甲基乙二醛存在下3-磷酸甘油醛脱氢酶的失活机制

Mechanism of inactivation of glyceraldehyde-3-phosphate dehydrogenase in the presence of methylglyoxal.

作者信息

Barinova K V, Serebryakova M V, Melnikova A K, Medvedeva M V, Muronetz V I, Schmalhausen E V

机构信息

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia.

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119991, Russia.

出版信息

Arch Biochem Biophys. 2023 Jan 1;733:109485. doi: 10.1016/j.abb.2022.109485. Epub 2022 Dec 5.

DOI:10.1016/j.abb.2022.109485

PMID:36481268

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to be one of the targets of methylglyoxal (MGO), a metabolite of glycolysis that increased in diabetes. However, the mechanism of GAPDH inactivation in the presence of MGO is unclear. The purpose of the work was to study the reaction of GAPDH with MGO and to identify the products of the reaction. It was shown that incubation of recombinant human GAPDH with MGO leads to irreversible inactivation of the enzyme, which is accompanied by a decrease in SH-group content by approximately 3.3 per tetramer GAPDH. MALDI-TOF MS analysis showed that the modification of GAPDH with MGO results in the oxidation of the catalytic cysteine residues (Cys152) to form cysteine-sulfinic acid. In addition, 2 arginine residues (R80 and R234) were identified that react with MGO to form hydroimidazolones. Incubation of SH-SY5Y neuroblastoma cells with MGO resulted in the inactivation of GAPDH and inhibition of glycolysis. The mechanism of GAPDH oxidation in the presence of MGO suggests the participation of superoxide anion, which is formed during the reaction of amino groups with methylglyoxal. The role of GAPDH in protection against the damaging effect of ROS in cells in the case of inefficiency of MGO removal by the GSH-dependent glyoxalase system is discussed.

摘要

甘油醛-3-磷酸脱氢酶（GAPDH）是甲基乙二醛（MGO）的作用靶点之一，MGO是糖酵解的一种代谢产物，在糖尿病中会增加。然而，在MGO存在的情况下GAPDH失活的机制尚不清楚。这项工作的目的是研究GAPDH与MGO的反应并鉴定反应产物。结果表明，重组人GAPDH与MGO孵育会导致该酶不可逆失活，同时每个四聚体GAPDH的SH基团含量减少约3.3。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析表明，MGO对GAPDH的修饰导致催化性半胱氨酸残基（Cys152）氧化形成半胱氨酸亚磺酸。此外，还鉴定出2个精氨酸残基（R80和R234）与MGO反应形成氢化咪唑酮。SH-SY5Y神经母细胞瘤细胞与MGO孵育导致GAPDH失活并抑制糖酵解。MGO存在时GAPDH氧化的机制表明超氧阴离子参与其中，超氧阴离子是在氨基与甲基乙二醛的反应过程中形成的。本文还讨论了在谷胱甘肽依赖性乙二醛酶系统清除MGO效率低下的情况下，GAPDH在保护细胞免受活性氧损伤作用中的作用。