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. 2023 Jan 20;43(1):29-38.
doi: 10.12122/j.issn.1673-4254.2023.01.04.

[Phosphoproteomic analysis of human umbilical venous endothelial cells with DENV-2 infection]

[Article in Chinese]
P Hu  1 , Y Cheng  1 , Y Wang  1 , X Gou  2 , H Liu  1 , L Zuo  2 , N Wu  3
Affiliations

[Phosphoproteomic analysis of human umbilical venous endothelial cells with DENV-2 infection]

[Article in Chinese]
P Hu et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

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.

目的: 对登革病毒-2（DENV-2）感染人脐静脉内皮细胞（HUVEC）的差异磷酸化蛋白质进行分析以及初步探究DENV-2感染的可能致病机制。

方法: 实验设置DENV-2感染HUVEC组和HUVEC空白对照组，每组3个重复。收集细胞沉淀后，利用SDT裂解法获取蛋白质，通过串联质谱标签（TMT）法对磷酸化蛋白质进行定性与定量分析，对鉴定到的差异磷酸化蛋白质进行氨基酸保守基序分析、亚细胞定位分析、蛋白质结构域分析、GO富集分析、KEGG通路分析和蛋白质互作（PPI）等生物信息学分析。利用Western blot检测JUN、MAP2K2和AKT1磷酸化蛋白的表达。

结果: 共检测到1385个差异蛋白质上的2918个修饰肽段，其中有1346个修饰肽段显著上调（FC > 1.2，P < 0.05），1572个修饰肽段显著下调（FC < 0.83，P < 0.05）。氨基酸保守基序分析共获得49个磷酸化保守基序。蛋白质结构域分析中富集到的差异磷酸化肽段最多是RNA识别基序、蛋白激酶结构域和PH结构域。通过亚细胞定位分析发现差异的修饰肽段主要定位在细胞核和细胞质中。GO富集和KEGG通路分析结果显示差异肽段主要在刺激反应的调节、含核碱基的小分子生物合成过程、吞噬体和白细胞跨内皮迁移处富集。对上调和下调的差异磷酸化蛋白分别进行蛋白质互作—KEGG联合分析，结果发现上调和下调的差异磷酸化蛋白质共富集到15条通路，且通过Western Blot检测与自噬途径相关的3个差异磷酸化蛋白质即JUN、MAP2K2和AKT1的表达，结果显示DENV-2组与空白组相比，p-JUN表达显著下调（1.48±0.01 vs 1.23±0.01，P < 0.0001）；p-AKT1表达显著上调（0.87±0.02 vs 1.01±0.01，P < 0.001）；p-MAP2K2表达显著上调（1.10±0.02 vs 1.29±0.05，P < 0.01）。

结论: DENV-2感染HUVEC存在较多差异表达蛋白，p-JUN的下调、p-MAP2K2和p-AKT1的上调提示3个能够调节自噬的磷酸化蛋白可能与DENV-2感染机制有关。

Keywords: DENV-2; human umbilical venous endothelial cells; phosphorylation omics.

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Figures

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修饰位点分布分析图 Distribution analysis of the modification sites. A: S/T/Y phosphorylation modification site distribution ratio. B: Number distribution of phosphorylation modification sites. C: Distribution frequency map of phosphorylation modification sites. Among all the identified modified proteins, the average number of phosphorylation modification sites per 100 amino acids is 0.47.
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磷酸化组学差异蛋白数量分析 Quantitative analysis of differentially phosphorylated proteins. A: Correlation coefficient diagram. B: Cluster analysis of differentially expressed phosphorylated peptides. C: Histogram of quantitative difference results of phosphorylated peptides. D: Volcano map.
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保守基序分析 Conserved motif analysis. A: Predicted conservative motif enrichment statistics (top 20). B: Prediction of the number of phosphorylated modified peptides corresponding to motif (top 20). C: Up-regulated and down-regulated phosphorylated peptides are significantly enriched in the top 3 motifs.
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差异表达修饰肽段所属蛋白亚细胞定位饼图 Pie chart of subcellular localization of proteins to which the differentially expressed modified peptides belong.
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结构域分析 Domain analysis. A: Analysis of protein domains of differentially expressed modified peptides (top 20). B: Domain enrichment analysis diagram.
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GO功能分析图 GO function analysis diagram. A: GO annotation statistics of proteins to which the differentially expressed modified peptide belongs. B: GO function enrichment bubble diagram (biological process, BP) under biological process classification. C: GO function enrichment bubble diagram (MF) under molecular function classification. D: GO function enrichment bubble diagram (cellular component, CC) under cell component classification.
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KEGG通路分析图 KEGG pathway analysis. A: KEGG pathway annotation statistics of differentially expressed proteins to which the modified peptides belong (top20). B: KEGG enrichment pathway map of the differentially expressed proteins to which the modified peptide belongs (top20).
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蛋白互作网络分析图及Western blot结果分析统计图 Protein interaction network analysis chart and Western blotting results. A: The interaction network of proteins to which the differentially expressed modified peptides belong. B: Significantly upregulated differentially phosphorylated protein network interaction map. C: Significantly downregulated differentially phosphorylated protein network interaction map. D: Effects of DENV-2 on p-JUN, p-MAP2K2 and p-AKT1 expressions. **P < 0.01, ***P < 0.001, ****P < 0.0001.
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