Hu P, Cheng Y, Wang Y, Gou X, Liu H, Zuo L, Wu N
Department of Biochemistry and Molecular Biology, School of Basic Medicine, Guizhou Medical University, Guiyang 550004, China.
Department of Immunology, School of Basic Medicine, Guizhou Medical University, Guiyang 550004, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2023 Jan 20;43(1):29-38. doi: 10.12122/j.issn.1673-4254.2023.01.04.
To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection.
The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs.
A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, < 0.05) and 1572 were significantly downregulated (FC < 0.83, < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated ( < 0.01).
DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.
分析登革病毒2型（DENV-2）感染的人脐静脉内皮细胞（HUVECs）中差异磷酸化蛋白，探讨DENV-2感染可能的致病机制。
采用SDT裂解方法从DENV-2感染的HUVECs和空白对照HUVECs中提取总蛋白。利用串联质谱（TMT）对磷酸化蛋白进行定性和定量分析。通过亚细胞定位分析、基因本体（GO）富集分析、京都基因与基因组百科全书（KEGG）通路分析和蛋白质-蛋白质相互作用（PPI）分析等生物信息学分析方法对鉴定出的差异磷酸化蛋白进行分析。采用蛋白质免疫印迹法检测DENV-2感染的HUVECs中磷酸化Jun、map2k2和AKT1蛋白的表达。
共检测到1385种不同蛋白质上的2918个修饰肽段（modification peptide），其中1346个显著上调（FC>1.2，P<0.05），1572个显著下调（FC<0.83，P<0.05）。通过氨基酸保守基序分析获得了49个磷酸化保守基序。蛋白质结构域分析中最丰富的差异磷酸化肽段包括RNA识别基序、蛋白激酶结构域和PH结构域。亚细胞定位分析表明，差异修饰肽段主要定位于细胞核和细胞质。GO富集分析和KEGG通路分析表明，差异肽段主要富集于刺激反应调节、含核碱基小分子的生物合成以及吞噬体和白细胞穿过内皮的迁移。PPI和KEGG联合分析表明，上调和下调的差异磷酸化蛋白富集于15条通路。在DENV-2感染的HUVECs中，蛋白质免疫印迹法检测到与自噬途径相关的磷酸化蛋白JUN、MAP2K2和AKT1的差异表达，其中p-JUN显著下调，p-AKT1和p-MAP2K2显著上调（P<0.01）。
DENV-2感染的HUVECs表现出众多差异表达的蛋白质。p-JUN的下调以及p-MAP2K2和p-AKT1的上调表明它们在调节自噬中可能发挥的作用，这可能参与了DENV-2感染的机制。
