Original ContributionOverexpression of MsrA protects WI-38 SV40 human fibroblasts against H2O2-mediated oxidative stress
Section snippets
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 μg/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
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
Discussion
In this study, we have shown that MsrA overexpression is able to protect immortalized WI-38 SV40 human 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,
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 (Université Paris 7) and by LVMH–Christian Dior Parfums (Convention Cifre No. 724/2002).
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2014, Molecular Aspects of MedicineCitation Excerpt :Surface-exposed methionyl residues can be easily oxidized by almost all kinds of ROS and then further reduced by Msr. through successive oxidation/reduction reactions that are trapping ROS and preventing further protein irreversible oxidative modifications (Picot et al., 2005). Hence, in the presence of active Msr, methionyl residues appear as built-in protein antioxidants.