Experimental Characterization of the Binding Affinities between Proapoptotic BH3 Peptides and Antiapoptotic Bcl-2 Proteins.

The Bcl-2 family proteins are key regulators of the intrinsic apoptotic pathway and are among the validated targets for developing anticancer drugs. Protein-protein interactions between the pro- and antiapoptotic members of this family determine mitochondrial outer-membrane permeabilization. Elucidating such protein-protein interactions in a quantitative way is helpful for network pharmacology studies on the Bcl-2 family, which, in turn, will provide valuable guidance for developing new anticancer therapies. In this study, the binding affinities of the BH3 peptides derived from eight proapoptotic BH3-only proteins (i.e., Bid, Bim, Puma, Noxa, Bad, Bmf, Bik, Hrk) against five well-studied antiapoptotic proteins (i.e., Bcl-xL , Bcl-2, Mcl-1, Bcl-w, Bfl-1) in the Bcl-2 family have been measured. Three different types of binding assay (i.e., surface plasmon resonance, fluorescence polarization, and homogeneous time-resolved fluorescence) were employed for cross-validation. The results confirmed that each proapoptotic BH3 peptide exhibited a distinct binding profile against the five antiapoptotic proteins. The binding data obtained herein serve as a fresh update or correction to existing knowledge. It is expected that such binding data will be helpful for building more accurate mathematical network models for depicting the complex protein-protein interactions within the Bcl-2 family.