Regulation of Apoptosis at the Mitochondrial Level by Bcl-2 Proteins

Abstract

During the life-time of higher organisms the turnover of the cell mass has to be highly regulated by apoptosis, one of the main types of programmed cell death. The anti-apoptotic integral membrane protein Bcl-2 belongs to the Bcl-2 protein family, which functions as a major gatekeeper in the mitochondrial apoptotic pathway. This protein has been found to be highly over-expressed in breast cancer tumors and also been shown to be involved in the inherent resistance to anti-cancer drugs. Our aim is to work with the full-length Bcl-2 protein and study its interplay with both, its mitochondrial membrane environment and the pro-apoptotic Bcl-2 family protein Bax. Bax is the counter-player of Bcl-2 and is upon activation translocated from the cytosol to the mitochondrial outer membrane where it forms oligomers. These oligomers function as pores which enable the release of cytochrome c to the cytosol, followed by initiation of the caspase activation cascade and subsequently cell death.So far we have been working on the expression and purification of Bax and Bcl-2, and managed to isolate full-length Bax using E. coli as expression system. For Bcl-2 we have performed cell free expression, and have succeeded in the purification of the full-length protein, solubilized with detergent. The next step is to characterize the proteins when they are reconstituted into model membrane, using spectroscopic methods such as CD and 31P solid state NMR spectroscopy. We are especially interested in how the presence of oxidized lipids influences the protein-protein and protein-membrane interactions, mimicking the apoptotic conditions triggered by reactive oxygen species. Our ultimate goal is to provide structural information of the membrane-mediated mechanism underlying the action of Bcl-2 as a potent inhibitor of cell death, information crucial for the development of new anti-cancer drugs.