Bak BH3 peptides antagonize Bcl-x(L) function and induce apoptosis through cytochrome c-independent activation of caspases

Abstract

The Bcl-2 homology 3 (BH3) domain is crucial for the death-inducing and dimerization properties of pro-apoptotic members of the Bcl-2 protein family, including Bak, Bax, and Bad. Here we report that synthetic peptides corresponding to the BH3 domain of Bak bind to Bcl-x(L), antagonize its anti- apoptotic function, and rapidly induce apoptosis when delivered into intact cells via fusion to the Antennapedia homeoprotein internalization domain. Treatment of HeLa cells with the Antennapedia-BH3 fusion peptide resulted in peptide internalization and induction of apoptosis within 2-3 h, as indicated by caspase activation and subsequent poly(ADP-ribose) polymerase cleavage, as well as morphological characteristics of apoptosis. A point mutation within the BH3 peptide that blocks its ability to bind to Bcl-x(L) abolished its apoptotic activity, suggesting that interaction of the BH3 peptide with Bcl- 2-related death suppressors, such as Bcl-x(L), may be critical for its activity in cells. While overexpression of Bcl-x(L) can block BH3-induced apoptosis, treatment with BH3 peptides resensitized Bcl-x(L)-expressing cells to Fas-mediated apoptosis. BH3-induced apoptosis was blocked by caspase inhibitors, demonstrating a dependence on caspase activation, but was not accompanied by a dramatic early loss of mitochondrial membrane potential or detectable translocation of cytochrome c from mitochondria to cytosol. These findings demonstrate that the BH3 domain itself is capable of inducing apoptosis in whole cells, possibly by antagonizing the function of Bcl-2- related death suppressors.