Cytoprotection by Bcl-2 Requires the Pore-forming α5 and α6 Helices
1998; Elsevier BV; Volume: 273; Issue: 47 Linguagem: Inglês
10.1074/jbc.273.47.30995
ISSN1083-351X
AutoresShigemi Matsuyama, Sharon L. Schendel, Zhihua Xie, John C. Reed,
Tópico(s)Antimicrobial Resistance in Staphylococcus
ResumoWe explored whether the putative channel-forming fifth and sixth α-helices of Bcl-2 and Bax account for Bcl-2-mediated cell survival and Bax-induced cell death in mammalian cells and in the yeast Saccharomyces cerevisiae. When α5-α6 were either deleted or swapped with each other, the Bcl-2Δα5α6 deletion mutant and Bcl-2-Bax(α5α6) chimeric protein failed to block apoptosis induced by either Bax or staurosporine in human cells and were unable to prevent Bax-induced cell death in yeast, implying that the α5-α6 region of Bcl-2 is essential for its cytoprotective function. Additional experiments indicated that, although α5-α6 is necessary, it is also insufficient for the anti-apoptotic activity of Bcl-2. In contrast, deletion or substitution of α5-α6 in Bax reduced but did not abrogate apoptosis induction in human cells, whereas it did completely nullify cytotoxic activity in yeast, implying that the pore-forming segments of Bax are critical for conferring a lethal phenotype in yeast but not necessarily in human cells. BaxΔα5α6 and Bax-Bcl-2(α5α6) also retained the ability to dimerize with Bcl-2. Bax therefore may have redundant mechanisms for inducing apoptosis in mammalian cells, based on its ability to form α5-α6-dependent channels in membranes and to dimerize with and antagonize anti-apoptotic proteins such as Bcl-2. We explored whether the putative channel-forming fifth and sixth α-helices of Bcl-2 and Bax account for Bcl-2-mediated cell survival and Bax-induced cell death in mammalian cells and in the yeast Saccharomyces cerevisiae. When α5-α6 were either deleted or swapped with each other, the Bcl-2Δα5α6 deletion mutant and Bcl-2-Bax(α5α6) chimeric protein failed to block apoptosis induced by either Bax or staurosporine in human cells and were unable to prevent Bax-induced cell death in yeast, implying that the α5-α6 region of Bcl-2 is essential for its cytoprotective function. Additional experiments indicated that, although α5-α6 is necessary, it is also insufficient for the anti-apoptotic activity of Bcl-2. In contrast, deletion or substitution of α5-α6 in Bax reduced but did not abrogate apoptosis induction in human cells, whereas it did completely nullify cytotoxic activity in yeast, implying that the pore-forming segments of Bax are critical for conferring a lethal phenotype in yeast but not necessarily in human cells. BaxΔα5α6 and Bax-Bcl-2(α5α6) also retained the ability to dimerize with Bcl-2. Bax therefore may have redundant mechanisms for inducing apoptosis in mammalian cells, based on its ability to form α5-α6-dependent channels in membranes and to dimerize with and antagonize anti-apoptotic proteins such as Bcl-2. transmembrane 1,2-dioleoylphosphatidylcholine 1,2-dioleoylphosphatidylglycerol green fluorescent protein glutathione S-transferase hemagglutinin polyacrylamide gel electrophoresis staurosporine. Bcl-2 family proteins play a pivotal role in the regulation of programmed cell death and apoptosis. Some members of this family such as Bcl-2 and Bcl-XL function as cell death suppressors, whereas others such as Bax and Bak induce apoptosis (1Reed J.C. Nature. 1997; 387: 773-776Crossref PubMed Scopus (1391) Google Scholar, 2Nuñuz G. Clarke M.F. Trends Cell Biol. 1994; 4: 399-403Abstract Full Text PDF PubMed Scopus (226) Google Scholar, 3Reed J.C. J. Cell Biol. 1994; 124: 1-6Crossref PubMed Scopus (2389) Google Scholar). At least three biochemical characteristics have been ascribed to various Bcl-2 family proteins, including: (a) dimerization with themselves and each other; (b) interactions with other types of proteins, ranging from protein kinases and phosphatases to proteins that bind cell death proteases of the caspase family; and (c) formation of pores or ion channels in membranes (1Reed J.C. Nature. 1997; 387: 773-776Crossref PubMed Scopus (1391) Google Scholar). The relative significance of these different functions remains to be clarified, but may depend on the precise repertoire of Bcl-2 family proteins expressed in cells and the type of cell death stimuli applied. The three-dimensional structure of one of the Bcl-2 family proteins, Bcl-XL, has been determined, revealing seven α-helices separated by flexible loops (4Muchmore S.W. Sattler M. Liang H. Meadows R.P. Harlan J.E. Yoon H.S. Nettesheim D. Changs B.S. Thompson C.B. Wong S. Ng S. Fesik S.W. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1289) Google Scholar). Some other members of the Bcl-2 family, including the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax, can be readily modeled on the Bcl-XL crystallographic coordinates, implying that they share a similar fold despite having opposing effects on cell life and death (5Schendel S. Montal M. Reed J.C. Cell Death Differ. 1998; 5: 372-380Crossref PubMed Scopus (277) Google Scholar). The C terminus of many Bcl-2 family proteins consists of a stretch of hydrophobic amino acids that serves the purpose of anchoring them within intracellular membranes, particularly the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope, with the bulk of the protein oriented toward the cytosol (6Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Reed J.C. Cancer Res. 1993; 53: 4701-4714PubMed Google Scholar, 7Lithgow T. van Driel R. Bertram J.F. Strasser A. Cell Growth Differ. 1994; 3: 411-417Google Scholar). Comparisons with other proteins for which structures are available revealed striking structural similarity of Bcl-XL to the pore-forming domains of certain bacterial toxins, including: (a) diphtheria toxin, which produces pores for transporting a polypeptide fragment of the toxin across lysosomal/endosomal membranes into the cytosol (8Kagan B.L. Finkelstein A. Colombini M. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4950-4954Crossref PubMed Scopus (246) Google Scholar, 9Donovan J.J. Simon M.I. Montal M. J. Biol. Chem. 1985; 260: 8817-8823Abstract Full Text PDF PubMed Google Scholar); and (b) the colicins, which form ion channels that kill sensitive Escherichia coliby depolarizing their inner membranes (10Cramer W.A. Heymann J.B. Schendel S.L. Deriy B.N. Cohen F.S. Elkins P.A. Stauffacher C.V. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 611-641Crossref PubMed Scopus (183) Google Scholar). Moreover, Bcl-2, Bcl-XL, and Bax have been reported to form ion channels in synthetic membranes in vitro, when tested under conditions similar to those required for channel formation by diphtheria toxin or the colicins (11Minn A.J. Velez P. Schendel S.L. Liang H. Muchmore S.W. Fesik S.W. Fill M. Thompson C.B. Nature. 1997; 385: 353-357Crossref PubMed Scopus (723) Google Scholar, 12Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (548) Google Scholar, 13Schlesinger P. Gross A. Yin X.-M. Yamamoto K. Saito M. Waksman G. Korsmeyer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11357-11362Crossref PubMed Scopus (443) Google Scholar, 14Antonsson B. Conti F. Ciavatta A. Montessuit S. Lewis S. Martinou I. Bernasconi L. Bernard A. Mermod J.-J. Mazzei G. Maundrell K. Gambale F. Sadoul R. Martinou J.-C. Science. 1997; 277: 370-372Crossref PubMed Scopus (932) Google Scholar). However, the characteristics of the channels formed in vitro by cytoprotective (Bcl-2, Bcl-XL) and cytotoxic (Bax) members of the Bcl-2 family differ. In general, Bcl-2 and Bcl-XL tend to form channels having low conductance, display modest cation selectivity, and exist in a mostly closed state, whereas Bax channels typically have 100–1000-fold larger conductances than Bcl-2 or Bcl-XLchannels, prefer anions, and dwell longer in an open state (reviewed in Ref. 5Schendel S. Montal M. Reed J.C. Cell Death Differ. 1998; 5: 372-380Crossref PubMed Scopus (277) Google Scholar). By analogy to structurally similar pore-forming domains from bacterial toxins, the predicted fifth and sixth α-helices of Bcl-2 and Bax are hypothesized to directly participate in channel formation. These α-helices are positioned in the core of these proteins (based on models derived from the Bcl-XL structure) and are believed to be inserted into the membrane bilayer perpendicular to the membrane surface, with the loop connecting α5 and α6 presumably protruding from the other side of the membrane (5Schendel S. Montal M. Reed J.C. Cell Death Differ. 1998; 5: 372-380Crossref PubMed Scopus (277) Google Scholar). Indeed, deletion of the α5-α6 regions from Bcl-2 abolishes its ability to form ion channels in synthetic membranes in vitro (12Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (548) Google Scholar). The structural basis for differences in the channels formed in vitro by Bcl-2 and Bax is unknown, but could be due at least in part to differences between the polar residues of the fifth and sixth α-helices of these proteins. Two acidic amino acids are predicted to be on the hydrophilic face of α5 in Bcl-2 and Bcl-XL, which would presumably line the lumen channel, compared with two basic amino acids in the corresponding position for the pro-apoptotic Bax and Bak proteins (reviewed in Ref. 5Schendel S. Montal M. Reed J.C. Cell Death Differ. 1998; 5: 372-380Crossref PubMed Scopus (277) Google Scholar). These differences in α5 and α6 might account for the relative cation specificity of the Bcl-2 and Bcl-XLchannels (11Minn A.J. Velez P. Schendel S.L. Liang H. Muchmore S.W. Fesik S.W. Fill M. Thompson C.B. Nature. 1997; 385: 353-357Crossref PubMed Scopus (723) Google Scholar, 12Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (548) Google Scholar), and the anion selectivity of the Bax channel (13Schlesinger P. Gross A. Yin X.-M. Yamamoto K. Saito M. Waksman G. Korsmeyer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11357-11362Crossref PubMed Scopus (443) Google Scholar). It remains to be determined whether channels are formed by Bcl-2 family proteins in vivo and whether this activity is critical for the biological functions of these proteins. However, intrinsic bioactivities for the Bcl-2 and Bax proteins have been demonstrated in yeast, where no Bcl-2 homologs apparently exist based on sequence homology searches of the now completed genome of Saccharomyces cerevisiae. The Bax and Bak proteins, for example, confer a lethal phenotype when ectopically expressed in either the budding yeastS. cerevisiae or the fission yeast Schizosaccharomyces pombe (15Sato T. Hanada M. Bodrug S. Irie S. Iwama N. Boise L.H. Thompson C.B. Golemis E. Fong L. Wang H.-G. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9238-9242Crossref PubMed Scopus (594) Google Scholar, 16Bodrug S.E. Aimé-Sempé C. Sato T. Krajewski S. Hanada M. Reed J.C. Cell Death Differ. 1995; 2: 173-182PubMed Google Scholar, 17Hanada M. Aimé-Sempé C. Sato T. Reed J.C. J. Biol. Chem. 1995; 270: 11962-11968Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 18Greenhalf W. Stephan C. Chaudhuri B. FEBS Lett. 1996; 380: 169-175Crossref PubMed Scopus (174) Google Scholar, 19Ink B. Zornig M. Baum B. Hajibagheri N. James C. Chittenden T. Evan G. Mol. Cell. Biol. 1997; 17: 2468-2474Crossref PubMed Scopus (125) Google Scholar, 20Jürgensmeier J.M. Krajewski S. Armstrong R. Wilson G.M. Oltersdorf T. Fritz L.C. Reed J.C. Ottilie S. Mol. Biol. Cell. 1997; 8: 229-325Crossref Scopus (149) Google Scholar, 21Zha H. Fisk H.A. Yaffe M.P. Mahajan N. Herman B. Reed J.C. Mol. Cell. Biol. 1996; 16: 6494-6508Crossref PubMed Scopus (267) Google Scholar). In contrast, mutants of Bax and Bak that lack the putative pore-forming α5 and α6 helices are devoid of cytotoxic activity in yeast. Bcl-2 and Bcl-XL can rescue yeast from the lethal effects of Bax and Bak, without necessity for dimerization between these proteins (22Zha H. Reed J.C. J. Biol. Chem. 1997; 272: 31482-31488Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Moreover, ectopic expression of Bcl-2 in the absence of Bax or Bak in certain mutant strains of yeast has also been shown to preserve cell viability under some circumstances (23Longo V.D. Ellerby L.M. Bredesen D.E. Valentine J.S. Gralla E.B. J. Cell Biol. 1997; 137: 1581-1588Crossref PubMed Scopus (176) Google Scholar), providing further evidence of an intrinsic function for this anti-apoptotic protein. In this report, we explored some of the structure-function relations of the Bcl-2 and Bax proteins that may be relevant to their similarity to pore-forming proteins, focusing specifically on the putative pore-forming α5 and α6 helices. The results provide further insights into the question of why Bcl-2 is cytoprotective and Bax is cytodestructive, and suggest that differences in the α5 and α6 helices of Bcl-2 and Bax are necessary but insufficient for determining the opposing phenotypes of these proteins. Human Bcl-2 and human Bax cDNAs were employed as the templates for the mutagenesis experiments. Mutations were created using a two-step polymerase chain reaction method (17Hanada M. Aimé-Sempé C. Sato T. Reed J.C. J. Biol. Chem. 1995; 270: 11962-11968Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 24Zha H. Fisk H.A. Yaffe M.P. Reed J.C. Mol. Cell. Biol. 1996; 16: 6494-6508Crossref PubMed Scopus (271) Google Scholar). All mutants were initially subcloned betweenEcoRI (5′ end) and XhoI (3′ end) sites in pEG202, pJG4–5, pcDNA3, or pcDNA3-HA plasmids. The following mutagenic primers were used in combination with the wild-type Bcl-2 forward (for pEG202, pJG4–5: 5′-GCGGAATTCATGGCGCACGCTGGGAGAACA-3; for pcDNA3: 5′-GCGGAATTCGCCACCATGGCGCACGCTGGGAGAACA-3′) and reverse (with C-terminal transmembrane domain (TM)1: 5′-ATTCTCGAGTCACTTGTGGCCCAGATAGGC-3′; without TM: 5′-CGCCTCGAGTCAAGTCTTCAGAGACAGCCAGGA-3′), for wild-type Bax forward (for pEG202, pJG4–5, or pcDNA3-HA: 5′-GCGGAATTCATGGACGGGTCCGGGGAGGAG-3′; for pcDNA3: 5′-GCGGAATTCGCCACCATGGACGGGTCCGGGGAGGAG-3′) and reverse (with TM: 5′-ATTCTCGAGTCAGCCCATCTTCTTCCAGAT-3′; without TM: 5′-ATTCTCGAGTCAGGGCGTCCCAAAGTAGGAGAG-3′), for Bcl-2Δα5α6, 5′-CTGCACACCTGGATCCAGGATAACGGA-3′ (forward) and 5′-CCAGGTGTGCAGCACCCCGTGCCTGAAGAGCTC-3′ (reverse), and for Bax2Δα5α6, 5′-GACGGCAACTTCGACCAGGGTGGTTGGGACGGC-3′ (forward) and 5′-GAAGTTGCCGTCAGAAAACATGTCAGC-3′ (reverse). For the construction of Bcl-2-Bax or Bax-Bcl-2 chimeras, first a SacI site was introduced into the Bax cDNA by two-step polymerase chain reaction using 5′-GCAGCTGAGCTCTTTTCTGACGGCAACTTCAAC-3′ (forward) and 5′-AGAAAAGAGCTCAGCTGCCACTCGGAAAAAGAC-3′ (reverse) with the above primers for wild-type Bax. Then, the region of the Bcl-2 cDNA and Bax cDNAs between the SacI and BamHI sites was swapped. For the production of recombinant GST-Bax(ΔTM) and GST-BaxΔα5α6(ΔTM), cDNAs encoding Bax(ΔTM) and BaxΔα5α6(ΔTM) were subcloned between EcoRI (5′ end) and XhoI (3′ end) sites in pGEX-4T-1 vector. 293T cells were cultured for 12 h in 60-mm diameter dishes in 5 ml of Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Fresh medium was exchanged, and 4 h later the cells were co-transfected with 0.5 μg of pEGFP (CLONTECH Laboratories, Inc.) and various plasmids encoding wild-type or mutants of Bcl-2 or Bax by a calcium phosphate precipitation method (total amount of DNA normalized to either 1.5 or 2.5 μg.). Four hours after transfections, fresh medium was exchanged and the cells were cultured for another 20 h before collecting both floating and adherent cells. Half of the recovered cells were used for immunoblot assays, and the remainder were stained with 4′,6-diamidino-2-phenylindole to determine the percentages of GFP-positive cells with apoptotic nuclei (25Matsuyama S. Xu Q. Velours J. Reed J.C. Mol. Cell. 1998; 1: 327-336Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). GM701 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) calf serum. Cells were transfected with pRC/CMV-hBcl2, pcDNA3-Bcl-2Δα5α6, or pcDNA3-Bcl-2-Bax(α5α6) by a calcium-phosphate precipitation method and selected in 1.4 mg/ml (active) G418. Pools of stable transfectants were passaged and then were cultured in 96-well plates for 12 h at a density of 1 × 104 cells/0.1 ml/well. Fresh medium was exchanged, and 1 μmstaurosporine (STS) was added to induce apoptosis. After 24 h, cell viability was determined by trypan blue dye exclusion assay. EGY48 strain cells were transformed by the lithium acetate method, using 1 μg of plasmid DNA (25Matsuyama S. Xu Q. Velours J. Reed J.C. Mol. Cell. 1998; 1: 327-336Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 26Schiestl R.H. Giest R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1776) Google Scholar). Cells were then plated on histidine-deficient glucose-based minimal medium supplemented with other essential amino acids. Colonies were counted after culturing at 30 °C for 3 days. For the examination of Bcl-2-mediated rescue of yeast from Bax-induced cell death, EGY48 cells were co-transformed with 1 μg of pGilda-Bax and 1 μg of pJG4–5-Bcl-2, pJG4–5-Bcl-2Δα5α6, pJG4–5-Bcl-2-Bax(α5α6), or pJG4–5-Bax-Bcl-2(α5α6), and plated on both histidine- and tryptophan-deficient glucose-based medium to select for the plasmids. Single colonies of transformed yeast cells were re-streaked on galactose-containing medium to induce the GAL-1 promoters in these plasmids and cultured for 4 days (25Matsuyama S. Xu Q. Velours J. Reed J.C. Mol. Cell. 1998; 1: 327-336Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Protein-protein interactions were evaluated by yeast two-hybrid assay as described previously, using EGY48 cells either for LEU2 or lacZ reporter gene assays, in conjunction with pEG202 (LexA DNA-binding domain) and pJG4–5 (B42 transactivation domain) plasmids (15Sato T. Hanada M. Bodrug S. Irie S. Iwama N. Boise L.H. Thompson C.B. Golemis E. Fong L. Wang H.-G. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9238-9242Crossref PubMed Scopus (594) Google Scholar, 17Hanada M. Aimé-Sempé C. Sato T. Reed J.C. J. Biol. Chem. 1995; 270: 11962-11968Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 27Zha H. Aime-Sempe C. Sato T. Reed J.C. J. Biol. Chem. 1996; 271: 7440-7444Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). Growth on leucine-deficient medium was scored 4 days after spotting on minimal medium plates containing 2% galactose and 1% raffinose to induce expression of the transactivation domain-containing proteins from the GAL1 promoter in pJG4–5. Filter assays were similarly performed for β-galactosidase measurements, using cells plated on either galactose- or glucose-containing minimal medium supplemented with leucine. Blue color development was scored at 2 h after adding 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). For co-immuno-precipitation experiments, 293T cells (2 × 106) were cultured for 12 h in 10 ml of medium. Fresh medium was exchanged, and 4 h later the cells were co-transfected with 10 μg of pRC/CMV-Bcl-2 and 10 μg of pcDNA3-HA-Bax, pcDNA3-HA-BaxΔα5α6, or pcDNA3-HA-Bax-Bcl-2(α5α6), or with 10 μg of pcDNA3-Bax and 10 μg of pRc-CMV-Bcl-2, pcDNA3-Bcl-2Δα5α6, or pcDNA3-Bcl-2-Bax(α5α6), by a calcium phosphate precipitation method. Four hours after transfections, fresh medium was exchanged and the cells were cultured for another 4 h before lysing in 0.6 ml of Nonidet P-40 lysis buffer (10 mm Hepes (pH 7.5) 142.5 mm KCl, 5 mm MgCl2, 1 mm EDTA, 0.2% Nonidet P-40), containing 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, and 5 μg/ml aprotinin. After preclearing with 50 μl of Protein G-Sepharose at 4 °C for 1 h, immnoprecipitations were performed by incubating 0.2 ml of lysate with 20 μl of Protein G-Sepharose preabsorbed with 5 μg of anti-Bcl-2 mouse monoclonal antibody ascites (clone 4D7) or 10 μl of anti-Bax rabbit serum at 4 °C for 2 h (28Reed J.C. Tanaka S. Cuddy M. Cho D. Smith J. Kallen R. Saragovi H.U. Torigoe T. Anal. Biochem. 1992; 205: 70-76Crossref PubMed Scopus (23) Google Scholar, 29Krajewski S. Blomvqvist C. Franssila K. Krajewska M. Wasenius V.-M. Niskanen E. Reed J.C. Cancer Res. 1995; 55: 4471-4478PubMed Google Scholar). After extensive washing in Nonidet P-40 lysis buffer, beads were boiled in 60 μl of Laemmli buffer and 20 μl of the eluted proteins were subjected to SDS-PAGE (12%) immunoblot analysis using anti-HA mouse monoclonal antibody conjugated with horseradish peroxidase (Boehringer Mannheim) or 4D7 anti-Bcl-2 mouse monoclonal antibody. For detection of Bcl-2, horseradish peroxidase-conjugated anti-mouse (Bio-Rad) antibody was employed. Immunodetection was achieved by using an enhanced chemiluminescence system (Amersham Pharmacia Biotech) with exposure to x-ray film. For immunoblot assays, whole cell lysates were normalized for total protein content, and immunoblot assays were performed as described previously using 0.1% (v/v) anti-LexA rabbit serum or either anti-Bax or anti-Bcl-2 rabbit serum (21Zha H. Fisk H.A. Yaffe M.P. Mahajan N. Herman B. Reed J.C. Mol. Cell. Biol. 1996; 16: 6494-6508Crossref PubMed Scopus (267) Google Scholar, 29Krajewski S. Blomvqvist C. Franssila K. Krajewska M. Wasenius V.-M. Niskanen E. Reed J.C. Cancer Res. 1995; 55: 4471-4478PubMed Google Scholar). Recombinant GST-Bax (ΔTM) and GST-BaxΔα5α6 (ΔTM) proteins were produced from pGEX-4T-1 in E. coli (BL21 (DE3) strain) bacteria and purified by glutathione-Sepharose affinity chromatography essentially as described (12Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (548) Google Scholar, 30Marzo I. Brenner C. Zamzami N. Susin S. Beutner G. Brdiczka D. Xie Z. Reed J. Kroemer G. J. Exp. Med. 1998; 187: 1261-1271Crossref PubMed Scopus (615) Google Scholar, 31Zamzami N. Marzo I. Susin S. Brenner C. Larochette N. Marchetti P. Reed J.C. Reinhard K. Kroemer G. Oncogene. 1998; 16: 1055-1063Crossref PubMed Scopus (146) Google Scholar). GST was removed by cleavage with thrombin, and the Bax(ΔTM) and BaxΔα5α6 (ΔTM) proteins were subsequently purified by ion-exchange chromatography (12Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (548) Google Scholar, 30Marzo I. Brenner C. Zamzami N. Susin S. Beutner G. Brdiczka D. Xie Z. Reed J. Kroemer G. J. Exp. Med. 1998; 187: 1261-1271Crossref PubMed Scopus (615) Google Scholar, 31Zamzami N. Marzo I. Susin S. Brenner C. Larochette N. Marchetti P. Reed J.C. Reinhard K. Kroemer G. Oncogene. 1998; 16: 1055-1063Crossref PubMed Scopus (146) Google Scholar) and dialyzed into 20 mm Tris-HCl, pH 8.0. Folding of the purified proteins was confirmed by circular dichroism measurements carried out on an AVIV 60DS spectropolarimeter. Proteins were assayed for channel activity on KCl-loaded unilammelar liposomes composed of 60% DOPC (1,2-dioleoylphosphatidylcholine) and 40% DOPG (1,2-dioleoylphosphatidylglycerol) at pH 4.0, measuring Cl− ion efflux as described (12Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (548) Google Scholar). To examine the biological significance of the putative pore-forming α5 and α6 helices within Bcl-2 and Bax, mutants having α5 and α6 deleted were prepared. Alternatively, the α5 and α6 helices were swapped, thus generating chimeric proteins in which the α5 and α6 helices of Bax were replaced with those from Bcl-2 and vice versa (Fig. 1). Previously, we demonstrated that deletion of the α5-α6 region from Bcl-2 abolishes the ability of the recombinant protein to form pH-dependent channels in liposomes in vitro(12Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (548) Google Scholar). To explore the relevance of the α5-α6 region of Bax to itsin vitro channel activity, recombinant Bax and BaxΔα5α6 proteins were produced in bacteria (without their C-terminal hydrophobic domains (ΔTM) for solubility purposes) and purified (data not shown). When applied at ∼150 ng/ml to KCl-loaded unilammelar liposomes under conditions previously shown to be permissive for channel formation by Bcl-2 family proteins (11Minn A.J. Velez P. Schendel S.L. Liang H. Muchmore S.W. Fesik S.W. Fill M. Thompson C.B. Nature. 1997; 385: 353-357Crossref PubMed Scopus (723) Google Scholar, 12Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (548) Google Scholar, 13Schlesinger P. Gross A. Yin X.-M. Yamamoto K. Saito M. Waksman G. Korsmeyer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11357-11362Crossref PubMed Scopus (443) Google Scholar, 14Antonsson B. Conti F. Ciavatta A. Montessuit S. Lewis S. Martinou I. Bernasconi L. Bernard A. Mermod J.-J. Mazzei G. Maundrell K. Gambale F. Sadoul R. Martinou J.-C. Science. 1997; 277: 370-372Crossref PubMed Scopus (932) Google Scholar), Bax (ΔTM) induced striking ion efflux (Fig. 2). In contrast, the BaxΔα5α6 (ΔTM) protein exhibited little or no channel activity under the same conditions. Additional experiments revealed that Bax channel formation was dependent on acidic pH (optimal pH ∼4.0) and the presence of acidic lipids within liposomes (DOPG), consistent with prior studies of Bax and other Bcl-2 family proteins (11Minn A.J. Velez P. Schendel S.L. Liang H. Muchmore S.W. Fesik S.W. Fill M. Thompson C.B. Nature. 1997; 385: 353-357Crossref PubMed Scopus (723) Google Scholar, 12Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (548) Google Scholar, 13Schlesinger P. Gross A. Yin X.-M. Yamamoto K. Saito M. Waksman G. Korsmeyer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11357-11362Crossref PubMed Scopus (443) Google Scholar, 14Antonsson B. Conti F. Ciavatta A. Montessuit S. Lewis S. Martinou I. Bernasconi L. Bernard A. Mermod J.-J. Mazzei G. Maundrell K. Gambale F. Sadoul R. Martinou J.-C. Science. 1997; 277: 370-372Crossref PubMed Scopus (932) Google Scholar). In contrast, the BaxΔα5α6 (ΔTM) protein induced either negligible ion-efflux or (at higher concentrations) exhibited only nonspecific effects, producing similar amounts of Cl– release at both neutral and acidic pH and regardless of whether lipsomes contained acidic lipids (DOPG) or were composed entirely of neutral lipids (DOPC) (data not shown). Although the absence of the C-terminal membrane anchoring domain may reduce the efficiency, these experiments nevertheless demonstrate α5-α6-dependent channel formation by Baxin vitro. When expressed in the human kidney epithelial cell line 293T by transient transfection, the wild-type Bax protein induced apoptosis in nearly half of the successfully transfected cells, as determined by 4′,6-diamidino-2-phenylindole staining of GFP-expressing cells (Fig. 3). Similarly, apoptosis was also induced by transfection with plasmids encoding either the BaxΔα5α6 or Bax-Bcl-2(α5α6) proteins into 293T cells. The BaxΔα5α6 and Bax-Bcl-2(α5α6) proteins consistently induced a lower percentage of the transiently transfected 293T cells to undergo apoptosis when compared with wild-type Bax in experiments where varying amounts of these plasmid DNAs were employed (1, 2, 4, and 8 μg). However, immunoblot analysis of lysates prepared from the transfected 293T cells suggested that these mutant proteins may be produced at somewhat lower levels than the wild-type Bax protein (Fig. 3 C; data not shown). These results indicate that the α5 and α6 helices of Bax are not absolutely required for apoptosis induction in 293T cells. Furthermore, introduction of the α5 and α6 helices from Bcl-2 into the Bax protein is insufficient to convert Bax from a killer to a protector protein. The bioactivities of Bcl-2 mutant proteins lacking either α5 and α6 (Bcl-2Δα5α6) or which contained the corresponding α5-α6 region from Bax (Bcl-2-Bax(α5α6)) were compared against the wild-type Bcl-2 protein in transient co-transfection assays to determine whether these proteins could suppress apoptosis induced by Bax. In contrast to wild-type Bcl-2, transfections performed with plasmids encoding the Bcl-2Δα5α6 or Bcl-2-Bax(α5α6) proteins failed to suppress Bax-induced apoptosis in 293T cells (Fig. 3 B). Immunoblot analysis of lysates prepared from these transiently transfected cells revealed at least comparable levels of production of the Bcl-2Δα5α6 and Bcl-2-Bax(α5α6) proteins compared with wild-type Bcl-2 (Fig. 3 D). Thus, removal of the α5-α6 region from Bcl-2 or replacement of the corresponding region from Bax abolishes the ability of Bcl-2 to block Bax-mediated apoptosis. When expressed in 293T cells without co-transfection of Bax, neither the Bcl-2Δα5α6 nor the Bcl-2-Bax(α5α6) protein induced significant apoptosis (Fig. 3 A), arguing that substitution of the α5-α6 region of Bax does not convert Bcl-2 into a killer protein. To further explore the function of the Bcl-2Δα5α6 and Bcl-2-Bax(α5α6) proteins, their ability to inhibit STS-induced apoptosis in GM701 cells was compared with the wild-type Bcl-2 protein. Treatment with this broad-specificity kinase inhibitor induced apoptosis in ∼70% of GM701 cells (Fig. 4). Wild-type Bcl-2 potently suppressed STS-induced apoptosis. In contrast, neither Bcl-2Δα5α6 nor Bcl-2-Bax(α5α6) interfered with STS-induced apoptosis (Fig. 4 A), despite expression of these mutant proteins at levels equivalent to or greater than the wild-type Bcl-2 protein (Fig. 4 B). Expression of Bcl-2Δα5α6 or Bcl-2-Bax(α5α6) in GM701 cells did not induce apoptosis in the absence of STS, indicating that these Bcl-2 mutant proteins are not intrinsically cytotoxic (data not shown). Taken together, these observations i
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