Identification of nitrated tyrosine residues of protein kinase G-Ia by mass spectrometry.
Identification of nitrated tyrosine residues of protein kinase G-Ia by mass spectrometry.
复制标题
通过质谱法鉴定蛋白激酶 G-Ia 的硝化酪氨酸残基。
DOI：
10.1007/s00216-013-7535-4
复制
发表时间：
2014
影响因子：
4.3
通讯作者：
Ito S
中科院分区：
文献类型：
--
作者：
Lu J;Yao I;Shimojo M;Katano T;Uchida H;Setou M;Ito S
酪氨酸硝化为3-硝基酪氨酸是一氧化氮对酪氨酸的氧化修饰，与许多疾病相关，靶向蛋白激酶G（PKG）-I代表了肺动脉高压和慢性疼痛的潜在治疗策略。由于目前蛋白质组学方法的低灵敏度，PKG-I的酪氨酸残基的直接分配仍有待进行。为了确定PKG-I的酪氨酸修饰残基，采用过氧亚硝基阴离子硝化法对昆虫Sf9细胞表达的PKG-I α进行了体外硝化，并对胰蛋白酶消化后的片段进行了基质辅助激光解吸/电离-飞行时间质谱和液相色谱-串联质谱分析。在PKG-I α的21个酪氨酸残基中，16个酪氨酸残基被分配到13个片段中;在过氧亚硝酸盐处理的样品中，6个酪氨酸残基被硝化，即Y71、Y141、Y212、Y336、Y345和Y567。Y71、Y212和Y336处酪氨酸残基突变为苯丙氨酸显著降低PKG-I α的硝化作用; Y71、Y141、Y212和Y336处的4个突变（Y4F突变体）使PKG-I α的硝化作用叠加降低。在30 μ M至1 mM范围内，过氧亚硝酸盐以浓度依赖性方式抑制PKG-I α活性，并且这种抑制作用在Y4F突变体中减弱。这些结果表明PKG-I α在多个酪氨酸残基处被硝化，并且其活性因这些残基的硝化而降低。
The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-Iα expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization–time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-Iα, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-Iα; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-Iα activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 μM to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-Iα was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.