Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation

Red blood cells (RBC) are thought to be well protected against oxidative stress by the antioxidant, cu-pro-zinc enzyme superoxide dismutase (CuZn SOD) which dismutates O2- to H2O2. CuZn SOD, however, is irreversibly inactivated by its product H2O2. Exposure of intact RBC to H2O2 resulted in the inactivation (up to 50%) of endogenous SOD in a concentration-dependent manner. When RBC were exposed to O2- and H2O2, generated by xanthine + xanthine oxidase, an even greater loss of SOD activity (approximately 75%) was observed. Intracellular proteolysis was markedly increased by exposure to these same oxidants; up to a 12-fold increase with H2O2 and a 50-fold increase with xanthine oxidase plus xanthine. When purified SOD was treated with H2O2, inactivation of the enzyme also occurred in a concentration-dependent manner. Accompanying the loss of SOD activity, the binding of the copper ligand to the active site of the enzyme diminished with H2O2 exposure, as evidenced by an increase in accessible copper. Significant direct fragmentation of SOD was evident only under conditions of prolonged exposure (20 h) to relatively high concentrations of H2O2. Gel electrophoresis studies indicated that under most experimental conditions (i.e. 1-h incubation) H2O2, O2-, and H2O2 + O2- treated SOD experienced charge changes and partial denaturation, rather than fragmentation. The proteolytic susceptibility of H2O2-modified SOD, during subsequent incubation with (rabbit, bovine or human) red cell extracts also increased as a function of pretreatment with H2O2. Both enzyme inactivation and altered copper binding appeared to precede the increase in proteolytic susceptibility (whether measured as an effect of H2O2 concentration or as a function of the duration of H2O2 exposure). These results suggest that SOD inactivation and modification of copper binding are prerequisites for increased protein degradation. Proteolytic susceptibility was further enhanced by H2O2 exposure under alkaline conditions, suggesting that the hydroperoxide anion is the damaging species rather than H2O2 itself. In RBC extracts, the proteolysis of H2O2-modified SOD was inhibited by sulfhydryl reagents, serine reagents, transition metal chelators, and ATP; suggesting the existence of an ATP-independent proteolytic pathway of sulfhydryl, serine, and metalloproteases, and peptidases. The proteolytic activity was conserved in a "Fraction II" of both human and rabbit RBC, and was purified from rabbit reticulocytes and erythrocytes to a 670-kDa proteinase complex, for which we have suggested the trivial name macroxyproteinase. In erythrocytes macroxyproteinase may prevent the accumulation of H2O2-modified SOD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Superoxide dismutase 1 protects retinal cells from oxidative damage

Bolstering the endogenous oxidative damage defense system is a good strategy for development of treatments to combat neurodegenerative diseases in which oxidative damage plays a role. A first step in such treatment development is to determine the role of various components of the defense system in cells that degenerate. In this study, we sought to determine the role of superoxide dismutase 1 (SOD1) in two models of oxidative damage-induced retinal degeneration. In one model, paraquat is injected into the vitreous cavity and then enters retinal cells and generates reactive oxygen species (ROS) that cause progressive retinal damage. Assessment of retinal function with serial electroretinograms (ERGs) showed that sod1 -/- mice were much more sensitive than sod1 +/+ mice to the damaging effects of paraquat, while sod1 +/- mice showed intermediate sensitivity. Compared to sod1 +/+ mice, sod1 -/- mice showed greater paraquat-induced oxidative damage and apoptosis. In the second model, mice were exposed to hyperoxia for several weeks, and sod1 -/- mice showed significantly greater reductions in ERG amplitudes than sod1 +/+ mice. In both of these models, transgenic mice carrying a sod1 transgene driven by a beta-actin promoter showed less oxidative stress-induced reduction in ERG amplitudes. These data demonstrate that SOD1 protects retinal cells against paraquat- and hyperoxia-induced oxidative damage and suggest that overexpression of SOD1 should be considered as one component of ocular gene therapy to prevent oxidative damage-induced retinal degeneration.     

Oxidative modification of neurofilament-L by the Cu,Zn-superoxide dismutase and hydrogen peroxide system

Neurofilament-L (NF-L) is a major element of neuronal cytoskeletons and known to be important for their survival in vivo. Since oxidative stress might play a critical role in the pathogenesis of neurodegenerative diseases, we investigated the role of Cu,Zn-superoxide dismutase (SOD) in the modification of NF-L. When disassembled NF-L was incubated with Cu,Zn-SOD and H2O2, the aggregation of protein was proportional to the concentration of hydrogen peroxide. Cu,Zn-SOD/H2O2-mediated modification of NF-L was significantly inhibited by radical scavenger, spin trap agents and copper chelators. Dityrosine crosslink formation was obtained in Cu,Zn-SOD/H2O2-mediated NF-L aggregates. Antioxidant molecules, carnosine and anserine significantly inhibited the aggregation of NF-L and the formation of dityrosine. This study suggests that copper-mediated NF-L modification may be closely related to oxidative reactions which play a critical role in neurodegenerative diseases.     

OGG1 is degraded by calpain following oxidative stress and cisplatin exposure

Deficient repair activity for 8-hydroxy-2'-deoxyguanine (8-oxoguanine), a premutagenic oxidative DNA damage, has been observed in affected tissues in neurodegenerative diseases of aging, such as Alzheimer's disease, and in ischemia/reperfusion injury, type 2 diabetes mellitus, and cancer. These conditions have in common the accumulation of oxidative DNA damage, which is believed to play a role in disease progression, and loss of intracellular calcium regulation. These observations suggest that oxidative DNA damage repair capacity may be influenced by fluctuations in cellular calcium. We have identified human 8-oxoguanine-DNA glycosylase 1 (OGG1), the major 8-oxoguanine repair activity, as a specific target of the Ca(2+)-dependent protease Calpain I. Protein sequencing of a truncated partially calpain-digested OGG1 revealed that calpain recognizes OGG1 for degradation at a putative PEST (proline, glutamic acid, serine, threonine) sequence in the C-terminus of the enzyme. Co-immunoprecipitation experiments showed that OGG1 and Calpain I are associated in human cells. Exposure of HeLa cells to hydrogen peroxide or cisplatin resulted in the degradation of OGG1. Pretreatment of cells with the calpain inhibitor calpeptin resulted in inhibition of OGG1 proteolysis and suggests that OGG1 is a target for calpain-mediated degradation in vivo during oxidative stress- and cisplatin-induced apoptosis. Polymorphic OGG1 S326C was comparatively resistant to calpain digestion in vitro, yet was also degraded by a calpain-dependent pathway in vivo following DNA damaging agent exposure. The degradation of OGG1 by calpain may contribute to decreased 8-oxoguanine repair activity and elevated levels of 8-oxoguanine reported in tissues undergoing chronic oxidative stress, ischemia/reperfusion, and other cellular stressors known to produce perturbations of intracellular calcium homeostasis which activate calpain.     

Petroselinum sativum protects HepG2 cells from cytotoxicity and oxidative stress induced by hydrogen peroxide

Molecular Biology Reports - A number of liver diseases are known to be caused by oxidative stress. Petroselinum sativum (P. sativum; parsley) is popular for its anti-inflammatory, antimicrobial,...     

Superoxide, hydrogen peroxide, and singlet oxygen in lipid peroxidation by a xanthine oxidase system.

1. Xanthine oxidase acting aerobically upon acetaldehyde was found to cause the peroxidation of linolenate. This was demonstrated by increased absorbance at 233 nm due to diene conjugation and by the detection of a lipid peroxide spot on the thin layer chromatograms. 2. Superoxide dismutase inhibited this lipid peroxidation, as did catalase, thus indicating that both O2- and H2O2 were essential intermediates. Scavengers of singlet oxygen also inhibited the peroxidation of linolenate, whereas scavengers of hydroxyl radical did not. These effects, which were observed in the absence of iron salts, led to the proposal that O2- and H2O2 can directly give rise to a singlet oxygen, as follows: O2- + H2O2 leads to OH- + OH. + O2. 3. This proposal was further supported through the use of 2,5-dimethylfuran, as an indicating scavenger of singlet oxygen. Thus, when this compound was exposed to a known source of singlet oxygen, it gave a product which was detectable by thin layer chromatography. This product was also observed when 2,5-dimethylfuran was exposed to the xanthine oxidase system, in which case its accumulation was prevented by superoxide dismutase or by catalase, but not by scavengers of hydroxyl radical.     

The permeability of human red blood cell membranes to hydrogen peroxide is independent of aquaporins

Hydrogen peroxide (H₂O₂) not only is an oxidant but also is an important signaling molecule in vascular biology, mediating several physiological functions. Red blood cells (RBCs) have been proposed to be the primary sink of H₂O₂ in the vasculature because they are the main cellular component of blood with a robust antioxidant defense and a high membrane permeability. However, the exact permeability of human RBC to H₂O₂ is neither known nor is it known if the mechanism of permeation involves the lipid fraction or protein channels. To gain insight into the permeability process, we measured the partition constant of H₂O₂ between water and octanol or hexadecane using a novel double-partition method. Our results indicated that there is a large thermodynamic barrier to H₂O₂ permeation. The permeability coefficient of H₂O₂ through phospholipid membranes containing cholesterol with saturated or unsaturated acyl chains was determined to be 4 × 10⁻⁴ and 5 × 10⁻³ cm s⁻¹, respectively, at 37 °C. The permeability coefficient of human RBC membranes to H₂O₂ at 37 °C, on the other hand, was 1.6 × 10⁻³ cm s⁻¹. Different aquaporin-1 and aquaporin-3 inhibitors proved to have no effect on the permeation of H₂O₂. Moreover, human RBCs devoid of either aquaporin-1 or aquaporin-3 were equally permeable to H₂O₂ as normal human RBCs. Therefore, these results indicate that H₂O₂ does not diffuse into RBCs through aquaporins but rather through the lipid fraction or a still unidentified membrane protein.     

Aggregation of alpha-synuclein induced by the Cu,Zn-superoxide dismutase and hydrogen peroxide system

Alpha-synuclein is a major component of the abnormal protein aggregation in Lewy bodies of Parkinson's disease (PD) and senile plaques of Alzheimer's disease (AD). Previous studies have shown that the aggregation of alpha-synuclein was induced by copper (II) and H(2)O(2) system. Since copper ions could be released from oxidatively damaged Cu,Zn-superoxide dismutase (SOD), we investigated the role of Cu,Zn-SOD in the aggregation of alpha-synuclein. When alpha-synuclein was incubated with both Cu,Zn-SOD and H(2)O(2), alpha-synuclein was induced to be aggregated. This process was inhibited by radical scavengers and spin trapping agents such as 5,5'-dimethyl 1-pyrolline N-oxide and tert-butyl-alpha-phenylnitrone. Copper chelators, diethyldithiocarbamate and penicillamine, also inhibited the Cu,Zn-SOD/H(2)O(2) system-induced alpha-synuclein aggregation. These results suggest that the aggregation of alpha-synuclein is mediated by the Cu,Zn-SOD/H(2)O(2) system via the generation of hydroxyl radical by the free radical-generating function of the enzyme. The Cu,Zn-SOD/H(2)O(2)-induced alpha-synuclein aggregates displayed strong thioflavin-S reactivity, reminiscent of amyloid. These results suggest that the Cu,Zn-SOD/H(2)O(2) system might be related to abnormal aggregation of alpha-synuclein, which may be involved in the pathogenesis of PD and related disorders.     

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.     

Microglia proliferation is regulated by hydrogen peroxide from NADPH oxidase

Microglia are resident brain macrophages that become activated and proliferate following brain damage or stimulation by immune mediators, such as IL-1beta or TNF-alpha. We investigated the mechanisms by which microglial proliferation is regulated in primary cultures of rat glia. We found that basal proliferation of microglia was stimulated by proinflammatory cytokines IL-1beta or TNF-alpha, and this proliferation was completely inhibited by catalase, implicating hydrogen peroxide as a mediator of proliferation. In addition, inhibitors of NADPH oxidase (diphenylene iodonium or apocynin) also prevented microglia proliferation, suggesting that this may be the source of hydrogen peroxide. IL-1beta and TNF-alpha rapidly stimulated the rate of hydrogen peroxide produced by isolated microglia, and this was inhibited by diphenylene iodonium, implying that the cytokines were acting directly on microglia to stimulate the NADPH oxidase. Low concentrations of PMA or arachidonic acid (known activators of NADPH oxidase) or xanthine/xanthine oxidase or glucose oxidase (generating hydrogen peroxide) also increased microglia proliferation and this was blocked by catalase, showing that NADPH oxidase activation or hydrogen peroxide was sufficient to stimulate microglia proliferation. In contrast to microglia, the proliferation of astrocytes was unaffected by the presence of catalase. In conclusion, these findings indicate that microglial proliferation in response to IL-1beta or TNF-alpha is mediated by hydrogen peroxide from NADPH oxidase.     

Proteins damaged by oxygen radicals are rapidly degraded in extracts of red blood cells

We have suggested that red blood cell proteolytic systems can degrade oxidatively damaged proteins, and that both damage and degradation are independent of lipid peroxidation (Davies, K. J. A., and Goldberg, A. L. (1987) J. Biol. Chem. 262, 8220-8226. These ideas have now been tested in cell-free extracts of rabbit erythrocytes and reticulocytes. Exposure to oxygen radicals or H2O2 increases the degradation of endogenous proteins in cell-free extracts, as in intact cells. Various radical-generating systems (acetaldehyde or xanthine + xanthine oxidase, ascorbic acid + iron, H2O2 + iron) and H2O2 alone enhanced the rates of proteolysis severalfold. Since these extracts were free of membrane lipids, protein damage and degradation must be independent of lipid peroxidation. An antioxidant buffer consisting of HEPES, glycerol, and dithiothreitol inhibited the increased proteolysis by 60-100%. Mannitol caused a 50-80% reduction in proteolysis suggesting that the hydroxyl radical (.OH), or a species with similar reactivity, may be the initiator of protein damage. When casein or bovine serum albumin were exposed to .OH (generated by H2O2 + Fe2+, or COCo radiation) these proteins were degraded up to 50 times faster than untreated proteins during subsequent incubations with red cell extracts. Mannitol inhibited this increase in proteolysis only if present during .OH exposure; mannitol did not affect the degradative system. Although ATP increased the degradation of untreated proteins 4- to 6-fold in reticulocyte extracts, it had little or no effect on the degradation of proteins exposed to .OH. ATP also did not stimulate hydrolysis of .OH-treated proteins in erythrocyte extracts. Leupeptin did not affect the degradative processes in either extract; thus lysosomal or Ca2+-activated thiol proteases were not involved. We propose that red cells contain a soluble, ATP-independent proteolytic pathway which may protect against the accumulation of proteins damaged by .OH or other active oxygen species.     

Enhanced oligomerization of the alpha-synuclein mutant by the Cu,Zn-superoxide dismutase and hydrogen peroxide system

The alpha-synuclein is a major component of Lewy bodies that are found in the brains of patients with Parkinson's disease (PD). Also, two point mutations in this protein, A53T and A30P, are associated with rare familial forms of the disease. We investigated whether there are differences in the Cu,Zn-SOD and hydrogen peroxide system mediated-protein modification between the wild-type and mutant alpha-synucleins. When alpha-synuclein was incubated with both Cu,Zn-SOD and H2O2, then the amount of A53T mutant oligomerization increased relative to that of the wild-type protein. This process was inhibited by radical scavenger, spin-trapping agent, and copper chelator. These results suggest that the oligomerization of alpha-synuclein is mediated by the generation of the hydroxyl radical through the metal-catalyzed reaction. The dityrosine formation of the A53T mutant protein was enhanced relative to that of the wild-type protein. Antioxidant molecules, carnosine, and anserine effectively inhibited the wild-type and mutant proteins' oligomerization. Therefore, these compounds may be explored as potential therapeutic agents for PD patients. The present experiments, in part, may provide an explanation for the association between PD and the alpha-synuclein mutant.     

Mechanism of oxidative damage to fish red blood cells by ozone

The present study was conducted to elucidate the adverse effects of ozone exposure on rainbow trout (Oncorhynchus mykiss) red blood cells (RBCs). We evaluated whether hemoglobin (Hb) or Hb-derived free iron could participate in the RBC damage using an in vitro ozone exposure system. Ozone exposure induced hemolysis, formation of methemoglobin, and RBC membrane lipid peroxidation. This RBC damage was not suppressed by the addition of a specific iron chelator (deferoxamine mesilate) to the medium but was suppressed by carbon monoxide (CO) treatment before ozone exposure. Generation of hydrogen peroxide (H2O2) in RBC was observed upon ozone exposure but was significantly suppressed by CO treatment before ozone exposure. Thus the Hb status (i.e., Hb redox condition) and H2O2 generation in RBC should play important roles in mediating RBC damage by ozone exposure. In other words, neither ozone nor its derivative directly attacked from the outside of the cell, but ozone that penetrated through the membrane derived the reactive oxygen species from Hb inside of the cell.     

Action of 1,1-dimethylhydrazine on bacterial cells is determined by hydrogen peroxide

Seven different recombinant bioluminescent strains of Escherichia coli containing, respectively, the promoters katG and soxS (responsive to oxidative damage), recA (DNA damage), fabA (membrane damage), grpE, and rpoE (protein damage) and lac (constitutive expression) fused to the bacterial operon from Photorhabdus luminescens, were used to describe the mechanism of toxicity of 1,1-dimethylhydrazine (1,1-DMH) on bacteria, as well as to determine whether bacteria can sensitively detect the presence of this compound. A clear response to 1,1-DMH was observed only in E. coli carrying the katG'::lux, soxS'::lux, and recA'::lux-containing constructs. Preliminary treatment with catalase of the medium containing 1,1-DMH completely diminished the stress-response of the P(katG), P(recA), and P(soxS) promoters. In the strain E. coli (pXen7), which contains a constitutive promoter, the level of cellular toxicity caused by the addition of 1,1-DMH was dramatically reduced in the presence of catalase. It is suggested that the action of 1,1-DMH on bacterial cells is determined by hydrogen peroxide, which is formed in response to reduction of the air oxygen level.     

Action of 1,1-dimethylhydrazine on bacterial cells is determined by hydrogen peroxide

Seven different recombinant bioluminescent strains of Escherichia coli containing, respectively, the promoters katG and soxS (responsive to oxidative damage), recA (DNA damage), fabA (membrane damage), grpE, and rpoE (protein damage) and lac (constitutive expression) fused to the bacterial operon from Photorhabdus luminescens, were used to describe the mechanism of toxicity of 1,1-dimethylhydrazine (1,1-DMH) on bacteria, as well as to determine whether bacteria can sensitively detect the presence of this compound. A clear response to 1,1-DMH was observed only in E. coli carrying the katG’::lux, soxS’::lux, and recA’::lux-containing constructs. Preliminary treatment with catalase of the medium containing 1,1-DMH completely diminished the stress-response of the P_katG, P_recA, and P_soxS promoters. In the strain E. coli (pXen7), which contains a constitutive promoter, the level of cellular toxicity caused by the addition of 1,1-DMH was dramatically reduced in the presence of catalase.It is suggested that the action of 1,1-DMH on bacterial cells is determined by hydrogen peroxide, which is formed in response to reduction of the air oxygen level.     

Copper, zinc superoxide dismutase plus hydrogen peroxide: a catalytic system for human lipoprotein oxidation

We report for the first time that bovine or human CuZnSOD plus H2O2 can catalyze human lipoprotein oxidation, inducing like free copper ions a typical oxidative kinetics with lag and propagation phases. Free copper released from CuZnSOD by H2O2, but not enzyme peroxidase activity and carbonate radical anion, is responsible for lipoprotein oxidation, which is indeed totally inhibited by copper chelators and BHT but unaffected by bicarbonate. Moreover, lipoprotein oxidation is significantly counteracted by the OH* scavengers formate and azide, which can enter the active site of CuZnSOD and decrease copper release through scavenging of copper-bound OH*; benzoate and ethanol, which cannot enter, are instead ineffective, indicating no oxidative involvement of free OH* escaped from the enzyme active site. The possibility of CuZnSOD/H2O2-catalyzed lipoprotein oxidation in vivo is discussed.     

Superoxide dismutase is a virulence factor produced by the coral bleaching pathogen Vibrio shiloi

Coral bleaching is a disease that threatens coral reefs throughout the world. The disease is correlated with higher-than-normal seawater temperatures. Data have been reported showing that bleaching of the coral Oculina patagonica during the summer in the Mediterranean Sea is the result of an infection with Vibrio shiloi. The summer temperatures induce the expression of virulence factors in the pathogen. We report here that V. shiloi produces an extracellular superoxide dismutase (SOD) at 30 degrees C, but not at 16 degrees C. An SOD(-) mutant was avirulent. The mutant adhered to corals, penetrated into coral cells, multiplied intracellularly for a short time, and then died. These data support the hypothesis that SOD protects the intracellular V. shiloi from oxidative stress caused by the high concentration of oxygen produced by intracellular zooxanthellae photosynthesis.