Modification and inactivation of Cu,Zn-superoxide dismutase by the lipid peroxidation product,acrolein

Acrolein is the most reactive aldehydic product of lipid peroxidation and is found to be elevated in the brain when oxidative stress is high. The effects of acrolein on the structure and function of human Cu,Zn-superoxide dismutase (SOD) were examined. When Cu,Zn-SOD was incubated with acrolein, the covalent crosslinking of the protein was increased, and the loss of enzymatic activity was increased in a dose-dependent manner. Reactive oxygen species (ROS) scavengers and copper chelators inhibited the acrolein-mediated Cu,Zn-SOD modification and the formation of carbonyl compound. The present study shows that ROS may play a critical role in acrolein-induced Cu,Zn-SOD modification and inactivation. When Cu,Zn-SOD that has been exposed to acrolein was subsequently analyzed by amino acid analysis, serine, histidine, arginine, threonine and lysine residues were particularly sensitive. It is suggested that the modification and inactivation of Cu,Zn-SOD by acrolein could be produced by more oxidative cell environments. [BMB Reports 2013; 46(11): 555-560]     

Modulation of NO and cytokines in microglial cells by Cu/Zn-superoxide dismutase.

The activation of microglial cells in response to neuropathological stimuli is one of the prominent features of human neurodegenerative diseases. Cytokines such as IL-1 beta and TNF-alpha and inflammation-related enzymes such as inducible nitric oxide synthase are usually induced during the activation of microglial cells. We investigated the modulation of the activation of microglial cell by transfecting a Cu/Zn-SOD cDNA into BV-2 cells. Parental and transfected BV-2 cells were then subjected to LPS stimulation. The results showed that in Cu/Zn-SOD-transfected BV-2 cells, the expression and activity of Cu/Zn-SOD increased. On the other hand, upon activation by LPS, these cells produced less NO, IL-1 beta, and TNF-alpha than the parental microglial cells. This finding suggests that superoxide may be an early signal triggering the induction of cytokines and that the transfected Cu/Zn-SOD may provide a neuroprotective function via suppression of microglial activation. In addition, this approach may provide a rationale for the development of treatments for neurodegenerative diseases.     

Lipid peroxidation induced by the Cu,Zn-superoxide dismutase and hydrogen peroxide system.

Cu,Zn-superoxide dismutase (SOD) can catalyze hydroxyl radical generation using H2O2 as a substrate. Lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system was investigated. When linoleic acids micelles or phosphatidylcholine liposomes were incubated with Cu,Zn-SOD and H2O2, lipid peroxidation was gradually increased in a time-dependent manner. The extent of lipid peroxidation was proportional to Cu,Zn-SOD and H2O2 concentrations. Hydroxyl radical scavengers and copper chelator inhibited lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system. These results suggest that lipid peroxidation is mediated by the Cu,Zn-SOD and H2O2 system via the generation of hydroxyl radicals by a combination of the peroxidative reaction of Cu,Zn-SOD and the Fenton-like reaction of free copper released from oxidatively damaged SOD.     

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 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.     

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.     

Regulation of Cu,Zn-superoxide dismutase in bovine pulmonary artery endothelial cells.

To evaluate the regulation of endothelial cell Cu,Zn-SOD, we have exposed bovine pulmonary artery endothelial cells in culture to hyperoxia and hypoxia, second messengers or related agonists, hormones, free radical generating systems, endotoxin, and cytokines and have measured Cu,Zn-SOD protein of these cells by an ELISA developed in our laboratory. Control preconfluent and confluent cells in room air contained 196 +/- 18 ng Cu,Zn-SOD/10(6) cells. A23187 (0.33 microM), forskolin (10 microM), isobutylmethylxanthine (0.1 mM), dexamethasone (1 microM), triiodothyronine (1 microM) and retinoic acid (1 microM) failed to alter this level of Cu,Zn-SOD. Exposure to anoxia and hyperoxia both elevated the level approximately 1.5-2.0-fold over 20% oxygen-exposed controls at 48-72 hr. Similarly, exposures to glucose oxidase (0.0075 units/ml), menadione (12.5 microM), xanthine-xanthine oxidase (10 microM, 0.03 units/ml) and H2O2 (0.0005%) increased the level up to two-threefold over controls at 24-48 hr. Lipopolysaccharide, TGF beta 1, TNF alpha, and Il-1 also increased levels of cellular Cu,Zn-SOD, but only in proliferating cells. Il-2, Il-4, interferon-gamma, and GM-CSF had no effect on Cu,Zn-SOD. All treatments that elevated SOD resulted in inhibition of cellular growth, but decreased growth of cells at confluence alone was not associated with increased Cu,Zn-SOD. We propose from these studies that Cu,Zn-SOD of endothelial cells is not under conventional second messenger or hormonal regulation, but that up-regulation of the enzyme is associated with (and perhaps stimulated by) free-radical or oxidant production that also may be influenced by availability of certain cytokines under replicating conditions.     

Overexpression of manganese superoxide dismutase attenuates neuronal death in human cells expressing mutant (G37R) Cu/Zn-superoxide dismutase

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor function and eventual death as a result of degeneration of motor neurons in the spinal cord and brain. The discovery of mutations in SOD1, the gene encoding the antioxidant enzyme Cu/Zn-superoxide dismutase (CuZnSOD), in a subset of ALS patients has led to new insight into the pathophysiology of ALS. Utilizing a novel adenovirus gene delivery system, our laboratory has developed a human cell culture model using chemically differentiated neuroblastoma cells to investigate how mutations in SOD1 lead to neuronal death. Expression of mutant SOD1 (G37R) resulted in a time and dose-related death of differentiated neuroblastoma cells. This cell death was inhibited by overexpression of the antioxidant enzyme manganese superoxide dismutase (MnSOD). These observations support the hypothesis that mutant SOD1-associated neuronal death is associated with alterations in oxidative stress, and since MnSOD is a mitochondrial enzyme, suggest that mitochondria play a key role in disease pathogenesis. Our findings in this model of inhibition of mutant SOD1-associated death by MnSOD represent an unique approach to explore the underlying mechanisms of mutant SOD1 cytotoxicity and can be used to identify potential therapeutic agents for further testing.     

Modification and inactivation of human Cu,Zn-superoxide dismutase by methylglyoxal

Methylglyoxal (MG) has been identified as an intermediate in non-enzymatic glycation, and increased levels have been reported in patients with diabetes. In this study, the effect of MG on the structure and function of human Cu,Zn-superoxide dismutase (SOD) was investigated. MG modifies Cu,Zn-SOD, as indicated by the formation of fluorescent products. When Cu, Zn-SOD was incubated with MG, covalent crosslinking of the protein increased progressively. MG-mediated modification of Cu,Zn-SOD led to loss of enzymatic activity and release of copper ions from the protein. Radical scavengers inhibited the crosslinking of Cu,Zn-SOD. When Cu,Zn-SOD that had been exposed to MG was analyzed, glycine, histidine, lysine, and valine residues were found to be particularly sensitive. It is suggested that oxidative damage to Cu,Zn-SOD by MG may perturb cellular antioxidant defense systems and damage cells. This effect may account, in part, for organ deterioration in diabetes.     

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.     

Mechanism of Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS

The mechanism for copper loading of the antioxidant enzyme copper, zinc superoxide dismutase (SOD1) by its partner metallochaperone protein is not well understood. Here we show the human copper chaperone for Cu,Zn-SOD1 (hCCS) activates either human or yeast enzymes in vitro by direct protein to protein transfer of the copper cofactor. Interestingly, when denatured with organic solvents, the apo-form of human SOD1 cannot be reactivated by added copper ion alone, suggesting an additional function of hCCS such as facilitation of an active folded state of the enzyme. While hCCS can bind several copper ions, metal binding studies in the presence of excess copper scavengers that mimic the intracellular chelation capacity indicate a limiting stoichiometry of one copper and one zinc per hCCS monomer. This protein is active and unlike the yeast protein, is a homodimer regardless of copper occupancy. Matrix-assisted laser desorption ionization-mass spectrometry and metal binding studies suggest that Cu(I) is bound by residues from the first and third domains and no bound copper is detected for the second domain of hCCS in either the full-length or truncated forms of the protein. Copper-induced conformational changes in the essential C-terminal peptide of hCCS are consistent with a "pivot, insert, and release" mechanism that is similar to one proposed for the well characterized metal handling enzyme, mercuric ion reductase.     

Altered expression of hypothetical proteins in hippocampus of transgenic mice overexpressing human Cu/Zn-superoxide dismutase 1

Background  

The rapid completion of genome sequences has created an infrastructure of biological information and provided essential information to link genes to gene products, proteins, the building blocks for cellular functions. In addition, genome/cDNA sequences make it possible to predict proteins for which there is no experimental evidence. Clues for function of hypothetical proteins are provided by sequence similarity with proteins of known function in model organisms.     

Superoxide dismutase inhibits lipid peroxidation in micelles.

Superoxide dismutase (SOD) taken in minor concentrations (a few U/ml) displays a pronounced inhibiting effect on the chain oxidation of methyl linoleate and methyl linolenate (but not methyl oleate) induced by 2,2'-azobis(2-amidinopropan) dihydrochloride (AAPH) in micellar solutions of sodium dodecyl sulfate and Triton X-100 in phosphate buffer, pH 7.40, at 37.0 degrees C. The inhibition is evidently caused by purging the system from O(2)*(-). The latter suggests the formation of O(2)*(-) (HO(2)* in the course of peroxidation, most likely, via beta-decay of lipid peroxy radical (LO(2)*. Thermodynamic estimations verify a rather high probability of beta-decay of LO(2)* produced from polyunsaturated fatty acids by contrast to that produced from saturated and monoenic fatty acids. It is speculated that O(2)*(-) (HO(2)*, being an amphiphilic, reactive and highly mobile species, participates in intermicellar (interliposomal) transfer of free valence during lipid peroxidation in microheterogeneous systems.     

Cu,Zn-superoxide dismutase is necessary for proper function of VDAC in Saccharomyces cerevisiae cells

Available data suggest that a copper-and zinc-containing dismutase (CuZnSOD) plays a significant role in protecting eukaryotic cells against oxidative modifications which may contribute to cell aging. Here we demonstrated that depletion of CuZnSOD in Saccharomyces cerevisiae cells (Δsod1 cells) affected distinctly channel activity of VDAC (voltage dependent anion selective channel) and resulted in a moderate reduction in VDAC levels as well as in levels of protein crucial for VDAC import into mitochondria, namely Tob55/Sam50 and Tom40. The observed alterations may result in mitochondriopathy and subsequently in the shortening of the replicative life span observed for S. cerevisiaeΔsod1 cells.     

Altered expression of hypothetical proteins in hippocampus of transgenic mice overexpressing human Cu/Zn-superoxide dismutase 1

Background  

Cu/Zn-superoxide dismutase 1 (SOD1), encoded on chromosome 21, is a key enzyme in the metabolism of reactive oxygen species (ROS) and pathogenetically relevant for several disease states including Down syndrome (DS; trisomy 21). Systematically studying protein expression in human brain and animal models of DS we decided to carry out "protein hunting" for hypothetical proteins, i.e. proteins that have been predicted based upon nucleic sequences only, in a transgenic mouse model overexpressing human SOD1.