Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation.
Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation.
复制标题
DOI：
10.1042/bj20100633
复制
发表时间：
2010-08-15
期刊：
影响因子：
--
通讯作者：
Murphy MP
中科院分区：
文献类型：
--
作者：
Chouchani ET;Hurd TR;Nadtochiy SM;Brookes PS;Fearnley IM;Lilley KS;Smith RA;Murphy MP
线粒体蛋白质的S亚硝化是NO代谢的结果，具有生理和病理意义。我们之前开发了一种线粒体靶向S亚硝硫醇(MitoSNO)，它可以选择性地使S亚硝化线粒体蛋白。为了鉴定这些S亚硝化的蛋白质，我们发展了一种选择性蛋白质组学方法，SNO-DIGE(S-亚硝硫醇差异凝胶电泳法)。阻断对照和MitoSnO处理样品中的蛋白质硫醇，然后与铜(II)和抗坏血酸孵育，选择性地还原S-亚硝硫醇。然后用硫醇反应性的Cy3(吲哚碳菁)或Cy5(吲哚二碳菁)荧光标记处理样品，混合在一起，用2D(二维)凝胶电泳法分辨单独的蛋白质斑点。对这些凝胶的荧光扫描显示，S亚硝化的蛋白质通过Cy5红色荧光的增加而被发现，这使得他们能够用MS进行鉴定。氧化还原-DGE平行分析使我们能够区分S亚硝化的硫醇蛋白质和那些由于没有代谢而被氧化的蛋白质。我们鉴定了13个S亚硝化的线粒体蛋白质，以及另外4个被氧化的蛋白质，可能是由于短暂的S亚硝化松弛到可逆的硫醇修饰所致。我们研究了S亚硝化对用SnO-DGE鉴定的三种酶(乌头酸酶、线粒体乙醛脱氢酶和α-酮戊二酸脱氢酶)的影响，发现S亚硝化对它们的活性有选择性和可逆性的抑制作用。我们的结论是，S亚硝化对酶活性的可逆调节修饰了线粒体代谢中心的酶，而对这些新靶点的鉴定和功能表征提供了对这种修饰所发挥的潜在生理和病理作用的机械性洞察。更广泛地说，SNO-DGE的发展促进了蛋白质S亚硝化跨蛋白质组的强有力的研究。
The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and α-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome.