Subcellular localization of methionine sulphoxide reductase A (MsrA): evidence for mitochondrial and cytosolic isoforms in rat liver cells
Subcellular localization of methionine sulphoxide reductase A (MsrA): evidence for mitochondrial and cytosolic isoforms in rat liver cells
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DOI：
10.1042/bj20030443
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发表时间：
2003-07-15
影响因子：
4.1
通讯作者：
Friguet, B
中科院分区：
文献类型：
--
作者：
Vougier, S;Mary, J;Friguet, B
蛋白质对活性氧物种很敏感，氧化蛋白质的积累与衰老过程和其他与年龄相关的病理过程有关。在蛋白质中，蛋氨酸残基对氧化特别敏感，导致S和R-蛋氨酸亚硫氧化物非对映异构体，它们的逆转分别通过蛋氨酸亚硫酸盐还原酶MSRA和MSRB实现。MSRA酶除了在修复中发挥作用外，还构成了在细胞抗氧化防御中重要的活性氧清除系统的一部分。MSRA存在于大多数活着的生物体中，哺乳动物的酶在所有被研究的组织中都被检测到。在本研究中，我们研究了MSRA在大鼠肝细胞中的亚细胞分布。由于MSRA可能定位于产生活性氧的区域，因此制备了大鼠肝脏线粒体基质和胞浆提取物。通过监测多肽蛋氨酸亚硫氧化物还原酶的活性、Western blotting和使用特定抗体的电子显微镜原位免疫定位来检测MSRA在这些亚细胞中的存在。此外，MS还在部分纯化的胞浆部分和线粒体基质粗提物中鉴定了MSRA。大鼠MSRA亚型由单个基因编码，推测线粒体形式的前体含有一个N端可切割的信号序列，该序列将MSRA定位于该细胞器。最后，对胞浆和线粒体中部分纯化的MSRA进行了双向凝胶电泳和Western-Blot分析，并与氧化重组MSRA的二维图谱进行了比较，发现半胱氨酸残基被氧化修饰。
Proteins are sensitive to reactive oxygen species, and the accumulation of oxidized proteins has been implicated in the aging process and in other age-related pathologies. In proteins, methionine residues are especially sensitive to oxidation, leading to S- and R-methionine sulphoxide diastereoisomers, the reversion of which is achieved by the peptide methionine sulphoxide reductases MsrA and MsrB respectively. The MsrA enzyme, in addition to its role in repair, forms part of the reactive oxygen species scavenging systems that are important in cellular antioxidant defence. MsrA is present in most living organisms, and the mammalian enzyme has been detected in all tissues investigated. In the present study, we investigated the subcellular distribution of MsrA in rat liver cells. Since it seemed likely that MsrA may be localized in areas where reactive oxygen species are produced, rat liver mitochondrial matrix and cytosolic extracts were prepared. The presence of MsrA was assayed in these subcellular compartments by monitoring peptide methionine sulphoxide reductase enzymic activity, by Western blotting and by in situ immunolocalization by electron microscopy using a specific antibody. Moreover, MsrA was identified by MS in a partially purified cytosolic fraction and in a mitochondrial matrix crude extract. Rat MsrA isoforms are encoded by a single gene, and it is suggested that the precursor of the mitochondrial form contains an N-terminal cleavable signal sequence that localizes the MsrA to this organelle. Finally, two-dimensional gel electrophoresis followed by Western-blot analysis of partially purified MsrA from the cytosol and mitochondria, and comparison with the two-dimensional patterns of oxidized recombinant MsrA, revealed oxidative modifications of cysteine residues.