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• W2117254651 abstract "Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling. Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling. Physiological and pathological cell death have been classified according to morphological criteria into at least three categories: type I cell death or apoptosis; type II cell death or autophagic cell death; and type III cell death or necrosis (1Clarke P.G. Developmental cell death: morphological diversity and multiple mechanisms.Anat. Embryol. (Berl.). 1990; 181: 195-213Crossref PubMed Scopus (1535) Google Scholar, 2Galluzzi L. Maiuri M.C. Vitale I. Zischka H. Castedo M. Zitvogel L. Kroemer G. Cell death modalities: classification and pathophysiological implications.Cell Death Differ. 2007; 14: 1237-1243Crossref PubMed Scopus (616) Google Scholar). Lysosomes were known to be involved in autophagy (3Mizushima N. Autophagy: process and function.Genes Dev. 2007; 21: 2861-2873Crossref PubMed Scopus (2931) Google Scholar, 4Denton D. Nicolson S. Kumar S. Cell death by autophagy: facts and apparent artefacts.Cell Death Differ. 2012; 19: 87-95Crossref PubMed Scopus (314) Google Scholar) and, after the massive rupture of lysosomes, in necrosis (5Guicciardi M.E. Leist M. Gores G.J. Lysosomes in cell death.Oncogene. 2004; 23: 2881-2890Crossref PubMed Scopus (612) Google Scholar). In response to lethal stimuli, partial and selective lysosomal membrane permeabilization (LMP) occurs in certain lysosomes (6Ono K. Kim S.O. Han J. Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor alpha-induced cell death.Mol. Cell. Biol. 2003; 23: 665-676Crossref PubMed Scopus (149) Google Scholar), and the redistributed lysosomal proteases induce apoptosis (7Boya P. Kroemer G. Lysosomal membrane permeabilization in cell death.Oncogene. 2008; 27: 6434-6451Crossref PubMed Scopus (1039) Google Scholar). Several lysosomal cathepsins (e.g., cathepsin B, D, L, etc.) have been implicated in apoptosis (8Repnik U. Stoka V. Turk V. Turk B. Lysosomes and lysosomal cathepsins in cell death.Biochim. Biophys. Acta. 2012; 1824: 22-33Crossref PubMed Scopus (287) Google Scholar, 9Masson O. Bach A.S. Derocq D. Prebois C. Laurent-Matha V. Pattingre S. Liaudet-Coopman E. Pathophysiological functions of cathepsin D: targeting its catalytic activity versus its protein binding activity?.Biochimie. 2010; 92: 1635-1643Crossref PubMed Scopus (82) Google Scholar). Recently, we found that, besides being a digestive enzyme, chymotrypsin is a novel member of lysosomal proteases (10Miao Q. Sun Y. Wei T. Zhao X. Zhao K. Yan L. Zhang X. Shu H. Yang F. Chymotrypsin B cached in rat liver lysosomes and involved in apoptotic regulation through a mitochondrial pathway.J. Biol. Chem. 2008; 283: 8218-8228Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Chymotrypsin release as a consequence of LMP in a small proportion of lysosomes appears to play an important role in cell apoptosis, irrespective of whether the apoptosis is triggered by extrinsic or intrinsic mediators (11Zhao K. Zhao X. Tu Y. Miao Q. Cao D. Duan W. Sun Y. Wang J. Wei T. Yang F. Lysosomal chymotrypsin B potentiates apoptosis via cleavage of Bid.Cell. Mol. Life Sci. 2010; 67: 2665-2678Crossref PubMed Scopus (19) Google Scholar). Further investigation indicated that, during the initiation of apoptosis, both the activation of caspase 8 and the existence of Bid are necessary for the induction of LMP, but the precise mechanism of LMP remains to be elucidated. The activation of caspase 8 leads to the proteolysis of Bid and the formation of caspase 8-cleaved Bid (tBid), which induces mitochondrial outer membrane permeabilization (MOMP) and results in the amplification of apoptotic signaling (12Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors.Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3076) Google Scholar, 13Li H. Zhu H. Xu C.J. Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis.Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3783) Google Scholar). The mechanism of MOMP has been studied extensively (14Tait S.W. Green D.R. Mitochondria and cell death: outer membrane permeabilization and beyond.Nat. Rev. Mol. Cell Biol. 2010; 11: 621-632Crossref PubMed Scopus (1843) Google Scholar). Mitochondrial membranes are enriched with cardiolipin, an acidic phospholipid that provides specificity for the targeting of tBid to mitochondria, leading to MOMP and ultimately to apoptosis (15Lutter M. Fang M. Luo X. Nishijima M. Xie X. Wang X. Cardiolipin provides specificity for targeting of tBid to mitochondria.Nat. Cell Biol. 2000; 2: 754-761Crossref PubMed Scopus (410) Google Scholar). Whether there are similar mechanisms underlying tBid-dependent LMP during apoptosis remains unclear. To reveal the precise mechanisms of tBid-dependent LMP, we began the present study by analyzing the phospholipid composition of lysosomal membrane. Besides sphingolipids and cholesterol, these membranes are rich in acidic phospholipids, especially phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylinositol (PI), but without cardiolipin. We demonstrated that tBid alone is sufficient to induce LMP in intact cells or in isolated lysosomes. Bax might cause an enhancement of tBid-induced LMP, but neither Bax nor Bak is crucial for this LMP. By using a cell-free model that involved large unilamellar vesicles (LUVs) mimicking the phospholipid composition of lysosomes, we found that tBid interacts with PA, which facilitates its insertion into model membrane and causes its conformation change. Size-exclusion chromatography and cross-linking assay indicated that tBid forms homo-oligomers in PA-containing membranes. 31P-NMR analysis showed that tBid promotes the formation of highly curved nonbilayer lipid phases via its interaction with PA. On the basis of these data and our previous reports (16Zhai D. Miao Q. Xin X. Yang F. Leakage and aggregation of phospholipid vesicles induced by the BH3-only Bcl-2 family member, BID.Eur. J. Biochem. 2001; 268: 48-55Crossref PubMed Scopus (29) Google Scholar, 17Yan L. Miao Q. Sun Y. Yang F. tBid forms a pore in the liposome membrane.FEBS Lett. 2003; 555: 545-550Crossref PubMed Scopus (19) Google Scholar), we proposed that tBid-PA interactions in the lysosomal membranes formed “lipidic pores,” which lead to the permeabilization of lysosomal or lysosomal-like membranes. As a result of LMP, lysosomal proteases are redistributed into the cytosol. We also found that chymBid, the proteolytic product of Bid by chymotrypsin released from lysosomes, exhibits more potent activity than that of tBid in binding with PA, inserting into the lipid bilayer, and promoting efficient leakage from lysosomal membranes. These events may play an important signal amplification role during the culmination of the mitochondrial-lysosomal apoptotic pathway. 1,2-Dioleoyl-sn-glycero-3-phosphate (PA), 1,2-dioleoyl-snglycero-3-[phospho-rac-(1-glycerol)] (PG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (PC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (PS), and 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (PI) were purchased from Avanti Polar Lipids (Alabaster, AL). N-(1-pyrenyl)maleimide (PM) and acridine orange (AO) were from Invitrogen (Eugene, OR). Fluorescein-conjugated dextran (FD)-20, FD-70, or FD-250 and Thesit were from Sigma-Aldrich (St. Louis, MO). Bis(2-(3-sulfo-N-succinimidyloxycarbonyloxy)ethyl)sulfone (Sulfo-BSOCOES) was purchased from Pierce (Rockford, IL). BioPORTER protein delivery reagent was purchased from Genlantis (San Diego, CA). 3-[(3-cholamidopropyl)-dimethyl-ammonio]1-propane sulfonate (CHAPS) was purchased from J. T. Baker Inc. (Princeton, NJ). Antibodies against Bid, cathepsin B, COX IV, and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against cytochrome c was purchased from BD Bioscience (Sparks, MD). Antibody against LAMP-1 was purchased from Cell Signaling Technology (Danvers, MA). Other reagents were manufactured in China and were of analytical grade. Recombinant full-length murine Bid with an N-terminal His6 tag and recombinant human activated caspase 8 were obtained as we described previously (16Zhai D. Miao Q. Xin X. Yang F. Leakage and aggregation of phospholipid vesicles induced by the BH3-only Bcl-2 family member, BID.Eur. J. Biochem. 2001; 268: 48-55Crossref PubMed Scopus (29) Google Scholar). Recombinant human chymotrypsin was obtained and activated as we described previously (11Zhao K. Zhao X. Tu Y. Miao Q. Cao D. Duan W. Sun Y. Wang J. Wei T. Yang F. Lysosomal chymotrypsin B potentiates apoptosis via cleavage of Bid.Cell. Mol. Life Sci. 2010; 67: 2665-2678Crossref PubMed Scopus (19) Google Scholar).Truncated Bid processed by caspase 8 (tBid; includes N-terminal fragment 1–59 and C-terminal fragment 60–195) was obtained by adding caspase 8 to the full-length Bid and then incubating overnight at 4°C (10Miao Q. Sun Y. Wei T. Zhao X. Zhao K. Yan L. Zhang X. Shu H. Yang F. Chymotrypsin B cached in rat liver lysosomes and involved in apoptotic regulation through a mitochondrial pathway.J. Biol. Chem. 2008; 283: 8218-8228Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). chymBid, which includes C-terminal fragment 68–195 only (the N-terminal fragment is degraded during chymotrypsin treatment), was obtained by adding chymotrypsin to the full-length Bid and then incubating at 30°C for 2 h (11Zhao K. Zhao X. Tu Y. Miao Q. Cao D. Duan W. Sun Y. Wang J. Wei T. Yang F. Lysosomal chymotrypsin B potentiates apoptosis via cleavage of Bid.Cell. Mol. Life Sci. 2010; 67: 2665-2678Crossref PubMed Scopus (19) Google Scholar). In some experiments, tBid was labeled with fluorescent probe N-(1-pyremyl)maleimide (PM) (18Lovell J.F. Billen L.P. Bindner S. Shamas-Din A. Fradin C. Leber B. Andrews D.W. Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax.Cell. 2008; 135: 1074-1084Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Before labeling, tBid was dialyzed exhaustively against Tris buffer (20 mM Tris-HCl [pH 7.5] and 50 mM NaCl) and then labeled with PM dye (3 mol of PM to 1 mol of tBid) in the same buffer with rotation at 30°C for 2 h in the dark. After the reaction, the protein sample was dialyzed against Tris buffer (20 mM Tris-HCl [pH 7.5] and 50 mM NaCl). Recombinant full-length human Bax with no additional amino acid residues was expressed in Escherichia coli and purified as an intein/chitin-binding domain fusion as described previously (19Suzuki M. Youle R.J. Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization.Cell. 2000; 103: 645-654Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). The plasmid pTYB1-Bax was kindly provided by Prof. Yigong Shi (Tsinghua University, China). Lysis of E. coli was achieved by ultrasonication, and at no point was the protein exposed to detergents that alter the native Bax conformation. After affinity chromatography with a chitin column (New England Biolabs, Ipswich, MA), intein self-cleavage and release of Bax from its fusion partner was initiated by incubation with buffer containing 100 mM 2-mercaptoethanol for 36 h. Then full-length human Bax was further purified by an ion-exchange chromatography on a mono-Q column (Amersham Pharmacia Biotech, Piscataway, NJ). Lysosomes were purified from the livers of Sprague-Dawley rats according to a method described previously (20Stoka V. Turk B. Schendel S.L. Kim T.H. Cirman T. Snipas S.J. Ellerby L.M. Bredesen D. Freeze H. Abrahamson M. et al.Lysosomal protease pathways to apoptosis. Cleavage of bid, not pro-caspases, is the most likely route.J. Biol. Chem. 2001; 276: 3149-3157Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar) with minor modifications. All steps were carried out at 4°C unless otherwise noted. Briefly, several livers were minced, rinsed with sucrose/PIPES buffer (250 mM sucrose, 20 mM PIPES [pH 7.2]), resuspended in 10 volumes of sucrose/PIPES buffer, and homogenized by two brief pulses from a Brinkman Polytron homogenizer. The homogenate was centrifuged for 10 min at 540 g to remove nuclei and particulates. CaCl2 (final concentration, 1 mM) was added to the supernatant, followed by incubation for 5 min at 37°C to disrupt the mitochondria. The supernatant was centrifuged for 10 min at 18,000 g, and the heavy membrane pellet was retained and resuspended in sucrose/PIPES buffer, centrifuged again for 10 min at 18,000 g, and resuspended in Percoll (40% w/v) in sucrose/PIPES. The Percoll solution was centrifuged for 30 min at 44,000 g to form a gradient, and 1-ml fractions were collected from the bottom of the tube and assayed for mitochondrial contamination by using the lysosomal and mitochondrial enzyme markers as described below. The lysosomal fractions were pooled, diluted in sucrose/PIPES (1:10 v/v) to decrease the Percoll, and pelleted by centrifugation at 17,000 g for 10 min. The lysosomes were washed, resuspended in an equal volume of sucrose/PIPES, and used immediately. Because the purity of lysosomes was crucial in this study, we analyzed the isolated lysosomes by immunoblotting with antibodies against LAMP1 (lysosomal marker), COX IV (mitochondrial marker), and GAPDH (cytosolic marker) to determine the mitochondrial or cytosolic contaminations. Only lysosomes without any detectable mitochondrial or cytosolic markers were used in the following experiments. Total lipids were obtained from lysosomal membranes by a two-step lipid extraction protocol (21Fraldi A. Annunziata F. Lombardi A. Kaiser H.J. Medina D.L. Spampanato C. Fedele A.O. Polishchuk R. Sorrentino N.C. Simons K. et al.Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders.EMBO J. 2010; 29: 3607-3620Crossref PubMed Scopus (163) Google Scholar), dried by evaporation under nitrogen, and stored dry at –20°C. Before analysis, the samples were diluted with hexane-isopropanol (3: 1, V/V). Sample aliquots (20 µl) were injected on an Astec Diol 5 mm diol-bonded silica normal-phase spherical HPLC column (250 × 4.6 mm), separated with a mobile phase of acetonitrile-methanol-85% phosphoric acid (100: 10: 0.8, V/V) flowing at 1.0 ml/min, and measured with a DAD detector at 205 nm. Purified rat liver lysosomes were suspended in buffer containing 250 mM sucrose and 20 mM HEPES (pH 7.2) and then incubated with the indicated concentrations of tBid for 30 min, followed by centrifugation at 18,000 g for 10 min. The pellets (lysosomes) were subjected to immunoblot analysis to detect the binding of tBid to lysosomal membranes. The supernatant was subjected to immunoblot analysis to assess the release of lysosomal contents. The percentage of cathepsin B released into the supernatant was used as an index of lysosomal membrane permeabilization (LMP). In some experiments, lysosomes were preincubated with 1 μg/μl PA or PC for 15 min before measuring the binding of tBid to lysosomal membranes and the tBid-induced LMP. Mitochondria were purified from the livers of Sprague-Dawley rats according to a method described previously (12Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors.Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3076) Google Scholar) with minor modifications. Mitochondria were suspended in buffer containing 400 mM mannitol, 5 mM succinate, 10 mM KH2PO4, and 50 mM Tris-HCl (pH 7.2) and then incubated with the indicated concentrations of tBid for 30 min, followed by centrifugation at 12,000 g for 10 min. The supernatant and the pellets (mitochondria) were subjected to immunoblotting analysis to detect the release of mitochondrial cytochrome c (10Miao Q. Sun Y. Wei T. Zhao X. Zhao K. Yan L. Zhang X. Shu H. Yang F. Chymotrypsin B cached in rat liver lysosomes and involved in apoptotic regulation through a mitochondrial pathway.J. Biol. Chem. 2008; 283: 8218-8228Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Lipids were mixed in the appropriate ratio from stocks dissolved in chloroform. The organic solvent was removed by evaporation under a stream of nitrogen gas, followed by incubation for 2 h in a vacuum to ensure complete solvent removal. Lipid films were resuspended in HEPES buffer (10 mM HEPES [pH 7.4], 50 mM NaCl, and 0.2 mM EDTA) and subjected to 10 freeze-thaw cycles. Large unilamellar vesicles (LUVs) were then formed by extrusion through 100-nm Nucleopore polycarbonate membranes. To prepare FITC-dextran (FD)-containing liposomes, 5 mg/ml of FITC-labeled FD-20, FD-70, or FD-250 (numbers = mol. wt. of dextran /1,000) was added to the HEPES buffer, and nonentrapped fluorophores were removed by centrifugation at 100,000 g for 30 min with a Beckman TL-100.3 ultracentrifuge. The leakage assay was performed as described previously (16Zhai D. Miao Q. Xin X. Yang F. Leakage and aggregation of phospholipid vesicles induced by the BH3-only Bcl-2 family member, BID.Eur. J. Biochem. 2001; 268: 48-55Crossref PubMed Scopus (29) Google Scholar). After incubation of increasing amounts of tBid with LUVs (phospholipids concentration, 100 μM) in 100 μl of HEPES buffer at 30°C for 30 min, the vesicles were sedimented by centrifugation (25 min, 35,000 rpm, 4°C). Half of the supernatant (S) was sampled out, and 350 μl HEPES buffer containing 0.1% Thesit was added to the remainder (R) in the centrifugation tube to dilute and dissolve the sample pellets. The control followed the same procedure except for the addition of buffer instead of protein. The FITC-dextran contained in each of these solutions was quantified using a Hitachi F-4500 spectrofluorometer with 5 nm bandwidths centered at 497 and 620 nm for excitation and emission, respectively. Fluorescent intensity was corrected for self-quenching according to a standard curve of fluorescence versus FITC-dextran concentration. The percentage of leakage was determined byLeakage(%)=[2S/(S+R)−B]×100where B is the extent of leakage by the control. The interaction between tBid and phospholipids was studied by surface plasmon resonance (22Liu J. Durrant D. Yang H.S. He Y. Whitby F.G. Myszka D.G. Lee R.M. The interaction between tBid and cardiolipin or monolysocardiolipin.Biochem. Biophys. Res. Commun. 2005; 330: 865-870Crossref PubMed Scopus (29) Google Scholar). The analysis was performed at 25°C using a BIAcore 2000 instrument equipped with a CM5 research-grade sensor chip. tBid proteins were immobilized on the sensor chip surface of one flow cell, and that of the second flow cell was left unmodified and served as a control. The lipids were suspended in buffer (10 mM HEPES [pH 7.4], 50 mM NaCl, 0.2 mM EDTA), and the kinetics of binding responses in this buffer were recorded as the lipids were injected and flowed across the two cells. Detection of the insertion of tBid into monolayers containing phosphatidic acid was performed by methods we described previously (17Yan L. Miao Q. Sun Y. Yang F. tBid forms a pore in the liposome membrane.FEBS Lett. 2003; 555: 545-550Crossref PubMed Scopus (19) Google Scholar). Briefly, 3 ml HEPES buffer was added into the mini-trough as a subphase followed by dropping the phospholipids on the buffer surface to form a monomolecular lipid layer, and the surface pressure was measured with a film balance. After the initial surface pressure stabilized at its plateau value, the appropriate amount of protein was injected to the mixing chamber with a magnetic stir bar through a 0.7 cm2 hole in the edge from where it rapidly diffused into the monolayer-spreading disk to cause an increase in surface pressure. All measurements were performed at room temperature. Fluorescence measurements were used to analyze the binding of tBid to LUV bilayer membranes (17Yan L. Miao Q. Sun Y. Yang F. tBid forms a pore in the liposome membrane.FEBS Lett. 2003; 555: 545-550Crossref PubMed Scopus (19) Google Scholar, 18Lovell J.F. Billen L.P. Bindner S. Shamas-Din A. Fradin C. Leber B. Andrews D.W. Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax.Cell. 2008; 135: 1074-1084Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). tBid was labeled with PM to form PM-labeled tBid and then incubated with PC/PE LUVs (PC, 80%; PE, 20%) or PA-containing LUVs (PC, 70%; PE, 20%; PA, 10%) in HEPES buffer (10 mM HEPES [pH 7.4], 50 mM NaCl, and 0.2 mM EDTA) at 30°C for 30 min. Fluorescence measurements were performed with a Hitachi F-4500 fluorescence spectrometer set to 340 nm excitation and 376 nm emission with 2.5 nm slit widths. The oligomerization of tBid was measured by size-exclusion chromatography. Experiments were performed in a Superdex-200 (1.5 × 45 cm) column equilibrated with 100 mM KCl, 10 mM HEPES, and 0.2 mM EDTA (pH 7.0) with or without 2% (w/v) CHAPS (J. T. Baker Inc.) at a 1 ml/min flow rate. The column was calibrated using protein gel filtration standards (Bio-Rad). Samples of 300 μl were loaded onto the column followed by collection of 2-ml elution fractions. Aliquots of individual fractions were subjected to SDS-PAGE in 15% Tris-glycine gels, followed by visualization of tBid using anti-Bid antibody. The oligomerization of tBid was further confirmed by a cross-linking assay. tBid was incubated with LUVs in buffer containing 20 mM HEPES (pH 7.0), 0.5 M NaCl, and 1 mM EDTA at 37°C for 30 min. At the end of the incubation, the samples were centrifuged at 35,000 g under cold, and the pellets and supernants were collected. Sulfo-BSOCOES (in dimethyl sulfoxide) was added to the pellets and the supernatants separately from a 10-fold stock solution to a final concentration of 10 mM. After incubation for 30 min at room temperature, the cross-linker was quenched by the addition of 1 M Tris-HCl (pH 7.5) to a final concentration of 20 mM. Then the samples were lysed and subjected to SDS-PAGE. Western blotting was performed to analyze the oligomerization of tBid. 31P-NMR was used to detect the formation of nonbilayer microdomains of phospholipids (23Epand R.F. Martinou J.C. Fornallaz-Mulhauser M. Hughes D.W. Epand R.M. The apoptotic protein tBid promotes leakage by altering membrane curvature.J. Biol. Chem. 2002; 277: 32632-32639Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Liposomes containing PC and PA were incubated with different concentrations of tBid or chymBid for 30 min. NMR spectra were recorded at 25°C on a Varian Inova 600 MHz NMR spectrometer equipped with an indirect detection probe. The proton resonance frequency was 599.82 MHz, and that for 31P was 242.80 MHz. A standard single pulse sequence was used with composite pulse decoupling operating on protons during the acquisition period. Typical parameters used were: spectral width, 4.8 kHz; time domain data points, 16,000; recycle delay, 1.2 s; number of scans, 10,000. All free induction decays were multiplied by an exponential function with a 100 (200) Hz line broadening factor before Fourier transformation. Wild-type and Bax−/−/Bak−/− mouse embryonic fibroblasts (MEFs) were generous gifts from Prof. Quan Chen (Institute of Zoology, CAS) and were maintained in DMEM supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated FBS (v/v). Recombinant tBid was delivered into MEFs by using the BioPORTER protein delivery kit (Sigma) according to the manufacturer's instructions. Briefly, 50 μl of tBid solution (1 μM) or a PBS control was used to hydrate the dried BioPORTER. The solution was pipetted up and down and incubated at room temperature for 5 min. Finally, the volume of the complexes was brought to 0.5 ml with serum-free medium and then transferred directly into the MEFs. After incubation for 5 h, 2 ml of serum-containing medium was added. The cells were incubated overnight and then subjected to the LMP assays. Lysosomal permeabilization was determined in intact healthy or apoptotic cells by staining with 5 μg/ml AO for 15 min. AO-emitted red (lysosomal) and green (nuclear and cytosolic) fluorescence was analyzed with an inverted laser scanning confocal microscope (model FV500; Olympus, Tokyo, Japan) or a flow cytometer (FACSCalibur; BD, Mountain View, CA). All data are expressed as the mean ± SD unless otherwise indicated. Differences between groups were compared by ANOVA followed by post hoc Bonferroni tests to correct for multiple comparisons. Differences were considered to be statistically significant at p < 0.05. We first measured the ability of tBid to induce LMP in a cell-free system. Lysosomes were isolated from rat livers, and the purity was assessed by Western blotting (Supplementary Fig. I). Lysosomes without detectable mitochondrial or cytosolic contaminations were incubated with tBid and the LMP was quantified by analyzing the percentage of the lysosomal cathepsin B (a well-accepted marker enzyme of lysosomes) that was released into the supernatant. Incubation of tBid with lysosomes caused its significant translocation to the lysosomal membranes and resulted in the loss of lysosomal membrane integrity in a dose-dependent manner (Fig. 1A). In contrast, uncleaved Bid could not bind to lysosomes and showed no LMP-inducing effects. The addition of Bax could potentiate the LMP induced by tBid, but Bax by itself could not induce significant LMP (Fig. 1B). We further investigated whether tBid is capable of inducing LMP in intact cells. We introduced the recombinant tBid protein into MEFs with BioPORTER reagent by methods we described previously (10Miao Q. Sun Y. Wei T. Zhao X. Zhao K. Yan L. Zhang X. Shu H. Yang F. Chymotrypsin B cached in rat liver lysosomes and involved in apoptotic regulation through a mitochondrial pathway.J. Biol. Chem. 2008; 283: 8218-8228Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). With the help of BioPORTER reagent, recombinant tBid could be uptaken into cells via endocytosis and then partially released from the endosomes into the cytosol, where they interact with intracellular structures and molecules. The lysosomotropic fluorophore AO was used to determine the degree of LMP. AO is a cell-permeable dye that gives rise to red fluorescence at high concentrations and green fluorescence at low concentrations. AO accumulates by proton trapping in intact lysosomes because it becomes positively charged in the acidic lysosomal milieu. After LMP, AO is released from lysosomes into the cytosol, where it emits enhanced green fluorescence that can be monitored by fluorescence microscopy. After delive" @default.
• W2117254651 created "2016-06-24" @default.
• W2117254651 creator A5005828971 @default.
• W2117254651 creator A5019643820 @default.
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• W2117254651 date "2012-10-01" @default.
• W2117254651 modified "2023-10-14" @default.
• W2117254651 title "Phosphatidic acid mediates the targeting of tBid to induce lysosomal membrane permeabilization and apoptosis" @default.
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