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.     

Isolation and Characterization of a New Peroxiredoxin from Poplar Sieve Tubes That Uses Either Glutaredoxin or Thioredoxin as a Proton Donor

A sequence coding for a peroxiredoxin (Prx) was isolated from a xylem/phloem cDNA library from Populus trichocarpa and subsequently inserted into an expression plasmid yielding the construction pET-Prx. The recombinant protein was produced in Escherichia coli cells and purified to homogeneity with a high yield. The poplar Prx is composed of 162 residues, a property that makes it the shortest plant Prx sequence isolated so far. It was shown that the protein is monomeric and possesses two conserved cysteines (Cys). The Prx degrades hydrogen peroxide and alkyl hydroperoxides in the presence of an exogenous proton donor that can be either thioredoxin or glutaredoxin (Grx). Based on this finding, we propose that the poplar protein represents a new type of Prx that differs from the so-called 2-Cys and 1-Cys Prx, a suggestion supported by the existence of natural fusion sequences constituted of a Prx motif coupled to a Grx motif. The protein was shown to be highly expressed in sieve tubes where thioredoxin h and Grx are also major proteins.     

Plasmodium falciparum 2-Cys peroxiredoxin reacts with plasmoredoxin and peroxynitrite

Thioredoxin peroxidase 1 (TPx1) of the malarial parasite Plasmodium falciparum is a 2-Cys peroxiredoxin involved in the detoxification of reactive oxygen species and - as shown here - of reactive nitrogen species. As novel electron acceptor of reduced TPx1, we characterised peroxynitrite; the rate constant for ONOO- reduction by the enzyme (1 x 10(6) M(-1) s(-1) at pH 7.4 and 37 degrees C) was determined by stopped-flow measurements. As reducing substrate of TPx1, we identified - aside from thioredoxin - plasmoredoxin; this 22-kDa protein occurs only in malarial parasites. When studying the potential roles of Cys74 and Cys170 of Tpx1 in catalysis, as well as in oligomerisation behaviour, we found that replacement of Cys74 by Ala influenced neither the dimerisation nor enzymatic activity of TPx1. In the C170A mutant, however, the kcat/Km for reduced Trx as a substrate was shown to be approximately 50-fold lower and, in contrast to the wild-type enzyme, covalently linked dimers were not formed. For the catalytic cycle of TPx1, we conclude that oxidation of the peroxidatic Cys50 by the oxidising substrate is followed by the formation of an intermolecular disulfide bond between Cys50 and Cys170' of the second subunit, which is then attacked by an external electron donor such as thioredoxin or plasmoredoxin.     

Comparative study of two thioredoxin peroxidases from disk abalone (Haliotis discus discus): cloning, recombinant protein purification, characterization of antioxidant activities and expression analysis

Thioredoxin peroxidase (TPx), also named peroxiredoxin (Prx), is an important peroxidase, which can protect organisms against various oxidative stresses. Two TPxs were isolated from a disk abalone (Haliotis discus discus) cDNA library, named as AbTPx1 and AbTPx2, respectively. AbTPx1 and AbTPx2 consist of 1315 and 1045 bp full-length cDNA with 753 and 597 bp open reading frames encoding 251 and 199 amino acids, respectively. The TPx signature motif 1 (FYPLDFTFVCPTEI) and motif 2 (GEVCPA) were conserved in both AbTPx1 and AbTPx2 amino acid sequences. Purified recombinant abalone TPx fusion proteins catalyzed the reduction of H2O2 and butyl hydroperoxide in peroxidase assays. Furthermore, both AbTPx fusion proteins were shown to protect super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Escherichia coli cells transformed with AbTPx1 and AbTPx2 coding sequences in pMAL-c2x showed resistance to H2O2 at 0.8 mM concentration by in vivo H2O2 tolerance assay. AbTPx1 and AbTPx2 mRNA were constitutively expressed in gill, mantle, abductor muscle and digestive tract in a tissue specific manner. Additionally, both TPxs mRNA were up-regulated in gill and digestive tract tissues against H2O2 at 3h post injection. The results indicate that AbTPx1 and AbTPx2 gene expressions are induced by oxidative stress and their respective proteins function in the detoxification of different ROS molecules to maintain efficient antioxidant defense in disk abalone.     

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.     

Molecular and enzymatic characterization of Schistosoma mansoni thioredoxin peroxidase

The ability of Schistosoma mansoni to escape oxidative damage from immune system-generated reactive oxygen intermediates has been extensively documented. The limiting step in the parasite's detoxification process appears to be at the level of hydrogen peroxide neutralization. In the present study, the possible role of a novel class of antioxidant enzymes, thioredoxin peroxidase (TPx), in hydrogen peroxide neutralization by schistosomes was investigated. An expressed sequence tag was characterized from the Schistosoma Genome Initiative with high similarity to TPx from other organisms. The gene encodes a polypeptide containing 2 conserved active-site cysteines and flanking amino acids, and 60-70% identity with previously characterized TPx proteins. Recombinant schistosome TPx was enzymatically active and found to have thioredoxin-dependent hydrogen peroxide reducing activity of 4500 nmol hydrogen peroxide/min/mg protein. Native TPx activity was determined to be 48.1 nmol hydrogen peroxide/min/mg protein in adult worm homogenates compared with 46.9 for glutathione peroxidase. TPx activity was precipitated from adult worm homogenates with antibodies prepared against the recombinant protein. Western blotting with antibodies made against recombinant protein showed that TPx was expressed in both male and female adult worms. This is the first demonstration of a TPx activity in schistosomes and our results suggest that TPx plays a significant role in schistosome-host interactions.     

NMR reveals a novel glutaredoxin-glutaredoxin interaction interface

Glutaredoxins (Grx) represent a large family of glutathione (GSH)-dependent oxidoreductases that catalyse the reduction of disulfides or glutathione mixed disulfide. Grx domains from pathogenic bacteria and plant Grxs have been recently reported to target specific peroxiredoxins (Prxs). The specificity that triggers the interaction between Grx and Prx is poorly understood and is only based on the structure of Haemophilus influenzae Prx-Grx hybrid (hyPrx5). We report here an NMR study of the Populus tremula Grx C4 that targets a P.tremula D-type II Prx. We show that Grx C4 specifically self-associates in a monomer-dimer equilibrium with an apparent K(d) of ca 2.6 mM. Grx C4 homodimer was docked under experimental restraints. The results reveal a novel Grx-Grx interface that is unrelated to the hyPrx5 Grx-Grx dimer interface. Chemical-shift perturbations and 15N spin-relaxation measurements show that the auto-association surface comprises both the active site and the GSH binding site. Reduced GSH is demonstrated to bind reduced Grx with a K(d) of ca 8.6 mM. The potential biological significance of the new Grx-Grx interaction interface is discussed.     

Structural and Biochemical Characterization of Yeast Monothiol Glutaredoxin Grx6

Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 Å resolution revealed a novel two-strand antiparallel β-sheet opposite the GSH binding groove. This extra β-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.     

Human glutaredoxin 2 affinity tag for recombinant peptide and protein purification

Recombinant proteins are commonly expressed in fusion with an affinity tag to facilitate purification. We have in the present study evaluated the possible use of the human glutaredoxin 2 (Grx2) as an affinity tag for purification of heterologous proteins. Grx2 is a glutathione binding protein and we have shown in the present study that the protein can be purified from crude bacterial extracts by a one-step affinity chromatography on glutathione-Sepharose. We further showed that short peptides could be fused to either the N- or C-terminus of Grx2 without affecting its ability to bind to the glutathione column. However, when Grx2 was fused to either the 27 kDa green fluorescent protein or the 116 kDa beta-galactosidase, the fusion proteins lost their ability to bind glutathione-Sepharose. Insertion of linker sequences between the Grx2 and the fusion protein did not restore binding to the column. In summary, our findings suggest that Grx2 may be used as an affinity tag for purification of short peptides and possibly also certain proteins that do not interfere with the binding to glutathione-Sepharose. However, the failure of purifying either green fluorescent protein or beta-galactosidase fused to Grx2 suggests that the use of Grx2 as an affinity tag for recombinant protein purification is limited.     

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.     

Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae

Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage. In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described. This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions. Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide. A significant increase in total protein carbonyl content is constitutively observed in grx5 cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant. The synthetic lethality of the grx5 and grx2 mutations on one hand and of grx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage. Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells.     

Elucidation of a novel Vibrio cholerae lipid A secondary hydroxy-acyltransferase and its role in innate immune recognition

Similar to most Gram-negative bacteria, the outer leaflet of the outer membrane of Vibrio cholerae is comprised of lipopolysaccharide. Previous reports have proposed that V. cholerae serogroups O1 and O139 synthesize structurally different lipid A domains, which anchor lipopolysaccharide within the outer membrane. In the current study, intact lipid A species of V. cholerae O1 and O139 were analysed by mass spectrometry. We demonstrate that V. cholerae serogroups associated with human disease synthesize a similar asymmetrical hexa-acylated lipid A species, bearing a myristate (C14:0) and 3-hydroxylaurate (3-OH C12:0) at the 2'- and 3'-positions respectively. A previous report from our laboratory characterized the V. cholerae LpxL homologue Vc0213, which transfers a C14:0 to the 2'-position of the glucosamine disaccharide. Our current findings identify V. cholerae Vc0212 as a novel lipid A secondary hydroxy-acyltransferase, termed LpxN, responsible for transferring the 3-hydroxylaurate (3-OH C12:0) to the V. cholerae lipid A domain. Importantly, the presence of a 3-hydroxyl group on the 3'-linked secondary acyl chain was found to promote antimicrobial peptide resistance in V. cholerae; however, this functional group was not required for activation of the innate immune response.     

Solution structure of Escherichia coli glutaredoxin-2 shows similarity to mammalian glutathione-S-transferases.

Glutaredoxin 2 (Grx2) from Escherichia coli is distinguished from other glutaredoxins by its larger size, low overall sequence identity and lack of electron donor activity with ribonucleotide reductase. However, catalysis of glutathione (GSH)-dependent general disulfide reduction by Grx2 is extremely efficient. The high-resolution solution structure of E. coli Grx2 shows a two-domain protein, with residues 1 to 72 forming a classical "thioredoxin-fold" glutaredoxin domain, connected by an 11 residue linker to the highly helical C-terminal domain, residues 84 to 215. The active site, Cys9-Pro10-Tyr11-Cys12, is buried in the interface between the two domains, but Cys9 is solvent-accessible, consistent with its role in catalysis. The structures reveal the hither to unknown fact that Grx2 is structurally similar to glutathione-S-transferases (GST), although there is no obvious sequence homology. The similarity of these structures gives important insights into the functional significance of a new class of mammalian GST-like proteins, the single-cysteine omega class, which have glutaredoxin oxidoreductase activity rather than GSH-S-transferase conjugating activity. E. coli Grx 2 is structurally and functionally a member of this new expanding family of large glutaredoxins. The primary function of Grx2 as a GST-like glutaredoxin is to catalyze reversible glutathionylation of proteins with GSH in cellular redox regulation including stress responses.     

Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe(3+)/O(2)) but not against an oxidation system without thiol. This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx). The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis. Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase. The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.     

Role of thioredoxin peroxidase in aging of stationary cultures of Saccharomyces cerevisiae

A soluble protein from Saccharomyces cerevisiae acts as a peroxidase but requires a NADPH-dependent thioredoxin system and was named thioredoxin peroxidase (TPx). The role of TPx in aging of stationary cultures of S. cerevisiae was investigated in a wild-type strain and a mutant yeast strain in which the tsa gene that encodes TPx was disrupted by homologous recombination. The occurrence of oxidative stress during aging of stationary cultures of the yeast has been proposed. Comparison of 5-day-old (young) stationary cultures of S. cerevisiae and of cultures aged for 3 months (old) revealed decreased viability, increased generation of reactive oxygen species, modulation of cellular redox status, and increased cellular oxidative damage reflected by increased protein carbonyl content and lipid peroxidation. The magnitude of this stress was augmented in yeast mutant lacking TPx. These results suggest that TPx may play a direct role in cellular defense against aging of stationary cultures presumably, functioning as an antioxidant enzyme.     

One single in-frame AUG codon is responsible for a diversity of subcellular localizations of glutaredoxin 2 in Saccharomyces cerevisiae

Glutaredoxins belong to a family of small proteins with glutathione-dependent disulfide oxidoreductase activity involved in cellular defense against oxidative stress. The product of the yeast GRX2 gene is a protein that is localized both in the cytosol and mitochondria. To throw light onto the mechanism responsible for the dual subcellular distribution of Grx2 we analyzed mutant constructs containing different targeting information. By altering amino acid residues around the two in-frame translation initiation start sites of the GRX2 gene, we could demonstrate that the cytosolic isoform of Grx2 was synthesized from the second AUG, lacking an N-terminal extension. Translation from the first AUG resulted in a long isoform carrying a mitochondrial targeting presequence. The mitochondrial targeting properties of the presequence and the influence of the mature part of Grx2 were analyzed by the characterization of the import kinetics of specific fusion proteins. Import of the mitochondrial isoform is relatively inefficient and results in the accumulation of a substantial amount of unprocessed form in the mitochondrial outer membrane. Substitution of Met(35), the second translation start site, to Val resulted in an exclusive targeting to the mitochondrial matrix. Our results show that a plethora of Grx2 subcellular localizations could spread its antioxidant functions all over the cell, but one single A to G [corrected] mutation converts Grx2 into a typical protein of the mitochondrial matrix. The "A" denotes adenine, rather than alanine, and the "G" refers to guanine, not glycine [corrected]     

Oligomerization and chaperone activity of a plant 2-Cys peroxiredoxin in response to oxidative stress

Plant 2-Cys peroxiredoxins (2-Cys Prxs) have been reported to localize to chloroplasts and perform antioxidative roles during plant development and photosynthesis. In this study, we identified that, in addition to the well-known function of thioredoxin (Trx)-dependent peroxidase, the plant 2-Cys Prx in Chinese cabbage 2-Cys Prx1, designated C2C-Prx1, also behaves as a molecular chaperone under oxidative stress conditions, like the yeast and mammalian 2-Cys Prxs. By the chaperone function of C2C-Prx1, the protein efficiently prevented the denaturation of citrate synthase and insulin from heat shock and dithiothreitol (DTT)-induced chemical stresses. Also, the protein structure of C2C-Prx1 was shown to have discretely sized multiple structures, whose molecular sizes were in the diverse ranges of low molecular weight (LMW) proteins to high molecular weight (HMW) protein complexes. The dual functions of C2C-Prx1 acting as a peroxidase and as a molecular chaperone are alternatively switched by heat shock and oxidative stresses, accompanying with its structural changes. The peroxidase function predominates in the lower MW forms, but the chaperone function predominates in the higher MW complexes. The precise regulation of C2C-Prx1 structures and functions may play a pivotal role in the protection of plant chloroplasts from photo-oxidative stress.