Critical Review. Peroxiredoxin, a Novel Family of Peroxidases

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1 IUBMB Life, 52: 35 41, /01 $ Critical Review Peroxiredoxin, a Novel Family of Peroxidases Sue Goo Rhee, 1 Sang Won Kang, 1 Tong-Shin Chang, 1 Woojin Jeong, 1 and Kanghwa Kim 2 1 Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA 2 Department of Food and Nutrition, College of Home Economics, Chonnam National University, Kwangju, Korea INTRODUCTION The incomplete reduction of oxygen to water during respiration results in the formation of the superoxide anions, which are spontaneously or enzymatically converted to H 2 O 2. Hydrogen peroxide itself is not very reactive, but can be further reduced to the extremely damaging hydroxyl radicals. The reactive oxygen species are also produced during the beta-oxidation of fatty acids, and upon exposure to radiation, light, metals, and redox drugs. Therefore, all aerobic cells are equipped with H 2 O 2 -removing enzymes, which include catalase, glutathione peroxidase, haem-containing peroxidase, and peroxiredoxin. Furthermore, substantial evidence suggests that H 2 O 2 is produced transiently in response to the activation of many cell surface receptors and serves as an intracellular messenger (32). Timely elimination of intracellular messengers after completion of their mission is critical for receptor singnaling. This would seem especially true for H 2 O 2, which is readily converted to deleterious hydroxyl radicals. Peroxiredoxin (Prx) is a family of peroxidases with molecular size of kda that are present in organisms from all kingdoms. Prx exists in multiple isoforms in all eukaryotic cells. By inspecting the recently available genome sequences, Saccharomyces cerevisiae (27 ) and Drosophila melanogaster (31) were shown to contain ve isoforms each, and human has at least 6 isoforms (33). Their amino acid sequences are aligned and divided into three subgroups in Fig. 1. Each of the isoforms is unique in that they exhibit different expression patterns during development, distribute differently in organelles, and undergo different reaction intermediates during catalysis. Some of these isoforms Received 5 August 2001; accepted 30 August This article is not subject to U.S. copyright laws. Address correspondence to Sue Goo Rhee, NHLBI, NIH, Lab Cell Signaling, Bldg. 50, Rm. 3523, Bethesda, MD Fax: (301) sgrhee@nih.gov provide defense against oxidative damage, yet others appear to participate in signaling by controlling H 2 O 2 concentration. General Properties of Prx Enzymes Prx was initially discovered in yeast as a 25-kDa enzyme that provides cellular components with protection against oxidative damages caused by the thiol-containing oxidation system (Fe 3C, O 2, and RSH) but not by the ascorbate-containing oxidation system (Fe 3C, O 2, and ascorbate) (21). The protection was possible because the 25-kDa protein was capable of reducing H 2 O 2 with the use of hydrogens provided by thiol. Cys 47 was the primary site of oxidation by H 2 O 2 produced from the thiolcontaining oxidation system (7 ). Because the peroxidase activity was supported by a variety of thiols but not by other reductants such as ascorbic acid, we named the yeast 25-kDa protein thiolspeci c anti-oxidant (TSA) (6 ). Subsequently, we found that the Cys47-SH is oxidized by H 2 O 2 to cysteine sulfenic acid (Cys47-SOH), which then reacts with Cys170-SH of the other subunit to produce an intermolecular disul de (3). The apparent thiol speci city observed for TSA is attributed to the fact that TSA disul de can be reduced by a thiol but not by ascorbate. We then showed physiologically that thioredoxin (Trx) together with thioredoxin reductase (TrxR) mediates the ow of electrons from NADPH toward the oxidized TSA intermediate (3). We thus renamed TSA thioredoxin peroxidase (TPx) in order to re ect that Trx is the immediate electron donor (3). Now, the TPx is called yctpx I to re ect that it is a cytosolic enzyme, which is one of ve distinct TPx isoforms in yeast (27 ). Although the designation TPx is meaningful, we were reluctant to refer to the entire family of peroxidases as the TPx family because not all members use Trx as the electron donor. Bacteria, plant, and mammalian cells all contain TPx homologs that are capable of reducing peroxide in the presence of dithiothreitol (DTT) but not in the presence of Trx (17 ). We thus proposed to call this family of homolgs Prx family (6). However, the TPx 35

2 36 RHEE ET AL. Figure 1. Amino acid sequences of human Prx (hprx), Drosophila Prx(dPrx), and yeast Prx(yPrx) are aligned according to a method described in (9). Prx enzymes are assorted into three subgroups as follows: 2-Cys (hprxi IV, dprx4783, dprx5037, dprx4156, yctpx1, and yctpxii), atypical 2-Cys (hprxv and yctpxiii), and 1-Cys (hprxvi, dprx2540, dprx6005, ymtpx, and yntpx) subgroups. The active site cysteine residue is shown in bold. The amino acid residues showing consensus value above 90% and 50% are in upper case and lower case, respectively. nomenclature is still used for yeast enzymes because all yeast Prx enzymes known to date are receiving the reducing equivalents from Trx (15, 27 ). All Prx proteins contain a conserved cysteine residue, which correspond to the Cys 47 in yeast ctpx I, in the NH 2 -terminal portion of the molecule, and most contain an additional conserved Cys in the COOH-terminal region, which correspond to the Cys 170 in yeast Prx (6). Both conserved Cys residues are required for their catalytic function. A small number of Prx proteins, with representatives from most phyla, lack the COOHterminal Cys. Members of the Prx family can thus be divided into two subgroups: 2-Cys Prx proteins, which contain both the NH 2 - and COOH-terminal Cys residues, and 1-Cys Prx proteins, which contain only the NH 2 -terminal Cys (6, 17 ). The presence or absence of the second conserved Cys is also correlated with the conservation of sequences surrounding the rst Cys, which resides in a signature motif of FTFVCPTEI in the 2-Cys Prx members and FTPVCTTEL in the 1-Cys Prx members (Fig. 1). The third group includes atypical Prx proteins that contain only the NH 2 -terminal conserved Cys but that require an additional Cys for their peroxidase activity. The crystal structures of 1-Cys and 2-Cys Prx enzymes revealed that both exist in a head-to-tail dimeric structure, as biochemically predicted (7, 8, 12). The N-terminal and C-terminal domains of the two subunits of human 1-Cys Prx (also called Prx VI, see later) form a narrow pocket with a diameter of approximately 4 ÊA and the catalytic Cys 47 is located at the bottom of this pocket (8). The reactive Cys 47 is thus protected from larger oxidant molecules that contain disul de linkages. The structure also explains why the Cys47-SH is preferentially oxidized by H 2 O 2. The cysteine thiolate anion (Cys-S ) is more readily oxidized by H 2 O 2 than is the Cys sulfhydryl group (Cys- SH). Because the pk a values of most protein Cys-SH residues are 8.5, few proteins would be expected to possess a Cys-SH

3 NOVEL FAMILY OF PEROXIDASES 37 residue that is readily susceptible to oxidation by H 2 O 2 in cells. Protein Cys residues exist as thiolate anions at neutral ph often because the negatively charged thiolate is stabilized by salt bridges to positively charged amino acid residues. The structure of 1-Cys Prx revealed that His 39 and the guanido group of Arg 132 are located close enough to stabilize the Cys 47 thiolate anion (8). In the 2-Cys Prx (rat Prx I), His 39 is replaced by Tyr but Arg 132 is conserved (12). Indeed, Arg 132 is conserved in all members of the Prx family (Fig. 1). 2-Cys Prx Subgroup Members Although Prx was identi ed initially in yeast, the presence of multiple isoforms and their characterization were achieved rst in mammalian cells. A mammalian (formerly TSA) Prx, which is 65% identical to yeast ctpxi in amino acid, was puri ed from bovine brain (6 ). A subsequent database search revealed 12 mammalian homologs: ve (TSA, PAG, NKEFA, NKEFB, and MER5) from human, four (TSA, MSP23, OSF3, and MER5) from mouse, two (TSA and HBP23) from rat, and one (SP22) from cow. With the exception of TSA, all these homologs were initially characterized without reference to peroxidase function: PAG was identi ed as the 22-kDa product of a gene whose expression increases during proliferation of human epithelial cells (30); NKEFA and NKEFB as cytosolic factors from human red blood cells that enhance the activity of natural killer cells (34); MER5 as the product of a housekeeping gene that is preferentially expressed in murine erythroleukemic cells during the early period of differentiation (38); MSP23 as a 23-kDa stressinduced protein in mouse peritoneal macrophages (13); OSF3 as the product of a gene that is speci cally expressed in mouse osteoblastic cells (19); SP22 as a substrate of a mitochondrial ATPdependent protease in bovine adrenal cortex (37 ); and HBP23 as a 23-kDa heme-binding protein puri ed from rat liver cytosol (14). Among the ve reported human Prx sequences, TSA and PAG were identical to NKEFB and NKEFA, respectively. Thus, there are three distinct human Prx proteins: PAG/NKEFA, TSA/NKEFB, and MER5. Similarly, because MSP23 is identical to OSF3, the four reported mouse sequences actually correspond to three distinct proteins: MSP23/OSF3, TSA, and MER5. The three human Prx proteins show only 60 to 80% sequence identity to each other, but each shares >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 three human or mouse proteins. Therefore, the nine mammalian Prx proteins can be divided into three types Prx I (human PAG/NKEFA, mouse MSP23/OSF3, and rat HBP23), Prx II (human TSA/NKEFB, mouse, and rat TSA), and Prx III (human MER5 and bovine SP22). Both Prx I and Prx II proteins consist of 199 residues and exist in cytosol. Prx III proteins include mouse and human MER5 and bovine SP22. The 257-amino acid sequence of MER5 (38) deduced from the cdna sequence is substantially larger than the 195-residue sequence of SP22 determined directly by peptide sequencing of SP22 puri ed from mitochondria of bovine adrenal cortex (37 ). The SP22 sequence aligns with 93% identity with the COOH-terminal 195 residues of MER5 and the additional 62 residues at the amino-terminus were proved to be the mitochondrial targeting sequence. Prx IV (AOE372) was identi ed as a protein that interacts with Prx I by the yeast two-hybrid assay (16). This proteinprotein interaction is probably because a small portion of Prx proteins form heterodimers. Prx IV contains the NH 2 -terminal hydrophobic stretch that is typical for the signal sequence for secretory proteins (25). Indeed, the 30-kDa Prx is found in the endoplasmic reticulum (ER) and culture medium of COS-1 cells, and its size is smaller than the predicted size from its cdna sequence (25). It is likely, therefore, Prx IV is posttrantionally processed in the ER and secreted to extracellular space. Mammalian Prx I, II, III, and IV all belong to the 2-Cys Prx subgroup and have the conserved NH 2 - and COOH-terminal Cys residues that are separated by 121 amino acid residues. As in the case of yeast yctpx I, the four Prx enzymes form reaction intermediates that contain an intermolecular disul de between the NH 2 - and COOH-terminal Cys residues that correspond to Cys 52 and Cys 173, respectively, of human Prx I, and the disul de is subsequently reduced by Trx (Fig. 2). Thus, mutant 2-Cys Prx proteins that lack either the NH 2 -terminal or COOH-terminal Cys residues do not exhibit Trx-coupled peroxidase activity. The reduction of the intermolecular disul de is speci c to Trx and could not be achieved by glutathione (GSH) or glutaredoxin. Mammalian cells contain mitochondria-speci c Trx and TrxR, suggesting that Prx III together with the mitochondria-speci c Trx (35) and TrxR (24) provide a primary line of defense against H 2 O 2 produced by the mitochondrial respiratory chain. Among the ve Drosophila Prx enzymes, three (dprx4783, dprx5037, and dprx4156) fall into the 2-Cys subgroup, while the other two (dprx2540 and dprx6005) belong to the 1-Cys subgroup (Fig. 1). They were named after the clot numbers, from which their cdna were identi ed (31). dprx4783 and dprx5037 are found in cytosol and mitochondria, respectively, whereas dprx4156 is predominantly found in the culture media. All three Drosophila 2-Cys enzymes exhibited Trx-dependent peroxidase activity. Two cytosolic yeast enzymes yctpxi (formerly TSA) and yctpxii are >50% identical to mammalian Prx I IV in amino Figure 2. Reaction mechanism of mammalian Prx I IV.

4 38 RHEE ET AL. acid sequence, form reaction intermediates with an intermolecular disul de, and depend on Trx for the supply of reducing equivalents. It is interesting to note, however, that their COOHterminal conserved Cys (Cys 170 of yctpxi) is removed from the predicted position by two residues (Fig. 1). Atypical 2-Cys Prx Subgroup Members The amino acid sequence identity among the four mammalian 2-Cys (Prx I to Prx IV) enzymes is >70%, with the homology being especially marked in the regions surrounding the conserved NH 2 - and COOH-terminal Cys residues (Fig. 1). Prx V was identi ed as the result of a human EST database search with the NH 2 -terminal conserved sequence (KGKYVVLFFY PLDFTFVCP) of the 2-Cys Prx enzymes (33). The 162-amino acid Prx V shares only 10% sequence identity with the four mammalian 2-Cys Prx proteins and the sequence surrounding the conserved NH 2 -terminal Cys (Cys48) (KGKKGVLFGVP GAFTPGCS) is only 52% identical (indicated in bold) to the search sequence. The COOH-terminal region of Prx V is smaller than those of 2-Cys Prx enzymes and lacks the conserved sequence containing the COOH-terminal Cys of the latter enzymes. Both human and mouse Prx V sequences contain Cys residues at positions 73 and 152 in addition to the conserved Cys48. However, the sequences surrounding Cys73 and Cys152 are not homologous to those surrounding the COOH-terminal conserved Cys residue of 2-Cys Prx enzymes, and the distances between Cys48 and these other two Cys residues are substantially smaller than the 120 to 123 residues that separate the two conserved Cys residues in typical 2-Cys Prx enzymes. Cys48 is the site of oxidation by peroxides, and that oxidized Cys48 reacts with the sulfhydryl group of Cys152 to form an intramolecular disul de linkage (Fig. 3). The oxidized intermediate of Prx V is thus distinct from those of other typical 2-Cys Prx enzymes, which form an intermolecular disul de. The disul de formed by Prx V is reduced by Trx, but not by glutaredoxin or GSH. Thus, Prx V mutants lacking Cys48 or Cys152 showed no detectable Trx-dependent peroxidase activity, whereas mutation of Cys73 had no effect on activity. Although only the NH 2 - terminal Cys residue is conserved in Prx V, it is designated 2-Cys Prx enzyme because its function is dependent on two Cys residues. Figure 3. Reaction mechanism of mammalian Prx V. PMP20 (39) and AOEB166 (23), other terms for Prx V, were cloned as the result of a search for mammalian homologs of a yeast peroxisomal protein PMP20 and as the result of an attempt to characterize a 17-kDa bronchoalveolar protein, respectively. It was observed that the 162-residue mammalian PMP20 (Prx V) protein contained a peroxisomal targeting sequence (Ser-Gln- Leu) at the COOH-terminus and that the AOEB166 cdna contains two potential initiation sites in the same reading frame, the use of one of which would result in the production of a 162-residue protein identical to Prx V (PMP20), and the use of the other would generate a polypeptide of 214 residues. The 52 amino acid residues at the NH 2 -terminus of the longer polypeptide were shown to constitute a mitochondrial presequence that is capable of importing a fusion protein of AOEB166 and green uorescent protein into mitochondria. Indeed, immunoblot analysis revealed that Prx V is localized intracellularly to cytosol, mitochondria, and peroxisomes (23). No homolog of Prx V has been found in Drosophila. Yeast contains a Prx V homolog, initially named type II TPx (15) and now renamed ctpx III because of its cytosolic localization (27 ). ctpx III was puri ed based on its capacity to reduce alkylhydroperoxides preferentially over H 2 O 2 (15). The amino acid sequence of 176 amino acids of ctpx III revealed no substantial homology to that of the 195 residues of ctpx I with the exception of a short segment preceding the catalytic site Cys62 of ctpx III, but showed 30% sequence identity to mammalian Prx V. Although, like mammalian Prx V, ctpx III contains two additional cysteine residues at positions 31 and 120, these two Cys residues do not correspond to Cys73 and Cys152 of Prx V. The reaction intermediate of ctpx III appears to be different from that of mammalian Prx V: The oxidized Cys62 of ctpx III reacts with the Cys120-SH group of another ctpx III molecule to form an intermolecular disul de linkage (15). The resulting disul de can then be reduced by Trx, but not by GSH. Thus, ctpx III mutants lacking Cys62 or Cys120 showed no detectable TPx activity, whereas mutation of Cys31 had no effect on TPx activity (15). 1-Cys Prx Subgroup Members The full-length cdna for a human 1-Cys Prx, also termed Prx VI for the mammalian enzymes, was identi ed without any reference to peroxidase activity as the result of a sequencing project with human myeloid cell cdna (26). The 1-cys Prx members are found in a variety of species including archaea, yeast, nematode, plant, and mammals (6, 17 ). The sequence identity among these 1-Cys Prx subgroup members is >60%, whereas that between human 1-Cys Prx and the four human 2-Cys Prx (Prx I to IV) enzymes is <30%. Upon exposure to H 2 O 2, the NH 2 -terminal Cys-SH of Prx VI, which corresponds to Cys 47 of human Prx VI, is readily oxidized. However, the resulting Cys-SOH does not form a disul de because of the unavailability of another Cys-SH nearby (8, 17 ) (Fig. 4). In addition to the Cys47 of human Prx VI, some 1-Cys Prx members contain other Cys residues, such as Cys91 of the human enzyme.

5 NOVEL FAMILY OF PEROXIDASES 39 Table 1 Amounts of various Prx isoforms in the indicated rat tissues 2-Cys Prx 1-Cys Prx Figure 4. Reaction mechanism of mammalian Prx VI. X denotes an unidenti ed hydrogen donor. However, neither Cys91 itself nor the sequence surrounding this residue is conserved among the 1-Cys Prx members (17 ). The Cys-SOH of oxidized 1-Cys Prx can be reduced by nonphysiological thiols such as DTT, but its physiological electron donor has not been well characterized. GSH has been suggested to be the physiological donor for 1-Cys Prx (10, 11). However, several laboratories failed to detect GSH-supported peroxidase activity for 1-Cys-Prx (17, 29). Human Prx VI was previously shown to be a calciumindependent phospholipase A 2 that exhibits maximal activity at ph 4 (22). Because human Prx VI contains a motif, Gly X Ser 32 X Gly, associated with the catalytic site of a serine hydrolase, Ser32 was proposed to be the primary site of catalysis. Moreover, because the Ca 2C -independent PLA 2 activity was optimal at ph 4 and negligible above ph 6, the enzyme was presumed to be a lysosomal protein (22). However, mutation of Ser32 or Cys47 had no effect on the lipase activity (17 ). Furthermore, Prx VI is not a lysosomal protein but is localized to the cytosol, the ph of which would be expected to prevent substantial manifestation of Ca 2C -independent PLA 2 activity. The 1-Cys Prx subgroup includes two Drosophila enzymes (dprx2540 and dprx6005) and two yeast enzymes (ymtpx and yntpx). Both dprx2540 and dprx6005 are cytosolic enzymes and show neither Trx- nor GSH-dependent peroxidase activity (31). ymtpx and yntpx are localized in the mitochondria and nucleus, respectively. Surprisingly, the two yeast enzymes have Trx-dependent peroxidase activity (27, 28). Although we included yntpx in the 1-Cys Prx subgroup, it is worthwhile to note that the amino acid sequence of yntpx is least homologous among those of the 1-Cys Prx members (Fig. 1). Distribution of Prx Isoform in Rat Tissues and Cultured Cells The amounts of all Prx isoforms, except the extracellular Prx IV, in various rat tissues (Table 1) and cultured mammalian cells (Table 2) were estimated by immunoblot analysis using isoform-speci c antibodies (4, 33). Prx enzymes are abundant in all the tissues and cultured cells examined. The sum of the ve types of Prx contained in 1 mg of soluble protein amounts to 1 to 10:microgram for tissues and cultured cell. Prx enzymes are more abundant in cultured (transformed) cells compared to rat tissues. Catalase and glutathione peroxidase are generally Tissues Prx I Prx II Prx III Prx V Prx VI Placenta < Thymus Testicle Thyroid Pancreas N.D. a N.D. a Adrenal > > Brain Hypothalamus Spleen Lung Kidney Liver Heart a N.D., not detectable. Data are expressed as micrograms of Prx per milligram of soluble protein. viewed as the major enzymes responsible for removal of cytotoxic H 2 O 2. The ubiquitous and abundant presence of Prx enzymes, however, indicates that Prx enzymes also play an antioxidant role. Cellular Functions of Prx Enzymes Soon after the discovery of ctpx I, the importance of this protein as anti-oxidant could be readily recognized as its synthesis in S. cerevisiae could be induced dramatically when oxidative Table 2 Amounts of various Prx isoforms in the indicated cultured mammalian cells 2-Cys Prx 1-Cys Prx Cell types Prx I Prx II Prx III Prx V Prx VI HaLa > >3.0 NIH3T A < A10 < N.D. a N.D. a K < >5.0 U < Ramos < Jurkat < HepG FRTL N.D. a KNRK N.D. a PC a N.D., not detectable. Data are expressed as micrograms of Prx per milligram of soluble protein.

6 40 RHEE ET AL. pressure was applied by incubating the cells under 100% O 2 (20). Furthermore, yeast strains harboring disrupted ctpx I gene exhibited much slower growth rate under various oxidative pressure (5). Recently physiological function of each of the ve yeast TPx isozymes in maintaining aerobic cycle was proposed based on their kinetic properties toward various peroxide substrates, their subcellular localization, and distinct physiology of each null mutant (27 ). Anti-oxidant function of mammalian Prx is also evident in that MSP23 (Prx I) was discovered as a protein that is induced in mouse peritoneal macrophages exposed to oxidative stresses (13). SP-22 (Prx III) was also induced when bovine aortic endothelial cells were exposed to various oxidative stresses, including mitochondrial respiratory inhibitors, which increase superoxide and H 2 O 2 generation in mitochondria (1). The bovine aortic endothelial cells with an elevated level of Prx III as the result of exposure to mild oxidative stress became more tolerant to subsequent intense oxidative stress (1). Although H 2 O 2 is generally considered a toxic by-product of respiration, increasing evidence suggests that the production of H 2 O 2 might be an integral component of membrane receptor signaling (32). In mammalian cells, a variety of extracellular stimuli, including peptide growth factors and cytokines, induce a transient increase in the intracellular concentration of H 2 O 2 (32). Inhibition of this increase by N-acetyl cysteine or catalase prevents the protein tyrosine phosphorylation induced by growth factors (2, 36) as well as the activation of the nuclear factor NF- B induced by interleukin-1, tumor necrosis factor-, or lysophosphatidic acid. The transient nature of the receptormediated increase in intracellular H 2 O 2 suggests that, in addition to its production, the rapid removal of this molecule is important for receptor signaling. Transient overexpression of Prx I or II in cultured broblasts showed that they were able to eliminate the intracellular H 2 O 2 generated in response to growth factors. Moreover, the activation of NF B induced by extracellularly added H 2 O 2 or tumor necrosis factor- was blocked by overproduction of Prx II (18). These results suggest that Prx I and II might participate in the signaling cascades of growth factors and tumor necrosis factor- by regulating the intracellular concentration of H 2 O 2. The cellular function of Prx II was also studied by overexpressing in Molt-4 leukemia cells. Prx II protected from apoptosis induced by serum starvation, ceramide, or etoposide. Prx II was able to inhibit release of cytochrome c from mitochondria to cytosol (40). 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