Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Free Radic Biol Med. 2010 Aug 15;49(4):641-8. doi: 10.1016/j.freeradbiomed.2010.05.017. Epub 2010 May 25.
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.
蛋白质中的蛋氨酸残基可被活性氧氧化，生成蛋氨酸亚砜。需氧生物具有能够将蛋氨酸亚砜还原为蛋氨酸的蛋氨酸亚砜还原酶。蛋氨酸亚砜还原酶 A 作用于蛋氨酸亚砜的 S-差向异构体，已知通过基因缺失或过表达改变其细胞水平对抵抗氧化应激和延长微生物到动物等物种的寿命有显著影响。在哺乳动物中，该酶存在于细胞质和线粒体中，本研究旨在评估每个亚细胞隔室的还原酶活性对抵抗氧化应激的贡献。非转基因小鼠胚胎成纤维细胞缺乏蛋氨酸亚砜还原酶 A 活性，为确定每个隔室中酶的表达对细胞的影响提供了便利的细胞类型。我们创建了将蛋氨酸亚砜还原酶 A 靶向细胞质、线粒体或两者的转基因小鼠，并研究了来自每条线的胚胎成纤维细胞。出乎意料的是，尽管在体外测定时表达的酶具有催化活性，但没有一种转基因细胞获得对各种氧化应激的抗性。鉴于体内活性需要硫氧还蛋白和硫氧还蛋白还原酶，我们测定了成纤维细胞中的这些蛋白水平，发现非转基因和转基因细胞中的这些蛋白水平都非常低。我们得出结论，蛋氨酸亚砜还原酶 A 的过表达并未赋予细胞对氧化应激的抗性，因为细胞缺乏构成功能性蛋氨酸亚砜还原系统所需的其他蛋白。
