Báo cáo khoa học: Peroxiredoxins as cellular guardians in Sulfolobus solfataricus – characterization of Bcp1, Bcp3 and Bcp4 pot

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Peroxiredoxins as cellular guardians in Sulfolobussolfataricus characterization of Bcp1, Bcp3 and Bcp4Danila Limauro1, Emilia Pedone2, Ilaria Galdi1and Simonetta Bartolucci11 Dipartimento di Biologia Strutturale e Funzionale, Universita`di Napoli ‘Federico II’, Complesso Universitario Monte S. Angelo, Naples, Italy2 Istituto di Biostrutture e Bioimmagini, CNR, Naples, ItalyTo maintain a proper intracellular redox environment,aerobic microorganisms use redox systems and antioxi-dants that protect cells from the attack of reactive oxy-gen species (ROS) such as superoxide anions, H2O2and hydroxyl radicals. Increased ROS concentrationinside a cell results in damage to the main biomole-cules and membranes and essential metabolic functions[1]; to maintain a low intracellular ROS level, cells areequipped with an array of antioxidant systems, in thefirst place superoxide dismutases (SODs), which cata-lyze the dismutation of superoxide anions into H2O2and oxygen. H2O2is reduced by various systems, inthe main by catalases and peroxidases. Peroxiredoxins(Prx) are thiol-peroxidases that scavenge peroxidesusing the enzyme-recycling thioredoxin (Trx) ⁄ thiore-doxin reductase (Tr) system as an electron donor [2,3].Keywordsantioxidant; archaea; disulfideoxidoreductase; oxidative stress;thiol-peroxidaseCorrespondenceSimonetta Bartolucci, Dipartimento diBiologia Strutturale e Funzionale, ComplessoUniversitario di Monte S. Angelo,Universita`di Napoli ‘Federico II’, Via Cinthia,80126 Naples, ItalyFax: +39 081679053Tel: +39 081679052E-mail: bartoluc@unina.it(Received 20 December 2007, revised 22February 2008, accepted 27 February 2008)doi:10.1111/j.1742-4658.2008.06361.xPeroxiredoxins are ubiquitous enzymes that are part of the oxidative stressdefense system. In the present study, we identified three peroxiredoxins[bacterioferritin comigratory protein (Bcp)1, Bcp3 and Bcp4] in the genomeof the aerobic hyperthermophilic archaeon Sulfolobus solfataricus. Basedon the cysteine residues conserved in the deduced aminoacidic sequence,Bcp1 and Bcp4 can be classified as 2-Cys peroxiredoxins and Bcp3 as a1-Cys peroxiredoxin. A comparative study of the recombinant Bcps pro-duced in Escherichia coli showed that these enzymes protect DNA plasmidfrom oxidative damage and remove both H2O2and tert-butyl hydroper-oxide, although at different efficiencies. We observed that all of them wereparticularly thermostable and that peak enzymatic activity fell within therange of the growth temperature of S. solfataricus. Furthermore, we dis-covered an alternative Bcp reduction system whose composition differsfrom that of the peroxiredoxin reduction system previously characterized inthe aerobic hyperthermophilic archaeon Aeropyrum pernix. Whereas thelatter uses the thioredoxin ⁄ thioredoxin reductase ⁄ NADPH system, thisalternative Bcp system is formed of the protein disulfide oxidoreducatase,SSO0192, the thioredoxin reductase, SSO2416, and NADPH. The role ofBcps in oxidative stress was investigated using transcriptional analysis.Different northern blot analysis responses suggested that the Bcp antioxi-dant system of S. solfataricus can both operate at the constitutive level,with Bcp1 and Bcp4 preventing endogenous peroxide formation, and at theinducible level, with Bcp3 and the already characterized Bcp2 protectingcells from the attack of external peroxides.AbbreviationsBcp, bacterioferritin comigratory protein; Cysp,peroxidatic cysteine; CysR,resolving cysteine; MCO, metal ion catalyzed oxidation; PDO,protein disulfide oxidoreductase; ROS, reactive oxygen species; SOD, superoxide dismutase; ssSOD, SOD from Sulfolobus solfataricus;t-BOOH, tert-butyl hydroperoxide; Tr, thioredoxin reductase; Trx, thioredoxin.FEBS Journal 275 (2008) 2067–2077 ª 2008 The Authors Journal compilation ª 2008 FEBS 2067Prxs are ubiquitous enzymes identified in eubacteria,archaea, yeast, algae, higher plants and animals [4,5].All Prxs share the same basic catalytic mechanisms,whereby a cysteine conserved in the N-terminalsequence, the peroxidatic cysteine (Cysp), is oxidized tosulfenic acid (Cys-SPOH) by a peroxide substrate.These enzymes are generally distinguished into 2-Cysor 1-Cys based on whether or not they contain theresolving cysteine (CysR). In 2-Cys Prxs, the Cys-SPOHand Cys-SRreact and form a disulfide (CP-S-S-CR):the stable disulfide form is then reduced by one of sev-eral disulfide oxidoreductases, completing the catalyticcycle. 2-Cys Prxs have been further distinguished intotypical or atypical, depending on the location of Cys-SRresidue. In typical 2-Cys-Prxs, the Cys-SPOH reactswith the Cys-SRresidue located in the C-terminalsequence of the other subunit of the antiparallelhomodimer. By contrast, in atypical 2-Cys Prx, theCys-SRresidue resides within the same subunit.Although catalases mainly detoxify high levels ofH2O2, the function of Prxs is to scavenge low levels ofH2O2[6]. It has been demonstrated that Prxs with aKMin the low lm range are kinetically more efficientscavengers of trace amounts of H2O2than catalases,and this is why they are probably the primary defenceagainst endogenous hydrogen peroxide. More than 40proteins have been found to perform a similar functionin a variety of organisms, ranging from bacteria andeukarya to archaea. A hexadecameric Prx [3] belongingto the 2-Cys Prx family has recently been isolated inthe archaeon Aeropyrum pernix and the enzyme wasfound to be dependent on the Trx ⁄ Tr ⁄ NADPH systemfor H2O2reduction.Another archaeal Prx from Pyrococcus horikoshiiPH1217 [7,8] has been characterized and subsumedwithin the 2-Cys family. Although it performs peroxi-dase activity, its electron donor partner may differfrom that found in A. pernix.On evaluation, the Sulfolobus solfataricus P2 genome[9] was found to contain no putative amino acidsequences that are homologues of catalases, but fourhomologues of Prxs, annotated as bacterioferritincomigratory protein (Bcp)1, Bcp2, Bcp3 and Bcp4.Indeed, the antioxidant system of S. solfataricus P2has so far been identified only in part, in that only twoenzymes have been characterized: SOD from S. solfa-taricus (SsSOD) [10] and a 1-Cys Prx (Bcp2) [11].SsSOD has a homodimeric structure that is sensitiveto inactivation by H2O2. Its half-life is 2 h at 100 °C[12]. SsSOD has been found both in the culture fluid ofS. solfataricus during growth on glucose-rich media andis associated with the cell surface. There is evidence thatcell-associated SsSOD protects both the cell surfaceenzyme glucose dehydrogenase and the integral-mem-brane enzyme succinate dehydrogenase against oxyradi-cal protein deactivation [13]. Transcriptional analysishas provided evidence of constitutive gene expression.Moreover, the high stability of the 2 h half-life mRNAsuggests that high SsSOD levels are likely to protect thecells from the superoxide anion generated in the naturaloxidant environment of S. solfataricus [14,15].Bcp2 is a recently characterized Prx; its transcriptionin S. solfataricus is up-regulated by various stressorsand the different kinetics observed in response to theseagents could imply different regulatory mechanisms, orat least variations in the same mechanism. Further-more, Bcp2 displays peroxidase activity at a tempera-ture optimum in the range 80–90 °C (i.e. the range inwhich S. solfataricus grows). The peroxidase activity ofBcp2 involves the single cysteine residue (Cys49) in thecatalytic activity, suggesting a mechanism wherebythe residue is oxidized by H2O2and then reduced bythe dithiothreitol used as electron donor in vitro [11].No physiological partner has so far been identified. Anew redox system formed of the protein disulfide oxi-doreductase (PDO) SSO0192 and the Tr SSO2416 hasrecently been characterized in S. solfataricus. SSO0192is a typical PDO, a member of the protein disulfideisomerase-like family [16], whose redox and chaperoneactivities confirm a central role in the biochemistry ofcytoplasmic disulfide bonds and suggest a potentialrole in intracellular protein stabilization, respectively[17]. Recent investigations of the genomic sequencedatabases of S. solfataricus led to the identification oftwo putative Trxs (i.e. TrxA1 and TrxA2; SSO0368and SSO2232, respectively). Unlike SSO0192, which ispart of the new thioredoxin system SSO0192 ⁄ SSO2416,both these Trxs proved to be inactive in reduction withSSO2416 [17].The present study aims to expand our knowledge ofthe antioxidant system in S. solfataricus. Accordingly,we characterize three Prxs deduced from the S. solfa-taricus P2 genome: we report on the cloning of bcp1,bcp3 and bcp4 and the characterization of the recombi-nant products and, next, we investigate the possiblerole of the SSO0192 ⁄ SSO2416 system as an in vivopartner of Bcps in enzyme recycling. Subsequently,transcriptional studies help to shed light on the tacticused by S. solfataricus during oxidative stress.ResultsSequence analysis of Bcp1, Bcp3 and Bcp4Four genes encoding the putative Prxs named Bcp1(SSO2071), Bcp2 (SSO2121), Bcp3 (SSO2255) andPeroxiredoxin antioxidant system D. Limauro et al.2068 FEBS Journal 275 (2008) 2067–2077 ª 2008 The Authors Journal compilation ª 2008 FEBSBcp4 (SSO2613) were identified in the S. solfataricusgenome database (http://www-archbac.u-psud.fr/projects/sulfolobus/). One of them, Bcp2 (215 amino acids,with a predicted molecular mass of 24 744.79 Da anda theoretical pI of 6.85), has recently been character-ized and classified as a 1-Cys Prx [11].bcp1 encodes a putative protein of 153 amino acidswith a predicted molecular mass of 17 460.12 Da anda theoretical pI of 7.73; bcp4 encodes a putative pro-tein of 156 amino acids with a predicted molecularmass of 17 461.28 Da and a theoretical pI of 7.65;bcp3 encodes a putative protein of 149 amino acidswith a predicted molecular mass of 16 976.42 Da anda theoretical pI of 8.84. Comparison of the sequencesof S. solfataricus Bcps revealed an approximate 35%identity for Bcp1, Bcp3 and Bcp4.A BlastP search against the Swiss-Prot ⁄ TrEMBLGenBank database identified numerous Prx homo-logues of Bcp1, Bcp3 and Bcp4. The highest identityof Bcps is found with archaeal thermophilic Prxs(Figs 1–3). The Bcp1 and Bcp4 amino acid sequencesinclude two cysteine residues at positions 45 and 50of the highly conserved N-terminal region and showsignificant identity with chloroplastic PrxQ, which isinvolved in antioxidant defence and in the redoxhomeostasis of photosynthesis [18]; by contrast, theBcp3 sequence only shows one cysteine at position 42in the N-terminal region.Expression and purification of Bcp1, Bcp3 andBcp4bcp1, bcp3 and bcp4 were amplified by PCR fromS. solfataricus genomic DNA and cloned into pET-30c(+). The genes were expressed in Escherichia colicells and the recombinant proteins were highly overex-pressed in soluble form, as fusions with a C-terminaleight-residue histidine tag (LEHHHHHH), with anapproximate 10% yield of homogeneous proteins.To purify the recombinant Bcps, the soluble frac-tions (140 mg) of the cell extracts were heated at 80 °Cfor 15 min; this heat-treatment removed approximately40% of E. coli proteins. Bcp1 was purified to homoge-neity in a one-stage process using affinity chromato-graphy on HisTrap HP. SDS ⁄ PAGE of the finalpreparation revealed a single band with a molecularFig. 1. Sequence alignment of Bcp1. CLUSTALW alignment of S. solfataricus (Bcp1) Q97WP9|SULSO, S. acidocaldarius Q4J6R7|SULAC,S. tokodaii Q974D1|SULTO, Picrophilus torridus Q6L214|PICTO, Anabaena variabilis Q3M6A0|ANAVT, and Synechococcus sp. Q31LU7|SYNP7.D. Limauro et al. Peroxiredoxin antioxidant systemFEBS Journal 275 (2008) 2067–2077 ª 2008 The Authors Journal compilation ª 2008 FEBS 2069mass of 18 ± 1 kDa. Bcp3 and Bcp4 required an addi-tional purification step with cation and anion-exchangechromatography, respectively.The molecular masses of the three recombinant pro-teins were determined using MS analysis as reportedin the Experimental procedures; the values reportedfor Bcp1 (18 525 Da), Bcp3 (18 041 Da) and Bcp4(18 526 Da) were in agreement with the correspondingtheoretical values. The quaternary structure of theenzymes was assessed via analytical gel filtration of thepurified proteins: Bcp1 and Bcp3 were eluted at a vol-ume consistent with a monomeric structure whereasBcp4 was eluted consistently with its dimeric structure.Peroxidase activityThe peroxidase activity of recombinant Bcps was testedin vitro via a metal ion catalyzed oxidation (MCO)assay. The assay was based on the ability to protect plas-mids against Fe3+-catalyzed reduction of O2to H2O2,which occurs in the presence of an electron donor, suchas dithiothreitol. Via the Fenton reaction, the H2O2formed in these conditions was further converted intoHO., which nicks supercoiled plasmid DNA. Accord-ingly, we investigated whether recombinant Bcp1, Bcp3and Bcp4 could protect DNA from oxidative damage interms of removing the H2O2, generated by the MCOsystem. As shown in Fig. 4A–C (lanes 2), in the pres-ence of the MCO system, the supercoiled form ofpUC19 was completely converted into nicked form,whereas the addition of Bcp1, Bcp3, or Bcp4 to the reac-tion mixture averted this damage. In more detail, 2 lmof Bcp3 was sufficient to remove the H2O2generated bythe MCO system and to preserve the supercoiled DNAplasmid form (Fig. 4B, lane 6); whereas 40 lm and20 lm of Bcp1 and Bcp4 (Fig. 4A, lane 6 and Fig. 4C,lane 6), were only partially able to convert nicked intosupercoiled plasmid; BSA and Bcp2 were used as nega-tive and positive controls, respectively.Subsequently, using dithiothreitol as electron donorfor enzyme recycling, we further investigated the anti-oxidant activity of Bcps by measuring both H2O2and the organic peroxide tert-butyl hydroperoxide(t-BOOH) removed. A non-enzymatic spectrophoto-metric assay was performed as described in the Experi-mental procedures. Bcp1, Bcp3 and Bcp4 were capableFig. 2. Sequence alignment of Bcp3. CLUSTALW alignment of S. solfataricus (Bcp3) Q97WG5|SULSO, S. tokodaii Q96YS1|SULTO, S. acido-caldarius Q4JCJ2|SULAC, A. pernix Q9YG15|AERPE, Pyrobaculum aerophilum Q8ZYA3|PYRAE, and Thermoplasma acidophilumQ9HL73|THEAC.Peroxiredoxin antioxidant system D. Limauro et al.2070 FEBS Journal 275 (2008) 2067–2077 ª 2008 The Authors Journal compilation ª 2008 FEBSof scavenging both H2O2and t-BOOH, although atdifferent efficiencies. The results shown in Fig. 5Ademonstrate that 2.5 lm of Bcp1 is sufficient toremove approximately 50% of the existing H2O2andthat Bcp4 at the same concentration and 1.4 lm Bcp3,respectively, remove all the peroxides. Figure 5B showsthe activity of the same enzymes when t-BOOH is usedas a substrate: 8 lm of Bcp1 and Bcp4 remove approx-imately 50% of the t-BOOH, a proportion which risesto approximately 90% when Bcp3 is used at the sameconcentration. In conclusion, Bcp1 Bcp3 and Bcp4 aremore efficient when H2O2is used as substrate in placeof t-BOOH.To determine the physiological partners involved inBcp reduction in vivo, we used the redox Trx-like systemof S. solfataricus comprising the PDO memberSSO0192, the Tr SSO2416 and NADPH [17]. The H2O2consumption rate at 80 °C was measured by monitoringthe decrease in A490, as in the previous assay. We foundthat Bcp1, Bcp3 and Bcp4 were also capable of perform-ing their peroxide reductase activity in the presence ofthe Trx-like system (Fig. 6), although at a slightly lesserefficiency compared to dithiothreitol. These results indi-cate that Bcp1, Bcp3 and Bcp4 are functional Trx-likeperoxidases, whereas Bcp2, which was previouslyreported to remove H2O2in the presence of dithiothrei-tol [11], is unable to eliminate H2O2in the presence ofthe SSO2416 ⁄ SSO0192 ⁄ NADPH system. In all likeli-hood, the different response observed for Bcp2, com-pared to the other three enzymes, either reflects adifferent oxidized catalytic center accessibility or adifferent reaction mechanism.Fig. 3. Sequence alignment of Bcp4. CLUSTALW alignment of S. solfataricus (Bcp4) Q97VL0|SULSO, S. acidocaldarius Q4J9Q3|SULACS. tokodaii Q96ZP9|SULTO, Thermotoga marittima Q9WZN7|THEMA, Orysa sativa PRXQ|ORSSJ, and Arabdopsis thaliana PRXQ|ARATH.D. Limauro et al. Peroxiredoxin antioxidant systemFEBS Journal 275 (2008) 2067–2077 ª 2008 The Authors Journal compilation ª 2008 FEBS 2071Temperature dependence and thermostabilityof Bcp1, Bcp3 and Bcp4To characterize the thermophilicity of recombinantBcp1, Bcp3 and Bcp4, we investigated the peroxidaseactivities of the enzymes by measuring H2O2removalat increasing temperatures. Bcp1 and Bcp3 activitywas shown to be highest at 85 °C, which is in theoptimum temperature range for S. solfataricus growth(Fig. 7A,B), whereas Bcp4 showed maximum peroxi-dase activity in the range 95–100 °C, the highesttemperature tested (Fig. 7C). At this point, we deter-mined the thermoresistance levels of the enzymes byincubating them for varying periods at 80, 90, 95 and100 °C and then assaying their respective residual per-oxidase activity (data not shown). Following a 2 hincubation at 80 °C, all the enzymes were shown tohave retained 100% of their initial activity; Bcp1 dis-played a half-life of 2 h at 95 °C but, after a 60 minincubation at 100 °C, its relative activity dropped to20% of the starting value. Bcp3 and Bcp4 are morethermostable than Bcp1: after 2 h at 100 °C, theyABCNFSF12 43657NFSF12 43657NFSF12 43657SFNFSF12 4365NFSF12 4365Fig. 4. DNA cleavage protection assay performed by Bcp1 (A),Bcp3 (B) and Bcp4 (C). Supercoiled pUC19 plasmid (lanes 1) wasexposed to the MCO system (dithiothreitol ⁄ Fe+3⁄ O2) alone (lanes 2)and with different Bcps concentrations. pUC19 plus the MCO sys-tem plus Bcp2 1 lM as positive control (lanes 3), pUC19 plus theMCO system plus BSA as negative control (lanes 7). pUC19 plusthe MCO system plus Bcp1 2 lM (lane 4A) or Bcp1 20 lM (lane 5A)or Bcp1 40 lM (lane 6A). pUC19 plus the MCO system plus Bcp31 lM (lane 4B) or Bcp3 1.5 lM (lane 5B) or Bcp3 2 lM (lane 6B).pUC19 plus the MCO system plus Bcp4 1 lM (lane 4C) or Bcp410 lM (lane 5C) or Bcp4 20 lM (lane 6C). NF, nicked form; SF,supercoiled form of pUC19 are indicated on the left by arrows.H 2 O 2 removal (%) Bcps (µM)0 20 40 60 80 100 A 0 1 2 3 4 5 Bcps (µM)B 0 20 40 60 80 100 t-BOOH removal (%) 0 4 8 12 16Fig. 5. Different efficiency of Bcps in removing H2O2(A) and t-BOOH (B). Peroxidase activity was measured using the ferrithiocyanatemethod at 80 °C as described in the Experimental procedures in the presence of dithiothreitol as an electron donor. Bcp1(d), Bcp3 (),Bcp4 ().02040608010012345Relative activity (%)Fig. 6. Peroxidase activity of recombinant Bcps in the presenceof the SSO2416 ⁄ SSO0192 ⁄ NADPH system of S. solfataricus.SSO0192 ⁄ SSO2416 ⁄ NADPH + H2O2negative control (1),SSO0192 ⁄ SSO2416 ⁄ NADPH + H2O2+ Bcp1 5 lM (2), SSO0192 ⁄SSO2416 ⁄ NADPH + H2O2+ Bcp2 5 lM (3), SSO0192 ⁄ SSO2416 ⁄NADPH + H2O2+ Bcp3 4 lM (4), SSO0192 ⁄ SSO2416 ⁄ NADPH +H2O2+ Bcp4 5 lM (5).Peroxiredoxin antioxidant system D. Limauro et al.2072 FEBS Journal 275 (2008) 2067–2077 ª 2008 The Authors Journal compilation ª 2008 FEBSboth showed similar half-lives but, after 2 h at 95 °C,relative activity had dropped to 92% for Bcp4 and to60% for Bcp3. Interestingly Bcp4 is found to befairly resistant in 6 m urea: after a 30 min incubation,the enzyme retained 50% of its activity (data notshown).Transcriptional analysis of bcp1, bcp3 and bcp4under oxidative stressThe involvement of bcp1, bcp3 and bcp4 in oxidativestress was investigated by assessing mRNA levels fol-lowing treatment of S. solfataricus cells with H2O2andt-BOOH as direct oxidants and with paraquat, whichwas used to generate superoxide anions [15]. To estab-lish the concentrations of agents capable of slowingdown or otherwise affecting growth, the cells weretreated with different amounts of stressors in the expo-nential phase [11]. Therefore, the S. solfataricus P2strain was grown until the early exponential phase (0.3A600) and then induced with 0.1 mm paraquat,0.05 mm H2O2and 0.05 mm t-BOOH for varyingperiods (Fig. 8A–C). The hybridizing bands in thenorthern analysis revealed the expected size of approxi-mately 500 bp, indicating that the genes aretranscribed as monocistronic mRNAs. When S. solfa-taricus cells were incubated with paraquat, H2O2andt-BOOH, the bcp1 and bcp4 mRNA levels did notincrease appreciably, whereas, 15 min after the addi-tion of H2O2, the level of bcp3 mRNA was found tohave risen approximately five-fold.DiscussionPrxs are recently identified thiol peroxidases and areubiquitous among both prokaryotes and eukaryotes.bcp116S rRNAbcp4 16S rRNA16S rRNA16S rRNAbcp316S rRNAparaquat0153060015ABC30 60H2O20153060t-BOOHparaquat01530600153060H2O20153060t-BOOH015 60paraquat06015 30t-BOOH0153060H2O2rRNA16S rRNAFig. 8. Transcriptional response of bcp1,bcp3 and bcp4 to oxidative stress. Culturesof S. solfataricus P2 were grown until themid-exponential phase and treated with0.05 mM H2O2, 0.1 mM paraquat, 0.05 mMt-BOOH for different times. RNAs wereobtained from culture harvested at the timeshown The arrows indicate the transcript ofbcp1 (A), bcp3 (B) and bcp4 (C). The tran-scripts of 16S rRNA were reported fornormalization.Temperature (°C)02040608010002040ABC60 80 100Temperature (°C)020406080100Temperature (°C)020406080100Relative activity (%)020406080100Relative activity (%)020406080100Relative activity (%)Fig. 7. Temperature dependence of Bcp1, Bcp3 and Bcp4. Bcp1 (A), Bcp3 (B) and Bcp4 (C) were incubated under the conditions describedin the Experimental procedures with 0.2 mM H2O2. The peroxidase activity was assayed at different temperatures using the ferrithiocyanatemethod. The non-enzymatic removal of H2O2by heat was performed in parallel.D. Limauro et al. Peroxiredoxin antioxidant systemFEBS Journal 275 (2008) 2067–2077 ª 2008 The Authors Journal compilation ª 2008 FEBS 2073They are classified into several subfamilies based onthe number and locations of the conserved cysteineresidues that they contain, on subunit composition, onthe nature of the electron donor involved in theirreduction, and on structural comparison analysis [19].Recent studies of multigenic Prx families presentin cyanobacteria, such as Synechococcus elongatusPCC7942 and Synechocystis sp. PCC6803 [18], and thecharacterization of Prxs from plants, have led to theidentification of a new class of Prxs named PrxQ, simi-lar to Bcps occurring in E. coli, [20]. This family ischaracterized by a Trx-dependent peroxidase activity,a primary structure in which the two conserved cyste-ine residues in the N-terminal region are separated byfive amino acids, and by a similar molecular weight ofapproximately 17 kDa.The discovery of Prxs in archaea, defined togetherwith cyanobacteria, the oldest evolutionary group oforganisms, emphasizes the ancient role of Prxs both indefence against ROS and in redox homeostasis.The system that we have elucidated in the presentstudy detoxifies S. solfataricus cells from peroxidesrelated to Prxs. S. solfataricus shows an array of Prxs,named Bcp1, Bcp2, Bcp3 and Bcp4, which are able toshield cells from the attack of peroxides in the absenceof catalases [9].Sequence analysis points to a significant identitybetween Bcp1 and Bcp4 and the PrxQ subfamilybecause of the presence of two conserved residues: Cys45 and Cys 50. By contrast, Bcp3 shows higher iden-tity values with archaeal Prxs and its functional rolediffers from those of Bcp1 and Bcp4. Our functionaldata point to a higher efficiency of Bcp3, compared toboth Bcp1 and Bcp4, in scavenging peroxides and inprotecting nucleic acid from oxidative damage. DNAcleavage assays performed with an MCO systemshowed that Bcp3 was able to protect plasmid 10-foldmore efficiently than either Bcp1 or Bcp4, in additionto ensuring DNA integrity at an efficiency level com-parable to that of the previously characterized Bcp2[11]. These findings are in agreement with the observa-tion that 1-Cys-Prxs protect nucleic acid both ineukaryotes and bacteria [18]. Furthermore, the rapidfive-fold increase in specific mRNA, as observed dur-ing the transcriptional analysis of Bcp3 15 min afterthe addition of H2O2, is comparable to that observedfor Bcp2, thus suggesting a role in response to oxida-tive stress. Based on our results, the physiological roleof Bcp3 appears to differ from those of Bcp1 andBcp4 despite their comparable molecular weights,significant sequence identity, and the use of theSSO0192 ⁄ SSO2416 ⁄ NADPH system for enzyme recy-cling. This reducing system involves, for the first time,an enzyme of the PDO family [21], namely SSO0192,in place of a Trx, associated with a Tr in the Prxreducing cascade, emphasizing the key role of the pro-tein both in antioxidant defence and in redox homeo-stasis. Hence, the PDO ⁄ Tr ⁄ NADPH is an alternativereduction system [17] of Prx with respect to the previ-ously characterized Trx (APE0641) ⁄ Tr (APE1061) ⁄NADPH system of ApPrx (APE2278) in the aerobicarchaeon A. pernix [3].The SSO0192 ⁄ SSO2416 ⁄ NADPH system regeneratesBcp1, Bcp3 and Bcp4, differently from previously char-acterized Bcp2 whose electron donor has so far notbeen identified. The presence of the conserved Cys45and Cys50 residues in Bcp1 and Bcp4 and the capabil-ity of the SSO0192 ⁄ SSO2416 ⁄ NADPH system withrespect to recycling oxidized enzymes suggests the fol-lowing catalytic mechanism: the putative peroxidaticcysteine, Cys45, could be transformed into a sulfenicacid intermediate that is reduced by the attack of thesecond cysteine, Cys50, to form an intramoleculardisulfide bridge. Subsequently, this disulfide is furtherreduced by SSO0192 and the oxidized form ofSSO0192 is eventually reduced by SSO2416 ⁄ NADPH.As for Bcp3, in which Cys42 is the only cysteine resi-due conserved, it is likely that the sulfenic acid isdirectly reduced by SSO0192 and that the resultingtransient intermolecular disulfide bridge is subse-quently reduced by SSO2416 ⁄ NADPH.The findings obtained in the present study suggestthat Bcp1 and Bcp4 protect cells from endogenous per-oxides formed during metabolism, whereas Bcp2 andBcp3 respond to oxidative stress. Furthermore, the dif-ferent enzyme recycling system adopted by the previ-ously characterized Bcp2, which, unlike the otherBcps, does not use the SSO0192 ⁄ SSO2416 ⁄ NADPHsystem (data not shown), may reflect different oxidizedcatalytic centre accessibility and ⁄ or different reactionmechanisms.Experimental proceduresConstruction and expression of recombinantproteinsGenomic DNA of S. solfataricus was prepared as describedin Arnold et al. [22]. Based on the bcp1, bcp3 and bcp4 nucle-otide sequences, the following oligonucleotides were designedand used as primers in the PCR gene amplification proce-dures. For bcp1, the forward primer 5¢-TATCTATCATATGGTAAAAGTGGGGGA-3¢ and the reverse primer5¢-AAGAAGGCCATCTCGAGAGCTGATCT-3¢ contain-ing the NdeI and XhoI restriction sites, respectively (under-lined), were used; the amplification was carried out at 94 °CPeroxiredoxin antioxidant system D. Limauro et al.2074 FEBS Journal 275 (2008) 2067–2077 ª 2008 The Authors Journal compilation ª 2008 FEBSfor 1 min, 50 °C for 1 min and 72 °C for 1 min, for 35 cyclesusing HF Taq DNA polymerase (Roche Applied Science,Monza, Italy). For bcp3, the forward primer 5¢-AAATTCATATGAACGTAGGAGAAGAAGCACCAG-3¢ and thereverse primer 5¢-GACTCGAGAGTTGAGTTTTGTCTCTTTATTATCTC-3¢ containing the NdeI and XhoI restric-tion sites, respectively (underlined), were used; the amplifi-cation was carried out at 94 °C for 1 min, 45 °C for 1 minand 72 °C for 1 min, for 35 cycles using HF Taq DNApolymerase (Roche). For bcp4, the forward primer5¢-CAAAATCTTTCATATGGTAGAAATAGG-3¢ and thereverse primer 5¢-GCCTAGCCATAACATCTCGAGAGATA-3¢ containing the NdeI and XhoI restriction sites,respectively (underlined), were used; the amplification wascarried out at 94 °C for 1 min, 48 °C for 1 min and 72 °C for1 min, for 35 cycles using HF Taq DNA polymerase (Roche).The PCR products were purified with QIAquick PCR purifi-cation kit (Quiagen Spa, Milan, Italy) and cloned inpGEMTeasy vector (Promega Italia Srl, Milan, Italy). Thenucleotide sequences of the inserted genes were determinedto ensure that no mutations were present in the genes.Then, NdeI-XhoI fragments were cloned into pET-30c(+)(Novagen, Darmstadt, Germany) giving the recombinantplasmids pETBcp1, pETBcp3 and pETBcp4, respectively,that were used to transform competent E. coli BL21-Codon-Plus (DE3)-RIL cells for expression purposes.Cells were grown to an A600nmof approximately 1 in LBmedia supplemented with kanamycin (50 lgÆmL)1) and chl-oramphenicol (33 lgÆmL)1)at37°C and were induced for3 h. The expression of Bcp3 and Bcp4 was induced by1mm isopropyl thio-b-d-galactoside (Inalco S.P.A., Milan,Italy) for 3 h, whereas the induction was carried out for6 h for Bcp1.Purification of Bcp1, Bcp3 and Bcp4 recombinantproteinsEscherichia coli cells containing the expressed recombinantproteins were harvested by centrifugation and pellets from1000 mL cultures were suspended in 20 mm Tris ⁄ HCl(pH 8.0) and disrupted by sonication with 20 min pulses at20 Hz (Sonicator Ultrasonic liquid processor; Heat SystemUltrasonics Inc., NY, USA). The suspensions were clarifiedby ultracentrifugation at 160 000 g for 30 min. The crudeextracts obtained were heated at 80 °C for 15 min and thencentrifugated at 15 000 g at 4 °C for 30 min, removingalmost 70% of the mesophilic host proteins. The extractwere concentrated (Amicon, Millipore Corp.; Bedford,MA, USA) and applied to a HisTrap HP (GE Healthcare,Healthcare Europe, GmbH, Milan, Italy) equilibratedwith 50 mm Tris ⁄ HCl (pH 8.0), 0.3 m NaCl (buffer A). Thecolumns were washed with buffer A with 20 mm imidazole,and proteins were eluted with the same buffer A, supple-mented with 250 mm imidazole. The active fractions werepooled and dialyzed against 20 mm Tris ⁄ HCl (pH 8.0). Theconcentrated samples of Bcp3 and Bcp4 were applied ontwo different ionic-exchange columns. The Bcp3 samplewas applied to a Resource S (GE Healthcare) in 20 mmsodium phosphate buffer (pH 6.5) connected to AKTAsystem (GE Healthcare) and eluted with a linear gradient0.1–0.4 m NaCl in 30 min at a flow rate of 1 mLÆmin)1.The active fractions were pooled, concentrated and exten-sively dialysed against 20 m m Tris ⁄ HCl (pH 8.0). Bcp4was applied to a Resource Q (GE Healthcare) in 50 mmTris ⁄ HCl (pH 9.0) connected to AKTA system (GEHealthcare) and eluted with a linear gradient 0.1–0.5 mNaCl for 30 min at a flow rate of 1 mLÆmin)1. The activefractions were pooled, concentrated and extensively dialysedagainst 20 mm Tris ⁄ HCl (pH 8.0).Determination of quaternary structureThe molecular mass of the Bcp1, Bcp3 and Bcp4 recombi-nant proteins were determined by gel-filtration chromato-graphy on a Superdex 75 PC (0.3 cm ⁄ 3.2 cm) connected toAKTA system (GE Healthcare). Proteins were eluted withbuffer 20 mm sodium phospate (pH 7.4), 0.2 m KCl at aflow rate of 0.04 mLÆmin)1. b-Amylase (200 kDa), alcoholdehydrogenase (150 kDa), BSA (65.4 kDa), ovalbumin(48.9 kDa), chimotrypsinogen (22.8 kDa) and the RNasi A(15.6 kDa) were used as molecular weight standards (GEHealthcare).Analytical methods for Bcp recombinant proteincharacterizationProteins concentration was determined using BSA as thestandard [23]. Protein homogeneity was estimated bySDS ⁄ PAGE [24] using 12.5% (w ⁄ v) acrylamide resolvinggel and 5% acrylamide stacking gel. Samples were heatedat 100 °C for 5 min in 2% SDS and 2% 2-mercaptoetha-nol and run in comparison with molecular weight stan-dards. Gels were stained with the Coomassie Brilliant Blueprocedure.The molecular mass of the Bcp1, Bcp3 and Bcp4 werealso estimated using electrospray mass spectra recorded ona Bio-Q triple quadrupole instrument (Micromass, Man-chester, UK). Samples were dissolved in 1% (v ⁄ v) aceticacid ⁄ 50% (v ⁄ v) acetonitrile and injected into the ion sourceat a flow rate of 10 lLÆmin)1using a Phoenix syringe pump(Carlo Erba Strumentazione, Milan, Italy). Spectra werecollected and elaborated using masslynx software providedby the manufacturer. Calibration of the mass spectrometerwas performed with horse heart myoglobin (16.9 kDa).DNA cleavage assay by the MCO systemThe ability of Bcps to protect DNA from oxidative nickingby hydroxyl radicals was determined as previouslyD. Limauro et al. Peroxiredoxin antioxidant systemFEBS Journal 275 (2008) 2067–2077 ª 2008 The Authors Journal compilation ª 2008 FEBS 2075described by Lim et al. [25]. A reaction mixture of 50 lLincluded 3 lm FeCl3,10mm dithiothreitol for the thiolMCO system, 100 mm Hepes (pH 7.0), different concentra-tions of recombinant Bcp1 or Bcp3 or Bcp4 or BSA as anegative control or Bcp2 as a positive control. The reactionwas initiated by incubating the mixture for 40 min at 37 °Cbefore adding 2 lg of plasmid pUC19 and developed foran additional 1 h at the same temperature. DNA bandswere evaluated on 0.8% (w ⁄ v) agarose gel after stainingwith ethidium bromide 5 lgÆmL)1.Assays of peroxidase activityBcps recombinant proteins were tested for their ability toremove peroxides in an in vitro non-enzymatic assay. Thereaction was started adding H2O2or t-BOOH at a finalconcentration of 0.2 mm to the reaction mixture containing50 mm Hepes (pH 7.0), 10 mm dithiothreitol in the presenceof different concentrations of Bcps in a final volume of0.1 mL. As an alternative to dithiothreitol as an electrondonor, a mixture containing 0.25 mm NADPH, 0.2 lmSSO2416, 20 lm SSO192, 0.05 mm FAD was used, forminga reducing cascade to recycle the enzymes. The reactionwas incubated at 80 °C for 1 min and stopped by adding0.9 mL of trichloroacetic acid solution (10%, w ⁄ v), as pre-viously described by Lim et al. [23]. Peroxidase activity wasdetermined from the amount of peroxide remaining, whichwas detected by measurement of the purple-colored ferri-thiocyanate complex developed after the addition of 0.2 mLof 10 mm Fe(NH4)2(SO4)2and 0.1 mL of 2.5 m KSCN,using H2O2as a standard. The amount of the ferrithiocya-nate complex present was determined by measurement ofA490. The percentage of peroxides removed was calculatedon the basis of the change in A490obtained with Bcps rela-tive to that obtained without Bcps. Experiments were per-formed in triplicate.Transcriptional analysisSulfolobus solfataricus P2 strain liquid cultures were grownaerobically at 80 °C in mineral medium supplemented with0.1% BactoÔ yeast extract (Becton Dickinson and Com-pany, Franklin Lakes, NJ, USA), 0.1% tryptone (Oxoid,Basingstoke, Hampshire, UK) and 0.2% sucrose (TYSmedium) in an orbital shaker. Oxidative stresses were per-formed by adding paraquat, H2O2or t-BOOH at a finalconcentration of 0.1 mm and 0.05 mm, respectively, toS. solfataricus cultures in early exponential growth phase(A600= 0.3). Aliquots of cultures were collected at differ-ent times by centrifugation at 5000 g for 10 min at 4 °C.Total RNA was extracted by the guanidinium isothiocya-nate method, as described in Sambrook et al. [26]. Theintegrity and concentration of total RNA were verified byelectrophoretic analysis by separating the total RNA on1% agarose gel containing formaldehyde.Northern blot analysis was performed to quantify theamount of bcp1, bcp3 and bcp4 mRNA in different stressconditions and to determine the size of the specific tran-scripts.The NdeI-XhoI fragments derived from digestion of pET-Bcp1, pETBcp3 and pETBcp4, purified from agarose gel,labelled with [a32P]dATP and with random primed DNAlabelling kit (Roche), were used to identify bcp1, bcp3 andbcp4 mRNA, respectively.AcknowledgementsThis work was supported by grants from MIUR(PRIN 2003-2004).References1 Imaly JA (2003) Pathways of oxidative damage. AnnuRev Microbiol 57, 395–418.2 Rho BS, Hung LW, Holton JM, Vigil D, Kim SI, ParkMS, Terwilliger TC & Pedelacq JD (2006) Functionaland structural characterization of a thiol peroxidase fromMycobacterium tuberculosis. 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Peroxiredoxins as cellular guardians in Sulfolobus solfataricus – characterization of Bcp1, Bcp3 and Bcp4 Danila Limauro1, Emilia
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