1 Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University , Kumamoto, Japan .
Antioxid Redox Signal. 2014 Jan 10;20(2):295-307. doi: 10.1089/ars.2012.4606. Epub 2012 Oct 31.
8-nitroguanosine 3',5'-cyclic monophosphate (8-Nitro-cGMP) is a nitrated derivative of cGMP that is formed via cross-talk of reactive oxygen species formed by NADPH oxidase 2 and mitochondria. This nitrated nucleotide can function as a unique electrophilic second messenger in regulation of redox signaling by inducing a post-translational modification of protein thiols via cGMP adduction (protein S-guanylation). With S-guanylation proteomics, we investigated endogenous mitochondrial protein S-guanylation.
We developed a new mass spectrometry (MS)-based proteomic method-S-guanylation proteomics-which comprised two approaches: (i) direct protein digestion followed by immunoaffinity capture of S-guanylated peptides that were subjected to liquid chromatography-tandem MS (LC-MS/MS); and (ii) two-dimensional (2D)-gel electrophoretic separation of S-guanylated proteins that were subjected to in-gel digestion, followed by LC-MS/MS. We thereby identified certain mitochondrial proteins that are S-guanylated endogenously during immunological stimulation, including mortalin and 60-kDa heat-shock protein (HSP60). Mortalin and HSP60 were recently reported to regulate mitochondrial permeability-transition pore (mPTP) opening, at least partly, by interacting with cyclophilin D, an mPTP component. Our data revealed that immunological stimulation and 8-nitro-cGMP treatment induced mPTP opening in a cyclophilin D-dependent manner.
Our S-guanylation proteomic method determined that mitochondrial HSPs may be novel targets for redox modification via protein S-guanylation that participates in mPTP regulation and mitochondrial redox signaling.
8-硝基鸟苷 3'，5'-环单磷酸（8-Nitro-cGMP）是 cGMP 的硝化衍生物，通过 NADPH 氧化酶 2 和线粒体形成的活性氧物质的串扰形成。这种硝化核苷酸可以作为一种独特的亲电第二信使，通过 cGMP 加合诱导蛋白质巯基的翻译后修饰（蛋白质 S-鸟苷化）来调节氧化还原信号。通过 S-鸟苷化蛋白质组学，我们研究了内源性线粒体蛋白质 S-鸟苷化。
我们开发了一种新的基于质谱（MS）的蛋白质组学方法-S-鸟苷化蛋白质组学，它包括两种方法：（i）直接蛋白质消化，然后通过免疫亲和捕获 S-鸟苷化肽，然后进行液相色谱-串联 MS（LC-MS/MS）；（ii）二维（2D）-凝胶电泳分离 S-鸟苷化蛋白质，然后进行胶内消化，然后进行 LC-MS/MS。因此，我们鉴定了某些在免疫刺激过程中内源性 S-鸟苷化的线粒体蛋白，包括 mortalin 和 60kDa 热休克蛋白（HSP60）。mortalin 和 HSP60 最近被报道通过与线粒体通透性转换孔（mPTP）组成部分 cyclophilin D 相互作用来调节 mPTP 的开放，至少部分是这样。我们的数据表明，免疫刺激和 8-Nitro-cGMP 处理以 cyclophilin D 依赖的方式诱导 mPTP 开放。
我们的 S-鸟苷化蛋白质组学方法确定线粒体 HSPs 可能是通过蛋白质 S-鸟苷化参与 mPTP 调节和线粒体氧化还原信号的新型氧化还原修饰靶标。
