Bax activation by engagement with, then release from, the BH3 binding site of Bcl-xL

Abstract

Bcl-2 homologues (such as Bcl-x(L)) promote survival in part through sequestration of "activator" BH3-only proteins (such as Puma), preventing them from directly activating Bax. It is thus assumed that inhibition of interactions between activators and Bcl-x(L) is a prerequisite for small molecules to antagonize Bcl-x(L) and induce cell death. The biological properties, described here of a terphenyl-based alpha-helical peptidomimetic inhibitor of Bcl-x(L) attest that displacement of Bax from Bcl-x(L) is also critical. Terphenyl 14 triggers Bax-dependent but Puma-independent cell death, disrupting Bax/Bcl-x(L) interactions without affecting Puma/Bcl-x(L) interactions. In cell-free assays, binding of inactive Bax to Bcl-x(L), followed by its displacement from Bcl-x(L) by terphenyl 14, produces mitochondrially permeabilizing Bax molecules. Moreover, the peptidomimetic kills yeast cells that express Bax and Bcl-x(L), and it uses Bax-binding Bcl-x(L) to induce mammalian cell death. Likewise, ectopic expression of Bax in yeast and mammalian cells enhances sensitivity to another Bcl-x(L) inhibitor, ABT-737, when Bcl-x(L) is present. Thus, the interaction of Bcl-x(L) with Bax paradoxically primes Bax at the same time it keeps Bax activity in check, and displacement of Bax from Bcl-x(L) triggers an apoptotic signal by itself. This mechanism might contribute to the clinical efficiency of Bcl-x(L) inhibitors.

FIG. 1.

Inhibition of Bcl-x_L binding to BH3 peptides by terphenyl 14 (Terph. 14). Fluorescence polarization-based competitive binding assays using fluorescein-labeled Bid-BH3, Bad-BH3, or Puma-BH3 (15 nM) in complex with Bcl-x_L (as a GST fusion protein; 100 nM) are shown for terphenyl 14 and respective nonfluorescent peptides at the indicated concentrations (C). Data are means (± SEM) of results from 3 independent experiments. mP, milli-polarization level.

FIG. 2.

Role of Puma and Bax in cell death induction of HCT116 p21^−/− cells by terphenyl 14. (A) Effect of terphenyl 14 on viability. Parental, Bax knockout, or Puma knockout HCT116 p21^−/− cells were treated with the indicated concentration of terphenyl 14 for 24 h prior to measurement of cell viability. Data are means (± SEM) of results from 3 independent experiments. (B) Effect of terphenyl 14 on Bax/Bcl-x_L and Puma/Bcl-x_L interactions. HCT116 p21^−/− cells were treated with terphenyl 14 (100 μM) (Ter. 14) or not treated (Unt.) for 24 h, and the resulting cell lysates were immunoprecipitated (I.P.) with an anti-Bcl-x_L antibody. Bcl-x_L, Bax, and Puma present in total extracts and immunoprecipitated fractions were analyzed by immunoblotting (W.B.). The amount of Bax or Puma that coimmunoprecipitated with Bcl-x_L under each condition was evaluated by densitometric analysis and normalized to the amount of protein that coimmunoprecipitated with Bcl-x_L in untreated cells. Data are means (± SE) of results from three independent experiments. P values were assessed using a Student t test.

FIG. 3.

Terphenyl 14 cooperates with Bcl-x_L to promote cell-free Bax activation. (A and B) Effect of terphenyl 14 on cell-free binding of Bax to Bcl-x_L. (A) In vitro-translated radiolabeled Bax (4 fmol) was incubated with the indicated His-tagged Bcl-x_L recombinant proteins (8 fmol). His-bound protein complexes were then centrifuged, and the presence of free ³⁵S-Bax in the resulting supernatant (F.) or ³⁵S-Bax bound to Bcl-x_L following three additional washes (B.) was analyzed by SDS-PAGE and autoradiography with a PhosphorImager. Autoradiograms representative of three independent experiments are shown. Input, 4 fmol of untreated ³⁵S-Bax was loaded for illustrative purposes. The average quantity of Bax bound to His complexes (mean ± SE of results from three independent experiments) was evaluated and is expressed as a percentage of the initial amount of ³⁵S-Bax (n.a., nonapplicable). wt, wild type. (B) His-Bcl-x_L wild type/³⁵S-Bax complexes were left untreated or treated with 10 μM terphenyl 14. The presence in the initial complexes (lane 1) and in the supernatant resulting either from a mock treatment (lane 2) or from terphenyl treatment (lane 3) of His-tagged Bcl-x_L and of ³⁵S-Bax was analyzed by Western blotting using an anti-His antibody (34660 from Qiagen) and by autoradiography, respectively. Data representative of three independent experiments are shown. The average quantity of Bax displaced from His complexes by 10 μM terphenyl 14 (mean ± SE of results from three independent experiments), expressed as a percentage of ³⁵S-Bax present in the initial His complexes, is indicated. (C) Effect of terphenyl 14 on Bax-induced mitochondrial permeabilization in the presence of Bcl-x_L. Left, ³⁵S-Bax (4 fmol) was incubated with His tagged-Bcl-x_L (8 fmol) prior to the addition of terphenyl 14. His-bound protein complexes were then centrifuged, and the presence of ³⁵S-Bax in the resulting supernatant (free ³⁵S-Bax) and in the pellet following three additional washes (Bcl-x_L-bound ³⁵S-Bax) was analyzed by SDS-PAGE and autoradiography with a PhosphorImager. Data representative of three independent experiments are shown. Right, ³⁵S-Bax (4 fmol) bound to His-tagged Bcl-x_L was treated with terphenyl 14 prior to its incubation with rat liver mitochondria (100 μg) for 1 h at 37°C. The presence of cytochrome c in the supernatant following centrifugation of mitochondria was analyzed by Western blotting. Where indicated, terphenyl 14 (10 μM) was directly added to 4 fmol ³⁵S-Bax in the absence of Bcl-x_L prior to incubation with isolated mitochondria. Western blots representative of three independent experiments are shown. (D) Effect of terphenyl 14 on Bax conformation in the presence of Bcl-x_L. ³⁵S-Bax was subjected to the indicated treatment prior to immunoprecipitation with the 2D2 or 6A7 anti-Bax antibody. 1, untreated ³⁵S-Bax; 2, ³⁵S-Bax treated with 10 μM terphenyl 14 in the absence of Bcl-x_L; 3, ³⁵S-Bax preincubated at pH 4; 4, ³⁵S-Bax preincubated with His-tagged Bcl-x_L and released from Bcl-x_L by 10 μM terphenyl 14. Data representative of three independent experiments are shown.

FIG. 4.

Expression of Bax and Bcl-x_L renders yeast cells sensitive to terphenyl 14. (A and B) Effect of terphenyl 14 treatment on Bax/Bcl-x_L interactions in yeast. The indicated strains were grown, and Bax and/or Bcl-x_L expression was induced by galactose prior to treatment with terphenyl 14 (100 μM) or mock treatment (DMSO plus 0.02% Tween 20) for an additional 6 h, followed by immunoprecipitation with anti-Bax antibodies and Western blotting (WB) of the resulting immunoprecipitates (A). The amounts of Bcl-x_L and of Bax that were immunoprecipitated by anti-Bax antibodies were evaluated by densitometric analysis. The Bcl-x_L/Bax ratio was normalized to 1 for mock conditions for each experiment. Data are the means (± SEM) of results from three independent experiments (B). (C) Effect of terphenyl 14 on the viability of yeast cells expressing Bax, Bcl-x_L, or both. The indicated strains were grown in the presence of galactose, and cell density was measured in each of the indicated yeast strains 12 h after treatment with terphenyl 14 (100 μM). It is expressed as a percentage of the density measured in the corresponding mock-treated samples. Data are means (± SEM) of results from 8 independent experiments. P values (*, P < 0.005) were assessed using a Student t test.

FIG. 5.

Bcl-x_L is involved in the induction of mitochondrial Bax activity by terphenyl 14. (A) Bcl-x_L contributes to cell death induction by terphenyl 14 in HCT116 p21^−/− Puma^−/− cells. HCT116 p21^−/− Puma^−/− cells were infected with a control recombinant lentivirus (shScr) or with a recombinant lentivirus expressing a short hairpin targeting Bcl-x_L (shBcl-x_L) before their treatment with terphenyl 14 (100 μM). Twenty-four hours later, cell death was assessed as described in the legend for Fig. 2. Data are means (± SEM) of results from 3 independent experiments. (B) Bcl-x_L is required for Bax-dependent induction of cytochrome c (cyt c) release and caspase 3 (casp3) activation by terphenyl 14. Immunostaining of cytochrome c and active caspase 3 in HeLa cells expressing a control plasmid (SCR), pSilencer Bax (shBax), or pSilencer BCL-X (shBcl-x) and treated with or without terphenyl 14 (100 μM) for 18 h was performed. The percentages of cells exhibiting mitochondrial cytochrome c release and active caspase 3 following treatment are indicated. Data shown are means (± SE) of results from 4 independent experiments. (C) Bcl-x_L is required for efficient induction of mitochondrial Bax insertion by terphenyl 14. The indicated HeLa cells were treated or not with terphenyl 14 (100 μM) for 18 h. Mitochondria were then isolated and left untreated or treated with alkaline carbonate. The amounts of mitochondrion-bound (untreated mitochondria) and membrane-inserted (alkaline-treated mitochondria) Bax and F1 ATPase (as a control mitochondrial membrane protein) were analyzed by Western blotting. The amount of Bax inserted into mitochondrial membranes (i.e., alkaline resistant) in each condition was evaluated by densitometric analysis and normalized to the amount of protein inserted into mitochondria from control cells treated with terphenyl 14. Data are means (± SE.) of results from four independent experiments. P values were assessed using a Student t test. (D) Mitochondria from HeLa cells are intrinsically sensitive to induction of Bcl-x_L-dependent cytochrome c release by terphenyl 14. Mitochondria isolated from the indicated HeLa cells were incubated with terphenyl 14 in standard mitochondrial buffer for 1 h at 37°C. The amounts of cytochrome c present in the mitochondrial fraction and in the mitochondrial supernatant (“released”) were then analyzed by Western blotting. Data representative of three independent experiments are shown. F1 ATPase was used as a control mitochondrial membrane protein.

FIG. 6.

The Bcl-x_L G138A mutant does not cooperate with terphenyl 14. (A and B) Transiently transfected Bcl-x_L G138A does not restore the sensitivity of Bcl-x_L knockdown HeLa cells (A) or HCT116 p21^−/− Puma^−/− cells (B) to terphenyl 14. Cells were infected with an shBcl-x_L lentivirus prior to transfection with plasmids expressing shRNA-resistant cDNAs encoding the indicated Bcl-x_L variant or with empty vector and treatment or not with terphenyl 14 (100 μM). Death rates in the resulting cells were evaluated 24 h later. Data are means (± SE) of results from three independent experiments. (Right) Western blot analysis of Bcl-x_L expression was performed to assess the efficiency of RNA interference (using cells infected with a control lentivirus, Scr, as a positive control) and of transduction in each condition. β-Tub., β-tubulin. (C) Microinjection of recombinant Bcl-x_L G138A does not restore the sensitivity of Bcl-x_L knockdown HeLa cells to terphenyl 14. Recombinant GST, GST-Bcl-x_L, or GST-Bcl-x_L G138A (1 μM) were microinjected together with the fluorescent marker FITC-Dextran 40S (0.5%) in the indicated HeLa cells 6 h prior to their treatment with terphenyl 14. The percentage of microinjected (i.e., fluorescent) cells exhibiting morphological features of cell death was assayed 18 h later. Data are means (± SE) of results from at least three independent experiments. (D) Resistance of Bcl-x_L^−/− cells to terphenyl 14. Wild-type and Bcl-x_L^−/− MEFs were treated with terphenyl 14 (100 μM) or DMSO carrier alone for 18 h. The activation of DEVDase (top) (A.U., arbitrary units) and the loss of cell viability (bottom) following treatment were then assayed. Data are means (± SEM) of results from 3 independent experiments. (E) Transiently transfected Bcl-x_L but not Bcl-x_L G138A cooperates with terphenyl 14 to promote cell death in Bcl-x_L knockout mouse embryonic fibroblasts. Bcl-x^−/− MEFs transfected with the indicated plasmids together with a plasmid encoding the red fluorescent protein (RFP) DsRed as a transfection marker were treated with terphenyl 14 for 18 h prior to morphological analysis of fluorescent cells. Data are means (± SE) of results from four independent experiments. P values were assessed using a Student t test.

FIG. 7.

ABT-737 promotes Bcl-x_L-dependent Bax activation. (A) ABT-737 impacts Bax conformation in the presence of Bcl-x_L in cell-free assays. Bottom, ³⁵S-Bax bound to recombinant His-Bcl-x_L was treated with terphenyl 14 (10 μM), ABT-737 (1 μM), or the indicated BH3 peptides. The resulting “free” Bax molecules were then immunoprecipitated with the 2D2 or 6A7 anti-Bax antibody. Top, ³⁵S-Bax was directly treated with the indicated compounds and peptides in the absence of Bcl-x_L prior to immunoprecipitation. (B) Expression of Bax and Bcl-x_L renders yeast cells sensitive to ABT-737. The effect of a 6-h ABT-737 treatment (20 μM) on Bax/Bcl-x_L interactions was evaluated and quantified as described in the legend for Fig. 4A and B. Data are means (± SE) of results from three independent experiments. The effect of the same treatment on the viability of yeast cells expressing the indicated protein was evaluated as described in the legend for Fig. 4C. Data are means (± SEM) of results from 8 independent experiments. P values (*, P < 0.005) were assessed using a Student t test. (C) Ectopic expression of Bax sensitizes HeLa cells to ABT-737 by a Bcl-x_L-dependent process. HeLa cells were infected with the indicated lentivirus, as described in the legend for Fig. 6A, transfected with the indicated plasmids, and treated or not with ABT-737 (5 μM) for 18 h prior to evaluation of cell death. Data are means (± SEM) of results from 3 independent experiments. Bottom insert, Western blot analysis of Bax expression was performed to assess transduction efficiency in each condition. (D) Acute knockdown of Bcl-x_L mitigates cell death induced by ABT-737 in combination with Mcl-1 downregulation. HeLa cells were transfected with the indicated sequence of siRNA prior to treatment with ABT-737 (1 μM), terphenyl 14 (100 μM), or vehicle alone for an additional 24 h, and loss of cell viability was evaluated. Cell death rates induced by each combination of siRNA in the absence of treatment are represented in white, while the specific cell death rates observed in the presence of compound are shown in gray and black (for ABT-737 and terphenyl 14, respectively). Data are means (± SE) of results from three independent experiments. Note that death rates measured in untreated siCtr/siCtr and siBcl-x_L/siCtr cells are shown twice, below ABT-737 and terphenyl 14-induced death rates, respectively. The bottom shows representative Western blotting, confirming the specificity of the RNA interference approach targeting Bcl-x_L and Mcl-1 expression. (E) Knockdown of Bax mitigates cell death induced by ABT-737 in combination with Mcl-1 downregulation. Control (Scr) or Bax knockdown (shBax, expressing pSilencer Bax) HeLa cells were transfected with control siRNA (siCtr) or siMcl-1 nucleotides 24 h before their treatment for an additional 24 h with ABT-737 (1 μM) or vehicle alone, followed by evaluation of loss of cell viability. Data (represented as in panel D) are means (± SE) of results from four independent experiments.

FIG. 8.

Model for Bax activation by its interaction with and then release by Bcl-x_L. Mitoch., mitochondrial. See the text for details.