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Archive > Volume 27 Issue 11 > 2020,27(11):1229-1238. DOI:10.3872/j.issn.1007-385X.2020.11.005 Prev Next
Basic Research
Phosphoproteomics analysis of the mechanism of antimicrobial peptide merecidin inhibiting the proliferation of lung adenocarcinoma A549 cells
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Abstract:
Objective: The phosphorproteomics technique was used to analyze the differential expression of phosphorylated proteins in the human lung adenocarcinoma A549 cells treated with antimicrobial peptide merecidin, and to explore the effect of merecidin on the protein activity and function of lung adenocarcinoma A549 cells and the signal pathway involved. Methods: A549 cells were treated with 9 μmol/L merecidin for 6 h. The total protein was collected and extracted, and SDS-PAGE experiment was used to test the total protein extraction efficiency. Pancreatin was added to digest the protein. The peptides obtained by enzymatic hydrolysis were labeled with TMT,fractionated by HPLC, enriching phosphorylated modified peptides by IMAC, and separate peptides by HPLC-MS/MS (liquid chromatographymass spectrometry) technology. The identified data were screened using the standard of localization probability>0.75, and the phosphoproteomics data were analyzed using GO (Gene Ontology) database, KEGG (Kyoto Encyclopedia of Genes and Genomes) database and STRING database. Results: SDS-PAGE results showed that the total protein separation of A549 cells treated with 9 μmol/L merecidin was clear and no obvious degradation, and there was significant difference in protein bands between the experimental group and the control group.A total of 10 320 phosphorylation modification sites on 3 089 proteins were identified by mass spectrometry, of which 753 proteins and 1 172 phosphorylation sites were screened out with |Fold Change| >1.5 and P<0.05 as the threshold. Protein function enrichment showed that functions of proteins with significant changes in phosphorylation level mainly focused on molecular binding, metabolic activity, molecular function regulation, cell process, biological function and other aspects. Bioinformatics results of integrated pathway showed that differentially expressed proteins were associated with Ras, PI3K/AKT, mTor, AMPK etc. COG database screening showed that the differentially expressed phosphorylated proteins were concentrated in cell signal transduction, processing and modification of RNA transcription and translation,protein synthesis of ribosome, formation of cytoskeleton proteins, intracellular substance transportation, secretion and vesicle transportation etc. At the protein interaction level, after merecidin treatment, an interaction network with MAPK1, RPL23A, SRSF3H, NCBP1, etc. as key proteins was formed in A549 cells; proteins such as ATG2B and ULK1 etc. were significantly up-regulated, while proteins such as MAPK1 and AKT1 etc. were significantly down-regulated. Conclusion: Phosphoproteomic analysis shows that the antimicrobial peptide merecidin may play a role in multiple biological functions and multiple signaling pathways through key proteins such as MAPK1, RPL23A, SRSF3H and AKT1, and promote the apoptosis and autophagy of lung adenocarcinoma A549 cells, thereby inhibiting cell proliferation.
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Project Supported:
Project supported by the National Natural Science Foundation of China (No. 81560573; No.81760661)
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