BH3-only proteins target BCL-xL/MCL-1, not BAX/BAK, to initiate apoptosis

Abstract

It has been widely accepted that mitochondria-dependent apoptosis initiates when select BH3-only proteins (BID, BIM, etc.) directly engage and allosterically activate effector proteins BAX/BAK. Here, through reconstitution of cells lacking all eight pro-apoptotic BH3-only proteins, we demonstrate that all BH3-only proteins primarily target the anti-apoptotic BCL-2 proteins BCL-xL/MCL-1, whose simultaneous suppression enables membrane-mediated spontaneous activation of BAX/BAK. BH3-only proteins' apoptotic activities correlate with affinities for BCL-xL/MCL-1 instead of abilities to directly activate BAX/BAK. Further, BID and BIM do not distinguish BAX from BAK or accelerate BAX/BAK activation following inactivation of BCL-xL/MCL-1. Remarkably, death ligand-induced apoptosis in cells lacking BH3-only proteins and MCL-1 is fully restored by BID mutants capable of neutralizing BCL-xL, but not direct activation of BAX/BAK. Taken together, our findings provide a "Membrane-mediated Permissive" model, in which the BH3-only proteins only indirectly activate BAX/BAK by neutralizing the anti-apoptotic BCL-2 proteins, and thus allowing BAX/BAK to undergo unimpeded, spontaneous activation in the mitochondrial outer membrane milieu, leading to apoptosis initiation.

Fig. 1

All BH3-only proteins can trigger BAX/BAK activation by targeting only BCL-xL/MCL-1. a Hypothesis of BH3-only-mediated BAX/BAK activation. b Dox-inducible expression of the BH3-only-GFP-fusion proteins was established in the listed four parental HCT116 knockout cell lines by puromycin selection. GFP is at the N-termini of most of the BH3-only proteins except for tBID, whose BH3 domain is at the N-terminus. Dox (2 µg/mL) was added to each of the selected pools expressing different BH3-only proteins as indicated. Eight hours after induction, cells were harvested for western blot analysis. The faster migrating protein species in lane 4 and 5 for the Octa/BAX/BAK KO cells are minor degradation products of G-PUMA and G-Bad3SA. c–e Cells in b were stained with Hoechst dye 8 h after Dox induction. The percentage of cells that underwent nuclear condensation or fragmentation was quantified. The results are averages with SEM of three independent experiments. f Diagram of the listed BH3-only proteins and their BADBH3 mutants. g Dox-inducible expression for the listed GFP or GFP-fusion proteins were established in Octa/MCL-1 KO and Octa/BCL-xL KO HCT116 cells. Cells were treated with Dox (2 µg/mL) for 3 h and were harvested for western blot analysis. h At the indicated time points after Dox addition, cells were harvested for western blot analysis. i Cells were treated with Dox (2 µg/mL) in the presence of z-VAD for the indicated amount of time before being harvested. Whole cell lysates were incubated with or without BMH (0.2 mM) at room temperature for 1 h. The reaction mixtures were then subjected to western blot analysis. j Cells in h were induced with Dox for the indicated amount of time. Apo-ONE Caspase substrate/buffer for Caspase 3/7 were added to cells at the end of the treatment, and the plates were analyzed on a fluorescence microplate reader. The results are averages with SEM of three independent experiments. k Stable and Dox-inducible expression of the indicated proteins were established in the listed knockout cell lines. Four hours after Dox addition, cells were harvested for western blot analysis. l Cells in k were stained with Hoechst dye 4 h after Dox induction. The percentage of cells that underwent nuclear condensation or fragmentation was quantified. The results are averages with SEM of three independent experiments. m Stable and Dox-inducible expression of the indicated proteins were established in the OctaKO cells. At different time points after the addition of Dox, cells were harvested for western blot analysis. Representatives from at least two independent experiments are shown for western blots

Fig. 2

BIM, tBID, and BAD do not distinguish BAX from BAK. a Generation of Octa/MCL-1/BAX KO and Octa/MCL-1/BAK KO cells. Whole cell lysates for the indicated cells were analyzed by western blot analysis. b Dox-inducible expression for the indicated proteins was established in Octa/MCL-1/BAX/BAK KO, Octa/MCL-1/BAX KO, and Octa/MCL-1/BAK KO cells. Four hours after Dox (2 µg/mL) addition, cells were harvested for western blot analysis. c Cells were treated the same way as in b and were stained with Hoechst dye after induction. The percentage of cells that underwent nuclear condensation or fragmentation was quantified. The results are averages with SEM of three independent experiments. d The Octa/MCL-1/BAX KO and Octa/MCL-1/BAK KO cells with the Dox-induced expression of the indicated proteins were harvested at different time points following the addition of Dox for western blot analysis. e Dox-inducible expression of the indicated proteins was established in Octa/BAX/BAK KO cells stably expressing comparable amounts of G-Bax or G-BAK. Four hours after induction, cells were harvested for western blot analysis. f Cells were treated the same way as in e and then stained with Hoechst dye. The percentage of cells that underwent nuclear condensation or fragmentation was quantified. The results are averages with SEM of three independent experiments. g Cells in e were harvested at the indicated time points after Dox addition for western blot analysis. Representatives from at least two independent experiments are shown for western blots

Fig. 3

BIM and tBID inactivate BCL-xL/MCL-1, but do not accelerate BAX/BAK activation in the absence of the anti-apoptotic BCL-2 proteins. a BAX/BAK/BCL-xL/BCL-2/BCL-w/MCL-1/A1 KO cells, which are named “2 + 5” cells, were generated with CRISPR/Cas9. Whole cell lysates were examined for the expression of the indicated proteins by western blot analysis. b Dox-inducible expression of G-Bax or G-BaxΔTM was established. At the indicated time points, cells were harvested for western blot analysis. c Cells were treated with Dox in the presence of z-VAD for the indicated amount of time before harvested. Whole cell lysates were incubated with or without BMH (0.2 mM) at room temperature for 1 h. The reaction mixtures were then subjected to western blot analysis. An asterisk indicates a non-specific protein. d The indicated cells were treated with TRAIL (25 ng/mL) for 3 h before they were harvested for western blot analysis. An asterisk indicates a non-specific protein. e Three hours after TRAIL treatment, Dox was added to the indicated cell lines with the Dox-inducible expression of either G-Bax or G-BaxΔTM. Cells were harvested at different time points after addition of Dox for western blot analysis. f Dox-inducible expression system for G-Bax was established in BCL-2 allKO cells expressing the indicated BH3-only proteins as GFP chimeras with or without the stable expression of BCL-xL. Dox (2 µg/mL) was added in the presence or absence of z-VAD (40 µM) for 2 h before the cells were harvested for western blot analysis. g Cells in f were stained by Hoechst dye 2 h after Dox induction. The percentage of cells that underwent nuclear condensation or fragmentation was quantified. The results are averages with SEM of three independent experiments. h At the indicated time points following the addition of Dox, cells from f with the expression of the indicated proteins were harvested for western blot analysis with PARP antibody. i Cells were treated with Dox in the presence of z-VAD for the indicated amount of time before harvested. Whole cell lysates were incubated with or without BMH at room temperature for 1 h. The reaction mixtures were then subjected to western blot analysis. Arrows indicate the protein bands of tBID-G. Representatives from at least two independent experiments are shown for western blots. An asterisk indicates a non-specific protein

Fig. 4

Reconstitution of the death receptor-mediated, BAX/BAK-dependent apoptosis pathway with BID and its direct activation-defective mutants in BH3-only-deficient cells. a Diagram of BID and its mutants. b Stable expression of BID and its mutants in OctaKO and Octa/MCL-1 KO cells following retroviral infection and flow cytometry sorting. c The indicated stable cell pools were treated with TRAIL (25 ng/mL) in the presence or absence of S63845 (1 µM) for 2.5 h and then harvested for western blot analysis. d, e Cells in c were stained by Hoechst dye 2.5 h after Dox induction. The percentage of cells that underwent nuclear condensation or fragmentation was quantified. The results are averages with SEM of three independent experiments. f The indicated stable cell pools were treated with TRAIL (25 ng/mL) for the indicated amounts of time and then harvested for western blot analysis. g, h Cells were treated with TRAIL (25 ng/mL) for the indicated amount of time. Apo-ONE Caspase substrate/buffer for Caspase 3/7 were added to cells at the end of the treatments, and the plates were analyzed on a fluorescence microplate reader. The results are averages with SEM of three independent experiments. i Diagram of the death receptor-mediated, mitochondria-dependent apoptosis pathway. Representatives from at least two independent experiments are shown for western blots

Fig. 5

A new model of BAX/BAK activation during apoptosis. a BH3-only-mediated BAX/BAK activation during mitochondria-dependent apoptotic signaling. In response to apoptotic signals, select groups of BH3-only proteins become activated, and the activated BH3-only proteins neutralize the anti-apoptotic BCL-2 proteins, allowing the free diffusion-driven, membrane-mediated, spontaneous activation of BAX/BAK through the mitochondrial outer membrane. While NOXA binds only MCL-1 and A1, and BAD binds only BCL-xL, BCL-2, and BCL-w, BID, BIM, PUMA, HRK, BMF, and BIK are able to bind all the anti-apoptotic BCL-2 proteins. b A mitochondrial outer membrane-mediated permissive model of BAX/BAK activation. BAX/BAK are normally inactive monomers or dimers in healthy cells. During apoptosis, activated BH3-only proteins bind to and neutralize the anti-apoptotic BCL-2 family proteins on the mitochondrial outer membrane, which then provides a permissive environment for the spontaneous activation of BAX/BAK, culminating in their homo-oligomerization and the formation of cytochrome c releasing pores