Both thioredoxin 2 and glutaredoxin 2 contribute to the reduction of the mitochondrial 2-Cys peroxiredoxin Prx3

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 μmol·liter(-1); V(max), 1.2 μmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 μmol·liter(-1); V(max), 1.1 μmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.     

Cloning and functional characterisation of a peroxiredoxin 1 (NKEF A) cDNA from Atlantic salmon (Salmo salar) and its expression in fish infected with Neoparamoeba perurans

Peroxiredoxin 1 (Prx 1), also known as natural killer enhancing factor A (NKEF A), has been implicated in the immune response of both mammals and fish. Amoebic gill disease (AGD), caused by Neoparamoeba perurans, is a significant problem for the Atlantic salmon (Salmo salar L.) aquaculture industry based in Tasmania, Australia. Here we have cloned and functionally characterized a Prx 1 open reading frame (ORF) from Atlantic salmon liver and shown that Prx 1 gene expression was down-regulated in the gills of Atlantic salmon displaying symptoms of AGD. The Prx 1 ORF encoded all of the residues and motifs characteristic of typical 2-Cys Prx proteins from eukaryotes and the recombinant protein expressed in Escherichia coli catalyzed thioredoxin (Trx)-dependent reduction of H(2)O(2), cumene hydroperoxide (CuOOH) and t-butyl hydroperoxide (t-bOOH) with K(m) values of 122, 77 and 91 μM, respectively, confirming that it was a genuine 2-Cys Prx. The recombinant protein also displayed a double displacement reaction mechanism and a catalytic efficiency (k(cat)/K(m)) with H(2)O(2) of 1.5 × 10(5) M(-1) s(-1) which was consistent with previous reports for the 2-Cys Prx family of proteins. This is the first time that a Prx 1 protein has been functionally characterized from any fish species and it paves the way for further investigation of this important 2-Cys Prx family member in fish.     

Redox atlas of the mouse : Immunohistochemical detection of glutaredoxin-, peroxiredoxin-, and thioredoxin-family proteins in various tissues of the laboratory mouse

Background

Oxidoreductases of the thioredoxin family of proteins have been thoroughly studied in numerous cellular and animal models mimicking human diseases. Despite of their well documented role in various disease conditions, no systematic information on the presence of these proteins is available.

Methods

Here, we have systematically analyzed the presence of some of the major constituents of the glutaredoxin (Grx)-, peroxiredoxin (Prx)-, and thioredoxin (Trx)-systems, i.e. Grx1, Grx2, Grx3 (TXNL-2/PICOT), Grx5, nucleoredoxin (Nrx), Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, Trx1, thioredoxin reductase 1 (TrxR1), Trx2, TrxR2, and γ-glutamyl cysteine synthetase (γ-GCS) in various tissues of the mouse using immunohistochemistry.

Results

The identification of the Trx family proteins in the central nervous system, sensory organs, digestive system, lymphatic system, reproductive system, urinary system, respiratory system, endocrine system, skin, heart, and muscle revealed a number of significant differences between these proteins with respect to their distribution in these tissues.

Conclusion

Our results imply more specific functions and interactions between the proteins of this family than previously assumed.

General significance

Crucial functions of Trx family proteins have been demonstrated in various disease conditions. A detailed overview on their distribution in various tissues will be helpful to fully comprehend their potential role and the interactions of these proteins in the most thoroughly studied model for human diseases—the laboratory mouse.This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.     

Glutaredoxin-dependent peroxiredoxin from poplar: protein-protein interaction and catalytic mechanism

Recently, a poplar phloem peroxiredoxin (Prx) was found to accept both glutaredoxin (Grx) and thioredoxin (Trx) as proton donors. To investigate the catalytic mechanism of the Grx-dependent reduction of hydroperoxides catalyzed by Prx, a series of cysteinic mutants was constructed. Mutation of the most N-terminal conserved cysteine of Prx (Cys-51) demonstrates that it is the catalytic one. The second cysteine (Cys-76) is not essential for peroxiredoxin activity because the C76A mutant retained approximately 25% of the wild type Prx activity. Only one cysteine of the Grx active site (Cys-27) is essential for peroxiredoxin catalysis, indicating that Grx can act in this reaction either via a dithiol or a monothiol pathway. The creation of covalent heterodimers between Prx and Grx mutants confirms that Prx Cys-51 and Grx Cys-27 are the two residues involved in the catalytic mechanism. The integration of a third cysteine in position 152 of the Prx, making it similar in sequence to the Trx-dependent human Prx V, resulted in a protein that had no detectable activity with Grx but kept activity with Trx. Based on these experimental results, a catalytic mechanism is proposed to explain the Grx- and Trx-dependent activities of poplar Prx.     

Segment-specific overexpression of redoxins after renal ischemia and reperfusion: protective roles of glutaredoxin 2, peroxiredoxin 3, and peroxiredoxin 6

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.     

Thioredoxin and glutaredoxin system proteins—immunolocalization in the rat central nervous system

Background

The oxidoreductases of the thioredoxin (Trx) family of proteins play a major role in the cellular response to oxidative stress. Redox imbalance is a major feature of brain damage. For instance, neuronal damage and glial reaction induced by a hypoxic–ischemic episode is highly related to glutamate excitotoxicity, oxidative stress and mitochondrial dysfunction. Most animal models of hypoxia–ischemia in the central nervous system (CNS) use rats to study the mechanisms involved in neuronal cell death, however, no comprehensive study on the localization of the redox proteins in the rat CNS was available.

Methods

The aim of this work was to study the distribution of the following proteins of the thioredoxin and glutathione/glutaredoxin (Grx) systems in the rat CNS by immunohistochemistry: Trx1, Trx2, TrxR1, TrxR2, Txnip, Grx1, Grx2, Grx3, Grx5, and γ-GCS, peroxiredoxin 1 (Prx1), Prx2, Prx3, Prx4, Prx5, and Prx6. We have focused on areas most sensitive to a hypoxia–ischemic insult: Cerebellum, striatum, hippocampus, spinal cord, substantia nigra, cortex and retina.

Results and conclusions

Previous studies implied that these redox proteins may be distributed in most cell types and regions of the CNS. Here, we have observed several remarkable differences in both abundance and regional distribution that point to a complex interplay and crosstalk between the proteins of this family.

General significance

We think that these data might be helpful to reveal new insights into the role of thiol redox pathways in the pathogenesis of hypoxia–ischemia insults and other disorders of the CNS.This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.     

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin

Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H₂O₂ and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m⁻¹ s⁻¹) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.     

Study of the thiol/disulfide redox systems of the anaerobe Desulfovibrio vulgaris points out pyruvate:ferredoxin oxidoreductase as a new target for thioredoxin 1

Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balance-controlling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent K(m) value for Trx1 of 8.9 μM. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (K(m) for NADPH, 743 μM; K(m) for NADH, 5.6 μM), and Trx3 was unable to reduce insulin. The K(m) value of TR3 for Trx3 was 1.12 μM. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1(C33S) as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme.     

The tetrameric structure of Haemophilus influenza hybrid Prx5 reveals interactions between electron donor and acceptor proteins

Cellular redox control is often mediated by oxidation and reduction of cysteine residues in the redox-sensitive proteins, where thioredoxin and glutaredoxin (Grx) play as electron donors for the oxidized proteins. Despite the importance of protein-protein interactions between the electron donor and acceptor proteins, there has been no structural information for the interaction of thioredoxin or Grx with natural target proteins. Here, we present the crystal structure of a novel Haemophilus influenza peroxiredoxin (Prx) hybrid Prx5 determined at 2.8-A resolution. The structure reveals that hybrid Prx5 forms a tightly associated tetramer where active sites of Prx and Grx domains of different monomers interact with each other. The Prx-Grx interface comprises specific charge interactions surrounded by weak interactions, providing insight into the target recognition mechanism of Grx. The tetrameric structure also exhibits a flexible active site and alternative Prx-Grx interactions, which appear to facilitate the electron transfer from Grx to Prx domain. Differences of electron donor binding surfaces in Prx proteins revealed by an analysis based on the structural information explain the electron donor specificities of various Prx proteins.     

New thioredoxins and glutaredoxins as electron donors of 3'-phosphoadenylylsulfate reductase

Reduction of inorganic sulfate to sulfite in prototrophic bacteria occurs with 3'-phosphoadenylylsulfate (PAPS) as substrate for PAPS reductase and is the first step leading to reduced sulfur for cellular biosynthetic reactions. The relative efficiency as reductants of homogeneous highly active PAPS reductase of the newly identified second thioredoxin (Trx2) and glutaredoxins (Grx1, Grx2, Grx3, and a mutant Grx1C14S) was compared with the well known thioredoxin (Trx1) from Escherichia coli. Trx1, Trx2, and Grx1 supported virtually identical rates of sulfite formation with a Vmax ranging from 6.6 units mg-1 (Trx1) to 5.1 units mg-1 (Grx1), whereas Grx1C14S was only marginally active, and Grx2 and Grx3 had no activity. The structural difference between active reductants had no effect upon Km PAPS (22.5 microM). Grx1 effectively replaced Trx1 with essentially identical Km-values: Km trx1 (13.7 microM), Km grx1 (14.9 microM), whereas the Km trx2 was considerably higher (34.2 microM). The results agree with previous in vivo data suggesting that Trx1 or Grx1 is essential for sulfate reduction but not for ribonucleotide reduction in E. coli.     

Nuclear and cytoplasmic peroxiredoxin-1 differentially regulate NF-kappaB activities

Peroxiredoxins (Prx) are widely distributed and abundant proteins, which control peroxide concentrations and related signaling mechanisms. Prx1 is found in the cytoplasm and nucleus, but little is known about compartmentalized Prx1 function during redox signaling and oxidative stress. We targeted expression vectors to increase Prx1 in nuclei (NLS-Prx1) and cytoplasm (NES-Prx1) in HeLa cells. Results showed that NES-Prx1 inhibited NF-kappaB activation and nuclear translocation. In contrast, increased NLS-Prx1 did not affect NF-kappaB nuclear translocation but increased activity of a NF-kappaB reporter. Both NLS-Prx1 and NES-Prx1 inhibited NF-kappaB p50 oxidation, suggesting that oxidation of the redox-sensitive cysteine in p50's DNA-binding domain is regulated via peroxide metabolism in both compartments. Interestingly, following treatment with H(2)O(2), nuclear thioredoxin-1 (Trx1) redox status was protected by NLS-Prx1, and cytoplasmic Trx1 was protected by NES-Prx1. Compartmental differences from increasing Prx1 show that the redox poise of cytoplasmic and nuclear thiol systems can be dynamically controlled through peroxide elimination. Such spatial resolution and protein-specific redox differences imply that the balance of peroxide generation/metabolism in microcompartments provides an important specific component of redox signaling.     

Aldehyde oxidase functions as a superoxide generating NADH oxidase: an important redox regulated pathway of cellular oxygen radical formation

The enzyme aldehyde oxidase (AO) is a member of the molybdenum hydroxylase family that includes xanthine oxidoreductase (XOR); however, its physiological substrates and functions remain unclear. Moreover, little is known about its role in cellular redox stress. Utilizing electron paramagnetic resonance spin trapping, we measured the role of AO in the generation of reactive oxygen species (ROS) through the oxidation of NADH and the effects of inhibitors of AO on NADH-mediated superoxide (O(2)(??)) generation. NADH was found to be a good substrate for AO with apparent K(m) and V(max) values of 29 μM and 12 nmol min(-1) mg(-1), respectively. From O(2)(??) generation measurements by cytochrome c reduction the apparent K(m) and V(max) values of NADH for AO were 11 μM and 15 nmol min(-1) mg(-1), respectively. With NADH oxidation by AO, ≥65% of the total electron flux led to O(2)(??) generation. Diphenyleneiodonium completely inhibited AO-mediated O(2)(??) production, confirming that this occurs at the FAD site. Inhibitors of this NADH-derived O(2)(??) generation were studied with amidone the most potent exerting complete inhibition at 100 μM concentration, while 150 μM menadione, raloxifene, or β-estradiol led to 81%, 46%, or 26% inhibition, respectively. From the kinetic data, and the levels of AO and NADH, O(2)(??) production was estimated to be ~89 and ~4 nM/s in liver and heart, respectively, much higher than that estimated for XOR under similar conditions. Owing to the ubiquitous distribution of NADH, aldehydes, and other endogenous AO substrates, AO is predicted to have an important role in cellular redox stress and related disease pathogenesis.     

GSH-dependent peroxidase activity of the rice (Oryza sativa) glutaredoxin,a thioltransferase

Glutaredoxin (Grx) is a 12-kDa thioltransferase that reduces disulfide bonds of other proteins and maintains the redox potential of cells. In addition to its oxidoreductase activity, we report here that a rice Grx (OsGrx) can also function as a GSH-dependent peroxidase. Because of this antioxidant activity, OsGrx protects glutamine synthetase from oxidative damage. Individually replacing the conserved Cys residues in OsGrx with Ser shows that Cys(23), but not Cys(26), is essential for the thioltransferase and GSH-dependent peroxidase activities. Kinetic characterization of OsGrx reveals that the maximal catalytic efficiency (V(max)/K(m)) is obtained with cumene hydroperoxide rather than H(2)O(2) or t-butyl hydroperoxide.     

Functional analysis and expression characteristics of chloroplastic Prx IIE

Peroxiredoxins (Prxs) are ubiquitous thiol-dependent peroxidases capable of eliminating a variety of peroxides through reactive catalytic cysteines, which are regenerated by reducing systems. Based on amino acid sequences and their mode of catalysis, five groups of thiol peroxidases have been distinguished in plants, and type II Prx is one of them with representatives in many sub-cellular compartments. The mature form of poplar chloroplastic Prx IIE was expressed as a recombinant protein in Escherichia coli . The protein is able to reduce H₂O₂ and tert-butyl hydroperoxide and is regenerated by both glutaredoxin (Grx) and thioredoxin (Trx) systems. Nevertheless, compared with Trxs, Grxs, and more especially chloroplastic Grx S12, are far more efficient reductants towards Prx IIE. The expression of Prx IIE at both the mRNA and protein levels as a function of organ type and abiotic stress conditions was investigated. Western blot analysis revealed that Prx IIE gene is constitutively expressed in Arabidopsis thaliana , mostly in young and mature leaves and in flowers. Under photo-oxidative treatment and water deficit, almost no change was observed in the abundance of Prx IIE in A.   thaliana , while the level of Prx Q (one of the two other chloroplastic Prxs with 2-Cys Prx) increased in response to both stresses, indicating that plastidic members of the Prx family exhibit specific patterns of expression under stress.     

A novel antioxidant mechanism of ebselen involving ebselen diselenide,a substrate of mammalian thioredoxin and thioredoxin reductase

The antioxidant mechanism of ebselen involves recently discovered reductions by mammalian thioredoxin reductase (TrxR) and thioredoxin (Trx) forming ebselen selenol. Here we describe a previously unknown reaction; ebselen reacts with its selenol forming an ebselen diselenide with a rate constant of 372 m(-1)s(-1). The diselenide also was a substrate of TrxR forming the selenol with K(m) of 40 microm and k(cat) of 79 min(-1) (k(cat)/K(m) of 3.3 x 10(4) m(-1)s(-1)). Trx increased the reduction because of its fast reaction with diselenide (rate constant 1.7 x 10(3) m(-1)s(-1)). Diselenide stimulated the H2O2 reductase activity of TrxR, even more efficiently with Trx present. Because the mechanism of ebselen as an antioxidant has been assumed to involve glutathione peroxidase-like activity, we compared the H2O2 reductase activity of ebselen with the GSH and Trx systems. TrxR at 50 nm, far below the estimated physiological level, gave 8-fold higher activity compared with 1 mm GSH; addition of 5 microm Trx increased this difference to 13-fold. The rate constant of ebselen selenol reacting with H2O2 was estimated to be faster than 350 m(-1)s(-1). We propose novel mechanisms for ebselen antioxidant action involving ebselen selenol and diselenide formation, with the thioredoxin system rather than glutathione as the predominant effector and target.     

Thioredoxin-dependent hydroperoxide peroxidase activity of bacterioferritin comigratory protein (BCP) as a new member of the thiol-specific antioxidant protein (TSA)/Alkyl hydroperoxide peroxidase C (AhpC) family

Escherichia coli bacterioferritin comigratory protein (BCP), a putative bacterial member of the TSA/AhpC family, was characterized as a thiol peroxidase. BCP showed a thioredoxin-dependent thiol peroxidase activity. BCP preferentially reduced linoleic acid hydroperoxide rather than H(2)O(2) and t-butyl hydroperoxide with the use of thioredoxin as an in vivo immediate electron donor. The value of V(max)/K(m) of BCP for linoleic acid hydroperoxide was calculated to be 5-fold higher than that for H(2)O(2), implying that BCP has a selective capability to reduce linoleic acid hydroperoxide. Replacement of Cys-45 with serine resulted in the complete loss of thiol peroxidase activity, suggesting that BCP is a new bacterial member of TSA/AhpC family having a conserved cysteine as the primary site of catalysis. BCP exists as a monomer, and its functional Cys-45 appeared to exist as cysteine sulfenic acid. The expression level of BCP gradually elevated during exponential growth until mid-log phase growth, beyond which the expression level was decreased. BCP was induced 3-fold by the oxidative stress given by changing the growth conditions from the anaerobic to aerobic culture. Bcp null mutant grew more slowly than its wild type in aerobic culture and showed the hypersensitivity toward various oxidants such as H(2)O(2), t-butyl hydroperoxide, and linoleic acid hydroperoxide. The peroxide hypersensitivity of the null mutant could be complemented by the expression of bcp gene. Taken together, these data suggest that BCP is a new member of thioredoxin-dependent TSA/AhpC family, acting as a general hydroperoxide peroxidase.     

Cloning, expression of a peroxiredoxin gene from Acinetobacter sp. SM04 and characterization of its recombinant protein for zearalenone detoxification

Zearalenone (ZEN) is a Fusarium mycotoxin, which has been associated with hyperestrogenism and other reproductive disorders in farm animals. ZEN-contaminated grains as well as its by-products had engendered numerous economic losses to farm animals' production, so the detoxification of ZEN-contaminated grains and its by-products would be necessary and beneficial. In this study, a peroxiredoxin (Prx) gene from Acinetobacter sp. SM04 was cloned, and over-expressed in Escherichia coli BL21 (DE3). The Prx gene of Acinetobacter sp. SM04 encodes a protein of 187 amino acids residues and NCBI BLAST program analysis of deduced amino acids shows high identity with 2-Cys Prx family. Interestingly, recombinant Prx show efficient ability to degrade ZEN using H(2)O(2). Results of MCF-7 cell proliferation assay also found ZEN were oxidized into little estrogenic metabolites by purified recombinant Prx plus H(2)O(2). Further, model experiments on decontamination of ZEN-contaminated corn using recombinant Prx were performed, and results found nearly 90% of ZEN was degraded when crushed ZEN-contaminated corn samples (nearly 1,000 μg ZEN per kg grain) were treated with purified recombinant Prx plus 0.09% (m/m) H(2)O(2) for 6h at 30°C. In addition, the optimum pH and temperature of purified recombinant Prx for ZEN degradation were 9.0 and 70°C respectively.