RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G(+)-bacterial toxins.
RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G(+)-bacterial toxins.
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RhoA S-亚硝基化作为调节机制影响内皮屏障功能以响应 G-细菌毒素。
DOI：
10.1016/j.bcp.2016.12.014
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发表时间：
2017-03-01
影响因子：
5.8
通讯作者：
Fulton DJR
中科院分区：
文献类型：
--
作者：
Chen F;Wang Y;Rafikov R;Haigh S;Zhi WB;Kumar S;Doulias PT;Rafikova O;Pillich H;Chakraborty T;Lucas R;Verin AD;Catravas JD;She JX;Black SM;Fulton DJR
革兰氏阳性（G +）细菌毒素引起的内皮屏障破坏是急性肺损伤（ALI）的一种主要并发症，并且可因刺激毒素释放的抗生素而进一步加重。肺内皮屏障的完整性由诸如小GTP酶RhoA等破坏力量和包括内皮衍生的一氧化氮（NO）在内的保护力量之间的平衡所介导。NO如何防止屏障功能障碍尚未完全了解，我们的目标是确定NO和S - 亚硝基化是否能够调节RhoA活性，以及这种机制对于G +毒素诱导的微血管通透性是否重要。我们发现G +毒素李斯特菌溶血素 - O（LLO）增加了RhoA活性，并且NO和S - NO供体抑制RhoA活性。通过生物素转换和汞柱分析确定RhoA被强烈地S - 亚硝基化。质谱分析显示三个主要的半胱氨酸残基被S - 亚硝基化，包括cys16、cys20和cys159。将这些残基突变为丝氨酸会减少对内源性NO的S - 亚硝基化，并且突变型RhoA对S - NO的抑制作用不太敏感。G +毒素刺激了RhoA的去亚硝基化，这不是由S - 亚硝基谷胱甘肽还原酶（GSNOR）、硫氧还蛋白（TRX）或巯基依赖性酶活性介导的，而是由升高的钙水平直接刺激的。钙促进了野生型（WT）而非突变型RhoA的直接去亚硝基化，并且突变型RhoA腺病毒在破坏人肺微血管内皮细胞的屏障完整性方面比野生型更有效。总之，我们揭示了一种新的机制，即NO和S - 亚硝基化通过该机制降低RhoA活性，这可能在G +毒素诱导的肺内皮通透性的处理中具有重要意义。
Disruption of the endothelial barrier in response to Gram positive (G+) bacterial toxins is a major complication of acute lung injury (ALI) and can be further aggravated by antibiotics which stimulate toxin release. The integrity of the pulmonary endothelial barrier is mediated by the balance of disruptive forces such as the small GTPase RhoA, and protective forces including endothelium-derived nitric oxide (NO). How NO protects against the barrier dysfunction is incompletely understood and our goal was to determine whether NO and S-nitrosylation can modulate RhoA activity and whether this mechanism is important for G+ toxin-induced microvascular permeability. We found that the G+ toxin listeriolysin-O (LLO) increased RhoA activity and that NO and S-NO donors inhibit RhoA activity. RhoA was robustly S-nitrosylated as determined by biotin-switch and mercury column analysis. MS revealed that three primary cysteine residues are S-nitrosylated including cys16, cys20 and cys159. Mutation of these residues to serine diminished S-nitrosylation to endogenous NO and mutant RhoA was less sensitive to inhibition by S-NO. G+-toxins stimulated the denitrosylation of RhoA which was not mediated by S-nitrosoglutathione reductase (GSNOR), thioredoxin (TRX) or thiol-dependent enzyme activity but was instead stimulated directly by elevated calcium levels. Calcium-promoted the direct denitrosylation of WT but not mutant RhoA and mutant RhoA adenovirus was more effective than WT in disrupting the barrier integrity of human lung microvascular endothelial cells. In conclusion, we reveal a novel mechanism by which NO and S-nitrosylation reduces RhoA activity which may be of significance in the management of pulmonary endothelial permeability induced by G+-toxins.
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