The measurement of protein degradation in response to oxidative stress.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
T Grune
Publications and source records attributed to T Grune.
Erythrocyte free radical and energy metabolism.
The erythrocyte is a highly specialized cell whose main functions are oxygen transport and the mediation of carbon dioxide transport. Energy production in the mature erythrocyte depends on glycolysis, with glucose as the principal substrate. Glycolysis and the oxidative pentose phosphate pathway generate NADH and NADPH to reduce methemoglobin, which is being continuously produced, and the antioxidant glutathione, which is present in high concentrations. Red blood cells are equipped with a highly effective antioxidant defense even without the glutathione system. Compared with other cell types, they possess high activities of the most important antioxidant enzymes. Most of the nonenzymatic antioxidant capacity of whole blood is likewise localized in the erythrocytes. Circulating red cells are mobile free radical scavengers and provide antioxidant protection to other tissues and organs. An imbalance between pro-oxidant reactions and antioxidant defense is described in patients with chronic renal failure. Oxidative stress increases as antioxidant defenses are weakened by pro-oxidant hemodialysis factors; it increases further still in renal anemia with a very low red cell count. Thus in terms of free radical metabolism, the only arguments remaining over the complete correction of renal anemia are those in favor, with none against.
Adenosine Triphosphate↗
Oxidative stress in anemia.
The increased formation of reactive oxygen species under hypoxic conditions often appears paradoxical. A prooxidant shift results from changes in cellular metabolism (especially energy metabolism), higher flux rates in catecholamine metabolism and permanent leukocyte activation. These mechanisms of increased free radical production then find themselves opposed by an antioxidant system that is markedly weakened by anemia. The erythrocytes represent an important component of the antioxidant capacity of blood, comprising in particular intracellular enzymes, e.g. superoxide dismutase and catalase, but also the glutathione system. It is thus possible that some complications of uremia are at least partly due to oxidative stress. These include cardiovascular complications, premature biological aging and increased susceptibility to infection. Strategies to strengthen the complex endogenous free radical defenses can thus be predicted to show long-term benefit. In this context the expansion of EPO therapy may well be a major step in stabilizing free radical metabolism in anemic patients.
Anemia↗
Does treatment of renal anemia with recombinant erythropoietin influence oxidative stress in hemodialysis patients?
Patients with chronic renal failure (CRF) undergoing hemodialysis (HD) are exposed to constant oxidative stress, as shown by elevated malondialdehyde (MDA) plasma concentrations in HD patients. The aim of our study was to investigate the role of renal anemia in oxidative stress. To this end, MDA and 4-hydroxynonenal (HNE) were measured in three groups of patients. Group I comprised 8 patients with hemoglobin (Hb) < 10 g/dl (mean Hb 8.1 +/- 1.3 g/dl) and group II 8 patients with Hb > 10 g/dl (mean Hb 12.4 +/- 1.9 g/dl). None of these 16 patients had been previously treated with recombinant erythropoietin (rhEPO). Group III comprised 27 patients with mean Hb 10.5 +/- 1.6 g/dl after long-term treatment with rhEPO. The plasma concentrations of both MDA and HNE in all 43 HD patients were significantly higher (p < 0.0001) than in 20 healthy controls (MDA 2.85 +/- 0.25 vs 0.37 +/- 0.03 microM, HNE 0.32 +/- 0.03 versus 0.10 +/- 0.01 microM). Comparison between the three groups showed that the HD patients with Hb < 10 g/dl had significantly higher plasma concentrations of lipid peroxidation products (MDA 3.81 +/- 0.86 microM, HNE 0.45 +/- 0.07 microM) than either HD patients with Hb > 10 g/dl (MDA 2.77 +/- 0.58 microM, HNE 0.25 +/- 0.05 microM) or HD patients treated with rhEPO (MDA 2.50 +/- 0.12 microM, HNE 0.29 micro 0.03 microM). An inverse correlation was also demonstrated between plasma HNE and Hb (r= 0.62, p < 0.0001). It follows that a substantial part of the oxidative stress is due to renal anemia. Treatment with rhEPO can therefore effectively reduce oxidative stress in HD patients.
Adult↗
Oxidation parameters in complete correction of renal anemia.
UNLABELLED: Chronic hemodialysis (HD) patients are more exposed to oxidative stress, with its adverse impact on many cell functions and not least on patient survival. There is evidence that partial correction of renal anemia by erythropoietin (rhEPO) therapy reduces oxidative stress. The aim of this study was to clarify whether complete correction of renal anemia with rhEPO reduces free radical generation in HD patients and increases antioxidant supply. The following parameters: malondialdehyde (MDA), reduced glutathione (GSH) and glutathione disulfide (GSSG) were investigated in patients with a hematocrit (Hct) normalized on rhEPO therapy (Hct > or = 0.4), and compared with those in anemic patients (Hct 0.3 - 0.39 and Hct < 0.3). The groups were similar in age, sex or body weight. Patients with normal Hct were significantly longer in the chronic HD program (74.0 +/- 70.3 vs. 23.0 +/- 30.9 and 30.6 +/- 34.8 months; p < 0.05) and received significantly lower doses of iron (35.7 +/- 19.5 vs. 55.4 +/- 26.0 and 80.0 +/- 47.1 mg/week; p < 0.05) and rhEPO (68.9 +/- 63.6 vs. 106.5 +/- 63.9 and 152.8 +/- 86.0 IU/kg/week; p < 0.05). MDA levels were significantly lower in the group with normal Hct than in the comparison groups (1.54 +/- 0.27 vs. 1.98 +/- 0.52 and 2.23 +/- 0.93 micromol/l; p < 0.01), but did not differ significantly between the anemic groups. GSH and GSSG concentrations corrected for erythrocyte levels showed no significant differences, but whole-blood levels in patients with Hct > or = 0.4 and 0.3 - 0.39 were significantly higher than in patients with Hct < 0.3 (GSH: 0.97 +/- 0.42 vs. 1.03 +/- 0.38 and 0.62 +/- 0.34 micromol/ml; GSSG: 14.57 +/- 6.06 vs 13.07 +/- 5.18 and 7.28 +/- 3.64 micromol/l; p < 0.05). CONCLUSION: After correction of renal anemia, MDA levels are significantly lower - reflecting decreased free radical generation - than in anemic HD patients. Whole-blood antioxidant capacity is significantly increased. Overall, rhEPO therapy has clearly positive effects on free radical metabolism.
Aged↗
Functional interaction of poly(ADP-ribose) with the 20S proteasome in vitro.
The nuclear enzyme poly(ADP-ribosyl) transferase (pADPRT) catalyzes the formation of poly(ADP-ribose) from NAD+. Several nuclear proteins and pADPRT itself are targets for the modification by poly(ADP-ribosyl)ation. It is demonstrated here that poly(ADP-ribose) or pADPRT automodified with poly(ADP-ribose) interacts noncovalently with the 20S proteasome in vitro. The interaction of pADPRT with the 20S proteasome requires the long ADP-ribose polymers formed by automodification of the pADPRT with poly(ADP-ribose). As a result pADPRT automodified with short ADP-ribose oligomers is unable to associate with the 20S proteasome. The interaction with poly(ADP-ribose) causes a specific stimulation of the peptidase activity of the 20S proteasome. Modified pADPRT does not serve as a substrate for the degradation by the 20S proteasome. No covalent modification of the 20S proteasome by ADP-ribosylation was observed. The results may point to a functional relationship between pADPRT and the 20S proteasome in a pathway protecting the cell from oxidative damage.
Cysteine Endopeptidases↗
Poly-ADP ribose polymerase activates nuclear proteasome to degrade oxidatively damaged histones.
The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified proteins in the cytoplasm. Nuclear proteins are also subject to oxidation, and the nucleus of mammalian cells contains proteasome. In human beings, tumor cells frequently are subjected to oxidation as a consequence of antitumor chemotherapy, and K562 human myelogenous leukemia cells have a higher nuclear proteasome activity than do nonmalignant cells. Adaptation to oxidative stress appears to be one element in the development of long-term resistance to many chemotherapeutic drugs and the mechanisms of inducible tumor resistance to oxidation are of obvious importance. After hydrogen peroxide treatment of K562 cells, degradation of the model proteasome peptide substrate suc-LLVY-MCA and degradation of oxidized histones in nuclei increases significantly within minutes. Both increased proteolytic susceptibility of the histone substrates (caused by modification by oxidation) and activation of the proteasome enzyme complex occur independently during oxidative stress. This rapid up-regulation of 20S proteasome activity is accompanied by, and depends on, poly-ADP ribosylation of the proteasome, as shown by inhibitor experiments, 14C-ADP ribose incorporation assays, immunoblotting, in vitro reconstitution experiments, and immunoprecipitation of (activated) proteasome with anti-poly-ADP ribose polymerase antibodies. The poly-ADP ribosylation-mediated activated nuclear 20S proteasome is able to remove oxidatively damaged histones more efficiently and therefore is proposed as an oxidant-stimulatable defense or repair system of the nucleus in K562 leukemia cells.
Adenosine Diphosphate Ribose↗
Influence of DNA binding on the degradation of oxidized histones by the 20S proteasome.
The 20S proteasome is localized in the cytosol and nuclei of mammalian cells. Previous work has shown that the cytosolic 20S proteasome is largely responsible for the selective recognition and degradation of oxidatively damaged cytosolic proteins. Since nuclear proteins are also susceptible to oxidative damage (e.g., from metabolic free radical production, ionizing radiation, xenobiotics, chemotherapy) we investigated the degradation of oxidatively damaged histones, in the presence and in the absence of DNA, by the 20S proteasome. We find that both soluble histones and DNA-bound histones are susceptible to selective proteolytic degradation by the 20S proteasome following mild oxidative damage. In contrast, more severe oxidative damage actually decreases the proteolytic susceptibility of histones. Soluble H1 showed the highest basal and maximal absolute proteolytic rates. Histone fraction H4 exhibited the greatest relative increase in proteolytic susceptibility following oxidation, almost 14-fold, and this occurred at a peroxide exposure of 5 mM. At the other end of the spectrum, histone H2A exhibited a maximal proteolytic response to H2O2 of only 6-fold, and this required an H2O2 exposure of 15 mM. An oxidation of reconstituted linear DNA plasmid-histone complex makes up to 95% of the histones bound to DNA susceptible to degradation, whereas undamaged protein-DNA complexes are not substrates for the proteasome. Severe oxidation by high concentrations of H2O2 appears to decreases the proteolytic susceptibility of histones due to the formation of cross-linked histone-DNA aggregates which appear to inhibit the proteasome. We conclude that the degradation of nuclear proteins is highly selective and requires prior damage of the substrate protein, such as that caused by oxidation.
Cysteine Endopeptidases↗
Oxidative stress in hemodialysis patients treated with a dialysis membrane which has alpha-tocopherol bonded to its surface.
BACKGROUND: MDA, a major product of LPO, was shown to be increased in plasma of patients with end-stage renal failure (ESRF) undergoing hemodialysis (HD). Elevated oxidative stress in ESRF patients is a result of multiple pathogenetic factors. HD treatment has been shown to be one important cause of accelerated radical generation. The aim of our study was to examine whether treatment with a dialysis membrane which has alpha-tocopherol hydrophobically bonded to its surface (Excebrane by Terumo, Japan) can decrease oxidative stress due to HD. METHODS: 10 ESRF patients undergoing HD three times weekly were examined. First, analysis was done when patients were still dialysed with the membranes used before the study. Thereafter, samples were taken when patients were dialysed first time with the Excebrane membrane, and six weeks after Excebrane treatment. Samples were collected always before and after HD session. A method with HPLC-separation and flourimetric detection was used to measure plasma concentration of MDA. RESULTS: After HD with the regularly used membranes MDA was found significantly increased (before HD 1.92 [1.81-2.02] microM (median and interquartile ranges) vs. after HD 2.26 [2.02-2.60] microM, p < 0.05) suggesting that MDA was produced during HD. The first time Excebrane was used MDA was decreased significantly (before HD 2.04 [1.95-2.88] microM vs. after HD 1.35 [1.19-1.92] microM, p < 0.05). After six weeks of Excebrane treatment, plasma MDA did not change significantly during HD (before HD 2.01 [1.69-2.62] microM vs. after HD 1.95 [1.42-2.20] microM). CONCLUSION: Oxidative stress due to HD might be significantly decreased by the Excebrane membrane. However, after six weeks of treatment with Excebrane no effect was seen on the initial plasma concentration of MDA compared to the time before.
Chromatography, High Pressure Liquid↗
Degradation of hypochlorite-damaged glucose-6-phosphate dehydrogenase by the 20S proteasome.
Glucose-6-phosphate dehydrogenase (G6PD) was treated with various concentrations of hypochlorite, which is produced by myeloperoxidase and is one of the most important oxidants during inflammatory processes. Inhibition of enzymatic activity, protein fragmentation, and proteolytic susceptibility toward the isolated 20S proteasome of G6PD were investigated. With rising hypochlorite concentrations, an increased proteasomal degradation of G6PD was measured. This occurred at higher hypochlorite concentrations than G6PD inactivation and at lower levels than G6PD fragmentation. The proteolytic activities of the 20S proteasome itself was determined by degradation of oxidized model proteins and cleavage of the synthetic proteasome substrate suc-LLVY-MCA. Proteasome activities remained intact at hypochlorite concentrations in which G6PD is maximally susceptible to proteasomal degradation. Only higher hypochlorite concentrations could decrease the proteolytic activities of the proteasome, which was accompanied by disintegration and fragmentation of the proteasome and proteasome subunits. Therefore, we conclude that the 20S proteasome can degrade proteins moderately damaged by hypochlorite and could contribute to an increased protein turnover in cells exposed to inflammatory stress.
Coumarins↗
Oxidized proteins as a marker of oxidative stress during coronary heart surgery.
The measurement of the degree of oxidative stress in patients often causes problems because of the lack of useful parameters. Therefore, we used an ELISA technique to evaluate serum protein carbonyls as a parameter of oxidative stress in patients during coronary heart surgery. Protein carbonyls were detected in serum samples of 14 patients undergoing coronary surgery and cardiopulmonary artery bypass grafting. A clear 2- to 3-fold increase in protein carbonyls in serum samples taken from human venous coronary sinus could be detected in the reperfusion period of the heart. We compared these data with markers of oxidative stress previously used, such as the glutathione status and the lipid peroxidation product malondialdehyde (MDA). Strong correlations of the protein carbonyl formation with MDA (r2 = 0.86) and oxidized glutathione (r2 = 0.81) were found in the early reperfusion stage. Increased levels of oxidized glutathione and MDA were detected only in the early reperfusion period. In contrast, the serum protein carbonyl content remained elevated for several hours, indicating a considerably slower serum clearance of oxidized proteins compared with that of lipid peroxidation products and the normalization of the glutathione status. We therefore concluded that the measurement of serum carbonyls by this ELISA technique is suitable to detect oxidative stress in serum samples of patients. The relative stability of the parameter makes the protein carbonyl detection even more valuable for clinical purposes.
Aged↗
Improved antioxidative protection in winter swimmers.
Adaptation to oxidative stress is an improved ability to resist the damaging effects of reactive oxygen species, resulting from pre-exposure to a lower dose. Changes in uric acid and glutathione levels during ice-bathing suggest that the intensive voluntary short-term cold exposure of winter swimming produces oxidative stress. We investigated whether the repeated oxidative stress in winter swimmers results in improved antioxidative adaptation. We obtained venous blood samples from winter swimmers and determined important components of the antioxidative defense system in the erythrocytes or blood plasma: reduced and oxidized glutathione (GSH and GSSG), and the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (Cat). The control group consisted of healthy people who had never participated in winter swimming. The baseline concentration of GSH and the activities of erythrocytic SOD and Cat, were higher in winter swimmers. We interpret this as an adaptative response to repeated oxidative stress, and postulate it as a new basic molecular mechanism of increased tolerance to environmental stress.
Adaptation, Physiological↗
Inhibition of tumor necrosis factor-alpha release in rat experimental endotoxemia by treatment with the 21-aminosteroid U-74389G.
OBJECTIVE: To determine the effect of the 21-aminosteroid U-74389G on tumor necrosis factor (TNF)-alpha release in experimental endotoxemia. DESIGN: Prospective, randomized, controlled animal study. SETTING: Experimental laboratory. SUBJECTS: Twenty-one male Wistar rats weighing 190+/-40 g. INTERVENTIONS: The rats were divided equally into 3 groups: a) control; b) endotoxemia (5 mg/kg lipopolysaccharide [LPS] from Escherichia coli 055:B5); and c) endotoxemia and U-74389G administration 30 mins before (3 mg/kg) and 60 mins after (1.5 mg/kg) endotoxin challenge. MEASUREMENTS AND MAIN RESULTS: At 0, 120, and 240 mins, serum levels of TNF-alpha were measured using a specific rat TNF-alpha ELISA kit. U-74389G-treated endotoxemic animals showed significantly reduced TNF-alpha release 120 mins after endotoxin challenge (control, 2.5+/-2.1 pg/mL; LPS, 4041+/-871 pg/mL; U-74389G, 1627+/-474 pg/mL [p < .05]). Two hundred forty minutes after LPS administration, TNF-alpha levels decreased, whereas values in the untreated LPS group remained twice as high as those in the U-74389G group (LPS, 863+/-182 pg/mL; U-74389G, 369+/-54 pg/mL [p < .05]). CONCLUSIONS: The study demonstrated that administration of U-74389G, which has radical-scavenging and membrane-stabilizing properties, decreased TNF-alpha release during endotoxemia. Thus, 21-aminosteroids may lend themselves to evaluation in the treatment of septic states.
Analysis of Variance↗
[Controlled reperfusion of ischemic extremity musculature to prevent free radical induced lesions].
Tissue injury following reperfusion represents an essential problem of reconstructive vascular surgery. Pathogenetically toxic oxygen radicals are considered to play a pivotal role. Pharmacotherapeutical approaches are based particularly on antioxidants and vasodilators. However, a standardized regimen is not yet clinically introduced. In 48 adult Lewis-rats lower limb ischemia was induced by aortal cross-clamping. Following 3.5 hours of ischemia intravascular flushing perfusion via the distal aorta with a heparinized electrolyte solution (group B). Group C received additionally oxypurinol, group D alprostadil and group E sodium selenite into the flushing solution. At 4 hours recirculation was established. After 10 min, 30 min and 24 hours of reperfusion we determined lactate, creatine kinase, lactate dehydrogenase, urea, malondialdehyde and the laser Doppler flux. At the end of the experiments biopsies were taken from M. tibialis anterior. In comparison to control animals (group A) we observed an attenuation of reperfusion injury in the groups treated with flushing perfusion. Free oxygen radical reactions measured by malondialdehyde release were significantly reduced (30 min: A-209.1 +/- 45.4, B-127.3 +/- 36.9, C-113.2 +/- 14.1, D-99.6 +/- 24.5, E-123.6 +/- 11.2 mmol/l, p < 0.05). The laser Doppler flux measurements corresponded with the biochemical analyses (30 min: A-52.4 +/- 11.1, B-48.0 +/- 11.0, C-72.6 +/- 12.0, D-74.4 +/- 13.3, E-62.6 +/- 10.8% of baseline). Histologically, treatment with alprostadil (PGE1) and oxypurinol revealed superior results. Standardized intraarterial flushing perfusion with antioxidants and vasodilators reduces reperfusion injury. Clinical trials are urgently required to confirm the experimental findings and to optimize the therapy of extremity ischemia/reperfusion injury in humans.
Animals↗
Proteasome-dependent degradation of oxidized proteins in MRC-5 fibroblasts.
Fibroblasts were exposed to various concentrations of hydrogen peroxide and the removal of oxidized proteins was followed by determining protein-bound carbonyls. Fibroblasts are able to increase the turnover of metabolically radiolabeled proteins after treatment with hydrogen peroxide. It was demonstrated for the first time, that the increased protein turnover was accompanied by a removal of protein-bound carbonyl groups. The proteasome-specific inhibitor lactacystin was able to inhibit the elimination of protein-bound carbonyl groups. Therefore, the key role of the proteasome in the degradation of oxidized proteins in fibroblasts could be demonstrated.
Acetylcysteine↗
Comparative resistance of the 20S and 26S proteasome to oxidative stress.
Oxidatively modified ferritin is selectively recognized and degraded by the 20S proteasome. Concentrations of hydrogen peroxide (H2O2) higher than 10 micromol/mg of protein are able to prevent proteolytic degradation. Exposure of the protease to high amounts of oxidants (H2O2, peroxynitrite and hypochlorite) inhibits the enzymic activity of the 20S proteasome towards the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-methylcoumarylamide (Suc-LLVY-MCA), as well as the proteolytic degradation of normal and oxidant-treated ferritin. Fifty per cent inhibition of the degradation of the protein substrates was achieved using 40 micromol of H2O2/mg of proteasome. No change in the composition of the enzyme was revealed by electrophoretic analysis up to concentrations of 120 micromol of H2O2/mg of proteasome. In further experiments, it was found that the 26S proteasome, the ATP- and ubiquitin-dependent form of the proteasomal system, is much more susceptible to oxidative stress. Whereas degradation of the fluorogenic peptide, Suc-LLVY-MCA, by the 20S proteasome was inhibited by 50% with 12 micromol of H2O2/mg, 3 micromol of H2O2/mg was enough to inhibit ATP-stimulated degradation by the 26S proteasome by 50%. This loss in activity could be followed by the loss of band intensity in the non-denaturing gel. Therefore we concluded that the 20S proteasome was more resistant to oxidative stress than the ATP- and ubiquitin-dependent 26S proteasome. Furthermore, we investigated the activity of both proteases in K562 cells after H2O2 treatment. Lysates from K562 cells are able to degrade oxidized ferritin at a higher rate than non-oxidized ferritin, in an ATP-independent manner. This effect could be followed even after treatment of the cells with H2O2 up to a concentration of 2mM. The lactacystin-sensitive ATP-stimulated degradation of the fluorogenic peptide Suc-LLVY-MCA declined, after treatment of the cells with 1mM H2O2, to the same level as that obtained without ATP stimulation. Therefore, we conclude that the regulation of the 20S proteasome by various regulators takes place during oxidative stress. This provides further evidence for the role of the 20S proteasome in the secondary antioxidative defences of mammalian cells.
Coumarins↗
Protective effects of the thiophosphate amifostine (WR 2721) and a lazaroid (U83836E) on lipid peroxidation in endothelial cells during hypoxia/reoxygenation.
Little is known about pharmacological interventions with thiophosphates or lazaroids in endothelial cells injured by hypoxia/reoxygenation with respect to membrane lipid peroxidation (LPO) caused by reactive oxygen species. Therefore, a cell line of bovine aortic endothelial cells was studied after 120-min hypoxia followed by 30-min reoxygenation, resulting in moderate and predominantly reversible injury (energy depression/cytosolic Ca2+-accumulation during hypoxia, which almost normalized during reoxygenation; membrane blebs, an increasing amount of lysosomes, vacuolization, lipofuscin formation, alterations in mitochondria size, some lyzed cells). 18.9 +/- 4.3% of the cells died. Radical-induced LPO measured as malondialdehyde continuously increased to 2.18 +/- 0.17 nmol/mg of protein after reoxygenation vs control (0.41 +/- 0.13, P < 0.05). Simultaneously, the content of 4-hydroxynonenal, a novel indicator of LPO, increased from 0.02 +/- 0.01 to 0.11 +/- 0.02 nmol/mg of protein (P < 0.01). The results support the assumption that reoxygenation injury is accompanied by an increase in membrane LPO, causing structural and functional disturbances in the monolayer. The thiophosphate WR 2721 [S-2-(3-aminopropylamino) ethylphosphorothioic acid] and the lazaroid U83836E [(-)-2-[[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl] methyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol (dihydrochloride)] were effective scavengers of .OH, being more efficient than trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbon acid) used as standard (EC50: 12, 5 and 15 microM, respectively, measured by electron spin resonance spectroscopy). One mM WR 2721, 10 microM U83836E, and 5 microM trolox C reduced formation of malondialdehyde during hypoxia/reoxygenation to 53 +/- 7, 51 +/- 10 and 48 +/- 6%, respectively (P < 0.05 each, versus control). In general, WR 2721 and U83836E prevent radical-induced membrane LPO in a model of endothelial cells injured by hypoxia/reoxygenation. The use of these two agents is a new approach to protect the endothelium against oxidative stress.
Amifostine↗
Peroxynitrite increases the degradation of aconitase and other cellular proteins by proteasome.
We report that exposure of aconitase to moderate concentrations of peroxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide- and nitric oxide-liberating substance), or hydrogen peroxide, inhibits the enzyme and enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, from these same agents, causes further inhibition of the enzymatic activity of aconitase but actually decreases its proteolytic breakdown by proteasome. It should be noted that the superoxide and nitric oxide liberated by SIN-1 decomposition react to form a steady flux of peroxynitrite. S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or less) and has no effect on the proteolytic susceptibility of the enzyme. Proteasome also seems to be the main protease in cell lysates that can degrade aconitase after it has been oxidatively modified by exposure to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates isolated from K562 cells treated for several days with an antisense oligodeoxynucleotide to the initiation codon region of the C2 subunit of proteasome (a treatment which diminishes proteasome activity by 50-60%), the enhanced degradation of moderately damaged aconitase was essentially abolished. Other model proteins as well as complex mixtures of proteins, such as cell lysates, also exhibit enhanced proteolytic susceptibility after moderate SIN-1 treatment. Therefore we conclude that peroxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by proteasome.
Aconitate Hydratase↗