Research ArticlePosttranslational Modifications

The coordination of S-sulfhydration, S-nitrosylation, and phosphorylation of endothelial nitric oxide synthase by hydrogen sulfide

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Sci. Signal.  09 Sep 2014:
Vol. 7, Issue 342, pp. ra87
DOI: 10.1126/scisignal.2005478

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Abstract

The gasotransmitter hydrogen sulfide (H2S), which is generated by cystathionine γ-lyase (CSE), signals by modifying proteins through S-sulfhydration and potentially other mechanisms. A target protein for H2S is endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide (NO), which causes vasodilation. We investigated whether H2S-induced S-sulfhydration affected the S-nitrosylation and phosphorylation of eNOS and the functional effects of changes in these posttranslational modifications on eNOS activity. In vitro, different NO donors induced the S-nitrosylation of eNOS without affecting its S-sulfhydration, whereas the H2S donor sodium hydrosulfide (NaHS) decreased the S-nitrosylation of eNOS. Cys443 was the primary S-sulfhydration site in eNOS and was one site that could be S-nitrosylated. Phosphorylation increases eNOS activity. Although exposure of eNOS-expressing HEK-293 cells to NaHS or vascular endothelial growth factor (VEGF) triggered the phosphorylation of wild-type and C443G-eNOS, VEGF did not affect the S-sulfhydration of eNOS and a mutant of eNOS that could not be phosphorylated was still S-sulfhydrated. eNOS can be present in cells in monomeric or dimeric form, but only eNOS dimers produce NO. In wild-type mice, eNOS proteins were predominantly dimerized, whereas eNOS from CSE-knockout (KO) mice, S-nitrosylated eNOS, and heterologously expressed C443G-eNOS was mostly monomeric. Accordingly, basal production of NO was lower in CSE-KO endothelial cells than in wild-type endothelial cells. Our data suggest that H2S increases eNOS activity by inducing the S-sulfhydration of eNOS, promoting its phosphorylation, inhibiting its S-nitrosylation, and increasing eNOS dimerization, whereas NO decreases eNOS activity by promoting the formation of eNOS monomers.

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