Molecular cloning and expression of a 2-Cys peroxiredoxin gene in the crustacean Eurypanopeus depressus induced by acute hypo-osmotic stress

Peroxiredoxins (Prxs) are a family of ubiquitous proteins that help minimize the harmful effects of oxidative stress by catalyzing the reduction of hydrogen peroxide (H₂O₂) and organic hydroperoxides to less harmful forms. A full-length cDNA corresponding to a 2-Cys Prx gene was isolated from the flatback mud crab Eurypanopeus depressus and designated as EdPrx-1 (GenBank accession no. EU684547). EdPrx-1 has a major open-reading frame of 594 bp and is capable of encoding a polypeptide of 198 amino acid residues. Like other 2-Cys Prxs, EdPrx-1 protein possesses two conserved cysteine residues that play an essential role for the antioxidant activity of the proteins. The EdPrx-1 protein, as deduced from the cDNA sequence, shows a high level (74–93%) of sequence similarity to the 2-Cys Prxs from other crustaceans as well as those from many arthropod species (73–76% similarity). It shares about 70% sequence similarity with homologs from mammalian species. EdPrx-1 gene is expressed at low level in the gill, hypodermis, and hepatopancreas tissues of the crab under non-stressed condition; however, its expression is elevated about three-fold in the gills under hypo-osmotic stress. This suggests a possible role in protecting against oxidative stress caused by the increased metabolic activities associated with hyperosmoregulation.     

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.     

Secretory expression and characterization of a novel peroxiredoxin for zearalenone detoxification in Saccharomyces cerevisiae

Zearalenone (ZEN) is a Fusarium mycotoxin, which is considered to be an oestrogenic endocrine disruptor found to cause severe morphological and functional disorders of reproductive organs in livestock. Increasing attention has been paid to the development of an effective strategy for ZEN decontamination. ZEN is oxidized into smaller estrogenic metabolites by a novel peroxiredoxin (Prx) isolated from Acinetobacter sp. SM04. The Prx coding gene was cloned in a secretory vector pYES2-alpha (pYα) with an alpha (α) signal peptide gene inserted into the multiple cloning site of pYES2. The recombinant Prx was secreted from Saccharomyces cerevisiae INVSc1 after inducing with 2% (w/v) galactose for 72 h, and was found to be nearly 20 kDa through 12% SDS-PAGE. The expressed amount of recombinant Prx was 0.24 mg/mL in the extracellular supernatant. Recombinant Prx showed a gradient increase at the beginning of ZEN degradation. The final ZEN degradation amount was 0.43 μg by one unit recombinant Prx after 12 h. Furthermore, the temperature, H₂O₂ concentration, and pH for highest peroxidase activity of recombinant Prx were 80 °C, 20 mM and 9.0, respectively. When compared with other peroxidases, the thermal stability and alkali resistance of recombinant Prx were much better. The results suggest that recombinant Prx is successfully expressed in S. cerevisiae.     

Hydrogen peroxide metabolism and sensing in human erythrocytes: A validated kinetic model and reappraisal of the role of peroxiredoxin II

Hydrogen peroxide (H₂O₂) metabolism in human erythrocytes has been thoroughly investigated, but unclear points persist. By integrating the available data into a mathematical model that accurately represents the current understanding and comparing computational predictions to observations we sought to (a) identify inconsistencies in present knowledge, (b) propose resolutions, and (c) examine their functional implications. The systematic confrontation of computational predictions with experimental observations of the responses of intact erythrocytes highlighted the following important discrepancy. The high rate constant (10⁷–10⁸ M⁻¹ s⁻¹) for H₂O₂ reduction determined for purified peroxiredoxin II (Prx2) and the high abundance of this protein indicate that under physiological conditions it consumes practically all the H₂O₂. However, this is inconsistent with extensive evidence that Prx2’s contribution to H₂O₂ elimination is comparable to that of catalase. Models modified such that Prx2’s effective peroxidase activity is just 10⁵ M⁻¹ s⁻¹ agree near quantitatively with extensive experimental observations. This low effective activity is probably due to a strong but readily reversible inhibition of Prx2’s peroxidatic activity in intact cells, implying that the main role of Prx2 in human erythrocytes is not to eliminate peroxide substrates. Simulations of the responses to physiological H₂O₂ stimuli highlight that a design combining abundant Prx2 with a low effective peroxidase activity spares NADPH while improving potential signaling properties of the Prx2/thioredoxin/thioredoxin reductase system.     

Molecular Basis for the Resistance of Human Mitochondrial 2-Cys Peroxiredoxin 3 to Hyperoxidation

Peroxiredoxins (Prxs) detoxify peroxides and modulate H₂O₂-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1–4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H₂O₂ to form Cys sulfinic acid (Cys-SO₂H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H₂O₂ and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation.     

Bombyx mori cathepsin D expression is induced by high temperature and H2O2 exposure

Cathepsin D is involved in the metamorphosis of the silkworm, Bombyx mori. Here, we show the expression profile of B. mori cathepsin D (BmCatD) in the fat body during exposure to stressors, such as high temperature and H₂O₂. Exposure of larvae in the fifth instar stage to high temperature (28 °C) led to accelerated metamorphosis and shortened larval stage compared to control larvae grown at 23 °C. Concomitantly, the expression level of BmCatD mRNA was greatly increased during exposure to high temperature. We also detected significantly elevated H₂O₂ levels in the hemolymph of larvae treated with high temperature. To confirm that oxidative stress induces BmCatD expression, B. mori larvae were injected with H₂O₂. As predicted, we observed increased expression of BmCatD following H₂O₂ exposure. Based on these results, we conclude that BmCatD expression is induced by high temperature and H₂O₂ exposure and that this stress-induced BmCatD expression leads to early metamorphosis.     

Microbial 2-Cys Peroxiredoxins: Insights into Their Complex Physiological Roles

The peroxiredoxins (Prxs) constitute a very large and highly conserved family of thiol-based peroxidases that has been discovered only very recently. We consider here these enzymes through the angle of their discovery, and of some features of their molecular and physiological functions, focusing on complex phenotypes of the gene mutations of the 2-Cys Prxs subtype in yeast. As scavengers of the low levels of H₂O₂ and as H₂O₂ receptors and transducers, 2-Cys Prxs have been highly instrumental to understand the biological impact of H₂O₂, and in particular its signaling function. 2-Cys Prxs can also become potent chaperone holdases, and unveiling the in vivo relevance of this function, which is still not established, should further increase our knowledge of the biological impact and toxicity of H₂O₂. The diverse molecular functions of 2-Cys Prx explain the often-hard task of relating them to peroxiredoxin genes phenotypes, which underscores the pleiotropic physiological role of these enzymes and complex biologic impact of H₂O₂.     

How pH Modulates the Dimer-Decamer Interconversion of 2-Cys Peroxiredoxins from the Prx1 Subfamily

2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H₂O₂ and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His¹¹³) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp⁷⁶ from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H₂O₂ can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H₂O₂ and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress.     

Enzymatic properties of a bee venom serine protease from the bumblebee Bombus ignitus

We previously reported that bumblebee (Bombus ignitus) venom serine protease (Bi-VSP) acts as a prophenoloxidase-activating factor in arthropods and a fibrin(ogen)olytic enzyme in mammals. In the present study, we characterized the enzymatic properties of Bi-VSP purified from B. ignitus venom. The 34-kDa active form of Bi-VSP was purified from the venom of B. ignitus worker bees. Glycoprotein staining showed that approximately 20% of the total molecular mass of Bi-VSP is due to carbohydrate moieties. Bi-VSP had an optimal pH and temperature of pH 9.0 and 40 °C, respectively, and was stable at 50 °C for at least 10 min. Bi-VSP activity decreased abruptly below pH 6.0, indicating that Bi-VSP activity is almost completely inhibited at pH 5.4 of B. ignitus venom. The protease activity of Bi-VSP was strongly inhibited by typical serine protease inhibitors such as phenylmethanesulfonyl fluoride, leupeptin, and soybean trypsin inhibitor.     

A 1-Cys Peroxiredoxin from a Thermophilic Archaeon Moonlights as a Molecular Chaperone to Protect Protein and DNA against Stress-Induced Damage

Peroxiredoxins (Prxs) act against hydrogen peroxide (H₂O₂), organic peroxides, and peroxynitrite. Thermococcus kodakaraensis KOD1, an anaerobic archaeon, contains many antioxidant proteins, including three Prxs (Tk0537, Tk0815, and Tk1055). Only Tk0537 has been found to be induced in response to heat, osmotic, and oxidative stress. Tk0537 was found to belong to a 1-Cys Prx6 subfamily based on sequence analysis and was named 1-Cys TkPrx. Using gel filtration chromatography, electron microscopy, and blue-native polyacrylamide gel electrophoresis, we observed that 1-Cys TkPrx exhibits oligomeric forms with reduced peroxide reductase activity as well as decameric and dodecameric forms that can act as molecular chaperones by protecting both proteins and DNA from oxidative stress. Mutational analysis showed that a cysteine residue at the N-terminus (Cys⁴⁶) was responsible for the peroxide reductase activity, and cysteine residues at the C-terminus (Cys²⁰⁵ and Cys²¹¹) were important for oligomerization. Based on our results, we propose that interconversion between different oligomers is important for regulating the different functions of 1-Cys TkPrx.     

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.     

A novel 1-Cys thioredoxin peroxidase gene in Apis cerana cerana: characterization of AccTpx4 and its role in oxidative stresses

Thioredoxin peroxidase (Tpx), also named peroxiredoxin (Prx), is an important peroxidase that can protect organisms against stressful environments. AccTpx4, a 1-Cys thioredoxin peroxidase gene from the Chinese honey bee Apis cerana cerana, was cloned and characterized. The AccTpx4 gene encodes a protein that is predicted to contain the conserved PVCTTE motif from 1-Cys peroxiredoxin. Quantitative real-time PCR (Q-PCR) and Western blotting revealed that AccTpx4 was induced by various oxidative stresses, such as cold, heat, insecticides, H₂O₂, and HgCl₂. The in vivo peroxidase activity assay showed that recombinant AccTpx4 protein could efficiently degrade H₂O₂ in the presence of DL-dithiothreitol (DTT). In addition, disc fusion assays revealed that AccTpx4 could function to protect cells against oxidative stresses. These results indicate that AccTpx4 plays an important role in oxidative stress responses and may contribute to the conservation of honeybees.     

A dissection of the effects of ethylene,H2O2 and high irradiance on antioxidants and several genes associated with stress and senescence in tobacco leaves

Ethylene and hydrogen peroxide are involved in the modulation of stress responses in plants, but their interrelation is not well understood. This work was designed to find differences between the actions of ethylene and H₂O₂ on antioxidants and senescence markers. Leaves of Nicotiana tabacum were sprayed with H₂O₂ or with ethephon (precursor of ethylene). To find the possible modulation of responses to acute abiotic stress, ethephon- and H₂O₂-sprayed leaves were further subjected to high irradiance (HL). The application of H₂O₂ strongly stimulated ethylene synthesis (ACC). Ethylene and H₂O₂, as single factors, stimulated the trolox equivalent antioxidant capacity (TEAC) and the activity of catalase (CAT), in contrast to HL alone (stimulation of nonspecific peroxidases and the total glutathione pool). However, after combined treatments (ethylene + HL and H₂O₂ + HL), the stimulatory action of H₂O₂ was related to TEAC and CAT activity, while the application of ethylene stimulated the total glutathione pool. Hydrogen peroxide enhanced the expression of the three CAT genes (Cat1, Cat2 and Cat3), in contrast to ethylene (Cat2 and Cat3) and HL (Cat1). In regard to the markers of senescence and pathogenesis the most pronounced difference between the actions of ethylene and H₂O₂, as single factors, was related to NPR1, whereas when leaf spraying was combined with HL, differences were found at WRKY53 and PR1a. HL reversed the stimulatory effects of H₂O₂/ethylene-driven enhancements of the expression of several genes (Cat1, Cat2, NPR1, WRKY53). These results show that multiple stressors, as usually encountered by plants in nature, may largely change those expression patterns of genes determined in a single factor analysis. Moreover, the actions of HL (often considered the internal H₂O₂ trigger) and of exogenous H₂O₂ on gene expression are clearly different.     

Effect of acetone extract from stem bark of Acacia species (A. dealbata,A. ferruginea and A. leucophloea) on antioxidant enzymes status in hydrogen peroxide-induced HepG2 cells

Acacia species are multipurpose trees, widely used in the traditional systems of medicine to treat various ailments. The major objective of the present study was to determine the gene expression of enzymatic antioxidants by acetone extract from the stem bark of three Acacia species (Acacia dealbata, Acacia ferruginea and Acacia leucophloea) in hydrogen peroxide (H₂O₂)-induced human hepatoma (HepG2) cells. The expression of antioxidant enzymes such as superoxide dismutase containing copper–zinc (CuZnSOD)/manganese (MnSOD), catalase (CAT) and glutathione peroxidase (GPx) in HepG2 cells was evaluated by real-time PCR. The results of antioxidant enzyme expression in real-time PCR study revealed that the H₂O₂ (200 μM) challenged HepG2 cells reduced the expression of enzymes such as SOD, GPx and CAT. However, the cells pre-treated with acetone extracts of all the three Acacia species significantly (P > 0.05) up-regulated the expression of antioxidant enzymes in a concentration dependent manner (25, 50 and 75 μg/mL). In conclusion, the findings of our study demonstrated that the acetone extract of Acacia species effectively inhibited H₂O₂ mediated oxidative stress and may be useful as a therapeutic agent in preventing oxidative stress mediated diseases.     

Kinetic and structural characterization of a typical two-cysteine peroxiredoxin from Leptospira interrogans exhibiting redox sensitivity

Little is known about the mechanisms by which Leptospira interrogans, the causative agent of leptospirosis, copes with oxidative stress at the time it establishes persistent infection within its human host. We report the molecular cloning of a gene encoding a 2-Cys peroxiredoxin (LinAhpC) from this bacterium. After bioinformatic analysis we found that LinAhpC contains the characteristic GGIG and YF motifs present in peroxiredoxins that are sensitive to overoxidation (mainly eukaryotic proteins). These motifs are absent in insensitive prokaryotic enzymes. Recombinant LinAhpC showed activity as a thioredoxin peroxidase with sensitivity to overoxidation by H₂O₂ (C_{hyp 1%} ~30 µM at pH 7.0 and 30 °C). So far, Anabaena 2-Cys peroxiredoxin, Helicobacter pylori AhpC, and LinAhpC are the only prokaryotic enzymes studied with these characteristics. The properties determined for LinAhpC suggest that the protein could be critical for the antioxidant defense capacity in L. interrogans.