Endogenous hydrogen sulfide sulfhydrates IKKβ at cysteine 179 to control pulmonary artery endothelial cell inflammation
Endogenous hydrogen sulfide sulfhydrates IKKβ at cysteine 179 to control pulmonary artery endothelial cell inflammation
复制标题
半胱氨酸 179 处的内源性硫化氢硫水合物 IKKβ 控制肺动脉内皮细胞炎症
作者：
Zhang D;Wang X;Chen S;Chen S;Yu W;Liu X;Yang G;Tao Y;Bu D;Zhang H;Kong W;Tang C;Huang Y;Du J;Jin H
背景：肺动脉内皮细胞（PAEC）炎症是肺动脉高压（PAH）发生的关键事件。然而PAEC炎症的发病机制仍不清楚。方法：本研究采用纯化的重组人κB激酶亚基β抑制剂(IKKβ)蛋白、人PAEC和野百合碱诱导的肺动脉高压大鼠。通过定点诱变、基因敲除或过表达来操纵靶蛋白的表达或活性。结果：我们发现，在野百合碱吡咯刺激或敲低胱硫醚γ-裂合酶（CSE）（一种 H2S 生成酶）诱导的人 PAEC 炎症细胞模型中，硫化氢 (H2S) 抑制 IKKβ 活化。从机制上讲，H2S 被证明可通过体外纯化的重组 IKKβ 蛋白中半胱氨酰残基 179 (C179) 处的 IKKβ 硫化作用直接抑制 IKKβ 活性，而硫醇还原剂二硫苏糖醇 (DTT) 可逆转 H2S 诱导的 IKKβ 失活。此外，为了证明 IKKβ 被 H2S 硫化在 PAEC 炎症发展中的重要性，我们将 IKKβ 中的 C179 突变为丝氨酸 (C179S)。在纯化的 IKKβ 蛋白中，IKKβ 的 C179S 突变消除了 H2S 诱导的 IKKβ 硫氢化和随后的 IKKβ 失活。在人类 PAEC 中，IKKβ 的 C179S 突变阻断了 H2S 抑制的 IKKβ 激活和 PAEC 炎症反应。在肺动脉高压大鼠中，IKKβ的C179S突变消除了H2S对IKKβ激活以及肺血管炎症和重塑的抑制作用。结论：总的来说，我们的体内和体外研究结果首次证明，内源性H2S通过Cys179处的IKKβ硫化物直接灭活IKKβ，从而抑制核因子-κB（NF-κB）通路激活，从而控制肺动脉高压大鼠的PAEC炎症。多环芳烃。
Background:Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear.Methods:Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein.Results:We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ proteinin vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling.Conclusion:Collectively, ourin vivoandin vitrofindings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.