Negative auto-regulation of sulfur dioxide generation in vascular endothelial cells: AAT1 S-sulfenylation

Recently, endogenous sulfur dioxide (SO₂) has been found to exert an important function in the cardiovascular system. However, the regulatory mechanism for SO₂ generation has not been entirely clarified. Hence, we aimed to explore the possible auto-regulation of endogenous SO₂ generation and its mechanisms in vascular endothelial cells. We showed that SO₂ did not affect the protein expression of aspartate aminotransferase 1 (AAT1), a major SO₂ 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 SO₂ 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, SO₂ induced S-sulfenylation of AAT1 in primary HUVECs and purified AAT1 protein. LC-MS/MS analysis indicated that SO₂ sulfenylated AAT1 at Cys192. Mechanistically, thiol reductant DTT treatment or C192S mutation prevented SO₂-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 SO₂ 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.