Reversible inactivation of yeast mitochondrial phenylalanyl-tRNA synthetase under oxidative stress
Reversible inactivation of yeast mitochondrial phenylalanyl-tRNA synthetase under oxidative stress
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氧化应激下酵母线粒体苯丙氨酰-tRNA合成酶的可逆失活
DOI：
10.1016/j.bbagen.2018.04.023
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Banerjee, Rajat
中科院分区：
文献类型：
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作者：
Chakraborty, Shruti;Ganguli, Sayak;Chowdhury, Aritra;Ibba, Michael;Banerjee, Rajat
研究背景在氧化胁迫下，胞质氨酰-tRNA合成酶（aaRSs）底物特异性受到损害，导致tRNA错装和蛋白质组的错误翻译。类似的过程是否发生在线粒体中，这是活性氧（ROS）的主要细胞来源，是未知的。然而，放宽了酵母线粒体苯丙氨酰-tRNA合成酶的底物特异性，方法非还原变性PAGE，半胱氨酸反应性研究，MALDI-TOF质谱，酶测定，蛋白质印迹，生长测定，圆二色性，用动态光散射和荧光光谱法研究了氧化应激对ScmitPheRS活性的影响。氧化应激氧化失活的靶点是两个保守的半胱氨酸残基，导致可逆的分子内二硫键形成。替换任何保守的半胱氨酸残基增加的活力在氧化stress. ConclusionFormationofintramolecular disulfide桥氧化应激下的生长过程中阻碍了tRNAPhe结合的酶，从而inactivatingScmitPheRSreversibly. GeneralsignificanceTheScmitPheRS活动是在氧化应激下，由于形成的分子内二硫键妥协。ScmitPheRS对氧化的敏感性可能为氧化应激下的易错翻译提供保护机制。
BackgroundUnder oxidative stress cytoplasmic aminoacyl-tRNA synthetase (aaRSs) substrate specificity can be compromised, leading to tRNA mischarging and mistranslation of the proteome. Whether similar processes occur in mitochondria, which are major cellular sources of reactive oxygen species (ROS), is unknown. However, relaxed substrate specificity in yeast mitochondrial phenylalanyl-tRNA synthetase (ScmitPheRS) has been reported to increase tRNA mischarging and blocks mitochondrial biogenesis.MethodsNon-reducing denaturing PAGE, cysteine reactivity studies, MALDI-TOF mass spectrometry, enzyme assay, western blot, growth assay, circular dichroism, dynamic light scattering and fluorescence spectroscopy were used to study the effect of oxidative stress onScmitPheRS activity.ResultsScmitPheRS is reversibly inactivated under oxidative stress. The targets for oxidative inactivation are two conserved cysteine residues resulting in reversible intra-molecular disulfide bridge formation. Replacement of either conserved cysteine residue increased viability during growth under oxidative stress.ConclusionFormation of intra-molecular disulfide bridge under oxidative stress hinders the tRNAPhebinding of the enzyme, thus inactivatingScmitPheRS reversibly.General significanceTheScmitPheRS activity is compromised under oxidative stress due to formation of intra-molecular disulfide bridge. The sensitivity ofScmitPheRS to oxidation may provide a protective mechanism against error-prone translation under oxidative stress.