Bcl-2 Family Member Bfl-1/A1 Sequesters Truncated Bid to Inhibit Its Collaboration with Pro-apoptotic Bak or Bax
2002; Elsevier BV; Volume: 277; Issue: 25 Linguagem: Inglês
10.1074/jbc.m201469200
ISSN1083-351X
AutoresArlette B. Werner, Evert de Vries, Stephen W. G. Tait, Ilja Bontjer, Jannie Borst,
Tópico(s)RNA Interference and Gene Delivery
ResumoFollowing caspase-8 mediated cleavage, a carboxyl-terminal fragment of the BH3 domain-only Bcl-2 family member Bid transmits the apoptotic signal from death receptors to mitochondria. In a screen for possible regulators of Bid, we defined Bfl-1/A1 as a potent Bid interacting protein. Bfl-1 is an anti-apoptotic Bcl-2 family member, whose preferential expression in hematopoietic cells and endothelium is controlled by inflammatory stimuli. Its mechanism of action is unknown. We find that Bfl-1 associates with both full-length Bid and truncated (t)Bid, via the Bid BH3 domain. Cellular expression of Bfl-1 confers protection against CD95- and Trail receptor-induced cytochrome c release.In vitro assays, using purified mitochondria and recombinant proteins, demonstrate that Bfl-1 binds full-length Bid, but does not interfere with its processing by caspase-8, or with its mitochondrial association. Confocal microscopy supports that Bfl-1, which at least in part constitutively localizes to mitochondria, does not impede tBid translocation. However, Bfl-1 remains tightly and selectively bound to tBid and blocks collaboration between tBid and Bax or Bak in the plane of the mitochondrial membrane, thereby preventing mitochondrial apoptotic activation. Lack of demonstrable interaction between Bfl-1 and Bak or Bax in the mitochondrial membrane suggests that Bfl-1 generally prevents the formation of a pro-apoptotic complex by sequestering BH3 domain-only proteins. Following caspase-8 mediated cleavage, a carboxyl-terminal fragment of the BH3 domain-only Bcl-2 family member Bid transmits the apoptotic signal from death receptors to mitochondria. In a screen for possible regulators of Bid, we defined Bfl-1/A1 as a potent Bid interacting protein. Bfl-1 is an anti-apoptotic Bcl-2 family member, whose preferential expression in hematopoietic cells and endothelium is controlled by inflammatory stimuli. Its mechanism of action is unknown. We find that Bfl-1 associates with both full-length Bid and truncated (t)Bid, via the Bid BH3 domain. Cellular expression of Bfl-1 confers protection against CD95- and Trail receptor-induced cytochrome c release.In vitro assays, using purified mitochondria and recombinant proteins, demonstrate that Bfl-1 binds full-length Bid, but does not interfere with its processing by caspase-8, or with its mitochondrial association. Confocal microscopy supports that Bfl-1, which at least in part constitutively localizes to mitochondria, does not impede tBid translocation. However, Bfl-1 remains tightly and selectively bound to tBid and blocks collaboration between tBid and Bax or Bak in the plane of the mitochondrial membrane, thereby preventing mitochondrial apoptotic activation. Lack of demonstrable interaction between Bfl-1 and Bak or Bax in the mitochondrial membrane suggests that Bfl-1 generally prevents the formation of a pro-apoptotic complex by sequestering BH3 domain-only proteins. cytochrome c Bcl-2 homology green fluorescent protein mitochondrion incubation buffer polyclonal antibody tumor necrosis factor monoclonal antibody phosphate-buffered saline 1,4-piperazinediethanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Whether induced by death receptors or by other stimuli, apoptosis signaling generally involves cytochrome c (Cytc)1 release from mitochondria. In the cytosol, Cyt c acts as cofactor in formation of a multimeric complex between the Apaf–1 scaffold protein and the initiator caspase-9. The ensuing proteolytic activation of caspase-9 allows for effector caspase processing and apoptotic execution (1Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6316) Google Scholar, 2Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (95) Google Scholar). Death receptors, which are apoptosis-inducing members of the tumor necrosis factor (TNF) receptor family, recruit and activate caspase-8 or –10. These enzymes process effector caspases, as well as the Bcl-2 family member Bid. A carboxyl-terminal fragment of Bid, termed truncated (t)Bid, subsequently translocates to mitochondria to promote Cyt c release (3Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3107) Google Scholar, 4Li H. Zhu H., Xu, C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3833) Google Scholar). Mitochondrial permeability also allows release of Smac/Diablo, which reverses caspase inhibition (5Du C. Fang M., Li, Y., Li, L. Wang X. 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Berberich-Siebelt F. Lindemann D. Schimpl A. Berberich I. Eur. J. Immunol. 1999; 29: 3077-3088Crossref PubMed Scopus (71) Google Scholar) and interleukin-3 withdrawal (9Lin E.Y. Orlowsky A. Berger M.S. Prystowsky M.B. J. Immunol. 1993; 151: 1979-1988PubMed Google Scholar). Systematic examination of Bfl-1 function in primary cells has not been documented. Germ-line gene inactivation is complicated by the presence of at least four a1 genes in the murine genome. Disruption of the a1-a gene accelerated spontaneous neutrophil apoptosis, but did not affect TNF-induced apoptosis of these cells (24Hamasaki A. Sendo F. Nakayama K. Ishida N. Negishi I. Nakayama K. Hatakeyama S. J. Exp. Med. 1998; 188: 1985-1992Crossref PubMed Scopus (185) Google Scholar). Human and mouse Bfl-1 share a BH1, BH2, and somewhat less conserved BH3 domain, but the limited homology to a BH4 domain found in human Bfl-1 is not present in the mouse. Protection by Bfl-1 impinges on intact BH1 and BH2 domains (23D'Sa-Eipper C. Chinnandurai G. Oncogene. 1998; 16: 3105-3114Crossref PubMed Scopus (59) Google Scholar, 25Zhang H. Cowan-Jacob S.W. Simonen M. Greenhalf W. Heim J. Meyhack B. J. Biol. Chem. 2000; 275: 11092-11099Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Whereas it is undisputed that Bfl-1 is a protective Bcl-2 type protein, its mechanism of action is unknown. The prototype members of this subgroup localize constitutively to membranes of mitochondria, endoplasmic reticulum, and nucleus, but Bfl-1 does not have a well defined hydrophobic carboxyl-terminal region, implicated in membrane anchoring. Whereas certain authors find Bfl-1 in mitochondria (21Duriez P.J. Wong F. Dorovini-Zis K. Shahidi R. Karsan A. J. Biol. 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The novel model emerging from these findings is that Cyt c release depends on interaction between a BH3 domain-only protein and a Bax-type partner, which allows formation of a Bax-type transmembrane pore. We show here that Bfl-1 acts by inhibiting the collaboration between the BH3 domain-only protein Bid and its pro-apoptotic partners Bax or Bak in the induction of Cyt c release. Bfl-1 does so by binding to full-length Bid via the Bid BH3 domain. It does not interfere with proteolytic activation of Bid, nor with its mitochondrial insertion, but remains selectively complexed to tBid in the mitochondrial membrane where it prevents the activity of a pro-apoptotic complex. Anti-human CD95 (APO-1/Fas) mAb 7C11 was purchased from Immunotech (Marseille, France). Rabbit anti-Bak pAb was from Sigma, rabbit anti-Bax (N-20) and anti-Bfl-1/A1 (FL-175) pAb were from Santa Cruz Biotechnology. Polyclonal rabbit anti-Bid serum was raised in our laboratory against a fusion protein of glutathioneS-transferase and full-length Bid. Anti-actin mAb C4 was obtained from Roche Molecular Biochemicals, anti-Cyt c mAb 7H8.2C12 from PharMingen, anti-HA mAb 3F10 was from Roche Molecular Biochemicals, and anti-HA mAb 12CA5 was isolated from hybridoma supernatant. Fluorescein isothiocyanate-conjugated goat anti-rat Ig was from Rockland (Gilbertsville, PA). Texas Red-conjugated rabbit anti-rat Ig, horseradish peroxidase-conjugated rabbit anti-mouse Ig, or swine anti-rabbit Ig were from DAKO A/S (Glostrup, Denmark). Etoposide was from Sigma. Soluble human recombinant Trail and enhancer were from Alexis (Läufelfingen, Switzerland). Recombinant human caspase-8 was obtained from PharMingen. Protein A- and G-Sepharose and the ECL kit were from Amersham Biosciences. The J16 clone was derived from the human T-acute lymphoblastic leukemia cell line Jurkat by limiting dilution and selected for CD95 sensitivity (40Tepper A.D. de Vries E. van Blitterswijk W. Borst J. J. Clin. Invest. 1999; 103: 971-978Crossref PubMed Scopus (166) Google Scholar, 41Tepper A.D. Ruurs P. Wiedmer T. Sims P.J. Borst J. van Blitterswijk W.J. J. Cell Biol. 2000; 150: 155-164Crossref PubMed Scopus (174) Google Scholar). J16 cells were cultured in Iscove's modified Dulbecco's medium, HeLa, COS-7, and Phoenix-Ampho cells in Dulbecco's modified medium, both supplemented with 8% fetal calf serum, 2 mm glutamine, and antibiotics. Prior to stimulation, J16 cells were suspended in serum-free Yssel's medium (49Yssel H. de Vries J.E. Koken M. van Blitterswijk W. Spits H. J. Immunol. 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Berberich (Institut für Virologie und Immunbiologie, Universität Würzburg, Germany). pET15b/Bid containing human full-length Bid cDNA was a gift from Dr. X. Wang (Howard Hughes Medical Institute, Dallas, TX). pMT2SM-Myc-Bid, pMD4-Bid, pEGFP-N1-Bid, and pET15b-tBid (nucleotides 181–588) vectors were made by introducing suitable restriction sites by PCR into this Bid cDNA. Modified pMT2 vectors with multiple cloning site and upstream Myc or HA tag sequence were constructed in our laboratory by Dr. M. Gebbink. pEGFP-N1 is a commercial vector (CLONTECH), which allows fusion of eGFP at the carboxyl terminus of the cDNA product of interest. Point mutations in the BH3 domain of tBid (pET15b/tBidM97A/D98A) as described (50Wang K. Yin X.-M. Chao D.T. Milliman C.L. Korsmeyer S.J. 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Virus-containing supernatant was harvested after 48 h and stored at −80 °C until further use. J16 cells were coated with RetroNectin (Takara) and transduced at a density of 0.5 × 106/ml of virus-containing supernatant. Supernatants were removed after overnight incubation, and cells were cultured in fresh medium. Selection of transduced cells was initiated after 48 h by the addition of 200 μg/ml Zeocin (Invitrogen). The selection process was completed within 2 weeks. Transduced cells were further cultured in the presence of 200 μg/ml Zeocin. To measure nuclear fragmentation, cells were lysed in 0.1% sodium citrate, 0.1% Triton X-100, 50 μg/ml propidium iodide (54Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4455) Google Scholar) as described earlier (40Tepper A.D. de Vries E. van Blitterswijk W. Borst J. J. Clin. Invest. 1999; 103: 971-978Crossref PubMed Scopus (166) Google Scholar, 41Tepper A.D. Ruurs P. Wiedmer T. Sims P.J. Borst J. van Blitterswijk W.J. J. Cell Biol. 2000; 150: 155-164Crossref PubMed Scopus (174) Google Scholar). Fluorescence intensity of propidium iodide-stained DNA was determined on a FACScan (Becton Dickinson, San Jose, CA) and data were analyzed using CellQuest software. Full-length Bid, tBid, Bfl-1, Bax, and Bak were in vitro transcribed and translated in the presence of [35S]methionine/cysteine from the appropriate cDNA vectors with the TNT Quick Coupled Transcription/Translation for genes cloned downstream from the T7 RNA polymerase promoter, according to instructions supplied by the manufacturer (Promega). COS-7 cells were lysed with 1% Brij-96 in 50 mm Tris, pH 7.6, 150 mm NaCl, 1 mm EDTA, 1 mmiodoacetamide, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, and 2.5 μg/ml aprotonin at 48 h after transfection. Cell lysates were clarified by centrifugation for 10 min at 14,000 rpm at 4 °C. Lysates were incubated for at least 2 h at 4 °C with either 12CA5 mAb or polyclonal anti-Bid serum. Immune complexes were incubated with protein A-Sepharose beads for an additional 2 h. Precipitated proteins were subjected to 15% SDS-PAGE and transferred to nitrocellulose. Blots were blocked in PBS containing 0.05% Tween 20 and 5% non-fat dry milk (Nutricia) and subsequently incubated with the appropriate primary and secondary antibodies. Immunostained proteins were visualized by ECL. After incubation with the appropriate stimuli, cells were washed twice with ice-cold PBS and resuspended in 100 μl of extraction buffer (50 mm PIPES-KOH, pH 7.4, 220 mm mannitol, 68 mm sucrose, 50 mm KCl, 5 mm EGTA, 2 mm MgCl2, 1 mm dithiothreitol and protease inhibitors) and allowed to swell on ice for 30 min (55Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1113) Google Scholar). Cells were homogenized by passing the suspension through a 25-gauge needle (10 strokes). Homogenates were centrifuged in a Beckman Airfuge at 100,000 × g for 15 min at 4 °C and supernatants were harvested and stored at −70 °C until analysis by gel electrophoresis. Ten μg of cytosolic protein, as determined by the Bio-Rad protein assay (Bio-Rad, München) were loaded per lane onto 12% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose sheets, which were blocked for 1 h in PBS, 0.05% Tween with 5% non-fat dry milk, and probed in PBS, 0.05% Tween with anti-Cyt c mAb (1:1000) and anti-actin mAb (1:10,000), to confirm equal loading. After incubation with a 1:7500 dilution of horseradish peroxidase-conjugated rabbit anti-mouse Ig, immunostained proteins were visualized by ECL. Mouse liver cells were lysed by Dounce homogenization in mitochondrion incubation buffer (MIB): 250 mm mannitol, 0.5 mm EGTA, 5 mmHepes pH 7.2, 0.1% (w/v) bovine serum albumin, 1 μg/ml leupeptin, and 0.1 mm phenylmethylsulfonyl fluoride as described (56Petit P.X. O'Connor J.E. Grunwald D. Brown S.C. Eur. J. Biochem. 1990; 194: 389-397Crossref PubMed Scopus (215) Google Scholar). Briefly, nuclei and debris were removed by a 5-min centrifugation at 600 × g at 4 °C, and a pellet containing mitochondria was obtained by a spin at 10,000 × g for 10 min at 4 °C. The pellet was resuspended in MIB and layered on a gradient consisting of four layers of 10, 18, 30, and 70% Percoll in 225 mm mannitol, 25 mm Hepes pH 7.2, 0.5 mm EGTA, and 0.1% (w/v) bovine serum albumin. Purified mitochondria were collected at the 30/70% Percoll interface after centrifugation in a SW-41 rotor for 35 min at 13,500 ×g at 4 °C. The harvested fraction was diluted in MIB, at least 5-fold, and centrifuged for 10 min at 6,300 × gat 4 °C. After two more washes in MIB, mitochondria were resuspended in Wang-buffer B (20 mm Hepes pH 7.5, 220 mmmannitol, 68 mm sucrose, 100 mm KCl, 1.5 mm MgCl2, 1 mm Na2EDTA, 1 mm Na2EGTA, 1 mm dithiothreitol, and 0.1 mm phenylmethylsulfonyl fluoride) (3Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3107) Google Scholar) to a protein concentration of 5 mg/ml. For monitoring Cytc release, mitochondria (25 μg/sample) were incubated in the presence or absence of the in vitrotranscription/translation mixture containing the indicated recombinant proteins (0.5–2 μl of tBid, 1 μl of Bid, Bax, Bak, or Bfl-1) in a final volume 30 μl of Wang-buffer B for 1 h at 30 °C, and then centrifuged for 10 min at 10,000 × g at 4 °C. Mitochondrial pellets, solubilized in SDS sample buffer at 12.5 μg/sample, and a corresponding volume of the supernatant fractions, were separated by 13% SDS-PAGE. Cyt c immunoblotting was performed as described above. Protein interaction studies were performed with 250 μg of mitochondria and 10 μl of the in vitro transcription/translation mixture in a final volume of 300 μl of Wang-buffer B. Proteins were targeted to the mitochondria by incubation for 30 min at 30 °C and then centrifuged for 10 min at 10,000 × g at 4 °C. Mitochondrial pellets were solubilized in 100 μl of RIPA buffer (10 mmtriethanolamine, pH 8.2, 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 5 mm EDTA, and protease inhibitors) followed by sonification. The solubilized proteins present in the supernatant (diluted in Wang-buffer B with 2% CHAPS) were precipitated with appropriate antibodies and Protein G-Sepharose beads and subjected to SDS-PAGE. Where indicated, transfected HeLa cells on coverslips were incubated in 150 nm Mitotracker Red (Molecular Probes) for 30 min prior to fixation. Cells were washed once in PBS and then fixed for 5 min with methanol kept at −20 °C. All subsequent incubations were at room te
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