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Investigating the Molecular Basis of c-FLIP/Calmodulin Interaction for Modulating Apoptosis
CC BY-NC-ND 4.0 · Indian J Med Paediatr Oncol 2022; 43(S 01): S1-S19
DOI: DOI: 10.1055/s-0042-1755515
Correspondence to: kbose@actrec.gov.in
Background: Overexpression of antiapoptotic protein c-FLIP in cancer and its interference with DISC leads to apoptosis evasion. Preventing c-FLIP recruitment to DISC is difficult due to its structural similarity with procaspase 8. The c-FLIP needs to bind to calmodulin to access the DISC. Thus, targeting c-FLIP/CaM interaction can be a good strategy.
Materials and Methods: The genes were cloned in expression vectors. Proteins were purified using Ni-NTA and amylose resins followed by circular dichroism and fluorescence spectroscopy. Interactions were checked using pull-down assays. Schrodinger software was used for in silico experiments. Calmodulin Sepharose resin was used to check interactions with mutant and wildtype c-FLIP.
Results: We confirmed the interaction between the two proteins and also the critical amino acid residues of the binding interface of CaM/c-FLIP using in silico methods, protein engineering, and biochemical tools. The structural conformation and thermal stability of the purified mutant and wild type proteins was determined, and they were found to be stable. Furthermore, using this information, we have designed peptides that interfere with this binding. Crystal trials for the c-FLIP DED domains showed microcrystal formation in few buffer systems.
Conclusion: This array of information will be crucial toward devising small molecules for therapeutic benefit after appropriate testing and validation.
Publication History
Article published online:
22 August 2022
© 2022. Indian Society of Medical and Paediatric Oncology. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)
Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, 2nd Floor, Sector 2, Noida-201301 UP, India
Correspondence to: kbose@actrec.gov.in
Background: Overexpression of antiapoptotic protein c-FLIP in cancer and its interference with DISC leads to apoptosis evasion. Preventing c-FLIP recruitment to DISC is difficult due to its structural similarity with procaspase 8. The c-FLIP needs to bind to calmodulin to access the DISC. Thus, targeting c-FLIP/CaM interaction can be a good strategy.
Materials and Methods: The genes were cloned in expression vectors. Proteins were purified using Ni-NTA and amylose resins followed by circular dichroism and fluorescence spectroscopy. Interactions were checked using pull-down assays. Schrodinger software was used for in silico experiments. Calmodulin Sepharose resin was used to check interactions with mutant and wildtype c-FLIP.
Results: We confirmed the interaction between the two proteins and also the critical amino acid residues of the binding interface of CaM/c-FLIP using in silico methods, protein engineering, and biochemical tools. The structural conformation and thermal stability of the purified mutant and wild type proteins was determined, and they were found to be stable. Furthermore, using this information, we have designed peptides that interfere with this binding. Crystal trials for the c-FLIP DED domains showed microcrystal formation in few buffer systems.
Conclusion: This array of information will be crucial toward devising small molecules for therapeutic benefit after appropriate testing and validation.
No conflict of interest has been declared by the author(s).
Publication History
Article published online:
22 August 2022
© 2022. Indian Society of Medical and Paediatric Oncology. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)
Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, 2nd Floor, Sector 2, Noida-201301 UP, India