Experimental and Clinical Research Center, a joint cooperation between the Charité and the Max-Delbrück Center for Molecular Medicine (MDC), Berlin, Germany.
MDC, Berlin, Germany.
Kidney Int. 2015 Oct;88(4):764-75. doi: 10.1038/ki.2015.159. Epub 2015 Jun 10.
Neutrophil serine proteases (NSPs) are released from activated neutrophils during inflammation. Here we studied the transfer of the three major NSPs, namely proteinase 3, human neutrophil elastase, and cathepsin G, from neutrophils to endothelial cells and used an unbiased approach to identify novel endothelial NSP substrates. Enzymatically active NSPs were released from stimulated neutrophils and internalized by endothelial cells in a dose- and time-dependent manner as shown by immunoblotting, flow cytometry, and the Boc-Ala substrate assay. Using terminal-amine isotopic labeling of substrates in endothelial cells, we identified 121 peptides from 82 different proteins consisting of 36 substrates for proteinase 3, 30 for neutrophil elastase, and 28 for cathepsin G, respectively. We characterized the extended cleavage pattern and provide corresponding IceLogos. Gene ontology analysis showed significant cytoskeletal substrate enrichment and confirmed several cytoskeletal protein substrates by immunoblotting. Finally, ANCA-stimulated neutrophils released all three active NSPs into the supernatant. Supernatants increased endothelial albumin flux and disturbed the endothelial cell cytoskeletal architecture. Serine protease inhibition abrogated this effect. Longer exposure to NSPs reduced endothelial cell viability and increased apoptosis. Thus, we identified novel NSP substrates and suggest NSP inhibition as a therapeutic measure to inhibit neutrophil-mediated inflammatory vascular diseases.
中性粒细胞丝氨酸蛋白酶（NSPs）在炎症期间从活化的中性粒细胞中释放出来。在这里，我们研究了三种主要的 NSP，即蛋白酶 3、人中性粒细胞弹性蛋白酶和组织蛋白酶 G，从中性粒细胞到内皮细胞的转移，并采用了一种无偏的方法来鉴定新的内皮 NSP 底物。如免疫印迹、流式细胞术和 Boc-Ala 底物测定所示，具有酶活性的 NSP 从受刺激的中性粒细胞中释放出来，并以内皮细胞依赖于剂量和时间的方式被内化。通过在内皮细胞中对末端胺同位素标记的底物进行分析，我们从 82 种不同的蛋白质中鉴定出 121 个肽段，分别由 36 种蛋白酶 3 的底物、30 种中性粒细胞弹性蛋白酶的底物和 28 种组织蛋白酶 G 的底物组成。我们对扩展的切割模式进行了表征，并提供了相应的 IceLogo。基因本体分析显示，细胞骨架底物明显富集，并通过免疫印迹证实了几种细胞骨架蛋白底物。最后，ANCA 刺激的中性粒细胞将所有三种活性 NSP 释放到上清液中。上清液增加了内皮细胞白蛋白的通量并破坏了内皮细胞的细胞骨架结构。丝氨酸蛋白酶抑制作用消除了这种影响。较长时间暴露于 NSP 会降低内皮细胞的活力并增加细胞凋亡。因此，我们鉴定了新的 NSP 底物，并提出了 NSP 抑制作为抑制中性粒细胞介导的炎症性血管疾病的治疗措施。
