Abstract
Selective targeting of apoptosis in vivo is a promising pharmacologic strategy for subverting cancer. BCL-2 family protein interactions constitute a critical control point for the regulation of apoptosis. Whereas multidomain anti-apoptotic proteins such as BCL-2 guard against cell death, multidomain pro-apoptotic proteins such as BAX constitute a gateway to cell death through mitochondrial damage. The BH3-only proteins function as death sentinels situated throughout the cell, poised to transmit signals of cell injury to multidomain members. BH3-only proteins deliver death signals via their alpha-helical BH3 domains, which are either neutralized by anti-apoptotic proteins or delivered, directly or indirectly, to the mitochondrial executioners BAX and BAK. By inserting hydrocarbon “staples” into native BH3 peptide sequences, we have generated a chemical toolbox of stabilized alpha-helices of BCL-2 domains (SAHBs) to dissect apoptotic pathways and develop prototype therapeutics. We previously demonstrated that a stapled peptide corresponding to the BID BH3 domain is a helical, protease-resistant, and cell-permeable compound that binds to anti-apoptotic targets with high affinity and exhibits anti-leukemic activity in vitro and in vivo. Using our expanded panel of compounds, we find that a stapled BAD BH3 likewise displays high affinity binding to select anti-apoptotic targets; however, BID SAHB is uniformly more potent than BAD SAHB in inducing apoptosis of a panel of leukemia cell lines. To explore the molecular mechanism underlying the differential potencies of BID and BAD SAHBs, we evaluated a model in which select BH3-only proteins directly engage pro-apoptotic multidomain proteins to trigger mitochondrial apoptosis. We detect and measure, for the first time, direct binding between select SAHBs, such as BID, and BAX. The observed interaction between BID SAHB and BAX triggered functional activation of BAX in vitro, resulting in mitochondrial cytochrome c release and FITC-dextran release from liposomes. The specificity of the BID SAHB-BAX interaction and its biochemical consequences is highlighted by abrogation of BID SAHB activity by point mutation and by competitive binding to anti-apoptotic BCL-XL. We confirmed the physiologic relevance of our observed in vitro interaction between BID SAHB and BAX by their co-immunoprecipitation from BID SAHB-treated leukemia cells. In contrast, BAD SAHB does not bind or activate multidomain pro-apoptotic BAX. These data provide an initial mechanistic explanation for the relative potency of BID SAHB in activating leukemia cell apoptosis. Thus, bifunctional SAHBs that directly engage both pro- and anti-apoptotic multidomain proteins may be more robust pro-apoptotic therapeutics, compared to compounds that selectively target anti-apoptotic proteins.
Identification and Analysis of the Interaction between Edc3 and Dcp2 in Saccharomyces cerevisiae