BH3 domains other than Bim and Bid can directly activate Bax/Bak

Abstract

Bcl-2 family proteins regulate a critical step in apoptosis referred to as mitochondrial outer membrane permeabilization (MOMP). Members of a subgroup of the Bcl-2 family, known as the BH3-only proteins, activate pro-apoptotic effectors (Bax and Bak) to initiate MOMP. They do so by neutralizing pro-survival Bcl-2 proteins and/or directly activating Bax/Bak. Bim and Bid are reported to be direct activators; however, here we show that BH3 peptides other than Bim and Bid exhibited various degrees of direct activation of the effector Bax or Bak, including Bmf and Noxa BH3s. In the absence of potent direct activators, such as Bim and Bid, we unmasked novel direct activator BH3 ligands capable of inducing effector-mediated cytochrome c release and liposome permeabilization, even when both Bcl-xL- and Mcl-1-type anti-apoptotic proteins were inhibited. The ability of these weaker direct activator BH3 peptides to cause MOMP correlated with that of the corresponding full-length proteins to induce apoptosis in the absence of Bim and Bid. We propose that, in certain contexts, direct activation by BH3-only proteins other than Bim and Bid may significantly contribute to MOMP and apoptosis.

FIGURE 1.

Cytochrome c release by BH3 ligands from Bim/Bid DKO MEFs. A, amino acid sequences of BH3 peptides (human) used in this study. B, Bim/Bid DKO MEFs were permeabilized with digitonin and incubated with cleaved Bid protein (N/C-Bid) or the BH3 peptides. The Bim BH3 peptide had the most potent cytochrome c releasing activity; the Bid, Bmf, and Puma BH3 peptides also had some activity at higher concentrations. Blots are representative of three independent experiments. C, BH3 peptides at 50 μm did not release cytochrome c in digitonin-permeabilized Bax/Bak DKO MEFs, therefore even at a relatively high concentration of 50 μm, cytochrome c release by BH3 peptides shown in B was Bax/Bak-dependent. Digitonin permeabilization was confirmed by cytochrome c release observed in the control in which Bax (480 nm) and N/C-Bid (45 nm) were added. D, Bcl-xL, Bcl-2 and Mcl-1 were immunoprecipitated from CHAPS or Nonidet P-40-solubilized Bim/Bid DKO MEFs and probed for Bax or Bak. L, lysate or supernatant; B, beads. Bax and Bak were found not associated with Bcl-xL or Bcl-2 in these cells with either detergent. Bax was not associated with Mcl-1, whereas ∼10–15% of Bak was bound to Mcl-1, estimated from the fact that 7.5% lysate and 50% beads samples were loaded on the gel (see also Fig. 6, B and C). E, Bcl-2 proteins detected in WT MEFs and Bim/Bid DKO MEFs. Protein was loaded at 50 μg per lane, and was detected by immunoblotting using specific antibodies. Equal loading was confirmed by immunoblotting for actin (bottom panels). Puma is not detectable in either WT MEFs or DKO MEFs (data not shown) (27). Bmf was barely detectable in the DKOs. There are no antibodies suitable for detection of mouse Noxa. Bad was not detectable in these cells by a few different antibodies (data not shown). The mRNA of A1, another anti-apoptotic Bcl-2 protein, is not present in MEFs (29). WT MEFs express similar levels of Bax and Bak, as estimated based on standards using recombinant Bax and cBak, respectively. The Bak and Bcl-2 levels in Bim/Bid DKO MEFs were found to be lower than those in the WT.

FIGURE 1.

Cytochrome c release by BH3 ligands from Bim/Bid DKO MEFs. A, amino acid sequences of BH3 peptides (human) used in this study. B, Bim/Bid DKO MEFs were permeabilized with digitonin and incubated with cleaved Bid protein (N/C-Bid) or the BH3 peptides. The Bim BH3 peptide had the most potent cytochrome c releasing activity; the Bid, Bmf, and Puma BH3 peptides also had some activity at higher concentrations. Blots are representative of three independent experiments. C, BH3 peptides at 50 μm did not release cytochrome c in digitonin-permeabilized Bax/Bak DKO MEFs, therefore even at a relatively high concentration of 50 μm, cytochrome c release by BH3 peptides shown in B was Bax/Bak-dependent. Digitonin permeabilization was confirmed by cytochrome c release observed in the control in which Bax (480 nm) and N/C-Bid (45 nm) were added. D, Bcl-xL, Bcl-2 and Mcl-1 were immunoprecipitated from CHAPS or Nonidet P-40-solubilized Bim/Bid DKO MEFs and probed for Bax or Bak. L, lysate or supernatant; B, beads. Bax and Bak were found not associated with Bcl-xL or Bcl-2 in these cells with either detergent. Bax was not associated with Mcl-1, whereas ∼10–15% of Bak was bound to Mcl-1, estimated from the fact that 7.5% lysate and 50% beads samples were loaded on the gel (see also Fig. 6, B and C). E, Bcl-2 proteins detected in WT MEFs and Bim/Bid DKO MEFs. Protein was loaded at 50 μg per lane, and was detected by immunoblotting using specific antibodies. Equal loading was confirmed by immunoblotting for actin (bottom panels). Puma is not detectable in either WT MEFs or DKO MEFs (data not shown) (27). Bmf was barely detectable in the DKOs. There are no antibodies suitable for detection of mouse Noxa. Bad was not detectable in these cells by a few different antibodies (data not shown). The mRNA of A1, another anti-apoptotic Bcl-2 protein, is not present in MEFs (29). WT MEFs express similar levels of Bax and Bak, as estimated based on standards using recombinant Bax and cBak, respectively. The Bak and Bcl-2 levels in Bim/Bid DKO MEFs were found to be lower than those in the WT.

FIGURE 2.

Recombinant cBak permeabilizes the liposome membrane and mitochondria. A, schematic diagrams of native Bak and cBak. Recombinant cBak lacks the N-terminal 15 amino acids and the C-terminal 25. B, cBak, N/C-Bid and Bcl-xL were incubated with fluorescein (F)-dextran-loaded liposomes, and dextran release was measured as reported previously (13). The liposomes were recovered by float-up centrifugation and Bak associated with the membranes was detected by immunoblotting. cBak association with the membrane was promoted by N/C-Bid, and this effect was blocked by Bcl-xL. Liposome permeabilization correlated with the amount of cBak in the membrane. Blots shown are representative of five experiments. C, N/C-Bid induced oligomerization of cBak in the membrane. cBak and N/C-Bid-incubated liposomes from the middle lane in Fig. 2B were solubilized in 2% CHAPS and fractionated by gel-filtration (N/C-Bid treated). As a control, total cBak that was incubated with liposomes in the absence of cleaved Bid was fractionated and found to be monomeric (Non treated). D, cBak was incubated with mouse liver mitochondria devoid of Bax and Bak. The Bim, Bid, and Bmf BH3 peptides stimulated robust cytochrome c release, and the Hrk, Puma, and Bik BH3 showed a modest release (upper panel). cBak oligomers were detected (lower panel). Note that cytochrome c release correlated well with the formation of cBak oligomers. E, BH3 peptides (25 μm) were added to mitochondria isolated from wild type mouse livers. The profiles of cytochrome c release in response to native Bak in combination with BH3 peptides was similar to those for cBak with BH3 peptides; Bim and Bid BH3 were highly potent, and Bmf and Puma BH3 also triggered moderate release consistently. We used mouse BH3 peptides for Bid and Bmf and the rest were human BH3s.

FIGURE 3.

Bax and cBak activation by BH3 ligands reveal low-grade direct activation. A, BH3 peptides alone do not trigger liposome permeabilization. Bax (120 nm), N/C-Bid (45 nm), or BH3 peptides (25 μm) were added to liposomes containing fluorescein (F)-dextran (10 kDa). B and C, direct activation of Bax or cBak by a panel of BH3 peptides. The potency of direct Bax activation ranked Bim BH3 as the strongest activator, followed by Bid, Bmf, and Noxa BH3 as moderate activators, Bik, Hrk, and Puma BH3 as weak activators and Bad BH3 as non-activating. D, difference in background release (Bax-alone release; black bars) does not lead to false direct activation. Bad BH3 peptide was consistently unable to directly activate Bax (gray bars) over a range (8–42%) of Bax alone-induced permeabilization. E, a time course of dextran release demonstrates low-grade direct activation by Bmf and Noxa BH3 (upper panel) and Bik, Hrk, and Puma BH3 (lower panel). Bim and Bmf BH3 peptides were included in both assays to show the relative potency between the peptides. Permeabilization induced by weak direct activators was slow, but notably higher than the Bax-alone background, which is consistent with the end-point assay. F, CD confirms that all the BH3 peptides used in this study were intrinsically disordered at 10 mm phosphate buffer of pH 7.0. The poor solubility of Bik BH3 in aqueous solutions required the use of 0.1% DMSO in the CD sample, which introduced significant noise in the data below 200 nm. Although the Bim and Noxa BH3 peptides apparently dissolved in water, they may be less soluble in the CD buffer, illustrated by a less pronounced ellipticity drop at 200 nm.

FIGURE 4.

BH3 ligands show direct activation when anti-apoptotic proteins are inhibited. A, in the presence of Bcl-xL, Bax was activated by the same set of BH3 peptides as in the absence of Bcl-xL, with the exception that Noxa BH3 was ineffective. B denotes the fact that this situation was addressed in the following panel, B. B, bad BH3 peptide presumably removed Bcl-xL inhibition, revealing direct activation of Bax by Noxa BH3. C, in the presence of Bcl-xL, cBak was only activated by Bim and Bmf BH3. D and E denote the fact that these situations were addressed in the following panels, D and E, respectively. D, bad BH3 interfered with Bcl-xL, revealing direct activation of cBak by Bid BH3. E, Bad BH3 inhibited Bcl-xL, uncovering direct activation of cBak by Noxa BH3. F and G, in the presence of Mcl-1, Bax and cBak were directly activated by the same peptides that directly activated them in the absence of Mcl-1, except for Puma BH3 exhibiting more potent direct activation in both cases and Noxa BH3 failing to activate cBak.

FIGURE 5.

BH3 ligands overcome effector inhibition by anti-apoptotics in liposomes. A, high levels of Bax (left panel) or cBak (right panel) led to significant permeabilization of liposomes, and this permeabilization was inhibited in a dose-dependent manner by Bcl-xL or Mcl-1. B, both Bad BH3 and its mimetic, ABT 737, were capable of interfering with the inhibition of Bax or cBak by Bcl-xL or Mcl-1.

FIGURE 6.

Bak dissociation from Mcl-1 correlates with cytochrome c release in permeabilized cells. A, cytochrome c release from mitochondria in Bim/Bid DKO MEFs, as in Fig. 1. Various BH3 peptides were added at 50 μm to digitonin-permeabilized Bim/Bid DKO MEFs for immunoprecipitation studies whose outcomes are shown in B and C. The combination of Noxa and Bad BH3 peptides triggered cytochrome c release, as previously reported. B, effects of Mcl-1 and Bak in the supernatant after immunoprecipitation with anti-Mcl-1 antibody. The cell pellet in A after the incubation with the peptide was dissolved in 1% CHAPS, and immunoprecipitation was performed. Mcl-1 was depleted from the lysate. The decrease in Bak levels was not detectable on the blot, presumably because only ∼10–15% of Bak was co-immunoprecipitated. C, Bak co-immunoprecipitation with Mcl-1 was inversely correlated to cytochrome c release; the less Mcl-1 associated with Bak, the more cytochrome c was released. D, apoptosis induced by overexpressing BH3-only protein correlated with cytochrome c release induced by BH3 peptides. We used mouse genes except for Bmf, which was human. The transfection efficiency across samples was comparable based on the FACS-based measurement of the expression of IRES-driven GFP expression from the same plasmid. All the data in this figure are representative of 3–7 experiments.

FIGURE 7.

Summary of interactions governing the Bcl-2 family proteins. We propose that there are three-way interactions between BH3-only proteins, anti-apoptotic proteins, and Bax/Bak. A, MOMP results when both the Bcl-xL- and Mcl-1-type anti-apoptotic Bcl-2 proteins are inhibited, and Bax/Bak are directly activated. BH3-only proteins displace activator BH3-only proteins and/or Bax/Bak. B, even when all anti-apoptotic proteins are inhibited and Bax/Bak are liberated, Bax/Bak still need to be directly activated before MOMP occurs. C, inhibiting only one type of pro-survival Bcl-2 protein is not sufficient to induce MOMP.