中性粒细胞胞外诱捕器(NETs)由嵌入胞外DNA网中的抗微生物分子组成。NETs的形成被认为是一种防御机制,利用中性粒细胞诱捕和杀死入侵的病原体,最近被称为NETosis。可以刺激中性粒细胞进行NETosis离体,预计含有高水平的丝氨酸蛋白酶,如中性粒细胞弹性蛋白酶(NE),组织蛋白酶G(CG)和蛋白酶3(PR3)。丝氨酸蛋白酶是中性粒细胞介导的免疫的重要效应,它们通过降解致病毒力因子直接起作用,并通过细胞因子的蛋白水解激活或失活间接起作用,趋化因子和受体。在这项研究中,我们利用了一个多样且无偏倚的肽文库来检测和分析与佛波醇-12-肉豆蔻酸酯-13-乙酸酯(PMA)诱导的NETs相关的蛋白酶活性。我们从源自健康供体嗜中性粒细胞的NET获得了“蛋白水解特征”,并使用蛋白质组学来协助鉴定这种蛋白水解活性的来源。此外,我们对每种中性粒细胞丝氨酸蛋白酶进行了分析,并包括了新鉴定的酶,中性粒细胞丝氨酸蛋白酶4(NSP4)。每种酶都具有重叠但不同的内肽酶活性,并且通常在同一肽底物内的独特位点处裂解。NETs中的主要蛋白水解活性归因于NE;然而,对应于CG和PR3活性的切割位点是明显的。当NE免疫耗尽时,其余活性归因于CG,程度较小的归因于PR3和NSP4.我们的结果表明,阻断NE活性将消除与NET相关的主要蛋白酶活性。此外,新鉴定的底物特异性特征将指导靶向NET相关蛋白酶的更多特异性探针和抑制剂的设计.
Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a \"proteolytic signature\" from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases.