Laboratory of Plant Redox Biology, Department of Pharmacy and Biotechnology, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy.
J Biol Chem. 2013 Aug 2;288(31):22777-89. doi: 10.1074/jbc.M113.475467. Epub 2013 Jun 7.
Nitrosylation is a reversible post-translational modification of protein cysteines playing a major role in cellular regulation and signaling in many organisms, including plants where it has been implicated in the regulation of immunity and cell death. The extent of nitrosylation of a given cysteine residue is governed by the equilibrium between nitrosylation and denitrosylation reactions. The mechanisms of these reactions remain poorly studied in plants. In this study, we have employed glycolytic GAPDH from Arabidopsis thaliana as a tool to investigate the molecular mechanisms of nitrosylation and denitrosylation using a combination of approaches, including activity assays, the biotin switch technique, site-directed mutagenesis, and mass spectrometry. Arabidopsis GAPDH activity was reversibly inhibited by nitrosylation of catalytic Cys-149 mediated either chemically with a strong NO donor or by trans-nitrosylation with GSNO. GSNO was found to trigger both GAPDH nitrosylation and glutathionylation, although nitrosylation was widely prominent. Arabidopsis GAPDH was found to be denitrosylated by GSH but not by plant cytoplasmic thioredoxins. GSH fully converted nitrosylated GAPDH to the reduced, active enzyme, without forming any glutathionylated GAPDH. Thus, we found that nitrosylation of GAPDH is not a step toward formation of the more stable glutathionylated enzyme. GSH-dependent denitrosylation of GAPC1 was found to be linked to the [GSH]/[GSNO] ratio and to be independent of the [GSH]/[GSSG] ratio. The possible importance of these biochemical properties for the regulation of Arabidopsis GAPDH functions in vivo is discussed.
亚硝基化是一种蛋白质半胱氨酸的可逆翻译后修饰，在许多生物体（包括植物）的细胞调节和信号转导中起着重要作用，在植物中，它被认为参与了免疫和细胞死亡的调节。给定半胱氨酸残基的亚硝基化程度受亚硝基化和脱亚硝基化反应之间的平衡控制。这些反应的机制在植物中仍未得到充分研究。在这项研究中，我们使用拟南芥的糖酵解 GAPDH 作为工具，通过结合使用活性测定、生物素开关技术、定点突变和质谱等方法，研究了亚硝基化和脱亚硝基化的分子机制。拟南芥 GAPDH 的活性可被催化半胱氨酸残基 Cys-149 的亚硝基化可逆抑制，这种亚硝基化可以通过强的 NO 供体化学诱导，也可以通过 GSNO 进行转亚硝基化。发现 GSNO 既能引发 GAPDH 的亚硝基化，也能引发谷胱甘肽化，尽管亚硝基化更为显著。发现拟南芥 GAPDH 可被 GSH 但不能被植物细胞质硫氧还蛋白还原。GSH 可将亚硝基化的 GAPDH 完全还原为具有活性的酶，而不会形成任何谷胱甘肽化的 GAPDH。因此，我们发现 GAPDH 的亚硝基化不是形成更稳定的谷胱甘肽化酶的步骤。发现 GAPC1 的 GSH 依赖性脱亚硝基化与[GSH]/[GSNO] 比值有关，与[GSH]/[GSSG] 比值无关。这些生化性质对调节拟南芥 GAPDH 体内功能的重要性进行了讨论。
