Ho Zoon Chae

Mammalian Peroxiredoxin Isoforms Can Reduce Hydrogen Peroxide Generated in Response to Growth Factors and Tumor Necrosis Factor-α

Mammalian tissues express three immunologically distinct peroxiredoxin (Prx) proteins (Prx I, II, and III), which are the products of distinct genes. With the use of recombinant proteins Prx I, II, and III, all have now been shown to possess peroxidase activity and to rely on Trx as a source of reducing equivalents for the reduction of H2O2. Prx I and II are cytosolic proteins, whereas Prx III is localized in mitochondria. Transient overexpression of Prx I or II in cultured cells showed that they were able to eliminate the intracellular H2O2 generated in response to growth factors. Moreover, the activation of nuclear factor κB (NFκB) induced by extracellularly added H2O2 or tumor necrosis factor-α was blocked by overproduction of Prx II. These results suggest that, together with glutathione peroxidase and catalase, Prx enzymes likely play an important role in eliminating peroxides generated during metabolism. In addition, Prx I and II might participate in the signaling cascades of growth factors and tumor necrosis factor-α by regulating the intracellular concentration of H2O2.

Regulatory Role for a Novel Human Thioredoxin Peroxidase in NF-κB Activation

Reduction-oxidation (redox) plays a critical role in NF-κB activation. Diverse stimuli appear to utilize reactive oxygen species (e.g. hydrogen peroxide) as common effectors for activating NF-κB. Antioxidants govern intracellular redox status, and many such molecules can reduce H2O2. However, functionally, it does appear that different antioxidants are variously selective for redox regulation of certain transcription factors such as NF-κB. For NF-κB, thioredoxin has been described to be a more potent antioxidant than either glutathione orN-acetylcysteine. Thioredoxin peroxidase is the immediate enzyme that links reduction of H2O2 to thioredoxin. Several putative human thioredoxin peroxidases have been identified using recursive sequence searches/alignments with yeast or prokaryotic enzymes. None has been characterized in detail for intracellular function(s). Here, we describe a new human thioredoxin peroxidase, antioxidant enzyme AOE372, identified by virtue of its protein-protein interaction with the product of a proliferation associationgene, pag, which is also a thiol-specific antioxidant. In human cells, AOE372 defines a redox pathway that specifically regulates NF-κB activity via a modulation of IκB-α phosphorylation in the cytoplasm. We show that AOE372 activity is regulated through either homo- or heterodimerization with other thiol peroxidases, implicating subunit assortment as a mechanism for regulating antioxidant specificities. AOE372 function suggests thioredoxin peroxidase as an immediate regulator of H2O2-mediated activation of NF-κB.

Reversible Oxidation of the Active Site Cysteine of Peroxiredoxins to Cysteine Sulfinic Acid IMMUNOBLOT DETECTION WITH ANTIBODIES SPECIFIC FOR THE HYPEROXIDIZED CYSTEINE-CONTAINING SEQUENCE

We previously suggested that oxidation of the active site cysteine of peroxiredoxin (Prx) I or Prx II to cysteine sulfinic acid in H2O2-treated cells is reversible (Woo, H. A., Chae, H. Z., Hwang, S. C., Yang, K.-S., Kang, S. W., Kim, K., and Rhee, S. G. (2003) Science 300, 653–656). In contrast, it was recently proposed that sulfinylation of Prx II, but not that of Prx I or Prx III, is reversible (Chevallet, M., Wagner, E., Luche, S., van Dorssealaer, A., Leize-Wagner, E., and Rabilloud, T. (2003) J. Biol. Chem. 278, 37146–37153). The detection of sulfinylated proteins in both of these previous studies relied on complex proteomics analysis. We now describe a simple immunoblot assay for the detection of sulfinylated Prx enzymes that is based on antibodies produced in response to a sulfonylated peptide modeled on the conserved active site sequence. These antibodies recognized both sulfinic and sulfonic forms of Prx equally well and allowed the detection of sulfinylated Prx enzymes in H2O2-treated cells with high sensitivity and specificity. With the use of these antibodies, we demonstrated that not only the cytosolic enzymes Prx I and Prx II but also the mitochondrial enzyme Prx III undergo reversible sulfinylation. The generation of antibodies specific for sulfonylated peptides should provide insight into protein function similar to that achieved with antibodies to peptides containing phosphoserine or phosphothreonine.

Irreversible Oxidation of the Active-site Cysteine of Peroxiredoxin to Cysteine Sulfonic Acid for Enhanced Molecular Chaperone Activity

The thiol (–SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (–SO2H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (–SO3H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H2O2 concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO3H) by mass spectrometry. Tsa1p-SO3H was not an autoxidation product of Tsa1p-SO2H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO3H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO3H multimer lost its peroxidase activity, it gained ∼4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO3H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO3H is a marker of cumulative oxidative stress in cells.

Novel Protective Mechanism against Irreversible Hyperoxidation of Peroxiredoxin Nα-TERMINAL ACETYLATION OF HUMAN PEROXIREDOXIN II

Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (CP), cycles between thiol (CP-SH) and disulfide (–S–S–) states via a sulfenic (CP-SOH) intermediate. Hyperoxidation of the CP thiol to its sulfinic (CP-SO2H) derivative has been shown to be reversible, but its sulfonic (CP-SO3H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with CP-SO3H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that Nα-terminal acetylation (Nα-Ac) occurred exclusively on Prx II after demethionylation. Nα-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-Nα-acetylated and Nα-terminal acetylated Prx II revealed that Nα-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of Tm from 59.6 to 70.9 °C. These findings suggest that the structural maintenance of Prx II by Nα-Ac may be responsible for preventing its hyperoxidation to form CP-SO3H.

PEROXIDASE ACTIVITY OF A TSA-LIKE ANTIOXIDANT PROTEIN FROM A PATHOGENIC AMOEBA

The 29 kDa surface protein of Entamoeba histolytica is an abundant antigenic protein expressed by pathogenic strains of this organism. The protein is a member of a widely-dispersed group of homologues which includes at least two cysteinyl peroxidases, Salmonella typhimurium alkyl hydroperoxidase C-22 protein (AhpC) and Saccharomyces cerevisiae thiol-specific antioxidant protein (TSA). Here, for the first time in a pathogenic eukaryote, we have demonstrated that the amoebic protein also possesses peroxidatic and antioxidant activities in the presence of reductants such as dithiothreitol or thioredoxin reductase plus thioredoxin. Although the S. typhimurium AhpF flavoprotein was not an effective reductant of the amoebic TSA protein, one inhibitory monoclonal antibody directed toward amoebic TSA was also partially inhibitory toward reduced but not oxidized bacterial AhpC. These antioxidant proteins are likely to be important not only in general cell protection, but also in the promotion of infection and invasion by these pathogenic organisms through protection against oxidative attack by activated host phagocytic cells.

Genetic mapping of six mouse peroxiredoxin genes and fourteen peroxiredoxin related sequences

Organisms living in aerobic environments require mechanisms that prevent or limit cellular damage caused by reactive oxygen species (O2 , H2O2, and HO) that arise from the incomplete reduction of oxygen during respiration. Alternatively, damage can result from exposure to external agents such as light, radiation, redox-cycling drugs, or stimulated host phagocytes (Sies 1993; Halliwell and Gutteridge, 1989). The reactive oxygen species cause damage to all major classes of biological macromolecules leading to protein oxidation, lipid peroxidation, and DNA base modifications and strand breaks. To guard against these destructive processes, organisms have developed a battery of antioxidant defenses (Halliwell and Gutteridge 1989; Amstad et al. 1991). The preventive antioxidant systems include enzymes that decompose peroxides and superoxide anion and compounds that sequester metal ions. These types of antioxidants reduce or eliminate the generation of free radicals. Chain-breaking antioxidants, such as ascorbate and a-tocopherol, scavenge transient free radicals and inhibit the attack of these reactive species on biological targets. We have previously purified a 25-kDa enzyme from yeast that prevents damage induced by the thiol oxidation system but not by the ascorbate oxidation system, despite the fact that the degree of oxidative stress is similar for the two systems as judged by the comparable extent of induced inactivation of glutamine synthetase (Kim et al. 1988). Thus, we originally named this protein thiolspecific antioxidant (TSA). Although the exact nature of the oxidant eliminated by TSA was not known at that time, the importance of TSA as an antioxidant was readily apparent as the application of oxidative pressure to yeast resulted in an increase in the synthesis of TSA, and TSA protein constituted 0.7% of total soluble protein from yeast grown aerobically (Kim et al. 1989). Yeast TSA gene was cloned and sequenced (Chae et al. 1993). It shows no significant homology to any known catalase, superoxide dismutase, or peroxidase enzymes. This lack of homology is consistent with the observation that TSA does not possess catalytic activity characteristic of conventional antioxidant enzymes. A yeast mutant that cannot produce TSA was constructed by homologous recombination (Chae et al. 1993). The mutant and wild-type trains grew at equal rates under anaerobic conditions. However, under aerobic conditions, especially under oxidative stress, the growth rate of mutant yeast was significantly lower than that of wild-type yeast. A database search revealed a number of proteins from a variety organisms that show similarity to TSA (Chae et al. 1994b). These homologous proteins have now been named the peroxiredoxin (PRDX) family. We recently demonstrated that the antioxidant activity of TSA is attributable to its ability to reduce H 2O2. The apparent specific requirement for a thiol for antioxidant function was due to the fact that an intermolecular disulfide linkage of oxidized TSA can be reduced by a thiol but not by ascorbate. We have shown that thioredoxin (TRX) is the physiological electron donor for the reduction of TSA (Chae et al. 1994a). TSA was thus the first peroxidase to be identified for which TRX is the immediate electron donor, and it was therefore renamed TRX peroxidase (TPX). Despite this finding, the TSA homologs (the PRDX gene family) were not termed the TPX family because not all members use TRX as the hydrogen donor. For example, enteric bacteria homolog AhpC and trypanosomatid homolog C22 receive electron from AhpF and C30 proteins, respectively, for the reduction of H2O2 (Jacobson et al. 1989; Montemartini et al. 1998). Furthermore, mammalian PRDX-V is capable of reducing H 2O2 in the presence of dithiothreitol but not in the presence of TPX (Kang et al. 1998). The complete amino acid sequences of 15 mammalian members of the PRDX family have been determined: six (PAG, NKEFA, NKEFB, TSA, MER5, and AOE372) from human, six (MSP23, OSF3, TSA, MER5, AOP1, and AOP2) from mouse, two (TSA and HBP23) from rat, and one (SP22) from cow. With the exception of TSA, all mammalian PRDX proteins were initially characterized without reference to antioxidant function (Chae et al., 1994c; Prosperi et al. 1993; Shau et al. 1994; Yamamoto et al. 1989; Jin et al. 1997). Among the six reported human PRDX sequences, there are four distinct human PRDX proteins: PAG/ NKEFA, TSA/NKEFB, MER5, and AOE372. Similarly, the six reported mouse sequences actually correspond to four distinct proteins: MSP23/OSF3, TSA, MER5/AOP1, and AOP2. The four human PRDX proteins show only 60–80% sequence identity to each other, but all except AOE372 share >90% identity with a corresponding mouse homolog. Each of the two rat proteins (HBP23 and TSA) and bovine SP22 show >92% sequence identity to one of the human or mouse proteins. Therefore, the mammalian PRDX proteins can be grouped into one of five types: PRDX-I, represented by PAGA; PRDX-II, represented by TSA; PRDX-III, represented by MER5; PRDX-IV, represented by AOE372; and * Present address:Laboratory of Population Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

Expression of peroxiredoxin and thioredoxin in dermatological disorders

SIR, Many thanks are due to Deirdre Buckley and Anthony Du Vivier for their extremely comprehensive and informative review of the use of topical contact sensitizers. I have been involved in a systematic review of randomized controlled trials for all local treatments for cutaneous warts; during our searches we discovered two randomized controlled trials of dinitrochlorobenzene (DNCB) which were subsequently included in our review. Neither trial was mentioned in the review by Buckley and Du Vivier and both are worthy of comment. The trials were small (80 patients in total) and of relatively low quality but they have the advantage of being properly randomized and hence less prone to bias. Data pooled from these trials showed cure in 32 of 40 (80%) with active treatment and 15 of 40 (38%) with placebo, odds ratio 6Æ67 (95% confidence interval 2Æ44–18Æ23), random effects model. There were no precise data concerning adverse effects in either of these trials but Rosado-Cancino et al. commented that six of their 20 participants treated with DNCB sensitized only after the second application of 2% DNCB to the warts. All of these patients subsequently experienced significant local irritation, with or without blistering, when they were treated with 1% DNCB. None withdrew from the study. In our review, cryotherapy, interestingly, fared slightly less well than simple topical treatments containing salicylic acid, with much more substantial evidence for the efficacy of the latter than the former. Two trials (involving a total of 320 participants) comparing these two treatments showed them to be only of equivalent efficacy, with cure rates of approximately 60–70%. Of all the treatments reviewed, DNCB came out with the highest odds ratio for cure of warts, albeit with rather wide confidence intervals because of the small numbers in these two trials. We thought it reasonable to conclude, however, that topical immunotherapy with sensitizers such as DNCB appears to be one of the more promising therapeutic avenues for refractory warts.