Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.
Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.
复制标题
DOI：
10.1074/jbc.m117.798744
复制
发表时间：
2017-09-01
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;James AM;Methner C;Pell VR;Prime TA;Erickson BK;Forkink M;Lau GY;Bright TP;Menger KE;Fearnley IM;Krieg T;Murphy MP
硝酸盐（NO3−）和亚硝酸盐（NO2−）是已知的心脏保护和改变体内能量代谢。NO3−的作用是由唾液细菌将其转化为NO2−产生的，但NO2−影响代谢的机制尚不清楚。NO2−可以通过s -亚硝化蛋白质硫醇起作用，从而改变蛋白质活性。但这是如何发生的，以及s -亚硝化位点在哺乳动物蛋白质组中的功能重要性，在很大程度上仍未被描述。在这里，我们使用定量蛋白质组学分析了小鼠心脏内的蛋白质硫醇，以确定s -亚硝化位点的占用。我们扩展了硫醇-氧化还原蛋白质组学技术，同位素编码亲和标签标记，以量化体内NO2−依赖的蛋白质硫醇s -亚硝化的程度。使用这种称为SNOxICAT （s -亚硝基硫醇氧化还原同位素编码亲和标签）的方法，我们发现在常温条件下暴露于NO2−或暴露于缺血单独导致蛋白质硫醇的最小s -亚硝化。然而，暴露于NO2−与缺血一起导致所有细胞区室中蛋白质硫醇的广泛s -亚硝化。在这些条件下，暴露于线粒体基质的几种线粒体蛋白硫醇被选择性地s -亚硝化，可能有助于NO2−对线粒体代谢的有益影响。线粒体内膜对HNO2的渗透性，而对NO2 -的渗透性，再加上缺氧或缺氧时缺乏s -亚硝化，限制了s -亚硝化在体内的发生方式及其对心脏保护和能量代谢调节的机制。现在，定量s -亚硝化蛋白硫醇可以测定蛋白质组中修饰的半胱氨酸，并鉴定那些最有可能对NO2−暴露的功能后果负责的半胱氨酸。
Nitrate (NO3−) and nitrite (NO2−) are known to be cardioprotective and to alter energy metabolism in vivo. NO3− action results from its conversion to NO2− by salivary bacteria, but the mechanism(s) by which NO2− affects metabolism remains obscure. NO2− may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2−-dependent S-nitrosation of proteins thiols in vivo. Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2− under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2− in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2− on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2−, combined with the lack of S-nitrosation during anoxia alone or by NO2− during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2− exposure.