Investing the Molecular Mechanism of Bcl-2, Bax and tBid in the Regulation of Cytochrome c Release from Mitochondria

Abstract

<p>Proteins in the Bcl-2 family are important regulators of apoptosis, but their exact mechanism of action remains unknown. To study the interactions of the anti-apoptotic protein Bcl-2 and the pro-apoptotic proteins Bax and tBid, a cell free assay was developed using mitochondria isolated from cultured cells. Using this assay we were able to show that tBid activates Bax, resulting in the oligomerization of Bax on the membrane. Bcl-2 inhibits Bax oligomerization by sequestering tBid-activated Bax.</p> <p>Bcl-2 is a transmembrane protein that adopts a tail-anchored topology with the hydrophobic carboxyl-terminus of helix 9 inserted into the membrane in healthy cells. After exposure to apoptotic stimuli, Bcl-2 changes conformation such that cysteine 158 that is located in helix 5 is inserted into the lipid bilayer. The experiments presented here show that exposure of Bcl-2 in isolated heavy membranes to either recombinant tBid or a peptide corresponding to the BH3 region of Bim triggered a similar conformational change. The data from the cell free assay showed that Bcl-2 proteins that have inserted cysteine 158 into the membrane are competent to prevent cytochrome c release <em>in vitro</em>. Taken together, these results suggest a model of Bcl-2 function, in which the conformationally modified form of Bcl-2 binds to Bax to prevent it from oligomerizing to form large molecular weight multimers that are competent release pro-apoptotic factors from the inter-membrane space of mitochondria. Thus these studies have provided valuable insight into both the complexity and molecular mechanisms ofthe Bcl-2 family ofproteins in the regulation of apoptosis.</p> <p>The ability of Bc1-2 expression to inhibit apoptosis has been linked to the expression ofthe proto-oncogene Myc. In cells, the absence ofmyc is associated with a marked decrease in the ability to undergo apoptosis. The effects of Myc were studied using the cell free assay with mitochondria isolated from Myc overexpressing and Myc null cells. The results show that mitochondria from Myc null cells are dramatically more resistant to tBid and Bax induced cytochrome c release than mitochondria from Myc overexpressing cells. Cytosol switching experiments confirmed that the defect in cytochrome c release after exposure to apoptotic stimuli in cells lacking myc is inherent to the mitochondria.</p>