8-Nitro-cGMP Enhances SNARE Complex Formation through S-Guanylation of Cys90 in SNAP25
8-Nitro-cGMP Enhances SNARE Complex Formation through S-Guanylation of Cys90 in SNAP25
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DOI：
10.1021/acschemneuro.5b00196
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发表时间：
2015-10-01
影响因子：
5
通讯作者：
Ihara, Hideshi
中科院分区：
文献类型：
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作者：
Kunieda, Kohei;Tsutsuki, Hiroyasu;Ihara, Hideshi
硝化鸟嘌呤核苷酸8-硝基鸟嘌呤3′，5′-环单磷酸（8-硝基- cgmp）由活性氧/氮种产生，引起蛋白质s -鸟嘌呤基化。然而，8-硝基- cgmp在正常大脑中的形成机制及其蛋白靶点尚未确定。在这里，我们研究了8-硝基cgmp在啮齿动物大脑中的生成和蛋白质s -鸟苷化。免疫组织化学表明，8-硝基cgmp是由锥体细胞和中间神经元等神经元产生的。采用液相色谱-串联质谱法测定脑内8-硝基cgmp水平为2.92 +/- 0.10 pmol/mg蛋白。基于s -鸟酰化蛋白质组学，我们鉴定了几种s -鸟酰化神经元蛋白，包括可溶性n -乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）复合物的核心成员SNAP25。SNAP25翻译后修饰，包括棕榈酰化、磷酸化和氧化，已知可调节神经传递。我们的研究结果表明，SNAP25的s -鸟苷化增强了SNARE复合物的稳定性，而神经元一氧化氮合酶的Ca2+依赖性激活进一步促进了SNARE复合物的稳定性。通过定点诱变，我们发现SNAP25半胱氨酸90是s -胍基化的主要靶点，这增强了SNARE复合物的稳定性。本研究揭示了神经系统中蛋白质s -鸟苷化氧化还原信号的新靶点，并首次提供了8-硝基- cgmp在神经系统中的功能的实质性证据。
Nitrated guanine nucleotide 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) generated by reactive oxygen/nitrogen species causes protein S-guanylation. However, the mechanism of 8-nitro-cGMP formation and its protein targets in the normal brain have not been identified. Here, we investigated 8-nitro-cGMP generation and protein S-guanylation in the rodent brain. Immunohistochemistry indicated that 8-nitro-cGMP was produced by neurons, such as pyramidal cells and interneurons. Using liquid chromatography-tandem mass spectrometry, we determined endogenous 8-nitro-cGMP levels in the brain as 2.92 +/- 0.10 pmol/mg protein. Based on S-guanylation proteomics, we identified several S-guanylated neuronal proteins, including SNAP25 which is a core member of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex. SNAP25 post-translational modification including palmitoylation, phosphorylation, and oxidation, are known to regulate neurotransmission. Our results demonstrate that S-guanylation of SNAP25 enhanced the stability of the SNARE complex, which was further promoted by Ca2+-dependent activation of neuronal nitric oxide synthase. Using site-directed mutagenesis, we identified SNAP25 cysteine 90 as the main target of S-guanylation which enhanced the stability of the SNARE complex. The present study revealed a novel target of redox signaling via protein S-guanylation in the nervous system and provided the first substantial evidence of 8-nitro-cGMP function in the nervous system.