Peroxiredoxin 1 and its role in cell signaling

Peroxiredoxins (Prdxs) are a family of small (22-27kDa) non-seleno peroxidases currently known to possess six mammalian isoforms. Although their individual roles in cellular redox regulation and antioxidant protection are quite distinct, they all catalyze peroxide reduction of H2O2, organic hydroperoxides and peroxynitrite. They are found to be expressed ubiquitously and in high levels, suggesting that they are both an ancient and important enzyme family. Prdxs can be divided into three major subclasses: typical 2-cysteine (2-Cys) Prdxs (Prdx1-4), atypical 2-Cys Prdx (Prdx 5) and 1-Cys Prdx (Prdx 6). Recent evidence suggests that 2-Cys peroxiredoxins are more than “just simple peroxidases”. This hypothesis has been discussed elegantly in recent review articles, considering “over”-oxidation of the protonated thiolate peroxidatic cysteine and post-translational modification of Prdxs as processes initiating a mechanistic switch from peroxidase to chaperon function. The process of over-oxidation of the peroxidatic cysteine (CP) occurs during catalysis in the presence of thioredoxin (Trx), thus rendering the sulfenic moiety to sulfinic acid , which can be reduced by sulfiredoxin (Srx). However, further oxidation to sulfonic acid is believed to promote Prdx degradation or, as recently shown, the formation of oligomeric peroxidase-inactive chaperones10 with questionable H2O2-scavenging capacity. In the light of this and given that Prdx1 has recently been shown by us and by others to interact directly with signaling molecules, we will explore the possibility that H2O2 regulates signaling in the cell in a temporal and spatial fashion via oxidizing Prdx1. Therefore, this review will focus on H2O2 modulating cell signaling via Prdxs by discussing: a) the activity of Prdxs towards H2O2; b) sub cellular localization and availability of other peroxidases, such as catalase or glutathione peroxidases; c) the availability of Prdxs reducing systems such as thioredoxin and sulfiredoxin and lastly, d) Prdx1 interacting signaling molecules.     

Analysis and expression of the class III peroxidase large gene family in Arabidopsis thaliana

Higher plants possess a large set of the classical guaiacol peroxidases (class III peroxidases, E.C. 1.11.1.7). These enzymes have been implicated in a wide array of physiological processes such as H(2)O(2) detoxification, auxin catabolism and lignin biosynthesis and stress response (wounding, pathogen attack, etc.). During the last 10 years, molecular cloning has allowed the isolation and characterization of several genes encoding peroxidases in plants. The achievement of the large scale Arabidopsis genome sequencing, combined with the DNA complementary to RNA (cDNA) expressed sequence tags projects, provided the opportunity to draw up the first comprehensive list of peroxidases in a plant. By screening the available databases, we have identified 73 peroxidase genes throughout the Arabidopsis genome. The evolution of the peroxidase multigene family has been investigated by analyzing the gene structure (intron/exon) in correlation with the phylogenetic relationships between the isoperoxidases. An evolutionary pattern of extensive gene duplications can be inferred and is discussed. Using a cDNA array procedure, the expression pattern of 23 peroxidases was established in the different organs of the plant. All the tested peroxidases were expressed at various levels in roots, while several were also detected in stems, leaves and flowers. The specific functions of these genes remain to be determined.     

The peroxiredoxin gene family in Drosophila melanogaster.

Five peroxiredoxin genes have been identified in Drosophila melanogaster on the basis of a genome-wide search. Three of the genes (DPx-4156, DPx-4783, and DPx-5037) fall into the 2-Cys subgroup, while the other two (DPx-2540 and DPx-6005) belong to the 1-Cys subgroup. Using cDNAs, all five were expressed in E. coli and the purified recombinant proteins were shown to reduce H(2)O(2) in the presence of dithiothreitol. The three 2-Cys Prx were also shown to be active in the thioredoxin system and were, consequently, classified as thioredoxin peroxidases. Antisera raised against the DPx-4783 recombinant protein crossreacted with all family members and recognized protein species of the predicted sizes (22-27 kD). All five family members, when individually overexpressed in Drosophila S2 cells, conferred some resistance to H(2)O(2) treatment, as measured by cell viability. Functional diversification of the Drosophila peroxiredoxin family members was suggested by two lines of evidence: (i) the patterns of mRNA accumulation varied for the different genes during development and (ii) recombinant proteins fused to an epitope tag and overexpressed in Drosophila cells, differed in subcellular localizations--three proteins occurred in the cytosol, one was localized to the mitochondria, and one was found to be secreted.     

Increased expression of mitochondrial peroxiredoxin-3 (thioredoxin peroxidase-2) protects cancer cells against hypoxia and drug-induced hydrogen peroxide-dependent apoptosis

Peroxiredoxin-3 (Prdx3) is a mitochondrial member of the antioxidant family of thioredoxin peroxidases that uses mitochondrial thioredoxin-2 (Trx2) as a source of reducing equivalents to scavenge hydrogen peroxide (H(2)O(2)). Low levels of H(2)O(2) produced by the mitochondria regulate physiological processes, including cell proliferation, while high levels of H(2)O(2) are toxic to the cell and cause apoptosis. WEHI7.2 thymoma cells with stable overexpression of Prdx3 displayed decreased levels of cellular H(2)O(2) and decreased cell proliferation without a change in basal levels of apoptosis. Prdx3-transfected cells showed a marked resistance to hypoxia-induced H(2)O(2) formation and apoptosis. Prdx3 overexpression also protected the cells against apoptosis caused by H(2)O(2), t-butylhydroperoxide, and the anticancer drug imexon, but not by dexamethasone. Thus, mitochondrial Prdx3 is an important cellular antioxidant that regulates physiological levels of H(2)O(2), leading to decreased cell growth while protecting cells from the apoptosis-inducing effects of high levels of H(2)O(2).     

The antioxidant systems in Toxoplasma gondii and the role of cytosolic catalase in defence against oxidative injury

Superoxide dismutase, catalase, glutathione peroxidase and peroxiredoxins form an antioxidant network protecting cells against reactive oxygen species (ROS). Catalase is a potent H2O2-detoxifying enzyme, which is unexpectedly absent in some members of the Kinetoplastida and Apicomplexa, but present in Toxoplasma gondii. In T. gondii, catalase appears to be cytosolic. In addition, T. gondii also possesses genes coding for other types of peroxidases, including glutathione/thioredoxin-like peroxidases and peroxiredoxins. This study presents a detailed analysis of the role of catalase in the parasite and reports the existence of antioxidant enzymes localized in the cytosol and the mitochondrion of T. gondii. The catalase gene was disrupted and, in addition, T. gondii cell lines overexpressing either catalase or a cytosolic 1-cys peroxiredoxin, TgPrx2, under the control of a strong promoter were created. Analysis of these mutants confirmed that the catalase activity is cytosolic and is encoded by a unique gene in T. gondii. Furthermore, the catalase confers protection against H2O2 exposure and contributes to virulence in mice. The overexpression of Prx2 also increases protection against H2O2 treatment, suggesting that catalase and other peroxidases function as a defence mechanism against endogenously produced reactive oxygen intermediates and the oxidative stress imposed by the host.     

Peroxidase-catalyzed S-oxygenation: mechanism of oxygen transfer for lactoperoxidase

The mechanism of organosulfur oxygenation by peroxidases [lactoperoxidase (LPX), chloroperoxidase, thyroid peroxidase, and horseradish peroxidase] and hydrogen peroxide was investigated by use of para-substituted thiobenzamides and thioanisoles. The rate constants for thiobenzamide oxygenation by LPX/H2O2 were found to correlate with calculated vertical ionization potentials, suggesting rate-limiting single-electron transfer between LPX compound I and the organosulfur substrate. The incorporation of oxygen from 18O-labeled hydrogen peroxide, water, and molecular oxygen into sulfoxides during peroxidase-catalyzed S-oxygenation reactions was determined by LC- and GC-MS. All peroxidases tested catalyzed essentially quantitative oxygen transfer from 18O-labeled hydrogen peroxide into thiobenzamide S-oxide, suggesting that oxygen rebound from the oxoferryl heme is tightly coupled with the initial electron transfer in the active site. Experiments using H2(18)O2, 18O2, and H2(18)O showed that LPX catalyzed approximately 85, 22, and 0% 18O-incorporation into thioanisole sulfoxide oxygen, respectively. These results are consistent with a active site controlled mechanism in which the protein radical form of LPX compound I is an intermediate in LPX-mediated sulfoxidation reactions.     

Saccharomyces cerevisiae expresses three phospholipid hydroperoxide glutathione peroxidases.

The GPX1, GPX2, and GPX3 genes of Saccharomyces cerevisiae have been reported previously to encode glutathione peroxidases (GPxs). We re-examined the sequence alignments of these proteins with GPxs from higher eukaryotes. Sequence identities, particularly with phospholipid hydroperoxide glutathione peroxidases (PHGPxs), were enhanced markedly by introduction to the yeast sequences of gaps that are characteristic of PHGPxs. PHGPx-like activity was detectable in extracts from wild-type S. cerevisiae and was diminished in extracts from gpx1 Delta, gpx2 Delta, and gpx3 Delta deletion mutants; PHGPx activity was almost absent in a gpx1 Delta/gpx2 Delta/gpx3 Delta triple mutant. Studies with cloned GPX1, GPX2, and GPX3 expressed heterologously in Escherichia coli confirmed that these genes encode proteins with PHGPx activity. An S. cerevisiae gpx1 Delta/gpx2 Delta/gpx3 Delta mutant was defective for growth in medium supplemented with the oxidation-sensitive polyunsaturated fatty acid linolenate (18:3). This sensitivity to 18:3 was more marked than sensitivity to H(2)O(2). Unlike H(2)O(2) toxicity, delayed toxicity of 18:3 toward gpx1 Delta/gpx2 Delta/gpx3 Delta cells was correlated with the gradual incorporation of 18:3 into S. cerevisiae membrane lipids and was suppressible with alpha-tocopherol, an inhibitor of lipid peroxidation. The results show that the GPX genes of S. cerevisiae, previously reported to encode GPxs, encode PHGPxs (PHGPx1, PHGPx2, and PHGPx3) and that these enzymes protect yeast against phospholipid hydroperoxides as well as nonphospholipid peroxides during oxidative stress. This is the first report of an organism that expresses PHGPx from more than one gene and produces PHGPx in the absence of a GPx.     

Water and bromide in the active center of vanadate-dependent haloperoxidases

Two aqua-oxovanadium complexes, viz. [A-VO(H2O)(sal-L-Leu)] (1) and [VO(H2O)2(5-Br-sal-Gly)] x H2O(2 x H2O), containing the water ligands in cis- and trans-positions to the oxo group at V-OH2 distances ranging from 2.008 to 2.228 A, have been structurally characterized in order to model the apical electron density feature found in the structures of fungal and algal vanadate-dependent peroxidases. Br K-edge XAS of bromide-treated bromoperoxidase from Ascophyllum nodosum and model compounds (including 2 x H2O) has been used to show that the substrate bromide does not bind to active site vanadium but to a light atom, possibly carbon, in its vicinity.     

On the role of the axial ligand in heme proteins: a theoretical study

We present a systematic investigation of how the axial ligand in heme proteins influences the geometry, electronic structure, and spin states of the active site, and the energies of the reaction cycles. Using the density functional B3LYP method and medium-sized basis sets, we have compared models with His, His+Asp, Cys, Tyr, and Tyr+Arg as found in myoglobin and hemoglobin, peroxidases, cytochrome P450, and heme catalases, respectively. We have studied 12 reactants and intermediates of the reaction cycles of these enzymes, including complexes with H(2)O, OH(-), O(2-), CH(3)OH, O(2), H(2)O(2), and HO(2)(-) in various formal oxidation states of the iron ion (II to V). The results show that His gives ~0.6 V higher reduction potentials than the other ligands. In particular, it is harder to reduce and protonate the O(2) complex with His than with the other ligands, in accordance with the O(2) carrier function of globins and the oxidative chemistry of the other proteins. For most properties, the trend Cys相似文献   

Peroxidases inhibit nitric oxide (NO) dependent bronchodilation: development of a model describing NO-peroxidase interactions

Recent studies demonstrate that nitric oxide (NO) serves as a physiological substrate for mammalian peroxidases [(2000) J. Biol. Chem. 275, 37524]. We now show that eosinophil peroxidase (EPO) and lactoperoxidase (LPO), peroxidases known to be enriched in airways of asthmatic subjects, function as a catalytic sink for NO, modulating its bioavailability and function. Using NO-selective electrodes and direct spectroscopic and rapid kinetic methods, we examined the interactions of NO with EPO and LPO compounds I and II and ferric forms and compared the results to those reported for myeloperoxidase. A unified kinetic model for NO interactions with intermediates of mammalian peroxidases during steady-state catalysis is presented that accommodates unique features observed with each member of the mammalian peroxidase superfamily. Potential functional consequences of peroxidase-NO interactions in asthma are investigated by utilizing organ chamber studies with tracheal rings. In the presence of pathophysiologically relevant levels of peroxidases and H(2)O(2), NO-dependent bronchodilation of preconstricted tracheal rings was reversibly inhibited. Thus, NO interaction with mammalian peroxidases may serve as a potential mechanism for modulating their catalytic activities, influencing the regulation of local inflammatory and infectious events in vivo.     

Proximal cavity, distal histidine, and substrate hydrogen-bonding mutations modulate the activity of Amphitrite ornata dehaloperoxidase

Dehaloperoxidase (DHP) from Amphitrite ornata is the first globin that has peroxidase activity that approaches that of heme peroxidases. The substrates 2,4,6-tribromophenol (TBP) and 2,4,6-trichlorophenol are oxidatively dehalogenated by DHP to form 2,6-dibromo-1,4-benzoquinone and 2,6-dichloro-1,4-benzoquinone, respectively. There is a well-defined internal substrate-binding site above the heme, a feature not observed in other globins or peroxidases. Given that other known heme peroxidases act on the substrate at the heme edge there is great interest in understanding the possible modes of substrate binding in DHP. Stopped-flow studies (Belyea, J., Gilvey, L. B., Davis, M. F., Godek, M., Sit, T. L., Lommel, S. A., and Franzen, S. (2005) Biochemistry 44, 15637-15644) show that substrate binding must precede the addition of H2O2. This observation suggests that the mechanism of DHP relies on H2O2 activation steps unlike those of other known peroxidases. In this study, the roles of the distal histidine (H55) and proximal histidine (H89) were probed by the creation of site-specific mutations H55R, H55V, H55V/V59H, and H89G. Of these mutants, only H55R shows significant enzymatic activity. H55R is 1 order of magnitude less active than wild-type DHP and has comparable activity to sperm whale myoglobin. The role of tyrosine 38 (Y38), which hydrogen bonds to the hydroxyl group of the substrate, was probed by the mutation Y38F. Surprisingly, abolishing this hydrogen bond increases the activity of the enzyme for the substrate TBP. However, it may open a pathway for the escape of the one-electron product, the phenoxy radical leading to polymeric products.     

Kinetic analysis of the acid-alkaline conversion of horseradish peroxidases.

The nature of the acid-alkaline conversion of horseradish peroxidases was studied by measuring four rate constants in reactions, E + H+ (k1) in equilibrium (k2) EH+ and E + H2O (k3) in equilibrium (k4) EH+ + OH-, where EH+ and E denote the acid and alkaline forms of the enzymes. The values of k1, (k2 + k3), and k4 were obtained by measuring the relaxation rates of the acid leads to alkaline and alkaline leads to acid conversions by means of th pH jump method with a stopped-flow apparatus. The value of k3 could also be obtained by measuring the rate of reactions between hydrogen peroxide and peroxidases at alkaline pH. The measurements were conducted with four peroxidases having different pKa values: peroxidase A )pKa = 9.3), peroxidase C (pKa = 11.1), diacetyldeuteroperoxidase A (pKa = 7.7), and diacetyldeuteroperoxidase C (pKa = 9.1). The value of k1 was about 10(10) M-1 s-1 in the reaction of the four enzymes while k4 was quite different between the enzymes. The pKa was determined by k3 and k4 for the natural peroxidases and by k1 and k2 for the diacetyldeuteroperoxidases. The mechanism of the acid-alkaline conversion was discussed in comparison with that of metmyoglobin.     

MacA is a second cytochrome c peroxidase of Geobacter sulfurreducens

The metal-reducing δ-proteobacterium Geobacter sulfurreducens produces a large number of c-type cytochromes, many of which have been implicated in the transfer of electrons to insoluble metal oxides. Among these, the dihemic MacA was assigned a central role. Here we have produced G. sulfurreducens MacA by recombinant expression in Escherichia coli and have solved its three-dimensional structure in three different oxidation states. Sequence comparisons group MacA into the family of diheme cytochrome c peroxidases, and the protein indeed showed hydrogen peroxide reductase activity with ABTS(-2) as an electron donor. The observed K(M) was 38.5 ± 3.7 μM H(2)O(2) and v(max) was 0.78 ± 0.03 μmol of H(2)O(2)·min(-1)·mg(-1), resulting in a turnover number k(cat) = 0.46 · s(-1). In contrast, no Fe(III) reductase activity was observed. MacA was found to display electrochemical properties similar to other bacterial diheme peroxidases, in addition to the ability to electrochemically mediate electron transfer to the soluble cytochrome PpcA. Differences in activity between CcpA and MacA can be rationalized with structural variations in one of the three loop regions, loop 2, that undergoes conformational changes during reductive activation of the enzyme. This loop is adjacent to the active site heme and forms an open loop structure rather than a more rigid helix as in CcpA. For the activation of the protein, the loop has to displace the distal ligand to the active site heme, H93, in loop 1. A H93G variant showed an unexpected formation of a helix in loop 2 and disorder in loop 1, while a M297H variant that altered the properties of the electron transfer heme abolished reductive activation.     

Biochemical mechanisms accounting for the toxic action of oxygen on living organisms: the key role of superoxide dismutase.

It seems that superoxide dismutase plays the key role in protecting aerobes against O2 toxicity, but there is a whole range of ancillary mechanisms: enzymes to remove H2O2 (catalase, peroxidases) and hence to control formation of .OH from O2, which requires H2O2; antioxidants (ascorbate, GSH, alpha-tocopherol, carotenoids), which also react with singlet oxygen and/or .OH and often inhibit lipid peroxidation and last, but not least in animals, glutathione peroxidase, which controls the rate of lipid peroxidation. These mechanisms cope well at normal O2 concentrations but are insufficient at higher levels.     

Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola.

The active oxygen species hydrogen peroxide (H2O2) was detected cytochemically by its reaction with cerium chloride to produce electron-dense deposits of cerium perhydroxides. In uninoculated lettuce leaves, H2O2 was typically present within the secondary thickened walls of xylem vessels. Inoculation with wild-type cells of Pseudomonas syringae pv phaseolicola caused a rapid hypersensitive reaction (HR) during which highly localized accumulation of H2O2 was found in plant cell walls adjacent to attached bacteria. Quantitative analysis indicated a prolonged burst of H2O2 occurring between 5 to 8 hr after inoculation in cells undergoing the HR during this example of non-host resistance. Cell wall alterations and papilla deposition, which occurred in response to both the wild-type strain and a nonpathogenic hrpD mutant, were not associated with intense staining for H2O2, unless the responding cell was undergoing the HR. Catalase treatment to decompose H2O2 almost entirely eliminated staining, but 3-amino-1,2,4-triazole (catalase inhibitor) did not affect the pattern of distribution of H2O2 detected. H2O2 production was reduced more by the inhibition of plant peroxidases (with potassium cyanide and sodium azide) than by inhibition of neutrophil-like NADPH oxidase (with diphenylene iodonium chloride). Results suggest that CeCl3 reacts with excess H2O2 that is not rapidly metabolized during cross-linking reactions occurring in cell walls; such an excess of H2O2 in the early stages of the plant-bacterium interaction was only produced during the HR. The highly localized accumulation of H2O2 is consistent with its direct role as an antimicrobial agent and as the cause of localized membrane damage at sites of bacterial attachment.