Department of Basic Medical Research, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, 511518, Guangdong, China.
Clinical Laboratory of Dongcheng People's Hospital, Dong guan, 523007, Guangdong, China.
Cell Commun Signal. 2020 Feb 4;18(1):19. doi: 10.1186/s12964-020-0515-3.
Neutrophils form the first line of innate host defense against invading microorganisms. We previously showed that F0F1 ATP synthase (F-ATPase), which is widely known as mitochondrial respiratory chain complex V, is expressed in the plasma membrane of human neutrophils and is involved in regulating cell migration. Whether F-ATPase performs cellular functions through other pathways remains unknown.
Blue native polyacrylamide gel electrophoresis followed by nano-ESI-LC MS/MS identification and bioinformatic analysis were used to identify protein complexes containing F-ATPase. Then, the identified protein complexes containing F-ATPase were verified by immunoblotting, immunofluorescence colocalization, immunoprecipitation, real-time RT-PCR and agarose gel electrophoresis. Immunoblotting, flow cytometry and a LPS-induced mouse lung injury model were used to assess the effects of the F-ATPase-containing protein complex in vitro and in vivo.
We found that the voltage-gated calcium channel (VGCC) α2δ-1 subunit is a binding partner of cell surface F-ATPase in human neutrophils. Further investigation found that the physical connection between the two proteins may exist between the F1 part (α and β subunits) of F-ATPase and the α2 part of VGCC α2δ-1. Real-time RT-PCR and PCR analyses showed that Ca2.3 (R-type) is the primary type of VGCC expressed in human neutrophils. Research on the F-ATPase/Ca2.3 functional complex indicated that it can regulate extracellular Ca influx, thereby modulating ERK1/2 phosphorylation and reactive oxygen species production, which are typical features of neutrophil activation. In addition, the inhibition of F-ATPase can reduce neutrophil accumulation in the lungs of mice that were intratracheally instilled with lipopolysaccharide, suggesting that the inhibition of F-ATPase may prevent neutrophilic inflammation-induced tissue damage.
In this study, we identified a mechanism by which neutrophil activity is modulated, with simultaneous regulation of neutrophil-mediated pulmonary damage. These results show that surface F-ATPase of neutrophils is a potential innate immune therapeutic target.
中性粒细胞构成了宿主抵御入侵微生物的第一道先天防御线。我们之前曾表明，广泛认为是线粒体呼吸链复合物 V 的 F0F1 ATP 合酶（F-ATPase）在人中性粒细胞的质膜中表达，并参与调节细胞迁移。但是否通过其他途径发挥细胞功能仍不清楚。
采用蓝Native 聚丙烯酰胺凝胶电泳结合纳升电喷雾 LC-MS/MS 鉴定和生物信息学分析来鉴定含 F-ATPase 的蛋白质复合物。然后，通过免疫印迹、免疫荧光共定位、免疫沉淀、实时 RT-PCR 和琼脂糖凝胶电泳来验证鉴定出的含 F-ATPase 的蛋白质复合物。免疫印迹、流式细胞术和 LPS 诱导的小鼠肺损伤模型用于评估体外和体内含 F-ATPase 蛋白质复合物的作用。
我们发现电压门控钙通道（VGCC）α2δ-1 亚基是人中性粒细胞表面 F-ATPase 的结合伴侣。进一步的研究发现，两种蛋白之间的物理连接可能存在于 F-ATPase 的 F1 部分（α和β亚基）和 VGCC α2δ-1 的α2 部分之间。实时 RT-PCR 和 PCR 分析表明，Ca2.3（R 型）是人中性粒细胞中主要表达的 VGCC 类型。对 F-ATPase/Ca2.3 功能复合物的研究表明，它可以调节细胞外 Ca2+内流，从而调节 ERK1/2 磷酸化和活性氧产生，这是中性粒细胞激活的典型特征。此外，抑制 F-ATPase 可减少脂多糖气管内滴注小鼠肺中的中性粒细胞积聚，提示抑制 F-ATPase 可能防止中性粒细胞炎症引起的组织损伤。
在这项研究中，我们确定了一种调节中性粒细胞活性的机制，同时调节了中性粒细胞介导的肺损伤。这些结果表明，中性粒细胞表面的 F-ATPase 是一种潜在的固有免疫治疗靶点。
