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. Author manuscript; available in PMC: 2010 Mar 24.
Published in final edited form as: Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2009 Aug;26(4):829–835.

The Bax BH3 Peptide H2–H3 Promotes Apoptosis by Inhibiting Bcl-2’s Pore-Forming and Anti-Bax Activities in the Membrane

Jun Peng 1,2, Suzanne M Lapolla 1, Zhi Zhang 1, Jialing Lin 1
PMCID: PMC2844869  NIHMSID: NIHMS185522  PMID: 19813621

Abstract

Pore-formation and protein-protein interactions are considered to play critical roles in the regulation of apoptosis by Bcl-2 family proteins. During the initiation of apoptosis, the anti-apoptotic Bcl-2 and the pro-apoptotic Bax form different pores to regulate the permeability of mitochondrial outer membrane, playing their opposite functions. Overexpression of Bcl-2 has been found in various cancer cells, therefore it is gaining widespread interest to discover small molecules to compromise Bcl-2 function for anti-cancer treatment. Since Bax binds to Bcl-2’s hydrophobic groove via its BH3 domain (composed of helices 2 and 3), by which their functions are inhibited each other, the H2–H3 peptide that contains the functional BH3 domain of Bax has been considered as a potential Bcl-2 antagonist. We recently reported that Bax peptide H2–H3 promotes cell death by inducing Bax-mediated cytochrome c release and by antagonizing Bcl-2’s inhibition on Bax. However, the mechanism of how H2–H3 inhibits the anti-apoptotic activity of Bcl-2 remains poorly understood. To address this question, we reconstituted the Bcl-2 or Bax pore-forming process in vitro. We found that H2–H3 inhibited Bcl-2’s pore formation and neutralized Bcl-2’s inhibition on Bax pore formation in the membrane, whereas the mutant H2–H3 peptide that does not induce apoptosis in cells was shown to have no effect on Bcl-2’s activities. Thus, inhibiting Bcl-2’s pore-forming and anti-Bax activities in the membrane is strongly correlated with H2–H3’s pro-apoptosis function in cells.

Keywords: Bcl-2, Bax, Bax peptide H2–H3, pore formation, oligomerization

INTRODUCTION

Bcl-2 family proteins play an important role in the regulation of apoptosis (1, 2). All members in this family contain at least one of the four conserved sequence elements called Bcl-2 homology (BH) motifs 1, 2, 3 and 4. Bcl-2 family proteins can be functionally divided into anti-apoptotic and proapoptotic proteins. According to the sequence homology, pro-apoptotic Bcl-2 family proteins can be further divided into Bax and BH3-only subfamilies. Members in Bax subfamily, such as Bax and Bak, contain BH1-3 motifs. The BH3-only subfamily includes Bid, Bim and Bad, sharing sequence homology only in the BH3 motif. In contrast, anti-apoptotic Bcl-2 subfamily proteins, Bcl-2, Bcl-XL, Bcl-w, Bfl-1 and Mcl-1 contain all the four BH motifs (1–5).

It is widely accepted that mitochondrial membrane permeability is altered during apoptosis by Bcl-2 family proteins including anti-apoptotic Bcl-2 and pro-apoptotic Bax and tBid, one of the mechanisms by which Bcl-2 family members regulate apoptosis (4–6). After binding to tBid, Bax forms large pores in mitochondrial outer membrane (MOM), releasing apoptogenic proteins such as cytochrome c and Diablo/Smac that trigger apoptosis by activating caspases and nucleases (7–13). Bcl-2 can inhibit Bax pore formation, preventing it from damaging the membrane (5, 13–15). Interestingly, consistent with that the 3-dimensional structures of Bax and Bcl-2 are strikingly similar to each other and to the pore-forming domains of bacterial toxins (16–19), Bcl-2 itself also possesses pore-forming activity in the membrane (20–23). However, Bcl-2 pore is much smaller than Bax pore, which only permeates molecules less than several kDa, close to the size of the molecules that are known to be freely crossing MOM, suggesting that Bcl-2 may play its anti-apoptotic function by forming small pores to maintain the normal permeability of membranes, as well as inhibiting the membrane disruption caused by Bax pores (23).

Bcl-2 subfamily proteins are commonly overexpressed in various cancers (24); and the overexpression of anti-apoptotic Bcl-2-like proteins not only confers a survival advantage to the cancer cells but also causes resistance to chemo/radio-therapies. Therefore, discovering small molecules to inhibit the anti-apoptosis activity of Bcl-2 subfamily proteins has been a major focus in development of new cancer therapies. Recently a Bax peptide, which contains Bax BH3 helix (helix 2) and residues in the down stream helix 3 (designated H2–H3 peptide), has been attracting our interest due to its pro-apoptotic activity in cells (25). It has been shown that the peptide H2–H3 can activate Bax to release cytochrome c from mitochondria (25). Although the Bax-mediated mitochondrial permeabilization is inhibited by Bcl-2, such inhibition can be overcome by higher concentrations of H2–H3 peptide (25). Therefore, Bax peptide H2–H3 induces cell death by activating Bax and antagonizing Bcl-2’s anti-Bax function. However, it is unclear how the H2–H3 peptide overrides Bcl-2-mediated inhibition of Bax. Does the peptide neutralizes Bcl-2 directly or indirectly through activation of more Bax? This question is difficult to address in the complex mitochondrial system, therefore in this study we used a simpler in vitro system using liposomes and purified peptides/proteins. We found that H2–H3 peptide could inhibit Bcl-2 oligomerization and block the association of Bcl-2 with liposomal membranes. Consequently, H2–H3 inhibited Bcl-2’s activity to form small pores in the membrane; and neutralized Bcl-2’s inhibition on the formation of Bax large pores. Mutation that abolishes the pro-apoptosis function of H2–H3 also abolished its inhibitory effect on Bcl-2, thereby correlating the in vitro anti-Bcl-2 activity with the in vivo function of H2–H3.

MATERIALS AND METHODS

Materials

The peptide containing helix 2 & 3 sequence of human Bax (designated Bax peptide H2–H3, 53DASTKKLSECLKRIGDELDSNMELQRMIAAVDTD86) and its mutant with the Gly replaced by Arg at the position 67 (H2–H3 G67R, 53DASTKKLSECLKRIRDELDSNMELQRMIAAVDTD86) were synthesized by Global Peptide Services (Fort Collins, CO). The molecular weight of peptides was verified by mass spectrometry, and purity was determined by HPLC to be > 90%. Peptides were dissolved in DMSO. All phospholipids were purchased from Avanti Polar Lipids. Fluorescent dye Cascade Blue (CB, Mr ~ 0.5 kDa), CB-labeled dextran of 10 kDa, and rabbit anti-CB polyclonal antibody were obtained from Molecular Probes. Bismaleimidohexane (BMH) was purchased from Pierces.

Preparation of plasmids and proteins

The construction of the plasmid pET22b-hBcl-2Δ22 encoding human Bcl-2 protein with LE (H)6 replacing the C-terminal 22 residues was generated as described (26). The sequence of all plasmids was verified by DNA sequencing. Expression and purification of His6-tagged Bcl-2ΔTM was done basically as described before (26). The purified proteins were stored in 50mM Tris-HCl (pH 8.5), 140mM NaCl, 2mM EDTA, 5mM DTT and 10% (v/v) glycerol at −80°C.

Preparation of Liposomes

Liposomes of MOM lipid composition and with CB or CB-dextrans encapsulated were prepared by the extrusion method as described (25). The plain liposomes without fluorophores were prepared similarly except that no fluorophores were in the citric-phosphate buffer (CPB) used for resuspension of the lipids and no gel filtration chromatography was needed after the extrusion.

Monitoring Bcl-2ΔTM pore formation in liposomal membranes by steady-state fluorescence spectroscopy

Steady-state fluorescence emission intensity was measured using the SLM-8100 spectrofluorometer as described before (25). Liposomes of 50 µM lipids (50 µM liposomes for short from here on) loaded with CB dyes or CB-labeled dextrans were mixed with 6 µg/ml anti-CB antibodies in 250 µl of citric-phosphate buffer (CPB). The initial emission intensity (F0) was determined after equilibration of the sample at 25 °C for 5 min. Then purified proteins were added into the liposome-containing sample and the emission intensity (F) was measured after 3 hours. Triton X-100 was then added to 0.1% for complete disruption of liposomes and release of all CB dyes. The emission intensity (Ft) was measures again. The quenching efficiency was calculated by ΔFProtein /ΔFTriton, where ΔFProtein = F0 − F and ΔFTriton = F0 − Ft. In experiments determining the effect of Bax peptide H2–H3 on Bcl-2 pore formation, Bcl-2ΔTM was pre-incubated with peptide for 1 hour before the protein was mixed with liposome solution.

Monitoring Bcl-2 homodimerization by cross-linking in solution

A fresh 5 mM stock solution of Bismaleimidohexane (BMH) in dimethyl sulfoxide (DMSO) was prepared. 2 µM purified His6-Bcl-2ΔTM proteins were incubated with different concentrations of Bax peptide H2–H3 in the CPB buffer (pH 5.0) containing 10 mM EDTA at 25 °C for 1 hour. After the addition of 10 µM BMH the sample was kept at 25 °C for another 30 min. The chemical reaction was stopped with 25 mM β-mercaptoethanol (β-Me). Proteins in the sample were precipitated by 25% TCA and analyzed by SDS-PAGE and Coomassie Brilliant Blue.

Monitoring Bcl-2-liposome association by sucrose-gradient float-up centrifugation

1 mM liposomes and 4 µM His6-Bcl-2ΔTM protein were incubated in 138 µl of CPB for 1 hour at 25°C at pH 5.0. The resultant sample was then mixed with 162 µl of 2.5 M sucrose to bring the final sucrose concentration to 1.35 M. The resulted sample was added to a centrifuge tube and overlaid with 500 µl of 0.8 M sucrose and then 200 µl of 0.25 M sucrose. The sample was centrifuged at 436,000 × g for 6 hrs at 4 °C. Four 250 µl fractions were drawn, starting from the top of the gradient, resulting in fractions S1–S4. The pellet was re-suspended in 250 µl of CPB buffer, resulting in fraction P. The distribution of the liposomes among these fractions was estimated by measuring the radioactivity of the 14C that resulted from the trace of [14C ]-PC incorporated into the liposomes. Proteins from each fraction were precipitated by 25% TCA and analyzed by SDS-PAGE and Coomassie Brilliant Blue. In experiments determining the effect of Bax peptide H2–H3 on Bcl-2’s association with liposomal membranes, Bcl-2ΔTM was pre-incubated with 40 µM peptide for 1 hour before the protein was mixed with liposome solution.

RESULTS

Bax peptide H2–H3 inhibits Bcl-2ΔTM pore formation in liposomal membrane

Previously we found that one of the possible mechanisms by which Bcl-2 exerts its anti-apoptotic function is forming small pores to maintain the normal permeability of mitochondria outer membrane (MOM) (23). We therefore wondered whether Bax peptide H2–H3 could inhibit Bcl-2 pore formation, thereby inducing apoptosis. Due to the high insolubility of the full-length Bcl-2 as an integral membrane protein, so far only the cytosolic domain of Bcl-2 (Bcl-2 lacking the transmembrane (TM) sequence, designated Bcl-2ΔTM) can be purified and used for in vitro studies. Additionally, Bcl-2ΔTM is structurally and functionally (in terms of pore-formation) related to the pore-forming domains of diphtheria toxin and E. coli colicins. Therefore in this study we used a recombinant His6-tagged Bcl-2ΔTM protein to study the effect of H2–H3 on Bcl-2 pore formation in membranes.

Purified Bcl-2ΔTM protein and/or different concentrations of Bax peptide H2–H3 were incubated at pH 5.0 for 1 hour, followed by the addition of MOM-liposomes loaded with 0.5 kDa fluorescent CB dye and incubation for another 3 hours. The release of CB dye from liposomes was monitored by the quenching of CB fluorescence emission by an anti-CB antibody presented outside of the liposome. As shown in Fig. 1, the Bcl-2ΔTM-mediated release of 0.5 kDa CB dyes was gradually reduced when Bcl-2ΔTM proteins were pre-incubated with increased concentrations of H2–H3, suggesting that Bcl-2ΔTM pore formation has been dose-dependently inhibited by H2–H3. Conversely, the mutant Bax peptide H2–H3 G67R that can not induce apoptosis in cells (25) had no effect on Bcl-2ΔTM pore formation even at the highest concentration of peptide tested (Fig.1). Therefore H2–H3’s inhibition on Bcl-2ΔTM pore formation in vitro is correlated with its pro-apoptosis activity in vivo.

Fig. 1. Bax H2–H3 peptide inhibits Bcl-2ΔTM pore formation in liposomal membrane.

Fig. 1

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Release of 0.5 kDa Cascade Blue (CB) dye from 50 µM MOM-mimic liposome by 0.5 µM Bcl-2ΔTM, in the presence of various concentrations of Bax H2–H3 peptide, was shown as the extent of the fluorescence quenching by the anti-CB antibody located outside of the liposome. The extent of protein-induced dye release is determined by the value of ΔFProtein /ΔFTriton, after a 3-hour incubation, in which ΔFProtein is the normalized reduction of fluorescence emission intensity after incubation of proteins or protein buffer with liposomes, and ΔFTriton is the same parameter determined after the liposomes are completely lysed by 0.1% Triton X-100 to release all CB dyes. Data shown are averages from three independent experiments with S.D. (error bars). The experiments were done at pH 5.0.

Bax peptide H2–H3 neutralizes Bcl-2’s inhibition on tBid-activated Bax in liposomal membrane

Unlike Bcl-2, the pro-apoptotic Bax activated by tBid forms large pores, releasing apoptogenic proteins such as cytochrome c from MOM to trigger apoptosis. Bcl-2 can inhibit Bax pore formation and Bax-mediated cytochrome c release, possibly by the interaction between Bcl-2 pore and Bax pore leading to the formation of non-functional complexes in the membrane. Recently we reported that Bax peptide H2–H3 could overcome Bcl-2’s inhibition on Bax-mediated cytochrome c release from mitochondria, with a poorly understood mechanism. To determine how H2–H3 suppresses Bcl-2’s anti-Bax activity, we tested whether the peptide could release Bcl-2’s inhibition on Bax pore formation in liposomal membranes. In this experiment, we performed the pore-forming assay at pH 7.4 using the liposomes loaded with CB-labeled 10 kDa dextrans that are similar in size to cytochrome c (12 kDa). We previously showed that at neutral pH activated Bax could form large pores in the liposomal membrane to permeate 10 kDa dextrans (25), but Bcl-2ΔTM did not show any pore-forming activity under the same condition (23). Therefore, the 10 kDa dextran-liposome system allowed us to focus on the tBid-induced, Bcl-2-inhibitable Bax membrane permeabilizing activity. As shown in Fig. 2, Bcl-2ΔTM did not change the membrane permeability at pH 7.4. In contrast, the tBid-activated Bax released 10 kDa CB-dextran from liposomes, which was significantly reduced by Bcl-2ΔTM. However, the inhibition of Bcl-2ΔTM on Bax pore formation was strongly overcome by Bax peptide H2–H3. As expected, the mutant peptide H2–H3 G67R that is nonfunctional in vivo did not affect Bcl-2’s anti-Bax activity in vitro. Taken together, H2–H3 could neutralize Bcl-2’s inhibition on Bax pore formation in the liposomal membrane.

Fig. 2. Bax H2–H3 peptide neutralizes Bcl-2’s inhibition on tBid-activated Bax in liposomal membrane.

Fig. 2

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Release of 10 kDa CB-labeled dextran from 50 µM MOM-mimic liposome by different combinations of proteins and peptide was monitored as Fig.1. Data shown are averages from three independent experiments with S.D. (error bars). The experiments were done at pH 7.4.

Bax peptide H2–H3 inhibits Bcl-2ΔTM homo-oligomerization

Recently we found that oligomerization is involved in Bcl-2 pore formation (23). It’s also been known that Bax can bind to Bcl-2 via its BH3 domain, by which their functions are inhibited each other. Some mutants of Bax like G67R can abolish the binding between Bcl-2 and Bax. We therefore predicted that H2–H3, as the functional domain of Bax, can bind to Bcl-2 thereby reducing the Bcl-2 oligomerization and pore formation.

Titration of Bax peptide H2–H3 experiment was performed first to determine its effect on the homo-association of Bcl-2ΔTM. We incubated Bcl-2ΔTM with different concentrations of Bax peptide H2–H3 at pH 5.0 for 1 hour, followed by the treatment with a homobifunctional sulfhydryl-reactive cross-linker BMH. Since each Bcl-2ΔTM protein contains only one cysteine residue at the position 158 and the cross-linker is cysteine-specific, only the dimer adduct can be generated although some proteins may be in a larger oligomeric form. Fig. 3A showed that with the increase of concentration of H2–H3peptide, Bcl-2ΔTM dimer adduct decreased gradually. A significant inhibition to Bcl-2ΔTM dimerization could be observed when the molar ratio of peptide to Bcl-2ΔTM was 10:1. On the contrary, the mutant peptide H2–H3 G67R did not have obvious effect on Bcl-2ΔTM dimerization even at the highest peptide to protein ratio tested. Therefore, it is suggested that H2–H3 peptide inhibits Bcl-2ΔTM pore formation by inhibiting Bcl-2ΔTM oligomerization.

Fig. 3. Bax H2–H3 peptide inhibits the homo-association Bcl-2ΔTM in solution.

Fig. 3

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A. The effect of Bax H2–H3 peptide on Bcl-2ΔTM homo-association in solution at pH 5.0 was monitored by BMH crosslinking. The peptide to protein ratio is indicated above the gel. B. Bcl-2ΔTM homo-association was monitored by BMH crosslinking in the presence of Bcl-2ΔTM alone (lane 1); Bcl-2ΔTM and wild-type H2–H3 peptide (lane 2); or Bcl-2ΔTM and the mutant peptide (G67R) (lane 3). The molar ratio of peptide to Bcl-2 was 10:1. Data shown are representative Coomassie stained SDS-PAGE gel from three experiments.

Bax peptide H2–H3 inhibits Bcl-2-liposome association

Furthermore, we tried to determine whether there were other ways by which H2–H3 inhibits Bcl-2ΔTM pore formation as well as inhibiting its anti-Bax activity. Since membrane insertion is required for Bcl-2ΔTM pore formation, we therefore examined the effect of H2–H3 on Bcl-2ΔTM association with liposomes. H2–H3 and Bcl-2ΔTM were incubated at a peptide to protein ration of 10:1, followed by the addition of liposomes. The membrane-bound Bcl-2ΔTM was then separated from soluble and aggregated Bcl-2ΔTM by sucrose-gradient float-up centrifugation. As shown in Fig. 4A–4C, the wild-type H2–H3 peptide but not the mutant G67R inhibited Bcl-2ΔTM-liposome association significantly, although the 14C radioactivity counting data showed that more than 65% of total loaded lipids floated to the top fraction in all tested samples. Together with above experiments, we conclude that Bax peptide H2–H3 inhibits Bcl-2ΔTM pore formation and its anti-Bax activity by inhibiting Bcl-2ΔTM oligomerization and blocking the Bcl-2ΔTM-liposome association.

Fig. 4. Bax H2–H3 peptide inhibits Bcl-2ΔTM-membrane association.

Fig. 4

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Bcl-2ΔTM-membrane association was examined by sucrose-gradient float-up centrifugation after incubation of the liposomes of 1 mM total lipids with Bcl-2ΔTM (4 µM) alone (A), Bcl-2ΔTM and wt Bax BH3 H2–H3 peptide (40 µM) (B), or Bcl-2ΔTM and mutant peptide (G67R) (C). The distribution of liposomes in the gradient is monitored by counting the 14C radioactivity of each sample and the results are shown below the gel. Data shown are representative of three experiments.

DISCUSSION

Apoptosis is regulated by a complicated series of interactions between Bcl-2 family proteins, which can influence the permeability of the mitochondrial outer membrane (MOM). These interactions include hetero- and homo-interactions of proteins containing either multiple Bcl-2 homology (BH) regions (BH1-3 or BH1-4) or a single (BH3) region. After receiving certain apoptotic stimuli, pro-apoptotic BH3-only proteins activate Bax subfamily members to release cytochrome c and other pro-apoptotic proteins from mitochondria, triggering apoptosis. The Bax-mediated cytochrome c release and cell death can be inhibited by Bcl-2 subfamily proteins. Consequently, overexpression of Bcl-2 subfamily proteins will confer a survival advantage to the cancer cells. Indeed Bcl-2-like proteins have been found to be commonly overexpressed in various cancers. Therefore, identifying small molecules inhibit the anti-apoptosis activity of Bcl-2 subfamily proteins has been a promising approach for anti-cancer therapy.

Recently, we reported that a Bax peptide containing helices 2 and 3 (designated H2–H3 peptide) can induce cell death, suggesting that it could be a potential candidate for anti-cancer reagents. Our previous study has shown that the H2–H3 peptide promotes apoptosis not only by directly activating Bax to release cytochrome c from mitochondria, but also by overcoming Bcl-2’s inhibition on Bax-mediated cytochrome c release. However, it also raised a question about how this peptide releases Bcl-2’s inhibition on Bax: by directly neutralizing Bcl-2 or indirectly by activating more Bax. To address this question, in this study we investigated the effect of H2–H3 on the pore formation of Bcl-2, an activity correlated with Bcl-2’s anti-apoptosis function in cells. We found that H2–H3 inhibited Bcl-2’s pore formation and neutralized Bcl-2’s inhibition on Bax pore formation in the membrane. Moreover, the mutant H2–H3 peptide that does not induce apoptosis in cells was shown to have no effect on Bcl-2 pore formation and its anti-Bax activity. Thus, inhibiting Bcl-2’s pore-forming and anti-Bax activities in the membrane could be one of the mechanisms by which H2–H3 peptide promotes apoptosis in cells.

ACKNOWLEDGMENTS

We thank Drs. Chibing Tan and Olga Nikolaeva for valuable suggestions, and NIH grant GM062964 to Jialing Lin for partial support.

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