抗菌肽merecidin作用于人肺腺癌A549细胞株的磷酸化蛋白质组学分析

Phospho-proteomic analysis of antibacterial peptide merecidin act on human lung adenocarcinoma A549 cell line

目的:观察抗菌肽merecidin作用于人肺腺癌A549细胞后其细胞内所有蛋白质的磷酸化水平变化,探究抗菌肽对A549细胞生物学功能的影响以及促进细胞凋亡可能涉及到的信号通路与作用靶点。方法:9μmol/L抗菌肽merecidin作用于肺腺癌A549细胞6 h后收集并提取总蛋白,通过胰酶酶解肽段,并对肽段进行TMT标记和HPLC分级,IMAC-Fe富集磷酸化肽段,利用高分辨质谱对富集肽段进行检测。利用MaxQuant软件对质谱分析得到的磷酸化肽段进行搜库鉴定和定量,结合生物信息技术分析差异磷酸化蛋白质的功能、通路等信息。结果:通过对对照组和抗菌肽merecidin处理组磷酸化蛋白进行IPA分析,以上调或下调倍数≥2倍且P<0.05的条件鉴定出抗菌肽merecidin处理组中差异磷酸化蛋白质共有753个,其中明显上调有229个、下调有417个;其中与细胞凋亡相关的差异蛋白质有RB1、MAPK1、ARAF、PTK2、FOXO、MARCKS等。生物进程分析结果显示差异磷酸化蛋白主要集中在细胞信号转导、核酸的降解转运以及细胞能量代谢、蛋白质的翻译合成、细胞骨架的形成等,富集结果显示差异磷酸化蛋白质主要参与凋亡相关信号通路有MAPK、ErbB、PI3K-Akt和Ras等,通过蛋白互作分析找出凋亡相关蛋白质PTK2,PRKCA、MA2PK2、MAPK1、LMNA等之间有关联。结论:抗菌肽merecidin可能通过影响RB1、MAPK1、ARAF、PTK2、FOXO、MARCKS等基因和信号通路诱导肺癌A549细胞的凋亡及其他细胞功能的改变。

Objective: To investigate the effects of antimicrobial peptides merecidin on the biological functions of human lung adenocarcinoma A549 cells and the potential signaling pathways and targets that involved in promoting apoptosis, by studying the changes of phosphorylation levels of proteins in A549 cells after merecidin treatment. Methods: The antibacterial peptide mericidin(9 μmol/L)was applied to treat A549 cells for 6 h, and the total protein was collected and extracted. The peptide was digested by trypsin and labeled with TMT, and then fractionated by HPLC. The phosphorylated peptides were enriched with IMAC-Fe, and finally subjected to mass spectrometry analysis. Library identification and quantification of phosphorylated peptides obtained by mass spectrometry were processed using MaxQuant software, to further analyze the functions and pathways of differentially expressed phosphorylated proteins by combining with bioinformatic analysis. Results: Through IPA analysis of phosphorylated proteins in the normal control group and the antibacterial peptide merecidin treatment group, 753 differentially phosphorylated proteins in mericidin treatment group were screened out under the conditions of |Fold Change|≥ 2 and P<0.05, including 229 significantly up-regulated genes and 417 down-regulated genes. Among them, the differentially expressed proteins related to apoptosis included RB1, MAPK1, ARAF, PTK2, FOXO,MARCKS and so on. The results of biological process analysis showed that differentially expressed phosphorylated proteins were mainly concentrated in cell signal transduction, degradation and transport of nucleic acid, and cellular energy metabolism, protein translation and synthesis, and cytoskeleton formation etc. The enrichment results showed that the differentially expressed phosphorylated proteins were mainly involved in apoptosis-related MAPK, ErbB, PI3 K-Akt, and Ras signaling pathways. Protein-protein interaction analysis indicated the associations among apoptosis-related proteins PTK2, PRKCA, MA2 PK2, MAPK1, and LMNA. Conclusion: The antibacterial peptide merecidin may induce apoptosis and alteration of other cell functions by affecting a variety of genes and signaling pathways such as RB1, MAPK1, ARAF, PTK2, FOXO and MARCKS etc.