Modulation of Kinase Activities In Vitro by Hepatitis C Virus Protease NS3/NS4A Mediated-Cleavage of Key Immune Modulator Kinases
Abstract
:1. Introduction
2. Experimental Procedures
2.1. Peptide Synthesis
2.2. Protein Expression and Purification of HCV NS3
2.3. Purification of Recombinant Human IKKα, IKKβ, IKKε, and TBK 1
2.4. Kinase Digestion Condition for Western Blot Analysis
2.5. MALDI-TOF and Nano-LC MS/MS Analysis
2.6. Kinase Digestion Conditions for Pre-Steady-State Kinetic Assays
2.7. Kinase Digestion Condition for Kinase Activity Assay
2.8. Protease Inhibition Assay
2.9. Kinase Activity Assay
3. Results
3.1. IKKα, IKKβ, IKKε, and TBK1 Are Substrates for NS3/NS4A Protease In Vitro
3.2. Estimation of the Substrate Specificity of IKKε by Pre-Steady-State Kinetic Methods
3.3. NS3/NS4A Modulates Kinase Activity
3.4. The NS3/NS4A Proteolytic Activity Is Essential for Modulation of the Kinase Activities of Both IKKε and TBK1
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Proteins | Residue Number of P6 | P6 P5 P4 P3 P2 P1 ↓ P1′ P2′ P3′ P4′ |
---|---|---|
NS4A/NS4B | D E M E E C ↓ A S H L | |
NS4B/NS5A | D C S T P C ↓ S G S W | |
NS5A/NS5B | E D V V C C ↓ S M S Y | |
IKKα | 173 | D Q G S L C ↓ T S F V |
374 | D G V R G C ↓ D S Y M | |
691 | D H S L S C ↓ V V T P | |
711 | E E N L N C ↓ L G H L | |
IKKβ | 174 | D Q G S L C ↓ T S F V |
711 | E A H N L C ↓ T L L E | |
IKKε | 84 | L V M E Y C ↓ S S G S |
504 | E V L S R C ↓ S Q N I | |
621 | L R L V G C ↓ S V A A | |
TBK1 | 262 | D M P V S C ↓ S L S R |
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Abdullah, M.A.F.; McWhirter, S.M.; Suo, Z. Modulation of Kinase Activities In Vitro by Hepatitis C Virus Protease NS3/NS4A Mediated-Cleavage of Key Immune Modulator Kinases. Cells 2023, 12, 406. https://doi.org/10.3390/cells12030406
Abdullah MAF, McWhirter SM, Suo Z. Modulation of Kinase Activities In Vitro by Hepatitis C Virus Protease NS3/NS4A Mediated-Cleavage of Key Immune Modulator Kinases. Cells. 2023; 12(3):406. https://doi.org/10.3390/cells12030406
Chicago/Turabian StyleAbdullah, Mohd Amir F., Sarah M. McWhirter, and Zucai Suo. 2023. "Modulation of Kinase Activities In Vitro by Hepatitis C Virus Protease NS3/NS4A Mediated-Cleavage of Key Immune Modulator Kinases" Cells 12, no. 3: 406. https://doi.org/10.3390/cells12030406
APA StyleAbdullah, M. A. F., McWhirter, S. M., & Suo, Z. (2023). Modulation of Kinase Activities In Vitro by Hepatitis C Virus Protease NS3/NS4A Mediated-Cleavage of Key Immune Modulator Kinases. Cells, 12(3), 406. https://doi.org/10.3390/cells12030406