In yeast redistribution of Sod1 to the mitochondrial intermembrane space provides protection against respiration derived oxidative stress

The antioxidative enzyme copper-zinc superoxide dismutase (Sod1) is an important cellular defence system against reactive oxygen species (ROS). While the majority of this enzyme is localized to the cytosol, about 1% of the cellular Sod1 is present in the intermembrane space (IMS) of mitochondria. These amounts of mitochondrial Sod1 are increased for certain Sod1 mutants that are linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). To date, only little is known about the physiological function of mitochondrial Sod1. Here, we use the model system Saccharomyces cerevisiae to generate cells in which Sod1 is exclusively localized to the IMS. We find that IMS-localized Sod1 can functionally substitute wild type Sod1 and that it even exceeds the protective capacity of wild type Sod1 under conditions of mitochondrial ROS stress. Moreover, we demonstrate that upon expression in yeast cells the common ALS-linked mutant Sod1^G93A becomes enriched in the mitochondrial fraction and provides an increased protection of cells from mitochondrial oxidative stress. Such an effect cannot be observed for the catalytically inactive mutant Sod1^G85R. Our observations suggest that the targeting of Sod1 to the mitochondrial IMS provides an increased protection against respiration-derived ROS.     

Cu, Zn Superoxide Dismutase and NADP(H) Homeostasis Are Required for Tolerance of Endoplasmic Reticulum Stress in Saccharomyces cerevisiae

Genome-wide screening for sensitivity to chronic endoplasmic reticulum (ER) stress induced by dithiothreitol and tunicamycin (TM) identified mutants deleted for Cu, Zn superoxide dismutase (SOD) function (SOD1, CCS1) or affected in NADPH generation via the pentose phosphate pathway (TKL1, RPE1). TM-induced ER stress led to an increase in cellular superoxide accumulation and an increase in SOD1 expression and Sod1p activity. Prior adaptation of the hac1 mutant deficient in the unfolded protein response (UPR) to the superoxide-generating agent paraquat reduced cell death under ER stress. Overexpression of the ER oxidoreductase Ero1p known to generate hydrogen peroxide in vitro, did not lead to increased superoxide levels in cells subjected to ER stress. The mutants lacking SOD1, TKL1, or RPE1 exhibited decreased UPR induction under ER stress. Sensitivity of the sod1 mutant to ER stress and decreased UPR induction was partially rescued by overexpression of TKL1 encoding transketolase. These data indicate an important role for SOD and cellular NADP(H) in cell survival during ER stress, and it is proposed that accumulation of superoxide affects NADP(H) homeostasis, leading to reduced UPR induction during ER stress.     

Proline 146 is critical to the structure, function and targeting of sod2, the Na/H exchanger of Schizosaccharomyces pombe

Sod2 is the Na⁺/H⁺ exchanger of the fission yeast Schizosaccharomyces pombe that is principally responsible for salt tolerance. We examined the role of nine polar, membrane associated amino acids in the ability of the protein to confer salt tolerance in S. pombe. Wild type sod2 protein with a C-terminal GFP tag effectively rescued salt tolerance in S. pombe with deleted endogenous sod2. Sod2 protein with the mutations P163A, P183A, D298N, D389N, E390Q, E392Q and E397Q also conveyed salt tolerance as effectively as the wild type sod2 protein. In contrast, the mutation P146A resulted in a protein that did not convey salt tolerance nearly as effectively as the wild type and did not extrude Na⁺ as well as the wild type. Mutation of Pro¹⁴⁶ to Ser, Asp or Lys had an intermediate effect. Mutation of Thr¹⁴² to Ser resulted in a slightly defective protein. Western blot analysis showed that all mutant proteins were expressed at similar levels as wild type sod2 protein. Examination of the localization of the proteins showed that wild type and most sod2 mutants were present in the plasma membrane while the P146A mutant had an intracellular localization. Limited tryptic digestion suggested that the P146A sod2 protein had a change in conformation in comparison to the wild type protein. The results suggest that Pro¹⁴⁶ is an amino acid critical to sod2 structure, function and localization.     

Melatonin and steroid hormones activate intermembrane Cu,Zn-superoxide dismutase by means of mitochondrial cytochrome P450

Melatonin and steroid hormones are cytochrome P450 (CYP or P450; EC 1.14.14.1) substrates that have antioxidant properties and mitochondrial protective activities. The mitochondrial intermembrane space (IMS) Cu,Zn-superoxide dismutase (SOD1) is activated after oxidative modification of its critical thiol moieties by superoxide anion (O₂^??). This study was aimed at investigating the potential association between the hormonal protective antioxidant actions in mitochondria and the regulation of IMS SOD1 activity. Melatonin, testosterone, dihydrotestosterone, estradiol, and vitamin D induced a sustained activation over time of SOD1 in intact mitochondria, showing a bell-shaped enzyme activation dose response with a threshold at 50 nM and a maximum effect at 1 μM concentration. Enzyme activation was not affected by furafylline, but it was inhibited by omeprazole, ketoconazole, and tiron, thereby supporting the occurrence of a mitochondrial P450 activity and O₂^?? requirements. Mitochondrial P450-dependent activation of IMS SOD1 prevented O₂^??-induced loss of aconitase activity in intact mitochondria respiring in State 3. Optimal protection of aconitase activity was observed at 0.1 μM P450 substrate concentration, evidencing a likely oxidative effect on the mitochondrial matrix by higher substrate concentrations. Likewise, enzyme activation mediated by mitochondrial P450 activity delayed CaCl₂-induced loss of transmembrane potential and decreased cytochrome c release. Omeprazole and ketoconazole abrogated both protecting mitochondrial functions promoted by melatonin and steroid hormones.     

Adaptative response to enhanced basal oxidative damage in sod mutants from Saccharomyces cerevisiae

We investigated the adaptative response of S. cerevisiae in sod mutants (sod1Δ, sod2Δ and sod1Δsod2Δ) after H₂O₂ treatment in the stationary phase. sod2Δ and sod1Δsod2Δ demonstrated the highest levels of GSH in the control, suggesting that pathways which include GSH protect these double mutants against oxidative stress. In addition, sod1Δ and sod1Δsod2Δ had higher iron levels than the wild-type, independently of H₂O₂ stress. Fe levels were increased in sod2Δ following H₂O₂ In addition, the sod2Δ mutant was more sensitive to H₂O₂ treatment than the wild-type. These results suggest that sod2Δ sensibility may be associated with •OH production by the Fenton reaction. This increased iron demand in the sod2Δ mutant may be a reflection of the cells’ efforts to reconstitute proteins that are inactivated in conditions of excess superoxide. MDA levels were assayed by HPLC in these mutants. The highest MDA levels could be observed after 10mM H₂O₂ treatment in the sod1Δsod2Δ double mutant. After treatment with a GSH inhibitor, the MDA level was still higher in the same strain. Thus, both direct and indirect GSH pathways are involved in the protection of lipid membranes and proteins in these mutants and may constitute an adaptative response to enhanced basal oxidative damage produced by superoxide.     

Endogenous Superoxide Dismutase Levels Regulate Iron-Dependent Hydroxyl Radical Formation in Escherichia coli Exposed to Hydrogen Peroxide

Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O₂^·− and H₂O₂. Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H₂O₂ but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical (^·OH) as a consequence of the reaction of H₂O₂ with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H₂O₂ in the presence of an α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)–ethanol spin-trapping system, the 4-POBN–^·CH(CH₃)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating ^·OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H₂O₂ in a similar manner, the magnitude of ^·OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of ^·OH spin trapped. Exogenous SOD failed to inhibit ^·OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in ^·OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H₂O₂-mediated killing seen in these mutants lacking SOD.     

The scavenging of superoxide radical by manganous complexes: in vitro

Dialyzable manganese has been shown to be present in millimolar concentrations within cells of Lactobacillus plantarum and related lactic acid bacteria. This unusual accumulation of Mn appears to serve the same function as Superoxide dismutase (SOD), conferring hyperbaric oxygen and Superoxide tolerance on these SOD-free organisms. The form of the Mn in the lactic acid bacteria and the mechanisms whereby it protects the cell from oxygen damage are unknown. This report examines the mechanisms by which Mn catalytically scavenges O₂^?, both in the xanthine oxidase/cytochrome c SOD assay and in a number of in vitro systems relevant to the in vivo situation. In all the reaction mixtures examined, Mn(II) is first oxidized by O₂^? to Mn(III), and H₂O₂ is formed. In pyrophosphate buffer the Mn(III) thus formed is re-reduced to Mn(II) by a second O₂^?, making the reaction a true metal-catalyzed dismutation like that catalyzed by SOD. Alternatively, if the reaction takes place in orthophosphate or a number of other buffers, the Mn(III) is preferentially reduced largely by reductants other than O₂^?, such as thiols, urate, hydroquinone, or H₂O₂. H₂O₂, a common product of the lactic acid bacteria, reacted rapidly with Mn(III) to form O₂, apparently without intermediate O₂ release. Free hexaquo Mn(II) ions were shown by electron spin resonance spectroscopy and activity assays in noncomplexing buffers to be poorly reactive with O₂^?. In contrast, Mn(II) formed complexes having a high catalytic activity in scavenging O₂^? with a number of organic acids, including malate, pyruvate, propionate, succinate, and lactate, with the Mn-lactate complex showing the greatest activity.     

The Effects of Exogenous Ascorbic Acid on the Mechanism of Physiological and Biochemical Responses to Nitrate Uptake in Two Rice Cultivars (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) Under Aluminum Stress

As a major antioxidant in plants, ascorbic acid (AsA) plays a very important role in the response to aluminum (Al) stress. However, the effect of AsA on the mitigation of Al toxicity and the mechanism of nitrate nitrogen (NO₃ ^?–N) uptake by plants under Al stress are unclear. In this study, a hydroponic experiment was conducted using peak 1 A rice (sterile line, Indica) with weaker resistance to Al and peak 1 superior 5 rice (F1 hybrid, Indica) with stronger resistance to Al to study the effects of exogenous AsA on the physiological and biochemical responses to NO₃ ^?–N uptake by rice roots exposed to 50 μmol L^?1 Al. Al stress induced increases in the concentrations of H₂O₂ and malondialdehyde (MDA) and in the activities of antioxidant enzymes [such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)]. Plasma membrane (PM) H⁺-ATPase and H⁺-pump activities, endogenous AsA content and NO₃ ^?–N uptake in rice roots decreased under Al stress. After treatment with 2 mmol L^?1 exogenous AsA combined with Al, concentrations of H₂O₂ and MDA in roots notably decreased, and endogenous AsA content and activities of SOD, POD, CAT, and APX in rice roots increased significantly; furthermore, the interaction of PM H⁺-ATPase and the 14-3-3 protein was also enhanced significantly compared with that in control plants without AsA treatment, which clearly increased NO₃ ^?–N uptake. Based on all of these data, the application of AsA significantly reduced the accumulation of H₂O₂ and MDA and increased the activities of PM H⁺-ATPase and the H⁺-pump by increasing the endogenous AsA content, the antioxidant enzyme activities, and the interaction of PM H⁺-ATPase and the 14-3-3 protein in the roots of the two rice cultivars under Al stress, thereby improving the uptake of NO₃ ^?–N in rice.     

Superoxide anion and hydrogen peroxide production by chemically elicited peritoneal macrophages--induction by multiple nonphagocytic stimuli

The ability of a number of stimulants to activate an oxidative burst (OB) in oil-elicited guinea pig peritoneal exudate macrophages (MPs) was examined. The parameters of the OB were the generation and extracellular release of Superoxide anions (O₂^?) and hydrogen peroxide (H₂O₂). We found that: (1) The cocarcinogen and skin irritant phorbol myristate acetate (PMA) was the most potent OB activator—The weak cocarcinogen 4-O-methyl PMA was a proportionally less effective OB activator; (2) The lectins concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean, Lotus, and pokeweed lectins, were also quite effective OB activators—The ability of Con A to stimulate O₂^? production was abolished by succinylation and could be prevented by the presence of α-methyl-D-mannoside; (3) Other stimulators of an OB in MPs were: N-formyl-methionyl peptides, opsonized zymosan, the Ca²⁺ ionophore A23187, phospholipase C, NaF, antimacrophage antibody, microtubule-disrupting drugs, and sodium nitroprusside—O₂^? generation induced by A23187 (but not that stimulated by PMA) was dependent on extracellular Ca²⁺; (4) The amount of O₂^? produced per cell was higher at low cell densities; (5) The addition of Superoxide dismutase (SOD) to the medium totally prevented the detection of O₂^? and augmented twice the amount of H₂O₂ recovered; (6) Pretreatment of MPs with the SOD inhibitor sodium diethyldithiocarbamate had no effect on the release of O₂^? but blocked H₂O₂ release in a dose-dependent manner. These data were interpreted as indicating that the bulk of H₂O₂ was derived by enzymatic dismutation of O₂^?; (7) The common mechanism by which such a variety of stimuli provoke an OB in MPs was not elucidated. No evidence was found to suggest a role for a cyclic nucleotide messenger.     

The system modulating ROS content in germinating seeds of two Brazilian savanna tree species exposed to As and Zn

The effects of increasing arsenic (0, 10, 50, 100 mg L^?1) and zinc (0, 50, 80, 120, 200 mg L^?1) doses on germination and oxidative stress markers (H₂O₂, MDA, SOD, CAT, APX, and GR) were examined in two Brazilian savanna tree species (Anadenanthera peregrina and Myracrodruon urundeuva) commonly used to remediate contaminated soils. The deleterious effects of As and Zn on seed germination were due to As- and Zn-induced H₂O₂ accumulation and inhibition of APX and GR activities, which lead to oxidative damage by lipid peroxidation. SOD and CAT did not show any As- and Zn-induced inhibition of their activities as was seen with APX and GR. We investigated the close relationships between seed germination success under As and Zn stress in terms of GR and, especially, APX activities. Increased germination of A. peregrina seeds exposed to 50 mg L^?1 of Zn was related to increased APX activity, and germination in the presence of As (10 mg L^?1) was observed only in M. urundeuva seeds that demonstrated increased APX activity. All the treatments for both species in which germination decreased or was inhibited showed decreases in APX activity. A. peregrina seeds showed higher Zn-tolerance than M. urundeuva, while the reverse was observed with arsenic up to exposures of 10 mg L^?1.     

Nitrogen fixation (acetylene reduction) inAquaspirillum magnetotacticum

Aquaspirillum magnetotacticum strain MS-1 and two nonmagnetic mutants derived from it reduced C₂H₂ microaerobically but not anaerobically even with NO₃ ^?. This organism apparently is not capable of NO₃ ^?-dependent nitrogen fixation. Cells ofA. magnetotacticum reduced C₂H₂ at rates comparable to those ofAzospirillum lipoferum grown under similar conditions, but much lower than that ofAzotobacter vinelandii grown aerobically. Cells ofA. magnetotacticum in anaerobic cultures lacking NO₃ ^? did not reduce C₂H₂ until O₂ was introduced. Optimum rates of C₂H₂ reduction byA. magnetotacticum were obtained at 200 Pa O₂. C₂H₂ reduction was inhibited by more than 1 kPa O₂ or 0.2 mM NO₃ ^? or NH₄ ⁺. These results suggest thatA. magnetotacticum fixes N₂ only under microaerobic, N-limited conditions.     

Plasmolysis effects and osmotic potential of two phylogenetically distinct alpine strains of Klebsormidium (Streptophyta)

The osmotic potential and effects of plasmolysis were investigated in two different Klebsormidium strains from alpine habitats by incubation in 300–2,000 (3,000) mM sorbitol. Several members of this genus were previously found to tolerate desiccation in the vegetative state yet information was lacking on the osmotic potentials of these algae. The strains were morphologically determined as Klebsormidium crenulatum and Klebsormidium nitens. These species belong to distinct clades, as verified by phylogenetic analysis of the rbcL gene. K. crenulatum is part of to the K. crenulatum/mucosum (‘F’ clade) and K. nitens of the ‘E2’ clade. Plasmolysis occurred in K. crenulatum at 800 mM sorbitol (961 mOsmol kg^?1, Ψ?=??2.09 MPa) and in K. nitens at 600 mM sorbitol (720 mOsmol kg^?1, Ψ?=??1.67 MPa). These are extraordinarily high osmotic values (very negative osmotic potentials) compared with values reported for other green algae. In K. crenulatum, the maximum photosynthetic rate (Pmax) in the light-saturated range was 116 μmol O₂ h^?1 mg^?1 chl a. Incubation in 1,000 mM sorbitol decreased Pmax to 74.1% of the initial value, whereas 2,000 mM sorbitol (Ψ?=??5.87 MPa) lead to an almost complete loss of oxygen production. In K. nitens, Pmax was 91 μmol O₂ h^?1 mg^?1 chl a under control conditions and incubation in 800 mM sorbitol did not decrease Pmax, 2,000 mM sorbitol decreased Pmax only to about 62.6% of the initial value whereas 3,000 mM sorbitol stopped oxygen evolution. This indicated a broader amplitude for photosynthesis in the examined strain of K. nitens. Control samples and samples plasmolysed for 3 h in 800 mM sorbitol (K. nitens), 1,000 mM sorbitol (K. crenulatum), or 2,000 mM sorbitol were investigated by transmission electron microscopy after chemical or high-pressure freeze fixation. In cells undergoing plasmolysis the protoplasts were retracted from the cell wall, the cytoplasm appeared dense, vacuoles were small and fragmented, and the cytoplasm was filled with ribosomes. Thin cytoplasmic strands were connected to the cell wall; 2,000 mM sorbitol increased the effect. The content of soluble carbohydrates in these two strains was investigated by HPLC, as this is one known mechanism for cells to maintain high osmotic pressure of the cytosol. Both Klebsormidium species contained diverse soluble carbohydrates, including a dominant mixed peak of unidentified oligosaccharides, and more minor amounts of raffinose, sucrose, glucose, xylose, galactose, mannose, inositol, fructose, glycerol, mannitol, and sorbitol. The total content of soluble carbohydrates was approximately 1.2% of the dry weight, indicating that this is not a major factor contributing to the high osmotic potential in these strains of Klebsormidium.     

Responses of the antioxidant defense system to drought stress in the leaves of Fargesia denudata seedlings,the staple food of the giant panda

The responses of the antioxidant defense system in plant species to drought stress are still relatively unknown. In order to further understand how the system responds to drought stress, the leaves of Fargesia denudata seedlings were investigated. Antioxidant enzyme activities, antioxidant contents, hydrogen peroxide (H₂O₂), superoxide anion (O ₂ ^·? ) and MDA contents in the seedling leaves were measured under well-watered (WW), moderate drought-stressed (MD), and severe drought-stressed (SD) treatments. Although drought stress significantly increased H₂O₂ and O ₂ ^·? levels in F. denudata leaves, only weak lipid peroxidation was observed. This is attributed to the higher superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR) activities in F. denudata leaves during the entire drought period. Reduced and oxidized ascorbate (AsA and DHA) contents were almost not affected by drought except that DHA under SD showed an obvious increase on day 30. Furthermore, reduced glutathione (GSH) content under drought stress significantly decreased, while oxidized glutathione (GSSG) markedly increased under SD on days 30 and 45 as well as under MD on day 30; as a result, the ratio GSH/GSSG declined considerably. These results indicated that GSH was involved in scavenging H₂O₂ and O ₂ ^·? under drought stress and it was more sensitive to drought stress in scavenging H₂O₂ and O ₂ ^·? than AsA. As a result, a highly efficient antioxidant defense system in drought-stressed F. denudate leaves operated mainly through the synergistic functioning of SOD, CAT, APX, MDHAR, DHAR, GR, and GSH against oxidative damage.