quantitatively for the total cellular Mn2+, however. A screen for soluble antioxidant activities in the Mn2+-supplemented cells detected O2- (superoxide) scavenging activity, with no change in catalase or peroxidase activities. This O2- scavenging activity was CN- and heat-resistant. No achromatic bands were revealed in nondenaturing gels of Mn2+- containing cell extracts stained for O2- scavenging activity. The Mn2+-dependent O2- scavenging activity in the cell extracts was quenched by EDTA and dialyzable. More than 60% of both the intracellular Mn2+ and the O2- scavenging activity was removed by 2-h dialysis. Dialyzed cells were not viable in air unless resupplemented with either Met or Mn2+. Although Mn2+ supported the aerobic growth of these mutants, excess Mn2+, which correlated with an elevated O2- scavenging activity, was toxic to both mutant and wild type. The results indicate that free or loosely bound Mn2+ ion protects the mutants against oxygen stress by providing an intracellular, presumably cytosolic, O2- scavenging activity which replaces the absent SOD-1.     

Overexpression of Cu,Zn superoxide dismutase attenuates oxidative inhibition of astrocyte glutamate uptake

Glutamate neurotoxicity in brain is normally prevented by rapid uptake of glutamate by astrocytes. Increased expression of Cu,Zn superoxide dismutase (SOD1) can increase resistance to cerebral ischemia and other oxidative insults, but the cellular mechanisms by which this occurs are not well established. Here we examine whether increased SOD1 expression can attenuate inhibition of astrocyte glutamate uptake by reactive oxygen species. Primary cortical astrocyte cultures were prepared from transgenic mice that overexpress human SOD1 and from nontransgenic littermate controls. Glutamate uptake was assessed after exposure of these cultures to xanthine oxidase plus hypoxanthine, an extracellular superoxide generating system, or to menadione, which generates superoxide in the cytosol. These treatments produced dose-dependent reductions in astrocyte glutamate uptake, and the reductions were significantly attenuated in the SOD1 transgenic astrocytes. A specific effect of reactive oxygen species on glutamate transporters was suggested by the much smaller inhibitory effects of xanthine oxidase/hypoxanthine and menadione on GABA uptake than on glutamate uptake. These findings suggest that the cerebroprotective effects of increased SOD1 expression during cerebral ischemia-reperfusion could be mediated in part by astrocyte glutamate transport.     

Divalent-metal-dependent nucleolytic activity of Cu, Zn superoxide dismutase

The known action of Cu, Zn superoxide dismutase (holo SOD) that converts O₂ ⁻ to O₂ and H₂O₂ plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction of holo SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much holo SOD may be injurious to the cells. In the in vitro study, we report a finding that the holo SOD from bovine erythrocytes and its apo form possess a divalent-metal-dependent nucleolytic activity, which was confirmed by UV–vis absorption titration of calf thymus DNA (ctDNA) with the holo SOD, quenching of holo SOD intrinsic fluorescence by ctDNA, and by gel electrophoresis monitoring conversion of DNA from the supercoiled DNA to nicked and linear forms, and fragmentation of a linear λDNA. Moreover, the DNA cleavage activity was examined in detail under certain reaction conditions. The steady-state study indicates that DNA cleavage supported by both forms of SOD obeys Michaelis–Menten kinetics. On the other hand, the assays with some other proteins indicate that this new function is specific to some proteins including the holo SOD. Therefore, this study reveals that the divalent-metal-dependent DNA cleavage activity is an intrinsic property of the holo SOD, which is independent of its natural metal (copper and zinc) sites, and may provide an alternative insight into the link between SOD enzymes and neurodegenerative disorders.     

Cu,Zn superoxide dismutase from chicken erythrocytes.

1. Copper, zinc superoxide dismutase (Cu,Zn SOD) has been purified to homogeneity from chicken erythrocytes by anion-exchange, immobilized metal affinity and size exclusion chromatography. 2. Molecular properties (amino acid composition, molecular mass, subunit composition and spec. act.) of the chicken enzyme are similar to those of a bovine erythrocyte Cu,Zn SOD. 3. The chicken and bovine enzymes are immunologically similar since antisera raised against each enzyme are cross-reactive.     

Preparation of reduced bovine Cu,Zn superoxide dismutase.

N.m.r. and e.p.r. were used to measure the oxidation state of copper in Cu,Zn superoxide dismutase treated with reducing agents such as NaBH4, K4Fe(CN)6, Na2S2O4 and H2O2. The activity and the electrophoretic pattern of the treated enzyme were also studied. On the basis of the reducing ability and of the absence of inactivating effects, NaBH4 was the most suitable reducer of those tested. Some characteristics of the reduction of superoxide dismutase by NaBH4 were further investigated. The results obtained indicate that NaBH4 can be used to prepare, in a few minutes, solutions of completely reduced enzyme without any apparent change of the activity and of the structure.     

O2-dependent methionine auxotrophy in Cu,Zn superoxide dismutase-deficient mutants of Saccharomyces cerevisiae.

Mutant strains of the yeast Saccharomyces cerevisiae which lack functional Cu,Zn superoxide dismutase (SOD-1) do not grow aerobically unless supplemented with methionine. The molecular basis of this O2-dependent auxotrophy in one of the mutants, Dscd1-1C, has been investigated. Sulfate supported anaerobic but not aerobic mutant growth. On the other hand, cysteine and homocysteine supported aerobic growth while serine, O-acetylserine, and homoserine did not, indicating that the interconversion of cysteine and methionine (and homocysteine) was not impaired. Thiosulfate (S2O3(2-] and sulfide (S2-) also supported aerobic growth; the activities of thiosulfate reductase and sulfhydrylase in the aerobic mutant strain were at wild-type levels. Although the levels of SO4(2-) and adenosine-5'-sulfate (the first intermediate in the SO4(2-) assimilation pathway) were elevated in the aerobically incubated mutant strain, this condition could be attributed to a decrease in protein synthesis caused by the de facto sulfur starvation and not to a block in the pathway. Therefore, the activation of SO4(2-) (to form 3'-phosphoadenosine-5'-phosphosulfate) appeared to be O2 tolerant. Sulfite reductase activity and substrate concentrations [( NADPH] and [SO3(2-)]) were not significantly different in aerobically grown mutant cultures and anaerobic cultures, indicating that SOD-1- mutant strains could reductively assimilate sulfur oxides. However, the mutant strain exhibited an O2-dependent sensitivity to SO3(2-) concentrations of less than 50 microM not exhibited by any SOD-1+ strain or by SOD-1- strains supplemented with a cytosolic O2(-)-scavenging activity. This result suggests that the aerobic reductive assimilation of SO4(2-) at the level of SO3(2-) may generate a cytotoxic compound(s) which persists in SOD-(1-) yeast strains.     

Effects of Cu/Zn superoxide dismutase on strain injury-induced oxidative damage to skeletal muscle in rats

This study was designed to determine whether the supplement of superoxide dismutase (SOD) could attenuate strain-induced oxidative damage to skeletal muscle in rats. Experimental animals were injured in right gastrocnemius muscles by a strain injury model. SOD-treated groups were given Cu/Zn SOD 10 000 U/kg body weight per day since injured, while control groups were given normal saline. Parameters of antioxidant and muscle damage were detected in plasma 3 and 7 days postinjury. The injured muscles were removed and fixed for histology observation and immunohisto-chemistry assay of desmin. The results showed that plasma levels of SOD, glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC) in SOD group were significantly higher than in the saline group on day 3 or 7, while the plasma creatine kinase (CK) and malondialdehyde (MDA) were lower in the SOD group than in the saline group. The histological examination of muscle sections revealed a lower degree of damage in the SOD group in which the expression level of desmin was higher than in the saline group. It is suggested that SOD supplement may attenuate strain-induced muscle damage and facilitate its regeneration.     

Cadmium-induced oxidative stress in Saccharomyces cerevisiae

The present study was undertaken to determine the effect of cadmium (Cd) on the antioxidant status of the yeast Saccharomyces cerevisiae. S. cerevisiae serves as a good eukaryotic model system for the study of the molecular mechanisms of oxidative stress. We investigated the adaptative response of S. cerevisiae exposed to Cd. Yeast cells could tolerate up to 100 microM Cd and an inhibition in the growth and viability was observed. Exposure of yeast cells to Cd showed an increase in malondialdehyde and glutathione. The activities of catalase, superoxide dismutase and glutathione peroxidase were also high in Cd-exposed cells. The incorporation of Cd led to significant increase in iron, zinc and inversely the calcium, copper levels were reduced. The results suggest that antioxidants were increased and are involved in the protection against macromolecular damage during oxidative stress; presumably, these enzymes are essential for counteracting the pro-oxidant effects of Cd.     

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.