Time and flowrate used for proteomics.

In cystic fibrosis (CF), there is abnormal translocation and function of the cystic fibrosis transmembrane conductance regulator (CFTR) and an upregulation of the epithelial sodium channel (ENaC). This leads to hyperabsorption of sodium and fluid from the airway, dehydrated mucus, and an increased risk of respiratory infections. In this study, we performed a proteomic assessment of differentially regulated proteins from CF and non-CF small airway epithelial cells (SAEC) that are sensitive to Mycobacterium avium. CF SAEC and normal non-CF SAEC were infected with M. avium before the cells were harvested for protein. Protein kinase C (PKC) activity was greater in the CF cells compared to the non-CF cells, but the activity was significantly attenuated in both cell types after infection with M. avium compared to vehicle. Western blot and densitometric analysis showed a significant increase in cathepsin B protein expression in M. avium infected CF cells. Myristoylated alanine rich C-kinase substrate (MARCKS) protein was one of several differentially expressed proteins between the groups that was identified by mass spectrometry-based proteomics. Total MARCKS protein expression was greater in CF cells compared to non-CF cells. Phosphorylation of MARCKS at serine 163 was also greater in CF cells compared to non-CF cells after treating both groups of cells with M. avium. Taken together, MARCKS protein is upregulated in CF cells and there is decreased phosphorylation of the protein due to a decrease in PKC activity and presumably increased cathepsin B mediated proteolysis of the protein after M. avium infection.

囊性纤维化（cystic fibrosis, CF）患者体内存在囊性纤维化跨膜传导调节蛋白（cystic fibrosis transmembrane conductance regulator, CFTR）的转位异常与功能异常，同时上皮钠通道（epithelial sodium channel, ENaC）表达上调。该病理改变可引发气道内钠与液体的过度吸收，形成黏液脱水状态，并显著增加呼吸道感染风险。本研究针对对鸟分枝杆菌（Mycobacterium avium）敏感的CF与非CF小气道上皮细胞（small airway epithelial cells, SAEC）中的差异调控蛋白开展了蛋白质组学评估。实验中，我们先分别用鸟分枝杆菌感染CF SAEC与正常非CF SAEC，随后收集细胞并提取蛋白进行后续分析。相较于非CF细胞，CF细胞内的蛋白激酶C（protein kinase C, PKC）活性更高；但与溶剂对照组相比，两种细胞在感染鸟分枝杆菌后，其PKC活性均显著降低。蛋白质免疫印迹（Western blot）结合光密度分析结果显示，感染鸟分枝杆菌的CF细胞内，组织蛋白酶B（cathepsin B）的蛋白表达水平显著升高。基于质谱的蛋白质组学鉴定发现，肉豆蔻酰化富含丙氨酸的C激酶底物（Myristoylated alanine rich C-kinase substrate, MARCKS）是各组间差异表达的蛋白之一。CF细胞内的总MARCKS蛋白表达量高于非CF细胞；在两组细胞均经鸟分枝杆菌处理后，CF细胞中MARCKS蛋白丝氨酸163位点的磷酸化水平同样高于非CF细胞。综上，CF细胞内MARCKS蛋白表达上调；而在鸟分枝杆菌感染后，由于PKC活性降低，且可能存在组织蛋白酶B介导的MARCKS蛋白水解增强，最终导致该蛋白的磷酸化水平下降。