Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119991, Russia.
Bach Institute of Biochemistry, Research Center of Biotechnology of Russian Academy of Sciences, Leninsky prospect 33, bld. 2, Moscow 119071, Russia; Koltzov Institute of Developmental Biology of Russian Academy of Sciences, ul. Vavilova 26, Moscow 119334, Russia.
Biochim Biophys Acta Gen Subj. 2023 Sep;1867(9):130418. doi: 10.1016/j.bbagen.2023.130418. Epub 2023 Jun 22.
The aim of this work was to compare the effect of reversible post-translational modifications, S-nitrosylation and S-glutathionylation, on the properties of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to reveal the mechanism of the relationship between these modifications. Comparison of S-nitrosylated and S-glutathionylated GAPDH showed that both modifications inactivate the enzyme and change its spatial structure, decreasing the thermal stability of the protein and increasing its sensitivity to trypsin cleavage. Both modifications are reversible in the presence of dithiothreitol, however, in the presence of reduced glutathione and glutaredoxin 1, the reactivation of S-glutathionylated GAPDH is much slower (10% in 2 h) compared to S-nitrosylated GAPDH (60% in 10 min). This suggests that S-glutathionylation is a much less reversible modification compared to S-nitrosylation. Incubation of HEK 293 T cells in the presence of HO or with the NO donor diethylamine NONOate results in accumulation of sulfenated GAPDH (by data of Western blotting) and S-glutathionylated GAPDH (by data of immunoprecipitation with anti-GSH antibodies). Besides GAPDH, a protein of 45 kDa was found to be sulfenated and S-glutathionylated in the cells treated with HO or NO. This protein was identified as beta-actin. The results of this study confirm the previously proposed hypothesis based on in vitro investigations, according to which S-nitrosylation of the catalytic cysteine residue (Cys152) of GAPDH with subsequent formation of cysteine sulfenic acid at Cys152 may promote its S-glutathionylation in the presence of cellular GSH. Presumably, the mechanism may be valid in the case of beta-actin.
这项工作的目的是比较可逆的翻译后修饰（S-亚硝基化和 S-谷胱甘肽化）对甘油醛-3-磷酸脱氢酶（GAPDH）性质的影响，并揭示这些修饰之间关系的机制。比较 S-亚硝基化和 S-谷胱甘肽化的 GAPDH 表明，这两种修饰都使酶失活并改变其空间结构，降低蛋白质的热稳定性并增加其对胰蛋白酶切割的敏感性。两种修饰在存在二硫苏糖醇的情况下都是可逆的，然而，在存在还原型谷胱甘肽和谷胱甘肽还原酶 1 的情况下，S-谷胱甘肽化 GAPDH 的再激活要慢得多（2 小时内 10%），与 S-亚硝基化 GAPDH（10 分钟内 60%）相比。这表明与 S-亚硝基化相比，S-谷胱甘肽化是一种不太可逆的修饰。在存在 HO 或 NO 供体二乙胺 NONOate 的情况下孵育 HEK 293T 细胞会导致磺基化 GAPDH（根据 Western blot 数据）和 S-谷胱甘肽化 GAPDH（根据用抗 GSH 抗体进行免疫沉淀的数据）的积累。除 GAPDH 外，还发现在用 HO 或 NO 处理的细胞中，一种 45 kDa 的蛋白质被磺基化和 S-谷胱甘肽化。该蛋白质被鉴定为β-肌动蛋白。这项研究的结果证实了先前基于体外研究提出的假设，根据该假设，GAPDH 催化半胱氨酸残基（Cys152）的 S-亚硝基化，随后在 Cys152 形成半胱氨酸亚磺酸，可能会在存在细胞 GSH 的情况下促进其 S-谷胱甘肽化。推测该机制在β-肌动蛋白的情况下可能是有效的。
