Département de Pharmacologie et Physiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Canada.
PhenoSwitch Bioscience, 975 Rue Léon-Trépanier, Sherbrooke, QC J1G 5J6, Canada.
Free Radic Biol Med. 2019 Sep;141:233-243. doi: 10.1016/j.freeradbiomed.2019.06.022. Epub 2019 Jun 19.
Ascorbic acid (vitamin C) plays a significant role in the prevention of oxidative stress. In this process, ascorbate is oxidized to dehydroascorbate (DHA). We have investigated the impact of DHA on peptide/protein intramolecular disulfide formation as well as S-glutathionylation and S-homocysteinylation. S-glutathionylation of peptides/proteins is a reversible, potential regulatory mechanism in oxidative stress. Although the exact role of protein S-homocysteinylation is unknown, it has been proposed to be of importance in pathobiological processes such as onset of cardiovascular disease. Using an in vitro model system, we demonstrate that DHA causes disulfide bond formation within the active site of recombinant human glutaredoxin (Grx-1). DHA also facilities the formation of S-glutathionylation and S-homocysteinylation of a model peptide (AcFHACAAK) as well as Grx-1. We discuss the possible mechanisms of peptide/protein S-thiolation, which can occur either via thiol exchange or a thiohemiketal intermediate. A thiohemiketal DHA-peptide adduct was detected by mass spectrometry and its location on the peptide/protein cysteinyl thiol group was unambiguously confirmed by tandem mass spectrometry. This demonstrates that peptide/protein S-thiolation mediated by DHA is not limited to thiol exchange reactions but also takes place directly via the formation of a thiohemiketal peptide intermediate. Finally, we investigated a potential reducing role of glutathione (GSH) in the presence of S-homocysteinylated peptide/protein adducts. S-homocysteinylated AcFHACAAK, human hemoglobin α-chain and Grx-1 were incubated with GSH. Both peptide and proteins were reduced, and homocysteine replaced with GS-adducts by thiol exchange, as a function of time.
抗坏血酸（维生素 C）在预防氧化应激中起着重要作用。在这个过程中，抗坏血酸被氧化为脱氢抗坏血酸（DHA）。我们研究了 DHA 对肽/蛋白质分子内二硫键形成以及 S-谷胱甘肽化和 S-同型半胱氨酸化的影响。肽/蛋白质的 S-谷胱甘肽化是氧化应激中一种可逆的、潜在的调节机制。虽然蛋白质 S-同型半胱氨酸化的确切作用尚不清楚，但它被认为在心血管疾病等病理生物过程中很重要。我们使用体外模型系统证明，DHA 会导致重组人谷胱甘肽还原酶（Grx-1）活性部位的二硫键形成。DHA 还促进模型肽（AcFHACAAK）和 Grx-1 的 S-谷胱甘肽化和 S-同型半胱氨酸化的形成。我们讨论了肽/蛋白质 S-巯基化的可能机制，其可以通过巯基交换或硫代半缩醛中间体发生。通过质谱检测到了 DHA-肽硫代半缩醛加合物，并通过串联质谱法明确确认了其在肽/蛋白质半胱氨酸巯基上的位置。这表明 DHA 介导的肽/蛋白质 S-巯基化不仅限于巯基交换反应，还可以直接通过形成硫代半缩醛肽中间体发生。最后，我们研究了在存在 S-同型半胱氨酸化肽/蛋白质加合物的情况下谷胱甘肽（GSH）的潜在还原作用。将 S-同型半胱氨酸化的 AcFHACAAK、人血红蛋白 α 链和 Grx-1 与 GSH 孵育。随着时间的推移，肽和蛋白质都被还原，同型半胱氨酸被巯基交换取代为 GS-加合物。
