Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H2O2-mediated oxidative stress

Free Radical Biology & Medicine 39 (2005) 1332 – 1341 www.elsevier.com/locate/freeradbiomed

Original Contribution

Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H2O2-mediated oxidative stress Ce´dric R. Picota,b, Isabelle Petropoulosa, Martine Perichona, Marielle Moreaub, Carine Nizardb, Bertrand Frigueta,* a

Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universite´ Paris 7-Denis Diderot, 2 Place Jussieu, Tower 33-23, First Floor, CC 7128, 75251 Paris Cedex 05, France b Laboratoires R&D, Branche Parfums-Cosme´tiques, LVMH-Recherches, 45804 Saint-Jean-de-Braye Cedex, France Received 7 January 2005; revised 22 June 2005; accepted 24 June 2005 Available online 23 September 2005

Abstract Proteins are modified by reactive oxygen species, and oxidation of specific amino acid residues can impair their biological functions, leading to an alteration in cellular homeostasis. Oxidized proteins can be eliminated through either degradation or repair. Repair is limited to the reversion of a few modifications such as the reduction of methionine oxidation by the methionine sulfoxide reductase (Msr) system. However, accumulation of oxidized proteins occurs during aging, replicative senescence, or neurological disorders or after an oxidative stress, while Msr activity is impaired. In order to more precisely analyze the relationship between oxidative stress, protein oxidative damage, and MsrA, we stably overexpressed MsrA full-length cDNA in SV40 T antigen-immortalized WI-38 human fibroblasts. We report here that MsrA-overexpressing cells are more resistant than control cells to hydrogen peroxide-induced oxidative stress, but not to ultraviolet A irradiation. This MsrA-mediated resistance is accompanied by a decrease in intracellular reactive oxygen species and is partially abolished when cells are cultivated at suboptimal concentration of methionine. These results indicate that MsrA may play an important role in cellular defenses against oxidative stress, by catalytic removal of oxidant through the reduction of methionine sulfoxide, and in protection against death by limiting, at least in part, the accumulation of oxidative damage to proteins. D 2005 Elsevier Inc. All rights reserved. Keywords: Methionine; Methionine sulfoxide reductase A; Human fibroblasts; Oxidative stress; Reactive oxygen species; Protein oxidation; Free radicals

Reactive oxygen species (ROS) produced within cells, or by external mechanisms such as ultraviolet light or inflammation or during the course of various pathologies, can affect cellular macromolecules. For example, proteins are modified in a variety of oxidative forms, including

Abbreviations: Msr, methionine sulfoxide reductase; ROS, reactive oxygen species; UV-A, ultraviolet A; WI-38 SV40, SV40 T antigenimmortalized WI-38; DMEM, Dulbecco’s modified Eagle’s medium; FITC, fluorescein isothiocyanate; SDS, sodium dodecyl sulfate; DCF-DA, dichlorodihydrofluorescein diacetate; FACS, fluorescence-activated cell sorter; Met R,S (O), methionine sulfoxide; SEM, standard error of the mean. * Corresponding author. Fax: +33 1 44 27 82 34. E-mail address: [email protected] (B. Friguet). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.06.017

oxidation of specific amino acids, formation of carbonyl groups, glycation, glycoxidation, or addition of lipid peroxidation products [1]. Oxidation of methionine, an amino acid highly susceptible to ROS, results in the formation of an asymmetric sulfur atom, leading to the appearance of two diastereoisomers, methionine-S and -R sulfoxides. The oxidized forms of methionine are reduced in eukaryotes as well as in bacteria by two enzymatic systems, MsrA and MsrB [2,3]. The MsrA protein is generated from a single gene and is localized in the cytosol and in the mitochondria of most eukaryotic cells [4 –6]. MsrB represents a family of four proteins described in humans and in mice [7– 9]. MsrB1 (SelR) is localized in the cytosol and nucleus, whereas MsrB2 (CBS-1) is targeted to mitochondria. Recently, two other proteins, MsrB3A and MsrB3B,

C.R. Picot et al. / Free Radical Biology & Medicine 39 (2005) 1332 – 1341

were found to be generated from the same gene by alternative splicing and were targeted to endoplasmic reticulum and mitochondria, respectively [9,10]. It has been proposed that these proteins not only act as repair enzymes, but also are involved in cellular protection against oxidative stress, likely due to the catalytic reversion of exposed methionine residues on proteins that can act as ROS scavengers [11]. We previously investigated the possibility that alteration of protein repair systems such as Msr systems could be involved in the age-related decline in protein maintenance. We reported that Msr activity and expression of MsrA decreased with age in rat organs [12] and in replicative senescence of WI-38 fibroblasts [13]. In the latter study, expression of MsrB2 (CBS-1) was also found to be decreased. This downregulation was accompanied by an increase in the level of protein oxidation, monitored by determining the protein carbonyl content. In addition, MsrA was shown to be involved in antioxidant defense in several organisms such as bacteria and yeast [14,15], as well as in mammals, and in longevity in Drosophila and mouse [16,17]. Moreover, a central role for MsrA in the protection of neuronal cells against hypoxia –reoxygenation-induced cell injury and in lens cell viability under oxidative stress has been recently reported [18,19]. To more precisely analyze the role of MsrA in mechanisms of cellular protection against oxidative stress, we transfected SV40 T antigen-immortalized WI-38 human fibroblast cells (WI-38 SV40) with the cDNA of rat MsrA. We demonstrate that overexpression of the MsrA enzyme protects cells after cytotoxic H2O2 treatment, lowers the level of intracellular ROS, and prevents accumulation of protein oxidative damage, as monitored by carbonyl content determination. This decreased susceptibility of oxidativestress-induced cell death is associated with weaker expression of the p21 protein in MsrA-overexpressing fibroblasts compared to control cells.

Materials and methods Transfection of WI-38 SV40 human fibroblasts by rat MsrA-cDNA WI-38 SV40 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 Ag/ml streptomycin at 37-C, 5% CO2, in a humidified incubator. The rat MsrA cDNA full sequence was cloned between the EcoRI and the BamHI restriction sites in the expression vector pCDNA3.1(+) (Invitrogen, Cergy Pontoise, France) under the control of the cytomegalovirus enhancer – promoter for high level expression. WI-38 SV40 fibroblasts were transfected either with the resulting plasmid or with pCDNA3.1 alone as control, using the liposome transfection reagent

1333

FuGene (Roche Diagnostics, Meylan, France). To select stable transfectants, cells were grown in complete medium supplemented with 200 Ag/ml Geneticin G418 antibiotics (Invitrogen) for 15 days. Transfectants were tested for functional protein expression levels by immunoblot and activity analysis. Immunofluorescence labeling Immunofluorescence labeling was carried out according to standard procedures, as previously described [13]. Briefly, a rabbit polyclonal anti-MsrA antibody or a mouse antibody against human mitochondria (Chemicon International, Temecula, CA, USA) was used to localize MsrA and mitochondria, respectively, by indirect immunofluorescence. Staining of MsrA was then performed using an anti-rabbit IgG/FITC-conjugated secondary antibody (Sigma – Aldrich, Saint Quentin Fallavier, France), and staining of mitochondria was detected with a rhodamineconjugated donkey anti-mouse secondary antibody (Chemicon International). The microscopy images were acquired with a Hammatsu camera and then treated with Adobe PhotoShop 6.0 software. Gel electrophoresis and immunoblotting Cellular homogenates were obtained after disruption of cells by sonication (3  10 s) in 10 mM Hepes, pH 8, 50 mM NaCl, 500 mM sucrose, 1 mM EDTA, 7.2 mM 2mercaptoethanol, and 0.2% Triton X-100 at 4-C. Cellular debris and organelles were removed from the crude extracts by centrifugation at 15,000g, 4-C, for 30 min and the protein concentration of the supernatant was determined by the Bradford method (Bio-Rad, Life Science Group, Marnes la Coquette, France). Total proteins were separated on 12% SDS – polyacrylamide gel under conditions described by Laemmli [20]. Then, gels were electrotransferred onto a Hybond nitrocellulose membrane (Amersham Biosciences, Orsay, France) for 1 h at room temperature. Western blotting experiments were performed with either anti-MsrA polyclonal antibodies [12] at a dilution of 1/40,000 or anti-p21 monoclonal antibodies (Sigma– Aldrich). For 2D gel electrophoresis, the first dimension was performed using ReadyStrips IPG strips (pH 3 – 10; 13 cm length, Bio-Rad) and the Multiphor II isoelectric focusing system (Amersham Biosciences). One hundred micrograms of the crude protein extracts was subjected to isoelectrofocusing followed by SDS – PAGE. Western blot analyses were carried out as for those performed after the 1D gel. Detection of carbonyl groups was performed with the OxyBlot oxidized protein detection kit (AbCys S.A., Paris, France), according to the manufacturer. Briefly, 2 Ag of total extract proteins was incubated for 15 min at room temperature with 2,4dinitrophenylhydrazine to form the carbonyl derivative dinitrophenylhydrazone before SDS – PAGE separation.

1334

C.R. Picot et al. / Free Radical Biology & Medicine 39 (2005) 1332 – 1341

After transfer onto nitrocellulose, modified proteins were revealed by anti-dinitrophenol antibodies. Blots were developed with chemiluminescence using the SuperSignal West Pico chemiluminescent substrate (Perbio Sciences, Brebie`res, France). Films were scanned and the amount of signal was quantified by densitometric analysis using MultiGauge quantification software (Fujifilm). Cell culture and oxidative stress WI-38 SV40 fibroblasts stably transfected with either pCDNA3.1(+)/msrA or pCDNA3.1(+) were grown in 75cm2 plastic flasks (Greiner, VWR, Issy-les-Moulineaux, France) at 37-C, 5% CO2, and 95% humidity, in DMEM (Sigma– Aldrich) supplemented with 10% fetal calf serum (Sigma– Aldrich), 2 mM l-glutamine, 100 U/ml penicillin, 100 Ag/ml streptomycin, and 200 Ag/ml Geneticin. For culture at suboptimal levels of methionine, cells were grown in DMEM containing 0.2 AM methionine, which was determined as suboptimal concentration, for 10 days before the stress. For oxidative stress, cells were grown to 85% confluence, then rinsed twice with PBS (1.3 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.2), and subjected to various concentrations of H2O2 (250, 500, and 750 AM) in DMEM without red phenol. After a 2-h stress, cells were rinsed with PBS and supplemented with complete medium. UV irradiation was performed at various doses of UV-A with an emission centered at 365 nm using a Bio-Sun RMX 3W (Vilber-Lourmat, France) as UV source. Then, the cells were rinsed and incubated in complete medium for 24 h. Determination of oxidized methionine in cellular homogenates Protein-bound Met(O) level in cellular extracts was determined by the method previously described [11] with minor modifications. Briefly, 200 Ag of cellular proteins was dialyzed against water for 3 h at 4-C and subjected to 200 mM CNBr. After lyophilization, the dried hydrolysates were dissolved in water and subjected to 6 N HCl hydrolysis in sealed glass tubes for 20 h at 110-C in the presence of 5 mM DTT and a known amount of norleucine as internal standard. After HCl evaporation the samples were analyzed on a L-8800 Hitachi amino acids analyzer (classical postcolumn derivatization with ninhydrin after ion-exchange chromatography separation). Measurements of cell viability Fibroblasts were plated in a 96-well plate at 4000 cells/ well density and subjected to oxidative treatment as described above. Cell viability was assayed 24 h after the stress, either by the trypan blue assay, wherein cells were diluted in 0.1% trypan blue solution (1:1) and then noncolored cells were counted, or by measuring the

mitochondrial activity of living cells with the cell proliferation kit II (XTT) (Boehringer Mannheim, Mannheim, Germany). Measurement of reactive oxygen species The fluorescent dye dichlorodihydrofluorescein diacetate (DCF-DA) was used to measure the total ROS level in fibroblasts. Cells were grown to 85% of confluence and were treated with various amounts of H2O2 for 2 h. Just after stress, cells were stained for 30 min with 80 AM DCF-DA at 37-C in the dark. After trypsinization and centrifugation at 700g for 10 min at 4-C, the pellet was diluted in a FACS (fluorescence-activated cell sorter) flow solvent (Becton – Dickinson) and cells were excited with a 488 nm argon laser in a cytometry system (FACSTAR Plus, Becton – Dickinson). The emission fluorescence was measured at 530 nm and the average fluorescence intensity was obtained from at least 104 cells and expressed in arbitrary units. Determination of Msr activity Msr enzymatic activity was determined in cellular homogenates using N-acetyl-[3H]Met R,S (O) as substrate as previously described [21]. One hundred micrograms of crude protein cellular extracts was incubated at 37-C for 45 min. Statistical analysis All results are expressed as the mean T SEM. Comparisons were analyzed with the Student t test and were assumed to be statistically significant if p  0.05.

Results Overexpression of MsrA in WI-38 SV40 human fibroblasts and characterization of the C3 clone WI-38 SV40 cells were transfected with the pCDNA3.1(+)/ msrA plasmid for stable expression of MsrA. After selection with Geneticin, resistant clones were isolated and individually expanded. Overexpression of MsrA was verified by Western blotting using an anti-MsrA antibody. As shown in Fig. 1A, several clones produced high levels of MsrA compared with cells transfected with the empty vector pCDNA3.1(+) (Fig. 1A, lanes C1, C2, and C3). Clone 3, which presented a higher expression of MsrA, was selected for further analysis. Methionine sulfoxide reductase activity, measured using Nacetyl-[3H]Met R,S (O) as substrate, showed a 2.5-fold increase in the C3 clone compared to control cells (Fig. 1B). Localization of MsrA in transfected cells from the C3 clone was determined by indirect immunofluorescence experiments performed with anti-MsrA or anti-mitochondrial antibodies. Results confirmed the overexpression of the protein in both

C.R. Picot et al. / Free Radical Biology & Medicine 39 (2005) 1332 – 1341

1335

cellular compartments, cytosolic and mitochondrial (Fig. 2A). Overexpressed MsrA isoforms were analyzed by 2D gel electrophoresis followed by Western blotting experiments (Fig. 2B). Different molecular weight and pI isoforms were present in WI-38 SV40 cells that overexpressed the protein. These isoforms most likely resulted from alternative translation initiation followed by proteolytic processing upon targeting to the mitochondria, as well as oxidative modifications of certain amino acids such as cysteine residues, as previously reported for rat liver MsrA [6]. Overexpressed MsrA increased survival and decreased intracellular ROS content after H2O2 oxidative stress Fig. 1. Overexpression of MsrA in WI-38 SV40 human fibroblasts. (A) MsrA overexpression was verified by Western blot analysis. 10 Ag of total soluble protein extracts from WI-38 SV40 cells transfected with the empty plasmid (CC, control cells) or with the plasmid containing rat MsrA cDNA (clones 1, 2, and 3) was subjected to 12% SDS – PAGE, electrotransferred, and immunoblotted with polyclonal rabbit anti-MsrA antibody. (B) Msr activity in the C3 clone and in control cells was measured using N-acetyl[3H]Met R,S (O) as substrate at 37-C in a 30-Al final volume reaction mixture containing 100 Ag of total protein extract. The activity of the control cells was taken as 100% (*p < 0.05). Error bars represent the SEM of four independent experiments.

To determine whether MsrA can protect cells against death induced by H2O2, WI-38 SV40 fibroblasts overexpressing MsrA, as well as control cells transfected with the empty vector, were exposed for 2 h to increasing concentrations of H2O2. Cell survival was assayed 24 h after stress by trypan blue staining and results were confirmed using the XTT test (data not shown). As shown in Fig. 3A, MsrA-overexpressing cells were more resistant to H2O2 treatment at 250 AM, and this resistance was more significant when the concentrations of H2O2 were increased.

Fig. 2. Characterization of overexpressed MsrA. (A) Immunolocalization of overexpressed MsrA. Cells were fixed, permeabilized, and incubated with a rabbit anti-MsrA antibody and with FITC-conjugated donkey anti-rabbit IgG, and mitochondria were detected with a rhodamine-coupled secondary antibody and analyzed by fluorescence microscopy. (B) Detection of overexpressed MsrA isoforms was performed by 2D gel electrophoresis. 100 Ag of total protein extract was separated by 2D gel electrophoresis, transferred to a nitrocellulose membrane, and immunodetected with rabbit polyclonal anti-MsrA antibody. The first dimension was performed using ReadyStrip IPG strips (pH 3 – 10; 13 cm length, Bio-Rad) and the second dimension was a 12% SDS – PAGE. The gel shown is representative of three separate protein extracts.

1336

C.R. Picot et al. / Free Radical Biology & Medicine 39 (2005) 1332 – 1341

Fig. 3. Cellular viability after H2O2 treatment or UV stress. The filled bars represent results for control cells and open bars represent results for MsrAoverexpressing cells. Viability was measured 24 h after stress by counting trypan blue-excluding cells. (A) Cells were plated at a density of 400 cells cm 2 and stressed for 2 h with 250, 500, and 750 AM H2O2. (B) Cells were cultivated in suboptimal concentrations of methionine before H2O2 treatment as indicated above. (C) Cells were plated at a density of 80% confluence and treated with 2, 4, or 6 J cm 2 of UV-A.

Indeed, at 750 AM H2O2, a 30% difference in cell survival was found. To further address the possibility that the MsrA protection against H2O2-induced oxidative stress was relying on the catalytic removal of oxidant through reduction of methionine sulfoxide, cells were cultivated at suboptimal levels of methionine before being exposed to H2O2-induced oxidative stress. As shown in Fig. 3B, depriving cells of methionine resulted in partially abolishing the protective effect of overexpressed MsrA, especially at 750 AM H2O2, indicating that cellular methionine availability can be a critical factor in MsrA-mediated protection against H2O2-induced cellular death. In contrast to H2O2-induced oxidative stress, no difference in survival was observed between control cells and MsrA-overexpressing cells after exposure to various doses of UV-A irradiation (Fig. 3C). These results suggest that cellular protection against UV-A irradiation does not rely on either the repair role of MsrA or its antioxidant properties.

Because it has been proposed that methionine sulfoxide reductases would serve as antioxidant enzymes through the reduction of exposed methionine sulfoxide in proteins, the ROS level was measured in cells after H2O2 treatment. As shown in Fig. 4A, the level of ROS, monitored by the fluorescent dye DCF-DA, was lower in MsrA-overexpressing cells for all the concentrations of H2O2 investigated and significantly at 500 AM H2O2. We analyzed the expression level of p21cip1/waf1, which is known to be a modulator of apoptosis and a cytoprotective agent in most cellular systems [22]. Oxidative stress, obtained by H2O2 or hyperoxia, leads to an induction of p21 in a p53-dependent manner, as well as in an independent manner [23,24]. Results from Western blotting experiments on total proteins from control and MsrAoverexpressing WI-38 SV40 cells showed that expression of p21 was also induced when cells were subjected to H2O2 treatment. Interestingly, the basal level of p21 was 60% less abundant in MsrA-overexpressing cells and

C.R. Picot et al. / Free Radical Biology & Medicine 39 (2005) 1332 – 1341

1337

Fig. 4. Overexpression of MsrA lowers ROS production in WI-38 cells. (A) Quantification of flow-cytometric analysis of ROS production of control cells (filled bars) and MsrA-overexpressing cells (open bars), before and after treatment with various concentrations of H2O2. MsrA-overexpressing cells stained with DCF-DA and the fluorescence of at least 104 cells was measured by FACS. Error bars represent the SEM of three independent experiments. (B) Expression of p21 protein. 10 Ag of cellular protein extracts was separated by SDS – PAGE, then transferred to a nitrocellulose membrane, and immunoblotted with a monoclonal human anti-p21 antibody. The gel shown is representative of three separate protein extracts each, for control and MsrA-overexpressing cells.

remained lower after oxidative treatment compared to control cells (Fig. 4B). Overexpression of MsrA prevents accumulation of oxidatively modified proteins after oxidative stress induced by H2O2 Because the ROS level was decreased in MsrA-overexpressing cells upon treatment with H2O2, a decreased content of methionine sulfoxide on proteins could have been expected. Cellular protein-bound methionine sulfoxide level was analyzed in both control and MsrA-overexpressing cells, before and after treatment with H2O2. No significant difference was observed in levels of methionine sulfoxide among the various conditions. This level was always between 10 and 14%, expressed as a percentage of the total methionine. However, the extent of protein oxidative modification can also be evaluated by the determination of carbonyl adducts in cellular proteins. To investigate the status of oxidatively modified proteins in MsrA-overexpressing cells and control cells, we monitored protein carbonyl content in cellular homogenates using OxyBlot. As shown in Fig. 5A, lanes 1 and 5, a decrease in highmolecular-weight oxidatively modified proteins was observed in clone 3 compared to the control, and quantification analysis confirmed a twofold decrease in protein carbonyl content in cellular protein homogenates (Fig. 5B). Exposure of WI-38 SV40 cells to oxidative stress induced by addition of different concentrations of H2O2 for 2 h led to an accumulation of modified proteins, as shown in experiments reported in Figs. 5A and 5B. This accumulation of

oxidatively modified proteins was correlated with a decrease in Msr activity, as shown in Fig. 5C. We subjected MsrAoverexpressing WI-38 SV40 cells to the same oxidative treatment. Results of OxyBlot experiments and relative quantification of total carbonyl derivatives indicated that the carbonyl content also increased after treatment with increased concentrations of H2O2. However, this level was significantly lower in proteins prepared from MsrA-overexpressing cells compared to control cells. In order to correlate the observed protection against protein oxidative damage with overall Msr activity, the reduction in the synthetic substrate N-acetyl-[3H] Met R,S (O) to N-acetyl[3H]Met was monitored. We showed that the activity increased after H2O2 treatment at 250 AM and was maintained at a level close to the basal level after treatment with 750 AM H2O2. These results indicate that overexpression of MsrA in WI-38 SV40 cells can prevent the accumulation of protein oxidative damage after H2O2mediated oxidative stress and may contribute to maintenance of cellular redox homeostasis. We also analyzed the protein carbonyl content in cells treated with UV-A irradiation. Fig. 6 clearly reveals that oxidized proteins increased in the same way in MsrA-overexpressing cells as in control cells, indicating that MsrA does not protect against UV-Amediated protein oxidative damage.

Discussion In this study, we have shown that MsrA overexpression is able to protect immortalized WI-38 SV40 human

1338

C.R. Picot et al. / Free Radical Biology & Medicine 39 (2005) 1332 – 1341

Fig. 5. Protein carbonyl content and Msr activity upon H2O2 treatment. (A) Oxidized proteins were determined by detecting carbonyl content with OxyBlot in control cells (lanes 1 – 4 and filled bars in B) and in clone 3 (lanes 5 – 8 and open bars in B) under basal conditions (lanes 1 and 5) and oxidative conditions achieved by H2O2 at 250 AM (lanes 2 and 6), 500 AM (lanes 3 and 7), and 750 AM (lanes 4 and 8). (B) Quantification of the blots was performed using MultiGauge software from Fujifilm. (C) Msr activity was measured in cellular extracts of control cells (filled bars) and MsrA-overexpressing cells (open bars) as described under Materials and methods 24 h after a 2-h H2O2 treatment performed using 250, 500, and 750 AM H2O2. Error bars represent SEM of four independent experiments.

fibroblasts from protein oxidative damage and cell death by, at least in part, lowering the content of intracellular ROS. We had previously demonstrated that MsrA activity, as well as mRNA and protein expression, declined in rat organs such as brain, liver, and heart during aging [12]. More recently, we reported downregulation of MsrA and MsrB2 (CBS-1), one of the members of the MsrB family, during replicative senescence of WI-38 fibroblasts [13]. This decline in the peptide methionine sulfoxide reductase function was associated with an accumulation of protein oxidative damage. Other studies have reported the involvement of the Msr system in the longevity of organisms such as yeast [25], Drosophila [16], and mouse [17] and in protection against oxidative-stress-induced cell death in T lymphocytes [25] and neuronal PC12 [18] and lens cells [19]. In order to assess the role of Msr in protein oxidative damage protection, we stably transfected the cDNA of rat MsrA into the immortalized WI-38 SV40 fibroblast cell line. Biochemical studies and immunofluorescence analyses

showed that the MsrA recombinant protein is expressed in both the mitochondria and the cytosol of transfected cells, as for the endogenous protein [5,6]. Moreover, both pI and molecular weight isoforms of MsrA proteins were detected in 2D gel electrophoresis followed by Western blotting. According to previous results obtained with rat liver MsrA, in which a similar pattern of MsrA isoforms was observed, these molecular weight isoforms could correspond to mitochondrial and cytosolic MsrA [6]. The differences between these two forms were explained by alternative use of Met-1 and Met-21 as the translation initiation codon and subsequent processing of the mitochondrial protein [12]. The pI isoforms may have originated from an oxidative posttranslational process that likely affects the cysteine residues [6]. Although the physiological relevance of these different oxidatively modified forms remains to be clarified, it is tempting to hypothesize that certain isoforms are less active or even inactive, especially if the catalytic cysteine residue is affected. This may explain why the WI-38 SV40 clone 3, which presents the highest overexpression of MsrA,

C.R. Picot et al. / Free Radical Biology & Medicine 39 (2005) 1332 – 1341

1339

Fig. 6. Protein carbonyl content upon UV-A irradiation. (A) Oxidized proteins were determined by detecting carbonyl content with OxyBlot in control cells and in clone 3 under basal (lanes 0) and oxidative conditions achieved by UV-A irradiation. (B) Quantification of blots was performed for control cells (filled bars) and clone 3 (open bars) using MultiGauge software from Fujifilm. Error bars represent SEM of three experiments.

exhibits only a 2.5-fold increase in peptide methionine sulfoxide reductase activity. It should be also pointed out that the Msr activity reflects the overall activity of both MsrA and MsrB, whereas the increase in Msr activity in transfected cells is dependent only on MsrA overexpression, which emphasizes the actual increase. Nevertheless, a bigger (10-fold) increase was obtained for MsrA-overexpressing PC12 cells compared to control cells [18]. We observed, in WI-38 SV40 fibroblasts, that overexpression of MsrA does confer resistance against H2O2mediated oxidative stress, and in particular, almost complete prevention of accumulation of oxidative damage to proteins, as evidenced by protein-bound carbonyl detection, whereas no significant change in protein-bound methionine sulfoxide was observed. Because the proteasome has been implicated in oxidized protein degradation [26 – 28], we measured proteasome peptidase activities in cellular extracts (data not shown). No significant difference in any of the three peptidase activities was observed in MsrA-overexpressing cells compared to control cells, indicating that the decrease in the oxidized protein level was not due to activation of the proteasome. However, when transfected cells are challenged with H2O2, the overexpression of MsrA was found to reduce the intracellular level of ROS as monitored by using a ROSsensitive intracellular fluorescent dye. In addition, the lower induction of the cell cycle inhibitor p21 upon H2O2 treatment in MsrA-overexpressing cells may also be taken as an indication of a decreased level of intracellular ROS.

As proposed earlier by Levine et al. [11], MsrA could reduce the intracellular level of ROS by scavenging them through cyclic oxidation/reduction of exposed methionine in proteins. The observation that depriving the cells of methionine partially abolished the protective effect of MsrA against oxidative stress-induced death of the transfected cells provides additional support to this hypothesis. However, the sensitivity of cells under methionine deprivation was observed only with treatment at high concentrations of H2O2. Thus, the protective effect of MsrA seems to result, at least in part, from an antioxidant mechanism, hence preventing the appearance of the protein carbonyls. Indeed, other studies have pointed out the protective role of MsrA against the deleterious effects of ROS in Drosophila and mammalian cells, emphasizing the important role of this enzyme in both maintenance of proteins under oxidative stress and overall redox cellular homeostasis [16,18,25]. In contrast, MsrA / knockout yeast and mice were found to be less resistant to oxidative stress [17,25]. Moreover, as already demonstrated for cysteine oxidation products such as sulfenic and sulfinic acids within proteins [29 – 31], oxidized methionines may also be critical components in redox signaling. For example, sulfiredoxin and sestrins that repair cysteine-sulfinic acid in peroxiredoxins are likely important not only for their antioxidant function, but also in signaling pathways sensitive to peroxiredoxin hyperoxidation [32 – 34]. Similarly, MsrA could modulate signal

1340

C.R. Picot et al. / Free Radical Biology & Medicine 39 (2005) 1332 – 1341

transduction through the regulation of methionine oxidation/ reduction within specific proteins and MsrA overexpression would be expected to impact such redox-sensitive signaling pathways. Interestingly, no protection against cell death or oxidatively modified protein accumulation was observed when WI-38 SV40 fibroblasts were irradiated with UV-A. UV-A induces the activation of photosensitizers that generate ROS such as hydroxyl radicals and singlet oxygen, which can affect DNA, lipids, and proteins [35,36]. Within proteins, tryptophan, histidine, tyrosine, methionine, and cysteine residues have been proposed as potential sites for singlet oxygen-mediated damage. Our results reporting the absence of MsrA-mediated protection against UV-A irradiation suggest that methionine is not the preferential target of UV-A-induced protein modifications. Finally, in the absence of exogenous stress, although no significant difference in cellular growth ability could be detected, we observed a decrease in the protein oxidation content in transfected cells. Very recently, the role of MsrA was investigated in lens cell viability, because oxidative stress is a major factor involved in the formation of cataracts [19]. It has been demonstrated that MsrA is important for cell viability even in the absence of oxidative stress, also indicating a determining role for MsrA in basal cellular homeostasis. We had previously shown that MsrA and MsrB2 (CBS-1) are affected during senescence of fibroblasts [13]. The next step will be to elucidate the roles of the different members of the MsrB family and their interplay with MsrA during aging and resistance to oxidative stress.

Acknowledgments We thank F. Groh and Dr. F. Baleux for amino acids determination and N. Kassis for FACS analysis. This work was supported by funds from the MENRT (Universite´ Paris 7) and by LVMH – Christian Dior Parfums (Convention Cifre No. 724/2002).

References [1] Berlett, B. S.; Stadtman, E. R. Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272:20313 – 20316; 1997. [2] Sharov, V. S.; Ferrington, D. A.; Squier, T. C.; Schoneich, C. Diastereoselective reduction of protein-bound methionine sulfoxide by methionine sulfoxide reductase. FEBS Lett. 455:247 – 250; 1999. [3] Grimaud, R.; Ezraty, B.; Mitchell, J. K.; Lafitte, D.; Briand, C.; Derrick, P. J.; Barras, F. Repair of oxidized proteins: identification of a new methionine sulfoxide reductase. J. Biol. Chem. 276:48915–48920; 2001. [4] Moskovitz, J.; Jenkins, N. A.; Gilbert, D. J.; Copeland, N. G.; Jursky, F.; Weissbach, H.; Brot, N. Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues. Proc. Natl. Acad. Sci. USA 93:3205 – 3208; 1996.

[5] Hansel, A.; Kuschel, L.; Hehl, S.; Lemke, C.; Agricola, H. J.; Hoshi, T.; Heinemann, S. H. Mitochondrial targeting of the human peptide methionine sulfoxide reductase (MSRA), an enzyme involved in the repair of oxidized proteins. FASEB J. 16:911 – 913; 2002. [6] Vougier, S.; Mary, J.; Friguet, B. Subcellular localization of methionine sulphoxide reductase A (MsrA): evidence for mitochondrial and cytosolic isoforms in rat liver cells. Biochem. J. 373:531–537; 2003. [7] Kryukov, G. V.; Kumar, R. A.; Koc, A.; Sun, Z.; Gladyshev, V. N. Selenoprotein R is a zinc-containing stereo-specific methionine sulfoxide reductase. Proc. Natl. Acad. Sci. USA 99:4245 – 4250; 2002. [8] Jung, S.; Hansel, A.; Kasperczyk, H.; Hoshi, T.; Heinemann, S. H. Activity, tissue distribution and site-directed mutagenesis of a human peptide methionine sulfoxide reductase of type B: hCBS1. FEBS Lett. 527:91 – 94; 2002. [9] Kim, H. Y.; Gladyshev, V. N. Methionine sulfoxide reduction in mammals: characterization of methionine-R-sulfoxide reductases. Mol. Biol. Cell 15:1055 – 1064; 2004. [10] Kim, H. Y.; Gladyshev, V. N. Characterization of mouse endoplasmic reticulum methionine-R-sulfoxide reductase. Biochem. Biophys. Res. Commun. 320:1277 – 1283; 2004. [11] Levine, R. L.; Mosoni, L.; Berlett, B. S.; Stadtman, E. R. Methionine residues as endogenous antioxidants in proteins. Proc. Natl. Acad. Sci. USA 93:15036 – 15040; 1996. [12] Petropoulos, I.; Mary, J.; Perichon, M.; Friguet, B. Rat peptide methionine sulphoxide reductase: cloning of the cDNA, and downregulation of gene expression and enzyme activity during aging. Biochem. J. 355:819 – 825; 2001. [13] Picot, C. R.; Perichon, M.; Cintrat, J. C.; Friguet, B.; Petropoulos, I. The peptide methionine sulfoxide reductases, MsrA and MsrB (hCBS-1), are downregulated during replicative senescence of human WI-38 fibroblasts. FEBS Lett. 558:74 – 78; 2004. [14] Moskovitz, J.; Rahman, M. A.; Strassman, J.; Yancey, S. O.; Kushner, S. R.; Brot, N.; Weissbach, H. Escherichia coli peptide methionine sulfoxide reductase gene: regulation of expression and role in protecting against oxidative damage. J. Bacteriol. 177: 502 – 507; 1995. [15] Moskovitz, J.; Berlett, B. S.; Poston, J. M.; Stadtman, E. R. The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo. Proc. Natl. Acad. Sci. USA 94:9585 – 9589; 1997. [16] Ruan, H.; Tang, X. D.; Chen, M. L.; Joiner, M. L.; Sun, G.; Brot, N.; Weissbach, H.; Heinemann, S. H.; Iverson, L.; Wu, C. F.; Hoshi, T.; Joiner, M. A. High-quality life extension by the enzyme peptide methionine sulfoxide reductase. Proc. Natl. Acad. Sci. USA 99: 2748 – 2753; 2002. [17] Moskovitz, J.; Bar-Noy, S.; Williams, W. M.; Requena, J.; Berlett, B. S.; Stadtman, E. R. Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals. Proc. Natl. Acad. Sci. USA 98:12920 – 12925; 2001. [18] Yermolaieva, O.; Xu, R.; Schinstock, C.; Brot, N.; Weissbach, H.; Heinemann, S. H.; Hoshi, T. Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia/reoxygenation. Proc. Natl. Acad. Sci. USA 101:1159 – 1164; 2004. [19] Kantorow, M.; Hawse, J. R.; Cowell, T. L.; Benhamed, S.; Pizarro, G. O.; Reddy, V. N.; Hejtmancik, J. F. Methionine sulfoxide reductase A is important for lens cell viability and resistance to oxidative stress. Proc. Natl. Acad. Sci. USA 101:9654 – 9659; 2004. [20] Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680 – 685; 1970. [21] Brot, N.; Werth, J.; Koster, D.; Weissbach, H. Reduction of N-acetyl methionine sulfoxide: a simple assay for peptide methionine sulfoxide reductase. Anal. Biochem. 122:291 – 294; 1982. [22] Coqueret, O. New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol. 13:65 – 70; 2003.

C.R. Picot et al. / Free Radical Biology & Medicine 39 (2005) 1332 – 1341 [23] Kemp, T. J.; Causton, H. C.; Clerk, A. Changes in gene expression induced by H(2)O(2) in cardiac myocytes. Biochem. Biophys. Res. Commun. 307:416 – 421; 2003. [24] O’Reilly, M. A.; Staversky, R. J.; Watkins, R. H.; Reed, C. K.; de Mesy Jensen, K. L.; Finkelstein, J. N.; Keng, P. C. The cyclindependent kinase inhibitor p21 protects the lung from oxidative stress. Am. J. Respir. Cell Mol. Biol. 24:703 – 710; 2001. [25] Moskovitz, J.; Flescher, E.; Berlett, B. S.; Azare, J.; Poston, J. M.; Stadtman, E. R. Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress. Proc. Natl. Acad. Sci. USA 95:14071 – 14075; 1998. [26] Davies, K. J.; Goldberg, A. L. Proteins damaged by oxygen radicals are rapidly degraded in extracts of red blood cells. J. Biol. Chem. 262:8227 – 8234; 1987. [27] Friguet, B.; Bulteau, A. L.; Chondrogianni, N.; Conconi, M.; Petropoulos, I. Protein degradation by the proteasome and its implications in aging. Ann. N. Y. Acad. Sci. 908:143 – 154; 2000. [28] Davies, K. J. Degradation of oxidized proteins by the 20S proteasome. Biochimie 83:301 – 310; 2001.

1341

[29] Finkel, T. Redox-dependent signal transduction. FEBS Lett. 476: 52 – 54; 2000. [30] Finkel, T. Oxidant signals and oxidative stress. Curr. Opin. Cell Biol. 15:247 – 254; 2003. [31] Poole, L. B.; Karplus, P. A.; Claiborne, A. Protein sulfenic acids in redox signaling. Annu. Rev. Pharmacol. Toxicol. 44:325 – 347; 2004. [32] Woo, H. A.; Chae, H. Z.; Hwang, S. C.; Yang, K. S.; Kang, S. W.; Kim, K.; Rhee, S. G. Reversing the inactivation of peroxiredoxins caused by cysteine sulfinic acid formation. Science 300:653 – 656; 2003. [33] Biteau, B.; Labarre, J.; Toledano, M. B. ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin. Nature 425: 980 – 984; 2003. [34] Budanov, A. V.; Sablina, A. A.; Feinstein, E.; Koonin, E. V.; Chumakov, P. M. Regeneration of peroxiredoxins by p53-regulated sestrins, homologs of bacterial AhpD. Science 304:596 – 600; 2004. [35] Davies, M. J.; Truscott, R. J. Photo-oxidation of proteins and its role in cataractogenesis. J. Photochem. Photobiol. B 63:114 – 125; 2001. [36] Davies, M. J. Singlet oxygen-mediated damage to proteins and its consequences. Biochem. Biophys. Res. Commun. 305:761 – 770; 2003.