Mapping the Endothelial Cell S -Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function

Abstract

Background: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H 2 S n ), that exert their biological actions via cysteine S -sulfhydration of target proteins. This study set out to map the " S -sulfhydrome" i.e. the spectrum of proteins targeted by H 2 S n in human endothelial cells. Methods: LC-MS/MS was used to identify S-sulfhydrated cysteines in endothelial cell proteins and β3 integrin intra-protein 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 knock out mice and a small collective of patients with endothelial dysfunction. Results: 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 H 2 S n donor, SG1002. The endothelial cell " S -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 S -sulfhydration affected intra-protein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin S -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 S -sulfhydration impaired interactions between β3 integrin and Gα13, resulting in the constitutive activation of RhoA and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H 2 S n generation, impaired flow-induced dilatation and a failure to detect β3 integrin S -sulfhydration, all of which were rescued following the administration of an H 2 S supplement. Conclusions: Vascular disease is associated with marked changes in the S -sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short term H 2 S n supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

Sodium thiosulfate acts as a hydrogen sulfide mimetic to prevent intimal hyperplasia via inhibition of tubulin polymerisation

“… 55 Several high throughput studies of post-translational modification of protein cysteinyl thiols (-SH) to persulfides (-SSH) demonstrated that cysteine residues in α- and β-tubulin are persulfidated in response to H 2 S donors in various cell types. 56 , 57 Given the prominent role of cytoskeleton dynamics and remodelling during mitosis and cell migration, we propose that STS/H 2 S-driven microtubule depolymerisation, secondary to cysteine persulfidation, contributes to cell cycle arrest and reduces migration in VSMC.…”

Sodium Thiosulfate acts as an H2S mimetic to prevent intimal hyperplasia via inhibition of tubulin polymerization

“…In particular, thiol™disulfide exchanges in intrachain disulfide bonds have been proposed to play a key role in microtubule assembly (42). Several high throughput studies of post-translational modification of protein cysteinyl thiols (-SH) to persulfides (-SSH) demonstrated that cysteine residues in α- and β-tubulin are persulfidated in response to H 2 S donors in various cell types (43, 44). Given the prominent role of cytoskeleton dynamics and remodelling during mitosis and cell migration, we propose that STS/H 2 S-driven microtubule depolymerisation, secondary to cysteine persulfidation, contributes to cell cycle arrest and reduces migration in VSMC.…”

Nanomaterial-based delivery vehicles for therapeutic cancer vaccine development

“…Although targeting TAAs has had some early clinical trials, to date, the vaccines lacked potency because of issues with their central or peripheral tolerance mechanisms 35 . In addition, although TAA analyses using sequencing and MS/MS techniques showed successful identification, the results were not supported by adequate clinical data 41 . Thus, more attention should be directed towards developing tumor-specific and potent therapeutic vaccines to favor effective immune responses.…”