From the
University of California, San Francisco Cancer Center, San Francisco, California 94123-0128,
¶ La Jolla Institute for Allergy and Immunology, San Diego, California 92121,
INSERM U419, 9 Quai Moncousu, 44035 Nantes, France
Received for publication, September 16, 2002
, and in revised form, February 10, 2003.
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ABSTRACT |
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INTRODUCTION |
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Bcl-2 family members are major regulators of mitochondrial integrity (2). Anti-apoptotic members such as Bcl-2 and Bcl-xL display sequence conservation throughout all four Bcl-2 homology domains (BH14), whereas the pro-apoptotic Bax and Bak possess homology in BH13 domains. These multidomain pro-apoptotic proteins have the innate ability to alter mitochondrial integrity, possibly via their ability to induce channels in the mitochondrial membrane (3). The ratio between anti-apoptotic and multidomain pro-apoptotic Bcl-2 family members helps determine the cellular susceptibility to death stimulation (4). NMR structural analysis of the Bcl-xL-BakBH3 peptide complex has revealed that the BH3 domain of Bak binds to a hydrophobic cleft formed by the BH1, BH2, and BH3 domains of Bcl-xL (5). Anti-apoptotic and multidomain pro-apoptotic Bcl-2 family members may thus engage, by a BH3 domain-dependent mechanism, in the formation of heterodimers in which they mutually antagonize each other's function (6).
The Bcl-2 family includes a third subgroup of pro-apoptotic members that display sequence homology only with the BH3 domain (7). These BH3 only proteins seem to act as the afferent effectors of various pro-apoptotic and anti-apoptotic signals (8). For instance, both Noxa and Puma are transcriptionally regulated by p53 (9, 10, 11); activation of Bid by caspase 8-mediated cleavage recruits the mitochondrial apoptotic pathway into death receptor signaling (12, 13), whereas the activity of Bad is negatively regulated by the Akt/protein kinase B survival signaling pathway (14). Murine cells lacking both multidomain Bax and Bak display long term resistance to mitochondrial damage and cell death induced by all BH3-only proteins tested (15, 16, 17). Thus, in mammals, activation of BH3 only proteins integrate diverse apoptotic stimuli into one single pathway by triggering Bax/Bak-mediated mitochondrial dysfunction.
All BH3-only proteins have the ability to bind to and to functionally antagonize anti-apoptotic Bcl-2 family members (8). The BH3 domain-dependent mechanism by which Bax/Bak pro-apoptotic function is recruited remains poorly characterized. Indeed, multidomain proteins, in viable cells, reside as inactive proteins located either at the mitochondria (Bak) or in the cytosol (Bax) (18). The direct binding of Bid to either Bax or Bak is sufficient to activate those proteins and allow their pro-apoptotic assembly in the mitochondrial membrane (19, 20, 21). Very recent evidence (22, 23) using synthetic peptides has indicated that the BidBH3 domain itself functions as a specific death ligand. These studies nevertheless revealed evidence for another functional subset of BH3 domains, which lack the ability to activate directly Bax/Bak but retain the ability to bind to anti-apoptotic Bcl-2 family members (22). It has remained unclear whether such BH3 domains can induce apoptosis and, if so, by which mechanism.
To explore this question further, we have analyzed in this study the apoptotic effects of a minimal BH3 domain synthetic peptide, comprising the critical 16 residues of the defined Bax BH3 domain (BaxBH3 (5772)). These 16 residues contain sufficient information to bind to (5), and functionally antagonize (24), Bcl-xL and to induce specifically Bax/Bak-mediated apoptosis (24). We show here that, despite these properties, BaxBH3 cannot efficiently induce Bax translocation to mitochondria, evoke a change in Bax conformation, nor stimulate the apoptotic activity of recombinant Bax, whereas it can stimulate that of a mutant form of Bax that exhibits an increased ability to homodimerize. BaxBH3 interferes with Bax/Bcl-xL interactions in cell-free experiments and stimulates the apoptotic activity of Bax combined with Bcl-xL. Similarly, a peptide derived from the BH3 domain of Bad was also unable to stimulate Bax activity by itself but cooperated with Bcl-xL to activate it. Taken together, these data support a model in which BH3 peptides that mainly act as antagonists of anti-apoptotic Bcl-2 family members can induce apoptosis by interfering with the interaction between multidomain pro- and anti-apoptotic proteins.
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MATERIALS AND METHODS |
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Peptides and Recombinant ProteinsThe high pressure liquid chromatography-purified BaxBH3 (KKLSECLKRIGDELDS), BaxBH3 L63A (KKLSECAKRIGDELDS), and BadBH3 (QRYGRELRRMSDEFVD) peptides were obtained from Genosys (Cambridge, UK).
Histidine-tagged human Bax, Bax
ART, and Bcl-xL were obtained by subcloning of the coding regions into pDEST17 plasmid (Gateway, Invitrogen) as described previously (25). His-tagged proteins were expressed in the Escherichia coli strain XL-1 blue and purified by chromatography on nickel-Sepharose acid resin according to the manufacturer's instructions. Recombinant proteins were dialyzed against PBS1 and stored at -80 °C until used. The purification of GST, GST-Bcl-xL, and tBid from bacterial lysates has been described previously (24, 26).
Microinjection ExperimentsRat-1 fibroblasts, grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS and 0.1 mM glutamine, were seeded on sterile coverslips the day prior to microinjection. Microinjection was performed as described previously (27) using an InjectMan NI2 micromanipulator and a FemtoJet injector from Eppendorf (Germany). Identical, standardized conditions of pressure (100 hPa) and time (0.1 s) were used in all experiments. Peptides and/or recombinant proteins were diluted in PBS together with dextran 70 kDa-conjugated lysine-fixable Oregon Green® (Molecular Probes, 0.5% final concentration) as a co-injection marker. Typically, 100 cells were microinjected for each condition in each experiment. The percentage of positive (i.e. fluorescent) cells exhibiting morphological features of apoptosis following microinjection was evaluated as described previously (27) using an inverted fluorescence microscope (DMIRE2, Leica, France).
ImmunocytochemistryImmunocytochemical staining of microinjected cells was performed as described previously (24). Both the primary polyclonal anti-Bax NT antibody and the anti-rabbit secondary antibodies were used at a 1:200 dilution. Images presented in this study were collected on a Leica TCS NT confocal microscope with a 100 x 1.3 NA Fluotar objective (Leica, France).
Cell-free AssaysMitochondria were isolated from normal rat liver as described previously (28). For mitochondrial targeting assays, [35S]Met-labeled proteins (Amersham Biosciences) were synthesized from cDNAs using the TNT-coupled Transcription/Translation system (Promega, France) and quantified as described previously (28). Radiolabeled Bax (4 fmol) was incubated with isolated mitochondria (2.5 mg proteins/ml) and with the indicated concentration of recombinant tBid or BaxBH3 at 30 °C for 1 h in 40 µl of standard import buffer (250 mM sucrose, 80 mM KCl, 10 mM MgCl2,10mM malic acid, 8 mM succinic acid, 1 mM ATP-Mg2+, 20 mM MOPS, pH7.5). When indicated, 35S-Bax was preincubated with unlabeled GST or GST-Bcl-xL (2 fmol) for 30 min prior to incubation with mitochondria in the presence or in the absence of BaxBH3 (200 nM). Radiolabeled proteins bound to the mitochondria were recovered by centrifugation of the incubation mixture at 8,000 x g for 10 min at 4 °C, separated by SDS-PAGE, and analyzed by scanning with a PhosphorImager (Amersham Biosciences) followed by quantification with IPLab gel program (Signal Analytics).
For cytochrome c release assays, recombinant Bax (5 nM) was preincubated with either GST or GST-BclxL (2.5 nM) for 30 min prior to its incubation with mitochondria (1 mg of proteins/ml) in the absence or in the presence of BaxBH3 (200 nM) for an additional hour in 100 µl of import buffer. Mitochondria were then recovered, and the amount of mitochondrial cytochrome c was analyzed by Western blotting and quantified with IPLab gel program (Signal Analytics).
Protein Binding ExperimentsIn vitro His-protein binding assays were performed as described previously (25). Briefly, each radiolabeled protein (4 or 8 fmol as indicated) was incubated with an equimolar concentration of either His-tagged Bcl-xL or Bax immobilized on nickel-Sepharose in 50 µl of binding buffer (142 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.4), 0.5 mM dithiothreitol, 1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, and a mixture of other protease inhibitors) at 4 °C for 2 h. BaxBH3 or BaxBH3 L63A (200 nM final concentration) was then added, and the mixture was incubated for an additional hour. Protein complexes were then centrifuged at 13,000 x g for 5 min at 4 °C, washed three times in binding buffer, eluted in elution buffer (50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, and 250 mM imidazole), separated by SDS-PAGE, and analyzed with a PhosphorImager as described above. For mild proteolysis experiments, both the pellet and the supernatant from the first centrifugation at 13,000 x g were treated with trypsin (1 mg/ml) for 15 min at 4 °C. Proteolysis was then stopped by the addition of 10 mg/ml soybean trypsin inhibitor prior to analysis as described above. To assess directly the sensitivity of radiolabeled Bax to trypsin treatment in the absence of His-tagged Bcl-xL, the radiolabeled protein (8 fmol) was incubated with tBid (8 fmol, 0.2 nM final concentration), the indicated peptides (200 nM), or was left untreated in 40 µl of binding buffer for 1 h at 30 °C prior to trypsin treatment as described above.
For immunoprecipitation experiments, soluble His-tagged Bcl-xL-radiolabeled Bax complexes were prepared as follows. Radiolabeled Bax (20 fmol) was incubated with 20 fmol of His tagged Bcl-xL immobilized on nickel-Sepharose in 250 µl of binding buffer at 4 °C for 2 h; protein complexes were then centrifuged at 13,000 x g for 5 min at 4 °C, washed three times in binding buffer, and then eluted in 20 µl of elution buffer. SDS-PAGE analysis and quantification using a PhosphorImager as described above showed that the resulting soluble complexes contained in average 4 fmol of radiolabeled Bax. These soluble complexes, or equivalent amounts of free radiolabeled Bax (4 fmol), were incubated with an equimolar amount of tBid (0.2 nM final concentration) or with the indicated peptides (200 nM) for 1 h at 30 °C in 40 µl of standard import buffer. The anti-Bax 2D2, 6A7, or TL41 antibodies were then added (1:10 final dilution) for an additional 1-h incubation at 4 °C. Antibody-protein complexes were then isolated by incubation with either Zysorbin-G (for the 6A7 and 2D2 antibodies) or Zysorbin (for the TL41 antibody) followed by centrifugation and washes according to the manufacturer's instructions (Zymed Laboratories Inc.). SDS-PAGE analysis and quantification of the immunoprecipitated and radiolabeled proteins using a PhosphorImager were performed as described above.
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RESULTS |
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We reasoned that if BaxBH3 can directly activate Bax, it should be more efficient in inducing apoptosis in cells with increased Bax levels; on the other hand, we would expect to see no increased sensitivity to BaxBH3 in these cells if BaxBH3 were unable to stimulate Bax function directly. We therefore made use of a recombinant full-length Bax (Bax) and assessed its effect on whole intact cells in the presence or in the absence of the BaxBH3 peptide. The recombinant protein used was prepared in the absence of detergent, in order to prevent the artifactual activation of Bax reported to occur during the purification procedure (30). This purified Bax
protein did not associate with Bax
in cell-free experiments ((25) see also Fig. 6). It is therefore unlikely to adopt a homodimeric structure. As shown in Fig. 1 (top left panel), microinjection of recombinant Bax
(0.5 nM) induced limited apoptosis even 4 h following injection. In order to check that this recombinant protein can be activated by certain stimuli, we co-injected Bax
with a recombinant protein equivalent to the caspase 8 cleavage product of Bid (tBid, 0.5 nM) (26). This led to far more widespread apoptosis than that observed upon microinjection of either Bax
or tBid alone (Fig. 1, top right panel). Apoptotic synergy between these two proteins was evident as early as 2 h after microinjection, strongly supporting the view that recombinant Bax
by itself is poorly apoptogenic but can be activated by signals such as Bid activation. Of note, the fact that subnanomolar quantities of tBid and Bax
efficiently induced apoptosis is consistent with a previous report (31) showing that similar concentrations of tBid elicit significant mitochondrial dysfunction and cell death.
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We then analyzed whether BaxBH3 stimulated the pro-apoptotic function of Bax. Microinjection of 0.5 nM BaxBH3 induced apoptosis to a certain degree (Fig. 1, bottom left panel), whereas microinjection of the mutant BaxBH3 L63A (0.5 nM), which does not interact with Bcl-xL, had only a limited effect on cell viability (Fig. 1, bottom right panel). In sharp contrast to what we observed for tBid, co-injection of Bax
and BaxBH3 did not result in an increased apoptosis in the microinjected cells (Fig. 1, bottom left panel). A similar lack of effect on Bax
activity was observed with the mutant BaxBH3 L63A peptide (Fig. 1, bottom right panel). Thus, BaxBH3 induces apoptosis, but it does not impact on the apoptotic activity of the native form of Bax.
BaxBH3 Neither Stimulates Bax Association with Mitochondria nor Significantly Modifies Its ConformationBax in its native conformation is only poorly targeted to mitochondria, and it acquires the ability to interact with its mitochondrial site of action only in response to certain death stimuli, including Bid activation (32). We therefore asked whether the lack of effect of BaxBH3 on Bax apoptotic function depended upon its inability to stimulate Bax
targeting to mitochondria. For this purpose, we used an in vitro system employing the 35S-labeled transcription-translation product of Bax cDNA in rabbit reticulocyte lysate together with purified mitochondria from rat liver. Previous analysis of Bax association with mitochondria in such a system has confirmed that Bax targeting to mitochondria is mostly inefficient but that it can be triggered by addition of cytosolic extracts from apoptotic cells (28, 33) or induced by specific mutations that serve to increase Bax apoptotic activity (25, 34). As a positive control, we checked that the addition of low concentrations (110 nM) of tBid stimulated the targeting of in vitro translated, radiolabeled (IVTR) Bax
to isolated mitochondria in a dose-dependent manner (Fig. 2A). In sharp contrast, the addition of BaxBH3 at 10 nM did not stimulate Bax
association to mitochondria (Fig. 2A). Increased concentrations of the peptide, up to 10 µM, failed to exhibit any effect on Bax
targeting (not shown, see also below in Fig. 5). We conclude that BaxBH3 is unable to stimulate Bax
association to mitochondria.
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The ability of certain stimuli, such as Bid activation, to induce translocation of Bax to mitochondria has been ascribed to their ability to induce a change in Bax conformation. Such change in Bax is accompanied by the exposure of an epitope located in its N terminus, which can be detected in whole cells by immunohistochemical analysis with a specific antibody (BaxNT (35)). To understand better the inability of BaxBH3 to induce Bax translocation, we asked whether BaxBH3 or tBid, when microinjected into Rat-1 cells, induced exposure of this epitope in endogenous Bax. We used subnanomolar doses of both tBid and BaxBH3, which we had shown by themselves induce only modest apoptosis 3 h following microinjection (see Fig. 1). Microinjection was performed in the presence of the broad range caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone (50 µM) to avoid caspase-induced changes in Bax conformation (32). Cells were fixed 3 h after microinjection with either tBid or BaxBH3 (0.5 nM) and immunostained using the conformation-specific BaxNT antibody as described previously (24). As shown in Fig. 2B, a significant portion of Rat-1 fibroblasts microinjected with tBid (0.5 nM) exhibited a marked increase in Bax NT immunostaining. This increased staining appeared as a punctate, extranuclear pattern consistent with a mitochondrial localization for the modified Bax molecules (see color panel in Fig. 2B). Careful quantification indicated that 25% of cells exhibited the change in Bax NT immunostaining 3 h after tBid microinjection (Fig. 2B). In sharp contrast, less than 10% of cells exhibited a similar feature 3 h after microinjection with BaxBH3 (0.5 nM). Thus, introduction of BaxBH3 into intact cells did not modify Bax conformation as efficiently as an equimolar amount of tBid. This suggests that BaxBH3 requires additional endogenous factors to induce efficiently a change in Bax conformation. Taken together, those results indicate that BaxBH3 is unable to induce a significant change in Bax conformation and to stimulate its association to mitochondria.
BaxBH3 Stimulates the Cytotoxic Activity of a Recombinant Bax Mutant Lacking Its N-terminal EndAlthough it is unable to stimulate Bax directly, BaxBH3 nevertheless recruits Bax function in cells (24). One possible explanation for these paradoxical results is that BaxBH3 can functionally cooperate with Bax, provided Bax is in receipt of a sufficient threshold of activation by other signals. To address this question directly, we tested the effect of BaxBH3 on the pro-apoptotic function of a recombinant mutant form of Bax, Bax
ART. This recombinant protein consists of a Bax protein lacking its N-terminal 19 residues. This extreme N terminus of Bax normally regulates the apoptotic function of Bax negatively, and its deletion consequently results in an increased ability of Bax to homodimerize, interact with mitochondria, and promote apoptosis (25, 32, 36). Microinjection of recombinant Bax
ART (0.5 nM) induced little apoptosis during the first 3 h following delivery (Fig. 3). However, by 4 h, a significant proportion of the microinjected cells exhibited morphological features of apoptosis. At this time, the extent of cell death observed in the microinjected population was 2-fold higher than that observed in control cells injected with the same amount of Bax
(compare Fig. 3 to Fig. 1A). This increased apoptosis in cells injected with Bax
ART, as opposed to cells injected with Bax
, was also observed at later time points (data not shown), indicating that, as expected, Bax
ART is more apoptogenic than Bax
. Co-injection of amounts of BaxBH3 that were unable to stimulate Bax
apoptotic activity (0.5 nM, see above in Fig. 1B) with Bax
ART led to a significant increase in the rate and the extent of cell death as early as 2 h following injection (Fig. 3). In sharp contrast, the mutant BaxBH3 L63A peptide had no effect on Bax
ART apoptotic activity (Fig. 3). Therefore, BaxBH3 can specifically sensitize cells to the deleterious effect of an activated form of Bax. This indicates that the inability of BaxBH3 to sensitize cells to Bax
relies on its inability to trigger a change in the conformation of the protein and not on its inability to sensitize cells to Bax apoptotic activity under the conditions used.
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BaxBH3 Stimulates the Cytotoxic Activity of Wild Type Bax in the Presence of Bcl-xLThe observation that BaxBH3 can promote Bax-dependent apoptosis without providing a threshold for the activation of Bax suggests that additional ratelimiting partners are required for BaxBH3 to promote Bax function. It is well established that BH3 peptides bind Bcl-xL, suggesting the possibility that BH3 peptides promote apoptosis by releasing Bax from dimers formed with its anti-apoptotic counterparts. Alternatively, additional factors, distinct from Bcl-xL itself, might instead be required for the peptide to activate Bax
indirectly. To distinguish between these two possibilities, we analyzed whether the presence of Bcl-xL suffices to allow our BH3 peptide to cooperate with Bax
.
We first analyzed the effect of BaxBH3 on mitochondrial targeting of Bax following its incubation with a recombinant GST-Bcl-xL fusion protein. As shown in Fig. 4A, IVTR Bax preincubated with the control recombinant protein GST (2:1 ratio) interacted poorly with mitochondria whether or not BaxBH3 (200 nM) was present. IVTR Bax
preincubated with GST-Bcl-xL (2:1 ratio) also interacted poorly with mitochondria in the absence of BaxBH3. It should be noted that the recombinant Bcl-xL used here lacks its C-terminal end and is therefore unable to interact with mitochondria itself (24). Addition of 200 nM of BaxBH3 resulted in a significant increase in the mitochondrial targeting of IVTR Bax
preincubated with GST-Bcl-xL (Fig. 4A). Thus, the BH3 peptide can stimulate Bax
association with mitochondria provided Bcl-xL is present.
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We next investigated whether the BaxBH3 peptide, when combined with Bcl-xL, elicits an increased ability of Bax to induce cytochrome c release from isolated mitochondria. Incubation of isolated mitochondria (1.5 mg of protein/ml) with recombinant Bax
(5 nM) plus GST (2.5 nM) for 1 h at 30 °C led to no significant cytochrome c release from the mitochondrial pellet (data not shown). As well, the presence of an excess of BaxBH3 peptide (200 nM) plus Bax
/GST did not significantly affect cytochrome c release (Fig. 4B). In contrast, the addition of BaxBH3 (200 nM) to a combination of Bax
and GST-Bcl-xL in a 2:1 ratio leads to a significant loss of cytochrome c from mitochondria (Fig. 4B). Incubation of the BH3 peptide in the presence of GST-Bcl-xL had no effect on mitochondrial cytochrome c (data not shown), indicating that induction of cytochrome c release by BaxBH3 under the conditions used depends on the presence of both Bax
and Bcl-xL. Thus, BaxBH3 stimulates Bax
-induced cytochrome c release provided Bcl-xL is present.
We next determined the ability of BaxBH3 to modulate Bax apoptotic activity in the presence GST-Bcl-xL. Bax (0.5 nM) combined with GST-Bcl-xL (0.25 nM) was even less efficient in inducing apoptosis than Bax
alone (compare Fig. 5A to Fig. 1, top left panel). This is consistent with the protective effect of microinjected GST-Bcl-xL against UV-induced apoptosis reported previously (24). This protective effect was further confirmed by the observation that BaxBH3 (0.5 nM) combined with GST-Bcl-xL was less efficient in inducing apoptosis than the peptide alone (compare Fig. 5A to Fig. 1, bottom left panel). In sharp contrast, addition of BaxBH3 (0.5 nM) to a mixture of Bax (0.5 nM) and GST-Bcl-xL (0.25 nM) led to a significantly more efficient induction of apoptosis than that observed in cells injected with BaxBH3 and Bax
in the absence of GST-Bcl-xL (Fig. 5A). This enhanced apoptosis was not observed when GST alone (0.25 nM) was used instead of GST-Bcl-xL, or when the mutant-inactive peptide BaxBH3 L63A (0.5 nM) was used instead of BaxBH3 (data not shown). Our recombinant Bcl-xL protein therefore diminishes the apoptotic activity of either BaxBH3 or Bax
alone, but it allows BaxBH3 to stimulate Bax
pro-apoptotic activity. In order to investigate whether this cooperativity between Bcl-xL and BaxBH3 can extend to other BH3 peptides, we used another synthetic 16-mer peptide encompassing the BH3 domain of Bad. It has been shown recently (22) that this BH3 domain is unable to activate Bax directly. Consistent with this observation, we observed that microinjection of BadBH3 (0.5 nM) alone or together with Bax
(0.5 nM) induced similar levels of apoptosis (Fig. 5B). BadBH3 (0.5 nM) combined with GST-Bcl-xL was less efficient in inducing apoptosis than the peptide alone (Fig. 5B). However, the addition of BadBH3 (0.5 nM) to a mixture of Bax (0.5 nM) and GST-Bcl-xL (0.25 nM) enhanced significantly the induction of apoptosis as compared with that observed with BadBH3 and Bax
in the absence of GST-Bcl-xL (Fig. 5B). Taken altogether, these data show that the presence of Bcl-xL is sufficient to allow both BaxBH3 and BadBH3 to stimulate the apoptotic activity of Bax
.
BaxBH3 Releases Bax from Heterodimers Formed with Bcl-xLThe previous results are mostly consistent with a model in which BaxBH3 releases an activated Bax from heterodimerization with Bcl-xL. To explore this idea, we analyzed the interaction between Bax and Bcl-xL in the presence or in the absence of BaxBH3. We used a cell-free assay in which tagged recombinant proteins were assayed for their abilities to pull-down IVTR proteins (25). As shown in Fig. 6A (left panel), recombinant Bax
was unable to associate with either IVTR Bax
or IVTR Bax
ART as reported previously (25). In sharp contrast, recombinant Bax
associated with IVTR Bcl-xL under the same conditions (Fig. 6A, left panel). This binding was inhibited by an excess of BaxBH3 peptide but not by the inactive peptide BaxBH3 L63A (Fig. 6A, left panel). Conversely, recombinant Bcl-xL significantly bound to IVTR Bax
, in a manner that could be specifically inhibited by BaxBH3 (Fig. 6A, right panel). Of note, the amount of Bax
bound to recombinant Bcl-xL could be further increased by preincubating IVTR Bax
with unlabeled tBid (Fig. 6A, right panel). This suggests that the affinity of Bax for Bcl-xL correlates with its ability to adopt an active configuration, a notion that was confirmed by the finding that Bax
ART bound more efficiently to Bcl-xL than Bax
under similar conditions (Fig. 6A, right panel). Thus, Bax
is unable to homodimerize, but it nonetheless associates with Bcl-xL, albeit with less efficiency than its activated forms. Furthermore, BaxBH3 displaces Bax from the heterodimers it forms with Bcl-xL.
We then investigated whether the binding of Bax to Bcl-xL, or its release from Bcl-xL upon treatment with BaxBH3, can significantly contribute to a change in its conformation and to its activation. In a first series of experiments, we analyzed the trypsin sensitivity of Bax following its association with Bcl-xL in cell-free pull-down assays. Previous experiments (28) have shown that incubation of Bax with apoptotic cytosols increases its resistance to mild proteolysis while concomitantly increasing its targeting to mitochondria. Moreover, mutants of Bax that exhibit an increased ability to homodimerize and to translocate to mitochondria also exhibit increased resistance to mild proteolysis (25). Consistent with the notion that a change in Bax sensitivity to proteolysis accompanies its activation, we observed that tBid-treated IVTR Bax
was highly resistant to mild proteolysis with trypsin, whereas untreated IVTR Bax
was not (Fig. 6B, top panel). In sharp contrast, Bax
sensitivity to proteolysis was neither affected by the presence of BaxBH3 nor by that of BaxBH3L63 (Fig. 6B, top panel). In a pull-down experiment using recombinant His-tagged Bcl-xL as described above, most Bcl-xL-bound IVTR Bax
exhibited resistance to trypsin treatment, whereas there was no detectable trypsin-resistant Bax
in the unbound fraction (compare Fig. 6B, bottom panel, to Fig. 6A). Addition of BaxBH3 resulted in a complete loss of Bcl-xL-bound, protease-resistant Bax
and the concomitant appearance of protease-resistant Bax
in the unbound fraction (Fig. 6B, bottom panel). In control experiments, no effect of the mutant BaxBH3 L63A was observed. Thus, Bcl-xL binding to Bax
is sufficient to modify its sensitivity to proteolysis, whereas the action of BaxBH3 is to trigger the release of a modified Bax from Bcl-xL. In a second series of experiments, we analyzed the ability of three epitope-specific antibodies to immunoreact with Bax in the presence of Bcl-xL, BH3 peptides, or both. The specific anti-Bax antibodies used were as follows: (i) the 2D2 monoclonal antibody that was raised against a peptide encompassing residues 316 of human Bax, an epitope which is exposed regardless of Bax conformation (37); (ii) the 6A7 monoclonal antibody that was raised against a peptide encompassing residues 1224, which only binds to active Bax (38); (iii) the TL41 polyclonal antibody that was produced against a peptide sequence (residues 5772), which represents the minimal Bax BH3 domain (24). In non-denaturing conditions, only the 2D2 antibody could immunoprecipitate IVTR Bax
that was left untreated (data not shown) or that was treated with the control peptide BaxBH3 L63A (Fig. 6C, left panel). In sharp contrast, all three antibodies could immunoprecipitate t-Bid treated IVTR Bax
(Fig. 6C, left panel). This indicates that the epitope for the 6A7 antibody, and the epitope for the TL41 antibody within the Bax BH3 domain, are normally hidden in native Bax but become exposed following activation of Bax by tBid. Treatment of IVTR Bax
with either BaxBH3 or BadBH3 had no effect on the immunoreactivity of the radiolabeled protein with either the 6A7 or the TL41 antibodies (Fig. 6C, left panel), further supporting the notion that these peptides cannot activate Bax by themselves. As shown in Fig. 6C (right panel), radiolabeled Bax complexed to Bcl-xL could be immunoprecipitated by the 2D2 antibody. This observation is consistent with the ability of an antibody raised against the equivalent peptide sequence to immunoreact with Bcl-xL-bound murine Bax reported previously (38). In sharp contrast, neither the 6A7 nor the TL41 conformation-specific antibodies could immunoprecipitate Bcl-xL-bound IVTR Bax
in native conditions (Fig. 6C), although they could do so in denaturing conditions (0.1% SDS, 0.1% Triton X-100, data not shown). Thus, the inability of these antibodies to immunoreact with Bax in non-denaturing conditions likely results from the inaccessibility of the corresponding epitopes in the Bcl-xL-bound protein. Moreover, when Bcl-xL-Bax complexes were treated with either BaxBH3 or BadBH3, but not with the mutant peptide BaxBH3 L63A, Bax
was significantly immunoprecipitated by the 2D2, the 6A7, and the TL41 antibodies (Fig. 6C, right panel). Similarly, IVTR Bax
directly released from nickel-Sepharose-bound His-tagged Bcl-xL by wild type BH3 peptides (as in Fig. 6B) also immunoreacted with all three antibodies (data not shown). Thus, both epitopes for the 6A7 antibody and the TL41 antibody become accessible when Bax molecules are specifically released from Bcl-xL by BH3 peptides. Taken altogether, these results indicate that, when it is directly activated by tBid, Bax becomes resistant to proteolysis, whereas its BH3 domain and a region in its N-terminal end (the 6A7 epitope) become exposed. Binding to Bcl-xL is sufficient to modify the sensitivity to proteolysis of Bax, but the release of Bax from Bcl-xL, induced by BH3 peptides, is necessary to produce an active Bax, as probed by the accessibility of the resulting protein to conformation-specific antibodies.
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DISCUSSION |
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In this study, we used a synthetic peptide representing the BH3 domain of Bax (BaxBH3), which composes the minimal sequence required to antagonize anti-apoptotic family members. We report here that BaxBH3 has no significant effect on either the apoptotic activity, the mitochondrial targeting, and/or the conformation of Bax (Figs. 1 and 2). We found that another synthetic peptide representing the minimal BH3 domain of Bad is also unable to stimulate Bax activity (Fig. 5B). Thus, BH3 peptides that contain sufficient information to occupy the surface pocket of the anti-apoptotic Bcl-2 family members do not all necessarily support direct activation of Bax/Bak. Consistent with this, BH3 peptides derived from Bad, Bik, or Noxa were shown to lack the ability to activate Bax/Bak (22), whereas small molecule non-peptidic ligands of Bcl-xL are reportedly unable to induce Bax insertion into mitochondrial membranes (6). These observations show that another subset of BH3 domains exists, which can function as survival antagonists but not as death agonists for Bax/Bak.
The lack of effect of BaxBH3 on Bax activity sharply contrasts with the ability of BaxBH3 to promote the apoptotic function of Bax
ART, an N-terminal deletion of Bax that displays an enhanced ability to homodimerize and to induce apoptosis (Fig. 3). The fact that the activity of Bax
ART can be further enhanced by BaxBH3 indicates that BaxBH3 can functionally cooperate with Bax, so long as Bax is provided with a sufficient trigger to lower its threshold of activation. Because BaxBH3 prevents Bax
ART binding to Bcl-xL (Fig. 6A), BaxBH3 is likely to enhance the apoptotic activity of Bax
ART by preventing the inhibitory association of this activated form of Bax with endogenous Bcl-xL and possibly Bcl-2. One interesting implication from this is that death agonists and survival antagonists (typified by our BH3 peptide) may activate cell death by mechanistically distinct, yet cooperative pathways. In agreement with this, we have observed that BH3 peptides can sensitize cells to apoptosis induction by microinjected tBid.2
Even though BaxBH3 is unable to activate Bax directly, the fact remains that it induces apoptosis (Fig. 1; see also Refs. 39 and 40). Our experiments therefore constitute proof of concept that the ability to function as a death ligand is not absolutely required for a BH3 domain to promote apoptosis. The observation that the BH3-only protein BimL lacks the ability to activate Bax directly but retains the ability to interact with Bcl-xL and induce apoptosis is consistent with this view (41). As BaxBH3 specifically induces apoptosis by a mechanism requiring the presence of either Bax or Bak (24), these experiments also imply that the ability of this peptide to promote Bax/Bak-mediated apoptosis must involve some other intermediaries. One possibility is that survival antagonists such as BaxBH3 promote the release from Bcl-xL/Bcl-2 of some death agonists such as Bid (17). It should be noted, however, that a mutant of Bcl-xL that cannot interact with Bax or Bak but interacts with Bid is less efficient than wild type Bcl-xL in preventing Bax-mediated mitochondrial dysfunction (42). Thus anti-apoptotic Bcl-2 family members may act on Bax/Bak directly, and their functional antagonists may promote Bax/Bak activity by suppressing this interaction. We found that addition of Bcl-xL was sufficient to allow BaxBH3 to stimulate the ability of Bax to interact with mitochondria, to induce cytochrome c release (Fig. 4), and to induce apoptosis upon microinjection (Fig. 5A). Similarly, we found that BadBH3 had no impact on the apoptotic activity of microinjected Bax by itself but that it could stimulate it when combined with Bcl-xL (Fig. 5B). Thus, induction of Bax-mediated apoptosis by a BH3 domain does not necessarily involve any other partner than Bcl-xL itself. This strongly favors a model in which survival antagonists promote apoptosis by inducing the release of Bax/Bak from heterodimers they engage with Bcl-xL/Bcl-2.
Most of the arguments against the aforementioned model arise from the observation that Bax/Bak and their anti-apoptotic counterparts exhibit only modest interaction in some cell types unless apoptosis is triggered (38, 43). We confirm that Bax binds to Bcl-xL with greater efficacy in its active conformation; its association to Bcl-xL is indeed stabilized by the addition of tBid or by a deletion in its N-terminal regulating domain (Fig. 6). However, we have also observed that, despite its inability to self-associate, Bax can bind to Bcl-xL (Fig. 6). This observation is consistent with whole cells studies in which Bax fused to a fluorescent protein, although inefficient at inducing apoptosis by itself, nonetheless exhibited significant interaction with Bcl-2 and Bcl-xL as assayed by fluorescence resonance energy transfer (6, 44).
Bax/Bcl-xL interactions were significantly inhibited by BaxBH3 (Fig. 4). Similarly, in vivo interactions between Bax and Bcl-xL were disrupted by small molecule ligands of the BH3-binding pocket of Bcl-xL (6). Thus, binding of the BH3 domain of Bax within the hydrophobic pocket of Bcl-xL is likely to be essential for the formation of a stable heterodimer. Residues within the BH3 domain of Bax that are critical for dimer formation are oriented toward the hydrophobic core of wild type Bax (46). Thus, major conformational modifications in Bax should accompany its BH3-dependent binding to Bcl-xL. Our observation that, upon its binding to Bcl-xL, Bax acquires resistance to mild proteolysis is consistent with this idea (Fig. 6). It is striking to note that protease resistance is a property that Bax also displays when it is activated by apoptotic cytosols (28), tBid (this study), or by site-directed mutagenesis (25). Thus, although the very nature of the protease-resistant, Bcl-xL-bound form of Bax requires further characterization, it is tempting to speculate that it may be close, on some aspects, to that of an active Bax. Microinjection experiments nevertheless suggest that, as long as it is bound to Bcl-xL, Bax does not exert its apoptotic activity (Fig. 5). Our immunoprecipitation experiments using conformation-specific antibodies further confirms this by showing that Bcl-xL-bound Bax does not fulfill all the criteria of an active Bax (Fig. 6). Indeed, tBid-activated Bax, but not native or Bcl-xL-bound Bax, can immunoreact with the well documented conformation-specific 6A7 antibody and with a polyclonal antibody raised against the minimal Bax BH3 domain (TL41). It was initially proposed that the epitope for the 6A7 antibody might be in the vicinity of the BH3 dimerization domain of Bax (38). Our observation that exposure of the 6A7 epitope occurs concomitantly with the exposure of an epitope within the BH3 domain of Bax is certainly consistent with this view. It also indicates that the BH3 domain of Bax is hidden within the native protein but is exposed when Bax is active. Dimerization of Bax with Bcl-xL may prevent both the 6A7 and the TL41 epitopes from being exposed, or alternatively, it may mask these epitopes on Bax (38). When specifically released from Bcl-xL by BH3 peptides, however, Bax recapitulates the features of tBid-activated Bax, as it becomes accessible to both conformation-specific antibodies and retains its resistance to mild proteolysis. This is most consistent with a model in which Bcl-xL binds to inactive Bax, modifies it, and then releases active Bax in response to a BH3 peptide. One provocative implication from this is that Bcl-xL should cooperate with ligands of its BH3 domain-binding pocket to induce Bax-mediated apoptosis, which may explain why cells overexpressing Bcl-xL are more sensitive to induction of apoptosis by a BH3 mimetic (47).
In summary, we have analyzed in this study the apoptotic function of a synthetic BH3 peptide comprising sufficient information to antagonize functionally anti-apoptotic Bcl-2 family members, and we found that it does not function as a death agonist of Bax. This BH3 peptide can nonetheless promote Bax-dependent apoptosis by interfering with the ability of Bax to interact with, and be suppressed by, Bcl-xL. There is a growing list of small molecules that have been selected for their ability to occupy the BH3-binding pocket of anti-apoptotic Bcl-2 family members and that act as true BH3 mimetics (6, 45, 47). Our results indicate that such molecules may efficiently induce apoptosis in cells expressing high levels of the anti-apoptotic Bcl-2 family members by triggering the release of active multidomain pro-apoptotic proteins from these survival proteins.
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FOOTNOTES |
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Supported by a fellowship from the Ligue Départementale Contre le Cancer Doubs/Montbeliard.
|| To whom correspondence should be addressed: INSERM U419, 9 Quai Moncousu, 44035 Nantes, France. Tel.: 33-24-008-4083; Fax: 33-24-008-4082; E-mail: pjuin{at}nantes.inserm.fr.
1 The abbreviations used are: PBS, phosphate-buffered saline; FCS, fetal calf serum; MOPS, 4-morpholinepropanesulfonic acid; IVTR, in vitro translated radiolabeled; GST, glutathione S-transferase.
2 C. Moreau, P.-F. Cartron, A. Hunt, K. Meflah, D. R. Green, G. Evan, F. M. Vallette, and P. Juin, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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