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Enhanced heterodimerization of Bax by Bcl-2 mutants improves irradiated cell survival

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Abstract

B Cell Lymphoma-2 (Bcl-2) protein suppresses ionizing radiation-induced apoptosis in hemato-lymphoid system. To enhance the survival of irradiated cells, we have compared the effects and mechanism of Bcl-2 and its functional variants, D34A (caspase-3 resistant) and S70E (mimics phosphorylation on S70). Bcl-2 and its mutants were transfected into hematopoietic cell line and assessed for cell survival, clonogenicity and cell cycle perturbations upon exposure to ionizing radiation. The electrostatic potential of BH3 cleft of Bcl-2/mutants and their heterodimerization with Bcl-2 associated X protein (Bax) were computationally evaluated. Correspondingly, these results were verified by co-immunoprecipitation and western blotting. The mutants afford higher radioprotective effect than Bcl-2 in apoptotic and clonogenic assays at D0 (radiation dose at which 37 % cell survival was observed). The computational and functional analysis indicates that mutants have higher propensity to neutralize Bax protein by heterodimerization and have increased caspase-9 suppression capability, which is responsible for enhanced survival. This study implies potential of Bcl-2 mutants or their chemical/peptide mimics to elicit radioprotective effect in cells exposed to radiation.

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Abbreviations

A:

Alanine

BH:

Bcl-2 Homology domain

D:

Aspartic acid

E:

Glutamic acid

G:

Glycine

K:

Lysine

L:

Leucine

N:

Asparagine

P:

Proline

Q:

Glutamine

R:

Arginine

S:

Serine

W:

Tryptophan

H:

Histidine

Bcl-2:

Wild-type protein

Variants or Mutants:

D34A and S70E

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Acknowledgments

Authors are thankful to Director R. P. Tripathi, INMAS, Delhi, India, for his support. Authors express gratitude to Mr. Rohit Mathur, Mr. J. S. Adhikari, Dr. B. S. Dwarakanath, Ms. Divya Khaitan and Mrs. Namita Kalra for flow cytometric measurements and Mr. Ravi Soni and Dr. Anant Narayan Bhatt for irradiation of cells. Authors are grateful to Prof. Jos Domen, Assistant Research Professor of Medicine, Department of Medicine, Division of Medical Oncology and Transplantation, University of Duke, Durham, North Carolina for gifting Bcl-2 cDNA. In addition, authors acknowledge Dr. Pallavi Gupta for proofreading of the manuscript. Director, Dr. B. R. Ambedkar Centre for Biomedical Research (ACBR), Delhi University, Delhi-110007, India, is appreciated for providing permission to use Accelrys Discovery Studio 2.0 system for 3D-modeling and EP calculations. This work was supported by Defence Research and Development Organisation (DRDO), Government of India.

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Authors and Affiliations

  1. Stem Cell and Gene Therapy Research Group, Institute of Nuclear Medicine and Allied Sciences (INMAS), Lucknow Road, Timarpur, Delhi, 110054, India

    Yogesh Kumar Verma, Pawan Kumar Raghav, Rajendra Prasad Tripathi & Gurudutta U. Gangenahalli

  2. Department of Biochemistry, VP Chest Institute, University of Delhi, Delhi, 110007, India

    Hanumantharao Guru Raj

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  1. Yogesh Kumar Verma
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  2. Pawan Kumar Raghav
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  3. Hanumantharao Guru Raj
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  4. Rajendra Prasad Tripathi
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  5. Gurudutta U. Gangenahalli
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Correspondence to Gurudutta U. Gangenahalli.

Additional information

Yogesh Kumar Verma and Gurudutta U. Gangenahalli are equally contributed authors.

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Verma, Y.K., Raghav, P.K., Raj, H.G. et al. Enhanced heterodimerization of Bax by Bcl-2 mutants improves irradiated cell survival. Apoptosis 18, 212–225 (2013). https://doi.org/10.1007/s10495-012-0780-8

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