BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.

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The multidomain pro-apoptotic proteins BAX and BAK constitute an essential gateway to mitochondrial dysfunction and programmed cell death. Among the "BCL-2 homology (BH) 3-only" members of pro-apoptotic proteins, truncated BID (tBID) has been implicated in direct BAX activation, although an explicit molecular mechanism remains elusive. We find that BID BH3 peptide alone at submicromolar concentrations cannot activate BAX or complement BID BH3 mutant-tBID in mitochondrial and liposomal release assays. Because tBID contains structurally defined membrane association domains, we investigated whether membrane targeting of BID BH3 peptide would be sufficient to restore its pro-apoptotic activity. We developed a Ni 2؉ -nitrilotriacetic acid liposomal assay system that efficiently conjugates histidine-tagged peptides to a simulated outer mitochondrial membrane surface. Strikingly, nanomolar concentrations of a synthetic BID BH3 peptide that is chemically tethered to the liposomal membrane activated BAX almost as efficiently as tBID itself. These results highlight the importance of membrane targeting of the BID BH3 domain in tBID-mediated BAX activation and support a model in which tBID engages BAX to trigger its pro-apoptotic activity.BCL-2 family proteins are essential regulators of programmed cell death or apoptosis and are composed of pro-and anti-apoptotic members that display sequence conservation in defined BCL-2 homology (BH) 3 domains (1-4). Whereas multidomain anti-apoptotic proteins such as BCL-2 and BCL-X L share up to four BH domains (BH1-4), multidomain pro-apoptotic proteins such as BAX and BAK contain three BH domains (BH1-3). The "BH3-only" proteins constitute a subset of pro-apoptotic proteins that display sequence conservation exclusively in the BH3 domain. BH3-only proteins antagonize anti-apoptotic proteins through direct interactions (5-8). A discrete subset of BH3-only members, including BIM and BID, are also believed to activate multidomain pro-apoptotic proteins BAX or BAK directly (5-14). BID is a BH3-only protein that connects the extrinsic TNFR1 and Fas death signaling pathway to the mitochondrial amplification cascade of the intrinsic death pathway (5). Engagement of TNFR1 and Fas activates caspase-8, which cleaves p22 BID into N-terminal 7-kDa and C-terminal 15-kDa fragments (6, 7, 15). The C-terminal fragment (p15 BID or tBID) is then myristoylated at the newly generated N-terminal glycine (16). Myristoylated tBID targets mitochondrial contact sites, which are enriched with cardiolipin (17-19). Membrane-targeted tBID triggers the homo-oligomerization of BAX or BAK in the mitochondrial outer membrane (MOM) (9,11,12,20), resulting in MOM permeabilization and release of intermembrane space proteins, including cytochrome c, into the cytosol (21). Released cytochrome c forms an apoptosome complex with Apaf-1 and caspase-9, leading to activation of effector caspases and commitment to cell death (22).The BH3 domain of BID is believed to play an essential role in BAX activation (5). Recent studies ...

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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.