Department of Physiology and Cell Biology, Dorothy M. Davis Heart and Lung Research Institute (L.L., J.M., N.S., S.J.T., Q.Y., J. He, J.Y., J.Z., Y.Z.), The Ohio State University, Columbus.
Department of Internal Medicine (J. Hu, R.K.M., J.Z., Y.Z.), The Ohio State University, Columbus.
Arterioscler Thromb Vasc Biol. 2023 May;43(5):674-683. doi: 10.1161/ATVBAHA.122.318894. Epub 2023 Mar 30.
NF-κB (nuclear factor kappa B) plays a pivotal role in endothelial cell (EC) inflammation. Protein ISGylation is regulated by E3 ISG15 (interferon-stimulated gene 15) ligases; however, ISGylation of NF-κBp65 and its role in EC functions have not been investigated. Here, we investigate whether p65 is ISGylated and the role of its ISGylation in endothelial functions.
In vitro ISGylation assay and EC inflammation were performed. EC-specific transgenic mice were utilized in a murine model of acute lung injury.
We find that NF-κBp65 is ISGylated in resting ECs and that the posttranslational modification is reversible. TNFα (tumor necrosis factor alpha) and endotoxin stimulation of EC reduce p65 ISGylation, promoting its serine phosphorylation through reducing its association with a phosphatase WIP1 (wild-type p53-induced phosphatase 1). Mechanistically, an SCF (Skp1-Cul1-F-box) protein E3 ligase SCF is identified as a new ISG15 E3 ligase that targets and catalyzes ISGylation of p65. Depletion of FBXL19 (F-box and leucine-rich repeat protein 19) increases p65 phosphorylation and EC inflammation, suggesting a negative correlation between p65 ISGylation and phosphorylation. Moreover, EC-specific FBXL19 overexpressing humanized transgenic mice exhibit reduced lung inflammation and severity of experimental acute lung injury.
Together, our data reveal a new posttranslational modification of p65 catalyzed by a previously unrecognized role of SCF as an ISG15 E3 ligase that modulates EC inflammation.
NF-κB（核因子 kappa B）在血管内皮细胞（EC）炎症中起着关键作用。蛋白质 ISG 化受 E3 ISG15（干扰素刺激基因 15）连接酶调节；然而，NF-κBp65 的 ISG 化及其在 EC 功能中的作用尚未被研究。在这里，我们研究 p65 是否被 ISG 化，以及其 ISG 化在血管内皮功能中的作用。
进行体外 ISG 化测定和 EC 炎症。利用急性肺损伤的小鼠模型，使用 EC 特异性转基因小鼠。
我们发现 NF-κBp65 在静息 EC 中被 ISG 化，并且这种翻译后修饰是可逆的。TNFα（肿瘤坏死因子-α）和内毒素刺激 EC 减少 p65 的 ISG 化，通过减少其与磷酸酶 WIP1（野生型 p53 诱导的磷酸酶 1）的结合来促进其丝氨酸磷酸化。从机制上讲，一种 SCF（Skp1-Cul1-F-box）蛋白 E3 连接酶 SCF 被鉴定为一种新的 ISG15 E3 连接酶，可靶向并催化 p65 的 ISG 化。FBXL19（F-box 和亮氨酸丰富重复蛋白 19）的耗竭增加了 p65 的磷酸化和 EC 炎症，表明 p65 的 ISG 化与磷酸化之间呈负相关。此外，EC 特异性 FBXL19 过表达的人源化转基因小鼠表现出肺炎症和实验性急性肺损伤的严重程度降低。
总的来说，我们的数据揭示了 p65 的一种新的翻译后修饰，该修饰由以前未被识别的 SCF 作为 ISG15 E3 连接酶发挥作用来调节 EC 炎症。
