Novel protein kinase C phosphorylated kinase inhibitor-matrine suppresses replication of hepatitis B virus via modulating the mitogen-activated protein kinase signal

Abstract

HBV (hepatitis B virus) infection still threatens human health. Therefore, it is essential to find new effective anti-HBV compounds. Here, we identified matrine as a novel inhibitor of PKC (protein kinase C) phosphorylated kinase by screening a natural compound library. After HepG2.215 cells were treated with matrine, we carried out a phosphorylated proteomics sequence study and analyzed the prediction of related kinase expression level. In the case of HBV infection, it was found that PKC kinase mediates the activation of mitogen-activated protein kinase (MAPK) signaling pathway known as son of sevenless (SOS) activation. It was also found that PKC kinase inhibits the expression of C-X-C Motif Chemokine Ligand 8 (CXCL8) by inhibiting the activity of activating transcription factor 2/ cAMP response element binding protein (ATF2/CREB), and this effect is independent of its activated MAPK signaling pathway. Finally, Western blot was used to detect the expression of MAPK, ATF2, CREB3 phosphorylation and nonphosphorylation in matrine-treated cells and PKC-treated cells. PKC phosphorylated kinase inhibitor-matrine suppresses the replication of HBV via modulating the MAPK/ATF2 signal. Matrine is a good clinical drug to enhance the autoimmunity in the adjuvant treatment of chronic HBV infection.

Keywords: HBV; MAPK signaling pathway; Matrine; PKC; phosphoproteome analysis.

Graphical abstract

Figure 1.

Matrine inhibits HBV DNA replication in HepG2.2.15 cells. (a) HepG2.2.15 cells in 24-well plates were cultured with 300 μL of 1 μM or 5 μM matrine MEM medium or ordinary MEM medium according to groups. After three days, the medium was changed, and 200 μL of supernatant was collected on the 7th day of the experiment. The quantity of HBV DNA in the samples was detected using a PCR fluorescence probe method. (b) The expression of CXCL8 was detected by ELISA.

Figure 2.

Analysis of the phosphorylated proteome in HepG2.215 cells treated with matrine. (a) Flow chart of sample quantitative phosphorylation proteome identification. HepG2.215 cells were cultured in 5 μM matrine medium and normal MEM medium respectively for 7 days. LC-MS/MS analysis, database search, and bioinformatics analysis were performed as described previously. (b) Pearson correlation coefficient thermogram of quantitative modification between two samples. (c) Two-dimensional scatter plot of PCA distribution of all samples using quantified proteins. (d) Box-plot graphs represent the distribution of sample means calculated for two repeated assays. (e) Peptide length distribution. (f)Distribution of modification sites.

Figure 3.

Matrine-induced differential phosphorylation of nucleoprotein. (a) The results of mass spectrum data are provided as a statistical chart. (b) Motif enrichment heat map of phosphorylation. Red color indicates that this amino acid was significantly enriched near the modification site, and green color indicates that the amount of this amino acid was significantly reduced near the modification site. (c) Histogram of differential modified protein and modified site number distribution: The quantitative value of modified peptides corresponding to each sample was detected by mass quantitative spectrometric analysis. For each repeated experiment, the ratio of the quantitative value of modified peptides between two different samples was taken as the differential expression of comparison group (ratio). For each comparison group, the average value of the two repeated ratios was taken as the comparison group’s ratio, and the coefficient of variation, CV of the two repeated ratios as the comparison group’s CV value. When CV value < 0.1, more than 1.2 of the differential expression was taken as the threshold of significant upregulation, and less than 1/1.2 was taken as the threshold of significant downregulation. (d) Subcellular localization of proteins corresponding to different phosphorylation sites.

Figure 4.

Differential modifications were significantly enriched in some functional types. (a) COG/KOG categories of differential modified proteins. (b) The proteins corresponding to different phosphorylation sites in Molecular Function. The vertical axis shows the functional classification or pathway, and the horizontal axis shows a log2-converted value of the proportion of differential protein in this functional type compared with the multiple of the proportion of identifying proteins. The circle color indicates the enrichment of significant P-value, and the circle size indicates the number of differential proteins in the functional class or pathway. (c–e) Visual display of significant enrichment of proteins corresponding to differential modification sites in a KEGG pathway. Red color indicates differently upregulated proteins; green color indicates differently downregulated proteins; yellow color indicates the presence of multiple proteins in this node, including differently upregulated and differently downregulated proteins. (f) The protein corresponding to different phosphorylation sites was enriched and distributed in KEGG pathway. (g) Cluster analysis heat map. The color blocks, corresponding to the differently expressed proteins and functional descriptions of different groups, indicate the degree of enrichment. Red color indicates strong enrichment, and blue color indicates weak enrichment.

Figure 4.

Continued.

Figure 5.

Matrine promotes the expression of CXCL8 through the PKC-dependent signaling pathway. (a) Protein–protein interaction network between kinase and substrate. The yellow octagon represents the kinase, and the circle represents the phosphorylation site. The red circle represents the upregulation site, and the blue circle represent the downregulation site. (b) Highly enriched kinase. (The NOM p-value was less than 0.01) Red was highly upregulated kinase, and blue was highly downregulated kinase. (c) The KEGG pathway. (d) Western blot was used to detect the expression of MAPK, ATF2, and CREB3 phosphorylation and nonphosphorylation in matrine-treated cells and PKC-treated cells.

Figure 6.

Mechanism showing that matrine increases the expression of inflammatory chemokine CXCL8 to inhibit HBV DNA replication through the PKC-MAPK-ATF2/CREB signaling pathway.