Phosphoproteomic analysis of protease-activated receptor-1 biased signaling reveals unique modulators of endothelial barrier function
Phosphoproteomic analysis of protease-activated receptor-1 biased signaling reveals unique modulators of endothelial barrier function
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DOI：
10.1073/pnas.1917295117
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发表时间：
2020-03-03
影响因子：
11.1
通讯作者：
Trejo, Joann
中科院分区：
文献类型：
--
作者：
Lin, Ying;Wozniak, Jacob M.;Trejo, Joann
凝血酶是一种促凝血酶，可裂解并激活蛋白酶激活的受体-1(PAR1)，从而促进炎症反应和内皮功能障碍。相比之下，活化蛋白C(APC)是一种抗凝血酶，通过一个独特的裂解位点激活PAR1，促进抗炎反应、生存和内皮屏障的稳定。凝血酶与APC通过切割PAR1形成不同的拴系配体，导致独特的活性受体构象，从而偏向PAR1信号转导。尽管在理解PAR1偏向信号方面取得了进展，但凝血酶和APC信号用于诱导相反的细胞功能的蛋白质和途径在很大程度上是未知的。在这里，我们报道了凝血酶和APC信号在血管内皮细胞中诱导的全球磷酸蛋白质组，并使用多重定量质谱仪对11,266个独特的磷酸肽进行了定量。我们的结果揭示了凝血酶和APC信号的独特动态磷酸蛋白质组图谱，丰富了相关的生物学功能，包括内皮屏障功能的关键调节因子，基因转录调节因子，以及被预测为介导PAR1偏向信号的特定激酶。利用小干扰RNA去除以前与凝血酶或APC信号无关的磷酸化蛋白的子集，揭示了afadin和adducin-1肌动蛋白结合蛋白在凝血酶诱导的内皮屏障破坏中的功能。Afadin耗竭导致凝血酶促进的屏障通透性增强，而Adducin-1耗竭完全消融凝血酶诱导的屏障破坏，而不影响p38信号转导。然而，内收蛋白-1的缺失阻断了APC诱导的Akt信号转导。这些研究确定了不同的凝血酶和APC动态信号特征，以及在内皮细胞中产生PAR1偏向信号的丰富蛋白质和生物通路。
Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.