Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress.
The yeast peptide-methionine sulfoxide reductase (MsrA) was overexpressed in a Saccharomyces cerevisiae null mutant of msrA by using a high-copy plasmid harboring the msrA gene and its promoter. The resulting strain had about 25-fold higher MsrA activity than its parent strain. When exposed to either hydrogen peroxide, paraquat, or 2,2'-azobis-(2-amidinopropane) dihydrochloride treatment, the MsrA overexpressed strain grew better, had lower free and protein-bound methionine sulfoxide and had a better survival rate under these conditions than did the msrA mutant and its parent strain. Substitution of methionine with methionine sulfoxide in a medium lacking hydrogen peroxide had little effect on the growth pattern, which suggests that the oxidation of free methionine in the growth medium was not the main cause of growth inhibition of the msrA mutant. Ultraviolet A radiation did not result in obvious differences in survival rates among the three strains. An enhanced resistance to hydrogen peroxide treatment was shown in human T lymphocyte cells (Molt-4) that were stably transfected with the bovine msrA and exposed to hydrogen peroxide. The survival rate of the transfected strain was much better than its parent strain when grown in the presence of hydrogen peroxide. These results support the proposition that the msrA gene is involved in the resistance of yeast and mammalian cells to oxidative stress.
Moskovitz
J
J
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20982-0342, USA. jmosko@helix.nih.gov
Flescher
E
E
Berlett
B S
BS
Azare
J
J
Poston
J M
JM
Stadtman
E R
ER
D000578:Amidines / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051150:Methionine Sulfoxide Reductases; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000502:physiology*; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D010269:Paraquat / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D013601:T-Lymphocytes; D014162:Transfection
D000578:Amidines; D016877:Oxidants; D011994:Recombinant Proteins; C046728:2,2'-azobis(2-amidinopropane); D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase; D010269:Paraquat