We will now consider the individual players ofthe Prdx-based system. Here, we will begin with Prdx itself. All members ofthe peroxiredoxin family have the structural thioredoxin fold. The fold has a central five-stranded ß-sheet surrounded by four a-helices (ß-a-ß-a-ß-a-ß-ß-a). The structural differences between the different members of the Prdx family are in extra domains or extensions outside this basic fold.

Peroxiredoxin-1 (Prdx 1, UniProt KB accession code Q06830) is also known as thioredoxin peroxidase 2, thioredoxin-dependent peroxide reductase 2, proliferation-associated gene product (PAG), natural killer cell-enhancing factor A and NKEF-A. It is a 199-amino-acid polypeptide with a molecular weight of 22 110 Da and a theoretical pi of 8.27 (unprocessed precursor). The encoding gene is on chromosome 1 (1p34.1). Prdx 1 is a 2-Cys Prdx with a conserved peroxidatic (Cys52) and a resolving (Cys173) cysteine residue. According to the X-ray crystal structure of the recombinant human enzyme, Prdx 1 exists as a decamer, composed of five homodimers, with the Cys83 residues forming disulfide bridges between the dimer-dimer interfaces within the decamer [21]. Probably due to the stabilizing effect of these interdimeric disulfide bonds, the molecular chaperone function of Prdx 1 (see below) is more pronounced than its antioxidant activity when compared with the very similar Prdx 2 - which, however, lacks the Cys83 residue. Furthermore, the peroxidatic cysteine of Prdx 1 is more easily overoxidized, probably owing to the surrounding hydrophobic residues [21]. With the exception of the dimer-dimer interface disulfide bond, Prdx 1 is structurally the same as Prdx 2.

Prdx 1 is a multifunctional enzyme. Beside its antioxidant and molecular chaperone activities, Prdx 1 also enhances the cytotoxicity of natural killer cells [22] and inhibits two oncogenes - c-myc [23] and c-Abl, a non-receptor tyrosine kinase [24]. In separate experiments, high levels of Prdx 1 promoted cell proliferation [25], and cells entering S phase displayed elevated Prdx 1 levels [26]. Therefore, Prdx 1 has been suggested to play an important role in cell proliferation [25, 26]. Prdx 1 knockout mice (Prdx suffer from severe hemolytic anemia, starting from the age of 9 months, due to shortened erythrocyte survival. Red cell destruction is caused by an increase in reactive oxygen species (ROS) levels leading to oxidation of red cell proteins, including hemoglobin. This in turn leads to precipitation of hemoglobin, formation of Heinz bodies and hemolysis. Prdx mice also develop multiple tumors, the most frequent types being histiocytic sarcoma, hepatocellular carcinoma, lymphoma and hemangiosarcoma. In fibroblast cell cultures, loss of Prdx function results in slower proliferation rates. The seemingly contradictory findings of increased tumor susceptibility in vivo and decreased cell proliferation in vitro due to loss of Prdx 1 may be explained by an increase in tumor development due to reduced tumor surveillance, attributable to compromised natural killer cell function in vivo [27].

Prdx 1 is located in the cytoplasm and the nucleus [17], and mitochondrial and peroxisomal localization was also described in rat liver [28]. In bovine aortic endothelial cells, Prdx 1 is present in the cytoplasm, mitochondria, Golgi and intermediate filaments [29]. Despite the fact that the protein does not contain any currently recognized secretory signals, its secretion has been shown in Prdx 1-overexpressing, transfected A549 lung adenocarcinoma cell cultures [30]. The secretion of Prdx 1 could be induced by transforming growth factor ß1 (TGFß1) and was not solely due to the overexpression (and thus cellular overload) of Prdx 1, as Prdx 1-overexpressing MCF7 breast cancer cell lines did not secrete the protein [30]. Prdx 1 is also present in the serum oflung cancer patients [31] where it probably increases antioxidant activity and contributes to the resistance of tumor cells to apoptosis-inducing agents.

Importantly, the ultimate function of Prdx 1 differs depending on the subcellular localization, that is, via the modulation of H2O2 concentration. Cytoplasmic Prdx 1, for instance, inhibits nuclear translocation of NF-kB (and so its transcriptional activity), while nuclear Prdx 1 enhances NF-kB activity and confers resistance to oxidant-induced apoptosis [32] (see also Chapters 17 and 18). As demonstrated by in vitro pull-down assays and immunoprecipitation, Prdx 1 interacts with and stimulates the androgen receptor, with implications in prostate cancer [33]. Prdx 1 contains a cyclin-dependent kinase (CDK) phosphorylation consensus sequence (Thr90-Pro-Lys-Lys), which is phosphorylated by cdc2 in the M (mitosis) phase of the cell cycle. Phosphorylation of Prdx 1 Thr90 leads to the marked reduction of its peroxidase activity [34], and induces structural changes leading to the formation of high molecular weight complexes which display chaperone activity [35].

Interestingly, Prdx 1 is also susceptible to phosphorylation by CDK4 and CDK6 in vitro [34]. The transcriptional regulation of Prdx 1 is incompletely known. The expression of the prdxl gene can be induced, however, by the transcription factor Nrf2 (nuclear factor erythroid-derived-related factor 2), which is upregulated by hypoxia/ reoxygenation [36] (see also Section 6.7 and Chapter 11).

Peroxiredoxin-2 (Prdx 2, UniProt KB accession code P32119), also known as thioredoxin peroxidase 1, thioredoxin-dependent peroxide reductase 1, thiol-specific antioxidant protein, natural killer cell-enhancing factor B, TSA, protector protein, PRP, NKEF-B, calpromotin, torin and band 8 protein, is a 198-amino-acid, 21892 Da polypeptide with a theoretical pi of 5.66 (unprocessed precursor). The gene encoding Prdx 2 is located on chromosome 19 (19p13.2). Prdx 2 is one of the best studied human peroxiredoxins, and with an approximate concentration of 5.6 mgml-1, one of the most abundant proteins in erythrocytes [37]. The X-ray crystallographic structure of Prdx 2 (PDB code 1QMV) purified from human erythrocytes, with the active site cysteine (Cys51) in the sulfinic acid state, was determined by Schroder et al. [38] and published in 2000. This is the only human Prdx crystal structure that is based on the native protein. The authors characterized the structure of the oxidized decamer and the structure of the monomeric and dimeric subunits within the decamer (Figure 6.3). The monomeric unit can be divided into a domain I (residues 2-169) which contains the thioredoxin fold, and a C-terminal arm (residues 170-198). The N-terminal methionine residue was not present and residue Ala2 was found to be posttranslationally modified by a carbamoylation [38]. The two monomers form an ellipsoidal dimer with the b-sheets of each subunit combine to form a 14-strand b-sheet. The dimer is held together by hydrogen bonds and hydrophobic interactions between residues in the seventh b-strand of each monomer unit [38]. The monomers come together in a "head-to-tail" arrangement, with the C-terminal arm of one monomer forming hydrogen bonds and hydrophobic interactions with domain I of the other monomer [38]. The decamer is composed of five dimers and forms a toroid-like structure, held together mainly by hydrophobic interactions at the dimer-dimer interfaces. The active site peroxidatic cysteine (Cys51) is buried in a hydrophobic pocket, while the resolving cysteine (Cys172)

Figure 6.3 Structure of the decameric, sulfinic acid is in domain 11, the C-terminal tail. (c)

monomeric and dimeric human erythrocyte Ribbon representation ofthedimeric Prdx2with

Prdx 2 as seen in the decameric structure [38]. the monomer units in a "head-to-tail"

(a) The decameric arrangement of the Prdx 2 arrangement. The peroxidatic cysteine of one monomer units. (b) Ribbon representation of the monomer and the resolving cysteine of the other monomer unit, the resolving and peroxidatic monomer form one of two active sites of the cysteine atoms are shown as balls, the peroxidatic dimer. All figures were created with PyMol [195].

Figure 6.3 Structure of the decameric, sulfinic acid is in domain 11, the C-terminal tail. (c)

monomeric and dimeric human erythrocyte Ribbon representation ofthedimeric Prdx2with

Prdx 2 as seen in the decameric structure [38]. the monomer units in a "head-to-tail"

(a) The decameric arrangement of the Prdx 2 arrangement. The peroxidatic cysteine of one monomer units. (b) Ribbon representation of the monomer and the resolving cysteine of the other monomer unit, the resolving and peroxidatic monomer form one of two active sites of the cysteine atoms are shown as balls, the peroxidatic dimer. All figures were created with PyMol [195].

is partially exposed [38]. In the crystal structure, the peroxidatic cysteine (Cys51) was found to be oxidized to the cysteine sulfinic acid state and the distance between the peroxidatic and resolving cysteine Sg atoms was around 13 A, implying that the two residues are too far apart for a disulfide bond to form between them [38]. In order for these two residues to interact the authors indicated that unwinding of two turns of helix a1 and extensive movements would be required [38]. According to Peskin et al. [39], the tertiary structure of Prdx 2 facilitates its interaction with H2O2, but restricts its reactivity with other thiol-containing agents.

Figure 6.4 summarizes the subcellular localization and the various functions of Prdx 2. The enzyme can be found in the cytosol, but it also associates with the cell membrane, at least in erythrocytes. The membrane-bound form (2.94 mgml-1 of blood) is only a fraction of the total Prdx 2 content of erythrocytes (5.6mgml_1 of blood) [40]. Membrane association is furthered by Ca2 + (at micromolar concentrations), and plays a role in the activation of the Gardos channels, promoting potassium

Figure 6.4 Subcellular localization and wide- presumably plays a role in angiotensin II (Ang II)

ranging functions of Prdx 2. (a) Role of Prdx 2 in signaling in vascular smooth muscle cells.

erythrocytes. Membrane-bound Prdx 2 (gray Activation ofthe G protein-coupled angiotensin II

donuts symbolize the decamer, and the white type 1 receptor (AT 1) leads to an increase of shape of five ovals and a donut symbolizes a high intracellular ROS levels and oxidation of Prdx 2.

molecular weight (HMW) oligomer) regulate the The details and significance of this mechanism activity of Gardos channels in erythrocytes. Prdx2 remain elusive. AsAT 1 activates numerous signal binds several membrane proteins (see text for transduction cascades, AT 1 receptor signaling is details). Note that the exact oligomeric form of not detailed in this figure (for a review see [196]).

the membrane-bound Prdx 2 is unknown. In the In Parkinson's disease, Prdx 2 is phosphorylated cytosol, Prdx 2 functions as H2O2-scavenger and and inactivated by the Cdk5-p25 complex (also by also as molecular chaperone. The true Cdk5-p35 complex) in dopaminergic neurons, significance of the high Prdx 2 concentration in leadingto increased ROS levels and increased cell erythrocytes is incompletely understood, but death. Prdx 2 also acts as H2O2-scavenger and peroxiredoxins probably have specific roles in presumably as molecular chaperone (not erythrocyte development (see text for details). (b) illustrated). Prdx2 influences a number of other

The role of Prdx 2 in nucleated cells. Prdx 2 is a processes which are not shown in this picture, negative regulator of PDGF signaling. Upon such asterminationofPLD! signaling, blockingof activation ofthe PDGF receptor by PDGF binding, AP1 activity, inhibition of monocyte recruitment

Prdx 2 is recruited to the intracellular part of the and adherence to endothelial cells, interaction

PDGF receptor. Prdx 2 scavenges H2O2 which with cyclophilin A, negative regulation ofthe LPS/

leads to the reactivaton of oxidatively inactivated TLR4 signaling pathway, and interference with protein tyrosine phosphatases (PTPs), which TNF-a-induced JNK/p38/ERKsignaling pathways subsequently dephosphorylate the PDGF (seetextformoredetail). PDGFsignalingdrawing receptors and terminate the PDGF signal. Prdx 2 based on [151].

efflux from the erythrocytes [41]. Gardos channels are Ca2 + -activated K + channels of erythrocytes. Overactivity of these channels results in increased K + efflux from erythrocytes, leading to dehydration of the erythrocytes - a major problem in sickle cell anemia patients. Membrane-associated Prdx 2 contributes to the modulation of the Gardos channels, and the channels are inactive in the absence of the protein [37, 42]. It is remarkable, however, that the K + /Na + content of erythrocytes is unchanged in Prdx mice [43]. Moore and Shriver [44] identified the Prdx 2-binding proteins of the erythrocyte membrane as stomatin (protein 7.2b), glycer-aldehyde-3-phosphate dehydrogenase (GAPDH), several unidentified small integral membrane proteins, and a 40 kDa protein. Spectrin and actin bound only weakly to a Prdx 2 affinity column.

Apparently, both the membrane-bound and the cytoplasmic forms of Prdx 2 exist in low and high molecular weight forms, but only the high molecular weight forms activate the Gardos channels. This process is enhanced by the calpain inhibitor leupeptin, and Plishker et al. [40] proposed that in the absence of leupeptin, calpain binds to the membrane in a Ca2 + -dependent manner, hydrolyzing membrane-associated proteins, including the high molecular weight form of Prdx 2 [40].

The high and low molecular weight forms of the protein isolated from erythrocytes were also studied by Kristensen et al. [45], who showed that the high molecular weight complex dissociated into the low molecular weight, dimeric form at high pH (>7.8) and high urea concentration (>2.5 M), suggesting that Prdx 2 molecules form strong associations and only disassociate upon unfolding.

In a pioneering study [46], the human endothelial cell line ECV304 was transfected with Prdx 2 and subjected to oxidized low-density lipoprotein (oxLDL) and lipopoly-saccharide (LPS) treatment. These cells were resistant to monocyte adhesion, showing that Prdx 2 inhibits inflammation-induced monocyte recruitment and adherence to endothelial cells. This, in turn, suggested that Prdx 2 may protect blood vessels from the development of atherosclerotic plaques. In the same study, Prdx 2 also protected cells against a chemotherapy agent (CT-2584) that stimulates ROS-mediated apoptosis [46]. Prdx 2 interacts with, and colocalizes with phospholi-pase Di (PLDX), a signal-transducing enzyme which is stimulated by H2O2. Overexpression of Prdx 2 reduces the response of PLDj to H2O2, and it was suggested that Prdx 2 maybe recruited to sites of H2O2-induced PLDj stimulation where it could act as a signal terminator of PLDj [47]. Prdx 2 knockout (Prdx mice display hemolytic anemia and splenomegaly [43]. In another experiment, Moon et al. [48] showed that both the number of splenocytes, especially CD3+ T cells, and the number of peripheral blood mononuclear cells were greater in Prdx 2 knockout mice. Furthermore, the differentiation rate of dendritic cells was enhanced. The lymphocytes were also more responsive to mitogenic stimulation in the knockout mutants.

This work suggests that Prdx 2 may generally be an inhibitor of immune cell responses [48]. In line with these results, Prdx 2 is a negative regulator of platelet-derived growth factor (PDGF) signaling [49] and also a negative regulator of the LPS-induced Toll-like receptor 4 (TLR4) inflammatory signaling pathway [50]. It is remarkable that extracellular Trx 1 acts as a chemoattractant for monocytes, and thus an enhancer of the immune response [51], whereas Prdx 2 is the inhibitor ofthe same.

Prdx 2 is probably involved in angiotensin II signaling; Tokutomi et al. [52] demonstrated an increase in Prdx 2 oxidation upon stimulation of type 1 angiotensin receptors (AT1) with angiotensin II (Ang II) inhuman coronary smooth muscle cells. Prdx 2 also influences downstream signaling of tumor necrosis factor a (TNF-a). TNF-a induces intracellular H2O2 production which activates JNK (c-jun NH2-terminal kinase) and p38 and inhibits ERK (extracellular signal-regulated kinase). Prdx 2 interferes with these pathways, inhibiting JNK and p38. This in turn inhibits apoptosis and enhances the activity of ERK, thereby transmitting cell proliferation and survival signals [53].

Peroxiredoxin-3 (Prdx 3, UniProt KB accession code P30048), also known as mitochondrial thioredoxin-dependent peroxide reductase, PrxIII, antioxidant protein 1, AOP-1, protein MER5 homolog and HBC189, is a 256-amino-acid protein with a molecular mass of 27 693 Da and a theoretical pi of 7.68 (unprocessed precursor). The prdx3 gene is located on chromosome 10 (10q25-q26). Prdx 3 is found exclusively in the mitochondria, and uses mitochondrial thioredoxins and thiore-doxin reductases (Trx 2 and TR 2, respectively) as electron donors for its peroxidase activity [54]. According to the model of Zhang et al. [55], the reaction mechanism of the reduction of disulfide state Prdx 3 by Trx 2 is very similar to the reaction between the peroxidatic and resolving cysteines of Prdx upon oxidation. Thus, Cys90 thiolate attacks the Prdx 3 disulfide and forms a mixed disulfide with Prdx. Subsequently, this disulfide is reduced by Cys93 of Trx 2, releasing the reduced form of Prdx 3 [55].

Approximately 30 times more abundant in mitochondria than glutathione peroxidase 1 (GPxl), Prdx 3 plays a critical role in the regulation of H2O2-mediated apoptosis signaling in mitochondria [56] (see Chapters 17 and 18). RNA interference-induced depletion of Prdx 3 resulted in increased H2O2 levels and enhanced sensitivity to TNF-a and staurosporine-induced apoptosis in HeLa cells [56]. In an earlier study, the abrin A-chain (ABRA) was shown to interact with Prdx 3, inhibiting its antioxidant activity and leading to increased cytochrome c release, the activation of the caspase cascade and apoptosis [57]. In Fas- and/or TNF-a-treated Jurkat cells, the majority of the Prdx 3 pool underwent overoxidation in the early phase of apoptosis, resulting in an increase of mitochondrial ROS [58]. Prdx 3- and Prdx 5-depleted neuroblastoma cells also exhibited increased sensitivity to apoptosis induced by the 1-methyl-4-phenylpyridinium ion MPP + [59], an ion implicated in chemically induced Parkinson's disease [60]. Prdx 3-overexpressing WEH172 mouse thymoma cells displayed decreased cell proliferation and decreased membrane potential. The cells were also protected against hypoxia-induced apoptosis via inhibition of hypoxia-induced increases of H2O2 levels, while the basal (untreated) levels of apoptosis did not change. These cells were also more resistant to H2O2, t-butyl hydroperoxide and imexon treatment (the latter is a drug inducing mitochondrial H2O2 increase). The cells were not resistant to dexamethasone, however, another apoptosis-inducing drug with an incompletely known reaction mechanism [61].

Prdx 3 is partly - but not exclusively - stimulated by the c-Myc transcription factor and thus contributes to functions mediated by c-Myc, such as normal mitochondrial function, including the maintenance of normal mitochondrial mass and membrane potential. Wonsey et al. [62] found that knockdown of Prdx 3 in the MCF7/ADR breast cancer cell line led to reduced mitochondrial membrane potential and increased c-Myc-mediated apoptosis upon glucose deprivation [62]. Liu et al. [63] used yeast two-hybrid screening, co-immunoprecipitation and a pull-down assay to demonstrate that Prdx 3 binds to RPK118, a sphingosinekinase-1-binding protein. The binding site was the pseudo-kinase domain of RPK118, and the two proteins colocalized in endosomes. Thus, this interaction might have implications in the mitochondrial transport of Prdx 3 via endosomal trafficking, where RPK118 might act as a specialized sorting protein [63]. Similar to Prdx 2, Prdx 3 forms dimers and decamers, and a small percentage of Prdx 3 molecules seem to exist as pairs of interlocked dodecamers [64, 65].

Like other 2-Cys peroxiredoxins, Prdx 3 is susceptible to overoxidation, but it is at present unknown if this overoxidized form can be recycled in the mitochondria [65].

Peroxiredoxin-4 (Prdx 4, UniProt KB accession code Q13162), also known as thioredoxin peroxidase AO372, thioredoxin-dependent peroxide reductase AO372, antioxidant enzyme AOE372, AOE372-2, thioredoxin peroxidase-related activator of NF-kB and c-Jun N-terminal kinase (TRANK), is a 271-amino-acid protein with a molecular mass of 30 540 Da and a theoretical pi of 5.86 (unprocessed precursor). The prdx4 gene is located on chromosome X (Xp22.11). Prdx 4 is a typical 2-Cys Prdx with Cys124 and Cys245 as the peroxidatic and resolving cysteines, respectively. Apparently, beside its major homodimeric state, it may also form heterodimers with

Prdx 1 [66]. The crystal structure of the complete human protein remains unknown. Interestingly, rat Prdx 4 can use either Trx or GSH as reducing agent [67]. Rat Prdx 4 - expressed in a baculovirus system - displayed 27 and 31 kDa molecular forms, and, using pulse-chase experiments, the former proved to be secreted. Within the cells, most of the secretable form could be detected in the endoplasmic reticulum (ER) where it colocalized with calreticulin [67]. Subsequently, human Prdx 4 was shown to be secreted by human Jurkat and HL-60 cells [68]. Prdx 4 contains an N-terminal signal sequence which apparently allows the extracellular secretion of the protein in certain cells. It has also been demonstrated recently that, at least in HT1080 cells, a 27 kDa cleaved product of Prdx 4 (cleavage site between amino acid residues 37 and 38) was cotranslationally transported to, and retained in, the ER. At the same time, an extracellular secretory form could not be detected at all [69]. This finding is all the more intriguing as the amino acid sequence of Prdx 4 does not contain a KDEL type ER retrieval motif. Moreover, it is likely that the protein forms homodecameric complexes within the ER [69]. A membrane-bound, 31 kDa form of Prdx 4 was found in rat testis, associated with the acrosomal vesicle membranes. This form has no peroxidase activity and is only detectable in elongated spermatids [70]. The 27kDa, peroxidase-active secretory form, was present throughout spermatogenesis and could be detected in ER, Golgi and the perinuclear space [70].

Co-immunoprecipitation experiments proved that Prdx 4 interacts with the b isoform of the thromboxane A2 receptor (TPb), a G protein-coupled receptor transmitting oxidative stress signals [71]. Prdx 4 suppressed signaling of TPb in HEK293 cells by inhibiting its cell surface expression. It is postulated that Prdx 4 achieves this inhibition while labeling TPb for degradation by the ER-associated degradation (ERAD) pathway. It is remarkable that Prdx 4 also colocalizes with TPb in the ER [71]. Prdx 4 induces NF-kB via the induction of phosphorylation (and thus degradation) of IkB-a (see Chapters 2, 11, 17 and 18). Through NF-kB induction, Prdx 4 also indirectly upregulates the expression of inducible nitric oxide synthase (iNOS) in rat astrocytes and intracellular adhesion molecule 1 (ICAM-1) in a human hybrid vascular endothelial cell line (A549 lung carcinoma cell line). Furthermore, Prdx 4 activates JNK. Considering these facts together, the activity of Prdx 4 is in some ways similar to that of inflammatory cytokines [68].

Peroxiredoxin-5 (Prdx 5, UniProt KB accession code P30044), also known as peroxisomal antioxidant enzyme, PLP, thioredoxin reductase, thioredoxin peroxidase PMP20, antioxidant enzyme B166, AOEB166, TPx typeVI or Alu corepressor 1, is a 214-amino-acid, 22 026 Da protein. Prdx 5 is a basic Prdx, with a theoretical pi of 8.85 (unprocessed precursor). The prdx5 gene, located on chromosome 11 (11q13), was first identified as a gene encoding a DNA-binding protein which inhibited the in vitro transcription of Alu repeats [72]. Prdx 5 is now known to contribute to the protection of the genome as an inhibitor of DNA double-strand breaks [73].

Initial studies have been carried out on the genetic characterization of prdx5. Several binding sites were identified in the promoter region of the gene, including ARE (antioxidant response element), AP-1 (activator-protein-1), NF-kB, InRE (insulin response element) and GRE (glucocorticoid response element) sites [74], but the significance of these findings is presently still unknown. Several single nucleotide polymorphisms (SNPs) were identified, six of which are in the coding region and cause amino acid substitutions. While these SNPs might be relevant in certain diseases, more studies are necessary to address their precise biochemical role(s) [74]. Multiple transciptional variants have also been identified, and two variants have been confirmed at the protein level: a short and a long form, with approximately 17 and 24kDa molecular masses, respectively. Only the long form (L-PRXV), which contains an N-terminal mitochondrial targeting signal, has been detected in mitochondria [75], and it is probably only this form that has DNA-binding activity [76]. There is experimental evidence that the transcriptional factors NFR-1 andNFR-2 (nuclear respiratory factor 1 and 2, respectively) play a major role in regulating Prdx 5 expression [76]. The same transcription factors also regulate mitochondrial biogenesis.

Prdx 5 is an atypical 2-Cys Prdx. During the second step ofthe peroxidatic catalytic mechanism, Prdx 5 builds an intramolecular disulfide bond between its peroxidatic (Cys47) and resolving (Cys151) cysteines. Prdx 5 is believed to occur mainly in its monomeric form, although recent crystallography studies suggest that monomeric and dimeric forms may exist in an equilibrium [77]. The X-ray crystal structures (1H4O, 1HD2, 1OC3, 1URM) of both the oxidized and reduced forms ofthe recombinant human Prdx 5 are known [77, 78]. The oxidized form of Prdx 5 forms homodimers via intermolecular disulfide bonds between Cys47 and Cys151 ofthe neighboring monomers [77]. Prdx 5 shows structural conservation with Prdx 1 and Prdx 2, but lacks the C-terminal arm. The kinetics of organic hydroperoxide, ONOO~ and H2O2 reduction by Prdx 5 have been characterized. The rate constants ofthe first two reactions are in the range of 106-107M_1 s while H2O2 reduction is slower, being about 105 M_1 s_1 [79].

Prdx 5 is unusual among the peroxiredoxins in that it is widely distributed in subcellular compartments: it can be found in the nucleus, the cytosol, the peroxi-somes and mitochondria [76].

Peroxiredoxin-6 (Prdx 6, UniProt KB accession code P30041), also known as non-selenium glutathione peroxidase, aiPLA2, acidic calcium-independent phospholi-pase A2, antioxidant protein 2,1-Cys peroxiredoxin), is a 224-amino-acid protein with a molecular mass of25 035 Da and a theoretical pi of 6 (unprocessed precursor). The X-ray structure ofthe molecule has been solved (1PRX) [80] and the protein was found to be a tightly associated dimer. As with Prdx 2, the monomer unit contains two domains, domain 1 containing the thioredoxin fold and domain 2 the C-terminal arm. The prdx6 gene is located on chromosome 1 (1q25.1).

Prdx 6 is a 1-Cys peroxiredoxin, with two presently recognized functions as a phospholipid peroxidase and a phospholipase A2 (PLA2). Unlike the other members ofthe Prdx family, Prdx 6 has only one conserved cysteine residue (Cys47), which is buried inside the protein globule and can be reduced by GSH via heterodimerization of Prdx 6 with a GSH-saturated p glutathione S-transferase (pGST). This, in turn, leads to spontaneous reduction of the mixed disulfide [18]. Although H2O2 is a potential substrate, Prdx 6 probably mainly protects cells from membrane oxidation through reduction of phospholipid hydroperoxides [18]. The presence of cyclophilin A enhances the antioxidant activity of Prdx 6, suggesting that it acts as an additional electron donor for Prdx 6 [20].

Prdx 6, like the other five peroxiredoxins, is sensitive to cysteine overoxidation and loss of peroxiredoxin function. Nevertheless, the phospholipase A2 activity of Prdx 6 is not influenced by the overoxidation (sulfinic acid formation) of the conserved site cysteine [81]. The PLA2 activity of Prdx 6 is Ca2 + -independent and has an important role in the metabolism of the phospholipids of lung surfactant [82], with the primary target of the enzyme being phosphatidylcholine. It is likely that - similar to other serine-based lipases - the S32/H26/D140 residues function as a catalytic triad [81].

Prdx 6 is an important cytoprotective enzyme in the skin [83]. It is overexpressed in keratinocytes in skin wounds [84] and in psoriatic epidermis [85]. Aged transgenic mice overexpressing Prdx 6 display a higher rate of wound closure than wild-type age-matched controls. This protective effect of Prdx 6 is likely due to the direct reduction of ROS, as Prdx 6 does not seem to influence the expression pattern of other skin repair proteins [83]. Beside this, a reduced number of apoptotic keratinocytes has been detected in the transgenic animals upon UVA and UVB exposure. A similar anti-apoptotic effect and increased ROS/RNS resistance has been observed in Prdx 6 overexpressing keratinocytes in vitro [83]. It is a matter of debate whether this protective effect of Prdx 6 in keratinocytes is beneficial to the organism in the long term, since Prdx 6 protection may contribute to the survival of malignant cells.

In the rat, Prdx 6 was shown to be secreted from olfactory epithelium [86]. (NB: Prdx 6, like peroxiredoxins 1, 2 and 5, does not contain a known secretory signal; yet there is strong evidence that all these isoforms are secreted from certain cells under certain conditions.)

Several Prdx 6 knockout studies have been published. Mo et al. [87] found that Prdx mice are viable and fertile, but showed increased sensitivity to 100% O2 exposure. In healthy, Prdx 6 +/ + animals, Prdx 6 displayed widespread tissue distribution, but the highest protein and mRNA levels were found in the lung. The authors noted the expression, at the level of mRNA, of an intronless gene termed 1-cysPrx-P1 - very similar to Prdx 6 - in the testis of the wild-type and knockout animals [87]. It is uncertain presently if the mRNA product of this intronless pseudogene has any influence on Prdx 6 function, although it does not seem to compensate for the lack of Prdx 6 in knockout mutants. In keeping with these data, Prdx 6-overexpressing transgenic mice (Prdx 6 protein levels threefold higher than wild type) showed longer survival, less epithelial cell necrosis, reduced perivascular edema and decreased inflammatory cell recruitment upon 100% O2 exposure compared with wild-type controls [88].

In an experiment by Wang et al. [89], Prdx 6 showed highest expression levels in lung, gastrointestinal, uterine and CNS epithelial cells, as well as inhepatocytes and immature spermatogenic cells of mice. Moreover, Prdx 6 was present in the cytosol but not in cell membranes, the nucleus or other cellular organelles. In agreement with the results of Mo et al. [87], Wang et al. [89] found that Prdx mice were viable and fertile, but were characterized by lower survival rates, more severe tissue damage and higher protein oxidation levels upon paraquat-induced oxidative stress when compared with controls. The organs most severely affected by oxidative stress were the lung, kidneys and liver, displaying thrombosis, hemorrhages, edema and epithelial necroses. Intriguingly, the authors demonstrated that mRNA expression of the other antioxidant enzymes - such as Prdx, GPx, catalase, superoxide dismutase (SOD), Trx and glutaredoxins (Grx) - was not enhanced in the Prdx mutants, contrary to the expectation that the loss of Prdx 6 would be compensated for by other antioxidant enzymes [89]. Prdx mouse hearts were highly susceptible to ischemia-reperfusion injury, resulting in increased myocardial infarct size, higher levels of apoptotic cardiomyocytes and reduced recovery of left ventricular function compared with controls. Remarkably, catalase and GPx -albeit abundantly present in the heart tissue - could not compensate for the absence of Prdx 6, underlining the unique and irreplacable role Prdx 6 plays in protecting the heart from oxidative stress injury [90].

It has recently been demonstrated that Prdx 6 interacts with saitohin. The saitohin gene is unique to humans and its most closely related primates, and the Q haplotype of saitohin - associated with a single polymorphism (Q7R) of the gene - results in increased susceptibility to several neurodegenerative disorders [91]. Transcriptional regulation of Prdx 6 was studied in mouse liver cells [92], and the promoter was found to contain Sp1, MYCandNrf2 consensus sites. Furthermore, NF-kB suppressed Prdx 6 expression [92].

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