Peroxiredoxin II restrains DNA damage-induced death in cancer cells by positively regulating JNK-dependent DNA repair

The 2-Cys peroxiredoxins (Prx) belong to a family of antioxidant enzymes that detoxify reactive oxygen and nitrogen species and are distributed throughout the intracellular and extracellular compartments. However, the presence and role of 2-Cys Prxs in the nucleus have not been studied. This study demonstrates that the PrxII located in the nucleus protects cancer cells from DNA damage-induced cell death. Although the two cytosolic 2-Cys Prxs, PrxI and PrxII, were found in the nucleus, only PrxII knockdown selectively and markedly increased cell death in the cancer cells treated with DNA-damaging agents. The increased death was completely reverted by the nuclearly targeted expression of PrxII in an activity-independent manner. Furthermore, the antioxidant butylated hydroxyanisole did not influence the etoposide-induced cell death. Mechanistically, the knockdown of Prx II expression impaired the DNA repair process by reducing the activation of the JNK/c-Jun pathway. These results suggest that PrxII is likely to be attributed to a tumor survival factor positively regulating JNK-dependent DNA repair with its inhibition possibly sensitizing cancer cells to chemotherapeutic agents.     

Reduction of cysteine sulfinic acid by sulfiredoxin is specific to 2-cys peroxiredoxins

Cysteine residues of certain peroxiredoxins (Prxs) undergo reversible oxidation to sulfinic acid (Cys-SO2H) and the reduction reaction is catalyzed by sulfiredoxin (Srx). Specific Cys residues of various other proteins are also oxidized to sulfinic acid, suggesting that formation of Cys-SO2H might be a novel posttranslational modification that contributes to regulation of protein function. To examine the susceptibility of sulfinic forms of proteins to reduction by Srx, we prepared such forms of all six mammalian Prx isoforms and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Purified sulfiredoxin reduced the sulfinic forms of the four 2-Cys members (Prx I to Prx IV) of the Prx family in vitro, but it did not affect those of Prx V, Prx VI, or GAPDH. Furthermore, Srx bound specifically to the four 2-Cys Prxs in vitro and in cells. Sulfinic forms of Prx I and Prx II, but not of Prx VI or GAPDH, present in H2O2-treated A549 cells were gradually reduced after removal of H2O2; overexpression of Srx increased the rate of the reduction of Prx I and Prx II but did not induce that of Prx VI or GAPDH. These results suggest that reduction of Cys-SO2H by Srx is specific to 2-Cys Prx isoforms. For proteins such as Prx VI and GAPDH, sulfinic acid formation might be an irreversible process that causes protein damage.     

Variable overoxidation of peroxiredoxins in human lung cells in severe oxidative stress

Peroxiredoxins (Prxs) are a group of thiol containing proteins that participate both in signal transduction and in the breakdown of hydrogen peroxide (H(2)O(2)) during oxidative stress. Six distinct Prxs have been characterized in human cells (Prxs I-VI). Prxs I-IV form dimers held together by disulfide bonds, Prx V forms intramolecular bond, but the mechanism of Prx VI, so-called 1-Cys Prx, is still unclear. Here we describe the regulation of all six Prxs in cultured human lung A549 and BEAS-2B cells. The cells were exposed to variable concentrations of H(2)O(2), menadione, tumor necrosis factor-alpha or transforming growth factor-beta. To evoke glutathione depletion, the cells were furthermore treated with buthionine sulfoximine. Only high concentrations (300 microM) of H(2)O(2) caused a minor increase (<28%, 4 h) in the expression of Prxs I, IV, and VI. Severe oxidant stress (250-500 microM H(2)O(2)) caused a significant increase in the proportion of the monomeric forms of Prxs I-IV; this was reversible at lower H(2)O(2) concentrations (< or =250 microM). This recovery of Prx overoxidation differed among the various Prxs; Prx I was recovered within 24 h, but recovery required 48 h for Prx III. Overall, Prxs are not significantly modulated by mild oxidant stress or cytokines, but there is variable, though reversible, overoxidation in these proteins during severe oxidant exposure.     

Glutathionylation induces the dissociation of 1-Cys D-peroxiredoxin non-covalent homodimer

1-Cys peroxiredoxins (1-Cys Prxs) are antioxidant enzymes that catalyze the reduction of hydroperoxides into alcohols using a strictly conserved cysteine. 1-Cys B-Prxs, homologous to human PrxVI, were recently shown to be reactivated by glutathione S-transferase (GST) pi via the formation of a GST-Prx heterodimer and Prx glutathionylation. In contrast, 1-Cys D-Prxs, homologous to human PrxV, are reactivated by the glutaredoxin-glutathione system through an unknown mechanism. To investigate the mechanistic events that mediate the 1-Cys D-Prx regeneration, interaction of the Prx with glutathione was studied by mass spectrometry and NMR. This work reveals that the Prx can be glutathionylated on its active site cysteine. Evidences are reported that the glutathionylation of 1-Cys D-Prx induces the dissociation of the Prx non-covalent homodimer, which can be recovered by reduction with dithiothreitol. This work demonstrates for the first time the existence of a redox-dependent dimer-monomer switch in the Prx family, similar to the decamer-dimer switch for the 2-Cys Prxs.     

Mitochondria of Saccharomyces cerevisiae contain one-conserved cysteine type peroxiredoxin with thioredoxin peroxidase activity

Peroxiredoxins are ubiquitously expressed proteins that reduce hydroperoxides using disulfur-reducing compounds as electron donors. Peroxiredoxins (Prxs) have been classified in two groups dependent on the presence of either one (1-Cys Prx) or two (2-Cys Prx) conserved cysteine residues. Moreover, 2-Cys Prxs, also named thioredoxin peroxidases, have peroxide reductase activity with the use of thioredoxin as biological electron donor. However, the biological reducing agent for the 1-Cys Prx has not yet been identified. We report here the characterization of a 1-Cys Prx from yeast Saccharomyces cerevisiae that we have named Prx1p. Prx1p is located in mitochondria, and it is overexpressed when cells use the respiratory pathway, as well as in response to oxidative stress conditions. We show also that Prx1p has peroxide reductase activity in vitro using the yeast mitochondrial thioredoxin system as electron donor. In addition, a mutated form of Prx1p containing the absolutely conserved cysteine as the only cysteine residue also shows thioredoxin-dependent peroxide reductase activity. This is the first example of 1-Cys Prx that has thioredoxin peroxidase activity. Finally, exposure of null Prx1p mutant cells to oxidant conditions reveals an important role of the mitochondrial 1-Cys Prx in protection against oxidative stress.     

Repeated Superovulation via PMSG/hCG Administration Induces 2-Cys Peroxiredoxins Expression and Overoxidation in the Reproductive Tracts of Female Mice

Superovulation induced by exogenous gonadotropin treatment (PMSG/hCG) increases the number of available oocytes in humans and animals. However, Superovulatory PMSG/hCG treatment is known to affect maternal environment, and these effects may result from PMSG/hCG treatment-induced oxidative stress. 2-Cys peroxiredoxins (2-Cys Prxs) act as antioxidant enzymes that protect cells from oxidative stress induced by various exogenous stimuli. Therefore, the objective of this study was to test the hypothesis that repeated PMSG/hCG treatment induces 2-Cys Prx expression and overoxidation in the reproductive tracts of female mice. Immunohistochemistry and western blotting analyses further demonstrated that, after PMSG/hCG treatment, the protein expression levels of 2-Cys Prxs increased most significantly in the ovaries, while that of Prx1 was most affected by PMSG/hCG stimulation in all tissues of the female reproductive tract. Repeated PMSG/hCG treatment eventually leads to 2-Cys Prxs overoxidation in all reproductive organs of female mice, and the abundance of the 2-Cys Prxs-SO_2/3 proteins reported here supports the hypothesis that repeated superovulation induces strong oxidative stress and damage to the female reproductive tract. Our data suggest that excessive oxidative stress caused by repeated PMSG/hCG stimulation increases 2-Cys Prxs expression and overoxidation in the female reproductive organs. Intracellular 2-Cys Prx therefore plays an important role in maintaining the reproductive organ environment of female mice upon exogenous gonadotropin treatment.     

The C-terminal extension of chloroplast 2-Cys peroxiredoxin is critical for interaction with ATP

2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous enzymes that have been implicated in peroxide-mediated signaling of markedly different processes, such as cancer and photosynthesis. A highly conserved C-terminal extension of eukaryotic homologues modulates both the overoxidation of cysteines and the formation of oligomers. Here, we reveal that the plant counterpart regulates the self-polymerization of 2-Cys Prx triggered by ATP and Mg(2+). This feature is of particular importance under oxidative stress because the interaction of ATP with 2-Cys Prx rapidly integrates nonredox chemistry of signaling pathways into a network hub governed by multiple redox transformations at cysteine residues.     

Seed 1-cysteine peroxiredoxin antioxidants are not involved in dormancy, but contribute to inhibition of germination during stress

Peroxiredoxins (Prx) are thiol-dependent antioxidants containing one (1-cysteine [-Cys]) or two (2-Cys) conserved Cys residues that protect lipids, enzymes, and DNA against reactive oxygen species. In plants, the 1-Cys Prxs are highly expressed during late seed development, and the expression pattern is dormancy related in mature seeds. We have expressed the Arabidopsis 1-Cys Prx AtPER1 in Escherichia coli and show that this protein has antioxidant activity in vitro and protects E. coli in vivo against the toxic oxidant cumene hydroperoxide. Although some 1-Cys Prxs are targeted to the nucleus, a green fluorescent protein-AtPER1 fusion protein was also localized to the cytoplasm in an onion epidermis subcellular localization assay. It has been proposed that seed Prxs are involved in maintenance of dormancy and/or protect the embryo and aleurone layer surviving desiccation against damage caused by reactive oxygen species. These hypotheses were tested using transgenic Arabidopsis lines overexpressing the barley (Hordeum vulgare) 1-Cys PER1 protein and lines with reduced levels of AtPER1 due to antisensing or RNA interference. We found no correlation between Prx levels and the duration of the after-ripening period required before germination. Thus, Prxs are unlikely to contribute to maintenance of dormancy. RNA interference lines almost devoid of AtPER1 protein developed and germinated normally under standard growth room conditions. However, seeds from lines overexpressing PER1 were less inclined to germinate than wild-type seeds in the presence of NaCl, mannitol, and methyl viologen, suggesting that Prx can sense harsh environmental surroundings and play a part in the inhibition of germination under unfavorable conditions.     

Chloroplast NADPH-Dependent Thioredoxin Reductase from Chlorella vulgaris Alleviates Environmental Stresses in Yeast Together with 2-Cys Peroxiredoxin

Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in Chlorella vulgaris. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from C. vulgaris and investigated the involvement of Chlorella NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced Chlorella 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature C. vulgaris NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature C. vulgaris 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, tert-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in Chlorella. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of mCvNTRC, and the elevated tolerances were more significant when both mCvNTRC and mCvPrx were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances.     

Distinct Characteristics of Two 2-Cys Peroxiredoxins of Vibrio vulnificus Suggesting Differential Roles in Detoxifying Oxidative Stress

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes reducing toxic peroxides. Two distinct 2-Cys Prxs, Prx1 and Prx2, were identified in Vibrio vulnificus, a facultative aerobic pathogen. Both Prxs have two conserved catalytic cysteines, C_P and C_R, but Prx2 is more homologous in amino acid sequences to eukaryotic Prx than to Prx1. Prx2 utilized thioredoxin A as a reductant, whereas Prx1 required AhpF. Prx2 contained GGIG and FL motifs similar to the motifs conserved in sensitive Prxs and exhibited sensitivity to overoxidation. MS analysis and C_P-SO₃H specific immunoblotting demonstrated overoxidation of C_P to C_P-SO₂H (or C_P-SO₃H) in vitro and in vivo, respectively. In contrast, Prx1 was robust and C_P was not overoxidized. Discrete expression of the Prxs implied that Prx2 is induced by trace amounts of H₂O₂ and thereby residential in cells grown aerobically. In contrast, Prx1 was occasionally expressed only in cells exposed to high levels of H₂O₂. A mutagenesis study indicated that lack of Prx2 accumulated sufficient H₂O₂ to induce Prx1. Kinetic properties indicated that Prx2 effectively scavenges low levels of peroxides because of its high affinity to H₂O₂, whereas Prx1 quickly degrades higher levels of peroxides because of its high turnover rate and more efficient reactivation. This study revealed that the two Prxs are differentially optimized for detoxifying distinct ranges of H₂O₂, and proposed that Prx2 is a residential scavenger of peroxides endogenously generated, whereas Prx1 is an occasional scavenger of peroxides exogenously encountered. Furthermore, genome sequence database search predicted widespread coexistence of the two Prxs among bacteria.     

Crystal structure of an archaeal peroxiredoxin from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1

Peroxiredoxins (Prxs) are thiol-dependent peroxidases that catalyze the detoxification of various peroxide substrates such as H2O2, peroxinitrite, and hydroperoxides, and control some signal transduction in eukaryotic cells. Prxs are found in all cellular organisms and represent an enormous superfamily. Recent genome sequencing projects and biochemical studies have identified a novel subfamily, the archaeal Prxs. Their primary sequences are similar to those of the 1-Cys Prxs, which use only one cysteine residue in catalysis, while their catalytic properties resemble those of the typical 2-Cys Prxs, which utilize two cysteine residues from adjacent monomers within a dimer in catalysis. We present here the X-ray crystal structure of an archaeal Prx from the aerobic hyperthermophilic crenarchaeon, Aeropyrum pernix K1, determined at 2.3 A resolution (Rwork of 17.8% and Rfree of 23.0%). The overall subunit arrangement of the A.pernix archaeal Prx is a toroid-shaped pentamer of homodimers, or an (alpha2)5 decamer, as observed in the previously reported crystal structures of decameric Prxs. The basic folding topology and the peroxidatic active site structure are essentially the same as those of the 1-Cys Prx, hORF6, except that the C-terminal extension of the A.pernix archaeal Prx forms a unique helix with its flanking loops. The thiol group of the peroxidatic cysteine C50 is overoxidized to sulfonic acid. Notably, the resolving cysteine C213 forms the intra-monomer disulfide bond with the third cysteine, C207, which should be a unique structural characteristic in the many archaeal Prxs that retain two conserved cysteine residues in the C-terminal region. The conformational flexibility near the intra-monomer disulfide linkage might be necessary for the dramatic structural rearrangements that occur in the catalytic cycle.     

A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement

2-Cys peroxiredoxin (Prx) is the major subgroup of a family of Prx enzymes that reduce peroxide molecules such as hydrogen peroxide (H₂O₂). 2-Cys Prxs are inactivated when their active site cysteine residue is hyperoxidized to sulfinic acid. Sulfiredoxin (Srx) is an enzyme that catalyzes reduction of hyperoxidized 2-Cys Prxs in the presence of ATP, Mg²⁺, and thiol equivalent. Therefore, Srx activity is crucial for cellular function of 2-Cys Prxs. The method currently available for the determination of Srx activity relies on immunoblot detection using antibodies to hyperoxidized enzymes. Here we introduce a simple quantitative assay for Srx activity based on the colorimetric determination of inorganic phosphate released in Srx-dependent reduction of hyperoxidized Prx using the malachite green. The colorimetric assay was used for high-throughput screening of 25,000 chemicals to find Srx inhibitors.     

Sulfiredoxin protein is critical for redox balance and survival of cells exposed to low steady-state levels of H2O2

Sulfiredoxin (Srx) is an enzyme that catalyzes the reduction of cysteine sulfinic acid of hyperoxidized peroxiredoxins (Prxs). Having high affinity toward H2O2, 2-Cys Prxs can efficiently reduce H2O2 at low concentration. We previously showed that Prx I is hyperoxidized at a rate of 0.072% per turnover even in the presence of low steady-state levels of H2O2. Here we examine the novel role of Srx in cells exposed to low steady-state levels of H2O2, which can be achieved by using glucose oxidase. Exposure of low steady-state levels of H2O2 (10-20 μm) to A549 or wild-type mouse embryonic fibroblast (MEF) cells does not lead to any significant change in oxidative injury because of the maintenance of balance between H2O2 production and elimination. In contrast, loss-of-function studies using Srx-depleted A549 and Srx-/- MEF cells demonstrate a dramatic increase in extra- and intracellular H2O2, sulfinic 2-Cys Prxs, and apoptosis. Concomitant with hyperoxidation of mitochondrial Prx III, Srx-depleted cells show an activation of mitochondria-mediated apoptotic pathways including mitochondria membrane potential collapse, cytochrome c release, and caspase activation. Furthermore, adenoviral re-expression of Srx in Srx-depleted A549 or Srx-/- MEF cells promotes the reactivation of sulfinic 2-Cys Prxs and results in cellular resistance to apoptosis, with enhanced removal of H2O2. These results indicate that Srx functions as a novel component to maintain the balance between H2O2 production and elimination and then protects cells from apoptosis even in the presence of low steady-state levels of H2O2.     

Regulation of peroxiredoxins by nitric oxide in immunostimulated macrophages

Reactive oxygen species and nitric oxide (NO) are capable of both mediating redox-sensitive signal transduction and eliciting cell injury. The interplay between these messengers is quite complex, and intersection of their signaling pathways as well as regulation of their fluxes requires tight control. In this regard, peroxiredoxins (Prxs), a recently identified family of six thiol peroxidases, are central because they reduce H2O2, organic peroxides, and peroxynitrite. Here we provide evidence that endogenously produced NO participates in protection of murine primary macrophages against oxidative and nitrosative stress by inducing Prx I and VI expression at mRNA and protein levels. We also show that NO prevented the sulfinylation-dependent inactivation of 2-Cys Prxs, a reversible overoxidation that controls H2O2 signaling. In addition, studies using macrophages from sulfiredoxin (Srx)-deficient mice indicated that regeneration of 2-Cys Prxs to the active form was dependent on Srx. Last, we show that NO increased Srx expression and hastened Srx-dependent recovery of 2-Cys Prxs. We therefore propose that modulation by NO of Prx expression and redox state, as well as up-regulation of Srx expression, constitutes a novel pathway that contributes to antioxidant response and control of H2O2-mediated signal transduction in mammals.     

Overoxidation of 2-Cys Peroxiredoxin in Prokaryotes: CYANOBACTERIAL 2-CYS PEROXIREDOXINS SENSITIVE TO OXIDATIVE STRESS*?

In eukaryotic organisms, hydrogen peroxide has a dual effect; it is potentially toxic for the cell but also has an important signaling activity. According to the previously proposed floodgate hypothesis, the signaling activity of hydrogen peroxide in eukaryotes requires a transient increase in its concentration, which is due to the inactivation by overoxidation of 2-Cys peroxiredoxin (2-Cys Prx). Sensitivity to overoxidation depends on the structural GGLG and YF motifs present in eukaryotic 2-Cys Prxs and is believed to be absent from prokaryotic enzymes, thus representing a paradoxical gain of function exclusive to eukaryotic organisms. Here we show that 2-Cys Prxs from several prokaryotic organisms, including cyanobacteria, contain the GG(L/V/I)G and YF motifs characteristic of sensitive enzymes. In search of the existence of overoxidation-sensitive 2-Cys Prxs in prokaryotes, we have analyzed the sensitivity to overoxidation of 2-Cys Prxs from two cyanobacterial strains, Anabaena sp. PCC7120 and Synechocystis sp. PCC6803. In vitro analysis of wild type and mutant variants of the Anabaena 2-Cys Prx showed that this enzyme is overoxidized at the peroxidatic cysteine residue, thus constituting an exception among prokaryotes. Moreover, the 2-Cys Prx from Anabaena is readily and reversibly overoxidized in vivo in response to high light and hydrogen peroxide, showing higher sensitivity to overoxidation than the Synechocystis enzyme. These cyanobacterial strains have different strategies to cope with hydrogen peroxide. While Synechocystis has low content of less sensitive 2-Cys Prx and high catalase activity, Anabaena contains abundant and sensitive 2-Cys Prx, but low catalase activity, which is remarkably similar to the chloroplast system.     

Crystal structure of Escherichia coli thiol peroxidase in the oxidized state: insights into intramolecular disulfide formation and substrate binding in atypical 2-Cys peroxiredoxins

Thioredoxin-dependent thiol peroxidase (Tpx) from Escherichia coli represents a group of antioxidant enzymes that are widely distributed in pathogenic bacterial species and which belong to the peroxiredoxin (Prx) family. Bacterial Tpxs are unique in that the location of the resolving cysteine (CR) is different from those of other Prxs. E. coli Tpx (EcTpx) shows substrate specificity toward alkyl hydroperoxides over H2O2 and is the most potent reductant of alkyl hydroperoxides surpassing AhpC and BCP, the other E. coli Prx members. Here, we present the crystal structure of EcTpx in the oxidized state determined at 2.2-A resolution. The structure revealed that Tpxs are the second type of atypical 2-Cys Prxs with an intramolecular disulfide bond formed between the peroxidatic (CP, Cys61) and resolving (Cys95) cysteine residues. The extraordinarily long N-terminal chain of EcTpx folds into a beta-hairpin making the overall structure very compact. Modeling suggests that, in atypical 2-Cys Prxs, the CR-loop as well as the CP-loop may alternately assume the fully folded or locally unfolded conformation depending on redox states, as does the CP-loop in typical 2-Cys Prxs. EcTpx exists as a dimer stabilized by hydrogen bonds. Its substrate binding site extends to the dimer interface. A modeled structure of the reduced EcTpx in complex with 15-hydroperoxyeicosatetraenoic acid suggests that the size and shape of the binding site are particularly suited for long fatty acid hydroperoxides consistent with its greater reactivity.     

Gene structure,immune response and evolution: Comparative analysis of three 2-Cys peroxiredoxin members of miiuy croaker,Miichthys miiuy

Peroxiredoxin family was a superfamily of selenium independent peroxidases. It was divided into six subtypes: Prx1-4 (typical 2-Cys), Prx5 (atypical 2-Cys) and Prx6 (1-Cys). This study reports the isolation and characterization three 2-Cys peroxiredoxin members of full cDNA and genomic clones from miiuy croaker (Miichthys miiuy). The genetic structure analysis showed that the C-terminal catalytic Cys positioned within GEVCPAXW. This sequence was different between Prx3 and Prx4, but was conservative in different species of the same gene, the X base was S in Prx3 but G in Prx4. Tissues expression analysis showed that the expressions of Prx3 in liver and brain were much higher than other tissues; the values of Prx4 in spleen, intestine and kidney were significantly higher than others; and the expression of Prx5 in muscle was higher than that of other tissues. Real-time PCR results showed that there were highest values of these three Prxs emerging with the time post challenge of Vibrio anguillarum in liver, spleen and kidney although the highest value time differed from each other and the expression of these three genes also changed with the change of infection time. These results indicated that expression analysis of these three genes play some positive function against pathogenic bacteria infection in miiuy croaker.