Department of Internal Medicine, College of Medicine Phoenix, University of Arizona, Phoenix, AZ, USA.
Division of Translational and Regenerative Medicine, Department of Medicine, College of Medicine Tucson, University of Arizona, Tucson, AZ, USA.
Redox Biol. 2021 Jan;38:101794. doi: 10.1016/j.redox.2020.101794. Epub 2020 Nov 13.
Acute lung injury (ALI), a devastating illness induced by systemic inflammation e.g., sepsis or local lung inflammation e.g., COVID-19 mediated severe pneumonia, has an unacceptably high mortality and has no effective therapy. ALI is associated with increased pulmonary microvascular hyperpermeability and alveolar flooding. The small Rho GTPases, RhoA and Rac1 are central regulators of vascular permeability through cytoskeleton rearrangements. RhoA and Rac1 have opposing functional outcome: RhoA induces an endothelial contractile phenotype and barrier disruption, while Rac1 stabilizes endothelial junctions and increases barrier integrity. In ALI, RhoA activity is increased while Rac1 activity is reduced. We have shown that the activation of RhoA in lipopolysaccharide (LPS)-mediated ALI, is dependent, at least in part, on a single nitration event at tyrosine (Y). Thus, the purpose of this study was to determine if the inhibition of Rac1 is also dependent on its nitration. Our data show that Rac1 inhibition by LPS is associated with its nitration that mass spectrometry identified as Y, within the switch I region adjacent to the nucleotide-binding site. Using a molecular modeling approach, we designed a nitration shielding peptide for Rac1, designated NipR2 (nitration inhibitor peptide for the Rho GTPases 2), which attenuated the LPS-induced nitration of Rac1 at Y, preserves Rac1 activity and attenuates the LPS-mediated disruption of the endothelial barrier in human lung microvascular endothelial cells (HLMVEC). Using a murine model of ALI induced by intratracheal installation of LPS we found that NipR2 successfully prevented Rac1 nitration and Rac1 inhibition, and more importantly attenuated pulmonary inflammation, reduced lung injury and prevented the loss of lung function. Together, our data identify a new post-translational mechanism of Rac1 inhibition through its nitration at Y. As NipR2 also reduces sepsis induced ALI in the mouse lung, we conclude that Rac1 nitration is a therapeutic target in ALI.
急性肺损伤 (ALI) 是一种由全身炎症引起的破坏性疾病，例如败血症，或局部肺部炎症，例如 COVID-19 介导的严重肺炎，其死亡率非常高，且目前尚无有效的治疗方法。ALI 与肺微血管通透性增加和肺泡积水有关。小的 Rho GTPases，RhoA 和 Rac1，是通过细胞骨架重排来调节血管通透性的核心调节剂。RhoA 和 Rac1 具有相反的功能结果：RhoA 诱导内皮收缩表型和屏障破坏，而 Rac1 稳定内皮连接并增加屏障完整性。在 ALI 中，RhoA 活性增加，而 Rac1 活性降低。我们已经表明，脂多糖 (LPS) 介导的 ALI 中 RhoA 的激活至少部分依赖于酪氨酸 (Y) 上的一个单一硝化事件。因此，本研究的目的是确定 Rac1 的抑制是否也依赖于其硝化。我们的数据表明，LPS 抑制 Rac1 与 Rac1 的硝化有关，质谱分析表明硝化位于核苷酸结合位点附近的开关 I 区域内的 Y。使用分子建模方法，我们设计了一种针对 Rac1 的硝化屏蔽肽，命名为 NipR2（Rho GTPases 2 的硝化抑制剂肽），它可减弱 LPS 诱导的 Rac1 硝化，保留 Rac1 活性，并减轻 LPS 介导的人肺微血管内皮细胞 (HLMVEC) 内皮屏障破坏。使用气管内安装 LPS 诱导的 ALI 小鼠模型，我们发现 NipR2 成功阻止了 Rac1 的硝化和抑制，更重要的是，它减轻了肺部炎症、降低了肺损伤并防止了肺功能丧失。总之，我们的数据确定了 Rac1 通过 Y 硝化抑制的新的翻译后机制。由于 NipR2 还减少了小鼠肺中败血症引起的 ALI，我们得出结论，Rac1 硝化是 ALI 的治疗靶点。
