Cytochrome c oxidase, Cu,Zn-superoxide dismutase, and ceruloplasmin activities in copper-deficient bovines

The activity of several cuproenzymes in relation to the immune system was examined in serum and blood cells from bovines with molybdenum-induced copper deficiency. Five female cattle were given molybdenum (30 ppm) and sulfate (225 ppm) to induce experimental secondary copper deficiency. Ceruloplasmin activity was determined in serum. The Cu,Zn-superoxide dismutase and cytochrome c oxidase activities were measured in peripheral blood lymphocytes, neutrophils, and monocyte-derived macrophages. Copper deficiency was confirmed from decreased serum copper levels and the animals with values less than 5.6 μmol/L were considered deficient. The content of intracellular copper decreased between 40% and 70% in deficient cells compared with the controls. In copper-deficient animals, the serum ceruloplasmin activity decreased to half of the control value. Both of them, the Cu,Zn-superoxide dismutase and the cytochrome c oxidase activities, undergo a significant reduction in leukocytes, showing differences among diverse cell populations. We concluded that the copper deficiency alters the activity of several enzymes, which mediate antioxidant defenses and ATP formation. These effects may impair the cell immune functionality, affecting the bactericidal capacity and making the animals more susceptible to infection.     

Leporipoxvirus Cu,Zn-superoxide dismutase (SOD) homologs are catalytically inert decoy proteins that bind copper chaperone for SOD

Many Chordopoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD). The biological function of these proteins is unknown, although the proteins encoded by Leporipoxviruses have been shown to promote a slow decline in the level of superoxide dismutase activity in virus-infected cells. To gain more insights into their function, we have further characterized the enzymatic and biochemical properties of a SOD homolog encoded by Shope fibroma virus. Shope fibroma virus SOD has retained the zinc binding properties of its cellular homolog, but cannot bind copper. Site-directed mutagenesis showed that it requires at least four amino acid substitutions to partially restore copper binding activity, but even these changes still did not restore catalytic activity. Reciprocal co-immunoprecipitation experiments showed that recombinant Shope fibroma virus SOD forms very stable complexes with cellular copper chaperones for SOD and these observations were confirmed using glutathione-S-transferase tagged proteins. Similar viral SOD/chaperone complexes were formed in cells infected with a closely related myxoma virus, where we also noted that some of the SOD antigen co-localizes with mitochondrial markers using confocal fluorescence microscopy. About 2% of the viral SOD was subsequently detected in gradient-purified mitochondria extracted from virus-infected cells. These poxviral SOD homologs do not form stable complexes with cellular Cu,Zn-SOD or affect its concentration. We suggest that Leporipoxvirus SOD homologs are catalytically inert decoy proteins that are designed to interfere in the proper metallation and activation of cellular Cu,Zn-SOD. This reaction might be advantageous for tumorigenic poxviruses, since higher levels of superoxide have been proposed to have anti-apoptotic and tumorigenic activity.     

A model for the incorporation of metal from the copper chaperone CCS into Cu,Zn superoxide dismutase.

BACKGROUND: Recent studies have identified the human copper chaperone CCS as the presumed factor responsible for copper incorporation into superoxide dismutase (SOD). A lack of knowledge of the chaperone's three-dimensional structure has prevented understanding of how the copper might be transferred. RESULTS: The three-dimensional structure of CCS was homology modelled using the periplasmic protein from the bacterial mercury-detoxification system and the structure of one subunit of the human SOD dimeric enzyme as templates. On the basis of the three-dimensional model, a mechanism for the transfer of copper from CCS to SOD is proposed that accounts for electrostatic acceptor recognition, copper storage and copper-transfer properties. CONCLUSIONS: The proposed model identifies a path for copper transfer based on the presence of different metal sites characterized by sulphur ligands. Such a model permits the development of strategies able to interfere with copper incorporation in SOD, providing a possible way to prevent or arrest degeneration in the fatal motor neuron disorder amyotrophic lateral sclerosis.     

Inhibition of chaperone activity is a shared property of several Cu,Zn-superoxide dismutase mutants that cause amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron degeneration, paralysis, and death. Mutant Cu,Zn-superoxide dismutase (SOD1) causes a subset of ALS by an unidentified toxic property. Increasing evidence suggests that chaperone dysfunction plays a role in motor neuron degeneration in ALS. To investigate the relationship between mutant SOD1 expression and chaperone dysfunction, we measured chaperone function in central nervous system tissue lysates from normal mice and transgenic mice expressing human SOD1 variants. We observed a significant decrease in chaperone activity in tissues from mice expressing ALS-linked mutant SOD1 but not control mice expressing human wild type SOD1. This decrease was detected only in the spinal cord, became apparent by 60 days of age (before the onset of muscle weakness and significant motor neuron loss), and persisted throughout the late stages. In addition, this impairment of chaperone activity occurred only in cytosolic but not in mitochondrial and nuclear fractions. Furthermore, multiple recombinant human SOD1 mutants with differing biochemical and biophysical properties inhibited chaperone function in a cell-free extract of normal mouse spinal cords. Thus, mutant SOD1 proteins may impair chaperone function independent of gene expression in vivo, and this inhibition may be a shared property of ALS-linked mutant SOD1 proteins.     

Increase of Cu,Zn-superoxide dismutase activity during differentiation of human K562 cells involves activation by copper of a constantly expressed copper-deficient protein.

Cu,Zn-superoxide dismutase activity, expressed on the basis of cell number, increased by 50% during sodium butyrate-induced differentiation of human K562 erythroleukemia cells. The increased enzyme activity was found to be concomitant with constant Cu,Zn-superoxide dismutase mRNA and immunoreactive protein levels and was accompanied by a rise in intracellular copper and glutathione. Incubation of K562 cell homogenates with copper caused an increase of Cu,Zn-superoxide dismutase activity which reached the levels observed after differentiation in the presence of sodium butyrate. The same treatment led to no significant activity increase in homogenates derived from differentiated cells. Externally added ceruloplasmin increased both intracellular copper levels and Cu,Zn-superoxide dismutase activity in undifferentiated cells to a level comparable with that observed after induction of differentiation. Both increments were abolished by depletion of cell glutathione. Cu,Zn-superoxide dismutase purified from control cells had both a lower kcat and a lower copper content than the enzyme purified from differentiated cells. From these data we conclude that: 1) Cu,Zn-superoxide dismutase is present in K562 cells also under the form of a less active copper-deficient enzyme, 2) the extent of enzyme activation is regulated post-translationally by differential delivery of copper as a function of differentiation stage, and 3) glutathione is likely to play a role in delivering copper to the copper-deficient protein in intact K562 cells.     

Putative denitrosylase activity of Cu,Zn-superoxide dismutase

The Cu,Zn-superoxide dismutase (SOD1) has been reported to exert an S-nitrosylated glutathione (GSNO) denitrosylase activity that was augmented by a familial amyotrophic lateral sclerosis (FALS)-associated mutation in this enzyme. This putative enzymatic activity as well as the spontaneous decomposition of GSNO has been reexamined. The spontaneous decomposition of GSNO exhibited several peculiarities, such as a lag phase followed by an accelerating rate plus a marked dependence on GSNO concentration, suggestive of autocatalysis, and a greater rate in polypropylene than in glass vessels. Dimedone caused a rapid increase in absorbance likely due to reaction with GSNO, followed by a slower increase possibly due to reaction with an intermediate such as glutathione sulfenic acid. SOD1 weakly increased the rate of decomposition of GSNO, but did so only when GSH was present; and FALS-associated mutant forms of SOD1 were not more active in this regard than was the wild type. Decomposed GSNO, when added to fresh GSNO, hastened its decomposition, in accord with autocatalysis, and when added to GSH, generated GSNO in accord with the presence of nitrite. A mechanism is proposed that is in accord with these observations.     

Release of copper ions from the familial amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutants

Point mutations of Cu,Zn-superoxide dismutase (SOD) have been linked to familial amyotrophic lateral sclerosis (FALS). We reported that the Swedish FALS Cu,Zn-SOD mutant, D90A, exhibited an enhanced hydroxyl radical-generating activity, while its dismutation activity was identical to that of the wild-type enzyme (Kim et al. 1998a; 1998b). Transgenic mice that express a mutant Cu,Zn-SOD, Gly93 --> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms. We cloned the cDNA for the FALS G93A mutant, overexpressed the protein in E. coli cells, purified the protein, and studied its enzymic activities. Our results showed that the G93A, the D90A, and the wild-type enzymes have identical dismutation activity. However, the hydroxyl radical-generating activity of the G93A mutant was enhanced relative to those of the D90A and the wild-type enzyme (wild-type < D90A < G93A). These higher free radical-generating activities of mutants facilitated the release of copper ions from their own molecules (wild-type < D90A < G93A). The released copper ions can enhance the Fenton-like reaction to produce hydroxyl radicals and play a major role in the oxidative damage of macromolecules. Thus, the FALS symptoms may be associated with the enhancements in both the free radical-generating activity and the releasing of copper ions from the mutant enzyme.     

Modification of Cysteine 111 in human Cu,Zn-superoxide dismutase

Human Cu,Zn-superoxide dismutase (hSOD1) has 4 cysteines per subunit. Cys57 and Cys148 are involved in an intrasubunit disulfide bond, while Cys6 and Cys111 are free. Cys6 is buried within the protein while Cys111 is on the surface, near the dimer interface. We examined by liquid chromatography-mass spectrometry the commercially purchased hSOD1 isolated from erythrocytes as well as hSOD1s isolated from human erythrocytes, brain, and hSOD1 expressed in Sf9, yeast, and E. coli. Our goal was to ascertain whether the Cys111 modification occurred naturally in vivo. Only the Sigma erythrocyte hSOD1 appeared to contain a trisulfide crosslink between the Cys111 residues. Thus it failed to react with N-ethylmaleimide, showed absorbtion at 325 nm that was eliminated by 2-mercaptoethanol, and had a mass 30 units more than expected for the native dimer. We examined the possibility that different purification methods might cause this modification in erythrocyte hSOD1. None of the procedures examined for hSOD1 purification produced such a trisulfide. In disagreement with Liu et al. [Biochemistry, 2000, 39, 8125-8132], complete derivitization of both Cys111s of hSOD1 from Sf9 cells with N-ethylmaleimide, 4-vinylpyridine, and by 5,5′-dithiobis(2-nitrobenzoic acid) were readily achieved; indicating that steric hindrance was not a problem.     

Proteome analysis in hippocampus of mice overexpressing human Cu/Zn-superoxide dismutase 1

Cu/Zn-superoxide dismutase 1 (SOD1), encoded on chromosome 21, is a key enzyme in metabolism of oxygen free radicals and oxidative stress. Transgenic mice overexpressing human SOD1 (Tg-hSOD1) are useful model for Down syndrome (trisomy 21) and familial amyotrophic lateral sclerosis (ALS). It was shown recently that Tg-hSOD1 mice develop a characteristic set of neurodegenerative changes in hippocampus and we therefore decided to study differential protein expression patterns, constructing a mouse hippocampal proteome map using two-dimensional electrophoresis (2-DE) with in-gel digestion of spots followed by matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) identification and quantitatively compared protein profiles between non-transgenic mice, hemizygous and homozygous Tg-hSOD1 mice. In total 1056 spots were analysed, resulting in the identification of 445 polypeptides that were the products of 157 different genes. Among these a series of proteins involved in scaffolding, metabolism, signaling and other functions were deranged. Our findings suggest that overexpressed SOD1 directly or by generating reactive oxygen species may lead to aberrant protein expressional patterns that in turn may lead to or reflect neurodegeneration observed in this animal model.     

Modification of Cu,Zn-superoxide dismutase by oxidized catecholamines

Oxidation of catecholamines may contribute to the pathogenesis of Parkinson's disease (PD). The effect of the oxidized products of catecholamines on the modification of Cu,Zn-superoxide dismutase (SOD) was investigated. When Cu,Zn-SOD was incubated with the oxidized 3,4-dihydroxyphenylalanine (DOPA) or dopamine, the protein was induced to be aggregated. The deoxyribose assay showed that hydroxyl radicals were generated during the oxidation of catecholamines in the presence of copper ion. Radical scavengers, azide, N-acetylcysteine, and catalase inhibited the oxidized catecholamine-mediated Cu,Zn-SOD aggregation. Therefore, the results indicate that free radicals may play a role in the aggregation of Cu,Zn-SOD. When Cu,Zn-SOD that had been exposed to catecholamines was subsequently analyzed by an amino acid analysis, the glycine and histidine residues were particularly sensitive. These results suggest that the modification of Cu,Zn-SOD by oxidized catecholamines might induce the perturbation of cellular antioxidant systems and led to a deleterious cell condition.     

Modification of cysteine 111 in human Cu,Zn-superoxide dismutase

Human Cu,Zn-superoxide dismutase (hSOD1) has 4 cysteines per subunit. Cys57 and Cys148 are involved in an intrasubunit disulfide bond, while Cys6 and Cys111 are free. Cys6 is buried within the protein while Cys111 is on the surface, near the dimer interface. We examined by liquid chromatography-mass spectrometry the commercially purchased hSOD1 isolated from erythrocytes as well as hSOD1s isolated from human erythrocytes, brain, and hSOD1 expressed in Sf9, yeast, and E. coli. Our goal was to ascertain whether the Cys111 modification occurred naturally in vivo. Only the Sigma erythrocyte hSOD1 appeared to contain a trisulfide crosslink between the Cys111 residues. Thus it failed to react with N-ethylmaleimide, showed absorbtion at 325 nm that was eliminated by 2-mercaptoethanol, and had a mass 30 units more than expected for the native dimer. We examined the possibility that different purification methods might cause this modification in erythrocyte hSOD1. None of the procedures examined for hSOD1 purification produced such a trisulfide. In disagreement with Liu et al. [Biochemistry, 2000, 39, 8125-8132], complete derivitization of both Cys111s of hSOD1 from Sf9 cells with N-ethylmaleimide, 4-vinylpyridine, and by 5,5′-dithiobis(2-nitrobenzoic acid) were readily achieved; indicating that steric hindrance was not a problem.     

Cu,Zn-superoxide dismutase is differently regulated by cadmium and lead in roots of soybean seedlings

The cadmium (Cd²⁺) and lead (Pb²⁺)-induced changes in Cu,Zn-SOD gene expression on the level of mRNA accumulation and enzyme activity were analyzed in roots of soybean (Glycine max) seedlings. The Cd²⁺ caused the induction of copper–zinc superoxide dismutase (Cu,Zn-SOD) mRNA accumulation, at each analyzed metal concentration (5–25 mg/l), whereas in Pb²⁺-treated roots this effect was observed only at the medium metal concentrations (50–100 mg/l of Pb²⁺). The analysis of Cu,Zn-SOD activity proved an increase in enzyme activity during Cd²⁺/Pb²⁺ stresses, however in Pb²⁺-treated plants the activity of enzyme was not correlated with respective mRNAs level. Presented data suggest that different metals may act on various level of Cu,Zn-SOD expression in plants exposed to heavy metals stress.     

Developmental regulation of rat lung Cu,Zn-superoxide dismutase.

In the present investigation we found that lung Cu,Zn-superoxide dismutase (SOD) activity (units/mg of DNA) increases steadily in the rat from birth to adulthood. The specific activity (units/micrograms of enzyme) of Cu,Zn-SOD was unchanged from birth to adulthood, excluding enzyme activation as a mechanism responsible for the increase in enzyme activity. Lung synthesis of Cu,Zn-SOD peaked at 1 day before birth and decreased thereafter to adult values. Calculations, based on rates of Cu,Zn-SOD synthesis and the tissue content of the enzyme, indicated that lung Cu,Zn-SOD activity increased during development owing to the rate of enzyme synthesis exceeding its rate of degradation by 5-10%. These calculations were supported by measurements of enzyme degradation in the neonatal (half-life, t1/2, = 12 h) and adult lung (t1/2 = greater than 100 h); the difference in half-life did not reflect the rates of overall protein degradation in the lung, since these rates were not different in lungs from neonatal and adult rats. We did not detect differences in the Mr or pI of Cu,Zn-SOD during development, but the susceptibility of the enzyme to inactivation by heat or copper chelation decreased with increasing age of the rats. We conclude that the progressive increase in activity of Cu,Zn-SOD is due to a rate of synthesis that exceeds degradation of the enzyme. The data also suggest that increased stabilization of enzyme conformation accounts for the greater half-life of the enzyme in lungs of adult compared with neonatal rats.     

Cardioprotection by Cu, Zn-superoxide dismutase is lost at high doses in the reoxygenated heart

Limited dose-response curves for superoxide dismutase (SOD) were assessed in isolated and in vivo hearts. SOD at 2.3, 7, 20, or 50 mg/L suppressed CK release in Langendorff rat hearts by 61%, 63%, 72%, and 30%, respectively. SOD at 0.5, 1, 5, and 50 mg/L suppressed LDH release in Langendorff rabbit hearts by 32%, 48%, 54%, and −12%, respectively. In rabbit hearts subjected to coronary artery ligation and reperfusion in vivo, SOD at 2, 5,or 15 mg/kg reduced infarct size by 10%, 30% or 19%, respectively, while 50 mg/kg increased infarct size by 28%. In conclusion, while SOD was protective at low doses in all models, protection was lost at higher doses in the isolated rat and rabbit hearts, and exacerbation of damage was seen in the in vivo rabbit hearts.