Glutathione- and glutaredoxin-dependent reduction of methionine sulfoxide reductase A

FEBS Letters 586 (2012) 3894–3899

journal homepage: www.FEBSLetters.org

Glutathione- and glutaredoxin-dependent reduction of methionine sulfoxide reductase A Jérémy Couturier a,⇑, Florence Vignols b, Jean-Pierre Jacquot a, Nicolas Rouhier a a b

UMR1136 Université de Lorraine-INRA, Interactions Arbres-Microorganismes, IFR 110, Faculté des Sciences, 54500 Vandoeuvre, France UMR186 Résistance des Plantes aux Bio-agresseurs, CNRS and Institut de Recherche pour le Développement, BP 64501, 34394 Montpellier Cedex 5, France

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Article history: Received 23 July 2012 Revised 18 September 2012 Accepted 18 September 2012 Available online 25 September 2012 Edited by Peter Brzezinski Keywords: Glutathione Glutaredoxin Methionine sufoxide reductase

a b s t r a c t A natural fusion occurring between two tandemly repeated glutaredoxin (Grx) modules and a methionine sulfoxide reductase A (MsrA) has been detected in Gracilaria gracilis. Using an in vivo yeast complementation assay and in vitro activity measurements, we demonstrated that this fusion enzyme was able to reduce methionine sulfoxide into methionine using glutathione as a reductant. Consistently, a poplar cytosolic MsrA can be regenerated in vitro by glutaredoxins with an efficiency comparable to that of thioredoxins, but using a different mechanism. We hypothesize that the glutathione/glutaredoxin system could constitute an evolutionary conserved alternative regeneration system for MsrA. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction In aerobic organisms, several metabolic pathways as well as stress conditions lead to the production and accumulation of various reactive oxygen and nitrogen species (ROS, RNS) that cause damages to cellular components. The oxidation of methionine (Met), one of the most sensitive amino acids to oxidation, into methionine sulfoxide (MetO), is a deleterious modification that can affect protein functioning, but which might also constitute a regulatory mechanism of the biological activity of some proteins [1]. Another possible function of methionine oxidation might be the scavenging of ROS, especially when Met residues are not involved in protein functioning [2]. Methionine sulfoxide reductases (Msrs) are present in most living organisms and are able to reduce free or peptide-bound MetO into Met. Hence, these enzymes are involved in protection mechanisms against oxidative damages having demonstrated roles in aging, bacterial adherence and virulence and more generally in stress response and signalling [3–5]. Molecular evolution has created two major classes of Msrs, called MsrA and MsrB, which display little if any sequence or structural homologies, and which are specific for the S- or R-epimers of MetO, respectively [6]. Enzymes of both classes contain a conserved catAbbreviations: GSH, glutathione; MetO, methionine sufoxide; Grx, glutaredoxin; Msr, methionine sulfoxide reductase; Trx, thioredoxin ⇑ Corresponding author. E-mail address: [email protected] (J. Couturier).

alytic cysteine or selenocysteine, which is transformed during catalysis into a sulfenic or selenenic acid after the nucleophilic attack of MetO [7,8]. Depending on the presence and position of resolving cysteines, the regeneration of these enzymes occurs either via the direct reduction of the sulfenic acid or via the reduction of one or sequentially of two intramolecular disulfide bridges [7,9]. Msrs forming a disulfide bond in their oxidized forms are usually regenerated using a thioredoxin (Trx)-recycling process [10]. There are a few reports, however, indicating that Trxs can also reduce the sulfenic acid intermediate of some mammalian and plant MsrB proteins, which do not possess any recycling cysteine [9,11–13]. Besides, a few Msrs such as Arabidopsis thaliana MsrB1 (AtMsrB1) and a Clostridium Sec-containing MsrA can be regenerated by a glutathione (GSH)/glutaredoxin (Grx) system [14–17]. For AtMsrB1, the sulfenic acid is reduced by glutathione forming a glutathionylated intermediate which is attacked by glutaredoxins [16]. We report here the occurrence in the red alga Gracilaria gracilis of a hybrid protein (GgMsrA for G. gracilis MsrA) consisting of two Grx domains fused to an MsrA domain and which is able to reduce MetO using GSH as an electron donor. This unusual specificity has been extended to a higher plant MsrA, which uses both Trx and GSH/Grx reducing systems with similar efficiencies. Hence, the present work illustrates how the identification of natural fusion proteins could constitute a tool to predict protein–protein interactions in organisms where the genes coding each domain are not fused.

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.09.020

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Transformed cells were grown on minimal YNB medium (0.7% yeast extract w/o amino acids, 2% glucose, 2% agar) with required amino acids (His, Ade, Lys, Met) at 30 °C for 2–4 days. Complementation of EMY63 defects is visualized by the ability of transformed cells to grow in the presence of 1 mM MetO, in the absence of Met or in the presence of 0.5 mM hydrogen peroxide.

2. Materials and methods 2.1. DNA constructs The open reading frame of GgMsrA was amplified by PCR using genomic DNA of G. gracilis as a template and cloned into the expression vector pET3d (Novagen), between NcoI and BamHI restriction sites (underlined) using the two primers 50 ccccccatgggaaacgttacacgaaaagctgccgtt30 and 50 ccccggatccttatcc ataacaacgaataga30 . For yeast complementation, the sequences coding for the fulllength protein (GgMsrA) or the MsrA domain (DGrx–GgMsrA) were cloned first into the pGEM-T Easy vector (Promega) and subcloned into the yeast expression vector pFL61 by NotI digestion. For the DGrx-GgMsrA construct, the sequence coding the first 246 amino acids (i.e., the two Grx domains) was removed during PCR cloning by using the following internal primer 50 ccccccatggctgcgtccgtttcatcg 30 . The Arabidopsis and yeast Trx-coding sequences were cloned into the yeast pFL61 shuttle vector [18].

2.3. Expression and purification of the recombinant proteins The Escherichia coli expression strain BL21(DE3) containing the helper plasmid pSBET was transformed with the recombinant plasmid pET3d–GgMsrA. A 2.4 L culture was typically induced at 37 °C in exponential phase using 100 lM isopropyl-b-D-thiogalactopyranoside (IPTG). All subsequent steps were performed at 4 °C. The cultures were centrifuged for 15 min at 4,400g and the pellets were resuspended in buffer A (30 mM Tris HCl pH 8.0, 1 mM EDTA, 200 mM NaCl). Cell lysis was performed by sonication (3  1 min) and the soluble and insoluble fractions were separated by centrifugation for 30 min at 27,000g. The pellet was resuspended in buffer A containing 20 mM DTT and 8 M urea at room temperature for 2 h and centrifuged again at 16,000g for 1 h. The supernatant was successively dialyzed against buffer A containing 500 mM urea (5 h or overnight) and then buffer A alone (5 h). After centrifugation, the supernatant

2.2. Yeast transformation Constructs were introduced into EMY63, a yeast strain carrying complete deletions of endogenous TRX1 and TRX2 genes [19].

GgMsrA

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Fig. 1. Amino acid sequence alignment of GgMsrA and PtMsrA2. The alignment was performed with ClustalW. The strictly conserved amino acids are depicted in white on black. Other conservative amino acid changes are indicated in black on gray. Two successive gray boxes indicate the two N-terminal Grx domains with the amino acids forming the active site and involved in glutathione binding surrounded. Black triangles and black circles indicate the cysteine residues involved or not involved in PtMsrA2 catalytic mechanism, respectively.

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was loaded first on an ACA 44 gel filtration column equilibrated with buffer A. The pooled fractions were dialyzed against buffer B (buffer A without NaCl) and loaded on a DEAE column equilibrated with buffer B. The fraction of interest was finally loaded on a Phenyl Sepharose (Amersham Pharmacia Biotech), equilibrated with buffer B containing 1.6 M NaCl. Elution was performed using a NaCl gradient from 1.6 to 0 M. As the protein was still retained, it was finally eluted using distilled water, dialyzed and concentrated in buffer B before storage at 20 °C. Purifications of poplar glutaredoxin C1 (GrxC1), C3 (GrxC3) and MsrA2 variants were performed as described previously [20,21].

GSH (0.25–5 mM) and L-Met-(R,S)-O (0–100 mM) and 10 lM GgMsrA. The reaction was started by adding GgMsrA after 2 min pre-incubation. Poplar MsrA2 activity was measured using a Trxor Grx-recycling system using conditions described previously [17,20]. The spontaneous reduction rate observed in absence of Msr was subtracted. Activity was expressed as nmol of NADPH oxidized per nmol enzyme per sec using a molar extinction coefficient of 6220 M1 cm1 at 340 nm for NADPH. Three independent experiments were performed at each substrate concentration and the apparent kcat and Km values were calculated by a non-linear regression using the program GraphPad Prism 4.

2.4. Enzymatic assays

3. Results and discussion

The reduction assays of insulin, dehydroascorbate (DHA), hydroxy-ethyl disulfide (HED) and poplar peroxiredoxin IIB (PrxIIB) were performed following procedures described in [22]. The Msr activity was measured by following NADPH oxidation at 340 nm in the presence of GSH and glutathione reductase (GR). A 500-ll cuvette contained 30 mM Tris–HCl pH 8.0, 1 mM EDTA, 200 lM NADPH, 0.5 units GR, varying concentrations of

3.1. G. gracilis possesses a functional glutaredoxin-methionine sulfoxide reductase hybrid protein

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A previously unrecognized sequence (accession No. AAD43253) in the eukaryotic red alga G. gracilis consists of two N-terminal Grx modules fused to an MsrA module and was named GgMsrA. The Grx domains display classical Grx sequence characteristics, with

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[DHA] (mM) Fig. 2. Reductase activity of the GgMsrA glutaredoxin domains. Insulin reduction was measured either using a DTT-based assay (A) in the presence of 2 lM poplar Trxh5 or 10 lM PtGrxC1 or GgMsrA, or using a GSH-based assay (B) containing a complete GSH regeneration system (NADPH/GR/GSH) and 10 lM PtGrxC1 or GgMsrA. Regeneration of poplar PrxIIB was measured using 1 lM Prx and 10 lM poplar GrxC1, GrxC3 or GgMsrA in presence of 250 lM H2O2 and 500 lM GSH (C). The data are represented as mean ± SD of three separate experiments. The HED (D) and DHA (E) reduction was achieved respectively using 20 nM or 1 lM GgMsrA in the presence of 2 mM GSH and either 0.7 mM HED or 1 mM DHA. The data are represented as mean ± SD of three separate experiments.

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CPYC or CPFC active site motifs and conserved residues involved in glutathione binding such as the TVP and GG motifs in their C-terminal region (Fig. 1). Concerning the MsrA domain, its sequence presents the highest similarity with higher plant MsrAs, ranging from 35% to 50% identity. Poplar MsrA2 contains 5 cysteine residues, the catalytic one being in position 31 and the two recycling ones in position 182 and 188 [20]. Indeed, the enzyme is regenerated in a two step reaction involving the successive formation and reduction of two different disulfides. Interestingly, there is no equivalent of Cys182 in GgMsrA (Fig. 1). Overall, both the specific domain arrangement and the conservation of catalytic cysteines suggest that the recycling of the MsrA domain could rely on a different system involving a GSH/Grx system. The full-length protein was produced as an untagged recombinant protein in E. coli. However, under standard conditions of growth (37 °C) and induction (100 lM IPTG), the protein was mostly insoluble. A low amount of soluble protein could however be obtained by growing the cells overnight at 20 °C. Alternatively, we have been able to purify to homogeneity higher protein amounts, after a denaturation/renaturation step followed by three chromatographies (gel filtration, anion exchange and phenylSepharose). Since the hybrid enzyme possesses two Grx domains, we first tested their disulfide reductase and deglutathionylation activities capacity by using classical substrates, i.e., insulin, DHA or HED, the two latter assays being associated to a NADPH-coupled spectrophotometric assay. The reduction of insulin was tested either

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in the presence of dithiothreitol (DTT) (Fig. 2A) or of a physiological NADPH/GR/GSH regeneration system (Fig. 2B). In the first case, GgMsrA was unable to reduce insulin disulfides contrary to poplar GrxC1 and poplar thioredoxin h5 (Trxh5) used as references. On the contrary, in the presence of a GSH regenerating system, GgMsrA became able to reduce insulin although less efficiently than GrxC1. As an alternative protein substrate, we observed that GgMsrA was also capable of regenerating poplar peroxiredoxin IIB (PrxIIB), a well characterized peroxiredoxin forming an intermediate glutathione adduct during the catalytic act, with efficiency comparable to that of plant Grxs (Fig. 2C). The kinetic analysis performed with both HED and DHA revealed an apparent Km value of 0.11 ± 0.02 mM for a glutathionylated b-mercaptoethanol (the adduct formed during the pre-incubation period in the HED assay) and a kcat of 24.6 ± 0.9 s1 and an apparent Km value of 0.44 ± 0.04 mM for DHA and a kcat of 0.55 ± 0.02 s1, respectively (Fig. 2D–E). These values are quite comparable to those reported for various plant and non-plant Grxs [22–25]. The functionality of the whole protein was first tested using in vitro activity measurements using a very similar NADPH-coupled spectrophotometric assay and free MetO as a substrate. The obtained results indicate that GgMsrA was able to reduce efficiently MetO in a GSH-dependent reaction (Fig. 3A–B). The kinetic analysis revealed apparent Km values of 3.8 ± 0.5 mM for MetO and 2.1 ± 0.3 mM for GSH and a kcat of 0.043 ± 0.002 s1. Next, we evaluated the capacity of this protein to complement the Dtrx1 Dtrx2 yeast mutant strain which is unable to grow in the presence of

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Fig. 3. Glutathione-dependence of GgMsrA for methionine sulfoxide reduction. The MetO reduction by GgMsrA was measured in the presence of varying GSH (A) and MetO (B) concentrations respectively. The data are represented as mean ± SD of three separate experiments. Complementation of a Dtrx1 Dtrx2 yeast strain (C). Cells expressing Arabidopsis thaliana Trxh2 (AtTrxh2) and Trxh3 (AtTrxh3), Saccharomyces cerevisiae TrxI (ScTrxI), GgMsrA or DGrx-GgMsrA, a truncated version of GgMsrA devoid of its Grx domains under the control of the yeast phosphoglycerate kinase promoter are plated as serial dilutions on different media. Complementation of EMY63 defects is visualized by the ability of transformed cells to grow in the presence of 1 mM MetO (+MetO, for Msr reduction), in the absence of methionine (-Met, for sulphate assimilation), and in the presence of 0.5 mM hydrogen peroxide (+H202, for sensitivity towards oxidants). A control assay shows all cells equally growing on complete medium containing methionine (+Met). AtTrxh2 and AtTrxh3 are used as positive controls for sulphate assimilation and for resistance to oxidative stress, respectively. ScTrxI is used as a positive control for all Dtrx1 Dtrx2 strain phenotypes analyzed here. Results shown here are the means of two independent experiments and correspond to cells grown for 4 days at 30 °C.

J. Couturier et al. / FEBS Letters 586 (2012) 3894–3899

A

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MetO [26]. Whereas it did not restore the capacity of this strain to assimilate sulphate and did not modify its sensitivity towards oxidants as measured by following growth in the absence of methionine and in the presence of hydrogen peroxide, the expression of GgMsrA fully restored the ability to grow in the presence of MetO after 4 days of growth (Fig. 3C, upper panel), despite with a slightly lower efficiency than that of other redoxins used as controls. Interestingly, a truncated version devoid of its Grx domains (DGrxGgMsrA) could not restore yeast growth in the presence of MetO (Fig. 3C, lower panel). This indicated that Grx domain(s) of GgMsrA are essential for methionine sulfoxide reductase activity and that endogenous yeast Grxs were not able to support this activity revealing some degree of specificity. Conversely, we cannot exclude that the Grx domains of GgMsrA can support the activity of yeast endogenous Msr. However as these Grx domains are very closely related to yeast Grxs of the same class (ScGrx1 and 2), it is rather unlikely that they could support the activity of yeast endogenous Msr given that endogenous yeast Grxs are unable to do it as deduced from the phenotype of the trx1 and trx2 mutant. Altogether, these results demonstrate that GgMsrA is a functional methionine sulfoxide reductase whose activity is dependent on GSH and on the presence of one or both N-terminal Grx domains. As there is no other identified Grx or MsrA genes to date in the genome of G. gracilis and as the Grx domain(s) possess their own activities, it might be that GgMsrA fulfills both MetO reduction and protein deglutathionylation functions.

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3.2. Grx-dependent recycling system of poplar MsrA2 The results obtained with an MsrA very similar to plant enzymes prompted us to investigate the ability of poplar GrxC1 to act as a reductant for poplar MsrA2 (previously referred to as cMsrA), whose Trx-dependent activity was shown to rely on three conserved cysteines [20]. Using a physiological NADPH/GR/GSH/ GrxC1 coupled system, we show that MsrA2 is indeed able to reduce MetO (Fig. 4A). The turnover number (kcat of 0.18 ± 0.01 s1) is quite comparable to the one obtained when using Trxh5 (kcat of 0.33 ± 0.02 s1) or Trxh1 as an electron donor (kcat of 1.20 s1) [20]. Apparent Km values for GSH and GrxC1 were 2.1 ± 0.5 mM and 19 ± 4 lM. The latter value is higher than those obtained with AtMsrB1 for other class I Grxs varying from 0.5 to 6.8 lM [16,17,22]. To better characterize the catalytic mechanism used by GrxC1 for the regeneration of MsrA2, MetO reduction by MsrA2 was recorded using mutated proteins for each of the three cysteines (Fig. 4A). Whereas GrxC1 C31S was expectedly inefficient in the reduction of MsrA2, both GrxC1 C34S and C88S kept the ability to regenerate MsrA2, GrxC1 C34S being more efficient as observed for the corresponding AtGrxC5 mutant in the AtMsrB1/AtGrxC5 couple [22]. Hence, these results indicate that MsrA2 can be recycled both by Trxs and Grxs and as already demonstrated for the Grx-dependent recycling of AtMsrB1, only the catalytic cysteine of Grx is required [16,22]. 3.3. Comparison between Trx and Grx-dependent recycling mechanisms As Grxs and Trxs usually employ different reducing mechanisms, the capacity of poplar Trxh5 and GrxC1 to serve as electron donors to MsrA2 cysteinic mutants was compared. As previously observed for Trxh1, Trxh5 is unable to regenerate MsrA2 variants mutated on either one of the two resolving cysteines (Cys182 and 188) as well as a variant (C66/85/182/188S) possessing only the catalytic cysteine Cys31, demonstrating that this Trx can efficiently reduce neither the sulfenic acid formed on Cys31 nor the disulfide bonds formed in the C182S and C188S variants (Fig. 4B and data not shown) [20]. Similarly, the weak activity obtained

Fig. 4. Comparison of PtMsrA2 Trx- and Grx-dependent recycling. The Grxdependent reduction of PtMsrA2 was estimated using 3 lM of PtMsrA2 in the presence of 25 lM GrxC1 variants, 5 mM GSH and 100 mM MetO (A). The data are represented as mean ± SD of three separate experiments. Activity of MsrA2 variants has been measured using 3 lM MsrA and 100 mM MetO with either 25 lM GrxC1 and 5 mM GSH (white bars) or 20 lM Trxh5 (black bars) (B). The data are represented as mean ± SD of three separate experiments.

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Grx Fig. 5. Proposed Grx-dependent regeneration of PtMsrA2. MetO reduction is concomitant to the formation of a sulfenic acid on catalytic cysteine (Cys31) and to the release of one mol of Met. The regeneration of a reduced cysteine occurs via the formation of two successive disulfides involving the two resolving cysteines (Cys182 and Cys188). As detailed in the text, the disulfide bond between Cys182 and Cys188 could be directly reduced by Grx as shown for Trx (1). Alternatively, once the Cys182–Cys188 disulfide is formed, regeneration of the sulfenic acid occurs through glutathionylation/deglutathionylation reactions (2).

for the MsrA2 C182S and C188S variants in the presence of GrxC1 likely indicates that neither GSH nor GrxC1 can reduce these disulfide bonds. In contrast, the activity obtained with the quadruple

J. Couturier et al. / FEBS Letters 586 (2012) 3894–3899

mutant is similar to the one recorded for the wt enzyme. Although we cannot completely rule out the possibility of direct reduction of the sulfenic acid by the catalytic Cys of Grx in this mutant, this result likely indicates that the reduction of the sulfenic acid proceeds via a glutathionylation step and subsequent deglutathionylation by Grx as already demonstrated for the 1-Cys AtMsrB1 [16]. Thus, there are only two possibilities to explain why wt MsrA2 is fully active, but not the MsrA2 C182S and C188S variants (Fig. 5). The disulfide bridge formed between Cys182 and Cys188 in the course of the catalytic mechanism of the wt enzyme is the only one efficiently regenerated by the GSH/Grx couple as demonstrated previously with Trxh1 and here with Trxh5 [20]. Alternatively, if this disulfide is not reduced by the GSH/Grx couple, the first catalytic turnover will lead indeed to the formation of a Cys182-Cys188 disulfide as it is probably favoured, but in subsequent turnovers, the enzyme catalyzes MetO reduction through glutathionylation/ deglutathionylation of the catalytic cysteine. Acknowledgements The authors would like to thank Marie-Christine Quillet (Univ. Lille Nord de France) for the generous gift of genomic DNA from Gracilaria gracilis. This work was supported by grants from Institut Universitaire de France to JPJ and NR. References [1] Stadtman, E.R., Moskovitz, J. and Levine, R.L. (2003) Oxidation of methionine residues of proteins: biological consequences. Antioxid. Redox Signal. 5, 577– 582. [2] Stadtman, E.R., Moskovitz, J., Berlett, B.S. and Levine, R.L. (2002) Cyclic oxidation and reduction of protein methionine residues is an important antioxidant mechanism. Mol. Cell. Biochem. 234–235, 3–9. [3] Moskovitz, J. (2005) Methionine sulfoxide reductases: ubiquitous enzymes involved in antioxidant defense, protein regulation, and prevention of agingassociated diseases. Biochim. Biophys. Acta 1703, 213–219. [4] Rouhier, N., Vieira Dos Santos, C., Tarrago, L. and Rey, P. (2006) Plant methionine sulfoxide reductase A and B multigenic families. Photosynth. Res. 89, 247–262. [5] Sasindran, S.J., Saikolappan, S. and Dhandayuthapani, S. (2007) Methionine sulfoxide reductases and virulence of bacterial pathogens. Future Microbiol. 2, 619–630. [6] Grimaud, R., Ezraty, B., Mitchell, J.K., Lafitte, D., Briand, C., Derrick, P.J. and Barras, F. (2001) Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase. J. Biol. Chem. 276, 48915–48920. [7] Boschi-Muller, S., Azza, S., Sanglier-Cianferani, S., Talfournier, F., Van Dorsselear, A. and Branlant, G. (2000) A sulfenic acid enzyme intermediate is involved in the catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli. J. Biol. Chem. 275, 35908–35913. [8] Kryukov, G.V., Kumar, R.A., Koc, A., Sun, Z. and Gladyshev, V.N. (2002) Selenoprotein R is a zinc-containing stereo-specific methionine sulfoxide reductase. Proc. Natl. Acad. Sci. USA 99, 4245–4250.

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