Protein tyrosine nitration of mitochondrial carbamoyl phosphate synthetase 1 and its functional consequences
Protein tyrosine nitration of mitochondrial carbamoyl phosphate synthetase 1 and its functional consequences
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DOI：
10.1016/j.bbrc.2012.02.114
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发表时间：
2012-03-30
影响因子：
3.1
通讯作者：
Nelson, Sidney D.
中科院分区：
文献类型：
--
作者：
Takakusa, Hideo;Mohar, Isaac;Nelson, Sidney D.
线粒体是产生活性氮物质（包括过氧亚硝酸盐）和随后的蛋白质酪氨酸硝化的主要场所。蛋白质酪氨酸硝化可能具有重要的功能和生物学后果，例如酶催化活性的改变。在本研究中，将小鼠肝脏线粒体与过氧亚硝酸盐一起孵育，并通过一维和二维凝胶电泳分离线粒体蛋白。用抗硝基酪氨酸抗体检测硝基酪氨酸化蛋白质。通过 LC-MS 蛋白质分析和蛋白质印迹鉴定，过氧亚硝酸盐硝化的主要蛋白质之一是氨基甲酰磷酸合成酶 1 (CPS1)。标准化为 CPS1 的硝化带强度以过氧亚硝酸盐浓度依赖性方式增加。此外，CPS1 活性通过过氧亚硝酸盐处理以过氧亚硝酸盐浓度和时间依赖性方式降低。还原型谷胱甘肽处理并未恢复降低的 CPS1 活性，这表明 CPS1 活性的降低是由于酪氨酸硝化而不是半胱氨酸氧化所致。对凝胶内消化样品进行 LC-MS 分析，以及基于 Popitam 的修饰搜索，定位到 CPS1 中 36 个酪氨酸残基中的 5 个被硝化。结合之前有关 CPS1 结构和功能的发现，小鼠 CPS1 的同源建模表明，变构域 a 螺旋中 Y1450 处的硝化可防止 CPS1 被其激活剂 N-乙酰基-L-谷氨酸激活。总之，本研究证明了过氧亚硝酸盐对 CPS1 的酪氨酸硝化及其功能后果。由于CPS1负责尿素循环中的氨去除，功能减弱的CPS1硝化可能与一些与线粒体相关的疾病和药物引起的毒性有关！功能障碍。 (C) 2012 Elsevier Inc. 保留所有权利。
Mitochondria are the primary locus for the generation of reactive nitrogen species including peroxynitrite and subsequent protein tyrosine nitration. Protein tyrosine nitration may have important functional and biological consequences such as alteration of enzyme catalytic activity. In the present study, mouse liver mitochondria were incubated with peroxynitrite, and the mitochondrial proteins were separated by 1D and 2D gel electrophoresis. Nitrotyrosinylated proteins were detected with an anti-nitrotyrosine antibody. One of the major proteins nitrated by peroxynitrite was carbamoyl phosphate synthetase 1 (CPS1) as identified by LC-MS protein analysis and Western blotting. The band intensity of nitration normalized to CPS1 was increased in a peroxynitrite concentration-dependent manner. In addition, CPS1 activity was decreased by treatment with peroxynitrite in a peroxynitrite concentration- and time-dependent manner. The decreased CPS1 activity was not recovered by treatment with reduced glutathione, suggesting that the decrease of the CPS1 activity is due to tyrosine nitration rather than cysteine oxidation. LC-MS analysis of in-gel digested samples, and a Popitam-based modification search located 5 out of 36 tyrosine residues in CPS1 that were nitrated. Taken together with previous findings regarding CPS1 structure and function, homology modeling of mouse CPS1 suggested that nitration at Y1450 in an a-helix of allosteric domain prevents activation of CPS1 by its activator, N-acetyl-L-glutamate. In conclusion, this study demonstrated the tyrosine nitration of CPS1 by peroxynitrite and its functional consequence. Since CPS1 is responsible for ammonia removal in the urea cycle, nitration of CPS1 with attenuated function might be involved in some diseases and drug-induced toxicities associated with mitochondria! dysfunction. (C) 2012 Elsevier Inc. All rights reserved.