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Sequence and helicity requirements for the proapoptotic activity of Bax BH3 peptides

Sanjeev Shangary, Christopher L. Oliver, Tommy S. Tillman, Michael Cascio and Daniel E. Johnson
DOI:  Published November 2004

Abstract

Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-XL is commonly observed in human malignancies and contributes to chemotherapy and radiation resistance. Bcl-2 and Bcl-XL inhibit apoptosis by binding to proapoptotic proteins such as Bax, thereby preventing chemotherapy-induced or radiation-induced release of cytochrome c from mitochondria and subsequent activation of the caspase protease cascade. Efforts to inhibit Bcl-2 or Bcl-XL function in tumor cells have focused on developing agents to inhibit the interactions of these proteins with proapoptotic proteins. Peptides derived from the BH3 domains of proapoptotic proteins have been shown to disrupt the interactions of Bcl-2 and Bcl-XL with key binding partners in cell-free reactions and to promote cellular apoptosis. However, less is known about the targets of BH3 peptides in intact cells as well as the sequence, length, and conformational requirements for peptide biological activity. In this report, we show that cell-permeable Bax BH3 peptides physically disrupt Bax/Bcl-2 heterodimerization in intact cells and that this disruption correlates with peptide-induced cell death. A point-mutant, control peptide that failed to disrupt intracellular Bax/Bcl-2 interactions also failed to promote apoptosis. To determine important sequence, length, and structural requirements for peptide activity, we generated and systematically analyzed the biological activities of 17 Bax BH3 peptide variants. Peptides were quantitatively examined for their ability to inhibit Bax/Bcl-2 and Bax/Bcl-XL heterodimerization in vitro and to promote cytochrome c release from mitochondria isolated from Jurkat, HL-60, U937, and PC-3 cells. Our results define 15 amino acids as the minimal length required for Bax BH3 peptide biological activity and show that amino acids COOH terminal to the BH3 core sequence are less critical than those located NH2 terminal to the core. In addition, circular dichroism spectroscopy revealed that high α-helical content generally correlated with, but was not sufficient for, peptide activity. Taken together, these studies provide a basis for future optimization of Bax BH3 peptide as a therapeutic anticancer agent.

Introduction

Apoptosis is a genetically defined form of cell suicide that is critically important for tissue homeostasis. The Bcl-2 protein family, which consists of both proapoptotic and antiapoptotic members, acts to regulate apoptosis via governance of the “intrinsic” pathway of cell death. The intrinsic or mitochondrial-mediated pathway is characterized by mitochondrial release of apoptogenic factors including cytochrome c, Smac/DIABLO, and apoptosis-inducing factor following treatment of cells with death stimuli such as chemotherapy drugs or ionizing radiation (1–5). The release of cytochrome c and Smac/DIABLO leads to subsequent activation of the caspase protease cascade, which promotes the internal destruction of the cell (1, 2, 4–7). Antiapoptotic Bcl-2 family members, including Bcl-2 and Bcl-XL, inhibit apoptosis by preventing the release of apoptogenic proteins into the cytosol (1–3), whereas proapoptotic proteins, including Bax and Bak, promote such release (8–10). In view of their ability to prevent apoptosis, it is not surprising that Bcl-2 and Bcl-XL are commonly overexpressed in human malignancies (11–14). Moreover, in several forms of cancer, Bcl-2 or Bcl-XL overexpression has been shown to correlate with chemotherapy and radiation resistance as well as with poor clinical prognosis (11–14). These observations highlight the need to develop effective agents that specifically target Bcl-2 or Bcl-XL in the tumor cells.

Antisense molecules directed against mRNA for Bcl-2 or Bcl-XL have been used to down-regulate the expression of these proteins in cell culture and in vivo (15–17). In particular, G3139, an 18-nucleotide antisense molecule, has been shown to down-regulate Bcl-2 expression in tumor cell lines (17) and has exhibited anticancer activity in clinical trials (13, 16, 18, 19). More recently, several agents have been developed that target Bcl-2 or Bcl-XL function. The application of a variety of techniques, including fluorescence polarization binding assays, nuclear magnetic resonance shift assays, computer modeling, and cell-based cytotoxicity assays, has led to the identification of several small organic molecules that bind and/or inhibit Bcl-2 and Bcl-XL, including HA-14-1 (20), antimycin A3 (21), BH3Is (22), gossypol (23), and compound 6 (ref. 24; small organic molecule inhibitors reviewed in refs. 13, 25, 26). In addition, increased understanding of the three-dimensional structures of Bcl-2 family members, and the interactions that occur between these proteins, has led to the design of peptide-based agents to inhibit Bcl-2 and Bcl-XL function (27–29).

Structural and functional studies have revealed that protein-protein interactions among Bcl-2 family members play a key role in the ability of these proteins to regulate apoptosis. Following treatment of cells with agents that activate the intrinsic pathway, Bax and/or Bak homooligomerize in the mitochondrial outer membrane and facilitate the release of apoptogenic factors (10, 30–32). Antiapoptotic Bcl-2 and Bcl-XL can inhibit this process by heterodimerizing with the Bax or Bak proteins (33–37). Mutational analyses have determined that the BH3 domain of proapoptotic proteins is necessary and sufficient for heterodimerization with antiapoptotic proteins (38–40). Thus, it has been reasoned that peptides based on the BH3 domains of proapoptotic proteins may have the capacity to disrupt physical interactions between proapoptotic and antiapoptotic Bcl-2 family members and thereby promote apoptosis in Bcl-2-overexpressing or Bcl-XL-overexpressing cells.

Previous studies have shown that peptides derived from the BH3 domains of Bak (19-mer), Bid (20-mer), Bad (21-mer), and Bax (20-mer) are capable of inducing apoptosis in a variety of cell line models (41–44). Peptide-induced apoptosis was associated with activation of the intrinsic cell death pathway and release of cytochrome c from the mitochondria (41, 43, 45). The ability of Bax, Bid, and Bim BH3 peptides to provoke cytochrome c release from purified mitochondria suggests that the peptides directly target Bcl-2 family members present in the outer mitochondrial membrane (41, 43, 45), although direct targeting of Bcl-2 family members in intact cells has not been firmly established. Furthermore, the ability of BH3 peptides to promote apoptosis in Bcl-2-overexpressing or Bcl-XL-overexpressing cells underscores the potential utility of these peptides as novel anticancer agents (41–44, 46). However, future clinical application of the BH3 peptides, or derivatives thereof, is hindered by a lack of understanding regarding the minimal sequence and structural characteristics needed for biological activity. Because shorter peptides are likely to exhibit improved delivery and stability, efforts are needed to needed to optimize the size and sequence composition of proapoptotic BH3 peptides. In this report, we used in vitro heterodimerization assays and cytochrome c release assays to determine the minimal length and essential sequence composition of Bax BH3 peptide. Additionally, we did circular dichroism (CD) spectroscopy to evaluate the relationship between α-helical content and the proapoptotic activity of Bax BH3 peptides. Our results define an optimized Bax BH3 peptide of 15 amino acids and indicate that α-helical content is important but not sufficient for potent proapoptotic activity.

Materials and Methods

Cell Lines and Reagents

Vector-transfected Jurkat T leukemic cells and Jurkat cells overexpressing Bcl-2 were generated as described previously (47). Jurkat cells overexpressing Bcl-XL were a kind gift of Dr. Craig Thompson (University of Pennsylvania, Philadelphia, PA). Transfected Jurkat cell lines were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 10% fetal bovine serum, 2 mmol/L l-glutamine, 1% penicillin/streptomycin, 0.2% fungizone, and 0.5 mg/mL G418 in a humidified atmosphere of 5% CO2 at 37°C. HL-60 and U937 cells were cultured in RPMI 1640 and PC-3 prostate cancer cells were maintained in F-12K medium (Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, 1% penicillin/streptomycin, and 0.2% fungizone.

Polyclonal anti-Bax antibody (N20) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), monoclonal anti-Bcl-2 antibody (clone 124) was from DAKO (Carpinteria, CA), polyclonal anti-caspase-3 was from Cell Signaling Technology (Beverly, MA), anti-tubulin (clone DM1A) was from Sigma Chemical Co. (St. Louis, MO), and anti–cytochrome c oxidase IV (clone 2OE8) was from Molecular Probes (Eugene, OR). Monoclonal antibodies recognizing poly(ADP-ribose) polymerase (clone 4C10-5) and cytochrome c (clone 7H8.2C12) were purchased from PharMingen (San Diego, CA).

Peptide Synthesis

Peptides were synthesized on a Pioneer peptide synthesizer (Applied Biosystems, Foster City, CA) using a fluoren-9-ylmethoxycarbonyl synthesis protocol (Peptide Synthesis Facility, Molecular Medicine Institute, University of Pittsburgh). Synthesis was done by stepwise addition of activated amino acids to the solid support (polyethylene glycol-polystyrene resin) starting from the COOH terminus. Activation of amino acids was done by DIPEA/HOBT/TBTU chemistry. Peptides were cleaved from the resin using reagent R (90% trifluoroacetic acid, 5% thioanisole, 3% ethanedithiol, 2% anisole) and subjected to multiple ether extractions. The crude peptides were purified to >95% purity by gel filtration (G-25 column) and reverse-phase high-performance liquid chromatography (486 and 600E; Waters Corporation, Milford, MA) on an acetonitrile/trifluoroacetic acid gradient. Correct mass was confirmed by electrospray mass spectroscopy (Quattro II, Fisons Inc., Valencia, CA). Peptides were reconstituted as 10 mmol/L stock solutions by dissolving in DMSO (Ant-Bax BH3 peptides) or 10 mmol/L Tris (pH 7.4; untagged peptides) and then stored as aliquots at −80°C.

Intracellular Heterodimerization

To examine the effects of the Ant-Bax BH3, Ant-Bax BH3 CS, and Ant-Bax BH3 CS/LE peptides on intracellular Bax/Bcl-2 heterodimerization, coimmunoprecipitation assays were done. HL-60 cells (2 × 106) were first incubated in the absence or presence of peptides for 12 hours at 37°C. Whole cell lysates were then prepared in coimmunuprecipitation lysis buffer [10 mmol/L HEPES, 140 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L EDTA (pH 7.4)] containing 0.2% NP40. Anti-Bax was added to a final concentration of 2 μg/mL and incubation was allowed to proceed for 2 hours at 4°C followed by addition of 20 μL of protein A-agarose beads for 2 hours. The immunoprecipitates were boiled, electrophoresed on 12.5% SDS-PAGE gels, transferred to nitrocellulose, and subjected to immunoblotting with anti-Bcl-2.

Protein Purification and In vitro Translations

Glutathione S-transferase (GST)-Bax protein was produced in Escherichia coli harboring pGEX-2T-Bax construct (41). Bacteria were grown to an A600 nm of 0.6 to 0.8, induced with 0.5 mmol/L isopropyl-l-thio-B-d-galactopyranoside for 3 hours at 37°C, and harvested by centrifugation. The pellet was resuspended in HKMEN buffer [10 mmol/L HEPES (pH 7.2), 140 mmol/L KCl, 5 mmol/L MgCl2, 2 mmol/L EDTA, 0.5% NP40] containing 0.03% SDS, 100 μg/mL lysozyme, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 mmol/L DTT, and 1 mmol/L phenylmethylsulfonyl fluoride. Following sonication, lysate was mixed with glutathione-agarose beads to pull-down GST-Bax. Purified protein was eluted with 20 mmol/L reduced glutathione prepared in 50 mmol/L Tris (pH 8.0) and then dialyzed against PBS.

In vitro translated 35S-Bcl-2 and 35S-Bcl-XL were synthesized from full-length cDNA (41, 48) using a TNT-coupled transcription/translation system (Promega, Madison, WI) according to the manufacturer's instructions.

Disruption of In vitro Heterodimerization Interactions

The effect of Bax BH3 peptides on Bax/Bcl-2 or Bax/Bcl-XL heterodimerization interactions was examined in in vitro assays as described previously (41). Briefly, for each assay, 5 μg of GST-Bax were incubated with 10 μL of in vitro translated 35S-Bcl-2 or 35S-Bcl-XL in a total reaction volume of 50 μL. Incubations were done for 2 hours at 4°C in the absence or presence of peptides. Glutathione-agarose beads were then added for 1 hour followed by washing of the beads with HKMEN buffer. The washed beads were boiled in SDS-PAGE sample buffer and the resulting supernatant proteins were electrophoresed on 12.5% SDS-PAGE gels and then transferred to nitrocellulose membranes. Autoradiography and densitometry were used to quantify the amount of radioactive protein bound to GST-Bax.

Isolation of Mitochondria and Cytochrome c Release Assays

To isolate mitochondria, cells were washed with PBS and then resuspended in homogenization buffer [20 mmol/L HEPES (pH 7.4), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 250 mmol/L sucrose] containing 1 mmol/L DTT, 1.5 mmol/L phenylmethylsulfonyl fluoride, 3 μg/mL leupeptin, and 20 μg/mL aprotinin. The resuspended cells were homogenized in a type B Dounce homogenizer (Wheaton, Millville, NJ). Nuclei and other debris were removed by centrifugation at 600 × g for 5 minutes at 4°C. Supernatants were subjected to a second centrifugation again for 30 minutes at 10,000 × g to pellet mitochondria, which were then resuspended in homogenization buffer containing protease inhibitors. Protein concentrations were determined using the Bio-Rad protein assay kit (Hercules, CA) and homogenization buffer was added to achieve a final concentration of 2 mg/mL. Cytochrome c release assays were done by incubating 100 μg of mitochondria in the absence or presence of peptides for 1 hour at 37°C. Following incubation, reaction mixtures were centrifuged at 10,000 × g for 30 minutes at 4°C to pellet mitochondria, whereas supernatants were carefully collected and subjected to a second centrifugation. Resulting supernatants and the mitochondrial pellets were analyzed by immunoblotting for release and depletion of cytochrome c, respectively.

CD Spectroscopy

Far UV CD spectra were recorded on a model 202 CD spectrometer (Aviv Instruments, Lakewood, NJ) with a 0.1 mm path length cuvette and thermostatically controlled temperature of 25°C. The spectra were collected with a 1-nm step size over the 280 to 185 nm wavelength range using a 1-second time constant. For each peptide, at least five spectra were averaged and the background signal of the buffer alone was subtracted. Peptide concentrations were determined by quantitative ninhydrin assay using leucine as a standard (49). The spectra were deconvoluted using a variable selection method, the CDSSTR algorithm (50–52), and a reference protein data set consisting of 42 proteins (5 denatured) in aqueous solution (protein reference data set 6; refs. 52, 53) using the DICHROWEB server (54).5

Results

Disruption of Intracellular Bax/Bcl-2 Heterodimerization and Activation of Apoptosis Signaling by Bax BH3 Peptides

Previously, we and others have shown that peptides derived from BH3 domains of proapoptotic proteins can physically disrupt heterodimerization interactions among Bcl-2 family members in cell-free systems (41, 55, 56). Specifically, we have shown that a 20–amino acid Bax BH3 peptide disrupts Bax/Bcl-2 heterodimerization interactions with an IC50 of 15 μmol/L and Bax/Bcl-XL interactions with an IC50 of 9.5 μmol/L (41). To verify that Bax BH3 peptides also disrupt heterodimerization interactions in whole cells, Bax BH3 peptides were introduced into whole cells. In prior studies, we have achieved successful intracellular delivery of peptides using a lipid-mediated delivery agent (BioPorter peptide delivery system, Gene Therapy Systems, San Diego, CA; ref. 41). However, long-term exposure of cells to the BioPorter agent resulted in high background levels of cell death. Therefore, for the current experiments, we employed Bax BH3 peptides fused to the 16–amino acid peptide transduction domain from Drosophila Antennapedia protein (termed Ant peptide; Fig. 1A). The Ant peptide was fused to the NH2 terminus of 20-mer Bax BH3 peptide to generate Ant-Bax BH3. In addition, to minimize the potential for intracellular oxidation of the peptide, we synthesized a variant peptide called Ant-Bax BH3 CS in which Cys62 (numbering based on human Bax sequence) in the BH3 domain was changed to Ser62. A control peptide, Ant-Bax BH3 CS/LE, was also synthesized in which Leu63 was mutated to Glu63. Mutation of Leu63 to Glu63 in the full-length protein has been shown to markedly reduce Bax function (57) and we have shown that peptides with this mutation are ineffective at disrupting heterodimerization interactions in vitro (41).

Figure 1.
Figure 1.

Cell-permeable Bax BH3 peptides disrupt intracellular Bax/Bcl-2 heterodimerization and activate apoptosis signaling in HL-60 cells. A, amino acid sequences of three cell-permeable BH3 peptides. Mutated amino acids are underlined. B, HL-60 cells (1 × 106 cells/lane) were plated at a density of 1 × 106 cells/mL and then treated in the absence (Control) or presence of 5 or 25 μmol/L of the indicated Ant-tagged peptides. After 12 hours at 37°C, whole cell extracts were prepared and cellular Bax immunoprecipitated with anti-Bax polyclonal antibody. The immunoprecipitated proteins were resolved on a 12.5% SDS-PAGE gel, transferred to nitrocellulose, and probed with anti-Bcl-2 monoclonal antibody. To verify equivalent immunoprecipitation of Bax in the different samples, the blot was also probed with anti-Bax polyclonal antibody. C, HL-60 cells were plated at 1 × 106 cells/mL and then treated in the absence (Control) or presence of 25 μmol/L of the indicated Ant-tagged peptides. Following a 12-hour incubation, aliquots of the cells were used to determine percentage cell death as assessed by trypan blue exclusion assay. In addition, whole cell lysates were prepared and subjected to immunoblotting with anti-caspase-3, anti–poly(ADP-ribose) polymerase (PARP), and anti-Bcl-2. The locations of intact and cleaved forms of the proteins are indicated. The blot was stripped and reprobed with anti-tubulin to verify equal loading.

In Fig. 1B, peptides were added to intact HL-60 cells for 12 hours followed by preparation of whole cell lysates and immunoprecipitation of cellular Bax. The immunoprecipitates were then analyzed by immunoblotting for the presence of coimmunoprecipitating Bcl-2. HL-60 cells were chosen for these experiments because they are known to express high levels of Bax and Bcl-2. As depicted, treatment of cells with 5 or 25 μmol/L Ant-Bax BH3 peptide efficiently disrupted intracellular interactions between Bax and Bcl-2. The Ant-Bax BH3 CS peptide was somewhat less effective than wild-type Ant-Bax BH3 peptide when used at 5 μmol/L but comparable with wild-type activity when used at 25 μmol/L. As expected, the Ant-Bax BH3 CS/LE mutant control peptide failed to disrupt Bax/Bcl-2 heterodimers even when used at 25 μmol/L. Taken together, these results verified that the ability of synthetic Bax BH3 peptides to disrupt heterodimerization interactions in cell-free reactions correlates with their ability to disrupt physiologically relevant interactions in whole cells.

The inability of Ant-Bax BH3 peptide to achieve complete disruption of intracellular interactions (Fig. 1B), compared with complete disruption of heterodimerization interactions observed previously in in vitro assays (41), may point to functional inactivation of the peptide in the complex intracellular milieu or to limited cellular permeability. At the same time, the ability of Ant-Bax BH3 peptide to cause at least partial disruption of intracellular interactions showed that fusion of the Ant peptide transduction domain to the NH2 terminus of the Bax BH3 peptide did not profoundly interfere with the ability of the peptide to bind to the BH3 binding domain on the surface of the Bcl-2 protein.

To determine whether Bax BH3 peptides activate apoptosis signaling in whole cells, the cell-permeable peptides were incubated with HL-60 cells for 12 hours followed by immunoblot analysis of caspase activation (Fig. 1C). Both Ant-Bax BH3 and Ant-Bax BH3 CS peptides induced processing of the 32-kDa pro-caspase-3 zymogen to an active enzyme form. The activation of caspase-3 by both peptides was accompanied by cleavage of the caspase-3 substrate proteins poly(ADP-ribose) polymerase and Bcl-2 to 85 and 23 kDa fragments, respectively. By contrast, Ant-Bax BH3 CS/LE mutant control peptide had no effect on caspase activation or the cleavage of caspase substrate proteins. Moreover, although Ant-Bax BH3 and Ant-Bax BH3 CS led to 62% and 79% cell death, respectively, the control peptide did not affect cell viability.

Disruption of Bax/Bcl-2 Heterodimerization by Bax BH3 Peptide Variants

We next sought to determine the minimal length and important sequence requirements for the biological activity of Bax BH3 peptide. Having verified that the activities of Bax BH3 peptides in cell-free assays accurately reflect activities observed in whole cells (ref. 41; Fig. 1), we did our studies in vitro using peptides lacking the Ant peptide transduction domain. A series of peptide variants (Table 1) were synthesized and compared with parental Bax BH3 20-mer (Bax 1) for biological activity. Because homology between BH3 domains from different proapoptotic proteins is highest in the central 7–amino acid BH3 core (italicized in Table 1), we focused on deleting less conserved amino acids outside of this core sequence. Briefly, peptide variants were generated containing deletions at the NH2 terminus, deletions in the COOH terminus, or a combination of deletions at both ends. In addition, several peptides were generated containing deletions of internal amino acids. Bax 2 was designed to substitute two NH2-terminal residues with alanine residues (ST/AA).

View this table:
Table 1.

Properties of the Bax BH3 peptides

The peptide variants were first examined for their ability to disrupt Bax/Bcl-2 heterodimerization interactions in vitro when used at 200 μmol/L (Fig. 2A). Peptides that displayed an ability to disrupt Bax/Bcl-2 were subsequently evaluated in greater detail to determine the IC50 value of the peptide for disrupting Bax/Bcl-2 interactions (Fig. 2B; Table 1). The findings from these experiments can be summarized as follows: First, deletion of two or more amino acids from the NH2 terminus abolished peptide activity. For example, Bax 9, with two amino acids deleted at the NH2 terminus, exhibited an IC50 value of ∼450.1 ± 30.3 μmol/L compared with an IC50 value of 4.7 ± 1.5 μmol/L for parental Bax 1 peptide. Interestingly, substitution of the two NH2-terminal amino acids with alanine residues did not affect activity, as Bax 2 exhibited an IC50 value of 3.3 ± 0.8 μmol/L. Second, deletion of up to four amino acids in the COOH terminus could be accomplished while still retaining an ability to disrupt Bax/Bcl-2. However, in these deletions, we found that it was important to retain the last COOH-terminal methionine residue (compare Bax 16 versus Bax 6 or 7). Third, all internal deletions abolished peptide activity. The internal deletion mutants were designed to remove amino acids that are less conserved among BH3 domains (57, 58). Up to three contiguous internal amino acids were deleted in an effort to preserve the amphipathic nature of the BH3 domain α-helix. However, none of the internal deletion mutants retained an ability to disrupt Bax/Bcl-2. In summary, these experiments defined a 16–amino acid peptide (Bax 7) as the shortest peptide capable of efficiently disrupting Bax/Bcl-2 interactions.

Figure 2.
Figure 2.

Disruption of Bax/Bcl-2 heterodimerization by Bax BH3 peptide variants. A, in vitro translated 35S-Bcl-2 (10 μL) was incubated with GST-Bax (5 μg) for 2 hours at 4°C in the absence (Control) or presence of 200 μmol/L of the indicated peptides. Glutathione-agarose was then used to pull down the GST-Bax along with bound 35S-Bcl-2. Samples were resolved on a 12.5% SDS-PAGE gel, transferred to nitrocellulose, and subjected to autoradiography. Lane 1, 20% of the input 35S-Bcl-2; lane 2, control lane reflects incubation in the absence of peptide. To determine the homogeneity of the purified fusion proteins used in the experiment, 5 μg of purified GST or purified GST-Bax were resolved on a 10% SDS-PAGE gel followed by staining with Coomassie blue. B, peptides exhibiting an ability to disrupt Bax/Bcl-2 interactions were examined in quantitative fashion to determine IC50 values. For each peptide tested, a dose-response curve was established for disruption of Bax/Bcl-2 interactions using the in vitro assay described in A. For each peptide concentration, densitometry of the autoradiograph was used to determine the percentage of Bax/Bcl-2 heterodimerization relative to that observed in the absence of peptide. Percentage disruption was calculated according to the equation: percentage disruption = (35S-Bcl-2 densitometric signal in the presence of peptide) / (35S-Bcl-2 densitometric signal in the absence of peptide) × 100. IC50 values were calculated from the point of 50% disruption of Bax/Bcl-2 heterodimerization and are shown in Table 1.

Disruption of Bax/Bcl-XL Heterodimerization by Bax BH3 Peptide Variants

We next examined whether the variant Bax BH3 peptides could disrupt Bax/Bcl-XL heterodimerization interactions (Fig. 3; Table 1). In general, the abilities of peptides to disrupt Bax/Bcl-XL correlated with their abilities to disrupt Bax/Bcl-2. However, all of the active peptides showed significantly lower IC50 values for disrupting Bax/Bcl-XL than for disrupting Bax/Bcl-2. Peptides that failed to disrupt Bax/Bcl-2 were found ineffective at disrupting Bax/Bcl-XL, with two notable exceptions. Bax 16, a 16–amino acid peptide, and Bax 17, a 15–amino acid peptide, while failing to disrupt Bax/Bcl-2 (Fig. 2), disrupted Bax/Bcl-XL with IC50 values of 20 ± 4.1 and 79 ± 18.3 μmol/L, respectively (Fig. 3; Table 1). Thus, two different 16–amino acid peptides (Bax 7 and Bax 16) and one 15–amino acid peptide (Bax 17) were capable of disrupting Bax/Bcl-XL interactions.

Figure 3.
Figure 3.

Disruption of Bax/Bcl-XL heterodimerization by Bax BH3 peptide variants. A, in vitro assays of Bax/Bcl-XL heterodimerization were done by incubating GST-Bax with 35S-Bcl-XL in the absence (Control) or presence of the indicated Bax BH3 peptides (200 μmol/L). Reaction mixtures were analyzed as described in Fig. 2A. B, dose-response curves of peptide-mediated disruption of Bax/Bcl-XL interactions were done as described in Fig. 2B and IC50 values for the peptides are shown in Table 1.

Induction of Cytochrome c Release by Bax BH3 Peptide Variants

During apoptosis mediated by the intrinsic mitochondrial pathway, the release of cytochrome c into the cytosol constitutes a key step toward activation of the caspase protease cascade and ultimate cellular demise (1, 2, 6, 7). Bcl-2 and Bcl-XL prevent apoptotic cell death by inhibiting cytochrome c release (1, 2, 59), whereas proapoptotic proteins such as Bax promote cytochrome c release (8, 30, 60). It is thought that Bcl-2 and Bcl-XL may prevent cytochrome c release by binding and inhibiting Bax or other proapoptotic proteins. Previously, we have shown that the parental 20–amino acid Bax BH3 peptide (Bax 1), in addition to disrupting Bax/Bcl-2 and Bax/Bcl-XL, can promote cytochrome c release from isolated mitochondria (41). Therefore, we examined the activity of the variant peptides in cytochrome c release assays.

Peptides were added for 1 hour to mitochondria isolated from Jurkat T leukemic cells and cytochrome c release was assessed by immunoblotting of supernatant proteins (Fig. 4; Table 1). To determine the abilities of the peptides to overcome the inhibitory effects of Bcl-2 or Bcl-XL overexpression, experiments were done using mitochondria isolated from vector-transfected Jurkat cells, Bcl-2-overexpressing Jurkat cells, and Bcl-XL-overexpressing Jurkat cells. As shown, each of the peptides that disrupted Bax/Bcl-2 or Bax/Bcl-XL interactions in vitro showed an ability to promote cytochrome c release. Similar profiles were seen when mitochondria from HL-60, U937, or PC-3 cells were used (Fig. 5; Table 1; data not shown). Expectedly, release of cytochrome c was accompanied by a corresponding loss of cytochrome c from the mitochondrial pellet. The specificity of the cytochrome c release was confirmed by immunoblotting for cytochrome c oxidase IV, a mitochondrial protein that is not released into the cytosol during apoptosis signal transduction.

Figure 4.
Figure 4.

Induction of cytochrome c release from isolated Jurkat mitochondria by Bax BH3 peptide variants. Mitochondria were isolated from vector-transfected Jurkat cells (A; Vector/Jurkat), Bcl-2-overexpressing Jurkat cells (B; Bcl-2/Jurkat), and Bcl-XL-overexpressing Jurkat cells (B; Bcl-XL/Jurkat) as described in Materials and Methods. Isolated mitochondria (100 μg/sample) were then incubated for 1 hour at 37°C in the absence (Control) or presence of the indicated peptides (200 μmol/L). Following incubation, the mitochondria were pelleted to obtain supernatant (released proteins) and pellet (mitochondria) fractions. The supernatant and pellet fractions were electrophoresed on 12.5% SDS-PAGE gels, transferred to nitrocellulose, and probed with anti–cytochrome c (Cyt c). The pellet fraction was also probed with anti–cytochrome c oxidase IV (Cyt c Ox IV) to verify the specificity of the cytochrome c release.

Figure 5.
Figure 5.

Induction of cytochrome c release from isolated HL-60 and U937 mitochondria. Isolated mitochondria (100 μg) were incubated with the indicated peptides for 1 hour at 37°C, and specific release of cytochrome c was assessed as described in Fig. 4.

The results shown in Fig. 4 also showed that peptides capable of promoting cytochrome c release from mitochondria of vector-transfected Jurkat cells also promoted release from mitochondria of Bcl-2-overexpressing or Bcl-XL-overexpressing cells (Fig. 4; Table 1). Thus, Bcl-2 and Bcl-XL, which normally inhibit cytochrome c release, are functionally inactivated by peptides that disrupt the interactions of these proteins with proapoptotic partners. Curiously, certain peptides (e.g., Bax 9 and Bax 17) with high IC50 values for disruption of heterodimerization interactions (Table 1) were still capable of promoting cytochrome c release, indicating that partial inhibition of Bcl-2 or Bcl-XL may be sufficient to trigger the release of cytochrome c.

Interestingly, both Bax 16 (16-mer) and Bax 17 (15-mer), which were capable of disrupting Bax/Bcl-XL but not Bax/Bcl-2, promoted cytochrome c release from Jurkat mitochondria (Fig. 4). In a dose-response experiment, Bax 17 (15-mer) exhibited nearly equal potency to that of Bax 1 (20-mer) parental (Fig. 6). Additional experiments showed that Bax 17 (15-mer) also promotes cytochrome c release from HL-60 mitochondria but not from mitochondria of U937 or PC-3 cells (Fig. 5; Table 1; data not shown). By contrast, Bax 16 (16-mer) promoted cytochrome c release from mitochondria of all four cell types (Jurkat, HL-60, U937, and PC-3). Thus, although Bax 17 is the shortest peptide capable of inducing cytochrome c release, the biological activity of this 15–amino acid peptide seems to depend on the specific cellular environment.

Figure 6.
Figure 6.

Parental Bax 1 (20-mer) and Bax 17 (15-mer) peptides exhibit similar potencies at overcoming Bcl-2 overexpression. Isolated mitochondria (100 μg) from Bcl-2-overexpressing Jurkat cells were incubated for 1 hour at 37°C in the absence or presence of increasing doses of Bax 1 or Bax 17 peptides. Cytochrome c release was assessed as described in Fig. 4.

The biological activity of Bax 9 also seems to be cell type specific. Surprisingly, Bax 9 promoted release of cytochrome c from Jurkat mitochondria despite only weak ability to disrupt Bax/Bcl-2 and Bax/Bcl-XL interactions (Figs. 2 and 3). In experiments with HL-60, U937, or PC-3 mitochondria, the Bax 9 peptide did not cause cytochrome c release (Fig. 5; Table 1; data not shown).

α-Helical Content of the Bax BH3 Peptide Variants

Nuclear magnetic resonance imaging and X-ray crystallography of full-length Bcl-XL, Bcl-2, and Bax proteins have revealed that BH3 domains exist in an α-helical conformation (27–29). However, short peptides, when taken out of context of the full-length protein, can be extremely flexible and often show little net structure in aqueous solution. To determine whether helical content of the Bax BH3 peptides correlated with biological activity, we used CD spectroscopy to analyze peptide α helicity. In 10 mmol/L potassium phosphate buffer (pH 7.4), the 20–amino acid Bax BH3 parental peptide showed a CD spectrum characteristic with that of a random coil, although we were unable to deconvolute it (data not shown). However, in a 40% solution of trifluoroethanol (TFE), this peptide showed a strong total helicity of 62%. TFE/H2O mixtures promote intrapeptide hydrogen bonds and are commonly used to determine the helix-forming propensities of small flexible peptides (61, 62). In previous studies, BH3-derived peptides were examined by CD spectroscopy in 40% (v/v) TFE to test whether the helical content of the peptide correlated with toxicity (44). Analogously, 40% TFE was used in our studies to compare the relative helical content of all the peptides. The CD spectra of the tested peptides are shown in Fig. 7. The percentage of α-helical content was quantified as described in Materials and Methods and is shown in Table 1. All values obtained had a normalized root mean square deviation (63) of <0.02, indicating that the reference database used in the CD studies was appropriate in fitting the experimentally determined CD spectra.

Figure 7.
Figure 7.

CD spectra of the Bax BH3 peptide variants. The CD spectrum of each peptide represents the average of at least five spectra of the peptide in 40% (v/v) TFE. For clarity, the spectra are divided arbitrarily into two panels. Optical signals are presented as Δε, which has units of mdeg mol/L−1 cm−1. The α-helical content was determined as described in Materials and Methods and is shown in Table 1.

The parental Bax BH3 peptide Bax 1 exhibited 62% α-helical content, whereas the α-helical content of the biologically active peptide variants Bax 2 to 7 ranged from 35% to 77%. The α-helical content of the inactive peptides tested (Bax 8, 10, 11, 12, 16, and 18) ranged from 16% to 46%. In general, the α-helical content of biologically active peptides (58 ± 16% average helicity for peptides testing positive for cytochrome c release in Jurkat cells) was higher than that observed in biologically inactive peptides (33 ± 11%). This suggests that α-helical content is important for the proapoptotic biological activity of the peptides. However, the results also indicate that α-helical content alone is not sufficient for biological activity. Bax 8, which was biologically inactive, exhibited higher helical content (46%) than the two active peptides [Bax 3 (35%) and Bax 7 (38%)] and similar content to a third active peptide [Bax 5 (45%)]. Moreover, two peptides that exhibited variable biological activities, Bax 16 and 17, were found to have high helical content. Bax 16, which was capable of disrupting Bax/Bcl-XL and promoting cytochrome c release in all cell lines tested but incapable of disrupting Bax/Bcl-2, exhibited 63% α helicity. Bax 17, which failed to disrupt Bax/Bcl-2 and only promoted cytochrome c release in certain cell types, exhibited the highest α-helical content at 82%. Thus, although helical content seems to roughly correlate with biological activity, it is not sufficient; the specific sequence composition of the peptide is also critical.

Discussion

Overexpression of antiapoptotic members of the Bcl-2 family protein family, including Bcl-2 and Bcl-XL, is common in human malignancies and contributes to chemotherapy and radiation resistance. In several cancers, overexpression of Bcl-2 or Bcl-XL has been shown to correlate with poor clinical prognosis (13). At the molecular level, overexpressed Bcl-2 or Bcl-XL binds to proapoptotic proteins such as Bax, thereby preventing cytochrome c release into the cytosol and activation of the intrinsic, mitochondrial-mediated apoptosis pathway (1, 2, 6, 10). Antisense-mediated down-regulation of Bcl-2 or Bcl-XL enhances the sensitivity of cancer cells to apoptotic stimuli, and Bcl-2 antisense (G3139) is currently being investigated in clinical trials (13, 16, 18, 19). The peptides we have characterized and optimized target the function, rather than the expression, of Bcl-2 and Bcl-XL. Using cytochrome c release assays, our results clearly show the ability of Bax BH3 peptides to overcome the antiapoptotic action of Bcl-2 and Bcl-XL.

Structural and molecular modeling studies have determined that the BH1, BH2, and BH3 domains of Bcl-2 and Bcl-XL form a hydrophobic groove on the surface of these proteins (27, 28). This groove is critical for the antiapoptotic activity of Bcl-2 and Bcl-XL and serves as a receptor-like binding pocket for BH3 domains of proapoptotic proteins (64–66). Thus, this BH3 binding pocket is an attractive target for drug discovery efforts aimed at inhibiting Bcl-2 or Bcl-XL function. We and others have shown that peptides derived from the BH3 domains of Bax, Bid, Bad, and Bak inhibit heterodimerization among Bcl-2 family members in in vitro assays and promote cellular apoptosis (41, 42, 46, 55, 56, 67). In addition, several small organic molecules have recently been identified that bind to Bcl-2 or Bcl-XL and promote apoptosis (20–24).

Although several studies have examined the ability of BH3 peptides to disrupt protein-protein interactions in cell-free systems, little is known about the ability of these peptides to directly target and interfere with antiapoptotic proteins in intact cells. Therefore, we first asked whether Bax BH3 domain peptides could disrupt intracellular heterodimerization between Bax and Bcl-2 and whether this correlates with the proapoptotic activities of the peptides. We found that cell-permeable Bax BH3 peptides that promoted caspase activation and loss of cell viability also disrupted Bax/Bcl-2 heterodimerization in intact cells. By contrast, a mutant peptide that failed to induce cell death also was deficient at disrupting Bax/Bcl-2 interactions. The ability of Bax BH3 peptide to disrupt Bax/Bcl-2 heterodimerization in intact cells argues strongly that the peptide directly targets Bcl-2 family members in the cell. In an attempt to improve the intracellular stability and activity of Bax BH3 peptide, we also changed Cys62 to Ser62. However, the serine-containing peptide was at best equivalent in activity to the wild-type cysteine-containing version, indicating no significant benefit to modifying this residue.

The usefulness of peptides as therapeutic agents is limited to some degree by the large size of peptide compounds. The large molecular weight and the chemical composition of peptides can contribute to poor solubility, cellular permeability, and stability. Thus, when considering a peptide-based compound as a therapeutic agent, it is first important to define the minimal size and essential sequence composition that is needed for biological activity. By reducing the size of the peptide compound, it also becomes more feasible to generate peptidomimetic derivatives with improved drug-like properties. Therefore, we undertook to optimize the size, sequence, and structural composition of biologically active Bax BH3 peptide.

To generate an optimized Bax BH3 peptide, we did a systematic analysis of peptide variants. We identified 15 amino acids as the shortest peptide length capable of promoting cytochrome c release. These results are consistent with those of Diaz et al. (55), who observed disruption of Bax/Bcl-XL heterodimerization in an in vitro plate binding assay using a 15–amino acid Bax BH3 peptide. The 15–amino acid peptide (Bax 17) we identified disrupted Bax/Bcl-XL but not Bax/Bcl-2 and promoted cytochrome c release from mitochondria of two of the four cell lines tested. A 16–amino acid peptide (Bax 7), however, disrupted both Bax/Bcl-XL and Bax/Bcl-2 and promoted release of cytochrome c from mitochondria of all four cell lines. Additional analyses indicated that deletion of amino acids COOH terminal to the BH3 core sequence was well tolerated but revealed a critical importance of Met74. Deletion of amino acids NH2 terminal to the BH3 core was not tolerated, although substitution of these residues with alanine residues may be sufficient to maintain activity.

The ability of the peptides to promote cytochrome c release from isolated mitochondria further supports the contention that Bcl-2 or Bcl-XL in the mitochondrial outer membrane are the direct targets of these agents. Moreover, peptides that failed to disrupt Bax/Bcl-XL were incapable of promoting cytochrome c release. Interestingly, Bax 16 (16-mer) and Bax 17 (15-mer), which disrupted Bax/Bcl-XL but not Bax/Bcl-2, retained the capacity to promote cytochrome c release from Jurkat and HL-60 mitochondria. This suggests that Bcl-XL represents the critical antiapoptotic protein in these cell lines. However, we cannot rule out the possibility that the Bax BH3 peptides may be binding and directly activating proapoptotic Bax or Bak. Letai et al. (43) have shown that Bid BH3 peptide binds directly to Bak, inducing oligomerization and proapoptotic activity. The inclusion of antibodies that block the BH3 binding domains of Bcl-2 and Bcl-XL in experiments involving BH3 peptide treatment of isolated mitochondria may help to resolve whether the peptides are directly targeting antiapoptotic (Bcl-2 or Bcl-XL) or proapoptotic (Bax or Bak) Bcl-2 family members on the surface of the mitochondria.

The role of α-helical content in the proapoptotic activity of BH3 peptides has been controversial. Structural analyses have revealed that BH3 domains exist in an α-helical conformation when binding to the BH3 binding pocket of antiapoptotic proteins (29, 64, 65). Moreover, the BH3 domain α helix is amphipathic, with hydrophobic residues interfacing with the binding pocket and polar residues exposed to the aqueous environment (57, 64, 65). Strikingly, a previous report indicated that cell death resulting from Bad BH3 peptide can be attributed solely to the helical content of the peptide in 40% TFE independent of any Bcl-2 binding capacity (44). Our results indicate a strong correlation between the ability of a peptide to disrupt either Bax/Bcl-XL or both Bax/Bcl-2 and Bax/Bcl-XL and the ability to promote cytochrome c release. In addition, although helical content generally correlated with biological activity, helical content alone was not sufficient to induce cytochrome c release. Bax 17 (15-mer), which exhibited the highest α-helical content (82%), was incapable of promoting cytochrome c release from mitochondria of two of the four cell lines tested. Thus, the precise sequence composition, length, and helical content all seem to play important roles in determining the biological activity of the peptides.

In summary, our studies have defined an optimal length as well as critical sequence and structural requirements of the Bax BH3 peptide, laying the groundwork for future optimization of the peptide, or derivatives thereof, as an anticancer agent. Future structural modifications, including lactam bridging (68), incorporation of non-natural amino acids, and generation of peptidomimetic derivatives, should help to improve stability and potency. Additionally, it will be informative to determine whether BH3 peptides synergize with any of the growing number of small molecule Bcl-2/Bcl-XL inhibitors to promote cell killing of Bcl-2-overexpressing or Bcl-XL-overexpressing tumors. Additionally, it will be informative to determine whether BH3 peptides synergize with any of the growing number of small molecule Bcl-2/Bcl-XL> inhibitors to promote cell killing of Bcl-2-overexpressing or Bcl-XL-overexpressing tumors. Moreover, it will be important to test whether sublethal doses of optimized BH3 peptides can be used to sensitize drug-resistant tumor cells to chemotherapeutic agents.

Acknowledgments

We thank the members of Dr. Thomas Smithgall's laboratory (University of Pittsburgh School of Medicine, Pittsburgh, PA) for providing assistance with the densitometric scanning.

Footnotes

  • ↵5 http://www.cryst.bbk.ac.uk/cdweb/html/home.html

  • Grant support: Leukemia and Lymphoma Society of America Translational Research Award 6456 and NIH grant RO1 CA86980 (D.E. Johnson).

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    • Accepted August 27, 2004.
    • Received July 13, 2004.
    • Revision received August 18, 2004.

References

  1. ↵
    Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 1997;275:1132–6.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 1997;275:1129–32.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Susin SA, Lorenzo HK, Zamzami N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999;397:441–6.
    OpenUrlCrossRefPubMed
  4. ↵
    Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 2000;102:33–42.
    OpenUrlCrossRefPubMed
  5. ↵
    Verhagen AM, Ekert PG, Pakusch M, et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 2000;102:43–53.
    OpenUrlCrossRefPubMed
  6. ↵
    Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997;91:479–89.
    OpenUrlCrossRefPubMed
  7. ↵
    Zou H, Li Y, Liu X, Wang X. An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 1999;274:11549–56.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Kuwana T, Mackey MR, Perkins G, et al. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 2002;111:331–42.
    OpenUrlCrossRefPubMed
  9. ↵
    Wei MC, Lindsten T, Mootha VK, et al. tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 2000;14:2060–71.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Gross A, Jockel J, Wei MC, Korsmeyer SJ. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J 1998;17:3878–85.
    OpenUrlAbstract
  11. ↵
    Gazitt Y, Fey V, Thomas C, Alvarez R. Bcl-2 overexpression is associated with resistance to dexamethasone, but not melphalan, in multiple myeloma cells. Int J Oncol 1998;13:397–405.
    OpenUrlPubMed
  12. ↵
    Friess H, Lu Z, Andren-Sandberg A, et al. Moderate activation of the apoptosis inhibitor bcl-xL worsens the prognosis in pancreatic cancer. Ann Surg 1998;228:780–7.
    OpenUrlCrossRefPubMed
  13. ↵
    Shangary S, Johnson DE. Recent advances in the development of anticancer agents targeting cell death inhibitors in the Bcl-2 protein family. Leukemia 2003;17:1470–81.
    OpenUrlCrossRefPubMed
  14. ↵
    Groeger AM, Esposito V, De Luca A, et al. Prognostic value of immunohistochemical expression of p53, bax, Bcl-2 and Bcl-xL in resected non-small-cell lung cancers. Histopathology 2004;44:54–63.
    OpenUrlCrossRefPubMed
  15. ↵
    Mercatante DR, Bortner CD, Cidlowski JA, Kole R. Modification of alternative splicing of Bcl-x pre-mRNA in prostate and breast cancer cells. analysis of apoptosis and cell death. J Biol Chem 2001;276:16411–7.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Webb A, Cunningham D, Cotter F, et al. BCL-2 antisense therapy in patients with non-Hodgkin lymphoma. Lancet 1997;349:1137–41.
    OpenUrlCrossRefPubMed
  17. ↵
    Kitada S, Miyashita T, Tanaka S, Reed JC. Investigations of antisense oligonucleotides targeted against bcl-2 RNAs. Antisense Res Dev 1993;3:157–69.
    OpenUrlPubMed
  18. ↵
    Marcucci G, Byrd JC, Dai G, et al. Phase 1 and pharmacodynamic studies of G3139, a Bcl-2 antisense oligonucleotide, in combination with chemotherapy in refractory or relapsed acute leukemia. Blood 2003;101:425–32.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Jansen B, Wacheck V, Heere-Ress E, et al. Chemosensitization of malignant melanoma by BCL2 antisense therapy. Lancet 2000;356:1728–33.
    OpenUrlCrossRefPubMed
  20. ↵
    Wang JL, Liu D, Zhang ZJ, et al. Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc Natl Acad Sci U S A 2000;97:7124–9.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Tzung SP, Kim KM, Basanez G, et al. Antimycin A mimics a cell-death-inducing Bcl-2 homology domain 3. Nat Cell Biol 2001;3:183–91.
    OpenUrlCrossRefPubMed
  22. ↵
    Degterev A, Lugovskoy A, Cardone M, et al. Identification of small-molecule inhibitors of interaction between the BH3 domain and Bcl-xL. Nat Cell Biol 2001;3:173–82.
    OpenUrlCrossRefPubMed
  23. ↵
    Kitada S, Leone M, Sareth S, Zhai D, Reed JC, Pellecchia M. Discovery, characterization, and structure-activity relationships studies of proapoptotic polyphenols targeting B-cell lymphocyte/leukemia-2 proteins. J Med Chem 2003;46:4259–64.
    OpenUrlCrossRefPubMed
  24. ↵
    Enyedy IJ, Ling Y, Nacro K, et al. Discovery of small-molecule inhibitors of Bcl-2 through structure-based computer screening. J Med Chem 2001;44:4313–24.
    OpenUrlCrossRefPubMed
  25. ↵
    Baell JB, Huang DC. Prospects for targeting the Bcl-2 family of proteins to develop novel cytotoxic drugs. Biochem Pharmacol 2002;64:851–63.
    OpenUrlCrossRefPubMed
  26. ↵
    Wang S, Yang D, Lippman ME. Targeting Bcl-2 and Bcl-XL with nonpeptidic small-molecule antagonists. Semin Oncol 2003;30:133–42.
    OpenUrlCrossRefPubMed
  27. ↵
    Muchmore SW, Sattler M, Liang H, et al. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature 1996;381:335–41.
    OpenUrlCrossRefPubMed
  28. ↵
    Petros AM, Medek A, Nettesheim DG, et al. Solution structure of the antiapoptotic protein bcl-2. Proc Natl Acad Sci U S A 2001;98:3012–7.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Suzuki M, Youle RJ, Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 2000;103:645–54.
    OpenUrlCrossRefPubMed
  30. ↵
    Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ, Schlesinger PH. Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ 2000;7:1166–73.
    OpenUrlCrossRefPubMed
  31. ↵
    Mikhailov V, Mikhailova M, Degenhardt K, Venkatachalam MA, White E, Saikumar P. Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release. J Biol Chem 2003;278:5367–76.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Wei MC, Zong WX, Cheng EH, et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 2001;292:727–30.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Ruffolo SC, Shore GC. BCL-2 selectively interacts with the BID-induced open conformer of BAK, inhibiting BAK auto-oligomerization. J Biol Chem 2003;278:25039–45.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Griffiths GJ, Dubrez L, Morgan CP, et al. Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J Cell Biol 1999;144:903–14.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Antonsson B, Montessuit S, Sanchez B, Martinou JC. Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J Biol Chem 2001;276:11615–23.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993;74:609–19.
    OpenUrlCrossRefPubMed
  37. ↵
    Mikhailov V, Mikhailova M, Pulkrabek DJ, Dong Z, Venkatachalam MA, Saikumar P. Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane. J Biol Chem 2001;276:18361–74.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Chittenden T, Flemington C, Houghton AB, et al. A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions. EMBO J 1995;14:5589–96.
    OpenUrlPubMed
  39. ↵
    Zha H, Fisk HA, Yaffe MP, Mahajan N, Herman B, Reed JC. Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells. Mol Cell Biol 1996;16:6494–508.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Simonen M, Keller H, Heim J. The BH3 domain of Bax is sufficient for interaction of Bax with itself and with other family members and it is required for induction of apoptosis. Eur J Biochem 1997;249:85–91.
    OpenUrlPubMed
  41. ↵
    Shangary S, Johnson DE. Peptides derived from BH3 domains of Bcl-2 family members: a comparative analysis of inhibition of Bcl-2, Bcl-x(L) and Bax oligomerization, induction of cytochrome c release, and activation of cell death. Biochemistry 2002;41:9485–95.
    OpenUrlCrossRefPubMed
  42. ↵
    Holinger EP, Chittenden T, Lutz RJ. Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases. J Biol Chem 1999;274:13298–304.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Letai A, Bassik MC, Walensky LD, Sorcinelli MD, Weiler S, Korsmeyer SJ. Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell 2002;2:183–92.
    OpenUrlCrossRefPubMed
  44. ↵
    Schimmer AD, Hedley DW, Chow S, Pham NA, Chakrabartty A, Bouchard D, et al. The BH3 domain of BAD fused to the Antennapedia peptide induces apoptosis via its α helical structure and independent of Bcl-2. Cell Death Differ 2001;8:725–33.
    OpenUrlCrossRefPubMed
  45. ↵
    Polster BM, Kinnally KW, Fiskum G. BH3 death domain peptide induces cell type-selective mitochondrial outer membrane permeability. J Biol Chem 2001;276:37887–94.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    Wang JL, Zhang ZJ, Choksi S, et al. Cell permeable Bcl-2 binding peptides: a chemical approach to apoptosis induction in tumor cells. Cancer Res 2000;60:1498–502.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    Dou QP, An B, Antoku K, Johnson DE. Fas stimulation induces RB dephosphorylation and proteolysis that is blocked by inhibitors of the ICE protease family. J Cell Biochem 1997;64:586–94.
    OpenUrlCrossRefPubMed
  48. ↵
    Maser RS, Antoku K, Scully WJ Jr, Cho RL, Johnson DE. Analysis of the role of conserved cysteine residues in the bcl-2 oncoprotein. Biochem Biophys Res Commun 2000;277:171–8.
    OpenUrlCrossRefPubMed
  49. ↵
    Tencza SB, Douglass JP, Creighton DJ Jr, Montelaro RC, Mietzner TA. Novel antimicrobial peptides derived from human immunodeficiency virus type 1 and other lentivirus transmembrane proteins. Antimicrob Agents Chemother 1997;41:2394–8.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    Compton LA, Johnson WC Jr. Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication. Anal Biochem 1986;155:155–67.
    OpenUrlCrossRefPubMed
  51. ↵
    Manavalan P, Johnson WC Jr. Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. Anal Biochem 1987;167:76–85.
    OpenUrlCrossRefPubMed
  52. ↵
    Sreerama N, Woody RW. Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 2000;287:252–60.
    OpenUrlCrossRefPubMed
  53. ↵
    Sreerama N, Venyaminov SY, Woody RW. Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis. Anal Biochem 2000;287:243–51.
    OpenUrlCrossRefPubMed
  54. ↵
    Lobley A, Whitmore L, Wallace BA. DICHROWEB: an interactive website for the analysis of protein secondary structure from circular dichroism spectra. Bioinformatics 2002;18:211–2.
  55. ↵
    Diaz JL, Oltersdorf T, Horne W, et al. A common binding site mediates heterodimerization and homodimerization of Bcl-2 family members. J Biol Chem 1997;272:11350–5.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Ottilie S, Diaz JL, Horne W, et al. Dimerization properties of human BAD. Identification of a BH-3 domain and analysis of its binding to mutant BCL-2 and BCL-XL proteins. J Biol Chem 1997;272:30866–72.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    Wang K, Gross A, Waksman G, Korsmeyer SJ. Mutagenesis of the BH3 domain of BAX identifies residues critical for dimerization and killing. Mol Cell Biol 1998;18:6083–9.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    Kelekar A, Chang BS, Harlan JE, Fesik SW, Thompson CB. Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL. Mol Cell Biol 1997;17:7040–6.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    Kharbanda S, Pandey P, Schofield L, et al. Role for Bcl-xL as an inhibitor of cytosolic cytochrome c accumulation in DNA damage-induced apoptosis. Proc Natl Acad Sci U S A 1997;94:6939–42.
    OpenUrlAbstract/FREE Full Text
  60. ↵
    Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998;281:1322–6.
    OpenUrlAbstract/FREE Full Text
  61. ↵
    Luo P, Baldwin RL. Mechanism of helix induction by trifluoroethanol: a framework for extrapolating the helix-forming properties of peptides from trifluoroethanol/water mixtures back to water. Biochemistry 1997;36:8413–21.
    OpenUrlCrossRefPubMed
  62. ↵
    Roccatano D, Colombo G, Fioroni M, Mark AE. Mechanism by which 2,2,2-trifluoroethanol/water mixtures stabilize secondary-structure formation in peptides: a molecular dynamics study. Proc Natl Acad Sci U S A 2002;99:12179–84.
    OpenUrlAbstract/FREE Full Text
  63. ↵
    Mao D, Wachter E, Wallace BA. Folding of the mitochondrial proton adenosine triphosphatase proteolipid channel in phospholipid vesicles. Biochemistry 1982;21:4960–8.
    OpenUrlCrossRefPubMed
  64. ↵
    Petros AM, Nettesheim DG, Wang Y, et al. Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies. Protein Sci 2000;9:2528–34.
    OpenUrlCrossRefPubMed
  65. ↵
    Sattler M, Liang H, Nettesheim D, et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 1997;275:983–6.
    OpenUrlAbstract/FREE Full Text
  66. ↵
    Yin XM, Oltvai ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994;369:321–3.
    OpenUrlCrossRefPubMed
  67. ↵
    Moreau C, Cartron PF, Hunt A, et al. Minimal BH3 peptides promote cell death by antagonizing anti-apoptotic proteins. J Biol Chem 2003;278:19426–35.
    OpenUrlAbstract/FREE Full Text
  68. ↵
    Yang B, Liu D, Huang Z. Synthesis and helical structure of lactam bridged BH3 peptides derived from pro-apoptotic Bcl-2 family proteins. Bioorg Med Chem Lett 2004;14:1403–6.
    OpenUrlCrossRefPubMed
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Molecular Cancer Therapeutics: 3 (11)
November 2004
Volume 3, Issue 11

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Sequence and helicity requirements for the proapoptotic activity of Bax BH3 peptides
Sanjeev Shangary, Christopher L. Oliver, Tommy S. Tillman, Michael Cascio and Daniel E. Johnson
Mol Cancer Ther November 1 2004 (3) (11) 1343-1354;
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