Abstract
The pro-apoptotic Bax protein and its counterplayer, the anti-apoptotic Bcl-2 integral membrane protein, exert their opposing actions at the mitochondrial outer membrane; a process crucial in the regulation of the mitochondrial apoptotic pathway. Overexpression of Bcl-2 causes severe failure in this regulation, leading not only to the development of cancer but especially to drug resistance of numerous tumours. But how Bax and Bcl-2 interact with each other to determine the fate of the cell is still an unsolved mystery. Bcl-2 has been suggested to inhibit apoptosis by directly binding to and sequestering Bax. However, other studies imply that Bcl-2 instead inhibits the BH3-only proteins which normally would activate pro-apoptotic proteins. Co-immunoprecipitation studies have indicated that Bax and Bcl-2 associate with each other, though the few biophysical studies that have been published previously have only involved truncated and/or otherwise mutated Bcl-2 variants. Here we present for the first time biophysical data for the interaction between full-length hydrophobic Bcl-2 and soluble Bax. By using CD spectroscopy protein-protein interactions were detected at low concentration of Brij-35. Fluorescence experiments supported this observation, where the presence of Bax prevented the binding of a Bcl-2-specific ligand to Bcl-2. In addition, a method for the reconstitution of Bcl-2 into proteoliposomes was elaborated. By using a mixture of Brij-35 and Triton X-100, it was possible to incorporate Bcl-2 into DMPC-vesicles, confirmed by ultracentrifugation followed by SDS-PAGE. In summary, this study provides further evidence for the putative interaction between Bax and Bcl-2. Furthermore, a method for reconstituting Bcl-2 into membranes is presented, a prerequisite for future studies of the protein in in vivo-mimicking membrane environment, potentially providing information crucial for understanding the regulation of apoptosis as well as for the design of novel anti-cancer drugs.
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