Institute for Vascular Signalling (S-I.B., J.H., J.W., M.K.D., V.R., F.D.L., B.F., S.Z., A.K., A.F.O.J., I.F.), Goethe University, Frankfurt am Main, Germany.
German Center of Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany (S-I.B., J.Hu, M.L., C.R., J.W., L.T., V.R., M.S.L., P.G., F.D.L., B.F., S.Z., A.K., A.F.O.J., J.Heidler, R.P.B., S.D., S.O., I.W., I.F.).
Circulation. 2021 Mar 2;143(9):935-948. doi: 10.1161/CIRCULATIONAHA.120.051877. Epub 2020 Dec 14.
In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (HS), that exert their biological actions via cysteine -sulfhydration of target proteins. This study set out to map the "-sulfhydrome" (ie, the spectrum of proteins targeted by HS) in human endothelial cells.
Liquid chromatography with tandem mass spectrometry was used to identify -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 HS donor, SG1002. The endothelial cell "-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 -sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin -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 -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 HS generation, impaired flow-induced dilatation, and failure to detect β3 integrin -sulfhydration, all of which were rescued after the administration of an HS supplement.
Vascular disease is associated with marked changes in the -sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term HS supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.
在血管内皮细胞中，胱硫醚γ裂解酶（CSE）通过半胱氨酸代谢生成与硫化氢相关的硫磺酸化合物（HS），这些化合物通过靶向蛋白的半胱氨酸 - 巯基化发挥其生物学作用。本研究旨在绘制人类内皮细胞中的“ - 巯基组”（即 HS 靶向的蛋白质谱）。
采用液相色谱 - 串联质谱法鉴定内皮细胞蛋白中的 - 巯基化半胱氨酸和β3 整合素蛋白内二硫键重排。功能研究包括内皮细胞黏附、切应力诱导的细胞排列、血压测量以及内皮细胞特异性 CSE 敲除小鼠和一小部分内皮功能障碍患者中的血流诱导血管扩张。
比较了三组配对样本：（1）来自无斑块肠系膜动脉（CSE 活性高）和斑块颈动脉（CSE 活性低）的原代人内皮细胞；（2）在静态条件下培养的人内皮细胞或暴露于流体切应力以降低 CSE 表达的人内皮细胞；以及（3）暴露于切应力以降低 CSE 表达并分别用溶剂或缓慢释放 HS 供体 SG1002 处理的培养内皮细胞。内皮细胞“- 巯基组”由 1591 种蛋白质中的 3446 个单独的半胱氨酸残基组成。改变最明显的蛋白质家族是整合素，详细研究β3 整合素发现，- 巯基化影响蛋白内二硫键的形成，并维持β 腿的伸展开放构象。β3 整合素 - 巯基化是体外内皮细胞力学转导以及鼠肠系膜动脉血流诱导扩张所必需的。在培养的细胞中，- 巯基化的丧失会损害β3 整合素与 Gα13（鸟苷酸结合蛋白亚基α13）之间的相互作用，导致 Ras 同源家族成员 A（ras homolog family member A）的组成型激活和血流诱导的内皮细胞重新排列受损。在患有动脉粥样硬化的人类中，内皮功能与低 HS 生成、血流诱导扩张受损以及未能检测到β3 整合素 - 巯基化相关，所有这些都在用 HS 补充剂治疗后得到挽救。
血管疾病与参与介导对血流反应的内皮细胞蛋白的 - 巯基化明显改变有关。短期 HS 补充剂改善了人类的血管反应性，突出了干扰该途径治疗血管疾病的潜力。
