Journal of Experimental Botany, Vol. 53, No. 372, pp. 1321-1329,
May 15, 2002
© 2002 Oxford University Press
Original Papers |
The function of the chloroplast 2-cysteine peroxiredoxin in peroxide detoxification and its regulation
University of Bielefeld, Department of Plant Physiology and Biochemistry/W5, 33501 Bielefeld, Germany
Received 3 April 2001; Accepted 21 December 2001
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Abstract |
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 Top Abstract Peroxiredoxins in non...Peroxiredoxins in plants and...The catalytic activity of...Structure of the 2-CPThe physiological function of...Antisense suppression of 2-CP...The role of 2-CP...Regulation of 2-CP gene...Perspectives: 2-CP, signalling...Note added in proofReferences |
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The Arabidopsis genome contains nine open reading frames with homology to members of the peroxiredoxin (prx) family: one 1-Cys-prx, two 2-Cys-prx, five type II-prx, and one peroxiredoxin Q. The function of the peroxiredoxins in plant metabolism is only slowly emerging. They are assumed to reduce toxic peroxides to their corresponding alcohols with a rather broad substrate specificity. The 2-Cys peroxiredoxins (2-CP) were recently identified as members of the antioxidant defence system of chloroplasts. Knock-out mutants of Synechocystis and antisense mutants of Arabidopsis have provided insight into the function of 2-CPs in the photosynthetic antioxidant network. This review summarizes present knowledge on the enzymatic mechanism, the physiological context and the genetic regulation of the 2-CPs in plants and cyanobacteria. In addition, an extrapolation on the metabolic role of the chloroplast 2-CP is attempted based on the molecular features of 2-CPs from other organisms.
Key words: Antioxidants, Arabidopsis, chloroplast peroxiredoxin, peroxide detoxification.
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Peroxiredoxins in non-photosynthetic bacteria, fungi and animals |
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TopAbstractPeroxiredoxins in non...Peroxiredoxins in plants and...The catalytic activity of...Structure of the 2-CPThe physiological function of...Antisense suppression of 2-CP...The role of 2-CP...Regulation of 2-CP gene...Perspectives: 2-CP, signalling...Note added in proofReferences |
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Peroxiredoxins have been identified in all groups of organisms and constitute a phylogenetically old group of enzymes with catalytic function in the detoxification of cell-toxic peroxides (Chae et al., 1993
; Prospéri et al., 1993; Storz et al., 1989; Lim et al., 1998). Related to the history of detection, the enzyme has received various names, for example alkyl hydroperoxide reductase (Ahp), protector protein, thiol-specific antioxidant (TSA) and, recently, peroxiredoxins (Prx). Within the peroxiredoxins, four clusters of related proteins are distinguished: 1-Cys peroxiredoxins (1-CP), 2-Cys peroxiredoxins (2-CP), YLR109-related peroxiredoxins (type II-Prx), and bacteriferritin-associated proteins (Prx Q). Biochemical data on type II-Prx and Prx Q are still scarce (Verdoucq et al., 1999). All Prx are characterized by one or two cysteine residue(s), respectively, located in a conserved structural environment of the protein and are central for the catalytic reaction (Chae et al., 1993; Baier and Dietz, 1996c; Verdoucq et al., 1999). The first peroxiredoxins were cloned from bacteria such as Salmonella typhimurium and E. coli (Storz et al., 1989). Since then, peroxiredoxin genes have been isolated from many organisms. For example, at least five 2-CP isoforms are present in the genomes of mouse and man (Prx I, II, III, IV, V; Seo et al., 2000). Sites of high levels of expression coincide with sites of active oxygen metabolism such as brain and erythrocytes. For example, in erythrocytes, with their main function in oxygen transport, the protein fraction represented by the 2-CP is the third most dominant protein with 14x106 copies in each red blood cell (Schröder et al., 2000).
The functional 2-CP is a homodimer with two reduced Cys residues per subunit. During the catalytic cycle, the peroxide substrate is reduced to the corresponding alcohol and the Cys residues oxidized to a disulphide bridge (Fig. 1). The regeneration of catalytically active 2-CP depends on reduction of the disulphide bridge. In most eukaryotic organisms, thioredoxin (Chae et al., 1999) or thioredoxin-related proteins such as trypathionin (in trypanosomes: Nogoceke et al., 1997; Montemartini et al., 1999; Lopez et al., 2000) act as electron donors. Recently, it was shown that the thioredoxin system also regenerates the mitochondrial 1-CP of Saccharomyces (Pedrajas et al., 2000) and the YLR109-related peroxiredoxins (type II-Prx) (Verdoucq et al., 1999).
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The substrate specificity of 2-CPs is rather broad and catalyses not only detoxification of H2O2 and alkyl hydroperoxides but also reduction of reactive nitrogen peroxides (Lim et al., 1993
; Bryk et al., 2000). Nitric oxide reacts with superoxide to form peroxynitrite which disproportionates to form nitrate and highly reactive hydroxyl radical and nitrogen dioxide radical (equations (1) and (2)).
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). Reductive detoxification of peroxynitrite is important to avoid destructive oxidation of cellular constituents.
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Peroxiredoxins in plants and their subcellular localization |
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TopAbstractPeroxiredoxins in non...Peroxiredoxins in plants and...The catalytic activity of...Structure of the 2-CPThe physiological function of...Antisense suppression of 2-CP...The role of 2-CP...Regulation of 2-CP gene...Perspectives: 2-CP, signalling...Note added in proofReferences |
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In the genome of Arabidopsis thaliana there are nine open reading frames with sequence similarity to peroxiredoxins (Table 1
; Fig. 1
). As stated above, peroxiredoxins can be grouped in 1-Cys Prx, 2-Cys Prx, type II Prx, and Prx Q. Members of each group have recently been identified in plants also. The first cDNA-sequence of a plant 1-cysteine peroxiredoxin (1-CP PER1) was identified as a ‘dormancy-related protein’ being expressed in the embryo and the aleurone layer of the barley caryopsis (Aalen et al., 1994). Immunocytochemistry revealed that the PER1 1-CP is preferentially localized in the nucleus, and there within the nucleolus (Stacy et al., 1996, 1999). Homologous sequences of PER1 were also isolated from Arabidopsis, rice, buckwheat, and wheat (Haslekas et al., 1998; Lewis et al., 2000; Majoul et al., 2000). The authors suggest a role of the 1-CP in protecting macromolecules from oxidative destruction during the period of drying and reswelling of the seeds and speculate on a role in maintaining dormancy.
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Plant 2-CPs were first cloned from barley and spinach (Baier and Dietz, 1996
a, b), and afterwards from Arabidopsis thaliana (Baier and Dietz, 1997), chinese cabbage (Cheong et al., 1999), rye (Berberich et al., 1998), and the liverwort Riccia fluitans (Horling et al., 2001). In the genome of A. thaliana there are two genes annotated encoding 2-CPs and one gene encoding a 1-CP (Table 1) (The Arabidopsis Genome Initiative, 2000). Compared to cytosolic animal and fungal 2-CPs and bacterial 2-CPs, all 2-CPs from higher plants cloned so far show an amino-terminal extension. Three pieces of evidence have proven that the pre-peptide mediates import into chloroplasts and, consequently, that plant 2-CP is a chloroplast enzyme: (1) 2-CP translated in a cell-free system was post-translationally imported by isolated intact chloroplasts. Following the import the targeting signal was cleaved off resulting in a mature protein similar to animal and bacterial 2-CP (Baier and Dietz, 1997). (2) During fractionation of mesophyll protoplasts, the 2-CP co-purified with chloroplasts. (3) In immunohistochemical studies of leaf cross-sections, the 2-CP was detected in chloroplasts where it was preferentially attached to the thylakoid membrane (M Baier, Kahmann, KJ Dietz, unpublished data).
The third class of plant peroxiredoxin-like proteins (type II peroxiredoxins) was identified in a screen for proteins binding to a modified thioredoxin h, first in yeast (YLR109) and, by means of sequence homology, in plants (in A. thaliana: AtTPX2) and animals (Verdoucq et al., 1999; Choi et al., 1999). This third class of peroxiredoxins is suggested to be targeted to the peroxisomes of eukaryotic cells and to be involved in detoxifying H2O2 that escapes from detoxification by catalase, or alkyl hydroperoxide produced by reactions of lipids with reactive oxygen species in the peroxisomal membrane. Alternatively, they may remain in the cytosol. The genome of Arabidopsis contains five genes coding for type II peroxiredoxins (Table 1). Recently, the fourth type of peroxiredoxin, a homologue of the bacteriferritin co-migratory protein of E. coli, was identified in Sedum lineare (Kong et al., 2000). One gene for a homologous protein is encoded in the A. thaliana genome (Table 1). In the comparatively small Prx Q-proteins the two catalytic Cys residues are spaced by only four amino acids. It has been suggested that the protein exists as a functional monomer and the disulphide bridge is formed between the two Cys-residues (Cys44 and Cys49) (Kong et al., 2000).
Homologues of the higher plant 2-CP were also found in the red algae Porphyra purpurea and the cyanobacterium Synechocystis PCC6803. The high similarity of the algal and cyanobacterial 2-CP with the plant 2-CPs suggests that the plastidic form was introduced into the plants via the photoautotrophic endosymbiont which later on developed into the chloroplast (Baier and Dietz, 1997). This hypothesis concurs with the genomic organization of the 2-CP gene. The chloroplast transit sequence is encoded by a separate exon.
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The catalytic activity of peroxiredoxins |
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TopAbstractPeroxiredoxins in non...Peroxiredoxins in plants and...The catalytic activity of...Structure of the 2-CPThe physiological function of...Antisense suppression of 2-CP...The role of 2-CP...Regulation of 2-CP gene...Perspectives: 2-CP, signalling...Note added in proofReferences |
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Peroxiredoxins reduce peroxide substrates to the corresponding alcohol. In the 1-CP, 2-CP, type II-prx, and prx Q, a Cys-residue in conserved structural environment constitutes the amino acid residue essential for the catalytic activity with the peroxide substrates. In addition to Cys64 (numbers as counted in the mature barley 2-CP: Baier and Dietz, 1996
), members of the 2-CP group contain an additional Cys residue at a more amino-terminal position (C185). During the catalytic cycle with two peroxides two disulphide bonds are successively formed with intermediate modification of one Cys residue to cysteine-sulphenic acid (Cys-OH) and subsequent release of H2O. Thus, three different conformational states of the 2-CP exist and can be detected in non-reducing denaturing gels as distinct bands which correspond to (i) the fully reduced 2-CP, the 2-CP with (ii) a single disulphide bridge and (iii) two disulphide linkages (Gommel et al., 1997; Baier and Dietz, 1998b). The shift in electrophoretic mobility under non-reducing conditions represents a convenient assay for reactivity with peroxide substrates (Fig. 2). Oxidation of the 2-CP also occurs during exposure to dissolved oxygen. The fully oxidized 2-CP loses its enzymatic activity and must be regenerated. For many peroxiredoxins it has been established that thioredoxins serve as the electron donor for the regeneration of the active form. In chloroplasts, thioredoxin f and m function in the regulation of redox-sensitive target proteins (Schürmann and Jacquot, 2000) and could be involved in the regeneration of oxidized 2-CP.
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The substrate specificity of the plant peroxiredoxins has not been investigated in any detail yet. The reconstitution of a functional redox cascade can be achieved for 2-CP with NADPH, E. coli thioredoxin reductase, E. coli thioredoxin, and peroxide substrates such as H2O2 (Fig. 3
), and butyl hydroperoxide or cumene hydroperoxide (data not shown). At 250 µM, the barley 2-CP reduced all three substrates at a similar rate (data not shown). The H2O2-reducing activity at 25 and 10 µM concentration was as high as in the presence of 50 and 100 µM H2O2 (Fig. 3). From kinetic analysis the KM(H2O2) was estimated to be below 2 µM (J König, unpublished data). At high H2O2 concentrations (
500 µM), the 2-CP was inactivated (Fig. 3). H2O2 and butyl hydroperoxide were also shown to be substrates of the cyanobacterial 2-CP. In the absence of other electron acceptors, the addition of either peroxide induced oxygen evolution in wild-type Synechocystis (Yamamoto et al., 1999). The onset of peroxide-induced oxygen liberation in photosynthesis was accompanied by quenching of chlorophyll a fluorescence, which indicated peroxide-dependent relief of thylakoid energization and oxidation of thylakoid electron transport carriers. The 2-CP-cDNA (C2C-Prx) was isolated from chinese cabbage and the protein characterized following recombinant production of the protein (Cheong et al., 1999). C2C-Prx protected DNA from oxidative damage, and glutamine synthase from inactivation in the presence of a mixed functional oxidation system consisting of Fe2+, O2 and thiols. The activity of peroxiredoxins towards H2O2 is well established. However, an analysis of the peroxiredoxin-activity towards lipid peroxides with physiological importance is not available for plants. Such detailed studies have only been performed for the trypanothionine-dependent tryparedoxin peroxidase of trypanosomatids (Nogoceke et al., 1997) and the bacterial alkyl hydroperoxide reductase (AhpC; Hillas et al., 2000). The tryparedoxin peroxidase reduced linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide with similar rate constants. It is likely, that the plant 2-CPs have a similar substrate specificities, but experimental proof needs to be provided.
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Structure of the 2-CP |
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TopAbstractPeroxiredoxins in non...Peroxiredoxins in plants and...The catalytic activity of...Structure of the 2-CPThe physiological function of...Antisense suppression of 2-CP...The role of 2-CP...Regulation of 2-CP gene...Perspectives: 2-CP, signalling...Note added in proofReferences |
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The human 2-CPs Tpx-B from erythrocytes (Schröder et al., 2000
) and the ‘proliferation associated gene’ pag (HBP 23, Hirotsu et al., 1999) were structurally resolved at 1.7 and 2.6 Å atomic resolution, respectively. These studies demonstrated that the functional 2-CP is a dimer. During catalysis, the more amino-terminally located conserved Cys-residue is oxidized followed by disulphide bridge formation and release of water. Under highly oxidizing conditions, the amino-terminally located Cys residue is further oxidized to sulphinic acid. In this ‘over-oxidized’ state the two catalytically interacting Cys residues, which are located on the different subunits of the dimer, are separated by more than 10 Å and the disulphide bridge cannot be formed. As a consequence, the enzyme is trapped in a conformation that cannot be reactivated by reduction. Concomitantly, the 2-CP aggregates as a decamer. Decamerization has been described for the human erythrocyte 2-CP (Schröder et al., 2000) and the Trypanosomas peroxiredoxin (Nogoceke et al., 1997).
The peroxide detoxification system was reconstituted with 2-CP of chinese cabbage and yeast thioredoxin/thioredoxin reductase and the peroxide reduction measured at a concentration of 1 mM H2O2 (Cheong et al., 1999). It will have to be established whether such high H2O2 concentrations are suitable for activity tests or lead to permanent inactivation by modification of the sulphydryl group to sulphinic acid. Figure 3 shows a decrease in the activity of the heterologously expressed barley 2-CP by elevated concentrations of H2O2. In this reaction, the peroxide reduction was coupled to NADPH, thioredoxin reductase and thioredoxin of E. coli.
In addition, the atomic structure at 1.7 Å resolution allowed refinement of the model of catalytic action. The midpoint potential of the barley 2-CP was determined to be more negative than -310 mV (J König, M Baier, KJ Dietz, unpublished results). Apparently, the specific molecular environment activates the Cys-residue. It was shown for the tryparedoxin peroxidase of Crithidia fasciculata that a Trp- and an Arg-residues are of critical importance for maintenance of the peroxiredoxin activity (Montemartini et al., 1999). Mutation of the corresponding amino acids (Arg 140 and Trp 100) of the barley 2-CP showed their importance for maintaining the catalytic activity.
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The physiological function of the 2-Cys peroxiredoxin |
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TopAbstractPeroxiredoxins in non...Peroxiredoxins in plants and...The catalytic activity of...Structure of the 2-CPThe physiological function of...Antisense suppression of 2-CP...The role of 2-CP...Regulation of 2-CP gene...Perspectives: 2-CP, signalling...Note added in proofReferences |
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The physiological function of 2-CPs in photosynthetic organisms has been investigated in insertion mutants of Synechocystis PCC 6803 (
2-CP) (Klughammer et al., 1998; Yamamoto et al., 1999) and in antisense mutants of A. thaliana. (Baier and Dietz, 1999b; Baier et al., 2000). The knock-out mutant strains of Synechocystis grew at the same rates as the wild type at low light intensities, but showed considerably decreased growth rates at high light intensities (Klughammer et al., 1998). Wild-type Synechocystis evolved O2 upon the addition of peroxides to the suspension medium, concomitantly, the chlorophyll a fluorescence was quenched. Both responses were not seen in the 2-CP cells indicating that the 2-CP is the only enzyme that scavenges H2O2 and alkyl hydroperoxides in Synechocystis (Yamamoto et al., 1999). Furthermore, wild type-like fast growth was restored for 2-CP cells when the Fe and trace element concentration was lowered in the nutrient medium to 1/10th (Klughammer et al., 1998). Transition metals such as Fe and Cu, but also other heavy metals cause oxidative stress either by direct transfer of electrons to oxygen or reactive oxygen species, and by means of the inhibition of metabolic reactions (Dietz et al., 1999). Thus micronutrients are both essential and toxic elements. Optimum growth at micromolar concentrations of Fe and Cu are intimately coupled to an efficient detoxification of reactive oxygen species by antioxidants. Elimination of the 2-CP as the main H2O2-detoxifying enzyme in Synechocystis can impair growth even at low metal concentrations.
The situation is considerably more complicated in higher plants. The site of most active oxygen metabolism in the light is the chloroplast. O2 is photo-reduced at photosystem I (PSI) and H2O2 is produced at appreciable rates. Attached to PSI is a scavenging complex for H2O2 consisting of CuZn superoxide dismutase (SOD), ascorbate peroxidase (Apx), ferredoxin (Fd), and Fd-dependent monodehydroascorbate reductase (Asada et al., 1998). The activity of this complex is believed to exceed the rate of O2-photoreduction more than 100-fold and, therefore, appears to be sufficiently active to detoxify H2O2 originating from the Mehler reaction (water/water cycle). In addition to detoxification of reactive oxygen species, the water/water cycle via ascorbate peroxidase serves the dissipation of excessively absorbed energy. The Prx-mediated detoxification pathway represents an alternative water/water cycle (Fig. 4). Similar to the Apx-dependent water/water cycle, the 2-CP-mediated detoxification of H2O2 is not only a scavenging mechanism for reactive oxygen species, but also allows dissipation of excess excitation energy and protection of the photosystems from photoinhibition.
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Chloroplastic Apx is highly sensitive to inactivation by reactive oxygen species. In tobacco, activity of chloroplast Apx was lost during a 48 h drought treatment at high light (Shikanai et al., 1998
). Simultaneously, the rates of photosynthetic CO2-fixation and activities of other oxidation-sensitive enzymes such as phosphoribulokinase (PRK) and glutathione reductase (GSHR) declined in the wilted and light-stressed leaves, however, to a lesser extent than Apx activity. Heterologous expression of a bacterial catalase in the chloroplast protected photosynthesis and PRK and GSHR from oxidative inhibition. In a converse manner, Apx activity also disappeared in these transgenic lines. Shikanai et al. concluded from their study that the ascorbate-mediated antioxidative system is insufficient to protect the photosynthetic apparatus from photoinhibition under conditions of (severe) oxidative stress (Shikanai et al., 1998). The 2-CP-dependent water/water cycle may be an important alternative metabolic pathway to detoxify H2O2 under normal conditions as well as under oxidative stress.
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Antisense suppression of 2-CP activity causes oxidation of ascorbate pool |
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TopAbstractPeroxiredoxins in non...Peroxiredoxins in plants and...The catalytic activity of...Structure of the 2-CPThe physiological function of...Antisense suppression of 2-CP...The role of 2-CP...Regulation of 2-CP gene...Perspectives: 2-CP, signalling...Note added in proofReferences |
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Circumstantial evidence for the role of 2-CP in H2O2 reduction and sparing of the ascorbate peroxidase system was obtained from Arabidopsis plants with decreased levels of 2-CP expression. The light stress sensitivity of the 2-CP-antisense plants was increased and their early seedling development disturbed (Baier and Dietz, 1998
c, 1999b). However, the most striking observation was an increased oxidation state of the ascorbate pool in the transgenic lines with decreased 2-CP levels (Baier et al., 2000). Concomitantly, expression and activities of stromal and thylakoid bound Apx and monodehydroascorbate reductase (MDHAR) were stimulated. Apparently, insufficient amounts of 2-CP directly fed back to the ascorbate system of the leaves. A possible explanation for that observation is the direct involvement of the 2-CP in H2O2 reduction as an alternative to the water/water pathway. Reduced activities of the 2-CP pathway would shift the burden of H2O2 reduction to the Apx-dependent water/water cycle and oxidation state of the ascorbate pool would increase. |
The role of 2-CP in alkyl hydroperoxide reduction |
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TopAbstractPeroxiredoxins in non...Peroxiredoxins in plants and...The catalytic activity of...Structure of the 2-CPThe physiological function of...Antisense suppression of 2-CP...The role of 2-CP...Regulation of 2-CP gene...Perspectives: 2-CP, signalling...Note added in proofReferences |
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Peroxiredoxins reduce a broad range of alkyl hydroperoxide substrates including short- and long-chain alkyl hydroperoxide, phospholipid peroxides and cholesterol peroxide (Nogoceke et al., 1997
; Hillas et al., 2000). For the time being, it can be assumed that plant Prx also shows little discrimination between various alkyl hydroperoxide substrates and reduce diverse peroxides. Alkyl hydroperoxides are produced by spontaneous chemical reactions of unsaturated organic substrates with reactive radicals such as OH·, or by enzymatic reaction of polyunsaturated lipids with lipoxygenases. Subsequently, alkyl hydroperoxides may initiate radical chain reactions and cause membrane destruction. Peroxides need to be eliminated to avoid severe damage of the cells. In addition to detoxification by non-enzymatic antioxidants, two types of enzymes exist in the chloroplasts which are suggested to catalyse the reduction of alkyl hydroperoxides (Baier and Dietz, 1999a, b): (i) The phospholipid hydroperoxide glutathione peroxidase (PHGPx) (Mullineaux et al., 1998) which reacts with peroxide substrates by concomitant oxidation of a cysteine residue. PHGPx is reactivated with reduced glutathione (Eshdat et al., 1997). (ii) The 2-CPs which have a similar substrate spectrum as PHGPxs and are regenerated by thioredoxin. Analysis of transgenic Arabidopsis with reduced 2-CP contents suggest that PHGPx cannot substitute for decreased 2-CP activity (Baier and Dietz, 1999b; Baier et al., 2000). (i) Antisense suppression of 2-CP caused increased damage of chloroplast proteins and impaired photosynthesis. (ii) Increased oxidation of the ascorbate pool and up-regulation of Apx and MDAR transcript level and activity indicated a major disturbance of antioxidant metabolism in the mutant plants, despite the fact that (iii) the PHGPx-transcript level, which was at least 100-fold lower than the 2-CP transcript level, was unchanged in the antisense mutants. A distinct sub-organellar compartmentalization may provide the explanation for the lack of interchangeability of PHGPx and 2-CP in the chloroplast. The PHGPx has been reported to be a soluble enzyme located in the stroma (Mullineaux et al., 1998) whereas the 2-CP is preferentially attached to the thylakoid membrane. |
Regulation of 2-CP gene expression |
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TopAbstractPeroxiredoxins in non...Peroxiredoxins in plants and...The catalytic activity of...Structure of the 2-CPThe physiological function of...Antisense suppression of 2-CP...The role of 2-CP...Regulation of 2-CP gene...Perspectives: 2-CP, signalling...Note added in proofReferences |
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The expression of the 2-CP genes is under developmental and redox control. The steady-state mRNA amount decreased with tissue age in barley (Baier and Dietz, 1996
), A. thaliana (Baier and Dietz, 1999b) and R. fluitans (Horling et al., 2001). Irrespective of the declining message abundance, the protein accumulated with tissue maturation and was unchanged during further ageing under normal growth conditions. However, the developmental responses of both, the 2-CP-mRNA and protein, still need to be tested under conditions of oxidative stress.
Redox-dependent changes in 2-CP gene expression were studied in the liverwort R. fluitans which was taken as a model plant due to the simple application protocol for effectors. The amount of 2-CP transcripts decreased rapidly and strongly upon application of ascorbate to the medium. A severe drop of transcript abundance was observed as early as 2 h after the start of treatment. 24 h after transfer of the liverwort to medium supplemented with 10 mM ascorbate, the 2-CP transcript was undetectable (Horling et al., 2001). The ascorbate-induced decline in 2-CP-mRNA was prevented after pretreating the thalli with the protein kinase inhibitor staurosporin. Apparently, ascorbate triggers a redox-signal transduction pathway which involves a protein phosphorylation step and suppresses 2-CP gene expression. Supplementing the medium with GSH caused a decrease in transcript amount at concentrations as low as 0.1 mM, but no further enhancement of the response at elevated GSH concentrations. In contrast to the liverwort, thiols fed to barley leaves through the petiole decreased the 2-CP transcript level of barley leaves in a concentration-dependent manner (Baier and Dietz, 1997). Establishing oxidizing conditions, for example by the administration of paraquat, stimulated 2-CP expression only slightly. The small response to oxidizing agents is interesting since the bacterial homologue of the 2-CP AhpC was initially identified by its strong response upon exposure to butyl hydroperoxide and H2O2 (Morgan et al., 1985). From all these studies it is concluded that the 2-CP is a constitutive enzyme of the chloroplast expressed at a high level under normal growth conditions. Although up-regulation of expression is not observed under stress, down-regulation is triggered under highly reducing conditions.
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Perspectives: 2-CP, signalling and future work |
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TopAbstractPeroxiredoxins in non...Peroxiredoxins in plants and...The catalytic activity of...Structure of the 2-CPThe physiological function of...Antisense suppression of 2-CP...The role of 2-CP...Regulation of 2-CP gene...Perspectives: 2-CP, signalling...Note added in proofReferences |
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Reactive oxygen species and peroxides are cell toxic on the one hand but they also provide valuable information on the state and metabolic performance of the cells on the other hand (Baier and Dietz, 1998
a). The reactive oxygen species O2·-and H2O2 have been proposed as components of signalling pathways (Noctor et al., 2000). Also alkylhydroperoxides are metabolic intermediates involved in the synthesis of signal molecules, for example, in the jasmonic acid pathway (Farmer et al., 1998). Hydroperoxides produced by lipoxygenase activity from linolenic or linoleic acid can either be cleaved by hydroperoxide lyase, are processed by allene oxide synthase or reduced by peroxygenase and reductases (Weichert et al., 1999). Prx with its broad substrate specificity may serve as reductase. The relative activity of the enzymes will determine the channelling of the substrate to the various pathways. Thus, the activity of peroxiredoxins in general, and chloroplast 2-CP in particular, will affect fatty acid signalling by compounds such as traumatin and jasmonic acid.
For animal cells, the relationship between the activity of peroxiredoxins and cellular signal transduction has been worked out in a series of publications. Over-expression of the peroxiredoxin Prx I suppressed the activation of the nuclear transcription factor NF-KB by externally applied tumour necrosis factor (Kang et al., 1998). The activation of the apoptosis programme via the p53-pathway was inhibited in cells over-expressing Prx V (Zhou et al., 2000). In another study, the expressional level and the subunit composition of the 2-CP dimer correlated with the propensity of the cells to activate the NF-KB-pathway (Jin et al., 1997). The authors of these studies concluded that peroxiredoxins participate in regulating intracellular redox signals and in antioxidative defence. Thus, the central role of peroxiredoxins in peroxide metabolism has unequivocally been established in bacteria and heterotrophic eukaryotes (Flohé, 1998; Rhee et al., 1999). Similar functions in accentuating or suppressing signalling pathways can be hypothesized for plant Prx.
Despite the progress made over the last seven years in understanding the activity and principal functions of peroxiredoxins in plants, important questions need to be addressed in the future, particularly, concerning physiological roles and regulation. Some topics are listed below and show the perspectives for the next years.
(1) The predicted chloroplast localization of the 2-CP-Prx encoded on chromosome 5 will have to be demonstrated unequivocally, similar to the proven location of 2-CP-Prx encoded on chromosome 3 (Baier and Dietz, 1997). The subcellular localization needs to be established for the type II Prx and PrxQ.
(2) Subunit oligomerization has been shown for Prx from other organisms such as trypanosomatids, mammals and yeast and reversible membrane association for the Prx of erythrocytes (Schröder et al., 2000). Both mechanisms represent intriguing possibilities of redox-dependent regulation of Prx activity and should be investigated in plants.
(3) Although the reconstitution of the redox chain with plant Prx, E. coli or yeast thioredoxin and thioredoxin reductase was successful in coupling NADPH oxidation to peroxide reduction in vitro (Cheong et al., 1999; Verdoucq et al., 1999; Fig. 3), the authentic endogenous electron donors of all plant peroxiredoxins still need to be identified and analysed, as well as their substrate spectrum with respect to physiologically relevant alkylhydroperoxides and peroxinitrite.
(4) Up to now, the physiological context of Prx activity has only been worked out for transgenic Arabidopsis with reduced levels of 2-CP and knockout mutants of Synechocystis (Baier and Dietz, 1999; Klughammer et al., 1998; Yamamoto et al., 1999). Similar studies will have to be performed for the various types of Prx and should include knockout mutants in order to separate completely the Prx function from similar metabolic reactions in antioxidant defence and signalling.
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Note added in proof |
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TopAbstractPeroxiredoxins in non...Peroxiredoxins in plants and...The catalytic activity of...Structure of the 2-CPThe physiological function of...Antisense suppression of 2-CP...The role of 2-CP...Regulation of 2-CP gene...Perspectives: 2-CP, signalling...Note added in proofReferences |
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The genome of Arabidopsis thaliana contains a tenth Prx gene denominated Prx II F (Acc. no. At3g06050) whose gene product is predicted to be targeted to the mitochondrion.
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Acknowledgments |
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Part of the work described here was performed within the framework of the Forschergruppe FOR 387, TP 3 and was supported by the grant Di 346/6 of the Deutsche Forschungsgemeinschaft.
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Notes |
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1 To whom correspondence should be addressed. Fax: +49 521106 6039. E-mail: karl\|[hyphen]\|josef.dietz{at}biologie.uni\|[hyphen]\|bielefeld.de
2 Present address: John Innes Centre, Norwich, UK.
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Abbreviations |
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AA, ascorbic acid; DHAP, dehydroascorbate; Fd, ferredoxin; PSI, photosystem I; PSII, photosystem II; Trx, thioredoxin..
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References |
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TopAbstractPeroxiredoxins in non...Peroxiredoxins in plants and...The catalytic activity of...Structure of the 2-CPThe physiological function of...Antisense suppression of 2-CP...The role of 2-CP...Regulation of 2-CP gene...Perspectives: 2-CP, signalling...Note added in proofReferences |
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Aalen RB, Opsahl-Ferstad HG, Linnestad C, Olsen OA. 1994. Transcripts encoding an oleosin and a dormancy-related protein are present in both the aleurone layer and the embryo of developing barley (Hordeum vulgare L.) seeds. The Plant Journal 5, 385–396.[ISI][Medline]
Asada K, Endo T, Mano J, Miyake C. 1998. Molecular mechanism for relaxation of and protection from light stress. In: Satoh K, Murata N, eds. Stress responses of photosynthetic organisms. Amsterdam: Elsevier Science BV, 37–52.
Baier M, Dietz K-J. 1996a. 2-Cys peroxiredoxin bas 1 from Arabidopsis thaliana. Plant Physiology—Plant Gene Register 111, 651.
Baier M, Dietz K-J. 1996b. Primary structure and expression of plant homologues of animal and fungal thioredoxin-dependent peroxide reductases and bacterial alkyl hydroperoxide reductases. Plant Molecular Biology 31, 553–564.[ISI][Medline]
Baier M, Dietz K-J. 1996c. The two-Cys peroxiredoxin Bas1: insight in a new family of plant peroxidases. In: Obinger C, Burner U, Ebermann R, Penel C, Greppin H, eds. Plant peroxidases biochemistry and physiology. Wien: University of Agriculture, 204–209.
Baier M, Dietz K-J. 1997. The plant 2-Cys peroxiredoxin BAS1 is a nuclear encoded chloroplast protein. Its expressional regulation, phylogenetic origin and implications for its specific physiological function in plants. The Plant Journal 12, 179–190.[ISI][Medline]
Baier M, Dietz K-J. 1998a. The costs and benefits of oxygen in photosynthetic plant metabolism. Progress in Botany 60, 283–314.
Baier M, Dietz K-J. 1998b. The plant 2-Cys peroxiredoxin BAS1 is a homodimer whose subunits are linked by disulphide bonds and non-covalent interactions. In: Noga G, Schmitz M, eds. Antioxidants in higher plants: biosynthesis, characteristics, actions and specific functions in stress defence. Aachen: Shaker Verlag, 31–37.
Baier M, Dietz K-J. 1998c. The plant 2-Cys peroxiredoxin protects chloroplasts from oxidative damage. In: Garab G, ed. Photosynthesis: mechanisms and effects. Proceedings of the XI International Congress on Photosynthesis, Budapest: Kluwer Academic Publishers, 2003–2006.
Baier M, Dietz K-J. 1999a. Alkyl hydroperoxide reductases: the way out of the oxidative breakdown of lipids in chloroplasts. Trends in Plant Science 4, 166–168.[ISI][Medline]
Baier M, Dietz K-J. 1999b. Protective function of chloroplast 2-Cys peroxiredoxin in photosynthesis: evidence from transgenic Arabidopsis thaliana. Plant Physiology 119, 1407–1414.
Baier M, Noctor G, Foyer CH, Dietz KJ. 2000. Antisense suppression of 2-Cys peroxiredoxin in Arabidopsis thaliana specifically enhances the activities and expression of enzymes associated with ascorbate metabolism but not glutathione metabolism. Plant Physiology 124, 823–832.
Berberich T, Uebeler M, Feierabend J. 1998. Cloning of a cDNA encoding a thioredoxin peroxidase (PTx) homolog from winter rye (Secale cereale L.) (Accession no. AF076920). Plant Physiology-Plant Gene Register 118, 98–167.
Bryk R, Griffin P, Nathan C. 2000. Peroxynitrite reductase activity of bacterial peroxiredoxins. Nature 407, 211–215.[Medline]
Chae HZ, Kang SW, Rhee SG. 1999. Isoforms of mammalian peroxiredoxin that reduce peroxides in presence of thioredoxin. Methods in Enzymology 300, 219–226.[ISI][Medline]
Chae HZ, Kim IH, Kim K, Rhee SG. 1993. Cloning, sequencing and mutation of thiol-specific antioxidant gene of Saccharomyces cerevisiae. The Journal of Biological Chemistry 268, 16815–16821.
Cheong NE, Choi YO, Lee KO, Kim WY, Jung BG, Chi YH, Jeong JS, Kim K, Cho MJ, Lee SY. 1999. Molecular cloning, expression and functional characterization of a 2Cys-peroxiredoxin in Chinese cabbage. Plant Molecular Biology 40, 825–834.[ISI][Medline]
Choi YO, Cheong NE, Lee KO, Jung BG, Hong CH, Jeong JH et al. 1999. Cloning and expression of a new isotype of the peroxiredoxin gene of chinese cabbage and its comparison to 2 cys-peroxiredoxin isolated from the same plant. Biochemical and Biophysical Research Communications 258, 768–771.[ISI][Medline]
Dietz K-J, Krämer U, Baier M. 1999. Free radicals and reactive oxygen species as mediators of heavy metal toxicity. In: Prasad MNV, Hagemeyer J, eds. Heavy metal stress in plants: from molecules to ecosystems. Springer-Verlag, 73–97.
Eshdat Y, Holland D, Faltin Z, Ben-Hayyim G. 1997. Plant glutathione peroxidases. Physiologia Plantarum 100, 234–240.
Farmer EE, Weber H, Vollenweider S. 1998. Fatty acid signalling in Arabidopsis. Planta 206, 167–174.[ISI][Medline]
Flohé L. 1998. The Achilles' heel of trypanosomatids: trypanothione-mediated hydroperoxide metabolism. Bio Factors 8, 87–91.
Gommel DU, Nogoceke E, Morr M, Kiess M, Kalisz HM, Flohé L. 1997. Catalytic characteristics of tryparedoxin. European Journal of Biochemistry 248, 913–918.[ISI][Medline]
Haslekas C, Stacy RAP, Nygaard V, Culianez-Macia FA, Aalen RB. 1998. The expression of a peroxiredoxin antioxidant gene, AtPer1, in Arabidopsis thaliana is seed-specific and related to dormancy. Plant Molecular Biology 36, 833–845.[ISI][Medline]
Hillas PJ, del Alba FS, Oyarzabal J, Wilks A, del Montellano PRO. 2000. The AhpC and AhpD antioxidant system of Mycobacterium tuberculosis. The Journal of Biological Chemistry 275, 18801–18809.
Hirotsu S, Abe Y, Okada K, Nagahara N, Hori H, Nishino T, Hakoshima T. 1999. Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product. Proceedings of the National Academy of Sciences, USA 96, 12333–12338.
Horling F, Baier M, Dietz K-J. 2001. Redox-regulation of expression of the peroxide-detoxifying chloroplast 2-cysteine peroxiredoxin in the liverwort Riccia fluitans. Planta 214, 304–313; online version DOI 10.1007/s004250100623.[ISI][Medline]
Jin D-Y, Chae HZ, Rhee SG, Jeang K-T. 1997. Regulatory role for a novel human thioredoxin peroxidase in NF-
B activation. The Journal of Biological Chemistry 272, 30952–30961.
Kang SW, Chae HZ, Seo MS, Kim K, Baines IC, Rhee SG. 1998. Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-
. The Journal of Biological Chemistry 273, 6297–6302.
Klughammer B, Baier M, Dietz K-J. 1998. Inactivation by gene disruption of 2-cysteine-peroxiredoxin in Synechocystis sp. PCC 6803 leads to increased stress sensitivity. Physiologia Plantarum 104, 699–70.
Kong W, Shiota S, Shi Y, Nakayama H, Nakayama K. 2000. A novel peroxiredoxin of the plant Sedum lineare is a homologue of Escherichia coli bacterioferritin co-migratory protein (Bcp). Biochemical Journal 351, 107–114.[ISI][Medline]
Lewis ML, Miki K, Ueda T. 2000. FePer 1, a gene encoding an evolutionarily conserved 1-Cys peroxiredoxin in buckwheat (Fagopyrum esculentum Moench), is expressed in a seed-specific manner and induced during seed germination. Gene 246, 81–91.[ISI][Medline]
Lim MJ, Chae HZ, Rhee SG, Yu DY, Lee K-K, Yeom YI. 1998. The type II peroxiredoxin gene family of the mouse: molecular structure, expression and evolution. Gene 216, 197–205.[ISI][Medline]
Lim Y-S, Cha MK, Kim HK, Uhm TB, Park JW, Kim K et al. 1993. Removals of hydrogen peroxide and hydroxyl radical by thiol-specific antioxidant protein as a possible role in vivo. Biochemical and Biophysical Research Communications 192, 273–280.[ISI][Medline]
Lopez JA, Carvalho TU, de Souza W, Flohe L, Guerrero SA, Montemartini M, Kalisz HM, Nogoceke E, Singh M, Alves MJM, Colli W. 2000. Evidence for a trypanothione-dependent peroxidase system in Trypanosoma cruzi. Free Radical Biology and Medicine 28, 767–772.[ISI][Medline]
Majoul T, Chahed K, Zamiti E, Ouelhazi L, Ghrir R. 2000. Analysis by two-dimensional electrophoresis of the effect of salt stress on the polypeptide patterns in roots of a salt-tolerant and a salt-sensitive cultivar of wheat. Electrophoresis 21, 2562–2565.[ISI][Medline]
Montemartini M, Kalisz HM, Hecht H-J, Steinert P, Flohé L. 1999. Activation of active-site cysteine residues in the peroxiredoxin-type tryparedoxin peroxidase of Crithidia fasciculata. European Journal of Biochemistry 264, 516–524.[ISI][Medline]
Morgan RW, Christman MF, Jacobson FS, Storz G, Ames BN. 1985. Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins. Proceedings of the National Academy of Sciences, USA 83, 8059–8063.
Mullineaux PM, Karpinski S, Jimanez A, Cleary SP, Robinson C, Creissen G. 1998. Identification of cDNAs encoding plastid-targeted glutathione peroxidase. The Plant Journal 13, 375–379.[ISI][Medline]
Noctor G, Veljovic-Jovanovic S, Foyer CH. 2000. Peroxide processing in photosynthesis: antioxidant coupling and redox signalling. Philosophical Society Transactions 355, 1465–1476.
Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohé L. 1997. A unique cascade of oxidoreductases catalyses trypanothione-mediated peroxide metabolism in Crithidia fasciculata. Biological Chemistry 378, 827–836.[ISI][Medline]
Pedrajas JR, Miranda-Vizuete A, Javanmardy N, Gustafsson JA, Spyrou G. 2000. Mitochondria of Saccharomyces cerevisiae contain one-conserved cysteine type peroxiredoxin with thioredoxin peroxidase activity. The Journal of Biological Chemistry 275, 16296–16301.
Prospéri MT, Ferbus D, Karczinski I, Goubin G. 1993. A human cDNA corresponding to a gene overexpressed during cell proliferation encodes a product sharing homology with amoebic and bacterial proteins. The Journal of Biological Chemistry 268, 11050–11056.
Rhee SG, Kang SW, Netto LE, Seo MS, Stadtman ER. 1999. A family of novel peroxidases, peroxiredoxins. Biofactors 10, 207–209.[ISI][Medline]
Schröder E, Littlechild JA, Lebedev AA, Errington N, Vagin AA, Isupov MN. 2000. Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 Å resolution. Structure 8, 605–615.[Medline]
Schürmann P, Jacquot J-P. 2000. Plant thioredoxin systems revisited. Annual Review of Plant Physiology and Plant Molecular Biology 51, 371–400.[ISI]
Seo MS, Kang SW, Kim K, Baines IC, Lee TH, Rhee SG. 2000. Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulphide as a reaction intermediate. The Journal of Biological Chemistry 275, 20346–20354.
Shikanai T, Takeda T, Yamauchi H, Sano S, Tomizawa KI, Yokota A, Shigeoka S. 1998. Inhibition of ascorbate peroxidase under oxidative stress in tobacco having bacterial catalase in chloroplasts. FEBS Letters 428, 47–51.[ISI][Medline]
Stacy RAP, Munthe E, Steinum T, Sharma B, Aaalen RB. 1996. A peroxiredoxin antioxidant is encoded by a dormancy-related gene, Per1, expressed during late development in the aleurone and embryo of barley grains. Plant Molecular Biology 31, 1205–1216.[ISI][Medline]
Stacy RAP, Nordeng TW, Culianez-Macia FA, Aalen RB. 1999. The dormancy-related peroxiredoxin anti-oxidant, PER1, is localized to the nucleus of barley embryo and aleurone cells. The Plant Journal 19, 1–8.[ISI][Medline]
Storz G, Jacobson FS, Tartaglia LA, Morgan RW, Silveira LA, Ames BN. 1989. An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp. Journal of Bacteriology 171, 2049–2055.
The Arabidopsis Genome Iniative. 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815.[Medline]
Van de Peer Y, De Wachter R. 1994. TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Computer Applications in Bioscience 10, 569–570.
Weichert H, Stenzel I, Berndt E, Wasternack C, Feussner I. 1999. Metabolite profiling of oxylipins upon salicylate treatment in barley leaves—preferential induction of the reductase pathway by salicylate. FEBS Letters 464, 133–137.[ISI][Medline]
Verdoucq L, Vignol F, Jacquot JP, Chartier Y, Meyer Y. 1999. In vivo characterization of a thioredoxin h target protein defines a new peroxiredoxin family. The Journal of Biological Chemistry 274, 19714–19722.
Yamamoto H, Miyake C, Dietz K-J, Tomizawa KI, Murata N, Yokota A. 1999. Thioredoxin peroxidase in the cyanobacterium Synechocystis sp. PCC 6803. FEBS Letters 447, 269–273.[ISI][Medline]
Zhou Y, Kok KH, Chun ACS, Wong CM, Wu HW, Lin MCM, Fung PCW, Kung HF, Jin DY. 2000. Mouse peroxiredoxin V is a thioredoxin peroxidase that inhibits p53-induced apoptosis. Biochemical and Biophysic