Phosphoproteomics Unravel HBV Triggered Rewiring of Host Phosphosignaling Events
Abstract
:1. Introduction
2. Results
2.1. Altered Phosphoproteome Landscape during HBV-Infection
2.2. Dysregulation of Biological Processes during HBV Infection
2.3. HBV Infection-Induced Host Kinome
2.4. Rewiring of Host Phosphosignaling Pathway during HBV Infection
2.5. Assessing Deregulated Kinases as Therapeutic Targets for HBV
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. HBV Infection
4.3. Extraction of RNA and DNA
4.4. Real-Time Quantitative PCR and HBeAg ELISA
4.5. Immunofluorescence
4.6. Cell Lysate Preparation
4.7. Reduction, Alkylation and Digestion
4.8. Tandem Mass Tag (TMT) Labelling
4.9. Phosphopeptide Enrichment
4.10. Mass Spectrometry Data Acquisition
4.11. Identification and Quantification of Phosphopeptides
4.12. Analysis of Differentially Regulated Phosphoproteins
4.13. Kinase–Substrate Prediction and Enrichment Assay
4.14. Cytotoxicity Assay
4.15. Kinase Inhibitor Treatment of HBV Infected HepG2-NTCP
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Kinases Inhibitor | Target | Reported Ki | IC50 |
---|---|---|---|
DMAT | CK2 | 40 nM | >500 µM |
RO-3306 | CDK1 | 35 nM | 164 µM |
PNU112455A11 | CDK2 and CDK5 | 3.2/3.6 µM (CDK2/5) | >500 µM |
MU1210 | CLKs | 23 nM | 16.7 mM |
MU140 | NA (Structural analog of NU1210) | NA | >5 00 µM |
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Lim, Z.; Mohd-Ismail, N.K.B.; Png, E.; Sze, C.W.; Lin, Q.; Hong, W.; Lim, S.G.; Tan, Y.-J.; Gunaratne, J. Phosphoproteomics Unravel HBV Triggered Rewiring of Host Phosphosignaling Events. Int. J. Mol. Sci. 2022, 23, 5127. https://doi.org/10.3390/ijms23095127
Lim Z, Mohd-Ismail NKB, Png E, Sze CW, Lin Q, Hong W, Lim SG, Tan Y-J, Gunaratne J. Phosphoproteomics Unravel HBV Triggered Rewiring of Host Phosphosignaling Events. International Journal of Molecular Sciences. 2022; 23(9):5127. https://doi.org/10.3390/ijms23095127
Chicago/Turabian StyleLim, ZiJie, Nur Khairiah Binte Mohd-Ismail, Evelyn Png, Ching Wooen Sze, Qifeng Lin, Wanjin Hong, Seng Gee Lim, Yee-Joo Tan, and Jayantha Gunaratne. 2022. "Phosphoproteomics Unravel HBV Triggered Rewiring of Host Phosphosignaling Events" International Journal of Molecular Sciences 23, no. 9: 5127. https://doi.org/10.3390/ijms23095127