Mapping the Endothelial Cell <i>S</i>-Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function.

Abstract

In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H₂S_n), that exert their biological actions via cysteine <i>S</i>-sulfhydration of target proteins. This study set out to map the "<i>S</i>-sulfhydrome" (ie, the spectrum of proteins targeted by H₂S_n) in human endothelial cells. Liquid chromatography with tandem mass spectrometry was used to identify <i>S</i>-sulfhydrated cysteines in endothelial cell proteins and β3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H₂S_n donor, SG1002. The endothelial cell "<i>S</i>-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on β3 integrin in detail we found that <i>S</i>-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin <i>S</i>-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of <i>S</i>-sulfhydration impaired interactions between β3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H₂S_n generation, impaired flow-induced dilatation, and failure to detect β3 integrin <i>S</i>-sulfhydration, all of which were rescued after the administration of an H₂S_n supplement. Vascular disease is associated with marked changes in the <i>S</i>-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H₂S_n supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.