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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 D(0) (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.

Bcl-2 (B cell lymphoma-2) is an anti-apoptotic member of Bcl-2 family and its overexpression causes development of several types of cancer. The BH3 domain of pro-apoptotic and BH3-only proteins is capable of binding to Bcl-2 protein to induce apoptosis. This binding is the basis for the development of novel anticancer drug which would likely antagonize Bcl-2 overexpression. In this study we have identified BH3 domain of Bax (Bax BH3) as potentially the best Bcl-2 antagonist by performing docking of BH3 peptides (peptides representing BH3 domain of pro-apoptotic and BH3-only proteins) into the Bcl-2 hydrophobic groove formed by BH3, BH1 and BH2 domains (also referred as BH3 cleft). To predict the best small antagonist for Bcl-2, three groups of small peptides (pentapeptide, tetrapeptide and tripeptide) were designed and screened against Bcl-2 which revealed the structural importance of a set of residues playing a vital role in interaction with Bcl-2. The docking and scoring function identified KRIG and KRI as specific peptides among the screened small peptides responsible for Bcl-2 neutralization and would induce apoptosis. The applied pharmacokinetic and pharmacological filters to all small peptides signify that only IGD has drug-like properties and displayed good oral bioavailability. However, the obtained binding affinity of IGD to Bcl-2 was diminutive. Hence deprotonation, amidation, acetylation, benzoylation, benzylation, and addition of phenyl, deoxyglucose and glucose fragments were performed to increase the binding affinity and to prevent its rapid degradation. Benzoylated IGD tripeptide (IGD(bzo)) was observed to have increased binding affinity than IGD with acceptable pharmacokinetic filters. In addition, stability of Bcl-2/IGD(bzo) complex was validated by Molecular Dynamics (MD) simulations revealing improved binding energy, salt bridges and strong interaction energies. This study suggests a new molecule that inhibits Bcl-2 associated cancer/tumor regression., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Bcl-2 (B Cell Lymphoma) protein is an anti-apoptotic member of Bcl-2 family, which is comprised of pro- and anti-apoptotic members. It regulates cellular proliferation and death by inter- and intra-family interactions. It has a potential to suppress apoptotic cell death under variety of stress conditions by modulating mitochondrial transmembrane potential. However, prevalence of constitutively activated Bcl-2 cellular activity is not always required in cells; a mechanism likely exists in cells, which controls its activity. When expression of Bcl-2 is unregulated, it generates lymphoma like, follicular B-cell lymphoma. This article reviews the structural and functional regulation of Bcl-2 activity at transcriptional, translational, domain, structural and post-translational level, which also accounts for the effects of its deletion and site-directed mutants in the regulation of cellular proliferation and differentiation in vitro and in vivo. This concisely reviewed information on Bcl-2 helps us to update our understanding of cell death and its modulation by Bcl-2 and its mutant's interaction, which has gained therapeutic benefits in cell growth and proliferation, particularly for sensitive human hematopoietic stem cells.

The sequence of Bcl-2 homology domains, BH1 and BH2, is known to be conserved among anti- and pro-apoptotic members of Bcl-2 family proteins. But structural conservation of these domains with respect to functionally active residues playing role in heterodimerization-mediated regulation of apoptosis has never been elucidated. Here, we have suggested the formation of an active site by structurally conserved residues in BH1 (glycine, arginine) and BH2 (tryptophan) domains of Bcl-2 family members, which also accounts for the functional effect of known mutations in BH1 (G145A, G145E) and BH2 (W188A) domains of Bcl-2.