Transgenic mice overexpressing methionine sulfoxide reductase A: Characterization of embryonic fibroblasts

Methionine residues in protein can be oxidized by reactive oxygen species to generate methionine sulfoxide. Aerobic organisms have methionine sulfoxide reductases capable of reducing methionine sulfoxide back to methionine. Methionine sulfoxide reductase A acts on the S-epimer of methionine sulfoxide, and it is known that altering its cellular level by genetic ablation or overexpression has notable effects on resistance to oxidative stress and on life span in species from microorganisms to animals. In mammals, the enzyme is present in both the cytosol and the mitochondria, and this study was undertaken to assess the contribution of each subcellular compartment's reductase activity to resistance against oxidative stresses. Nontransgenic mouse embryonic fibroblasts lack methionine sulfoxide reductase A activity, providing a convenient cell type to determine the effects of expression of the enzyme in each compartment. We created transgenic mice with methionine sulfoxide reductase A targeted to the cytosol, mitochondria, or both and studied embryonic fibroblasts derived from each line. Unexpectedly, none of the transgenic cells gained resistance to a variety of oxidative stresses even though the expressed enzymes were catalytically active when assayed in vitro. Noting that activity in vivo requires thioredoxin and thioredoxin reductase, we determined the levels of these proteins in the fibroblasts and found that they were very low in both the nontransgenic and the transgenic cells. We conclude that overexpression of methionine sulfoxide reductase A did not confer resistance to oxidative stress because the cells lacked other proteins required to constitute a functional methionine sulfoxide reduction system.