Coordination of oxidized protein hydrolase and the proteasome in the clearance of cytotoxic denatured proteins

Intracellular accumulation of denatured proteins impairs cellular function. The proteasome is recognized as an enzyme responsible for the effective clearance of those cytotoxic denatured proteins. As another enzyme that participates in the destruction of damaged proteins, we have identified oxidized protein hydrolase (OPH) and found that OPH confers cellular resistance to various kinds of oxidative stress. In this study, we demonstrate the roles of the proteasome and OPH in the clearance of denatured proteins. The inhibition of proteasome activity results in the elevation of protein carbonyls in cells under oxidative stress. On the other hand, cells overexpressing OPH retain higher resistance to oxidative stress, even though the proteasome activity is inhibited. Furthermore, upon inhibition of the proteasome activity, OPH is recruited to a novel organelle termed the aggresome where misfolded or denatured proteins are processed. Thus, OPH and the proteasome coordinately contribute to the clearance of cytotoxic denatured proteins.     

Modulation of Lon protease activity and aconitase turnover during aging and oxidative stress

We compared Lon protease expression in murine skeletal muscle of young and old, wild-type and Sod2(-/+) heterozygous mice, and studied Lon involvement in the accumulation of damaged (oxidized) proteins. Lon protease protein levels were lower in old and oxidatively challenged animals, and this Lon deficiency was associated with increased levels of carbonylated proteins. We identified one of these proteins as aconitase, and another as an aconitase fragmentation product, which we can also generate in vitro by treating purified aconitase with H(2)O(2). These results imply that aging and oxidative stress down-regulate Lon protease expression which, in turn, may be responsible for the accumulation of damaged proteins, such as aconitase, within mitochondria.     

Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals

The disaccharide trehalose, which accumulates dramatically during heat shock and stationary phase in many organisms, enhances thermotolerance and reduces aggregation of denatured proteins. Here we report a new role for trehalose in protecting cells against oxygen radicals. Exposure of Saccharomyces cerevisiae to a mild heat shock (38 degrees C) or to a proteasome inhibitor (MG132) induced trehalose accumulation and markedly increased the viability of the cells upon exposure to a free radical-generating system (H(2)O(2)/iron). When cells were returned to normal growth temperature (28 degrees C) or MG132 was removed from the medium, the trehalose content and resistance to oxygen radicals decreased rapidly. Furthermore, a mutant unable to synthesize trehalose was much more sensitive to killing by oxygen radicals than wild-type cells. Providing trehalose exogenously enhanced the resistance of mutant cells to H(2)O(2). Exposure of cells to H(2)O(2) caused oxidative damage to amino acids in cellular proteins, and trehalose accumulation was found to reduce such damage. After even brief exposure to H(2)O(2), the trehalose-deficient mutant exhibited a much higher content of oxidatively damaged proteins than wild-type cells. Trehalose accumulation decreased the initial appearance of damaged proteins, presumably by acting as a free radical scavenger. Therefore, trehalose accumulation in stressed cells plays a major role in protecting cellular constituents from oxidative damage.     

Age-dependent effect of oxidative stress on cardiac sarcoplasmic reticulum vesicles

The oxidative stress hypothesis of aging suggests that accumulation of oxidative damage is a key factor of the alterations in physiological function during aging. We studied age-related sensitivity to oxidative modifications of proteins and lipids of cardiac sarcoplasmic reticulum (SR) isolated from 6-, 15- and 26-month-old rats. Oxidative stress was generated in vitro by exposing SR vesicles to 0.1 mmol/l FeSO4/EDTA + 1 mmol/l H2O2 at 37 degrees C for 60 min. In all groups, oxidative stress was associated with decreased membrane surface hydrophobicity, as detected by 1-anilino-8-naphthalenesulfonate as a probe. Structural changes in SR membranes were accompanied by degradation of tryptophan and significant accumulation of protein dityrosines, protein conjugates with lipid peroxidation products, conjugated dienes and thiobarbituric acid reactive substances. The sensitivity to oxidative damage was most pronounced in SR of 26-month-old rat. Our results indicate that aging and oxidative stress are associated with accumulation of oxidatively damaged proteins and lipids and these changes could contribute to cardiovascular injury.     

The impact of oxidative stress on Arabidopsis mitochondria

Treatment of Arabidopsis cell culture for 16 h with H2O2, menadione or antimycin A induced an oxidative stress decreasing growth rate and increasing DCF fluorescence and lipid peroxidation products. Treated cells remained viable and maintained significant respiratory rates. Mitochondrial integrity was maintained, but accumulation of alternative oxidase and decreased abundance of lipoic acid-containing components during several of the treatments indicated oxidative stress. Analysis of the treatments was undertaken by IEF/SDS-PAGE, comparison of protein spot abundances and tandem mass spectrometry. A set of 25 protein spots increased >3-fold in H2O2/menadione treatments, a subset of these increased in antimycin A-treated samples. A set of 10 protein spots decreased significantly during stress treatments. A specific set of mitochondrial proteins were degraded by stress treatments. These damaged components included subunits of ATP synthase, complex I, succinyl CoA ligase, aconitase, and pyruvate and 2-oxoglutarate dehydrogenase complexes. Nine increased proteins represented products of different genes not found in control mitochondria. One is directly involved in antioxidant defense, a mitochondrial thioredoxin-dependent peroxidase, while another, a thioredoxin reductase-dependent protein disulphide isomerase, is required for protein disulfide redox homeostasis. Several others are generally considered to be extramitochondrial but are clearly present in a highly purified mitochondrial fraction used in this study and are known to play roles in stress response. Using H2O2 as a model stress, further work revealed that this treatment induced a protease activity in isolated mitochondria, putatively responsible for the degradation of oxidatively damaged mitochondrial proteins and that O2 consumption by mitochondria was significantly decreased by H2O2 treatment.     

Oxidative stress induces protein kinase D activation in intact cells. Involvement of Src and dependence on protein kinase C

Protein kinase D (PKD) is a protein serine kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids, and activated by phorbol esters, neuropeptides, and platelet-derived growth factor via protein kinase C (PKC) in intact cells. Recently, oxidative stress was shown to activate transfected PKC isoforms via tyrosine phosphorylation, but PKD activation was not demonstrated. Here, we report that oxidative stress initiated by addition of H(2)O(2) (0.15-10 mm) to quiescent Swiss 3T3 fibroblasts activates PKD in a dose- and time- dependent manner, as measured by autophosphorylation and phosphorylation of an exogenous substrate, syntide-2. Oxidative stress also activated transfected PKD in COS-7 cells but not a kinase-deficient mutant PKD form or a PKD mutant with critical activating serine residues 744 and 748 mutated to alanines. Genistein, or the specific Src inhibitors PP-1 and PP-2 (1-10 micrometer) inhibited H(2)O(2)-mediated PKD activation by 45%, indicating that Src contributes to this signaling pathway. PKD activation by H(2)O(2) was also selectively potentiated by cotransfection of PKD together with an active form of Src (v-Src) in COS-7 cells, as compared with PDB-mediated activation. The specific phospholipase C inhibitor, partly blocked H(2)O(2)-mediated but not PDB-mediated PKD activation. In contrast, PKC inhibitors blocked H(2)O(2) or PDB-mediated PKD activation essentially completely, suggesting that whereas Src mediates part of its effects via phospholipase C activation, PKC acts more proximally as an upstream activator of PKD. Together, these studies reveal that oxidative stress activates PKD by initiating distinct Src-dependent and -independent pathways involving PKC.     

Oxidatively denatured proteins are degraded by an ATP-independent proteolytic pathway in Escherichia coli

E. coli contains a soluble proteolytic pathway which can recognize and degrade oxidatively denatured proteins and protein fragments, and which may act as a "secondary antioxidant defense." We now provide evidence that this proteolytic pathway is distinct from the previously described ATP-dependent, and protease "La"-dependent, pathway which may degrade other abnormal proteins. Cells (K12) which were depleted of ATP, by arsenate treatment or anaerobic incubation (after growth on succinate), exhibited proteolytic responses to oxidative stress which were indistinguishable from those observed in cells with normal ATP levels. Furthermore, the proteolytic responses to oxidative damage by menadione or H2O2 were almost identical in the isogenic strains RM312 (a K12 derivative) and RM1385 (a lon deletion mutant of RM312). Since the lon (or capR) gene codes for the ATP-dependent protease "La," these results indicate that neither ATP nor protease "La" are required for the degradation of oxidatively denatured proteins. We next prepared cell-free extracts of K12, RM312, and RM1385 and tested the activity of their soluble proteases against proteins (albumin, hemoglobin, superoxide dismutase, catalase) which had been oxidatively denatured (in vitro) by exposure to .OH, .OH + O2- (+O2), H2O2, or ascorbate plus iron. The breakdown of oxidatively denatured proteins was several-fold higher than that of untreated proteins in extracts from all three strains, and ATP did not stimulate degradation. Incubation of extracts at 45 degrees C, which inactivates protease "La," actually stimulated the degradation of oxidatively denatured proteins. Although Ca2+ had little effect on proteolysis, serine reagents, transition metal chelators, and hemin effectively inhibited the degradation of oxidatively denatured proteins in both intact cells and cell-free extracts. Degradation of oxidatively denatured proteins in cell-free extracts was maximal at pH 7.8, and was unaffected by dialysis of the extracts against membranes with molecular weight cutoffs as high as 50,000. Our results indicate the presence of a neutral, ATP- and calcium- independent proteolytic pathway in the E. coli cytosol, which contains serine- and metallo- proteases (with molecular weights greater than 50,000), and which preferentially degrades oxidatively denatured proteins.     

Hydrogen peroxide-induced carbonylation of key metabolic enzymes in Saccharomyces cerevisiae: the involvement of the oxidative stress response regulators Yap1 and Skn7

H(2)O(2) induces a specific protein oxidation in yeast cells, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Tdh) is a major target. Using a 2D-gel system to study protein carbonylation, it is shown in this work that both Tdh2p and Tdh3p isozymes were oxidized during exposure to H(2)O(2). In addition, we identified two other proteins carbonylated and inactivated: Cu,Zn-superoxide dismutase and phosphoglycerate mutase. The oxidative inactivation of Cu,Zn-superoxide dismutase decreases the antioxidant capacity of yeast cells and probably contributes to H(2)O(2)-induced cell death. Cyclophilin 1 was also carbonylated, but CPH1 gene disruption did not affect peroxide stress sensitivity. The correlation between H(2)O(2) sensitivity and the accumulation of oxidized proteins was evaluated by assaying protein carbonyls in mutants deficient in the stress response regulators Yap1p and Skn7p. The results show that the high sensitivity of yap1delta and skn7delta mutants to H(2)O(2) was correlated with an increased induction of protein carbonylation. In wild-type cells, the acquisition of stress resistance by pre-exposure to a sublethal H(2)O(2) stress was associated with a lower accumulation of oxidized proteins. However, pre-exposure of yap1delta and skn7delta cells to 0.4 mM H(2)O(2) decreased protein carbonylation induced by 1.5 mM H(2)O(2), indicating that the adaptive mechanism involved in the protection of proteins from carbonylation is Yap1p- and Skn7p-independent.     

Mechanism of the formation and proteolytic release of H2O2-induced dityrosine and tyrosine oxidation products in hemoglobin and red blood cells

Oxyhemoglobin exposed to a continuous flux of H(2)O(2) underwent oxidative modifications, including limited release of fluorescent fragmentation products. The main fragments formed were identified as oxidation products of tyrosine, including dopamine, dopamine quinone, and dihydroxyindol. Further release of these oxidation products plus dityrosine was only seen after proteolytic degradation of the oxidatively modified hemoprotein. A possible mechanism is proposed to explain the formation of these oxidation products that includes cyclization, decarboxylation, and further oxidation of the intermediates. Release of dityrosine is proposed as a useful technique for evaluating selective proteolysis after an oxidative stress, because dityrosine is metabolically stable, and it is only released after enzymatic hydrolysis of the oxidatively modified protein. The measurement can be accomplished by high performance liquid chromatography with fluorescence detection or by high efficiency thin layer chromatography. Comparable results, in terms of dityrosine release, were obtained using red blood cells of different sources after exposing them to a flux of H(2)O(2). Furthermore, dityrosine has been reported to occur in a wide variety of oxidatively modified proteins. These observations suggest that dityrosine formation and release can be used as a highly specific marker for protein oxidation and selective proteolysis.     

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.     

Identification of oxidized protein hydrolase of human erythrocytes as acylpeptide hydrolase

Partial amino acid sequence of 80 kDa oxidized protein hydrolase (OPH), a serine protease present in human erythrocyte cytosol (Fujino et al., J. Biochem. 124 (1998) 1077-1085) that is adherent to oxidized erythrocyte membranes and preferentially degrades oxidatively damaged proteins (Beppu et al., Biochim. Biophys. Acta 1196 (1994) 81-87; Fujino et al., Biochim. Biophys. Acta 1374 (1998) 47-55) was determined. The N-terminal amino acid of diisopropyl fluorophosphate (DFP)-labeled OPH was suggested to be masked. Six peptide fragments of OPH obtained by digestion of DFP-labeled OPH with lysyl endopeptidase were isolated by use of reverse-phase high-performance liquid chromatography, and the sequence of more than eight amino acids from the N-terminal position of each peptide was determined. Results of homology search of amino acid sequence of each peptide strongly suggested that the protein was identical with human liver acylpeptide hydrolase (ACPH). OPH showed ACPH activity when N-acetyl-L-alanine p-nitroanilide and N-acetylmethionyl L-alanine were used as substrates. Glutathione S-transferase (GST)-tagged recombinant ACPH (rACPH) was prepared by use of baculovirus expression system as a 107-kDa protein from cDNA of human erythroleukemic cell line K-562. rACPH reacted with anti-OPH antiserum from rabbit. rACPH showed OPH activity when hydrogen peroxide-oxidized or glycated bovine serum albumin was used as substrates. As well as the enzyme activities of OPH, those of rACPH were inhibited by DFP. The results clearly demonstrate that ACPH, whose physiological function has not yet been well characterized, can play an important role as OPH in destroying oxidatively damaged proteins in living cells.     

Effects of aging and hyperoxia on oxidative damage to cytochrome c in the housefly, Musca domestica

Cytochrome c is a component of the mitochondrial electron transport chain, where it transfers electrons from ubiquinol-cytochrome c reductase to cytochrome c oxidase. Autoxidation of some of the components of the electron transport chain is the main source of intracellular O(2)(-*)/H(2)O(2) production in aerobic organisms. Because cytochrome c is located on the outer surface of the inner mitochondrial membrane, it is likely to be constantly exposed to H(2)O(2), secreted by mitochondria into the cytosol. The specific objective of this study was to determine whether cytochrome c in the flight muscle mitochondria of the housefly is oxidatively damaged during aging and/or under severe oxidative stress induced by exposure of flies to 100% oxygen. Results of two independent methods, namely tritiated borohydride labeling for determining carbonylation and mass spectral analysis for the measurement of molecular mass, indicated that neither the carbonyl level nor the molecular mass of cytochrome c was affected by aging or hyperoxia. Thus, either cytochrome c is resistant to oxidative damage in vivo or the oxidized cytochrome c is promptly degraded. These findings also support the concept that protein oxidative damage during aging and under oxidative stress is selective.     

Proteolytic response to oxidative stress in mammalian cells

Oxidative stress in mammalian cells is an inevitable consequence of their aerobic metabolism. The production of reactive oxygen and nitric oxide species causes oxidative modifications of proteins often combined with a loss of their biological function. Like most partially denatured proteins, moderately oxidized proteins are more sensitive to proteolytic attack by proteases. The diverse cellular proteolytic systems are an important secondary defense against oxidative stress by degrading oxidized and damaged proteins, thereby preventing their intracellular accumulation. In mammalian cells, a range of proteases exists which are distributed throughout the cell. In this review we summarize the function of the cytosolic (proteasome and calpains), the lysosomal, the mitochondrial and the nuclear proteolytic pathways in response to oxidative stress. Particular emphasis is given to the proteasomal system, since this pathway appears to be the most important proteolytic system involved in the removal of oxidatively modified or damaged proteins.     

Superoxide dismutase is preferentially degraded by a proteolytic system from red blood cells following oxidative modification by hydrogen peroxide

The cupro-zinc enzyme superoxide dismutase (SOD) undergoes an irreversible (oxidative) inactivation when exposed to its product, hydrogen peroxide (H2O2). Recent studies have shown that several oxidatively modified proteins (e.g., hemoglobin, albumin, catalase, etc.) are preferentially degraded by a novel proteolytic pathway in the red blood cell. We report that bovine SOD is oxidatively inactivated by exposure to H2O2, and that the inactivated enzyme is selectively degraded by proteolytic enzymes in cell-free extracts of bovine erythrocytes. For example, 95% inactivation of SOD by 1.5 mM H2O2 was accompanied by a 106 fold increase in the proteolytic susceptibility of the enzyme during (a subsequent) incubation with red cell extract. Both SOD inactivation and proteolytic susceptibility increased with H2O2 concentration and/or time of exposure to H2O2. Pre-incubation of red cell extracts with metal chelators, serine reagents, or sulfhydryl reagents inhibited the (subsequent) preferential degradation of H2O2-modified SOD. Furthermore, a slight inhibition of degradation was observed with the addition of ATP. We suggest that H2O2-inactivated SOD is recognized and preferentially degraded by the same. ATP-independent, metallo- serine- and sulfhydryl- proteinase pathway which degrades other oxidatively denatured red cell proteins. Related work in this laboratory suggests that this novel proteolytic pathway may actually consist of a 700 kDa enzyme complex of proteolytic activities. Mature red cells have no capacity for de novo protein synthesis but do have extremely high concentrations of SOD. Red cell SOD generates (and is, therefore, exposed to) H2O2 on a continuous basis, by dismutation of superoxide (from hemoglobin autooxidation and the interaction of hemoglobin with numerous xenobiotics).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transcriptional regulation of the antioxidant protein 2 gene, a thiol-specific antioxidant, by lens epithelium-derived growth factor to protect cells from oxidative stress.

Antioxidant protein 2 (AOP2), a member of the newly defined family of thiol-specific antioxidant proteins, has been shown to remove H(2)O(2) and protect proteins and DNA from oxidative stress. Here we report that LEDGF is one of the regulatory factors for the AOP2 gene. We found that LEDGF bound to the heat shock element and to stress-related elements in the AOP2 promoter. It trans-activated expression of AOP2-CAT in COS-7 cells and lens epithelial cells overexpressing LEDGF. Mutations in the heat shock element and stress-related elements of the AOP2 promoter reduced LEDGF-dependent trans-activation. Lens epithelial cells showed a higher level of AOP2 mRNA in the presence of LEDGF. Cells overexpressing LEDGF exhibited a higher level of AOP2 protein, the level of which was directly related to the increase in cellular protection. Thus, LEDGF, by activating the AOP2 gene, protected and enhanced the survival of cells under oxidative stress.     

Promotion of oxidative lipid membrane damage by amyloid beta proteins

Senile plaques in the cerebral parenchyma are a pathognomonic feature of Alzheimer's disease (AD) and are mainly composed of aggregated fibrillar amyloid beta (Abeta) proteins. The plaques are associated with neuronal degeneration, lipid membrane abnormalities, and chemical evidence of oxidative stress. The view that Abeta proteins cause these pathological changes has been challenged by suggestions that they have a protective function or that they are merely byproducts of the pathological process. This investigation was conducted to determine whether Abeta proteins promote or inhibit oxidative damage to lipid membranes. Using a mass spectrometric assay of oxidative lipid damage, the 42-residue form of Abeta (Abeta42) was found to accelerate the oxidative lipid damage caused by physiological concentrations of ascorbate and submicromolar concentrations of copper(II) ion. Under these conditions, Abeta42 was aggregated, but nonfibrillar. Ascorbate and copper produced H(2)O(2), but Abeta42 reduced H(2)O(2) concentrations, and its ability to accelerate oxidative damage was not affected by catalase. Lipids could be oxidized by H(2)O(2) and copper(II) in the absence of ascorbate, but only at significantly higher concentrations, and Abeta42 inhibited this reaction. These results indicate that the ability of Abeta42 to promote oxidative damage is more potent and more likely to be manifest in vivo than its ability to inhibit oxidative damage. In conjunction with prior results demonstrating that oxidatively damaged membranes cause Abeta42 to misfold and form fibrils, these results suggest a specific chemical mechanism linking Abeta42-promoted oxidative lipid damage to amyloid fibril formation.     

The reaction of hydrogen peroxide with hemoglobin induces extensive alpha-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways

Cell-free hemoglobin (Hb) enhances the oxidation-related toxicity associated with inflammation, ischemia, and hemolytic disorders. Hb is highly vulnerable to oxidative damage, and irreversible structural changes involving iron/heme oxidation, heme-adduct products, and amino acid oxidation have been reported. Specific structural features of Hb, such as unconstrained alpha-chains and molecular size, determine the efficiency of interactions between the endogenous Hb scavengers haptoglobin (Hp) and CD163. Using HPLC, mass spectrometry, and Western blotting, we show that H(2)O(2)-mediated Hb oxidation results in the formation of covalently stabilized globin multimers, with prominent intramolecular crosslinking between alpha-globin chains. These structural alterations are associated with reduced Hp binding, reduced CD163 interaction, and severely impaired endocytosis of oxidized Hb by the Hp-CD163 pathway. As a result, when exposed to oxidized Hb, CD163-positive HEK293 cells and human macrophages do not increase hemeoxygenase-1 (HO-1) expression, the physiological anti-oxidative macrophage response to Hb exposure. Failed Hb clearance, inadequate HO-1 expression, and the subsequent accumulation of oxidatively damaged Hb species might thus contribute to pathologies related to oxidative stress.     

Proteasomal degradation of oxidatively damaged endogenous histones in K562 human leukemic cells

A number of antitumor drugs act via the oxidation of nuclear material in the tumor cell. It is therefore important to know if tumor cells can effectively and precisely cope not only with oxidatively induced DNA damage, but also with nuclear protein oxidation. In this study, we investigated the endogenous degradation of oxidatively damaged histones in K562 human leukemic cells after oxidative challenge and demonstrated a link to the overall cellular stress response pathways by poly-ADP-ribose-polymerase (PARP). After an oxidative challenge, endogenous nuclear protein degradation, as well as histone degradation, was enhanced. Among the histone fractions, histone H1 revealed the highest degradation rate, and more than 85% of the total degraded H1 disappeared in the first 30 min after oxidative challenge. Short-term degradation of histones up to 30 min, as well as long-term degradation up to 48 h after oxidative challenge, was significantly reduced in the presence of the PARP inhibitor 3-aminobenzamide, and nearly completely abrogated by the selective proteasome inhibitor lactacystin. Immunoprecipitation experiments indicated that the proteasome specifically degraded oxidized histones. Thus, we show that the nuclear proteosome system in tumor cells is capable of preventing the accumulation of oxidized proteins in this compartment and may suggest further treatment strategies to effectively interfere with the protein "repair" and replacement strategies of tumor cells.     

Protein oxidation and proteolysis

One of the hallmarks of chronic or severe oxidative stress is the accumulation of oxidized proteins, which tend to form high-molecular-weight aggregates. The major proteolytic system responsible for the removal of oxidized cytosolic and nuclear proteins is the proteasome. This complicated proteolytic system contains a core proteasomal form (20S proteasome) and several regulators. All of these components are affected by oxidative stress to various degrees. The ATP-stimulated 26S proteasome is sensitive to oxidative stress, whereas the 20S form seems to be more resistant. The nuclear proteasome selectively degrades oxidatively damaged histones in the nuclei of mammalian cells, where it is activated and regulated by automodified PARP-1 after oxidative challenge. In this brief review we highlight the proteolysis and its regulatory effects during oxidative stress.     

Bcl-2 family proteins regulate mitochondrial reactive oxygen production and protect against oxidative stress

Bcl-2 family proteins protect against a variety of forms of cell death, including acute oxidative stress. Previous studies have shown that overexpression of the antiapoptotic protein Bcl-2 increases cellular redox capacity. Here we report that cell lines transfected with Bcl-2 paradoxically exhibit increased rates of mitochondrial H(2)O(2) generation. Using isolated mitochondria, we determined that increased H(2)O(2) release results from the oxidation of reduced nicotinamide adenine dinucleotide-linked substrates. Antiapoptotic Bcl-2 family proteins Bcl-xL and Mcl-1 also increase mitochondrial H(2)O(2) release when overexpressed. Chronic exposure of cells to low levels of the mitochondrial uncoupler carbonyl cyanide 4-(triflouromethoxy)phenylhydrazone reduced the rate of H(2)O(2) production by Bcl-xL overexpressing cells, resulting in a decreased ability to remove exogenous H(2)O(2) and enhanced cell death under conditions of acute oxidative stress. Our results indicate that chronic and mild elevations in H(2)O(2) release from Bcl-2, Bcl-xL, and Mcl-1 overexpressing mitochondria lead to enhanced cellular antioxidant defense and protection against death caused by acute oxidative stress.