Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
Biochem Biophys Res Commun. 2011 Jan 7;404(1):482-7. doi: 10.1016/j.bbrc.2010.12.007. Epub 2010 Dec 6.
Peptides simultaneously produced during maturation and degradation of peptidergic hormones and functional proteins have recently become a great interest because they display unpredictably different biological roles than the parent proteins. Namely, we discovered two novel functional cryptic peptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial cytochrome c oxidase and cytochrome b, that efficiently induced neutrophilic migration and activation at nanomolar concentrations. We named these functional "cryptic" peptides hidden in protein structures as "cryptides." In this study, we investigated the receptor molecules and cellular signaling mechanisms for neutrophil-activating N-formylated cryptide MCT-2. In order to identify the receptor molecules, we established HEK-293 cells stably expressing either formyl-peptide receptor (FPR) or its homologue FPR-like 1 (FPRL1), because neutrophilic cells express these receptor molecules which recognize N-formylated peptides. We observed that MCT-2 directly bound to FPRL1 and promoted an increase in intracellular Ca(2+) concentration (Ca(2+)), and neither interacted with nor activated FPR, demonstrating that MCT-2 is a specific agonist for FPRL1. Moreover, MCT-2 induced not only Ca(2+) increase and phosphorylation of extracellular signal-regulated protein kinases 1 and 2, but also β-hexosaminidase release in neutrophilic/granulocytic cells differentiated from HL-60 cells. Such signaling events were diminished by pretreatment with pertussis toxin, indicating that MCT-2-promoted neutrophilic function is a consequence of G(i)- or G(o)-type G protein-dependent intracellular signaling events via FPRL1 activation. These findings suggest that MCT-2, a cryptide derived from mitochondrial cytochrome b, is a specific endogenous agonist for FPRL1 which is proposed to play key roles in inflammatory responses but whose physiological agonists are equivocal.
最近，在神经肽类激素和功能性蛋白质的成熟和降解过程中同时产生的肽引起了极大的关注，因为它们显示出与母体蛋白质不同的不可预测的生物学作用。具体来说，我们发现了两个隐藏在线粒体细胞色素 c 氧化酶和细胞色素 b 中的新型功能性隐匿肽，即 mitocryptide-1（MCT-1）和 mitocryptide-2（MCT-2），它们在纳摩尔浓度下有效地诱导中性粒细胞的迁移和激活。我们将这些隐藏在蛋白质结构中的功能性“隐匿”肽命名为“cryptides”。在这项研究中，我们研究了激活中性粒细胞的 N-甲酰化 cryptide MCT-2 的受体分子和细胞信号转导机制。为了鉴定受体分子，我们建立了稳定表达甲酰肽受体（FPR）或其同源物 FPRL1 的 HEK-293 细胞，因为中性粒细胞表达识别 N-甲酰化肽的这些受体分子。我们观察到 MCT-2 直接与 FPRL1 结合并促进细胞内 Ca2+浓度（[Ca2+]（i））增加，并且既不与 FPR 相互作用也不激活 FPR，表明 MCT-2 是 FPRL1 的特异性激动剂。此外，MCT-2 诱导不仅[Ca2+]（i）增加和细胞外信号调节蛋白激酶 1 和 2 的磷酸化，而且还诱导 HL-60 细胞分化的中性粒细胞/粒细胞细胞中β-己糖胺酶的释放。用百日咳毒素预处理可减少这种信号事件，表明 MCT-2 促进中性粒细胞功能是通过 FPRL1 激活的 G(i)-或 G(o)-型 G 蛋白依赖性细胞内信号事件的结果。这些发现表明，源自线粒体细胞色素 b 的 cryptide MCT-2 是 FPRL1 的特异性内源性激动剂，它被认为在炎症反应中发挥关键作用，但生理激动剂尚不清楚。
