Regeneration of peroxiredoxins during recovery after oxidative stress: only some overoxidized peroxiredoxins can be reduced during recovery after oxidative stress

Peroxiredoxins (prx) are redox enzymes using an activated cysteine as their active site. This activated cysteine can be easily overoxidized to cysteine sulfinic acid or cysteine sulfonic acid, especially under oxidative stress conditions. The regeneration of peroxiredoxins after a short, intense oxidative stress was studied, using a proteomics approach. Important differences in regeneration speed were found, prx2 being the fastest regenerated protein, followed by prx1, whereas prx3 and prx6 were regenerated very slowly. Further study of the mechanism of this regeneration by pulse-chase experiments using stable isotope labeling and cycloheximide demonstrated that the fast-regenerating peroxiredoxins are regenerated at least in part by a retroreduction mechanism. This demonstrates that the overoxidation can be reversible under certain conditions. The pathway of this retroreduction and the reasons explaining the various regeneration speeds of the peroxiredoxins remain to be elucidated.     

Bacterial defenses against oxidants: mechanistic features of cysteine-based peroxidases and their flavoprotein reductases

Antioxidant defenses include a group of ubiquitous, non-heme peroxidases, designated the peroxiredoxins, which rely on an activated cysteine residue at their active site to catalyze the reduction of hydrogen peroxide, organic hydroperoxides, and peroxynitrite. In the typical 2-Cys peroxiredoxins, a second cysteinyl residue, termed the resolving cysteine, is also involved in intersubunit disulfide bond formation during the course of catalysis by these enzymes. Many bacteria also express a flavoprotein, AhpF, which acts as a dedicated disulfide reductase to recycle the bacterial peroxiredoxin, AhpC, during catalysis. Mechanistic and structural studies of these bacterial proteins have shed light on the linkage between redox state, oligomeric state, and peroxidase activity for the peroxiredoxins, and on the conformational changes accompanying catalysis by both proteins. In addition, these studies have highlighted the dual roles that the oxidized cysteinyl species, cysteine sulfenic acid, can play in eukaryotic peroxiredoxins, acting as a catalytic intermediate in the peroxidase activity, and as a redox sensor in regulating hydrogen peroxide-mediated cell signaling.     

Complementary antioxidant defense by cytoplasmic and mitochondrial peroxiredoxins in Leishmania infantum

In Kinetoplastida 2-Cys peroxiredoxins are the ultimate members of unique enzymatic cascades for detoxification of peroxides, which are dependent on trypanothione, a small thiol specific to these organisms. Here we report on two distinct Leishmania infantum peroxiredoxins, LicTXNPx and LimTXNPx, that may be involved in such a pathway. LicTXNPx, found in the cytoplasm, is a typical 2-Cys peroxiredoxin encoded by LicTXNPx, a member of a multicopy gene family. LimTXNPx, encoded by a single copy gene, LimTXNPx, is confined to the mitochondrion and is unusual in possessing an Ile-Pro-Cys motif in the distal redox center, replacing the common peroxiredoxin Val-Cys-Pro sequence, apart from an N-terminal mitochondrial leader sequence. Based on sequence and subcellular localization, the peroxiredoxins of Kinetoplastida can be separated in two distinct subfamilies. As an approach to investigate the function of both peroxiredoxins in the cell, L. infantum promastigotes overexpressing LicTXNPx and LimTXNPx were assayed for their resistance to H(2)O(2) and tert-butyl hydroperoxide. The results show evidence that both enzymes are active as peroxidases in vivo and that they have complementary roles in parasite protection against oxidative stress.     

Active site mutagenesis and phospholipid hydroperoxide reductase activity of poplar type II peroxiredoxin

The nature of the active site and the substrate specificity of poplar type II peroxiredoxin, an enzyme which preferentially uses glutaredoxin as an electron donor, were investigated in this study. The type II peroxiredoxin is able to use phospholipid hydroperoxide nearly as efficiently as hydrogen peroxide. Two of the hyper-conserved amino acid residues in peroxiredoxins have been altered, by site-directed mutagenesis, generating the mutants T48V and R129Q. The two mutant proteins are inactive with hydrogen peroxide or tertiary butyl hydroperoxide as substrates. On the other hand, the mutant enzymes catalyse the degradation of cumene hydroperoxide with low efficiency. This suggests that the thiol-dependent regeneration process of the catalytic cysteine is not affected by the mutations and that all substrates are not accommodated identically in the active site.     

Oxidation of active center cysteine of bovine 1-Cys peroxiredoxin to the cysteine sulfenic acid form by peroxide and peroxynitrite.

Peroxiredoxins are antioxidant enzymes whose peroxidase activity depends on a redox-sensitive cysteine residue at the active center. In this study we investigated properties of the active center cysteine of bovine 1-Cys peroxiredoxin using a recombinant protein (BRPrx). The only cysteine residue of the BRPrx molecule was oxidized rapidly by an equimolar peroxide or peroxynitrite to the cysteine sulfenic acid. Approximate rates of oxidation of BRPrx by different peroxides were estimated using selenium glutathione peroxidase as a competitor. Oxidation of the active center cysteine of BRPrx by H2O2 proceeded only several times slowly than that of the selenocysteine of glutathione peroxidase. The rate of oxidation varied depending on peroxides tested, with H2O2 being about 7 and 80 times faster than tert-butyl hydroperoxide and cumene hydroperoxide, respectively. Peroxynitrite oxidized BRPrx slower than H2O2 but faster than tert-butyl hydroperoxide. Further oxidation of the cysteine sulfenic acid of BRPrx to higher oxidation states proceeded slowly. Oxidized BRPrx was reduced by dithiothreitol, dihydrolipoic acid, and hydrogen sulfide, and demonstrated peroxidase activity (about 30 nmol/mg/min) with these reductants as electron donors. beta-Mercaptoethanol formed a mixed disulfide and did not support peroxidase activity. Oxidized BRPrx did not react with glutathione, cysteine, homocysteine, N-acetyl-cysteine, and mercaptosuccinic acid.     

Structure and mechanism of the alkyl hydroperoxidase AhpC, a key element of the Mycobacterium tuberculosis defense system against oxidative stress

The peroxiredoxin AhpC from Mycobacterium tuberculosis (MtAhpC) is the foremost element of a NADH-dependent peroxidase and peroxynitrite reductase system, where it directly reduces peroxides and peroxynitrite and is in turn reduced by AhpD and other proteins. Overexpression of MtAhpC in isoniazid-resistant strains of M. tuberculosis harboring mutations in the catalase/peroxidase katG gene provides antioxidant protection and may substitute for the lost enzyme activities. We report here the crystal structure of oxidized MtAhpC trapped in an intermediate oligomeric state of its catalytic cycle. The overall structure folds into a ring-shaped hexamer of dimers instead of the usual pentamer of dimers observed in other reduced peroxiredoxins. Although the general structure of the functional dimer is similar to that of other 2-Cys peroxiredoxins, the alpha-helix containing the peroxidatic cysteine Cys61 undergoes a unique rigid-body movement to allow the formation of the disulfide bridge with the resolving cysteine Cys174. This conformational rearrangement creates a large internal cavity enclosing the active site, which might be exploited for the design of inhibitors that could block the catalytic cycle. Structural and mutagenesis evidence points to a model for the electron transfer pathway in MtAhpC that accounts for the unusual involvement of three cysteine residues in catalysis and suggests a mechanism by which MtAhpC can specifically interact with different redox partners.     

Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses

The chloroplastic drought-induced stress protein of 32 kDa (CDSP32) is a thioredoxin induced by environmental stress conditions. To gain insight into the function of CDSP32, we applied two strategies to analyze its targets. First, using affinity chromatography with an immobilized CDSP32 active site mutant, we identified six plastidic targets of CDSP32. Three of them are involved in photosynthetic processes: ATP-ase gamma-subunit, Rubisco and aldolase. The three others participate in the protection against oxidative damage: two peroxiredoxins, PrxQ and the BAS1 2-Cys peroxiredoxin, and a B-type methionine sulfoxide reductase. Then, we developed a novel strategy to trap targets directly in leaf extracts. The method, based on co-immunoprecipitation using extracts from plants overexpressing Wt CDSP32 or CDSP32 active site mutant, confirmed the interaction in vivo between CDSP32 and the PrxQ and BAS1 peroxiredoxins. We showed that CDSP32 is able to form heterodimeric complexes with PrxQ and that the peroxiredoxin displays CDSP32-dependent peroxidase activity. Under photooxidative stress induced by methyl viologen, plants overexpressing CDSP32 active site mutant exhibit decreased maximal PSII photochemical efficiency and retain much less chlorophyll compared with Wt plants and with plants overexpressing Wt CDSP32. We propose that the increased sensitivity results from trapping in planta of the targets involved in the protection against oxidative damage. We conclude that CDSP32, compared with other plant thioredoxins, is a thioredoxin more specifically involved in plastidic responses against oxidative stress.     

Overexpression of human peroxiredoxin 5 in subcellular compartments of Chinese hamster ovary cells: effects on cytotoxicity and DNA damage caused by peroxides

Peroxiredoxin 5 is a mammalian thioredoxin peroxidase ubiquitously expressed in tissues. Peroxiredoxin 5 can be intracellularly localized to mitochondria, peroxisomes, the cytosol, and, to a lesser extent, the nucleus. This remarkably wide subcellular distribution compared with the five other mammalian peroxiredoxins prompted us to further investigate the antioxidant protective function of peroxiredoxin 5 in mammalian cells according to its subcellular localization. Chinese hamster ovary cells overexpressing human peroxiredoxin 5 in the cytosol, in mitochondria, or in the nucleus were established by stable transfection. Cells overexpressing peroxiredoxin 5 were exposed for 1 h to low or acute oxidative stress with exogenously added hydrogen peroxide or tert-butylhydroperoxide. Cell protection conferred by peroxiredoxin 5 was evaluated by clonogenicity and lactate dehydrogenase assays. Overexpressing peroxiredoxin 5 in either the cytosolic, mitochondrial, or nuclear compartment significantly reduced cell death, with more effective protection with overexpression of peroxiredoxin 5 in mitochondria, confirming that this organelle is a major target of peroxides. Moreover, we evaluated, with the comet assay, nuclear DNA damage induced by hydrogen peroxide or tert-butylhydroperoxide. Overexpression of peroxiredoxin 5 in the nucleus significantly decreased DNA damage induced by both peroxides. In conclusion, the present study suggests that multiple subcellular targeting of peroxiredoxin 5 in mammalian cells can be implicated in antioxidant protective mechanisms under nonpathological conditions but also during acute oxidative stress caused by peroxides occurring in pathophysiological situations.     

Extraordinary μs-ms backbone dynamics in Arabidopsis thaliana peroxiredoxin Q

Peroxiredoxin Q (PrxQ) isolated from Arabidopsis thaliana belongs to a family of redox enzymes called peroxiredoxins, which are thioredoxin- or glutaredoxin-dependent peroxidases acting to reduce peroxides and in particular hydrogen peroxide. PrxQ cycles between an active reduced state and an inactive oxidized state during its catalytic cycle. The catalytic mechanism involves a nucleophilic attack of the catalytic cysteine on hydrogen peroxide to generate a sulfonic acid intermediate with a concerted release of a water molecule. This intermediate is subsequently relaxed by the reaction of a second cysteine, denoted the resolving cysteine, generating an intramolecular disulfide bond and release of a second water molecule. PrxQ is recycled to the active state by a thioredoxin-dependent reduction. Previous structural studies of PrxQ homologues have provided the structural basis for the switch between reduced and oxidized conformations. Here, we have performed a detailed study of the activity, structure and dynamics of PrxQ in both the oxidized and reduced states. Reliable and experimentally validated structural models of PrxQ in both oxidation states were generated using homology based modeling. Analysis of NMR spin relaxation rates shows that PrxQ is monomeric in both oxidized and reduced states. As evident from R(2) relaxation rates the reduced form of PrxQ undergoes unprecedented dynamics on the slow μs-ms timescale. The ground state of this conformational dynamics is likely the stably folded reduced state as implied by circular dichroism spectroscopy. We speculate that the extensive dynamics is intimately related to the catalytic function of PrxQ.     

Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate

Peroxidases of the peroxiredoxin (Prx) family contain a Cys residue that is preceded by a conserved sequence in the NH(2)-terminal region. A new type of mammalian Prx, designated PrxV, has now been identified as the result of a data base search with this conserved Cys-containing sequence. The 162-amino acid PrxV shares only approximately 10% sequence identity with previously identified mammalian Prx enzymes and contains Cys residues at positions 73 and 152 in addition to that (Cys(48)) corresponding to the conserved Cys. Analysis of mutant human PrxV proteins in which each of these three Cys residues was individually replaced with serine suggested that the sulfhydryl group of Cys(48) is the site of oxidation by peroxides and that oxidized Cys(48) reacts with the sulfhydryl group of Cys(152) to form an intramolecular disulfide linkage. The oxidized intermediate of PrxV is thus distinct from those of other Prx enzymes, which form either an intermolecular disulfide or a sulfenic acid intermediate. The disulfide formed by PrxV is reduced by thioredoxin but not by glutaredoxin or glutathione. Thus, PrxV mutants lacking Cys(48) or Cys(152) showed no detectable thioredoxin-dependent peroxidase activity, whereas mutation of Cys(73) had no effect on activity. Immunoblot analysis revealed that PrxV is widely expressed in rat tissues and cultured mammalian cells and is localized intracellularly to cytosol, mitochondria, and peroxisomes. The peroxidase function of PrxV in vivo was demonstrated by the observations that transient expression of the wild-type protein, but not that of the Cys(48) mutant, in NIH 3T3 cells inhibited H(2)O(2) accumulation and activation of c-Jun NH(2)-terminal kinase induced by tumor necrosis factor-alpha.     

Methamphetamine downregulates peroxiredoxins in rat pheochromocytoma cells

Methamphetamine (METH) is an abusive psychostimulant that induces neuronal cell death/degeneration in experimental animals and humans. METH-induced apoptosis in rat pheochromocytoma cells was utilized to study the neurotoxic mechanism. During METH intoxication, we found that peroxiredoxins and thioredoxins/thioredoxin reductases (peroxiredoxin reducing systems) which are known to prevent oxidative stress and apoptosis were differentially downregulated and upregulated, respectively. We also found not only the free radicals but also the oxidative forms of peroxiredoxin and thioredoxin were increased, indicating the dysfunction of these enzymes. Thus, METH-induced differential regulation and oxidation of peroxiredoxins and thioredoxin may be an important mechanism for apoptosis.     

Crystal structure of a novel Plasmodium falciparum 1-Cys peroxiredoxin

Plasmodium falciparum, the causative agent of malaria, is sensitive to oxidative stress and therefore the family of antioxidant enzymes, peroxiredoxins (Prxs) represent a target for antimalarial drug design. We present here the 1.8 A resolution crystal structure of P.falciparum antioxidant protein, PfAOP, a Prx that in terms of sequence groups with mammalian PrxV. The structure is compared to all 11 known Prx structures to gain maximal insight into its properties. We describe the common Prx fold and show that the dimeric PfAOP can be mechanistically categorized as a 1-Cys Prx. In the active site the peroxidatic Cys is over-oxidized to cysteine sulfonic acid, making this the first Prx structure seen in that state. Now with structures of Prxs in Cys-sulfenic, -sulfinic and -sulfonic acid oxidation states known, the structural steps involved in peroxide binding and over-oxidation are suggested. We also describe that PfAOP has an alpha-aneurism (a one residue insertion), a feature that appears characteristic of the PrxV-like group. In terms of crystallographic methodology, we enhance the information content of the model by identifying bound water sites based on peak electron densities, and we use that information to infer that the oxidized active site has suboptimal interactions that may influence catalysis. The dimerization interface of PfAOP is representative of an interface that is widespread among Prxs, and has sequence-dependent variation in geometry. The interface differences and the structural features (like the alpha-aneurism) may be used as markers to better classify Prxs and study their evolution.     

Reversible oxidation of mitochondrial peroxiredoxin 3 in mouse heart subjected to ischemia and reperfusion

Peroxiredoxins decompose peroxides through reversible oxidation of their active site cysteines. The redox state of the 2-Cys peroxiredoxins, 1, 2 and 3, was investigated in mouse hearts undergoing ischemia and reperfusion in a Langendorff system. The peroxiredoxins were predominantly reduced in control hearts. Mitochondrial peroxiredoxin 3 underwent significant oxidation to its disulfide-linked dimer during ischemia. Oxidation was largely reversed during reperfusion. No redox changes in cytoplasmic peroxiredoxins 1 and 2 were apparent. Peroxiredoxin 3 oxidation suggests localized mitochondrial generation of reactive oxidants during ischemia. This local antioxidant activity of peroxiredoxin 3 may have a role in maintaining cardiac function.     

Lack of an Efficient Endoplasmic Reticulum-localized Recycling System Protects Peroxiredoxin IV from Hyperoxidation

Typical 2-Cys peroxiredoxins are required to remove hydrogen peroxide from several different cellular compartments. Their activity can be regulated by hyperoxidation and consequent inactivation of the active-site peroxidatic cysteine. Here we developed a simple assay to quantify the hyperoxidation of peroxiredoxins. Hyperoxidation of peroxiredoxins can only occur efficiently in the presence of a recycling system, usually involving thioredoxin and thioredoxin reductase. We demonstrate that there is a marked difference in the sensitivity of the endoplasmic reticulum-localized peroxiredoxin to hyperoxidation compared with either the cytosolic or mitochondrial enzymes. Each enzyme is equally sensitive to hyperoxidation in the presence of a robust recycling system. Our results demonstrate that peroxiredoxin IV recycling in the endoplasmic reticulum is much less efficient than in the cytosol or mitochondria, leading to the protection of peroxiredoxin IV from hyperoxidation.     

In vivo targets of S-thiolation in Chlamydomonas reinhardtii

Glutathionylation is the major form of S-thiolation in cells. This reversible redox post-translational modification consists of the formation of a mixed disulfide between a free thiol on a protein and a molecule of glutathione. This recently described modification, which is considered to occur under oxidative stress, can protect cysteine residues from irreversible oxidation, and alter positively or negatively the activity of diverse proteins. This modification and its targets have been mainly studied in non-photosynthetic organisms so far. We report here the first proteomic approach performed in vivo on photosynthetically competent cells, using the eukaryotic unicellular green alga Chlamydomonas reinhardtii with radiolabeled [(35)S]cysteine to label the glutathione pool and diamide as oxidant. This method allowed the identification of 25 targets, mainly chloroplastic, involved in various metabolic processes. Several targets are related to photosynthesis, such as the Calvin cycle enzymes phosphoglycerate kinase and ribose-5-phosphate isomerase. A number of targets, such as chaperones and peroxiredoxins, are related to stress responses. The glutathionylation of HSP70B, chloroplastic 2-Cys peroxiredoxin and isocitrate lyase was confirmed in vitro on purified proteins and the targeted residues were identified.     

Peroxiredoxin-null yeast cells are hypersensitive to oxidative stress and are genomically unstable

Peroxiredoxins are a family of abundant peroxidases found in all organisms. Although these antioxidant enzymes are thought to be critically involved in cellular defense and redox signaling, their exact physiological roles are largely unknown. In this study, we took a genetic approach to address the functions of peroxiredoxins in budding yeast. We generated and characterized a yeast mutant lacking all five peroxiredoxins. The quintuple peroxiredoxin-null mutant was still viable, though the growth rate was lower under normal aerobic conditions. Although peroxiredoxins are not essential for cell viability, peroxiredoxin-null yeast cells were more susceptible to oxidative and nitrosative stress. In the complete absence of peroxiredoxins, the expression of other antioxidant proteins including glutathione peroxidase and glutathione reductase was induced. In addition, the quintuple mutant was hypersensitive to glutathione depletion. Thus, the glutathione system might cooperate with other antioxidant enzymes to compensate for peroxiredoxin deficiency. Interestingly, the peroxiredoxinnull yeast cells displayed an increased rate of spontaneous mutations that conferred resistance to canavanine. This mutator phenotype was rescued by yeast peroxiredoxin Tsa1p, but not by its active-site mutant defective for peroxidase activity. Our findings suggest that the antioxidant function of peroxiredoxins is important for maintaining genome stability in eukaryotic cells.