BIM and tBID Are Not Mechanistically Equivalent When Assisting BAX to Permeabilize Bilayer Membranes
2008; Elsevier BV; Volume: 283; Issue: 12 Linguagem: Inglês
10.1074/jbc.m708814200
ISSN1083-351X
AutoresOihana Terrones, Aitor Etxebarria, Ane Landajuela, Olatz Landeta, Bruno Antonsson, Gorka Basáñez,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoBIM and tBID are two BCL-2 homology 3 (BH3)-only proteins with a particularly strong capacity to trigger BAX-driven mitochondrial outer membrane permeabilization, a crucial event in mammalian apoptosis. However, the means whereby BIM and tBID fulfill this task is controversial. Here, we used a reconstituted liposomal system bearing physiological relevance to explore systematically how the BAX-permeabilizing function is influenced by interactions of BIM/BID-derived proteins and BH3 motifs with multidomain BCL-2 family members and with membrane lipids. We found that nanomolar dosing of BIM proteins sufficed to reverse completely the inhibition of BAX permeabilizing activity exerted by all antiapoptotic proteins tested (BCL-2, BCL-XL, BCL-W, MCL-1, and A1). This effect was reproducible by a peptide representing the BH3 motif of BIM, whereas an equivalent BID BH3 peptide was less potent and more selective, reversing antiapoptotic inhibition. On the other hand, in the absence of BCL-2-type proteins, BIM proteins and the BIM BH3 peptide were inefficient, directly triggering the BAX-permeabilizing function. In contrast, tBID alone potently assisted BAX to permeabilize membranes at least in part by producing a structural distortion in the lipid bilayer via BH3-independent interaction of tBID with cardiolipin. Together, these results support the notion that BIM and tBID follow different strategies to trigger BAX-driven mitochondrial outer membrane permeabilization with strong potency. BIM and tBID are two BCL-2 homology 3 (BH3)-only proteins with a particularly strong capacity to trigger BAX-driven mitochondrial outer membrane permeabilization, a crucial event in mammalian apoptosis. However, the means whereby BIM and tBID fulfill this task is controversial. Here, we used a reconstituted liposomal system bearing physiological relevance to explore systematically how the BAX-permeabilizing function is influenced by interactions of BIM/BID-derived proteins and BH3 motifs with multidomain BCL-2 family members and with membrane lipids. We found that nanomolar dosing of BIM proteins sufficed to reverse completely the inhibition of BAX permeabilizing activity exerted by all antiapoptotic proteins tested (BCL-2, BCL-XL, BCL-W, MCL-1, and A1). This effect was reproducible by a peptide representing the BH3 motif of BIM, whereas an equivalent BID BH3 peptide was less potent and more selective, reversing antiapoptotic inhibition. On the other hand, in the absence of BCL-2-type proteins, BIM proteins and the BIM BH3 peptide were inefficient, directly triggering the BAX-permeabilizing function. In contrast, tBID alone potently assisted BAX to permeabilize membranes at least in part by producing a structural distortion in the lipid bilayer via BH3-independent interaction of tBID with cardiolipin. Together, these results support the notion that BIM and tBID follow different strategies to trigger BAX-driven mitochondrial outer membrane permeabilization with strong potency. One of the decisive events in mammalian apoptosis is the MOMP 3The abbreviations used are: MOMPmitochondrial outer membrane (MOM) permeabilizationFD70fluorescein isothiocyanate-labeled dextrans of 70 kDaPCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolCLcardiolipinDOGS-NTA-Ni1,2,-dioleoyl-sn-glycero-3-{[N-5-amino-1-carboxylpentyl)-iminodiacetic acid]succinyl}LUVlarge unilamellar vesiclesBHBCL-2 homology. 3The abbreviations used are: MOMPmitochondrial outer membrane (MOM) permeabilizationFD70fluorescein isothiocyanate-labeled dextrans of 70 kDaPCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolCLcardiolipinDOGS-NTA-Ni1,2,-dioleoyl-sn-glycero-3-{[N-5-amino-1-carboxylpentyl)-iminodiacetic acid]succinyl}LUVlarge unilamellar vesiclesBHBCL-2 homology. resulting in release of multiple apoptogenic factors, including cytochrome c, from the intermembrane space into the cytosol (1Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2774) Google Scholar). The BCL-2 protein family has emerged as the master regulator of this crucial step in the intracellular apoptotic cascade (Refs. 1Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2774) Google Scholar, 2Youle R.J. Strasser A. Nat. Rev. Mol. Cell Biol. 2008; 9: 47-59Crossref PubMed Scopus (3465) Google Scholar, 3Leber B. Lin J. Andrews D. Apoptosis. 2007; 12: 897-911Crossref PubMed Scopus (301) Google Scholar but see Ref. 4Mizuta T. Shimizu S. Matsuoka Y. Nakagawa T. Tsujimoto Y. J. Biol. Chem. 2007; 282: 16623-16630Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Based on sequence homology and functional criteria, this family can be divided into three main subclasses (2Youle R.J. Strasser A. Nat. Rev. Mol. Cell Biol. 2008; 9: 47-59Crossref PubMed Scopus (3465) Google Scholar). One subclass includes the antiapoptotic proteins BCL-2, BCL-W, BCL-XL, MCL-1, BFL-1/A1, and BCL-B, all of which possess four BCL-2 homology domains (BH1–4) and inhibit MOMP. A second subclass is represented by proapoptotic BAX and BAK, which contain BH1–3 domains and are directly responsible for MOMP. Finally, members of the more distantly related BH3-only subclass (tBID, BIM, PUMA, BAD, NOXA, HRK, BIK, and BMF) display sequence homology only within the third BH domain and promote apoptosis by triggering BAX-driven MOMP.It is agreed that BAX-type proteins exist in an inactive state in healthy cells and that BH3-only proteins funnel multiple apoptotic signals to activate the permeabilizing function of BAX/BAK. However, the means whereby BH3-only members perform this task is a subject of current debate (2Youle R.J. Strasser A. Nat. Rev. Mol. Cell Biol. 2008; 9: 47-59Crossref PubMed Scopus (3465) Google Scholar, 3Leber B. Lin J. Andrews D. Apoptosis. 2007; 12: 897-911Crossref PubMed Scopus (301) Google Scholar, 5Galonek H.L. Hardwick J.M. Nat. Cell Biol. 2006; 8: 1317-1319Crossref PubMed Scopus (107) Google Scholar, 6Youle R.J. Science. 2007; 315: 776-777Crossref PubMed Scopus (49) Google Scholar). BIM and tBID are two well known BH3-only members with a particularly high potency for triggering BAX-driven MOMP and apoptotic cell death. Two main models evolved in the last few years to explain the highly lethal activity of BIM and tBID. On the one hand, the so-called “displacement model” postulates that BIM and tBID physically interact with all five BCL-2-type proteins to competitively displace BAX/BAK, thereby indirectly activating the permeabilizing function of BAX-type proteins (7Willis S.N. Fletcher J.I. Kaufmann T. van Delft M.F. Chen L. Czabotar P.E. Ierino H. Lee E.F. Fairlie W.D. Bouillet P. Strasser A. Kluck R.M. Adams J.M. Huang D.C. Science. 2007; 315: 856-859Crossref PubMed Scopus (927) Google Scholar). Indeed, multiple binding assays (7Willis S.N. Fletcher J.I. Kaufmann T. van Delft M.F. Chen L. Czabotar P.E. Ierino H. Lee E.F. Fairlie W.D. Bouillet P. Strasser A. Kluck R.M. Adams J.M. Huang D.C. Science. 2007; 315: 856-859Crossref PubMed Scopus (927) Google Scholar, 8Chen L. Willis S.N. Wei A. Smith B.J. Fletcher J.I. Hinds M.G. Colman P.M. Day C.L. Adams J.M. Huang D.C.S. Mol. Cell. 2005; 17: 393-403Abstract Full Text Full Text PDF PubMed Scopus (1506) Google Scholar, 9Letai A. Bassik M.C. Walensky L.D. Sorcinelli M.D. Weiler S. Korsmeyer S.J. Cancer Cell. 2002; 2: 183-192Abstract Full Text Full Text PDF PubMed Scopus (1342) Google Scholar, 10Kuwana T. Bouchier-Hayes L. Chipuk J.E. Bonzon C. Sullivan B.A. Green D.R. Newmeyer D.D. Mol. Cell. 2005; 17: 525-535Abstract Full Text Full Text PDF PubMed Scopus (982) Google Scholar, 11Certo M. Del Gaizo Moore V. Nishino M. Wei G. Korsmeyer S.J. Armstrong S.A. Letai A. 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However, although it is firmly established that BIM binds the whole panel of antiapoptotic proteins with low nanomolar affinity constants via its BH3 domain, evidence indicating that tBID engages the complete set of BCL-2-type proteins with strong affinity is tenuous. Alternatively, according to the “direct model” the potent proapoptotic activity of BIM and tBID originates from the exclusive capacity of their BH3 domains to directly bind to and activate the permeabilizing function of BAX/BAK (9Letai A. Bassik M.C. Walensky L.D. Sorcinelli M.D. Weiler S. Korsmeyer S.J. Cancer Cell. 2002; 2: 183-192Abstract Full Text Full Text PDF PubMed Scopus (1342) Google Scholar, 10Kuwana T. Bouchier-Hayes L. Chipuk J.E. Bonzon C. Sullivan B.A. Green D.R. Newmeyer D.D. Mol. Cell. 2005; 17: 525-535Abstract Full Text Full Text PDF PubMed Scopus (982) Google Scholar, 11Certo M. Del Gaizo Moore V. Nishino M. Wei G. Korsmeyer S.J. Armstrong S.A. Letai A. 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Yet, it remains to be clarified how closely the behavior of such chemically modified peptides reflects that of parent proteins as well as the exact molecular pathway by which the BIM/BID BH3-BAX interaction leads to MOMP.Albeit for completely different reasons, the aforementioned two models share the view that BIM and tBID are mechanistically equivalent, triggering the functional activation of BAX. However, evidence exists contradicting this notion. First, pioneering work by Martinou and co-workers (19Terradillos O. Montessuit S. Huang D.C. Martinou J.C. FEBS Lett. 2002; 522: 29-34Crossref PubMed Scopus (45) Google Scholar) showed that recombinant BIML and tBID did not elicit the release of cytochrome c from isolated mitochondria in the same manner. In addition, structural studies revealed that tBID displays a helical globular fold similar to that found in multidomain BCL-2 family members and certain pore-forming toxins, whereas BIM is an intrinsically disordered protein (2Youle R.J. 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Here, we have attempted to elucidate the apoptotic mode of BIM and tBID action by thoroughly characterizing the effects of various recombinant BCL-2 family proteins and selected BH3 peptides on a reconstituted liposomal system bearing physiological significance. The results obtained indicate that BIM and tBID use different molecular mechanisms to effectively trigger the permeabilizing function of BAX.EXPERIMENTAL PROCEDURESMaterials—KCl, HEPES, EDTA, MgCl2, dodecyl octaethylene glycol monoether (C12E8), melittin, Staphylococcus aureus α-toxin (α-toxin), tetanolysin, and fluorescein isothiocyanate-labeled dextrans of 70 kDa (FD70) were obtained from Sigma. Egg phosphatidylcholine (PC), egg phosphatidylethanolamine (PE), liver phosphatidylinositol (PI), heart cardiolipin (CL), and 1,2,-dioleoyl-sn-glycero-3-{[N-5-amino-1-carboxylpentyl)-iminodiacetic acid]succinyl} (nickel salt) (DOGS-NTA-Ni) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Disuccinimidyl suberate was purchased from Molecular Probes (Eugene, OR).Protein Production—All proteins, including BAX, were purified from soluble fractions of bacterial extracts obtained in the absence of detergents and were >90% pure electrophoretically (supplemental Fig. 1). Recombinant His6-tagged full-length monomeric human BAX (BAX) (36Roucou X. Montessuit S. Antonsson B. Martinou J-C. Biochem. J. 2002; 368: 915-921Crossref PubMed Scopus (169) Google Scholar), His6-tagged human monomeric BAX lacking the C-terminal 20 amino acids (BAXΔC) (35Antonsson B. Montessuit S. Lauper S. Eskes R. Martinou J-C. Biochem. J. 2000; 345: 271-278Crossref PubMed Scopus (557) Google Scholar), His6-tagged murine BID (BID) and its mutants 97A98A and 94A (41Desagher S. Osen-Sand A. Nichols A Eskes R Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.-C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1090) Google Scholar), His6-tagged human BIMEL lacking the C-terminal 22 amino acids (BIMELΔC) (42Yamaguchi H. Wang H.G. J. Biol. Chem. 2002; 277: 41604-41612Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and His6-tagged human BCL-2 lacking the C-terminal 24 amino acids (BCL-2ΔC) (41Desagher S. Osen-Sand A. Nichols A Eskes R Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.-C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1090) Google Scholar) were obtained as previously described. To obtain caspase-8-cleaved BID (tBID), granzyme B-cleaved BID (tgBID), and calpain-cleaved BID (tcalBID), indicated proteases were incubated with BID in appropriate buffers at 1/100 (mol/mol) ratio for 1 h at 37°C. Recombinant murine BIML (BIMLΔC) was either (i) purchased from R&D systems with a His6 tag and lacking the C-terminal 20 amino acids (assays corresponding to results shown in Figs. 2, B and C) or (ii) obtained from bacterial expression with a glutathione S-transferase tag and lacking the C-terminal 27 amino acids (all other assays). Recombinant His6-tagged human BCL-W lacking the C-terminal 29 amino acids (BCL-WΔC), human glutathione S-transferase (GST)-tagged BCL-XL lacking the C-terminal 24 amino acids (BCL-XLΔC), mouse GST-tagged A1 lacking the C-terminal 20 amino acids (A1ΔC), and mouse GST-tagged MCL-1 lacking the N-terminal 151 amino acids and the C-terminal 23 amino acids (MCL-1ΔC) were obtained as described previously (8Chen L. Willis S.N. Wei A. Smith B.J. Fletcher J.I. Hinds M.G. Colman P.M. Day C.L. Adams J.M. Huang D.C.S. Mol. Cell. 2005; 17: 393-403Abstract Full Text Full Text PDF PubMed Scopus (1506) Google Scholar, 43Day C.L. Dupont C. Lackmann M. Vaux D.L. Hinds M.G. Cell Death Differ. 1999; 6: 1125-1132Crossref PubMed Scopus (44) Google Scholar) and purified by affinity chromatography followed by Superdex 200 size-exclusion chromatography. High performance liquid chromatography-purified 21-mer BIM/BID BH3 peptides and the 29-mer BID BH3 peptide were obtained from Abgent (San Diego, CA). Peptide identity was confirmed by electrospray mass spectroscopy.Cytochrome c Release Assays—Mitochondria were isolated from livers of male Harlan Sprague-Dawley rats as described previously (30Basañez G. Zhang J. Chau B.N. Maksaev G.I. Frolov V.A. Brandt T.A. Burch J. Hardwick J.M. Zimmerberg J. J. Biol. Chem. 2001; 276: 31083-31091Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and used within 3 h. Isolated mitochondria (500 μg of protein/ml) were incubated for 20 min at 37 °C with recombinant proteins in 50 μl of 125 mm KCl, 5 mm KH2PO4, 25 μm EGTA, 5 mm succinate, 5 μm rotenone, and 10 mm HEPES-KOH, pH 7.2. Reaction mixtures were centrifuged at 14,000 × g for 10 min to isolate the pellet and supernatant fractions, and cytochrome c contents were determined quantitatively using a colorimetric enzyme-linked immunosorbent assay (R&D Systems). The percentage of cytochrome c released into the supernatant (% cytochrome c) was calculated according to the equation, % cytochrome c = ([cytochrome csup - cytochrome cbackgr]/[cytochrome ctotal - cytochrome cbackgr)] × 100, where background release represents cytochrome c detected in the supernatant of buffer-treated samples, and total release represents cytochrome c measured in Triton X-100-treated samples. Absorbance measurements were carried on a Biotek Synergy HT fluorescence microplate reader (Winooski, VT).Liposome Preparation—Unless otherwise stated, liposomes were prepared with lipid composition resembling that of mitochondrial outer membrane (MOM) contact sites ((40 PC/35 PE/10 PI/15 CL (mol/mol)] (MOM-mimetic liposomes)). Organic solvents were removed by evaporation under a N2 stream followed by incubation under vacuum for 2 h. Dry lipid films were resuspended by vigorous vortexing in 100 mm KCl, 10 mm Hepes, pH 7.0, 0.1 mm EDTA (KHE buffer) resulting in the generation of multilamellar vesicles. For assays of vesicle contents, release KHE buffer was supplemented with 100 mg/ml FD70. Multilamellar vesicles were then subjected to five freeze/thaw cycles. These frozen/thawed liposomes were used in assays of direct protein binding assays and lipid morphological and structural studies. Large unilamellar vesicles (LUV) were produced by extrusion through two polycarbonate membranes of 0.2-μm pore size (Nucleopore, San Diego, CA). Untrapped FD70 was removed by gel filtration in Sephacryl S-500 HR columns.Fluorimetric Measurements of Vesicular Contents Release—Release of LUV-encapsulated FD70 was monitored in an SLM-2 Aminco-Bowman luminescence spectrometer (Spectronic Instruments, Rochester, NY) in a thermostatted 1-cm path length cuvette with constant stirring at 37 °C (Vfinal = 1 ml). λex was 490, and λem was 530 nm (slits, 4 nm). A 515-nm cut-off filter was placed between the sample and the emission monochromator to avoid scattering interferences. The extent of marker release was quantified on a percentage basis according to the equation (Ft - F0/F100 - F0) = 100 where Ft is the measured fluorescence of protein-treated LUV at time t, F0 is the initial fluorescence of the LUV suspension before protein addition, and F100 is the fluorescence value after complete disruption of LUV by addition of C12E8 (final concentration, 0.5 mm). Lipid concentration was 50 μm.Binding of Proteins to MOM-mimetic Liposomes—To examine the binding of different BCL-2-family members to liposomes in a direct manner, proteins and freeze/thawed MOM-mimetic liposomes (500 μm lipid) were incubated in KHE buffer for 20 min at 37 °C (Vfinal = 100 μl). The mixture was centrifuged for 30 min at 14,000 × g, 4 °C. Supernatant and pellet fractions contained 14 ± 4 and 83 ± 7% of the total lipid content. Equivalent aliquots were taken from supernatant (corresponding to free protein) and pellet fractions (corresponding to liposome-bound protein), then samples were subjected to reducing SDS-PAGE on 15% Tris-glycine gels. After electrophoresis, proteins from the gel were electroblotted onto 0.2-μm nitrocellulose membranes followed by visualization by Western blotting using appropriate primary antibodies, peroxidase-conjugated secondary antibodies, and enhanced-chemiluminescent (ECL) kit substrates (Pierce). Primary antibodies used for BIM, tBID, and BAX detection were anti-BIM B7929 polyclonal antibody (Sigma), anti-BID MAB860 monoclonal antibody (R&D systems), and anti-BAX N20 polyclonal antibody (Santa Cruz), respectively.Assays of BAX Oligomerization at MOM-mimetic LUV—For studies of BAX oligomerization, apoptotic proteins were first incubated with MOM-mimetic LUV (100 μm) for 20 min at 37 °C (Vfinal = 100 μl). Then, the amine-reactive disuccinimidyl suberate cross-linker was added at a 0.1 mm concentration followed by incubation of the mixture for 30 min at room temperature and quenching of free cross-linker by the addition of 0.1 volume of 2 m Tris-HCl, pH 7.4. Finally, samples were examined for BAX immunoreactivity as described above.Monolayer Surface Pressure Measurements—Surface pressure measurements were carried out with a MicroTrough-S system from Kibron (Helsinki, Finland) at 37 °C with constant stirring. Lipid monolayers resembling the composition of MOM contact sites were used ((40 PC/35 PE/10 PI/15 CL (mol/mol)) (MOM-mimetic monolayers). The lipid, dissolved in chloroform and methanol (2:1), was gently spread over the surface of 1 ml of KHE buffer and kept at a constant surface area. The desired initial surface pressure was attained by changing the amount of lipid applied to the air-water interface. After 10 min to allow for solvent evaporation, the protein/peptide was injected through a hole connected to the subphase. The change in surface pressure was recorded as a function of time until a stable signal was obtained. Maximal surface pressures obtained after injecting an excess of protein/peptide in the absence of a lipid monolayer were below initial monolayer pressure values examined.Far-UV Circular Dichroism (CD) Measurements—Far-UV CD spectra were recorded at 37 °C on a Jasco J-810 spectrapolarimeter (Jasco Spectroscopic Co. Ltd., Hachioji City, Japan) equipped with a Jasco PTC-423S temperature control unit using a 1-mm path length cell. Data were collected every 0.2 nm at 50 nm/min from 250 to 200 nm with a bandwidth of 2 nm, and results were averaged from 20 scans. All samples were allowed to equilibrate for 15 min before measurement. Peptides (5 μm) were mixed with MOM liposomes (500 μm) in 50 mm Na2HPO4, 20 mm KCl, pH 7.0. Each spectrum represents the average of three distinct spectral recordings. The contribution of buffer and lipid to the measured ellipticity was subtracted as blank.Measurements of LUV Size by Quasi-elastic Light Scattering—Vesicle size was determined by quasi-elastic light scattering at a fixed angle of 90° and room temperature using a Malvern Zetasizer 4 instrument (Malvern, UK). A 64-channel correlator was used capable of estimating particle sizes in the range from 5 to 5000 nm. Data were analyzed by the cumulant method using Malvern Application Software. The hydrodynamic radius of the particle was obtained from the first cumulant.31P NMR Measurements—Samples for 31P NMR were prepared by dispersing 15 μmol of dry MOM-mimetic lipid mixtures in either 0.5 ml of KHE buffer alone or 0.5 ml of KHE buffer containing the protein/peptide of interest at 150 μm concentration. Multilamellar vesicle suspensions were freezethawed 3 times in liquid N2 to disperse the added proteins in the lipid membranes, and the liposomes were spun down in an Eppendorf centrifuge (14,000 rpm, 15 min, 4 °C). Pellets were loaded directly into 5-mm Pyrex NMR tubes. Samples were equilibrated for 20 min at each temperature before data acquisition. High power, proton noise-decoupled 31P NMR spectra were recorded on a Bruker AV-500 spectrometer operating at 202.4 MHz using 5-mm broadband inverse probes with z-gradient equipment. 1024 free induction decays were averaged using a 2-s recycle delay. Spectra were processed and evaluated using TOPSPIN 1.3 (Bruker) and plotted with 80-kHz line broadening.RESULTSTruncated BID but Not BIM Potently Triggers the Permeabilizing Function of BAX at Nanomolar Dosing—We previously showed that the BAX-driven MOM permeabilization pathway triggered by tBID can be reconstituted in MOM-mimetic LUV containing entrapped FD70 (32Terrones O. Antonsson B. Yamaguchi H. Wang H-G. Liu Y. Lee R.M. Herrmann A. Basañez G. J. Biol. Chem. 2004; 279: 30081-30091Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). To gain more insigh
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