Background: Hydrogen sulfide (H2S) is one of the endogenous gaseous molecules promoting the production of nitric oxide (NO) which has cardioprotective functions. However, the role of the H2S-mediated protein S-nitrosoproteome and its subsequent physiological response remains unclear.
Methods: Endothelial cells EAhy 926 were treated with 50 μM of H2S for 2 hours. The NO bound S-nitrosoproteins were purified by a biotin-switch and then digested by trypsin. Resulting peptides from control and H2S treatment were separately labeled by isobaric tag for relative and absolute quantitation 114/115, quantified by liquid chromatography tandem-mass spectrometry and analyzed by ingenuity pathway analysis (IPA) software. The microP software was applied to analyze the morphological changes of mitochondria.
Results: With the treatment of H2S, 416 S-nitrosylated proteins were identified. IPA analysis showed that these proteins were involved in five signaling pathways. The NO-bound cysteine residues and the S-nitrosylation levels (115/114) were shown for ten S-nitrosoproteins. Western blot further verified the S-nitrosylation of thioredoxin-dependant peroxide reductase, cytochrome c oxidase and cytochrome b-c1 complex that are involved in the mitochondrial signaling pathway. H2O2-induced mitochondrial swelling can be reduced by the pretreatment of H2S.
Conclusions: The H2S-mediated endothelial S-nitrosoproteome has been confirmed. In the present study, we have proposed the cardioprotective role of H2S via maintaining mitochondrial homeostasis.
Keywords: Endothelial cell; Hydrogen sulfide; MicroP; Mitochondria; Mitophagy; S-nitrosoproteome.