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.     

Modified peroxiredoxins as prototypes of drugs with powerful antioxidant action

The possibility of using modified peroxiredoxins as powerful antioxidant agents has been considered. Peroxiredoxins immobilized on perfluorocarbon emulsions and PTD-modified peroxiredoxins have been studied. It has been shown that peroxiredoxins efficiently bind to particles of perfluorocarbon emulsions, while maintaining their antioxidant properties. A panel of PTD-modified peroxiredoxins has been created and peroxiredoxins most effective both in antioxidant properties and the ability to penetrate cells have been selected. The modified peroxiredoxins obtained may serve as the basis for the design of drug with powerful antioxidant action.     

Peroxiredoxins from Trypanosoma cruzi: virulence factors and drug targets for treatment of Chagas disease?

Cytosolic and mitochondrial Trypanosoma cruzi tryparedoxin peroxidases belong to the family of 2-Cys peroxiredoxins. These enzymes play an essential role as antioxidants by their peroxidase and peroxynitrite reductase activities. TXNPx are key components of the trypanosomatid peroxide detoxification pathways. The aim of this work was to determine the role of TXNPx as virulence factors in the parasite, and whether these enzymes are good candidates for drug design. We observed that peroxiredoxins are not highly abundant proteins expressed at similar levels throughout the T. cruzi life cycle. In order to study the role of c-TXNPx and m-TXNPx in invasion and infectivity, parasites overexpressing TXNPx were produced, and infection experiments were carried out using phagocytic and non-phagocytic cells. Parasites overexpressing peroxiredoxins showed a significant increase in infectivity with respect to the control ones. The results presented in this work point out that the T. cruzi peroxiredoxins are important in survival, replication and differentiation of T. cruzi and could constitute virulence factors. Moreover, their expression in the infective forms of the life cycle and their low intracellular concentration make them good candidates to become targets for drug design.     

Proteomics analysis of cellular response to oxidative stress. Evidence for in vivo overoxidation of peroxiredoxins at their active site

The proteomics analysis reported here shows that a major cellular response to oxidative stress is the modification of several peroxiredoxins. An acidic form of the peroxiredoxins appeared to be systematically increased under oxidative stress conditions. Peroxiredoxins are enzymes catalyzing the destruction of peroxides. In doing so, a reactive cysteine in the peroxiredoxin active site is weakly oxidized (disulfide or sulfenic acid) by the destroyed peroxides. Cellular thiols (e.g. thioredoxin) are used to regenerate the peroxiredoxins to their active state. Tandem mass spectrometry was carried out to characterize the modified form of the protein produced in vivo by oxidative stress. The cysteine present in the active site was shown to be oxidized into cysteic acid, leading to an inactivated form of peroxiredoxin. This strongly suggested that peroxiredoxins behave as a dam upon oxidative stress, being both important peroxide-destroying enzymes and peroxide targets. Results obtained in a primary culture of Leydig cells challenged with tumor necrosis factor alpha suggested that this oxidized/native balance of peroxiredoxin 2 may play an active role in resistance or susceptibility to tumor necrosis factor alpha-induced apoptosis.     

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.     

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.     

How pH Modulates the Dimer-Decamer Interconversion of 2-Cys Peroxiredoxins from the Prx1 Subfamily

2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H₂O₂ and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His¹¹³) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp⁷⁶ from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H₂O₂ can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H₂O₂ and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress.     

Leishmania mitochondrial peroxiredoxin plays a crucial peroxidase-unrelated role during infection: insight into its novel chaperone activity

Two-cysteine peroxiredoxins are ubiquitous peroxidases that play various functions in cells. In Leishmania and related trypanosomatids, which lack catalase and selenium-glutathione peroxidases, the discovery of this family of enzymes provided the molecular basis for peroxide removal in these organisms. In this report the functional relevance of one of such enzymes, the mitochondrial 2-Cys peroxiredoxin (mTXNPx), was investigated along the Leishmania infantum life cycle. mTXNPx null mutants (mtxnpx(-)) produced by a gene replacement strategy, while indistinguishable from wild type promastigotes, were found unable to thrive in a murine model of infection. Unexpectedly, however, the avirulent phenotype of mtxnpx(-) was not due to lack of the peroxidase activity of mTXNPx as these behaved like controls when exposed to oxidants added exogenously or generated by macrophages during phagocytosis ex vivo. In line with this, mtxnpx(-) were also avirulent when inoculated into murine hosts unable to mount an effective oxidative phagocyte response (B6.p47(phox-/-) and B6.RAG2(-/-) IFN-γ(-/-) mice). Definitive conclusion that the peroxidase activity of mTXNPx is not required for parasite survival in mice was obtained by showing that a peroxidase-inactive version of this protein was competent in rescuing the non-infective phenotype of mtxnpx(-). A novel function is thus proposed for mTXNPx, that of a molecular chaperone, which may explain the impaired infectivity of the null mutants. This premise is based on the observation that the enzyme is able to suppress the thermal aggregation of citrate synthase in vitro. Also, mtxnpx(-) were more sensitive than controls to a temperature shift from 25°C to 37°C, a phenotype reminiscent of organisms lacking specific chaperone genes. Collectively, the findings reported here change the paradigm which regards all trypanosomatid 2-Cys peroxiredoxins as peroxide-eliminating devices. Moreover, they demonstrate, for the first time, that these 2-Cys peroxiredoxins can be determinant for pathogenicity independently of their peroxidase activity.     

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.     

Plant peroxiredoxins: alternative hydroperoxide scavenging enzymes

The role of plant peroxiredoxins in the detoxification systems is discussed in relation with the existence of many isoforms of this protein in distinct plant compartments. Phylogenetic analyses indicate that plant peroxiredoxins can be divided into four classes. Two of these classes correspond to chloroplastic enzymes. All isoforms contain at least one conserved catalytic cysteine. The enzymes belonging to the 1-Cys Prx class seem to be seed restricted and to play a role of detoxification during the germination process. At least one putative cytosolic isoform can use both thioredoxin and glutaredoxin as an electron donor, but the chloroplastic isoforms characterized depend on reduced thioredoxin. Mutagenesis and plant transformation studies support the proposal that the chloroplastic peroxiredoxins play an important role in combating the ROS species generated at the level of the chloroplastic electron transfer chain. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of sestrin2 in peroxide signaling in macrophages

Reactive oxygen species not only serve as signaling molecules, they also contribute to oxidative stress and cell damage. The thioredoxin and glutaredoxin systems form along with peroxiredoxins a precisely regulated defense system to maintain the cellular redox homeostasis. There is evidence that nitric oxide (NO) protects cells from oxidative stress by preventing inactivation of peroxiredoxins by sulfinylation. Here we demonstrate that NO and hypoxia upregulate Sestrin2 by HIF-1-dependent and additional mechanisms and that Sestrin2 contributes to preventing peroxiredoxins from sulfinylation. We conclude that Sestrin2 plays a role in peroxide defense as a reductase for peroxiredoxins.     

The plant multigenic family of thiol peroxidases

Thiol peroxidases are ubiquitous recently characterized heme-free peroxidases, which catalyze the reduction of peroxynitrites and of various peroxides by catalytic cysteine residues and thiol-containing proteins as reductants. In plants, five different classes can be distinguished, according to the number and the position of conserved catalytic cysteines. Four classes are defined as peroxiredoxins and were already identified by phylogenetic sequence analysis, 1-Cys, 2-Cys, type II, and type Q peroxiredoxins, and the fifth is represented by glutathione peroxidases, which were recently shown to possess a thioredoxin-dependent activity in plants. Since the discovery of peroxiredoxins in plants in 1996, a lot of work has been devoted to the biochemical and functional characterization of the different peroxiredoxin isoforms, but in contrast, few structural data are available. The analysis of the Arabidopsis thaliana genome indicates that at least 17 isoforms of thioredoxin-dependent peroxidases are expressed in various plant compartments. The role of these proteins is discussed in terms of electron donor and substrate specificities and in light of their expression and localization. These enzymes are expressed in many plant tissues and are involved notably in the protection of the photosynthetic apparatus, in the response to various biotic or abiotic stresses by fighting reactive oxygen or nitrogen species and lipid peroxidation.     

Peroxiredoxins play a major role in protecting Trypanosoma cruzi against macrophage- and endogenously-derived peroxynitrite

There is increasing evidence that Trypanosoma cruzi antioxidant enzymes play a key immune evasion role by protecting the parasite against macrophage-derived reactive oxygen and nitrogen species. Using T. cruzi transformed to overexpress the peroxiredoxins TcCPX (T. cruzi cytosolic tryparedoxin peroxidase) and TcMPX (T. cruzi mitochondrial tryparedoxin peroxidase), we found that both cell lines readily detoxify cytotoxic and diffusible reactive oxygen and nitrogen species generated in vitro or released by activated macrophages. Parasites transformed to overexpress TcAPX (T. cruzi ascorbate-dependent haemoperoxidase) were also more resistant to H2O2 challenge, but unlike TcMPX and TcCPX overexpressing lines, the TcAPX overexpressing parasites were not resistant to peroxynitrite. Whereas isolated tryparedoxin peroxidases react rapidly (k=7.2 x 10(5) M(-1) x s(-1)) and reduce peroxynitrite to nitrite, our results demonstrate that both TcMPX and TcCPX peroxiredoxins also efficiently decompose exogenous- and endogenously-generated peroxynitrite in intact cells. The degree of protection provided by TcCPX against peroxynitrite challenge results in higher parasite proliferation rates, and is demonstrated by inhibition of intracellular redox-sensitive fluorescence probe oxidation, protein 3-nitrotyrosine and protein-DMPO (5,5-dimethylpyrroline-N-oxide) adduct formation. Additionally, peroxynitrite-mediated over-oxidation of the peroxidatic cysteine residue of peroxiredoxins was greatly decreased in TcCPX overexpressing cells. The protective effects generated by TcCPX and TcMPX after oxidant challenge were lost by mutation of the peroxidatic cysteine residue in both enzymes. We also observed that there is less peroxynitrite-dependent 3-nitrotyrosine formation in infective metacyclic trypomastigotes than in non-infective epimastigotes. Together with recent reports of up-regulation of antioxidant enzymes during metacyclogenesis, our results identify components of the antioxidant enzyme network of T. cruzi as virulence factors of emerging importance.     

Multiple Roles of Peroxiredoxins in Inflammation

Inflammation is a pathophysiological response to infection or tissue damage during which high levels of reactive oxygen and nitrogen species are produced by phagocytes to kill microorganisms. Reactive oxygen and nitrogen species serve also in the complex regulation of inflammatory processes. Recently, it has been proposed that peroxiredoxins may play key roles in innate immunity and inflammation. Indeed, peroxiredoxins are evolutionarily conserved peroxidases able to reduce, with high rate constants, hydrogen peroxide, alkyl hydroperoxides and peroxynitrite which are generated during inflammation. In this minireview, we point out different possible roles of peroxiredoxins during inflammatory processes such as cytoprotective enzymes against oxidative stress, modulators of redox signaling, and extracellular pathogen- or damage-associated molecular patterns. A better understanding of peroxiredoxin functions in inflammation could lead to the discovery of new therapeutic targets.     

Cloning, overexpression, and characterization of peroxiredoxin and NADH peroxiredoxin reductase from Thermus aquaticus

The genes for peroxiredoxin (Prx) and NADH:peroxiredoxin oxidoreductase (PrxR) have been cloned from the thermophilic bacterium Thermus aquaticus. prx is located upstream from prxR, the two genes being separated by 13 bases. The amino acid sequences show that Prx is related to two-cysteine peroxiredoxins from a range of organisms and that PrxR resembles NADH-dependent flavoenzymes that catalyze the reduction of peroxiredoxins in mesophilic bacteria. The sequence of PrxR also resembles those of thioredoxin reductases (TrxR) from thermophiles but with an N-terminal extension of about 200 residues. PrxR has motifs for two redox-active disulfides, one in the FAD-binding site, as occurs in TrxR, and the other in the N-terminal extension. The molecular masses of the monomers of Prx and PrxR are 21.0 and 54.9 kDa, respectively; both enzymes exist as multimers. The recombinant flavoenzyme requires 3 mol equivalents of dithionite for full reduction, as is consistent with 1 FAD and 2 disulfides per monomer. PrxR and Prx together catalyze the anaerobic reduction of hydrogen peroxide. The activity of Prx is much less than has been observed with homologous proteins. Prx appears to be inactivated by cumene hydroperoxide. PrxR itself has low peroxidase activity.