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• W2018972223 abstract "During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid, activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a single-spanning to a multispanning conformation that binds the active Bax and inhibits cytochrome c release. However, it is not known whether other mitochondrial proteins are required to elicit the tBid-induced Bcl-2 conformational alteration. To define the minimal components that are required for the functionally important Bcl-2 conformational alteration, we reconstituted the reaction using purified proteins and liposomes. We found that purified tBid was sufficient to induce a conformational alteration in the liposome-tethered, but not cytosolic Bcl-2, resulting in a multispanning form that is similar to the one found in the mitochondrial outer membrane of drug-treated cells. Mutations that abolished tBid/Bcl-2 interaction also abolished the conformational alteration, demonstrating that a direct tBid/Bcl-2 interaction at the membrane is both required and sufficient to elicit the conformational alteration. Furthermore, active Bax also elicited the Bcl-2 conformational alteration. Bcl-2 mutants that displayed increased or decreased activity in the conformational alteration assay showed corresponding activities in inhibiting pore formation by Bax in vitro and in preventing apoptosis in vivo. Thus, there is a strong correlation between the direct interaction of membrane-bound Bcl-2 and tBid with activation of Bcl-2 in vitro and in vivo. During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid, activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a single-spanning to a multispanning conformation that binds the active Bax and inhibits cytochrome c release. However, it is not known whether other mitochondrial proteins are required to elicit the tBid-induced Bcl-2 conformational alteration. To define the minimal components that are required for the functionally important Bcl-2 conformational alteration, we reconstituted the reaction using purified proteins and liposomes. We found that purified tBid was sufficient to induce a conformational alteration in the liposome-tethered, but not cytosolic Bcl-2, resulting in a multispanning form that is similar to the one found in the mitochondrial outer membrane of drug-treated cells. Mutations that abolished tBid/Bcl-2 interaction also abolished the conformational alteration, demonstrating that a direct tBid/Bcl-2 interaction at the membrane is both required and sufficient to elicit the conformational alteration. Furthermore, active Bax also elicited the Bcl-2 conformational alteration. Bcl-2 mutants that displayed increased or decreased activity in the conformational alteration assay showed corresponding activities in inhibiting pore formation by Bax in vitro and in preventing apoptosis in vivo. Thus, there is a strong correlation between the direct interaction of membrane-bound Bcl-2 and tBid with activation of Bcl-2 in vitro and in vivo. Proteins of the Bcl-2 family are key regulators of apoptosis that function either as promoters or inhibitors and that display homology in one to four short sequences termed Bcl-2 homology (BH) 3The abbreviations used are: BH, Bcl-2 homology; CB, Cascade Blue; Bcl-2ΔTM, Bcl-2 lacking the C-terminal transmembrane sequence thus equivalent to the cytosolic domain; 7-doxyl-PC, 1-palmitoyl-2-stearoyl-(7-doxyl)-sn-glycero-3-phosphotidylcholine; IASD, 4-acetamido-4′-[(iodoacetyl)amino]stilbene-2,2′-disulfonic acid; MOM, mitochondrial outer membrane; NBD, 7-nitrobenz-2-oxa-1,3-diazole; PARP, poly(ADP-ribose) polymerase. motifs (1Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4060) Google Scholar, 2Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2831) Google Scholar, 3Sharpe J.C. Arnoult D. Youle R.J. Biochim. Biophys. Acta. 2004; 1644: 107-113Crossref PubMed Scopus (343) Google Scholar). Anti-apoptotic subfamily proteins such as Bcl-2 and Bcl-xL contain four BH motifs (BH1–4). Pro-apoptotic proteins are grouped into either multiple or single BH motif subfamilies. The former subfamily includes the proteins Bax and Bak that display homology in BH1–3 motifs. The latter includes proteins such as Bid, Bim, and Bad that are similar only in the limited BH3 motif. Despite the limited sequence homology, the three-dimensional structures determined for seven Bcl-2 family proteins including members from all three subfamilies are very similar, consisting of a hydrophobic core of one to three helices wrapped by five to eight amphipathic helices and their connecting loops. This structure is strikingly similar to the structure of the pore-forming domains of diphtheria toxin and Escherichia coli colicins. In addition there is an obvious hydrophobic groove on the surface of all of the anti-apoptotic Bcl-2 family proteins (4Petros A.M. Olejnicizak E.T. Fesik S.W. Biochim. Biophys. Acta. 2004; 1644: 83-94Crossref PubMed Scopus (604) Google Scholar, 5Woo J.S. Jung J.S. Ha N.C. Shin J. Kim K.H. Lee W. Oh B.H. Cell Death Differ. 2003; 10: 1310-1319Crossref PubMed Scopus (37) Google Scholar, 6Day C.L. Chen L. Richardson S.J. Harrison P.J. Huang D.C. Hinds M.G. J. Biol. Chem. 2005; 280: 4738-4744Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). From decades of extensive studies, two properties are well known to be shared by most, if not all, Bcl-2 family proteins: homo- or hetero-binding and pore formation in membranes. More experiments have been done on the homo- or hetero-binding of Bcl-2 family proteins, resulting in the following major conclusions. 1) The hetero-binding between different subfamily proteins is important for their function. Most BH3-only proteins have been proposed to bind to Bcl-2-like proteins following various death signals, either neutralizing the pro-survival function or inhibiting the anti-death activity of Bcl-2-like proteins. A few BH3-only proteins have also been shown to bind Bax-like proteins and activate their pro-death function (7Chen 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 (1533) Google Scholar, 8Cheng E.H. Wei M.C. Weiler S. Flavell J.K. Mak T.W. Lindsten T. Korsmeyer S.J. Mol. Cell. 2001; 8: 705-711Abstract Full Text Full Text PDF PubMed Scopus (1439) Google Scholar, 9Kuwana 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 (997) Google Scholar). 2) Binding between two different subfamily proteins is mediated by different motifs. The interaction of a BH3-only protein and a Bcl-2-like protein is likely accomplished by the binding of the BH3-motif helix of the former into the hydrophobic groove of the latter (10Petros A.M. Nettesheim D.G. Wang Y. Olejnicizak E.T. Meadows R.P. Mack J. Swift K. Matayoshi E.D. Zhang H. Thompson C.B. Fesik S.W. Protein Sci. 2000; 9: 2528-2534Crossref PubMed Scopus (371) Google Scholar, 11Liu X. Dai S. Zhu Y. Marrack P. Kappler J.W. Immunity. 2003; 19: 341-352Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 12Yan N. Gu L. Kokel D. Chai J. Li W. Han A. Chen L. Xue D. Shi Y. Mol. Cell. 2004; 15: 999-1006Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). A similar interface may be formed in the complex between a Bax-like protein and a Bcl-2-like protein (13Sattler M. Liang H. Nettesheim D. Meadows R.P. Harlan J.E. Eberstadt M. Yoon H.S. Shuker S.B. Chang B.S. Minn A.J. Thompson C.B. Fesik S.W. Science. 1997; 275: 983-986Crossref PubMed Scopus (1301) Google Scholar). 3) The interface for homobinding of Bcl-2 is different from that for Bcl-xL. Whereas a Bcl-2 dimer is formed asymmetrically by an acceptor surface including the BH1–3 hydrophobic groove and a donor surface including BH4 helix, a Bcl-xL dimer is formed symmetrically by inserting the C-terminal tail of one protomer into the hydrophobic groove of the other and vice versa (14Zhang Z. Lapolla S.M. Annis M.G. Truscott M. Roberts G.J. Miao Y. Shao Y. Tan C. Peng J. Johnson A.E. Zhang X.C. Andrews D.W. Lin J. J. Biol. Chem. 2004; 279: 43920-43928Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 15Jeong S.Y. Gaume B. Lee Y.J. Hsu Y.T. Ryu S.W. Yoon S.H. Youle R.J. EMBO J. 2004; 23: 2146-2155Crossref PubMed Scopus (139) Google Scholar). Interestingly, in healthy cells Bcl-2 is exclusively bound to membranes in a conformation in which the C-terminal tail is inserted into the lipid bilayer, and therefore not available for binding to the hydrophobic groove in the cytosolic domain of other Bcl-2 proteins (16Chen-Levy Z. Cleary M.L. J. Biol. Chem. 1990; 265: 4929-4933Abstract Full Text PDF PubMed Google Scholar, 17Janiak F. Leber B. Andrews D.W. J. Biol. Chem. 1994; 269: 9842-9849Abstract Full Text PDF PubMed Google Scholar). In contrast, a substantial fraction of Bcl-xL is found either in the cytosol or loosely bound to membranes in healthy cells. The C-terminal tail of the soluble Bcl-xL could therefore, be involved in homobinding (18Hsu Y.T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1032) Google Scholar). During the induction of apoptosis Bcl-xL inserts into intracellular membranes. A recent crystallography study suggests that the membrane-bound Bcl-xL may swap helices 6–8 to form a symmetrical homodimer (19O'Neill J.W. Manion M.K. Maguire B. Hockenbery D.M. J. Mol. Biol. 2006; 356: 367-381Crossref PubMed Scopus (86) Google Scholar). Because Bax can release cytochrome c from purified mitochondria, pore formation by Bax has been studied extensively. The following major conclusions have been reached. 1) tBid protein or BH3 peptide from Bid or Bim was required for Bax to release cytochrome c from purified mitochondria, and to release dextrans up to 2 MDa from mitochondrial outer membrane (MOM) vesicles or liposomes of MOM lipids (MOM-liposomes) (9Kuwana 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 (997) Google Scholar, 20Kuwana T. Mackey M.R. Perkins G. Ellisman M.H. Latterich M. Schneiter R. Green D.R. Newmeyer D.D. Cell. 2002; 111: 331-342Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar). Pore formation is dependent on the mitochondrial specific lipid, cardiolipin, and can be inhibited by Bcl-xL. 2) The putative Bax pore that can release 2-MDa dextrans should have a radius more than 273 Å, suggesting that massive Bax oligomerization may be involved (21Venturoli D. Rippe B. Am. J. Physiol. 2005; 288: F605-F613Crossref PubMed Scopus (368) Google Scholar). 3) Oligomerization of Bax is required for Bax to permeabilize the MOM, thereby releasing proapoptotic factors such as cytochrome c and apoptosis-inducing factor that activate caspases and nucleases that eventually dismantle the cell (20Kuwana T. Mackey M.R. Perkins G. Ellisman M.H. Latterich M. Schneiter R. Green D.R. Newmeyer D.D. Cell. 2002; 111: 331-342Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar, 22Antonsson B. Montessuit S. Lauper S. Eskes R. Martinou J.C. Biochem. J. 2000; 345: 271-278Crossref PubMed Scopus (563) Google Scholar, 23Desagher 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 (1093) Google Scholar, 24Arnoult D. Gaume B. Karbowski M. Sharpe J.C. Cecconi F. Youle R.J. EMBO J. 2003; 22: 4385-4399Crossref PubMed Scopus (370) Google Scholar). The oligomerization occurs after Bax is inserted into the membrane as a multispanning monomer with helices 5, 6, and 9 buried in the bilayer (25Annis M.G. Soucie E.L. Dlugosz P.J. Cruz-Aguado J.A. Penn L.Z. Leber B. Andrews D.W. EMBO J. 2005; 24: 2096-2103Crossref PubMed Scopus (326) Google Scholar). The oligomerization of Bax-like proteins requires their BH3 motif and can be inhibited by Bcl-2-like proteins (26Zha 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, 27Simonian P.L. Grillot D.A. Andrews D.W. Leber B. Nunez G. J. Biol. Chem. 1996; 271: 32073-32077Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 28Willis S.N. Chen L. Dewson G. Wei A. Naik E. Fletcher J.I. Adams J.M. Huang D.C.S. Genes Dev. 2005; 19: 1294-1305Crossref PubMed Scopus (1058) Google Scholar, 29Dlugosz P.J. Billen L. Annis M.G. Zhu W. Zhang Z. Lin J. Leber B. Andrews D.W. EMBO J. 2006; 25: 2287-2296Crossref PubMed Scopus (223) Google Scholar). 4) The BH3-activated Bax-like proteins can activate more Bax-like proteins to amplify the initial prodeath signal produced by BH3-only proteins (30Ruffolo S. Shore G.C. J. Biol. Chem. 2003; 278: 25039-25045Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 31Tan C. Dlugosz P.J. Peng J. Zhang Z. Lapolla S.M. Plafker S.M. Andrews D.W. Lin J. J. Biol. Chem. 2006; 281: 14764-14775Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Only a few studies have examined pore formation by Bcl-2 and Bcl-xL proteins, resulting in the following conclusions (19O'Neill J.W. Manion M.K. Maguire B. Hockenbery D.M. J. Mol. Biol. 2006; 356: 367-381Crossref PubMed Scopus (86) Google Scholar, 32Schendel 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, 33Schlesinger P.H. Gross A. Yin X.M. Yamamoto K. Saito M. Waksman G. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11357-11362Crossref PubMed Scopus (443) Google Scholar, 34Minn 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, 35Kim K.M. Giedt C.D. Basanez G. O'Neil J.W. Hill J.J. Han Y.-H. Tzung S.-P. Zimmerberg J. Hockenbery D.M. Zhang K.Y.J. Biochemistry. 2001; 40: 4911-4922Crossref PubMed Scopus (84) Google Scholar, 36Lam M. Bhat M.B. Nunez G. Ma J. Distelhorst C.W. J. Biol. Chem. 1998; 273: 17307-17310Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). 1) Both Bcl-2 and Bcl-xL form pores in anionic lipid containing liposomal membranes at extreme acidic pH. The pore-forming activity can be enhanced by a trans-negative membrane potential. 2) In planar lipid bilayers both proteins form ion-conducting channels that display a linear current-voltage relationship, following the law of Ohm. The channels have discrete conductances ranging from 20 to 90 pS for Bcl-2 and 80 to 276 pS for Bcl-xL, suggesting that they may have different structures. Unlike the pores in liposomal membranes that are only detectable at acidic pH, the channels formed in planar bilayers can be detected at both neutral and acidic pH. But the channels open more frequently at acidic pH. The channels are cation selective. 3) Deletion of helices 5 and 6 eliminates the channel activity for Bcl-2, and replacing helices 5 and 6 of Bcl-xL with those from Bax alters the channel property, suggesting that the two hydrophobic helices are required for channel formation by the two anti-apoptosis proteins. 4) During induction of apoptosis, Bcl-2 changes from the tail-anchored conformation to a multispanning one in which in addition to the C-terminal tail (helix 9) both helices 5 and 6 are inserted into the membrane (37Kim P.K. Annis M.G. Dlugosz P.J. Leber B. Andrews D.W. Mol. Cell. 2004; 14: 523-529Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). This conformational alteration can be induced in isolated mitochondria by tBid and is required for anti-Bax activity (29Dlugosz P.J. Billen L. Annis M.G. Zhu W. Zhang Z. Lin J. Leber B. Andrews D.W. EMBO J. 2006; 25: 2287-2296Crossref PubMed Scopus (223) Google Scholar). Thus, only the conformationally altered Bcl-2 can form a stable complex with the active, multispanning Bax, thereby inhibiting its oligomerization and membrane permeabilization. It is intriguing that both pro- and anti-apoptotic multi-BH motif proteins can be switched to a similar multispanning conformation by the BH3-only protein, tBid. Perhaps they form different structures in the membrane to fulfill their opposite functions. The multispanning Bax is known to form large oligomeric pores. Acidic pH induces the Bcl-2 pore formation in liposomal membranes, but the relationship between the acidic pH-induced pore formation and the tBid-induced conformational alteration remains elusive. Moreover, the correlation between the in vitro pore-forming activity and the in vivo anti-apoptotic activity of Bcl-2 has yet to be established. Finally, although tBid can induce the Bcl-2 conformational alteration in isolated mitochondria, the minimal components required for the alteration needs to be defined. In this study we used the MOM-liposomes with encapsulated fluorescent dyes and purified cytosolic domain of Bcl-2 (Bcl-2ΔTM) to reconstitute the pore-forming process in vitro. We found that acidic pH induced a conformational alteration in soluble Bcl-2ΔTM, exposing a hydrophobic core helix to facilitate the membrane insertion and pore formation. In contrast, tBid did not induce the pore formation by the soluble Bcl-2ΔTM. However, when Bcl-2ΔTM was tethered to the liposome, it changed conformation and formed pores after interacting with tBid. Furthermore, the pore formed by the tBid-activated Bcl-2ΔTM is smaller than that formed by the tBid-activated Bax. Most importantly, Bcl-2ΔTM inhibited the large pore formation by the activated Bax only when it was also activated by either tBid or tBid-activated Bax. Mutations that enhanced or reduced the tBid-mediated Bcl-2ΔTM activation in liposomes also enhanced or reduced Bcl-2 anti-apoptosis function in cells, respectively, thereby correlating the in vitro detected conformational alteration with the in vivo function. Materials—All phospholipids and lipid analogs were purchased from Avanti Polar Lipids. Cascade Blue (CB), CB-labeled dextrans, and rabbit anti-CB polyclonal antibody were from Molecular Probes. Peptides were synthesized by Global Peptide Services (Fort Collins, CO) and their sequences were as described (31Tan C. Dlugosz P.J. Peng J. Zhang Z. Lapolla S.M. Plafker S.M. Andrews D.W. Lin J. J. Biol. Chem. 2006; 281: 14764-14775Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Preparation of Plasmids and Proteins—Construction of pET22b(+)-based plasmid encoding His6-tagged Bcl-2ΔTM with either Gly154–Gly155 replaced by two alanines or Val159 replaced by an aspartate (designated G154A/G155A or V159D) was done using appropriate primers and overlapping PCR-based mutagenesis as described (38Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar). Construction of pBabe/hygromycin-based plasmid encoding each corresponding full-length Bcl-2 mutant was done by replacing the corresponding DNA segment with the one containing the mutation. The sequence of all the plasmids was verified by DNA sequencing. Expression and purification of the His6-tagged Bcl-2ΔTM and mutant, tBid and mutant with Met97–Asp98 replaced by two alanines (designated M97A/D98A), and Bax proteins were as described (14Zhang Z. Lapolla S.M. Annis M.G. Truscott M. Roberts G.J. Miao Y. Shao Y. Tan C. Peng J. Johnson A.E. Zhang X.C. Andrews D.W. Lin J. J. Biol. Chem. 2004; 279: 43920-43928Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 39Yethon J.A. Epand R.F. Leber B. Epand R.M. Andrews D.W. J. Biol. Chem. 2003; 278: 48935-48941Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Preparation of Liposomes—Liposomes of MOM lipid composition and with CB or CB-dextrans encapsulated were prepared by the extrusion method as described (31Tan C. Dlugosz P.J. Peng J. Zhang Z. Lapolla S.M. Plafker S.M. Andrews D.W. Lin J. J. Biol. Chem. 2006; 281: 14764-14775Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The plain liposomes without fluorophores were prepared similarly except that no fluorophores were in the buffer A used for resuspension of the lipids and no gel filtration chromatography was needed after the extrusion. The liposomes containing lipophilic quenchers were prepared similarly except that 30 mol % of phosphatidylcholine (PC) was replaced by a doxyl-labeled PC (1-palmitoyl-2-stearoyl-(7-doxyl)-sn-glycero-3-PC,7-doxyl-PC). The Ni2+-chelating liposomes were prepared similarly except that 1 mm Ni2+-chelating lipid analog, 1,2-dioleoyl-sn-glycero-3-{[N(5-amino-1-carboxypentyl)iminodiacetic acid]-succinyl} (nickel salt), was added to 20 mm MOM lipids before resuspension in buffer A. Isolation of Liposome-bound Proteins or Peptides—For Figs. 2A or 3B, 400 or 200 nm Bcl-2ΔTM protein, respectively, was incubated with liposomes of 50 μm lipids (50 μm liposomes for short from here on) in 116 μl of buffer A with certain pH values at 25 °C for 1–2 h. For Fig. 3D, 50 nm Bcl-2ΔTM, 20 nm tBid, and/or 50 nm Bax were incubated with 12.5 μm Ni2+-liposomes. When indicated, 5 mm EDTA or 200 mm imidazole were included either during or after the incubation. The samples were then subjected to sucrose gradient float-up centrifugation as described (39Yethon J.A. Epand R.F. Leber B. Epand R.M. Andrews D.W. J. Biol. Chem. 2003; 278: 48935-48941Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). For the sample containing EDTA or imidazole, the same concentration of corresponding chemical was also presented in the sucrose gradient. Four 250-μl fractions were drawn, starting from the top of the gradient, resulting in fractions S1–S4. The pellet was re-suspended in 250 μl of buffer A, resulting in fraction P. The Bcl-2ΔTM proteins in these fractions were analyzed by SDS-PAGE and immunoblotting with sheep anti-Bcl-2 polyclonal antibody. The distribution of the liposomes among these fractions was estimated by scintillation counting of the [14C]PC of each fraction as described (31Tan C. Dlugosz P.J. Peng J. Zhang Z. Lapolla S.M. Plafker S.M. Andrews D.W. Lin J. J. Biol. Chem. 2006; 281: 14764-14775Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar).FIGURE 3Effect of tBid on the pore formation by soluble and membrane-tethered Bcl-2ΔTM. A, extent of CB dye release by the buffer control (minus tBid and Bcl-2), the soluble Bcl-2ΔTM and/or tBid at pH 7.4 was monitored as above. Data shown are averages of two to three independent experiments with S.D. (error bars). B, binding of the C-terminal His6-tagged Bcl-2ΔTM with regular or Ni2+-liposome (with 0 or 5% Ni2+-lipid, respectively) in the absence or presence of EDTA or imidazole was assayed by the float-up centrifugation. Data shown are immunoblots of the Bcl-2ΔTM that was recovered from the S1 fraction after centrifugation. Similar results were obtained in other two independent repeats. C, extent of CB dye release by the buffer control, tBid, or mutant (M97A/D98A, mut), and/or His6-tagged Bcl-2ΔTM or mutant (G145A, V159D, or G154A/G155A) from Ni2+-liposome at pH 7.4 was monitored as above. Data shown are averages of two to three independent experiments with S.D. (error bars). D, insertion of Bcl-2ΔTM into the membrane was monitored by float-up centrifugation after incubating the protein with Ni2+-liposomes in the absence or presence of tBid and/or Bax and then treating with EDTA. Data shown are representative immunoblots of Bcl-2ΔTM bound to the liposomes in the S1 fraction of the float-up centrifugation. Similar data were obtained in other two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Assay of CB or CB-dextran Release from Liposomes by Fluorescence Quenching—For Fig. 2C, 50 μm liposomes with CB dyes encapsulated were mixed with 6 μg/ml anti-CB antibodies in 250 μl of buffer A of certain pH values that were adjusted by varying the concentration of Na2HPO4 and citric acid. The initial emission intensity (F0) was determined after equilibrating the sample at 25 °C for 5 min. Purified Bcl-2ΔTM protein (400 nm) was then added. For other figures, if indicated, 12.5 μm liposomes with CB dyes or CB-dextrans were added, and after F0 was taken, 50 nm Bcl-2ΔTM or mutant protein, 20 nm tBid or mutant protein, and/or 50 nm Bax were added. The first fluorescence intensity measurement was started exactly 20 s after the addition of the protein(s) and followed by multiple measurements in a predetermined time interval for 5 h, resulting in multiple intermediate intensities (F). At the end of the time course 0.1% Triton X-100 was added and the final measurement was taken, resulting in the final intensity (Ft). The extent of CB or CB-dextran release is proportional to the extent of fluorescence quenching that is equal to ΔFProtein/ΔFTriton, where ΔFProtein = F0 – F, and ΔFTriton = F0 – Ft. All fluorescence intensities were measured using the SLM-8100 fluorometer as described (31Tan C. Dlugosz P.J. Peng J. Zhang Z. Lapolla S.M. Plafker S.M. Andrews D.W. Lin J. J. Biol. Chem. 2006; 281: 14764-14775Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Collisional Quenching of Soluble NBD-labeled Bcl-2ΔTM by Iodide Ions—NBD (7-nitrobenz-2-oxa-1,3-diazole) labeling of Bcl-2ΔTM with a single Cys (Cys158) was done as described (40Shepard L.A. Heuck A.P. Hamman B.D. Rossjohn J. Parker M.W. Ryan K.R. Johnson A.E. Tweten R.K. Biochemistry. 1998; 37: 14563-14574Crossref PubMed Scopus (271) Google Scholar). The NBD fluorescence emission scan was carried out at 1-nm intervals between 500 and 600 nm with excitation at 468 nm. The spectrum was integrated to determine the intensity. To determine the pH effect on iodide ion accessibility to the NBD, the initial net fluorescence intensity (F0) for a sample with 100 nm NBD-Bcl-2ΔTM was determined in buffer A of the indicated pH by subtracting the signal of a control without the NBD-Bcl-2ΔTM. The sample and control were titrated with aliquots of 1 m KI and 1 mm Na2S2O3. The intensities of sample and control were measured after each addition of KI/Na2S2O3 and corrected for dilution to obtain the net intensity (F). In parallel, another pair of sample and control was titrated with 1 m KCl and 1 mm Na2S2O3 for correcting the effect of ionic strength on the NBD fluorescence. In each experiment, the net change in fluorescence due to the quenching at each KI/KCl concentration was determined by the equation F0/F = (F0/F)KI/(F0/F)KCl, which was used to plot against the KI concentration to obtain the Stern-Volmer plot as described (40Shepard L.A. Heuck A.P. Hamman B.D. Rossjohn J. Parker M.W. Ryan K.R. Johnson A.E. Tweten R.K. Biochemistry. 1998; 37: 14563-14574Crossref PubMed Scopus (271) Google Scholar). Fluorescence Intensity of NBD-labeled Bcl-2ΔTM Inserting into Liposomal Membranes—The initial fluorescence intensity (F0) was determined in the absence of liposomes with 100 nm NBD-Bcl-2ΔTM in buffer A at the indicated pH. Emission intensity (F) was monitored exactly 20 s after addition of 100 μm liposomes to the sample, and continuously monitored until the intensity reached a plateau. The liposomes used are either the plain liposomes or the liposomes containing the lipophilic quencher 7-doxyl-PC. Other Fluorescence Methods—The tryptophan fluorescence intensity and anisotropy of Bcl-2ΔTM, and the anisotropy of carboxyfluorescein-labeled Bax H2 peptide were measured as described (31Tan C. Dlugosz P.J. Peng J. Zhang Z. Lapolla S.M. Plafker S.M. Andrews D.W. Lin J. J. Biol. Chem. 2006; 281: 14764-14775Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Assay Bcl-2 Mutants in Rat-1MycERTAM Cell Line—Rat-1MycERTAM cell culture, retroviral infection, and apoptosis induction were conducted as described (37Kim P.K. Annis M.G. Dlugosz P.J. Leber B. Andrews D.W. Mol. Cell. 2004; 14: 523-529Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 41Zhu W. Cowie A. Wasfy G.W. Penn L.Z. Leber B. Andrews D.W. EMBO J. 1996; 15: 4130-4141Crossref PubMed Scopus (284) Google Scholar, 42Fiebig A.A. Zhu W. Hollerbach C. Leber B. Andrews D.W. BMC Cancer. 2006; 6: 213Crossref PubMed Scopus (84) Google Scholar). Briefly, the Rat-1 cells stably expressing Bcl-2, Bcl-2 mutant, or vector were first treated with tamoxifen that induces the c-myc protooncogene activity and sensitizes the cells to apoptotic signals. The cells were then treated with either 6 μm etoposide for 12 and 18 h or low serum (0.03% fetal bovine serum) for 24 and 48 h. The cleavage of poly(ADP-ribose) polymerase (PARP) by caspases was used to assess the drug-induced apoptosis as described (29Dlugosz P.J. Billen L. Annis M.G. Zhu W. Zhang Z. Lin J. Leber B. Andrews D.W. EMBO J. 2006; 25: 2287-2296Crossref PubMed Scopus (223) Google Scholar). The labeling of Cys158 in helix 5 by 4-acetamido-4′-[(iodoacetyl)amino]stilbene-2,2′-disulfonic acid (IASD), a bilayer-impermeant, sulfhydryl-specific reagent, was used to monitor the conformational alteration of Bcl-2 as described (29Dlugosz P.J. Billen L. Annis M.G. Zhu W. Zhang Z. Lin J. Leber B. Andrews D.W. EMBO J. 2006; 25: 2287-2296Crossref PubMed Scopus (223) Google Scholar, 37Kim P.K. Annis M.G. Dlugosz P.J. Leber B. Andrews D.W. Mol. Cell. 2004; 14: 5" @default.
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