Proteomic identification of S-nitrosylated proteins in Arabidopsis
Proteomic identification of S-nitrosylated proteins in Arabidopsis
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DOI：
10.1104/pp.104.058719
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发表时间：
2005-03-01
期刊：
影响因子：
7.4
通讯作者：
Durner, J
中科院分区：
文献类型：
--
作者：
Lindermayr, C;Saalbach, G;Durner, J
虽然在过去的几年中，一氧化氮（NO）已经成长为植物中的一个关键信号分子，但对NO如何调节植物中的不同事件知之甚少。动物系统中NO依赖性过程的分析表明，蛋白质半胱氨酸（Cys）残基的S-亚硝基化是许多动物蛋白质的主要调节机制之一。对于植物，S-亚硝基化的原理仍有待阐明。我们产生的S-亚硝基硫醇处理提取物从拟南芥（拟南芥）细胞悬浮培养与NO-供体S-亚硝基谷胱甘肽。此外，用气态NO处理拟南芥植物，以分析S-亚硝基化是否可以在植物细胞体内的特定氧化还原环境中发生。通过生物素转换方法检测S-亚硝基化蛋白，将S-亚硝基化Cys转换为生物素化Cys。生物素标记的蛋白质进行纯化和分析，使用纳米液相色谱结合质谱。我们从细胞培养物中鉴定了63种蛋白质，从叶片中鉴定了52种蛋白质，这些蛋白质代表S-亚硝基化的候选者，包括应激相关、氧化还原相关、信号传导/调节、细胞骨架和代谢蛋白。引人注目的是，这些蛋白质中的许多先前已被鉴定为动物中S-亚硝基化的靶点。在酶的水平上，一个案例研究表明，NO依赖可逆抑制植物甘油醛-3-磷酸脱氢酶，这表明这种酶可能受到S-亚硝基化。这项工作的结果是进一步研究的出发点，以了解信号通路和其他细胞过程调节的蛋白质S-亚硝基化在植物中。
Although nitric oxide (NO) has grown into a key signaling molecule in plants during the last few years, less is known about how NO regulates different events in plants. Analyses of NO-dependent processes in animal systems have demonstrated protein S-nitrosylation of cysteine (Cys) residues to be one of the dominant regulation mechanisms for many animal proteins. For plants, the principle of S-nitrosylation remained to be elucidated. We generated S-nitrosothiols by treating extracts from Arabidopsis (Arabidopsis thaliana) cell suspension cultures with the NO-donor S-nitrosoglutathione. Furthermore, Arabidopsis plants were treated with gaseous NO to analyze whether S-nitrosylation can occur in the specific redox environment of a plant cell in vivo. S-Nitrosylated proteins were detected by a biotin switch method, converting S-nitrosylated Cys to biotinylated Cys. Biotin-labeled proteins were purified and analyzed using nano liquid chromatography in combination with mass spectrometry. We identified 63 proteins from cell cultures and 52 proteins from leaves that represent candidates for S-nitrosylation, including stress-related, redox-related, signaling/regulating, cytoskeleton, and metabolic proteins. Strikingly, many of these proteins have been identified previously as targets of S-nitrosylation in animals. At the enzymatic level, a case study demonstrated NO-dependent reversible inhibition of plant glyceraldehyde-3-phosphate dehydrogenase, suggesting that this enzyme could be affected by S-nitrosylation. The results of this work are the starting point for further investigation to get insight into signaling pathways and other cellular processes regulated by protein S-nitrosylation in plants.