Nitric oxide inhibits myocardial apoptosis by preventing caspase-3 activity via S-nitrosylation
Nitric oxide inhibits myocardial apoptosis by preventing caspase-3 activity via S-nitrosylation
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DOI：
10.1016/j.yjmcc.2004.10.012
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发表时间：
2005-01-01
影响因子：
5
通讯作者：
Isobe, M
中科院分区：
文献类型：
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作者：
Maejima, Y;Adachi, S;Isobe, M
磷酸化和S-亚硝基化两种蛋白质信号系统影响细胞生理学的大多数方面。在半胱氨酸残基上产生亚硝基硫醇键的S-亚硝基化由一氧化氮（NO）引起。NO被认为是通过抑制心肌细胞中的半胱天冬酶活性而起抗凋亡剂的作用，但对此几乎没有直接证据。我们研究了在阿霉素（DOX）诱导的心肌细胞凋亡中，NO对凋亡的抑制是否涉及半胱天冬酶的S-亚硝基化。用1 μ mol/mol DOX处理心肌细胞以诱导凋亡。用NO供体S-亚硝基-N-乙酰青霉胺（SNAP）预处理可减少细胞凋亡。用100 μ mol/l的二氯化汞（HgCl 2）（一种脱亚硝基化试剂）处理可减弱这种效应。DOX处理24小时后，SNAP降低了63%的caspase-3活性，这种作用被HgCl 2处理逆转。免疫印迹分析表明，裂解的caspase-3蛋白，一种活性形式，诱导细胞凋亡的积累被抑制SNAP显着。为了阐明NO对caspase-3的亚硝基硫醇形成，我们做了几个实验。先我们制备了胱天蛋白酶-3的免疫沉淀物，并测量了HgCl从沉淀复合物中释放的NO浓度。一下S-亚硝基化的蛋白质，通过免疫沉淀的半胱天冬酶-3纯化，生物素化和生物素浓度通过免疫印迹法估计。三分之用S-亚硝基半胱氨酸和半胱天冬酶-3的抗体进行双重免疫荧光染色。结果显示，与未处理的心肌细胞相比，在DOX处理的心肌细胞中，与SNAP相比，caspase-3中亚硝基硫醇的形成显著增加。我们在这里报道，外源性NO产生的抗凋亡作用，通过抑制半胱天冬酶活性，通过S-亚硝基化心肌细胞。(C)2004爱思唯尔有限公司保留所有权利。
Two protein signaling systems, phosphorylation and S-nitrosylation, influence most aspects of cellular physiology. S-nitrosylation, which generates a nitrosothiol linkage on cysteine residues, is caused by nitric oxide (NO). NO is believed to act as an anti-apoptotic agent by inhibiting caspase activity in cardiomyocytes, but there is little direct evidence for this. We investigated whether apoptosis inhibition by NO involved S-nitrosylation of caspases in doxorubicin (DOX)-induced myocardial apoptosis. Cardiomyocytes were treated with 1 mumol/mol of DOX to induce apoptosis. Pretreatment with an NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) reduced the apoptosis. This effect was attenuated by treatment with 100 mumol/l of mercury dichloride (HgCl2), which is an agent of denitrosylation. After 24 h DOX-treatment, SNAP reduced the increased caspase-3 activity by 63%, and this effect was reversed by treatment with HgCl2. Immunoblot analysis showed that accumulation of the cleaved caspase-3 protein, an active form that induces apoptosis was inhibited significantly by SNAP. To elucidate nitrosothiol formation on caspase-3 by NO, we did several experiments. First. we prepared an immunoprecipitate of caspase-3 and measured the concentration of NO released from the precipitated complex by HgCl,. Second. S-nitrosylated proteins, purified by immunoprecipitation of caspase-3, were biotinylated and the biotin concentration was estimated by immunoblotting. Third. dual immunofluorescent staining was done with antibodies for S-nitrosocysteine and caspase-3. Results showed that formation of nitrosothiol in caspase-3 in DOX-treated cardiomyocytes with SNAP was increased significantly compared with untreated cardiomyocytes. We reported here that exogenous NO produces an anti-apoptotic effect by suppression of caspase activity via S-nitrosylation in cardiomyocytes. (C) 2004 Elsevier Ltd. All rights reserved.