Song Yunjia, Peng Hanlin, Bu Dingfang, Ding Xiang, Yang Fuquan, Zhu Zhigang, Tian Xiaoyu, Zhang Lulu, Wang Xiuli, Tang Chaoshu, Huang Yaqian, Du Junbao, Jin Hongfang
Department of Pediatrics, Peking University First Hospital, Beijing, 100034, China; Research Unit of Clinical Diagnosis and Treatment of Pediatric Syncope and Cardiovascular Diseases, Chinese Academy of Medical Sciences, China.
Department of Pediatrics, Peking University First Hospital, Beijing, 100034, China.
Biochem Biophys Res Commun. 2020 Feb 19. doi: 10.1016/j.bbrc.2020.02.040.
Recently, endogenous sulfur dioxide (SO) has been found to exert an important function in the cardiovascular system. However, the regulatory mechanism for SO generation has not been entirely clarified. Hence, we aimed to explore the possible auto-regulation of endogenous SO generation and its mechanisms in vascular endothelial cells. We showed that SO did not affect the protein expression of aspartate aminotransferase 1 (AAT1), a major SO synthesis enzyme, but significantly inhibited AAT activity in primary human umbilical vein endothelial cells (HUVECs) and porcine purified AAT1 protein. An AAT1 enzymatic kinetic study showed that SO reduced the Vmax (1.89 ± 0.10 vs 2.55 ± 0.12, μmol/mg/min, P < 0.05) and increased the Km (35.97 ± 9.54 vs 19.33 ± 1.76 μmol/L, P < 0.05) values. Furthermore, SO induced S-sulfenylation of AAT1 in primary HUVECs and purified AAT1 protein. LC-MS/MS analysis indicated that SO sulfenylated AAT1 at Cys192. Mechanistically, thiol reductant DTT treatment or C192S mutation prevented SO-induced AAT1 sulfenylation and the subsequent inhibition of AAT activity in purified AAT1 protein and primary HUVECs. Our findings reveal, for the first time, a mechanism of auto-regulation of SO generation through sulfenylation of AAT1 at Cys192 to suppress AAT activity in vascular endothelial cells. These findings will greatly deepen the understanding of regulatory mechanisms in the cardiovascular homeostasis.
最近，人们发现内源性二氧化硫（SO）在心血管系统中发挥着重要作用。然而，SO生成的调节机制尚未完全阐明。因此，我们旨在探讨血管内皮细胞中内源性SO生成的可能自我调节及其机制。我们发现，SO不影响主要的SO合成酶天冬氨酸转氨酶1（AAT1）的蛋白表达，但能显著抑制原代人脐静脉内皮细胞（HUVECs）和猪纯化AAT1蛋白中的AAT活性。一项AAT1酶动力学研究表明，SO降低了Vmax（1.89±0.10对2.55±0.12，μmol/mg/min，P<0.05）并增加了Km（35.97±9.54对19.33±1.76μmol/L，P<0.05）值。此外，SO在原代HUVECs和纯化的AAT1蛋白中诱导AAT1的S-亚磺酰化。液相色谱-串联质谱（LC-MS/MS）分析表明，SO使AAT1的半胱氨酸192位点发生亚磺酰化。从机制上讲，硫醇还原剂二硫苏糖醇（DTT）处理或C192S突变可防止SO诱导的AAT1亚磺酰化以及随后对纯化的AAT1蛋白和原代HUVECs中AAT活性的抑制。我们的研究结果首次揭示了一种通过AAT1半胱氨酸192位点的亚磺酰化来抑制其活性从而实现SO生成自我调节的机制。这些发现将极大地加深对心血管稳态调节机制的理解。
