Proc. Natl. Acad. Sci. USA Vol. 95, pp. 14071–14075, November 1998 Biochemistry Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress JACKOB MOSKOVITZ* † ,ELIEZER FLESCHER ‡ ,BARBARA S. BERLETT*, JANEEN AZARE § , J. MICHAEL POSTON*, AND EARL R. STADTMAN* *Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20982-0342; ‡ Department of Human Microbiology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel; and § New York University Medical Center, Nelson Institute of Environmental Medicine, Long Meadow Road, Tuxedo, NY 10987 Contributed by Earl R. Stadtman, September 21, 1998 ABSTRACT The yeast peptide-methionine sulfoxide re- ductase (MsrA) was overexpressed in a Saccharomyces cerevi- siae 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 treat- ment, 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 methi- onine 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. Ultra- violet A radiation did not result in obvious differences in survival rates among the three strains. An enhanced resis- tance 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 per- oxide. These results support the proposition that the msrA gene is involved in the resistance of yeast and mammalian cells to oxidative stress. Oxidation of methionine to methionine sulfoxide can occur in the presence of various oxidants, such as hydrogen peroxide, hypochlorite, peroxynitrite, hydroxyl radicals, and ozone, as well as metal-catalyzed oxidation systems. Most, if not all, cells contain methionine sulfoxide reductases (MsrA) that catalyze the thioredoxin-dependent reduction of protein methionine sulfoxide (MetO) residues back to Met. The oxidation of methionine residues of some proteins may lead to either activation or inactivation of their biological activities (1–3), whereas the oxidation of one or more methionine residues in other proteins may have little or no effect on biological functions (4). This idea has led to the suggestion that MsrA may have multiple biological functions: (i) It may serve to repair oxidative protein damage in some proteins. (ii) It may play an important role in the regulation of enzyme (protein) activities by facilitating the interconversion of specific methi- onine residues of these proteins between oxidized and reduced forms [as occurs, for example, in regulating the ability of bacteria to adhere to prokaryotic cells (5)], in the regulation of various plasma proteinase activities and hormones [re- viewed by Vogt (3), Brot and Weissbach (6), and Swaim and Pizzo (7)] in the calmodulin-dependent activation of plasma membrane Ca-ATPase (8), and in the modulation of potas- sium channel function (9). (iii) MsrA might also serve as an antioxidant enzyme to protect some enzymes from oxidative damage by various reactive oxygen species (4). A yeast null mutant of msrA was shown to accumulate both free and protein-bound MetO, and its growth was found to be more severely inhibited by H 2 O 2 treatment than was that of the wild-type parent strain (2). A null mutant of msrA in Esche- richia coli was also more susceptible to being killed by hydro- gen peroxide than its parent and msrA overproducing strains, but only when grown on plates; however, these E. coli strains were shown to be equally sensitive to methyl viologen (para- quat) treatment (10). E. coli and S. cerevisiae both contain at least two methionine sulfoxide reductases. One (msrA) is able to reduce both free and protein-bound MetO, and the other can reduce only free MetO. The peptide-methionine sulfoxide reductase was highly expressed in macrophages (11) and neutrophils (12). The high level of msrA may reflect the need to repair oxidatively damaged Met residues of endogenous proteins during periods of enhanced oxidative stress associated with oxidative burst activity. It has been shown that preexposure to oxidative stress suppresses the ability of T lymphocytes to respond to mito- genic stimulation in terms of proliferation, interleukin 2 biosynthesis, and signal transduction and that N-acetyl cysteine protected them against the stress (13–15). In the present study, we determine the localization of the promoter for the msrA gene in S. cerevisiae and show that overexpression of MsrA in yeast and in human T lymphocytes leads to a decrease in the cellular levels of both free and protein-bound MetO and to enhanced survival of these cells under conditions of oxidative stress. MATERIALS AND METHODS Materials. Hydrogen peroxide was purchased from Fisher. Dabsyl chloride was purchased from Pierce. Methyl viologen (paraquat) was purchased from Sigma. The compound 2,2'- azobis-(2-amidinopropane) dihydrochloride (AAPH), was purchased from Wako Chemicals USA (Richmond, VA). G418 sulfate, a generic form of Geneticin, was purchased from GIBCOBRL. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424989514071-5$2.000 PNAS is available online at www.pnas.org. Abbreviations: UVA, ultraviolet A radiation; MetO, methionine sulfoxide; MsrA, peptide-methionine sulfoxide reductase enzyme; AAPH, 2,2'-azobis-(2-amidinopropane) dihydrochloride; Molt-4, hu- man T lymphocyte cells; yeast strain msrA::URA3, yeast null mutant of peptide-methionine sulfoxide reductase gene. † To whom reprint requests should be addressed at Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 3, Room 207, Bethesda, MD 20982- 0342. e-mail: jmosko@helix.nih.gov. 14071