Phosphoproteomic analysis of human umbilical venous endothelial cells with DENV-2 infection / 南方医科大学学报

OBJECTIVE@#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.@*METHODS@#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.@*RESULTS@#A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 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 (P < 0.01).@*CONCLUSION@#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.

Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

The objective of this study was to observe the infection of human cytomegalovirus (HCMV) to human umbilical vein endothelial cells, and its effect on the expression of single-stranded DNA-binding protein (SSBP1) and on lipid metabolism in endothelial cells. We screened the differential expression of mRNAs after HCMV infection by suppression subtractive hybridization and the expression levels of SSBP1 mRNA and protein after HCMV infection by real-time PCR and western blot. After verification of successful infection by indirect immunofluorescent staining and RT-PCR, we found a differential expression of lipid metabolism-related genes including LDLR, SCARB, CETP, HMGCR, ApoB and LPL induced by HCMV infection. The expression levels of SSBP1 mRNA and protein after HCMV infection were significantly down-regulated. Furthermore, we found that upregulation of SSBP1 inhibited the expression of atherosclerosis-associated LDLR, SCARB, HMGCR, CETP as well as the accumulation of lipids in the cells. The results showed that the inhibition of SSBP1 by HCMV infection promotes lipid accumulation in the cells.