Peroxiredoxins: Structure, Mechanism, and Regulation of These Antioxidant Enzymes, Lab Reports of Pharmacology

Download Peroxiredoxins: Structure, Mechanism, and Regulation of These Antioxidant Enzymes and more Lab Reports Pharmacology in PDF only on Docsity! Structure, mechanism and regulation of peroxiredoxins Zachary A. Wood1, Ewald Schröder2, J. Robin Harris3 and Leslie B. Poole2 1Institute of Molecular Biology, Howard Hughes Medical Institute, University of Oregon, Eugene, OR 97403, USA 2Department of Biochemistry, Wake Forest (University School of Medicine, Winston-Salem, NC 27157, USA 3Institute of Zoology, University of Mainz, D-55099 Mainz, Germany Peroxiredoxins (Prxs) are a ubiquitous family of antioxi- dant enzymes that also control cytokine-induced per- oxide levels which mediate signal transduction in mammalian cells. Prxs can be regulated by changes to phosphorylation, redox and possibly oligomerization states. Prxs are divided into three classes: typical 2-Cys Prxs; atypical 2-Cys Prxs; and 1-Cys Prxs. All Prxs share the same basic catalytic mechanism, in which an active- site cysteine (the peroxidatic cysteine) is oxidized to a sulfenic acid by the peroxide substrate. The recycling of the sulfenic acid back to a thiol is what distinguishes the three enzyme classes. Using crystal structures, a detailed catalytic cycle has been derived for typical 2- Cys Prxs, including a model for the redox-regulated oli- gomeric state proposed to control enzyme activity. Peroxiredoxins (Prxs) [1,2] have received considerable attention in recent years as a new and expanding family of thiol-specific antioxidant proteins, also termed the thio- redoxin peroxidases and alkyl-hydroperoxide-reductase- C22 proteins. Prxs exert their protective antioxidant role in cells through their peroxidase activity (ROOH þ 2e2 ! ROH þ H2O), whereby hydrogen peroxide, peroxynitrite and a wide range of organic hydroperoxides (ROOH) are reduced and detoxified [3–7]. Indeed, these enzymes are truly ubiquitous having been identified in yeast, plant and animal cells, including both protozoan and helminth parasites, and most, if not all, eubacteria and archaea. Although located primarily in the cytosol, Prxs are also found within mitochondria, chloroplasts and peroxisomes, associated with nuclei and membranes, and, in at least one case, exported [3,8]. Prxs are produced at high levels in cells: they are among the ten most abundant proteins in Escherichia coli [9], the second or third most abundant protein in erythrocytes [10] and compose 0.1–0.8% of the soluble protein in other mammalian cells [11]. Many organisms produce more than one isoform of Prx, including at least six Prxs identified in mammalian cells (PrxI– PrxVI; Table 1). Recently, a range of other cellular roles have also been ascribed to mammalian Prx family members, including the modulation of cytokine-induced hydrogen peroxide levels, which have been shown to mediate signaling cascades leading to cell proliferation, differentiation and apoptosis [3,8,12,13]. The peroxidatic functions of Prxs probably overlap to some extent with those of the better known glutathione peroxidases and catalases, although it has been suggested that their moderate catalytic efficiencies (,105 M21 s21) compared with those of glutathione peroxidases (,108 M21 s21) [3] and catalases (,106 M21 s21) [14] makes their importance as peroxidases questionable [3]. Nonetheless, the high abundance of Prxs in a wide range of cells and a recent finding that a bacterial Prx [alkyl hydroperoxide reductase C22 (AhpC)] and not catalase is responsible for reduction of endogenously generated H2O2 [15] argue that Prxs are indeed important players in peroxide detoxification in cells. Prxs use redox-active cysteines to reduce peroxides and were originally divided into two categories, the 1-Cys and 2-Cys Prxs, based on the number of cysteinyl residues directly involved in catalysis [1]. Structural and mechan- istic data now support the further division of the 2-Cys Prxs into two classes called the ‘typical’ and ‘atypical’ 2-Cys Prxs. The peroxidase reaction is composed of two steps centered around a redox-active cysteine called the peroxidatic cysteine. Based on existing data [16,17], all three Prx classes appear to have the first step in common, in which the peroxidatic cysteine (Cys–SPH) attacks the peroxide substrate and is oxidized to a cysteine sulfenic acid (Cys–SOH) (Fig. 1) [16,18]. The peroxide decompo- sition probably requires a base to deprotonate the peroxidatic cysteine as well as an acid to protonate the poor RO2 leaving group, but these catalysts have yet to be identified. All Prxs to date conserve an active-site Arg, which would lower the pKa of the peroxidatic cysteine somewhat by stabilizing its thiolate form (Fig. 1). The second step of the peroxidase reaction, the resolution of the cysteine sulfenic acid, distinguishes the three Prx classes. The typical 2-Cys Prxs are the largest class of Prxs and are identified by the conservation of their two redox-active cysteines, the peroxidatic cysteine (generally near residue 50) and the resolving cysteine (near residue 170) [3]. Typical 2-Cys Prxs are obligate homodimers containing two identical active sites [19–22]. In the second step of the peroxidase reaction, the peroxidatic cysteine sulfenic acid (Cys–SPOH) from one subunit is attacked by the resolving cysteine (Cys–SRH) located in the C terminus of the other subunit (Fig. 1). This condensation reaction results in the formation of a stable intersubunit disulfide bond, which is then reduced by one of several cell-specific disulfideCorresponding author: Leslie B. Poole (lbpoole@wfubmc.edu). Review TRENDS in Biochemical Sciences Vol.28 No.1 January 200332 http://tibs.trends.com 0968-0004/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S0968-0004(02)00003-8 oxidoreductases (e.g. thioredoxin, AhpF, tryparedoxin or AhpD [23–25]), completing the catalytic cycle. The second class of Prxs are the atypical 2-Cys Prxs, which have the same mechanism as typical 2-Cys Prxs but are functionally monomeric [26,27]. In these Prxs, both the peroxidatic cysteine and its corresponding resolving cysteine are contained within the same polypeptide, with the condensation reaction resulting in the formation of an intramolecular disulfide bond (Fig. 1). Although the resolving cysteines of typical and atypical 2-Cys Prxs are not conserved in sequence, they are functionally equival- ent. To recycle the disulfide, known atypical 2-Cys Prxs appear to use thioredoxin as an electron donor [26]. The last class of Prxs, the 1-Cys Prxs, conserve only the peroxidatic cysteine and do not contain a resolving cysteine (Fig.1) [17].Theircysteinesulfenicacidgeneratedonreaction with peroxides is presumably reduced by a thiol-containing electron donor, but the identity of this redox partner is not yet clear (although proposed electron donors have included glutathione, lipoic acid and cyclophilin [3,7,28,29]). By ana- logy, one donor thiol probably forms a transient mixed disulfide bond with the enzyme, followed by its reduction by a second donor thiol, thus recycling the enzyme. Recently, studies of several typical 2-Cys Prxs have revealed dramatic changes in oligomeric state (dimers and decamers) linked to changes in redox state like those occurring during the catalytic cycle. A combination of biophysical techniques has been used to examine the various oligomeric forms of several these enzymes, revealing an intimate connection between the oxidation state of the peroxidatic cysteine and the preferred oligomeric state of the enzyme. Although we are at an early stage of understanding the link between the oligomeric state of Prxs and their function, the recent biophysical studies have provided some important insights to the field and are the subject of this article. Dimers, decamers and redox-dependent oligomerization The first reports of Prx oligomerization came in the late 1960s, when transmission electron microscopy (TEM) studies of torin, an abundant protein isolated from human erythrocytes, revealed discrete complexes with apparent tenfold symmetry (Fig. 2a) [30]. Later, in the 1980s, bacterial and yeast Prxs were identified based on their antioxidant properties [31,32]. Torin has since been identified as mammalian PrxII, a typical 2-Cys Prx [33]. In the TEM reports, it was observed that under certain conditions, PrxII and the related PrxIII could also form higher-order multimers by stacking into columns of various lengths (Fig. 2b) [33,34]. The physiological rele- vance of these columns, if any, is not known. Recently, single-particle TEM analysis of negatively stained PrxII particles enabled the three-dimensional reconstruction of the toroid to ,20-Å resolution (Fig. 2c) [33]. Importantly, the surface-rendered TEM reconstruction correlated well with the solvent-accessible surface revealed by the X-ray crystal structure of PrxII (Fig. 2d) [20]. The oligomeric properties of several typical 2-Cys Prxs in solution have been studied using gel filtration [35–39], light scattering [22,37] and analytical ultracentrifugation [20,22]. Factors shown to promote oligomerization in typical 2-Cys Prxs include high [37] or low [34,39] ionic strength, low pH [40], high magnesium [34] or calcium [41,42] concentrations, reduction of the redox-active disul- fide center [20,22,36], and ‘overoxidation’ of the peroxi- datic cysteine to a sulfinic acid (Cys–SO2H) [20]. Reduction of the active-site disulfide of typical 2-Cys Prxs is emerging as the primary factor in the stabilization of the decameric forms of these enzymes; a direct link between redox state and oligomerization state was recently established through analytical ultracentrifugation of sev- eral bacterial 2-Cys Prxs [22,43] (L.B. Poole, unpublished) and human PrxII [20], as well as earlier gel-filtration Table 1. Six subclasses of Peroxiredoxins (Prxs) from mammals Prx subtype PrxI (2-Cys) PrxII (2-Cys) PrxIII (2-Cys) PrxIV (2-Cys) PrxV (atypical 2-Cys) PrxVI (1-Cys) Previous nomenclature TPx-A TPx-B AOP-1 AOE372 AOEB166 ORF06 NKEF A NKEF B SP22 TRANK PMP20 LTW4 MSP23 PRP MER5 AOPP AOP2 OSF-3 Calpromotin HBP23 Torin PAG Band-8 TSA Polypeptide length 199 aa 198 aa 256 aa (cleaved at 63–64)a 271 aa (cleaved at 36–37)a 214 aa (cleaved at 52–53)a 224 aa Human chromosomal location 1q34.1 13q12 10q25–q26 10p22.13 11q13 1q23.3 Cellular location Cytosol, nucleus Cytosol, membrane Mitochondria Cytosol, Golgi, secreted Mitochondria, peroxisome, cytosol Cytosol Genbank AAA50464 AAA50465 BAA08389 AAB95175 AAF03750 BAA03496 SwissProt tdx2_human tdx1_human tdxm_human tdxn_human aopp_human aop2_human P35703 P32119 P30048 Q13162 P30044 P30041 Interactions with proteins and other ligands c-Abl Presenilin-1 Heme Macrophage migration inhibitory factor Cyclophilin Protein 7.2b (stomatin) Presenilin-1 Erythrocyte membrane Cyclophilin Cyclophilin Abrin A-chain Heparin Cyclophilin DNA Cyclophilin Cyclophilin Abbreviation: aa, amino acids. aThese proteins are post-translationally processed. Review TRENDS in Biochemical Sciences Vol.28 No.1 January 2003 33 http://tibs.trends.com the adjacent dimer (Fig. 5a). The remaining portion of the loop–helix motif, the Cp loop (Fig. 4a), contains the peroxidatic cysteine and has been identified as the molecular switch responsible for the redox-sensitive oligomerization of 2-Cys Prxs [22]. In the reduced (thiol) state, the Cp loop forms a helix, positioning the peroxidatic cysteine in the active-site pocket and packed against region I, thus buttressing region I against region II in the interface (Fig. 5bi). During peroxide decomposition, the peroxidatic cysteine is oxi- dized to a cysteine sulfenic acid, burying the sulfur atom with the sulfenic-acid oxygen (Fig. 5bii). Local unfolding of the active site converts the Cp loop into a solvent-exposed loop, making the sulfenate sulfur accessible for disulfide- bond formation (Fig. 5biii). It is notable that such an unfolding in the absence of a suitable reductant could have deleterious consequences by making the sulfenate accessible to further oxidation by peroxide to yield inactive sulfinic (–SO2H) or sulfonic (–SO3H) acid forms (Fig. 5bvi). By maintaining a locally high concentration of reduced thiol (the resolving cysteine), 2-Cys Prxs can avoid this fate by forming a stable disulfide bond (Fig. 5biv). Still, overoxidation of some yeast Prxs and mammalian Prxs I–IV and VI has been observed after exposure of cells to peroxides [35,47,48]. In the crystal structure of PrxII, the peroxidatic cysteine is present as cysteine sulfinic acid (Cys–SO2H), trapping this protein as a decamer owing to its inability to form a disulfide bond. Overoxidation of this protein might even promote further aggregation (Fig. 2b) [34]. In this structure, the Cp loop containing the peroxidatic cysteine sulfinic acid maintains a fold very similar to that of a reduced Prx and also buttresses the dimer–dimer interface. In addition, the folded conformation of the Cp loop in the PrxII structure might Fig. 3. Peroxiredoxin (Prx) structural classes. Ribbon models and associated topology diagrams for the crystal structures representing the three Prx classes: the typical 2- Cys Prxs (rat PrxI [19], human PrxII [20], Crithidia fasciculata TryP [21] and Salmonella typhimurium AhpC [22]); the atypical 2-Cys Prxs (human PrxV) [27]; and the 1-Cys Prxs (human PrxVI) [17]. Ribbon models are progressively colored from N to C terminus (blue to red) with consistent coloring for conserved structural elements. The top- ology diagrams (N and C for N and C terminus, respectively) depict the basic functional unit as domain-swapped dimers (red and blue lines for subunits) or a monomer in the case of PrxV. The conserved thioredoxin fold is identified as red-filled circles and triangles for helices and strands, respectively. The peroxidatic cysteine is identified as thiol (black SP), sulfenic- or sulfinic-acid states (red SP), or as a disulfide (connecting bar) with the resolving cysteine (SR). The oxidation state of the peroxidatic cysteines of the different structures is identified in parentheses as thiol (–SH), sulfenic acid (–SOH), sulfinic acid (–SO2H) or disulfide (–S–S–). Asterisks signify the truncation of the model owing to disorder. This figure was generated using MOLSCRIPT [55] and TOPS [56]. Review TRENDS in Biochemical Sciences Vol.28 No.1 January 200336 http://tibs.trends.com be favored by the salt bridge between the sulfinic acid and the conserved Arg127 in the active site [20]. Interestingly, PrxII shows a well-ordered helix at its C terminus, which has been proposed to stabilize the C-terminal arm, reducing its mobility [20]. Because disulfide-bond for- mation requires the C-terminal arm to unfold, it was proposed that this added stability might slow down this step and allow further oxidation of the reactive Cys–SOH species [22]. The predicted influence of C-terminal-tail interactions on susceptibility to overoxidation is supported by experimental observations of a typical 2-Cys Prx from Schizosaccharomyces pombe, in which C-terminal trunca- tion converts the overoxidation-sensitive enzyme to an overoxidation-resistant form [49]. In the above model, the Cp loop acts a lynchpin holding the decamer together. In the folded conformation, it stabilizes the decamer interface, whereas disulfide-bond formation traps the Cp loop in the unfolded state, in effect removing the lynchpin supporting the interface and weakening the decamer (Fig. 5biv). Despite the instability of the disulfide-bonded form of the decamer, the high protein concentration during the crystallization of this form of AhpC favored decamer formation, allowing this important intermediate to be trapped [22]. At physiologi- cally relevant concentrations, as more active sites in the decamer form disulfide bonds, the instability reaches a critical point and the decamer breaks down into free dimers (Fig. 5bv). In doing so, region I collapses into the active-site pocket vacated by the peroxidatic cysteine, restructuring the oligomerization interface. The catalytic cycle is completed with the reduction of the redox-active disulfide bond. Although the loop-helix structure of the active site is conserved in all Prxs, the region-I–Cp-loop and region-II sequence motifs have only been identified in typical 2-Cys Prxs (Fig. 4b). Given this sequence con- servation and the observation that typical 2-Cys Prxs from groups as diverse as bacteria and mammals have been reported to undergo redox-sensitive oligomerization, it is tempting to speculate that this might be a property of this class in general. Regulation of Prx activity Prxs have received a great deal of attention recently owing to their role in regulating levels of hydrogen peroxide, an intracellular signaling molecule common to many cytokine- induced signal-transduction pathways [3,8,12,13]. As noted above, some Prxs are themselves sensitive to inactivation by hydrogen peroxide and perhaps peroxyni- trite through irreversible oxidation of their peroxidatic cysteine. Indeed, regulation of redox signaling through cysteine modification by peroxides and peroxynitrite has been reported for a growing number of enzymes and transcriptional regulators [50]. It was recently shown that the overoxidation of PrxII is likely to be physiologically relevant, in that its peroxidatic cysteine is oxidized to sulfinic (–SO2H) or sulfonic (–SO3H) acid forms in vivo upon exposure of Leydig cells to tumor necrosis factor [51]. It has been proposed that Prxs in mammalian cells act as a dam against oxidative stress, and that the ratio of active to inactive enzyme might play a role in whether cells are susceptible to cytokine-induced apoptosis [51]. In addition to overoxidation, Prx activity has also been shown to be regulated by phosphorylation and proteolysis [36,49,52,53]. Recently, phosphorylation of mammalian PrxI, PrxII, PrxIII and PrxIV at the conserved residue Thr89 (PrxII numbering) by cyclin-dependent kinases was shown to decrease the peroxidase activity of the Prxs [52]. In the case of PrxI, this phosphorylation was observed to occur in vivo during mitosis. The authors concluded that the phosphorylated Thr89 had an unfavorable electrostatic effect on the peroxidatic active site. However, analysis of the mammalian crystal structures show Thr89 to be solvent exposed and too distant (.16 Å) to interfere with the active site. An examination of the structure of the PrxII decamer reveals that a phosphorylated Thr89 would introduce unfavorable electrostatic interactions within the dimer–dimer interface by placing two negatively Fig. 4. Peroxiredoxin (Prx) active sites. (a) The reduced Prx active site (PrxV) and the conserved hydrogen-bonding network (dotted lines). (b) The illustration is an alignment of selected residues representing the consensus sequences for the three Prx classes. In both the illustration and the alignment, the active-site residues conserved in all Prxs are colored red and the loop-helix region blue. Also identified in the alignment are regions I and II, which are necessary for decamer formation. (c) The dramatic conformational changes in which the conserved loop–helix motif (cyan) undergoes a local unfolding required for disulfide-bond formation. The figure shows PrxI and PrxII, dimeric 2-Cys Prxs (individual monomers colored gray or green) in which the peroxidatic cysteine (SP) is present as an overoxidized sulfi- nic acid (ball and stick) or in a disulfide with the resolving cysteine (SR). The con- served Arg is also shown to ease comparisons. It is notable that the sulfinic acid form still adopts the folded conformation seen in the thiol form of Prxs. Review TRENDS in Biochemical Sciences Vol.28 No.1 January 2003 37 http://tibs.trends.com charged phosphates in close proximity (Fig. 5a). Indeed, a reasonable alternative interpretation is that phosphoryl- ation of Thr89 attenuates the enzyme activity by disrupt- ing the decameric structure (Fig. 5a). Several researchers have reported that dimeric forms of Prxs exhibit less activity than decameric forms [23,37,39]. This observation is supported by the crystal structures, which show that the active sites of the typical 2-Cys Prxs are adjacent to and stabilized by the dimer–dimer interface of the decamer. It is notable that these two control mechanisms, phosphoryl- ation and overoxidation, probably favor different oligo- meric states (dimer and decamer, respectively). Another mechanism proposed to regulate peroxidase activity in vivo entails specific proteolysis of the C termini of Prxs, preventing peroxide-mediated inactivation in response to rising levels of peroxide [49]. In studies of a typical 2-Cys Prx from yeast, a portion of the enzyme was found to have a truncated C-terminal following purifi- cation [49]. In follow-up mutagenesis studies, C-terminally truncated forms of the enzyme were found to be more resistant to peroxide overoxidation and inactivation than the sensitive wild-type enzyme [49]. A similar truncation of PrxII that removed the C-terminal 13 residues (includ- ing the last a helix) has also been observed during the isolation of the enzyme from erythrocytes [53]. Interest- ingly, the regulatory protease calpain is present in erythrocytes and will specifically cleave this region of PrxII in vitro [36]. Proteolysis would make the enzyme resistant to overoxidation but leave it susceptible to inactivation by phosphorylation. Conclusions The ubiquitous Prxs appear to be diverse in function, ranging from antioxidant enzymes to regulators of signal transduction. This diversity is reflected in slight evolu- tionary modifications in sequence and structure, built around a common peroxidatic active site. The literature within the Prx field is currently focused on their more recently identified roles as regulators of redox-sensitive signaling [3,8]. Although the precise relationship between the peroxidase activity and the oligomeric status of these enzymes is currently unclear, the two appear to be closely linked. Here, we have highlighted the current state of our understanding of Prx mechanism, structure and regulation. † There are three classes of Prx, distinguished by the number and location of catalytic cysteines – the typical 2-Cys, atypical 2-Cys and 1-Cys Prxs. † Despite differences in quaternary structure and cataly- tic cycle, all three classes share the same peroxidatic active-site structure. † Some bacterial and mammalian typical 2-Cys Prxs undergo redox-sensitive oligomerization, and this might be a property of typical 2-Cys Prxs in general. † Prx peroxidase activity might be regulated in vivo by cysteine oxidation, phosphorylation and limited proteolysis. Future research should aim to improve our under- standing of the influence of changes in oligomeric structure and post-translational modifications upon the peroxidatic and signaling activities of Prxs. Fig. 5. Redox-sensitive oligomerization. (a) The interface between adjacent dimers in the Peroxiredoxin (Prx) II decamer. The loop-helix (including region I), region II and the C-terminal arm (C-term) are colored cyan, blue and green, respectively. Also depicted in ball and stick are the peroxidatic cysteine (SP), the resolving cysteine (SR), and Thr89, the site of phosphorylation by regulatory kinases [52]. (b) The catalytic cycle of some typical 2-Cys Prxs with relevant species indicated by roman numerals. The loop-helix, region II and the C-terminal arm are colored as in (a). The different oxidation states of the peroxidatic cysteine are identified as thiol [SPH; species (i)], sulfenic [SPOH; species (ii) and species (iii)] or sulfinic [SPO2H; species (vi)] acid forms, or disulfide bonded with the resolving cysteine [SR; species (iv) and (v)]. The loop-helix (cyan) is depicted in folded [cylinder and solid line; species (i), (ii) and (vi)] or unfolded [dashed line; species (iii) and (iv)] conformations, or with the peroxidatic active site restructured [species (v)]. Unidirectional arrows indicate changes in redox state and bidirectional arrows represent dynamic equilibrium. Ti BS HOOH HOH Peroxidation Catalytic cycle Resolution Reduction and decamerization Decamer to dimer transition 2 RSH RSSR HOOH HOH Overoxidation of select Prxs HOH SPH C-term HSR C-term HSR SPOH HOSP C-term HSR C-term HSR SPO2H SP SR C-term SR C-term SP (a) (b) (i) (ii) (iii) (v) (iv) (vi) Review TRENDS in Biochemical Sciences Vol.28 No.1 January 200338 http://tibs.trends.com