Physiological and pathological views of peroxiredoxin 4

Free Radical Biology and Medicine 83 (2015) 373–379

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Physiological and pathological views of peroxiredoxin 4 Junichi Fujii a,n, Yoshitaka Ikeda b, Toshihiro Kurahashi a, Takujiro Homma a a b

Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, 2-2-2 Iidanishi, Yamagata 990-9585, Japan Division of Molecular Cell Biology, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan

art ic l e i nf o

a b s t r a c t

Article history: Received 3 November 2014 Received in revised form 21 January 2015 Accepted 23 January 2015 Available online 2 February 2015

Peroxiredoxins (PRDXs) form an enzyme family that exhibits peroxidase activity using electrons from thioredoxin and other donor molecules. As the signaling roles of hydrogen peroxide in response to extracellular stimuli have emerged, the involvement of PRDX in the hydrogen peroxide-mediated signaling has become evident. Among six PRDX members in mammalian cells, PRDX4 uniquely possesses a hydrophobic signal peptide at the amino terminus, and, hence, it undergoes either secretion or retention by the endoplasmic reticulum (ER) lumen. The role of PRDX4 as a sulfoxidase in ER is now attracting much attention regarding the oxidative protein folding of nascent proteins. Contrary to this role in the ER, the functional significance of PRDX4 in the extracellular milieu is virtually unknown despite its implications as a biomarker under pathological conditions in some diseases. Other than its systemically expressed form, a variant form of PRDX4 is transcribed from the upstream promoter/exon 1 of the systemic promoter/exon 1 and is uniquely expressed in sexually matured testes. Circumstantial evidence, together with deduced functions from the systemic form, suggests that there are potential roles for testicular PRDX4 in the reproductive processes such as the regulation of hormonal signals and the oxidative packaging of sperm chromatin. Elucidation of these PRDX4 functions under in vivo situations is expected to show the whole picture of how PRDX4 has evolved in multicellular organisms. & 2015 Elsevier Inc. All rights reserved.

Keywords: Peroxiredoxin Hydrogen peroxide signal Oxidative protein folding Sulfoxidase

Introduction The vital activities of living organisms largely depend on oxidationreduction (redox) reactions for survival. Many small molecules and proteins such as glutathione and thioredoxin (Trx), respectively, are involved in redox reactions [1,2]. New redox family proteins were discovered about a quarter-century ago [3] and were tentatively designated as thiol-specific antioxidants [4]. The name peroxiredoxin has been assigned to the genetic family named PRDX [4,5]. For the sake of simplicity in this paper, we used PRDX to identify both proteins and genes. A majority of PRDX members exhibit peroxidase activity using Trx as a preferred electron donor. The resultant oxidized Trx is recycled by Trx reductase, which is a member of the glutathione reductase family, and also utilizes NADPH as the primary electron donor (Fig. 1). Six members that possess high similarities in their molecular structures are regarded as conventional PRDX in mammals [6–8]. PRDX1 to PRDX4 are typical members of the 2-Cys PRDX class,

Abbreviations: ER, endoplasmic reticulum; ERO1, endoplasmic reticulum oxidoreductin 1; GCSF, granulocyte colony-stimulating factor; PDI, protein disulfide isomerase; PRDXs, peroxiredoxins; PTP, phosphotyrosine phosphatases; ROS, reactive oxygen species; GPX, glutathione peroxidase. n Corresponding author. Fax: þ 81 23 628 5230. E-mail address: [email protected] (J. Fujii). http://dx.doi.org/10.1016/j.freeradbiomed.2015.01.025 0891-5849/& 2015 Elsevier Inc. All rights reserved.

exhibiting high similarities across the catalytic domain. They exist as homodimers with a head-to-tail type of association and transiently form disulfide bridges during catalysis [6–8]. PRDX5 and PRDX6 are monomeric enzymes that show more divergent structures. Reductive detoxification of peroxides is an established catalytic activity, but additional enzymatic activities are also known [8]. Roles in signal regulation by reactive oxygen species (ROS), particularly hydrogen peroxide, would be the most profound function of PRDX [9]. Moreover, chaperone-like activity has been found in PRDX1 and 2 [10,11], although no consensus exists as to the relationship between peroxidase activity and the chaperone function. Here we focus on mammalian PRDX4 from the viewpoint of its physiological and pathological roles. Because unicellular organisms such as yeast do not possess PRDX4 [12], the gene appears to have evolved with the advent of multicellular organisms, which require intercellular communication via secreted products. Compared with other mammalian PRDX members, the reports of the localization and roles of PRDX4 are conflicting [13–16]. Recently, an understanding of the roles of PRDX4 has markedly advanced, particularly concerning the sulfoxidase function in the endoplasmic reticulum (ER) [17]. Because PRDX4 is also present extracellularly, we are proposing a hypothetical function acknowledging that defining its function remains a future challenge. We also discuss a variant form, designated as PRDX4t, that is uniquely expressed in sexually matured testes.

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Glutathione peroxidase (GPX)/Glutathione reductase system H2O2 LOOH

Reduced

GPX H2O LOH

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NADP+

Glutathione reductase NADPH

Oxidized Direct electron donor

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Peroxiredoxin (PRDX)/Thioredoxin reductase system H2O2

PRDX H2O

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Thioredoxin

Thioredoxin reductase

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NADPH

Direct electron donor

Primary electron source

Fig. 1. Comparison of electron transfer systems in the GPX- and PRDX-catalyzed peroxidase reactions. GPX reduces hydrogen peroxide and lipid peroxides (LOOH) to water and corresponding alcohol, respectively, using electrons from reduced glutathione. Oxidized glutathione is then reduced back by glutathione reductase via the reducing power of NADPH. PRDX reduces mainly hydrogen peroxide using electrons from reduced Trx. Oxidized Trx is then reduced back by Trx reductase using the reducing power of NADPH.

Structure and enzymatic reactions of PRDX4 Structure and molecular organization of PRDX4 PRDX4 is a member of the 2-Cys-type peroxiredoxin family and is homologous to typical 2-Cys peroxiredoxins such as PRDX1 and PRDX2, thus serving as a Trx-dependent peroxidase via the same catalytic mechanism as other 2-Cys peroxiredoxins [6–8]. PRDX4 has an additional N-terminal region, which is unique to this enzyme, following a signal peptide that is responsible for translocation across the ER membrane into the luminal space (Fig. 2A) [18,19]. The signal sequence is cleavable and allows the enzyme to be extracellularly released. In cultured cells, secretion of PRDX4 protein into the medium has been observed, and significant levels of PRDX4 were also detected in human and rat serum by immunological assay using specific antibodies [20,21]. PRDX4, like PRDX1 and PRDX2, forms homodimers with subunits assembling in a head-to-tail manner. These form disulfide links within the dimeric subunits during catalytic peroxidase activity [22]. The dimers are also structurally organized into homodecamers, each carrying five dimeric units (Fig. 2B) [23–25]. However, in contrast to PRDX1 and PRDX2, the five homodimeric units of PRDX4 are further covalently linked by unique cysteine residues found in the N-terminal extension of PRDX4 [19,25,26]. Trx-dependent peroxidase activity of PRDX4 PRDX4 is capable of catalyzing the detoxification of peroxides, wherein Trx is used as an electron donor in vitro, as with other typical 2-Cys types of peroxiredoxins [16,26,27]. This peroxiredoxin shows significant structural similarities to PRDX1 and PRDX2, and two cysteine residues that are catalytically essential for 2-Cys types of PRDX are perfectly conserved in PRDX4, consistent with its Trx-dependent reduction of peroxides. In a typical reaction with peroxides, the conserved peroxidatic cysteine, which is one of the two catalytic cysteine residues and is located more N-terminally, first reacts with hydroperoxides, forming cysteine sulfenic acid (Cys-SOH), while the substrate peroxide is reduced by two electrons. The resultant cysteine sulfenic acid then reacts with the other catalytic cysteine, the resolving Cys, to form

Fig. 2. Schematic presentation of the structure and molecular organization of PRDX4. (A) The heavily shaded region of PRDX4 is homologous to PRDX1 and 2, and contains two catalytic cysteine residues, peroxidatic Cys and resolving Cys. The lightly shaded portion represents the signal sequence that allows translocation across the ER membrane and thereafter should be cleaved off. (B) The homodecameric structure consists of five units of the functional dimer and reveals a toroidal organization. The disulfide bonds shown are formed between the cysteine residues that are contained by the N-terminal extension. In the oxidized state, the functional dimer units are covalently linked by these disulfides. The disulfide bridges by the catalytic Cys residues in the active sites are not represented here.

their disulfide and generate a water molecule. The reaction with Trx as an electron donor finally reduces the disulfide, thus completing the catalytic cycle of PRDX4 in the detoxification of peroxide. Hyperoxidation of PRDX The catalytic cycle of PRDX4 as well as other 2-Cys peroxiredoxins involves the formation of Cys-SOH, which subsequently reacts with another catalytic Cys-SH. Alternatively, the Cys-SOH can further react with peroxide and is “hyperoxidized” or “overoxidized” into cysteine sulfinic acid, Cys-SO2H [28,29]. Thus, the interchain disulfide formation competes with hyperoxidation for the Cys-SOH. The disulfide is reduced generally by Trx to maintain the catalytic cycle. However, the hyperoxidation into Cys-SO2H, or possibly cysteine sulfonic acid by further oxidation, terminates the catalytic cycle because Trx is incapable of restoring Cys-SH from these oxidized forms. It is probable that a sulfiredoxin family protein regenerates such an inactivated form of PRDX4 in an ATP-dependent manner, which is known to happen with other peroxiredoxins [30,31]. In general, when peroxiredoxins are inactivated by the formation of Cys-SO2H, the enzymes no longer exhibit any significant reducing activities of peroxides. This inactivation process is more prominent with higher concentrations of peroxide as the substrate. In the case of PRDX4, however, activity was observed even after hyperoxidation of the catalytic Cys [29]. It appears that the enzyme still functions, albeit with altered kinetic properties, probably by an alternative catalytic mechanism in which the resolving Cys residue directly reacts with peroxide and interacts with Trx. This altered form of PRDX4 shows a substrate preference that differs from that of an intact fully active enzyme. The altered form displayed higher activity toward hydrogen peroxide compared to t-butyl hydroperoxide while the intact acts more actively on t-butyl hydroperoxide [29]. Structural features associated with hyperoxidation While eukaryotic 2-Cys peroxiredoxins readily undergo inactivation due to the hyperoxidation of the catalytic Cys, the prokaryotic

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orthologs of 2-Cys peroxiredoxin are quite resistant to inactivation and retain their activity even in the presence of a relatively higher concentrations of hydroperoxide [24]. The vulnerability of eukaryotic peroxiredoxins has been attributed mostly to two common structural motifs: the Tyr-Phe (YF) motif and Gly-Gly-Leu-Gly (GGLG). The YF motif is contained in the most C-terminal helix, and the GGLG motif is a part of the loop structure that is located in the middle of the two catalytic cysteine residues. In fact, the susceptibility of PRDX4 to hyperoxidation is suppressed by deletion of the YF motif or dissociation into a dimeric form, probably via increases in the flexibility of the peroxidatic Cys residue [25]. Structural analyses have suggested that 2-Cys peroxiredoxins including PRDX4 transit between at least two structural states: a “fully folded (FF)” conformation, in which the YF and GGLG motifs are packed up to bury the peroxidatic Cys; and, a “locally unfolded (LU)” conformation, in which the YF motif containing a helix is more flexible. In the FF conformation, the two catalytic cysteines do not form a disulfide and are positioned apart from each other. During the catalytic cycle, the sulfenic acid of the peroxidatic Cys occupies the position where the aromatic rings of the YF motif locate. As a result of a “clashing” of the Cys and the YF motif and the accompanying shift of the Cys-SOH, the two cysteines locate in close vicinity to each other, and are then allowed to react to form a disulfide [25]. The YF motif serves to decrease the dynamics of catalysis and thereby restricts the conformational change from the FF to the LU, thus increasing the chance of overoxidation by further reaction with hydroperoxide. The presence of the YF motif and the sensitivity of the 2-Cys peroxiredoxins have little association with the catalytic mechanism to reduce peroxides. Therefore, the “floodgate hypothesis” proposes that the enzymes were altered by evolution for involvement in the signal transduction that is related to hydrogen peroxide [24]. The recycling system that drives the catalytic cycle of the peroxiredoxins, however, is almost absent from the ER, and it seems probable that PRDX4 does not suffer from hyperoxidation in vivo [32]. It remains uncertain why PRDX4 has such vulnerable or susceptible properties despite being disadvantageous to the efficient detoxification of peroxide. Although PRDX4 also has the YF and the GGLG motifs that are common among eukaryotic 2-Cys peroxiredoxins, their roles may be different from those in the cytosolic homologues. The motifs of PRDX4 may play a specific role, for example, in oxidative protein folding in the ER, rather than being involved in signal transduction.

The surveillance mechanism that regulates hydrogen peroxide to proper levels appears to be one of the pivotal functions of PRDX family members. Extensive characterization of PRDX1 has revealed a several fold mortgage in the regulation of hydrogen peroxide during signal transduction [36]. The phosphorylation of a tyrosine residue in PRDX1 by Src or other tyrosine kinases suppresses peroxidase activity, which enables the transient transmission of the phosphorylation signal.

Roles of PRDX in the regulation of hydrogen peroxide signaling

Roles of PRDX4 in the ER

ROS have been regarded as deleterious molecules since their discovery, but a transient elevation in hydrogen peroxide levels occurs when cells are stimulated by growth factors such as EGF and PDGF [33]. These antithetical roles of hydrogen peroxide have long been a mystery when studied from the viewpoint of redox biology, but a theoretical explanation has recently been offered [34].

Because more than 90% of molecular oxygen is consumed by the respiratory chain, there is a high risk to produce superoxide by the reaction with leaked electrons. Thus the mitochondria have been regarded as a major source of ROS in most cells. However, a recent study using fluorescent protein-based hydrogen peroxide sensors in various intracellular compartments has indicated that the ER is the organelle that produces the highest levels of hydrogen peroxide [42]. The major source for ROS is thought to be hydrogen peroxide that is produced by the reaction of endoplasmic reticulum oxidoreductin 1 (ERO1) during oxidative disulfide formation of newly synthesized proteins in the lumen of the ER. Both secretory proteins and membrane proteins are synthesized and subjected to oxidative protein folding in the ER (Fig. 3). Only strict control of the structures of these proteins can enable proper interaction between the ligands and their receptors. The protein conformation is properly maintained by several types of chemical bonds, among which the disulfide bond is covalently formed between and within polypeptide chains, and, hence, is a principle determinant of the protein structure. ERO1 uses molecular oxygen

Modulation of hydrogen peroxide signaling as a common function of PRDXs Hydrogen peroxide under oxidative stress can cause a permanent inactivation of phosphotyrosine phosphatases (PTP) by oxidation and will continue to stimulate cell proliferation in an uncontrollable manner, which can result in tumorigenic proliferation over a long period of time. The significance of the PTP inactivation in signaling is supported by findings that indicate a linkage of hereditary disorders with some PTP variants that possess a low or null degree of activity [35].

PRDX4 also modulates hydrogen peroxide signaling According to the structural basis of PRDX4, most reports show a localization of mature PRDX4 in the ER lumen or in extracellular spaces. However, cytoplasmic localization of PRDX4 has also been reported under certain circumstances such as the binding to the cytosolic domain by the thromboxane A2 receptor [37] and by the granulocyte colony-stimulating factor (GCSF) receptor [38]. Precise analysis of the PRDX4 function has indicated that PRDX4 suppresses GCSF receptor function [38]. Deletion of the amino-terminal domain of PRDX4, which is a unique extension not found in other family members (Fig. 2), results in a loss of interaction and, hence, a loss of the suppressive effects. Thus, PRDX4 binds by the amino-terminal domain to the cytosolic C-terminal portion of the GCSF receptor and efficiently removes excessive hydrogen peroxide in the surrounding microenvironment, which results in the fine-tuning of hydrogen peroxide-mediated PTP suppression during stimulus. Different from cytosolic PRDXs, there remains a serious problem regarding the PRDX4-mediated signal regulation of membrane receptors. The N-terminal hydrophobic amino acid stretch encoded by the PRDX4 mRNA is used for translocation of the PRDX4 protein into the ER lumen. To regulate the plasma membrane receptors by binding their cytoplasmic domains, mature PRDX4 must be retrotranslocated to the cytoplasm. However, it is ambiguous how PRDX4 is retrotranslocated to the cytoplasm from the ER lumen. Although a proper explanation has not been provided for cytoplasmic PRDX4, there are other examples that show similar unusual cellular localization, such as calreticulin, BiP, and protein disulfide isomerase (PDI) [39]. Among them, extensive analysis has been performed for calreticulin, a molecular chaperone for sugar chains in the ER lumen [40]. A linkage between inflammatory stimuli and the cytosolic localization of PDI has also been implicated [41], which may provide clues to understanding the mechanism of the retrotranslocation of the proteins from the ER to the cytoplasm.

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Inactivation

O2

SO2/O3H

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H 2O 2 Gene expression

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SOH PRDX

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P

Ligand

Growth stimuli

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O2 ERO1 NOX4 PRDX4

H2 O2

Oxidative Protein folding

ROS Mitochondrion

ER

Fig. 3. A common role for PRDXs in the ROS signaling and a specific role for PRDX4 in oxidative protein folding in the ER. When a growth factor binds receptors for tyrosine kinase (RTK), tyrosine phosphorylation occurs on the receptor. Phosphotyrosine phosphatase (PTP) in its active form dephosphorylates RTK and suppresses the phosphorylation signals to the nucleus. When NADPH oxidase (NOX) in the plasma membrane is activated by ligand binding, superoxide is simultaneously produced and dismutated to hydrogen peroxide. The resultant hydrogen peroxide moves into the cell and oxidizes the catalytic cysteine sulfhydryl in PTP to cysteine sulfenic acid (-SOH), which leads to the transient inactivation of PTP. Cys-SOH can be further oxidized to sulfinic/sulfonic acid (-SO2/SO3H) by excess hydrogen peroxide, which irreversibly inactivates PTP. PRDX fine-tunes the ROS signal by eliminating the excess hydrogen peroxide. ERO1 introduces a disulfide bond in the PDI family proteins (not presented in the figure) in the ER by using the oxidizing power of oxygen, which simultaneously results in the production of hydrogen peroxide. PRDX4 uses the oxidizing power of hydrogen peroxide to introduce a disulfide bond in the nascent protein via oxidation of the PDI family proteins. NOX4 is a NADPH oxidase in the ER membrane and may provide hydrogen peroxide to PRDX4. ROS may also come from a mitochondria-associated membrane (MAM).

to introduce a disulfide bond in PDI and its family members and converts the oxygen to hydrogen peroxide [43]. PDI family proteins consisting of more than 20 members then introduce disulfide bonds in nascent target proteins. Thus, the ER in cells that produce a large body of secretory proteins is a potential source for hydrogen peroxide production [42]. Abundant expression of PRDX4 is found in some tissues such as the pancreas, wherein protein secretion is critical for both exocrine and endocrine secretion. The transgenic expression of PRDX4 in mice protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines [44]. The overexpression of PRDX4 in insulin-secreting cells also improves insulin synthesis and secretion [45]. Considering the peroxidase activity of PRDX4, the elimination of hydrogen peroxide, which is largely produced by ERO1, is an expected function in the ER. However, different from catalase, an electron donor is required for the PRDX4-mediated peroxidase reaction. While hyperoxidation occurs dominantly to PRDX1, 2, and 3, hyperoxidized PRDX4 is rarely detected in vivo [32,46]. Because hyperoxidation occurs during catalytic reaction of PRDX with Trx as the preferred electron donor, this could be explained by an insufficient electron-donating system in the ER that includes the Trx-Trx reductase system and the NADPH generating system. ERO1 is the primary oxido-reductase in the ER and is an evolutionarily ancient enzyme that is found in primitive eukaryotic cells. However, its deficiency causes only a moderate phenotypical abnormality in mice [47], which suggests the presence of a redundant system in mammals. Zito et al. [12] have demonstrated that PRDX4 uses hydrogen peroxide to catalyze the formation of disulfide bonds in PDI [12,48]. PRDX4 rescues the ERO1-deficient yeast by utilizing hydrogen peroxide to oxidize reactive sulfhydryls

in PDI, which consequently introduces disulfide bonds in newly synthesized proteins [12]. It is now the consensus that the principle role of PRDX4 in the ER is the sulfoxidation of PDI family members during oxidative protein folding. Different from ERO1, PRDX4-mediated disulfide formation accompanies the elimination of hydrogen peroxide. Thus, PRDX4 exerts two advantages in cells: sulfoxidation of newly synthesized proteins and suppression of oxidative stress by eliminating hydrogen peroxide. Based on the structural basis and in vitro experiments, the precise molecular machinery has been proposed using purified ERO1, PRDX4, and members of the PDI protein family [48]. Thus, the coordinated function of ERO1 and PRDX4 during oxidative protein folding in the ER constitutes a safe and efficient system in mammalian cells. The mechanism of this system is discussed in detail in this issue of the journal [17,43]; hence, we need go no further. Regarding oxidation power other than the ERO1 function, ROS appear to come from local mitochondria at the interface with the ER, which is referred to as the mitochondrial associated membrane (MAM) [49]. In addition, the participation of NOX4, which is present in the ER membrane, may also supply hydrogen peroxide to sulfoxidation [50]. NOX4 releases hydrogen peroxide rather than superoxide. Hydrogen peroxide is produced by a two-electron reduction in an NADPHdependent manner inside the ER lumen, whereas other NOX members primarily produce superoxide. Thus, the resultant hydrogen peroxide is directly utilized by PRDX4 and other peroxidase-like sulfoxidases. NOX4 is known to play a crucial role in the regulation of growth factor signaling via the suppression of PTPs such as PTP1B [51]. PRDX4 is induced during the terminal differentiation of B cells to plasma cells by lipopolysaccharide treatment [52]. A large aggregation of IgM is abundantly formed in PRDX4-deficient plasma cells, which is consistent with the role of PRDX4 in oxidative protein folding. In the processes of oxidative protein folding in the ER, a portion of the nascent proteins is misfolded, and the accumulation induces an unfolded protein response (UPR) [53], which is a selfprotective mechanism whereby cells attempt to rescue the ER function. However, cell death is induced when the accumulation of misfolded proteins cannot be properly handled under extreme situations. PRDX4 deficiencies in mice nonetheless show apparently normal ER functions probably due to a functional redundancy to other molecules in the ER [54]. However, a deficiency of both ERO1 and PRDX4 results in atypical scurvy [55] due to the consumption of ascorbic acid, which is caused by a large production of sulfenic acid in the ER by the cells lacking these sulfoxidases. Thereafter, GPX7 and GPX8 were also found to partially account for the sulfoxidase activity of the ER [56,57]. Functional redundancy collectively supports oxidative protein folding in the ER and participates in maintaining the homeostasis of the body.

A testis-specific PRDX4 variant and a hypothetical role The PRDX4 gene exhibits complex characteristics compared with other PRDX members and appears to play a unique role in male reproduction [58]. The presence of some PRDX members in the testes is known, but the roles of PRDX in the male reproductive system and sperm functions are unclear [59]. There are two forms of PRDX4 expressed in the testes, and these two differ in their electrophoretic mobility in SDS-polyacrylamide gel electrophoresis: one has ordinary electrophoretic mobility, whereas the mobility in the other is slightly lower [18,60]. Now the low-mobility form has been identified as the variant PRDX4t that is transcribed from the upstream promoter/exon 1 only in sexually mature testes (Fig. 4A) [46]. Thus, two splice variants, the systemically expressed form with a hydrophobic signal peptide and the testis-specific form without the signal peptide, are transcribed from the PRDX4 gene, which is localized in the X chromosome [15]. Because the N-terminal signal

J. Fujii et al. / Free Radical Biology and Medicine 83 (2015) 373–379

deleted Ex1

Ex1t

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Ex3

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Ex4

Ex5

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PRDX4 Catalytic center SH

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Chromatin DNA Protamine

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Transition

testis HS

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SH SH SH

H2O2

HS

Given the oxidative sperm chromatin packaging and the role of PRDX4 as sulfoxidase in the oxidative protein folding in the ER, it has been hypothesized that PRDX4t may be involved in the introduction of a disulfide bridge in protamine during the spermatogenic process in the testes. The majority of the sulfoxidation in protamines occurs in the epididymis where the maturation of sperm proceeds. In addition, PRDX4 rapidly, and promiscuously, mediates oxidative protein folding in the ER [48], which implies that PRDX4 may facilitate the initial compaction of protamines in the early spermatogenic process. The phospholipid hydroperoxide glutathione peroxidase that is encoded by GPX4 is richly present in the midpiece of sperm as capsule material [62] and is also known to play a role in the sulfoxidation of the sperm protamines. In fact, a defect in the GPX4 gene has been associated with male infertility [63,64]. While information on the responsible proteins in the oxidative protamine folding during spermatogenesis is limited, the testis-specific protein disulfide isomerase homolog (PDILT) plays an indispensable role in the disulfide-bond formation and folding of ADAM3 in the ER, which functions on the sperm surface [65]. Thus, PRDX4t and GPX4 may function as sulfoxidases during spermatogenesis in a coordinated manner. Destruction of the testis-specific variant expression could be a key to understanding the role of PRDX4t in this highly differentiated process.

SH

SH SH SH

Extracellular PRDX4

PRDX4t

Sulfoxidation H2O

377

GPX4

epididymis

Fig. 4. A hypothetical role for testis-specific PRDX4t in male reproduction. (A) The upper portion indicates the structure of the PRDX4 gene. Promoter/exon 1 is required for the systemic expression, but is deleted in PRDX4-knockout (KO) mice. A testis-specific PRDX4 variant is transcribed from the upper promoter/exon 1. The middle blue lines indicate positions for the peptides that are used in the production of antibodies. The lower portion indicates the immunoblots of the proteins in the testes during the sexual maturation of wild-type (WT) and KO mice. (B) The schematic diagram shows the conversion of the chromatin structure during spermatocyte differentiation to sperm and the redox state of chromatin during sperm maturation. Histones in chromatin are replaced by protamines that are rich in sulfhydryl residues (-SH). A testis-specific PRDX4t together with GPX4 may be involved in the introduction of the disulfide bond (–S–S–) in protamines. The maturation of sperm is accomplished by full sulfoxidation in the epididymis.

peptide determines the localization of PRDX4 to be an ER resident and/or a part of the extracellular milieu, the absence of it would also affect the localization and physiological roles of the variant PRDX4t. While knockout mice show nearly normal phenotypes in most organs and tissues, an abnormally high rate of spermatogenic cell death has been observed in the PRDX4 knockout mice [61]. After meiosis, spermatogenic cells experience spermiogenesis that includes tremendous changes in gene expression, morphogenesis, and the redox environment. Basic protein histones constitute the chromatin of somatic cells and stabilize the DNA in ordinary somatic cells. However, histones are replaced by transition proteins and ultimately by protamines during the spermatogenic process (Fig. 4B). While histones contain mostly lysine and arginine and only a scant amount of cysteine, the cysteine predominates next to arginine in the protamines of rodents and humans. The sulfoxidation of cysteines to cysteine disulfide occurs in the protamines in the epididymis, which furnishes chromatin with resistance against oxidative stress and compacts the sperm nucleus, to about one-twentieth that of somatic cells.

Discovery of the amino-terminal signal peptide revealed the role of PRDX4 in the extracellular space [15], and the pulse–chase experiment demonstrated secretion using PRDX4-overexpressing COS1 cells [18]. Immunological assay using specific antibodies has enabled quantification of the plasma levels of PRDX4 [20,21]. However, the fate of PRDX4, either as ER resident or in secretion, appears to depend on the types of the cells, because no secretion has been observed in some types of cells [19]. In fact, neither ERO1 nor PRDX4 possess an ER retention signal, and PDI and ERp44 prevent their secretion by sequential interactions with each other [66]. Thus, an abundance of PDI family members in the ER may determine the fate of PRDX4 in individual cells. Recent studies have proposed the usefulness of extracellular PRDX4 as biomarkers for some diseases [67], which include sepsis [68], type 2 diabetes [69,70], and microalbuminemia [71]. When PRDX4 is overexpressed, beneficial effects have been reported such as protection against streptozotocin-induced diabetes [44], attenuation of atherosclerosis [72], amelioration of nonalcoholic steatohepatitis, prevention of type 2 diabetes [73], and improved insulin secretion [45]. Moreover, elevated tissue levels of PRDX4 have been found in cancer cells including glioma [74–76], lung cancer [77], leukemia [78], prostate cancer [79], colorectal cancer [80], and multiple myeloma [81]. A defect in the PRDX4 gene has been identified in rare cases of the malignancy [82,83]. Thus, PRDX4 may also play roles in these malignant cells, but these roles remain mostly unknown.

Redox regulation in the extracellular milieu, a future perspective Other than PRDX4, there are some extracellular antioxidant enzymes that are translated into the ER via N-terminal hydrophobic signal peptides such as extracellular SOD (SOD3) and plasma glutathione peroxidase (GPX3). As of this writing, no solid evidence was available regarding the function of extracellular PRDX4. The redox state in the extracellular space is more oxidized compared with that inside the cells, which places most sulfhydryls in the oxidized state. Proteins and other

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thiol compounds form stable disulfide bonds under extracellular oxidative conditions. However, even under such oxidative conditions, thiol exchange can occur. In fact, thiol isomerases such as PDI are present extracellularly and are involved in a rearrangement of the disulfide bonds in the membrane proteins, which results in the regulation of their functional states. For example, when enveloped viruses use the membrane protein CD4 for their entry into cells, their disulfide bond is rearranged by the PDI that is present on the cell surface [84]. Extracellular disulfide bonds in integrins, which are used for attachment by platelets and leukocytes, are also rearranged by the function of PDI family member ERp57 [85,86]. ERO1 is also reportedly present on the platelet surface and is involved in the activation of integrin [87]. Because PRDX4 coordinates with ERO1 and PDI in the ER, it may also play similar roles in the rearrangement of disulfide bonds in coordination with such partner molecules.

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