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The dithiocarbamate fungicide maneb (MB) has attracted interest due to increasing concern of the negative health effects of pesticides, as well as its association with Parkinson’s disease (PD). Our laboratory has previously reported distinct phenotypic changes of neuroblastoma cells exposed to acute, sub-toxic levels of MB, including decreased mitochondrial respiration, altered lactate dynamics, and metabolic stress. In this study, we aimed to further define the specific molecular mechanisms of MB toxicity through the comparison of several thiol-containing compounds and their effects on cellular energy metabolism and thiol redox nodes. Extracellular flux analyses and stable isotope labeled tracer metabolomics were employed to evaluate alterations in energy metabolism of SK-N-AS human neuroblastoma cells after acute exposure of an array of compounds, including dithiocarbamates (maneb, nabam, zineb) and other thiol-containing small molecules (glutathione, N-acetylcysteine). These studies revealed MB and its methylated form (MeDTC) as unique toxicants with significant alterations to mitochondrial respiration, proliferation, and glycolysis. We observed MB to significantly impact cellular thiol redox status by oxidizing cellular glutathione and altering the thiol redox status of peroxiredoxin 3 (Prx3, mitochondrial) after acute exposure. Redox Western blotting revealed a MB-specific modification of cellular Prx3, strengthening the argument that MB can preferentially target mitochondrial enzymes containing reactive cysteine thiols. Further, stable isotope tracer metabolomics confirmed our energetics assessments, and demonstrated that MB exposure results in acute derangement of central carbon metabolism. Specifically, we observed shunting of cellular glucose into the pentose-phosphate pathway and reduction of TCA intermediates derived from glucose and glutamine. Also, we report novel lactate utilization for TCA enrichment and glutathione synthesis after MB exposure. In summary, our results further confirm that MB exerts its toxic effects via thiol modification, and significantly transforms central carbon metabolism.

Responding appropriately to exercise is essential to maintenance of skeletal muscle mass and function at all ages and particularly during aging. Here, a hypothesis is presented that a key component of the inability of skeletal muscle to respond effectively to exercise in aging is a denervation-induced failure of muscle redox signalling. This novel hypothesis proposes that an initial increase in oxidation in muscle mitochondria leads to a paradoxical increase in the reductive state of specific cysteines of signalling proteins in the muscle cytosol that suppresses their ability to respond to normal oxidising redox signals during exercise. The following are presented for consideration:Transient loss of integrity of peripheral motor neurons occurs repeatedly throughout life and is normally rapidly repaired by reinnervation, but this repair process becomes less efficient with aging. Each transient loss of neuromuscular integrity leads to a rapid, large increase in mitochondrial peroxide production in the denervated muscle fibers and in neighbouring muscle fibers. This peroxide may initially act to stimulate axonal sprouting and regeneration, but also stimulates retrograde mitonuclear communication to increase expression of a range of cytoprotective proteins in an attempt to protect the fiber and neighbouring tissues against oxidative damage. The increased peroxide within mitochondria does not lead to an increased cytosolic peroxide, but the increases in adaptive cytoprotective proteins include some located to the muscle cytosol which modify the local cytosol redox environment to induce a more reductive state in key cysteines of specific signalling proteins. Key adaptations of skeletal muscle to exercise involve transient peroxiredoxin oxidation as effectors of redox signalling in the cytosol. This requires sensitive oxidation of key cysteine residues. In aging, the chronic change to a more reductive cytosolic environment prevents the transient oxidation of peroxiredoxin 2 and hence prevents essential adaptations to exercise, thus contributing to loss of muscle mass and function. Experimental approaches suitable for testing the hypothesis are also outlined., Graphical abstract Image 1, Highlights • It is hypothesised that denervation leads to failed redox signalling and attenuated muscle exercise responses in aging. • Loss of peripheral motor neurons leads to increased mitochondrial peroxide in denervated and neighbouring muscle fibers. • This peroxide stimulates increased expression of cytoprotective proteins to protect the fiber against oxidative damage. • Increased mitochondrial peroxide does not increase cytosolic peroxide, but adaptations reduce the cytosol redox state. • Key adaptations of skeletal muscle to exercise involve transient peroxiredoxin oxidation in the cytosol. • Peroxiredoxin oxidation is suppressed during aging by a chronic change to a more reductive local cytosolic environment.

Skeletal muscle generates superoxide during contractions which is rapidly converted to H2O2. This molecule has been proposed to activate signalling pathways and transcription factors that regulate key adaptive responses to exercise but the concentration of H2O2 required to oxidise and activate key signalling proteins in vitro is much higher than the intracellular concentration in muscle fibers following exercise. We hypothesised that Peroxiredoxins (Prx), which reacts with H2O2 at the low intracellular concentrations found in muscle, would be rapidly oxidised in contracting muscle and hence potentially transmit oxidising equivalents to downstream signalling proteins as a method for their oxidation and activation. The aim of this study was to characterise the effects of muscle contractile activity on the oxidation of Prx1, 2 and 3 and determine if these were affected by aging. Prx1, 2 and 3 were all rapidly and reversibly oxidised following treatment with low micromolar concentrations of H2O2 in C2C12 myotubes and also in isolated mature flexor digitalis brevis fibers from adult mice following a protocol of repeated isometric contractions. Significant oxidation of Prx2 was seen within 1 min (i.e. after 12 contractions), whereas significant oxidation was seen after 2 min for Prx1 and 3. In muscle fibers from old mice, Prx2 oxidation was significantly attenuated following contractile activity. Thus we show for the first time that Prx are rapidly and reversibly oxidised in response to contractile activity in skeletal muscle and hypothesise that these proteins act as effectors of muscle redox signalling pathways which are key to adaptations to exercise that are attenuated during aging., Graphical abstract Image 1, Highlights • Hydrogen peroxide is generated by skeletal muscle during contractions. • Peroxiredoxins (Prx) react with H2O2 at the physiological levels generated during contractions. • Prx 1–3 are all oxidised by H2O2 and contractile activity in muscle fibers. • Prx2 oxidation during contractions is attenuated in muscle fibers from old mice. • Prx may act as effectors in activation of redox-regulated adaptations to contractile activity in muscle.

Ebselen is an organoselenium compound exhibiting hydroperoxide- and peroxynitrite-reducing activity, acting as a glutathione peroxidase and peroxiredoxin enzyme mimetic. Ebselen reacts with a multitude of protein thiols, forming a selenosulfide bond, which results in pleiotropic effects of antiviral, antibacterial and anti-inflammatory nature. The main protease (Mpro) of the corona virus SARS-CoV-2 is a potential drug target, and a screen with over 10,000 compounds identified ebselen as a particularly promising inhibitor of Mpro (Jin, Z. et al. (2020) Nature 582, 289–293). We discuss here the reaction of ebselen with cysteine proteases, the role of ebselen in infections with viruses and with other microorganisms. We also discuss effects of ebselen in lung inflammation. In further research on the inhibition of Mpro in SARS-CoV-2, ebselen can serve as a promising lead compound, if the inhibitory effect is confirmed in intact cells in vivo. Independently of this action, potential beneficial effects of ebselen in COVID-19 are ascribed to a number of targets critical to pathogenesis, such as attenuation of inflammatory oxidants and cytokines., Graphical abstract Image 1, Highlights • Ebselen, an organoselenium compound, exhibits anti-inflammatory and antiviral activity. • Ebselen is a glutathione peroxidase and peroxiredoxin mimetic. • Ebselen reacts with a multitude of protein thiols, resulting in pleiotropic effects. • Ebselen is an effective inhibitor of Mpro, the main protease of SARS-CoV-2. • Ebselen may serve as lead compound for drugs targeting COVID-19.

Hydrogen peroxide undergoes an equilibrium reaction with bicarbonate/CO2 to produce peroxymonocarbonate (HCO4-). Peroxymonocarbonate is more reactive with thiols than H2O2 but it makes up only a small fraction of the H2O2 in physiological bicarbonate buffers so the increase in rate of oxidation of low molecular weight thiols is modest. However, for some thiol proteins such as protein tyrosine phosphatases, the rate enhancement is very much greater. We have investigated the effect of bicarbonate/CO2 on the oxidation of peroxiredoxins (Prdxs) 2 and 3. Using an assay in which reduced Prdx2 inhibits oxidation of horseradish peroxidase by H2O2, we saw no difference between phosphate and bicarbonate buffers (pH 7.4). However, hyperoxidation of both Prdxs in bicarbonate was considerably enhanced. Hyperoxidation involves the reaction of the sulfenic acid formed at the active site with a second H2O2, and prevents its condensation to a disulfide. Using LC/MS analysis, we determined that the presence of 25 mM bicarbonate/CO2 increased the ratio of hyperoxidation compared with condensation 6-fold for Prdx2 and 11-fold for Prdx3. These results imply that Prdx hyperoxidation will occur more readily under physiological conditions than appreciated from in vitro experiments, which seldom use bicarbonate buffers. They also raise the possibility that variations in bicarbonate concentration could provide a mechanism for regulating the cellular level of active Prdxs.

Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (C(P), C(R)). The first step in catalysis, the reduction of peroxide substrate, is fast, 10(7) − 10(8) M(−1)s(−1) for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also as central players in redox signaling pathways. These biological functions can be affected by post-translational modifications that could alter the peroxidase activity and/or interaction with other proteins. In particular, inactivation by hyperoxidation of C(P), which occurs when a second molecule of peroxide reacts with the C(P) in the sulfenic acid form, modulates their participation in redox signaling pathways. The higher sensitivity to hyperoxidation of some Prx has been related to the presence of structural motifs that disfavor disulfide formation at the active site, making the C(P) sulfenic acid more available for hyperoxidation or interaction with a redox protein target. We previously reported that treatment of human Prx2 with peroxynitrite results in tyrosine nitration, a post-translational modification on non-catalytic residues, yielding a more active peroxidase with higher resistance to hyperoxidation. In this work, studies on different mutants of hPrx2 confirm that the presence of the tyrosyl side-chain of Y193, belonging to the C-terminal YF motif of eukaryotic Prx, is necessary to observe the increase in Prx2 resistance to hyperoxidation. Moreover, our results underline the critical role of this structural motif on the rate of disulfide formation that determines the differential participation of Prx in redox signaling pathways.

Mitochondria are main sites of peroxynitrite formation. While at low concentrations mitochondrial peroxynitrite has been associated with redox signaling actions, increased levels can disrupt mitochondrial homeostasis and lead to pathology. Peroxiredoxin 3 is exclusively located in mitochondria, where it has been previously shown to play a major role in hydrogen peroxide reduction. In turn, reduction of peroxynitrite by peroxiredoxin 3 has been inferred from its protective actions against tyrosine nitration and neurotoxicity in animal models, but was not experimentally addressed so far. Herein, we demonstrate the human peroxiredoxin 3 reduces peroxynitrite with a rate constant of 1 × 107 M−1 s−1 at pH 7.8 and 25 °C. Reaction with hydroperoxides caused biphasic changes in the intrinsic fluorescence of peroxiredoxin 3: the first phase corresponded to the peroxidatic cysteine oxidation to sulfenic acid. Peroxynitrite in excess led to peroxiredoxin 3 hyperoxidation and tyrosine nitration, oxidative post-translational modifications that had been previously identified in vivo. A significant fraction of the oxidant is expected to react with CO2 and generate secondary radicals, which participate in further oxidation and nitration reactions, particularly under metabolic conditions of active oxidative decarboxylations or increased hydroperoxide formation. Our results indicate that both peroxiredoxin 3 and 5 should be regarded as main targets for peroxynitrite in mitochondria.

Thioredoxin system is a ubiquitous disulfide reductase system evolutionarily conserved through all living organisms. It contains thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH. TrxR can use NADPH to reduce Trx which passes the reducing equivalent to its downstream substrates involved in various biomedical events, such as ribonucleotide reductase for deoxyribonucleotide and DNA synthesis, or peroxiredoxins for counteracting oxidative stress. Obviously, TrxR stays in the center of the system to maintain the electron flow. Mammalian TrxR contains a selenocysteine (Sec) in its active site, which is not present in the low molecular weight prokaryotic TrxRs. Due to the special property of Sec, mammalian TrxR employs a different catalytic mechanism from prokaryotic TrxRs and has a broader substrate-spectrum. On the other hand, Sec is easily targeted by electrophilic compounds which inhibits the TrxR activity and may turn TrxR into an NADPH oxidase. Ebselen, a synthetic seleno-compound containing selenazol, has been tested in several clinical studies. In mammalian cells, ebselen works as a GSH peroxidase mimic and mainly as a peroxiredoxin mimic via Trx and TrxR to scavenge hydrogen peroxide and peroxynitrite. In prokaryotic cells, ebselen is an inhibitor of TrxR and leads to elevation of reactive oxygen species (ROS). Recent studies have made use of the difference and developed ebselen as a potential antibiotic, especially in combination with silver which enables ebselen to kill multi-drug resistant Gram-negative bacteria. Collectively, Sec is important for the biological functions of mammalian TrxR and distinguishes it from prokaryotic TrxRs, therefore it is a promising drug target.

Intracellular redox status influences the oxidation and enzyme activity of the tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). Cumene hydroperoxide (CuHP), an organic hydroperoxide, is a known tumor promoter. However, molecular targets and action mechanism of CuHP in tumor promotion have not been well characterized. In this study, we investigated the effect of CuHP on the redox state of PTEN in HeLa cells. In addition, the intracellular reducing system of oxidized PTEN was analyzed using a biochemical approach and the effect of CuHP on this reducing system was also analyzed. While PTEN oxidized by hydrogen peroxide is progressively converted to its reduced form, PTEN was irreversibly oxidized by exposure to CuHP in HeLa cells. A combination of protein fractionation and mass analysis showed that the reducing system of PTEN was comprised of NADPH, thioredoxin reductase (TrxR), and thioredoxin (Trx). Although CuHP-mediated PTEN oxidation was not reversible in cells, CuHP-oxidized PTEN was reactivated by the exogenous Trx system, indicating that the cellular Trx redox system for PTEN is inactivated by CuHP. We present evidence that PTEN oxidation and the concomitant inhibition of thioredoxin by CuHP results in irreversible oxidation of PTEN in HeLa cells. In addition, ablation of peroxiredoxin (Prdx) enhanced CuHP-induced PTEN oxidation in cells. These results provide a new line of evidence that PTEN might be a crucial determinant of cell fate in response to cellular oxidative stress induced by organic hydroperoxides.

The Peroxiredoxin Q (PrxQ) proteins are thiol-based peroxidases that are important for maintaining redox homeostasis in several organisms. Activity of PrxQs is mediated by two cysteines, peroxidatic (Cp) and resolving (Cr), in association with a reducing partner. A PrxQ, Alr3183, from the cyanobacterium, Anabaena PCC 7120, was characterized in this study. Alr3183, which required thioredoxin A (TrxA) for peroxidase activity, was an intramolecular disulfide bond-containing monomeric protein. However, Alr3183 lacking Cp (Alr3183C46S) or Cr (Alr3183C51S) formed intermolecular disulfide linkages and was dimeric. Alr3183C46S was completely inactive, while Alr3183C51S required higher concentration of TrxA for peroxidase activity. Surface plasmon resonance analysis showed that unlike Alr3183 or Alr3183C46S, Alr3183C51S bound rather poorly to TrxA. Also, compared to the oxidized protein, the DTT-treated (reduced) Alr3183 displayed decreased interaction with TrxA. In vivo, Alr3183 was found to be induced in response to γ-radiation. On exposure to H2O2, Anabaena strain over-expressing Alr3183 showed reduced formation of ROS, intact photosynthetic pigments and consequently better survival than the wild-type, whereas overproduction of Alr3183C46S did not provide any protection. Significantly, this study (1) reveals the importance of Cr for interaction with thioredoxins and (2) demonstrates that over-expression of PrxQs can protect cyanobacteria from oxidative stresses.

A broad range of redox-regulated proteins undergo reversible disulfide bond formation on oxidation-prone cysteine residues. Heightened reactivity of the thiol groups in these cysteines also increases susceptibility to modification by organic electrophiles, a property that can be exploited in the study of redox networks. Here, we explored whether divinyl sulfone (DVSF), a thiol-reactive bifunctional electrophile, cross-links oxidant-sensitive proteins to their putative redox partners in cells. To test this idea, previously identified oxidant targets involved in oxidant defense (namely, peroxiredoxins, methionine sulfoxide reductases, sulfiredoxin, and glutathione peroxidases), metabolism, and proteostasis were monitored for cross-link formation following treatment of Saccharomyces cerevisiae with DVSF. Several proteins screened, including multiple oxidant defense proteins, underwent intermolecular and/or intramolecular cross-linking in response to DVSF. Specific redox-active cysteines within a subset of DVSF targets were found to influence cross-linking; in addition, DVSF-mediated cross-linking of its targets was impaired in cells first exposed to oxidants. Since cross-linking appeared to involve redox-active cysteines in these proteins, we examined whether potential redox partners became cross-linked to them upon DVSF treatment. Specifically, we found that several substrates of thioredoxins were cross-linked to the cytosolic thioredoxin Trx2 in cells treated with DVSF. However, other DVSF targets, like the peroxiredoxin Ahp1, principally formed intra-protein cross-links upon DVSF treatment. Moreover, additional protein targets, including several known to undergo S-glutathionylation, were conjugated via DVSF to glutathione. Our results indicate that DVSF is of potential use as a chemical tool for irreversibly trapping and discovering thiol-based redox partnerships within cells.

Mitochondria produce hydrogen peroxide (H2O2) during energy metabolism in most mammalian cells as well as during the oxidation of cholesterol associated with the synthesis of steroid hormones in steroidogenic cells. Some of the H2O2 produced in mitochondria is released into the cytosol, where it serves as a key regulator of various signaling pathways. Given that mitochondria are equipped with several H2O2-eliminating enzymes, however, it had not been clear how mitochondrial H2O2 can escape destruction by these enzymes for such release. Peroxiredoxin III (PrxIII) is the most abundant and efficient H2O2-eliminating enzyme in mitochondria of most cell types. We found that PrxIII undergoes reversible inactivation through hyperoxidation of its catalytic cysteine residue to cysteine sulfinic acid, and that release of mitochondrial H2O2 likely occurs as a result of such PrxIII inactivation. The hyperoxidized form of PrxIII (PrxIII-SO2H) is reduced and reactivated by sulfiredoxin (Srx). We also found that the amounts of PrxIII-SO2H and Srx undergo antiphasic circadian oscillation in mitochondria of the adrenal gland, heart, and brown adipose tissue of mice maintained under normal conditions. Cytosolic Srx was found to be imported into mitochondria via a mechanism that requires formation of a disulfide-linked complex with heat shock protein 90, which is likely promoted by H2O2 released from mitochondria. The imported Srx was found to be degraded by Lon protease in a manner dependent on PrxIII hyperoxidation state. The coordinated import and degradation of Srx underlie Srx oscillation and consequent PrxIII-SO2H oscillation in mitochondria. The rhythmic change in the amount of PrxIII-SO2H suggests that mitochondrial release of H2O2 is also likely a circadian event that conveys temporal information on steroidogenesis in the adrenal gland and on energy metabolism in heart and brown adipose tissue to cytosolic signaling pathways.

Microglial activation is a hallmark of neurodegenerative diseases. ROS activates microglia by regulating transcription factors to express pro-inflammatory genes and is associated with disruption of Ca2+ homeostasis through thiol redox modulation. Recently, we reported that Prx5 can regulate activation of microglia cells by governing ROS. In addition, LPS leads to excessive mitochondrial fission, and regulation of mitochondrial dynamics involved in a pro-inflammatory response is important for the maintenance of microglial activation. However, the precise relationship among these signals and the role of Prx5 in mitochondrial dynamics and microglial activation is still unknown. In this study, we demonstrated that Ca2+/calcineurin-dependent de-phosphorylation of Drp1 induces mitochondrial fission and regulates mitochondrial ROS production, which influences the expression of pro-inflammatory mediators in LPS-induced microglia cells. Moreover, it is likely that cytosolic and Nox-derived ROS were upstream of mitochondrial fission and mitochondrial ROS generation in activated microglia cells. Prx5 regulates LPS-induced mitochondrial fission through modulation of Ca2+/calcineurin-dependent Drp1 de-phosphorylation by eliminating Nox-derived and cytosolic ROS. Therefore, we suggest that mitochondrial dynamics may be essential for understanding pro-inflammatory responses and that Prx5 may be used as a new therapeutic target to prevent neuroinflammation and neurodegenerative diseases.

Redox signaling is now recognized as an important regulatory mechanism for a number of cellular processes including the antioxidant response, phosphokinase signal transduction and redox metabolism. While there has been considerable progress in identifying the cellular machinery involved in redox signaling, quantitative measures of redox signals have been lacking, limiting efforts aimed at understanding and comparing redox signaling under normoxic and pathogenic conditions. Here we have outlined some of the accepted principles for redox signaling, including the description of hydrogen peroxide as a signaling molecule and the role of kinetics in conferring specificity to these signaling events. Based on these principles, we then develop a working definition for redox signaling and review a number of quantitative methods that have been employed to describe signaling in other systems. Using computational modeling and published data, we show how time- and concentration- dependent analyses, in particular, could be used to quantitatively describe redox signaling and therefore provide important insights into the functional organization of redox networks. Finally, we consider some of the key challenges with implementing these methods.

Alzheimer's disease (AD), a neurodegenerative disorder, is caused by amyloid-beta oligomers (AβOs). AβOs induce cell death by triggering oxidative stress and mitochondrial dysfunction. A recent study showed that AβO-induced oxidative stress is associated with extracellular signal-regulated kinase (ERK)-dynamin related protein 1 (Drp1)-mediated mitochondrial fission. Reactive oxygen species (ROS) are regulated by antioxidant enzymes, especially peroxiredoxins (Prxs) that scavenge H2O2. These enzymes inhibit neuronal cell death induced by various neurotoxic reagents. However, it is unclear whether Prx5, which is specifically expressed in neuronal cells, protects these cells from AβO-induced damage. In this study, we found that Prx5 expression was upregulated by AβO-induced oxidative stress and that Prx5 decreased ERK-Drp1-mediated mitochondrial fragmentation and apoptosis of HT-22 neuronal cells. Prx5 expression was affected by AβO, and amelioration of oxidative stress by N-acetyl-L-cysteine decreased AβO-induced Prx5 expression. Prx5 overexpression reduced ROS as well as RNS and apoptotic cell death but Prx5 knockdown did not. In addition, Prx5 overexpression ameliorated ERK-Drp1-mediated mitochondrial fragmentation but Prx5 knockdown did not. These results indicated that inducible Prx5 expression by AβO plays a key role in inhibiting both ERK-Drp1-induced mitochondrial fragmentation and neuronal cell death by regulating oxidative stress. Thus, Prx5 may be a new therapeutic agent for treating AD.

Hydrogen peroxide (H2O2) acts as a signaling molecule via its reactions with particular cysteine residues of certain proteins. Determining the roles of direct oxidation by H2O2 versus disulfide exchange reactions (i.e. relay reactions) between oxidized and reduced proteins of different identities is a current focus. Here, we use kinetic modeling to estimate the spatial and temporal localization of H2O2 and its most likely oxidation targets during a sudden increase in H2O2 above the basal level in the cytosol. We updated a previous redox kinetic model with recently measured parameters for HeLa cells and used the model to estimate the length and time scales of H2O2 diffusion through the cytosol before it is consumed by reaction. These estimates were on the order of one micron and one millisecond, respectively. We found oxidation of peroxiredoxin by H2O2 to be the dominant reaction in the network and that the overall concentration of reduced peroxiredoxin is not significantly affected by physiological increases in intracellular H2O2 concentration. We used this information to reduce the model from 22 parameters and reactions and 21 species to a single analytical equation with only one dependent variable, i.e. the concentration of H2O2, and reproduced results from the complete model. The reduced kinetic model will facilitate future efforts to progress beyond estimates and precisely quantify how reactions and diffusion jointly influence the distribution of H2O2 within cells.

Peroxynitrite [ONOO(H)] is an oxidant associated with deleterious effects in cells. Because it is an inorganic peroxide that reacts rapidly with peroxidases, we speculated that cells may respond to ONOO(H) and H2O2 challenge in a similar manner. We exposed yeast cells to SIN-1, a well-characterized ONOO(H) generator, and observed stimulation of catalase and peroxiredoxin (Prx) activities. Previously, we reported that H2O2 challenge increases these activities in wild-type cells and in cells producing the hyperactive mutant H2O2 sensor Ccp1(W191F) but not in Ccp1-knockout cells (ccp1Δ). We find here that the response of ccp1Δ and ccp1(W191F) cells to SIN-1 mirrors that to H2O2, identifying Ccp1 as a sensor of both peroxides. SIN-1 simultaneously releases (•)NO and O2(•-), which react to form ONOO(H), but exposure of the three strains separately to an (•)NO donor (spermine-NONOate) or an O2(•-) generator (paraquat) mainly depresses catalase or Prx activity, whereas co-challenge with the NONOate and paraquat stimulates these activities. Because Ccp1 appears to sense ONOO(H) in cells, we examined its reaction with ONOO(H) in vitro and found that peroxynitrous acid (ONOOH) rapidly (k210(6)M(-1)s(-1)) oxidizes purified Ccp1 to an intermediate with spectral and ferrocytochrome-oxidizing properties indistinguishable from those of its well-characterized compound I formed with H2O2. Importantly, the nitrite released from ONOOH is not oxidized to (•)NO2 by Ccp1(׳)s compound I, unlike peroxidases involved in immune defense. Overall, our results reveal that yeast cells mount a common antioxidant response to ONOO(H) and H2O2, with Ccp1 playing a pivotal role as an inorganic peroxide sensor.

Life under aerobic conditions has shaped peroxiredoxins (Prx) as ubiquitous thiol-dependent hydroperoxidases and redox sensors. Structural features that balance the catalytically active or inactive redox states of Prx, and, therefore, their hydroperoxidase or sensor function, have so far been analyzed predominantly for Prx1-type enzymes. Here we identify and characterize two modulatory residues of the Prx5-type model enzyme PfAOP from the malaria parasite Plasmodium falciparum. Gain- and loss-of-function mutants reveal a correlation between the enzyme parameters and the inactivation susceptibility of PfAOP with the size of residue 109 and the presence or absence of a catalytically relevant but nonessential cysteine residue. Based on our kinetic data and the crystal structure of PfAOPL109M, we suggest a novel mechanism for balancing the hydroperoxidase activity and inactivation susceptibility of Prx5-type enzymes. Our study provides unexpected insights into Prx structure–function relationships and contributes to our understanding of what makes Prx good enzymes or redox sensors.

The diterpenoid, adenanthin, represses tumor growth and prolongs survival in mouse promyelocytic leukemia models (Liu et al., Nat. Chem. Biol. 8, 486, 2012). It was proposed that this was done by inactivating peroxiredoxins (Prxs) 1 and 2 through the formation of an adduct specifically on the resolving Cys residue. We confirmed that adenanthin underwent Michael addition to isolated Prx2, thereby inhibiting oxidation to a disulfide-linked dimer. However, contrary to the original report, both the peroxidatic and the resolving Cys residues could be derivatized. Glutathione also formed an adenanthin adduct, reacting with a second-order rate constant of 25±5 M(-1) s(-1). With 50 µM adenanthin, the peroxidatic and resolving Cys of Prx2 reacted with half-times of 7 and 40 min, respectively, compared with 10 min for GSH. When erythrocytes or Jurkat T cells were treated with adenanthin, we saw no evidence for a reaction with Prxs 1 or 2. Instead, adenanthin caused time- and concentration-dependent loss of GSH followed by dimerization of the Prxs. Prxs undergo continuous oxidation in cells and are normally recycled by thioredoxin reductase and thioredoxin. Our results indicate that Prx reduction was inhibited. We observed rapid inhibition of purified thioredoxin reductase (half-time 5 min with 2 µM adenanthin) and in cells, thioredoxin reductase was much more sensitive than GSH and loss of both preceded accumulation of oxidized Prxs. Thus, adenanthin is not a specific Prx inhibitor, and its reported antitumor and anti-inflammatory effects are more likely to involve more general inhibition of thioredoxin and/or glutathione redox pathways.

Peroxiredoxin-2 (Prx2), a typical two-cysteine peroxiredoxin, is the third most abundant protein in red cells. Although progress has been made in the functional characterization of Prx2, its role in red cell membrane protein homeostasis is still under investigation. Here, we studied Prx2(-/-) mouse red cells. The absence of Prx2 promotes (i) activation of the oxidative-induced Syk pathway; (ii) increased band 3 Tyr phosphorylation, with clustered band 3; and (iii) increased heat shock protein (HSP27 and HSP70) membrane translocation. This was associated with enhanced in vitro erythrophagocytosis of Prx2(-/-) red cells and reduced Prx2(-/-) red cell survival, indicating the possible role of Prx2 membrane recruitment in red cell aging and in the clearance of oxidized hemoglobin and damaged proteins through microparticles. Indeed, we observed an increased release of microparticles from Prx2(-/-) mouse red cells. The mass spectrometric analysis of erythroid microparticles found hemoglobin chains, membrane proteins, and HSPs. To test these findings, we treated Prx2(-/-) mice with antioxidants in vivo. We observed that N-acetylcysteine reduced (i) Syk activation, (ii) band 3 clusterization, (iii) HSP27 membrane association, and (iv) erythroid microparticle release, resulting in increased Prx2(-/-) mouse red cell survival. Thus, we propose that Prx2 may play a cytoprotective role in red cell membrane protein homeostasis and senescence.

Peroxiredoxin 5 is a member of the peroxiredoxin family, which has been shown to act as an antioxidant whose main function is to reduce reactive oxygen species in cells. Peroxiredoxin 5 has been found to be abnormally elevated in human osteoarthritic chondrocytes. However, the detailed mechanism by which peroxiredoxin 5 modulates human osteoarthritic chondrocytes' survival has not been elucidated. In the current study, we demonstrated that peroxiredoxin 5 knockdown activated osteoarthritic chondrocytes apoptosis, and decreased scavenging of endogenous reactive oxygen species. Furthermore, silencing of peroxiredoxin 5 resulted in an altered expression of proteins associated with Wnt signaling. Collectively, these results demonstrated that the regulatory effects of peroxiredoxin 5 can be partially attributed to Wnt/β-catenin signaling.

The thioredoxin (Trx) system, which is composed of NADPH, thioredoxin reductase (TrxR), and thioredoxin, is a key antioxidant system in defense against oxidative stress through its disulfide reductase activity regulating protein dithiol/disulfide balance. The Trx system provides the electrons to thiol-dependent peroxidases (peroxiredoxins) to remove reactive oxygen and nitrogen species with a fast reaction rate. Trx antioxidant functions are also shown by involvement in DNA and protein repair by reducing ribonucleotide reductase, methionine sulfoxide reductases, and regulating the activity of many redox-sensitive transcription factors. Moreover, Trx systems play critical roles in the immune response, virus infection, and cell death via interaction with thioredoxin-interacting protein. In mammalian cells, the cytosolic and mitochondrial Trx systems, in which TrxRs are high molecular weight selenoenzymes, together with the glutathione-glutaredoxin (Grx) system (NADPH, glutathione reductase, GSH, and Grx) control the cellular redox environment. Recently mammalian thioredoxin and glutathione systems have been found to be able to provide the electrons crossly and to serve as a backup system for each other. In contrast, bacteria TrxRs are low molecular weight enzymes with a structure and reaction mechanism distinct from mammalian TrxR. Many bacterial species possess specific thiol-dependent antioxidant systems, and the significance of the Trx system in the defense against oxidative stress is different. Particularly, the absence of a GSH-Grx system in some pathogenic bacteria such as Helicobacter pylori, Mycobacterium tuberculosis, and Staphylococcus aureus makes the bacterial Trx system essential for survival under oxidative stress. This provides an opportunity to kill these bacteria by targeting the TrxR-Trx system.

Chagas’ disease (CD), caused by the protozoan Trypanosoma cruzi, remains a major public health concern. During invasion, T. cruzi must deal with the oxidative environment found inside the mammalian host in order to spread and persist in the infected tissues for years. The parasite antioxidant armamentarium is critical for parasite survival either in cell (macrophage/cardiomyocyte) or animal models. In this work, we used a human isolated pathogenic T. cruzi clone (C8C3) that was maintained for years by regular mice passages preserving its virulence (C8C3hvir) or, in axenic culture, turning it avirulent (C8C3lvir). To study if the antioxidant armamentarium influences this virulence behaviour, we searched for the expression of different antioxidant enzymes. Specifically, we found that the virulent cell line expresses higher levels of mitochondrial (TcMPX) and cytosolic (TcCPX) peroxiredoxins (Prx) respect to the avirulent cell line. To determine if these changes in Prx expression may be involved in parasite virulence, we genetically-modified the avirulent cell line in order to overexpress TcCPX. Avirulent parasites overexpressing TcCPX restored infectivity to macrophages and cardiomyocytes in culture and, importantly, in the mouse model of CD.

Hydrogen sulfide (H2S) has been proposed to be a signaling agent. However, its mechanisms of action are usually unknown. H2S can modify cysteine residues to persulfides (RSSH), poorly characterized species proposed to mediate the signaling effects of H2S. The successful intracellular pathogen Mycobacterium tuberculosis (Mt), the causative agent of tuberculosis disease, is able to produce H2S and to import it from the host, although the effects of H2S on Mt physiology and virulence are still unknown. In this work, we utilized the one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mt (MtAhpE-S-) as a model to study the formation and reactions of protein persulfides. The peroxidatic cysteine in MtAhpE is oxidized by different peroxides to a stable sulfenic acid (MtAhpE-SOH), which reacts with H2S to form a persulfide derivative (MtAhpE-SS-). The formation of MtAhpE-SS- was evidenced by treatment with iodoacetamide and detection of the alkyl derivative (MtAhpE-SS-R) by mass spectrometry. The kinetics of persulfide formation reaction was determined (103 M-1s-1 at pH 7.4, 25oC) by two independent competition assays. In addition, MtAhpE catalyzed the reduction of H2O2 by H2S indicating that H2S was able to complete the catalytic cycle of MtAhpE. However, H2S is not expected to be the preferred reducing substrate in vivo, since it would not compete with mycothiol/mycoredoxin-1. Reactivity of MtAhpE-SS- was compared with that of MtAhpE-S-: MtAhpE-SS- reacted faster in non-specific nucleophilic reactions (such as with the disulfide 4,4’-dithiodipyridine) but lost the specific fast reactivity with hydroperoxides (H2O2 and peroxynitrite). Computational simulations indicated that persulfidation of peroxidatic cysteine leads to a disruption of the hydrogen bond network at the active site, which is required for fast hydroperoxide reduction. Overall, the data indicate that persulfuration is a posttranslational modification that can alter the reactivities of cysteine residues in different directions depending on their specific roles and protein microenvironments.

Oxidative stress, mediated by reactive oxygen species (ROS) like superoxide and H2O2, is thought to promote pancreatic β-cell dysfunction and contribute to type 1 and type 2 diabetes. When blood glucose is chronically elevated, it is thought that increased oxidative phosphorylation results in accumulation of ROS in β-cells due to electron leak from the mitochondrial electron transport chain. However, early reports that β-cells express low levels of detoxifying enzymes, including catalase and glutathione peroxidases, suggested that β-cells are ill-equipped to detoxify ROS. This notion has been propagated by the common use of bolus H2O2 delivery to cells. However, the logic supporting the “vulnerable β-cell” model is unclear when considering the importance of β-cells to survival of the organism as well as the tight coupling of oxidative phosphorylation to insulin secretion. Here, using glucose oxidase to deliver H2O2 continuously over time, rather than the more traditional bolus delivery, we found that β-cells are capable of detoxifying micromolar levels of this oxidant. Treatment with H2O2 bolus, but not glucose oxidase, results in DNA damage, activation of the DNA damage response, and depletion of cellular energy stores. We find that β-cells readily express peroxiredoxins, thioredoxins, and thioredoxin reductase, essential components of the peroxiredoxin/thioredoxin antioxidant system. Either inhibition or specific knockdown of thioredoxin reductase or peroxiredoxins 1 or 3 sensitizes β-cells to continuously-generated H2O2, suggesting that this mechanism is essential for β-cells to detoxify this ROS. Finally, inhibition of thioredoxin reductase sensitizes β-cells to peroxynitrite, a potent RNS generated by the reaction of superoxide and nitric oxide. Together, these studies, which directly contradict the current dogma, suggest that β-cells are able to detoxify H2O2 and peroxynitrite through a peroxiredoxin-dependent mechanism, and may not be so vulnerable after all.

Down syndrome (DS) results from the triplication of whole or part of chromosome 21. Individuals with DS experience a variety of comorbidities at a higher rate than the euploid population, and many of these pathologies involve oxidative stress as an etiological factor. Blood and postmortem tissue samples from DS individuals indicate elevated basal levels of oxidative stress, potentially due to mitochondrial dysfunction. Due to these observations we hypothesized that cells from DS individuals would display greater basal oxidation in the cytoplasmic and mitochondrial compartments, and that manipulation of the thioredoxin (Trx) or glutathione (GSH) systems would exacerbate this oxidation. To investigate this, we employed four pairs of patient-derived euploid or DS fibroblasts, and utilized auranofin or BSO to selectively modulate the Trx and GSH systems, respectively. Additionally, peroxiredoxin (Prx) redox Western blots were used to assess cytoplasmic (Prx1) and mitochondrial (Prx3) redox status. Results from these studies showed that inhibition of the Trx system via auranofin induced significant oxidation of both cytoplasmic and mitochondrial compartments. Interestingly, depletion of GSH by BSO did not cause significant oxidation of either Prx1 or Prx3 in DS vs. euploid controls. Mechanistically, increased sensitivity in compartmental oxidation observed in DS may be explained by our observation of significantly decreased protein levels of thioredoxin reductase (TrxR) 1 and 2, as well as significantly decreased TrxR activity in DS. Together, these data indicate that DS individuals may be highly susceptible to exposures or treatments known to oxidize the thiol redox proteome, thereby enhancing the risk of initiation and progression of DS-related comorbidities.

Hydrogen peroxide is widely viewed as the main second messenger in redox signaling, and it has been proposed that deactivation of the antioxidant peroxiredoxin (Prdx) enzymes allows free peroxide to accumulate and directly oxidize target proteins (the floodgate model). We assessed the role of cytosolic Prdxs 1 and 2 in peroxide-induced activation of the apoptosis signaling kinase 1 (ASK1)/p38 signaling pathway, in which oxidation of ASK1 is required for phosphorylation of p38. In response to peroxide, Prdx1 catalyzed oxidation of ASK1 to a disulfide-linked multimer, and this occurred via transient formation of a Prdx1-ASK1 mixed disulfide intermediate. Oxidation of ASK1 and phosphorylation of p38 were inhibited by knockdown of Prdx1, but also by overexpression of Prdx2. This suggests that these two cytosolic Prdxs have distinct roles in the cellular peroxide response and compete for available peroxide substrate. These data imply that Prdx1 can function as a peroxide receptor in response to extracellular H(2)O(2), receiving the peroxide signal and transducing it into a disulfide bond that is subsequently transmitted to the substrate, ASK1, resulting in p38 phosphorylation. Interestingly, in response to peroxide, Prdx1 and Prdx3 transiently formed reducible higher molecular weight complexes, suggesting that multiple proteins are targets for Prdx-mediated oxidation via a disulfide-exchange mechanism. This model of active peroxide signal distribution via disulfide exchange is consistent with Prdx function in yeast and explains how peroxides may trigger specific disulfide bond formation in mammalian cells.

Peroxiredoxins (Prxs) constitute a family of peroxidases in which cysteine serves as the primary site of oxidation during the reduction of peroxides. Members of the 2-Cys Prx subfamily of Prxs (Prx I to IV in mammals) are inactivated via hyperoxidation of the active-site cysteine to sulfinic acid (Cys-SO(2)H) during catalysis and are reactivated via an ATP-consuming reaction catalyzed by sulfiredoxin (Srx). This reversible hyperoxidation reaction has been proposed to protect H(2)O(2) signaling molecules from premature removal by 2-Cys Prxs or to upregulate the chaperone function of these enzymes. In addition to its sulfinic acid reductase activity, Srx catalyzes the removal of glutathione (deglutathionylation) from modified proteins. The physiological relevance of both the reversible hyperoxidation of 2-Cys Prxs and the deglutathionylation catalyzed by Srx remains unclear. Recent findings have revealed that Srx expression is induced in mammalian cells under a variety of conditions, such as in metabolically stimulated pancreatic β cells, in immunostimulated macrophages, in neuronal cells engaged in synaptic communication, in lung cells exposed to hyperoxia or cigarette smoke, in hepatocytes of ethanol-fed animals, and in several types of cells exposed to chemopreventive agents. Such induction of Srx in mammalian cells is regulated at the transcriptional level, predominantly via activator protein-1 and/or nuclear factor erythroid 2-related factor 2. Srx expression is also regulated at the translational level in Saccharomyces cerevisiae.

Hexavalent chromium [Cr(VI)] compounds are highly redox active and have long been recognized as potent cytotoxins and carcinogens. The intracellular reduction of Cr(VI) generates reactive Cr intermediates, which are themselves strong oxidants, as well as superoxide, hydrogen peroxide, and hydroxyl radical. These probably contribute to the oxidative damage and effects on redox-sensitive transcription factors that have been reported. However, the identification of events that initiate these signaling changes has been elusive. More recent studies show that Cr(VI) causes irreversible inhibition of thioredoxin reductase (TrxR) and oxidation of thioredoxin (Trx) and peroxiredoxin (Prx). Mitochondrial Trx2/Prx3 are more sensitive to Cr(VI) treatment than cytosolic Trx1/Prx1, although both compartments show thiol oxidation with higher doses or longer treatments. Thiol redox proteomics demonstrate that Trx2, Prx3, and Trx1 are among the most sensitive proteins in cells to Cr(VI) treatment. Their oxidation could therefore represent initiating events that have widespread implications for protein thiol redox control and for multiple aspects of redox signaling. This review summarizes the effects of Cr(VI) on the TrxR/Trx system and how these events could influence a number of downstream redox signaling systems that are influenced by Cr(VI) exposure. Some of the signaling events discussed include the activation of apoptosis signal regulating kinase and MAP kinases (p38 and JNK) and the modulation of a number of redox-sensitive transcription factors including AP-1, NF-κB, p53, and Nrf2.

Not all cigarette smokers develop chronic obstructive pulmonary disease, and discovering susceptibility factors is an important research priority. The oxidative burden of smoking may overwhelm antioxidant defenses, and vulnerabilities may exist as a result of sequence variants in genes encoding antioxidant enzymes. This study explored the association between genetic variation in a network of antioxidant enzymes and lung phenotypes. Linear models evaluated single-locus marker associations in 2387 European American and African American participants in the Health, Aging, and Body Composition Study. After corrections were made for multiple comparisons, 15 statistically significant associations were identified, all of which were for SNP by smoking interactions. The most statistically significant findings were for genes encoding members of the isocitrate dehydrogenase gene family ( IDH3A, IDH3B, IDH2 ). For rs6107100 ( IDH3B ) the variant genotype was associated with a difference of 6% in the FEV 1 /FVC ratio in African American current smokers, but the SNP had little or no association with FEV 1 /FVC in former and never smokers (nominal p interaction = 5 × 10 − 6 ). A variant of the peroxiredoxin gene (rs9787810, PRDX5 ) was associated with lower percentage predicted FEV 1 and a lower ratio in European American current smokers, with little or no association in other smoking groups (nominal p interaction = 0.0001 and 0.0003, respectively). The studied genes have not been reported in previous candidate gene association studies, and thus the findings suggest novel mechanisms and targets for future research and provide evidence for a contribution of sequence variation in genes encoding antioxidant enzymes to susceptibility in smokers.

Protein sulfenic acids are essential cysteine oxidations in cellular signaling pathways. The thermodynamics that drive protein sulfenylation are not entirely clear. Experimentally, sulfenic acid reduction potentials are hard to measure, because of their highly reactive nature. We designed a calculation method, the reduction potentials from electronic energies (REE) method, to give for the first time insight into the thermodynamic aspects of protein sulfenylation. The REE method is based on the correlation between reaction path-independent reaction energies and free energies of a series of analogous reactions. For human peroxiredoxin (Tpx-B), an antioxidant enzyme that forms a sulfenic acid on one of its active-site cysteines during reactive oxygen scavenging, we found that the reduction potential depends on the composition of the active site and on the protonation state of the cysteine. Interaction with polar residues directs the RSO−/RS− reduction to a lower potential than the RSOH/RSH reduction. A conserved arginine that thermodynamically favors the sulfenylation reaction might be a good candidate to favor the reaction kinetics. The REE method is not limited to thiol sulfenylation, but can be broadly applied to understand protein redox biology in general.

Peroxiredoxin 6 (Prx 6) is a bifunctional enzyme with both glutathione peroxidase and acidic Ca(2+)-independent phospholipase A(2) activities. We have recently shown that exposure of murine bone marrow-derived macrophages to LPS and IFN-gamma leads to induction of COX-2 expression and secretion of PGE(2), up-regulating Prx 6 mRNA levels. This study was designed to investigate various prostaglandins (PGs) for their ability to induce gene expression of Prxs, in particular Prx 6, and to determine the underlying regulatory mechanisms. We provide evidence that both conventional and cyclopentenone PGs enhance Prx 6 mRNA expression. Treatment with either activators or inhibitors of adenylate cyclase as well as cAMP analogs indicated that Prx 6 gene expression is regulated by adenylate cyclase in response to PGD(2) or PGE(2). Furthermore, our study revealed that JAK2, PI3K, PKC, and p38 MAPK contribute to the PGD(2)- or PGE(2)-dependent Prx 6 induction. Using stimulated macrophages from Nrf2-deficient mice or activators of Nrf2 and PPARgamma, we found that Nrf2, but not PPARgamma, is involved in the PG-dependent increase in Prx 6 mRNA expression. In summary, our data suggest multiple signaling pathways of Prx 6 regulation by PGs and identified Nrf2 as a critical player mediating transcriptional induction.

The disruption of redox control, i.e., oxidative stress, is one of the most destructive causes of ischemia-reperfusion (IR) injury. Thioredoxin (Trx) family proteins play a major role in the cellular response to oxidative stress. Here, we systematically investigated the levels and tissue distribution of 15 members of this family (Trx and TrxR 1 and 2, Nrx, Prx 1-6, and Grx 1-3 and 5) in mouse kidneys after induction of IR by comparing control, clamped, and contralateral organs. After IR, levels of various redoxins were quantified. Immunohistochemical analysis revealed segment-specific alterations induced by the ischemic insult. Grx2, Prx3, and Prx6 were highly expressed in proximal tubule cells. Overexpression of these proteins in HEK293 and HeLa cells subjected to hypoxia and reoxygenation revealed higher survival and proliferation rates and lower oxidative damage compared to controls. Furthermore, we report for the first time the accumulation of Grx1 at the apical side of distal convoluted cells and the specific secretion of Grx1 into the urine after IR. The differences in both the basal equipment and the segment-specific responses of the antioxidant proteins may contribute to the distinct susceptibilities and regeneration processes of the various segments of the nephron to the IR insult.

Drosophila mitochondria contain two peroxidases, peroxiredoxin 3 (dPrx3) and peroxiredoxin 5 (dPrx5), which together constitute the sole known intramitochondrial mechanism for the catalytic removal of hydrogen and organic peroxides. dPrx3 exists exclusively within mitochondria, whereas dPrx5 is also present in some other intracellular compartments. Levels of these two peroxiredoxins were genetically manipulated, singly and together, in D. melanogaster, for the purpose of understanding their respective functions. Underexpression of dPrx3 by 90-95% had no discernable effect on life span under normal or oxidative stress conditions; the dPrx5 null flies were previously reported to exhibit a 10% shortening of mean life span and an increase in sensitivity to oxidative stress. Flies underexpressing both dPrx3 and dPrx5 showed an 80% decrease in life span, a severe disruption in thiol homeostasis, and a massive induction of apoptosis in the muscle and digestive system tissues. The early mortality in flies underexpressing both peroxiredoxins was partially offset by overexpression of thioredoxin reductase but not mitochondrion-targeted catalase. These results suggest that mitochondrial peroxiredoxins confer specific protection for thioredoxin/glutathione systems, play a critical role in the maintenance of global thiol homeostasis, and prevent the age-associated apoptosis and premature death.

The ratio of reduced glutathione to its oxidized form (GSH/GSSG) is essential for cell viability including its role in redox-dependent regulation of gene expression. The aim of our study was to estimate GSH/GSSG ratio and expression of redox-dependent genes (glutaredoxin, thioredoxin, peroxiredoxin) under development of cancer cell resistance to cisplatin (CDDP) possessed pro-oxidant action. Under development of resistance of human erythroleukemia K562 and ovarian carcinoma SKOV-3 cells to CDDP co-ordinative enhanced expression of genes encoding glutathione synthetase (GS), subunits of γ-glutamylcysteine synthetase (γ-GCSH, γ-GCSL) was found. In addition, the growth of GSH/GSSG, enhanced expression of glutaredoxin (GLRX1, GLRX2), thioredoxin (TRX2), peroxiredoxin (PRDX6) genes as well as elevated level of transcription factor Nrf2 were observed in both types of resistant cells. We suggest that mechanism of development of cancer cell resistance to CDDP can include activation of GSH synthesis de novo, redox-dependent increase in expression of GLRX1, GLRX2, TRX2, PRDX6 genes which are co-ordinative regulated by redox-dependent transcription factor Nrf2 and elevated level of GSH/GSSG. The publication was prepared with the support of the “RUDN University Program 5–100”.

The etiological agent of Chagas disease, Trypanosoma cruzi contains 2-Cys peroxiredoxins (Prx) localized in mitochondria (TcMPX) and cytosol (TcCPX) as part of its antioxidant armamentarium. The participation of these Prx in the defense towards the host and parasite-derived oxidative stress led us to postulate them as virulence factors. In addition to its peroxidase activity, 2-Cys Prx have alternative functions. In this work, we studied the peroxidase, holdase and signaling functions of TcMPX in the context of parasite-macrophage and drug interactions (nifurtimox, NFX). Parasites overexpressing TcMPX were more infective compared to wild type (wt) under macrophage-derived O2•- and/or ONOO- generation indicating the protective role of TcMPX during infection. Additionally, TcMPX overexpressers also contain higher holdase activity and were more resistant to NFX-treatment. Importantly, wt parasites treated with NFX increase TcMPX protein content suggesting the involvement of the holdase activity in the stress response towards NFX. Finally, under stress conditions, TcMPX protein-disulfide adducts were detected indicating additional redox protein partners which may participate in the biological actions of TcMPX.

Acute pancreatitis (AP) is an acute inflammatory process of the pancreatic gland. The aim of this work was to evaluate the role of thiol oxidation of key proteins that can be involved in the regulation of the inflammatory process during AP. AP was induced in C57BL/6 mice by 7 hourly subcutaneous injections of cerulein (50 ug/kg bw). Animals were sacrificed after 1, 3, 5 and 7 injections of cerulein. One hour after the first injection, hyperoxidation of peroxiredoxin 1–4 was detected coinciding with a H2O2 peak. Three hours later, a marked up-regulation of mRNA and protein expression of sulfiredoxin, partially mediated by Nrf-2, takes place. The up-regulation of sulfiredoxin seems to be responsible for the decrease in hyperoxidation of peroxiredoxin 1–4 to allow Prdx1 and Prdx2 reduce H2O2 production in the middle phase of AP induction. During the induction of AP we have detected an overoxidation of protein phosphatases PP2A and SHP2. Inactivation of PP2A and SHP2 during acute pancreatitis by intramolecular disulphide bridges would be favour the early activation of MAPKs JNK, ERK1/2 and p38. Therefore, we conclude that modulation of the redox state of PP2A and SHP2 and peroxiredoxins by sulfiredoxin may play a key role in the up-regulation of the inflammatory cascade.

Protein tyrosine phosphatases need to be inhibited by oxidation of their active site cysteine during growth factor signaling. It is not clear how this inhibition is mediated but bicarbonate (HCO3-) has been shown to potentiate PTP1B inactivation. Therefore we investigated the effects of HCO3- and H2O2 on PTP1B activity in the presence of a reducing system constituted of Prx2, Trx1 and TrxR1. Recombinant PTP1B, TrxR1, Trx1 and Prx2 together with NADPH were used to mimic cellular growth factor-dependent PTP1B regulation in vitro. Effects of HCO3- and H2O2 exposure were assessed by PTP1B activity analysis. We found that HCO3-- and H2O2 inactivated PTP1B also in the presence of the complete TrxR1 / Trx1 / Prx system. Interestingly, a dynamic PTP1B inactivation pattern mimicking an oxidative burst was observed. The capacity for PTP1B reactivation was proportional to either TrxR1 or Trx1 concentrations. In summary, HCO3- and H2O2 in combination potently negatively regulates PTP1B activity. TrxR1 was previously shown to prevent H2O2 dependent PTP1B inactivation. Here we found that HCO3- with H2O2 together potently overcomes TrxR1- and Trx1 -mediated protection of PTP1B and that its subsequent reactivation is dependent upon Prx2, where H2O2 clearance becomes a key regulatory event.

Peroxiredoxin (Prx) enzymes are considered major systems for reduction of peroxides in mitochondria. Although the import of Prx from cytosolic ribosomes to distinct mitochondrial subcompartments is driven by a cleavable N-terminal presequence, the mechanisms underlying this process are still elusive. Here, we show by cell fractionation/western blot analysis that yeast mitochondrial Prx (Prx1) displays dual localization: matrix and intermembrane space. Prx1 sorting into the intermembrane space likely involves the release of the precursor protein within the lipid bilayer of the inner membrane, followed by cleavage by the inner membrane peptidase complex (IMP). Alternatively, during its translocation into the matrix, Prx1 is sequentially cleaved by mitochondrial processing peptidase (MPP) and then by octapeptidyl aminopeptidase 1 (Oct1). Oct1 removed eight amino acid residues from the N-terminal region of Prx1, but its physiological role is still elusive. Remarkably, the processing of Prx proteins by Oct1 appears to be evolutionarily conserved, since yeast Oct1 could also cleave the human mitochondrial peroxiredoxin Prx3 when this thiol peroxidase was expressed in yeast. In addition, the alignment of the N-terminal amino acid sequences of Prx3 from mammals indicated the putative cleavage sites by MPP and Oct1 proteases. Remarkably, there are orthologues of IMP and Oct1 proteins in human cells and their malfunctioning was associated with diseases. For instance, loss of Oct1 provokes disturbances in iron metabolism and loss of mitochondrial DNA. Currently, we are investigating the import of mammalian Prx enzymes into distinct mitochondrial subcompartments and the involvement of IMP and Oct1orthologues.

Normal red blood cells (RBCs) are subject to a high level of oxidative stress as a result of the continuous production of the superoxide anion that accompanies hemoglobin (Hb) autoxidation. The superoxide anion is dismutated to hydrogen peroxide (H2O2), which is further converted to the hydroxyl radical through the Fenton reaction in the presence of iron. To cope with oxidative stress, RBCs are equipped with Cu and Zn-dependent SOD (SOD1), catalase, glutathione peroxidase 1 (GPx1), and three isoforms of peroxiredoxin (Prx I, Prx II, and Prx VI). SOD1 converts the superoxide anion to H2O2, which is then removed by catalase, GPx1 and the Prxs. Prx II, the third most abundant protein in RBCs, is responsible for eliminating low concentrations of H2O2, whereas catalase scavenges H2O2 efficiently at high concentrations of the oxidant. Mice that lack Prx II thus develop hemolytic anemia, whereas RBC-related defects are not apparent in catalase-deficient mice. Deficiency of GPx1 renders human RBCs susceptible to oxidant stress. The primary physiologic substrate of GPx1 in RBCs is lipid hydroperoxide. GPx1 is susceptible to irreversible inactivation by its own substrates, likely as a consequence of the irreversible conversion of the active-site selenocysteine residue to dehydroalanine (DHA). Prx enzymes also are inactivated occasionally during catalysis because the active-site cysteine undergoes oxidation to sulfinic acid (Cys–SO2H). Reactivation of Prx I and Prx II is achieved by reduction of the sulfinic moiety catalyzed by sulfiredoxin. No mechanism has been identified for reactivation of the sulfinic form of Prx VI, however. Catalase is resistant to inactivation by its own substrate. In addition to Hb autoxidation, oxidative stress on RBCs can be derived from superoxide anion–generating enzymes (NADPH oxidase, xanthine oxidase) present in the endothelium and leukocytes. Catalase, glutathione peroxidase 1 (GPx1), and Prxs are responsible for the elimination of H2O2 in RBCs. Compared with catalase and GPx, which do not have a specific binding site for H2O2, Prxs function more effectively at low H2O2 concentrations because they do possess a high-affinity binding site for this molecule. Prx II is also much more abundant than the other two enzymes. RBCs express three Prx isoforms: PrxI, PrxII, and PrxVI. During my talk, I will discuss specific roles of PrxII, catalase, and Gpx1 in RBCs, based on the results obtained using RBCs obtained from various KO mice.

Cytosolic 2-cys peroxiredoxin (2-cysPrx) exhibiting thioredoxin-dependent hydroperoxide reductase activity has been demonstrated to be involved in a number of signaling processes, such as receptor tyrosine kinase and MAP kinase activation. However, its role in the cell death pathway has yet to be elucidated. Here we show that cytosolic 2-cysPrx suppresses the TNF-alpha-induced apoptosis of human cervical cancer cells in a caspase-8-dependent manner. The HeLa cervical cancer cells expressing a dominant negative mutant (DN) of a cytosolic 2-cysPrx manifested remarkable increase in intracellular reactive oxygen species level, which was counteracted by catalase administration, and apoptotic cell death induced by combined treatment of TNF-alpha and cycloheximide compared to the control (CT) cells. Similarly, the DN cells were also susceptible to apoptosis induced by the TNF-related apoptosis-inducing ligand. The apoptosis enhanced by DN expression was shown to be dependent on a typical FADD/caspase pathway. The DN cells undergoing apoptosis showed enhanced caspase-8 and -3 activations, as compared to the CT cells. In contrast, there was no difference observed in the sustained JNK activation between CT and DN cells. Thus, this study illustrates that intracellular reactive oxygen species regulated by cytosolic 2-cysPrx is involved in the TNF-alpha-induced apoptotic cell death via controlling caspase activation.

We analyzed the role of the peroxisomal peroxiredoxin Pmp20 of the yeast Hansenula polymorpha. Cells of a PMP20 disruption strain (pmp20) grew normally on substrates that are not metabolized by peroxisomal enzymes, but showed a severe growth defect on methanol, the metabolism of which involves a hydrogen peroxide producing peroxisomal oxidase. This growth defect was paralleled by leakage of peroxisomal matrix proteins into the cytosol. Methanol-induced pmp20 cells accumulated enhanced levels of reactive oxygen species and lipid peroxidation products. Moreover, the fatty acid composition of methanol-induced pmp20 cells differed relative to WT controls, suggesting an effect on fatty acid homeostasis. Plating assays and FACS-based analysis of cell death markers revealed that pmp20 cells show loss of clonogenic efficiency and membrane integrity, when cultured on methanol. We conclude that the absence of the peroxisomal peroxiredoxin leads to loss of peroxisome membrane integrity and necrotic cell death. (C) 2008 Elsevier Inc. All Fights reserved

Oxidants are produced as a by-product of aerobic metabolism, and organisms ranging from prokaryotes to mammals have evolved with an elaborate and redundant complement of antioxidant defenses to confer protection against oxidative insults. Compelling data now exist demonstrating that oxidants are used in physiological settings as signaling molecules with important regulatory functions controlling cell division, migration, contraction, and mediator production. These physiological functions are carried out in an exquisitely regulated and compartmentalized manner by mild oxidants, through subtle oxidative events that involve targeted amino acids in proteins. The precise understanding of the physiological relevance of redox signal transduction has been hampered by the lack of specificity of reagents and the need for chemical derivatization to visualize reversible oxidations. In addition, it is difficult to measure these subtle oxidation events in vivo. This article reviews some of the recent findings that illuminate the significance of redox signaling and exciting future perspectives. We also attempt to highlight some of the current pitfalls and the approaches needed to advance this important area of biochemical and biomedical research.

The components of the redox metabolism in Entamoeba histolytica have been recently revisited by Arias et al. (Free Radic. Biol. Med. 42:1496-1505; 2007), after the identification and characterization of a thioredoxin-linked system. The present work deals with studies performed for a better understanding of the localization and identification of different components of the redox machinery present in the parasite. The gene encoding for amoebic thioredoxin 8 was cloned and the recombinant protein typified as having properties similar to those of thioredoxin 41. The ability of these thioredoxins and the specific reductase to assemble a system utilizing NADPH to metabolize hydroperoxides in association with a peroxiredoxin has been kinetically characterized. The peroxiredoxin behaved as a typical 2 cysteine enzyme, exhibiting a ping-pong mechanism with hyperbolic saturation kinetics for thioredoxin 8 (Km = 3.8 μM), thioredoxin 41 (Km = 3.1 μM), and tert-butyl hydroperoxide (Km about 35 μM). Moreover, the tandem system involving thioredoxin reductase and either thioredoxin proved to be operative for reducing low molecular weight disulfides, including putative physiological substrates as cystine and oxidized trypanothione. Thioredoxin reductase and thioredoxin 41 (by association also the functional redox system) have been immunolocalized underlying the plasma membrane in Entamoeba histolytica cells. These findings suggest an important role for the metabolic pathway involving thioredoxin as a redox interchanger, which could be critical for the maintenance and virulence of the parasite when exposed to highly toxic reactive oxygen species. Fil: Arias, Diego Gustavo. Universidad Nacional del Litoral; Argentina Fil: Carranza, Pedro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Lujan, Hugo Daniel. Universidad Católica de Córdoba. Facultad de Medicina; Argentina Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina

It is hypothesized that activation of death receptors disrupts the redox homeostasis of cells and that this contributes to the induction of apoptosis. The redox status of the peroxiredoxins, which are extremely sensitive to increases in H2O2 and disruption of the thioredoxin system, were monitored in Jurkat T lymphoma cells undergoing Fas-mediated apoptosis. The only detectable change during the early stages of apoptosis was oxidation of mitochondrial peroxiredoxin 3. Increased H2O2 triggers peroxiredoxin overoxidation to a sulphinic acid; however during apoptosis peroxiredoxin 3 was captured as a disulfide, suggesting impairment of the thioredoxin system responsible for maintaining peroxiredoxin 3 in its reduced form. Peroxiredoxin 3 oxidation was an early event, occurring within the same timeframe as increased mitochondrial oxidant production, caspase activation and cytochrome c release. It preceded other major apoptotic events including mitochondrial permeability transition and phosphatidylserine exposure, and glutathione depletion, global thiol protein oxidation and protein carbonylation. Peroxiredoxin 3 oxidation was also observed in U937 cells stimulated with TNF-alpha. We hypothesize that the selective oxidation of peroxiredoxin 3 leads to an increase in mitochondrial H2O2 and that this may influence the progression of apoptosis.

The Anabaena sp. (ANASP) genome contains seven open reading frames with homology to thiol peroxidase (TPx), also known as peroxiredoxin (Prx). Based on sequence similarities among putative TPx's derived from various cyanobacteria genomes, we designated the seven putative TPx members as VCP, VCT, TCS, and GCT clusters according to the sequence of their conserved catalytic motif. The GCT cluster consists of four members, named GCT1, GCT2, GCT3, and GCT4. The ANASP GCT-TPx genes were recombinantly expressed in Escherichia coli. The purified proteins were characterized with an emphasis on the ability to destroy various peroxides, the electron donor, and the conserved cysteine structure as a catalytic intermediate. All GCT members, as an atypical 2-Cys TPx family, exerted the highest peroxidase activity toward a lipid hydroperoxide using an electron from thioredoxin. Periplasmic protein analysis revealed that GCT2 and GCT4 are distributed in the cytoplasm, whereas GCT1 and GCT3, homologues of E. coli bacterioferritin comigratory protein/plant PrxQ, are localized in the periplasmic space. Immunoblots of the heterocystic proteins showed that the level of GCT2 in the heterocyst is comparable to that in the vegetative cell, whereas the other GCT members were not significantly detected in the heterocyst. The transcriptional responses of ANASP GCT genes to various oxidative stresses and growth environments were multifarious. Their intrinsic differences in transcriptional responsiveness and cellular localization suggest that this large GCT cluster is designed as an adaptive strategy to efficiently combat lipid hydroperoxide in Anabaena sp. that perform oxygenic photosynthesis and N(2) fixation.

The genome of the cyanobacterium Anabaena PCC 7120 encodes seven polypeptides showing sequence similarities with peroxiredoxins (Prx-s). One of them, prxQ-A (alr2503), which encodes a Prx Q homologue, is located in the same gene cluster as pkn22, which encodes a Ser/Thr kinase. Here we report that the pkn22-knockout mutant (Mp22) is sensitive to oxidative stress because it fails to synthesize PrxQ-A; the expression of prxQ-A is significantly induced under oxidative stress conditions. The hypersensitivity of the Mp22 mutant to oxidative stress was restored by inducing the expression of the prxQ-A gene in trans. The recombinant PrxQ-A protein shows antioxidant activity protecting the DNA from being degraded by reactive oxygen species, catalyzes the reduction of H2O2 in the presence of DTT, and shows thioredoxin-dependent peroxidase activity in vitro. The conserved Cys47 residue is the peroxide oxidation site, since the replacement of Cys47 by a Ser residue completely abolished the peroxidase activity. All these data suggest that PrxQ-A may efficiently protect this organism from oxidative stress.

The mammalian 1-Cys peroxiredoxin (Prdx6) is a unique member of the peroxiredoxin family of proteins capable of protecting cells from metal-catalyzed oxidative damage. We recently identified Prdx6 as a candidate for the quantitative trait locus Ath1, a gene responsible for a difference in diet-induced atherosclerosis susceptibility in mice. To investigate the role of Prdx6 in atherosclerosis, we generated transgenic mice that overexpress the Prdx6 allele from the Ath1-resistant 129/SvJ strain on an Ath1-susceptible C57BL/6J background. These mice expressed significantly elevated levels of Prdx6 mRNA and protein in multiple tissues including liver, aorta, and peritoneal macrophages, which accumulated significantly lower levels of hydrogen peroxide, revealing an enhanced antioxidant activity in these mice. However, overexpression of Prdx6 had no protective effect on LDL oxidation in vitro, and transgenic mice fed an atherogenic diet for 10 weeks did not possess an increased resistance to atherosclerosis nor did they maintain the high prediet plasma HDL levels consistent with the Ath1-resistant phenotype. In addition, the Prdx6 allele from the susceptible strain was shown to have a higher antioxidant activity than that of the resistant strains. These data suggest that the increased peroxidase activity attributable to Prdx6 overexpression in transgenic mice is not sufficient to protect mice from atherosclerosis, and that Prdx6 is not likely to be the gene underlying Ath1.