Calpain-1 Regulates Bax and Subsequent Smac-dependent Caspase-3 Activation in Neutrophil Apoptosis
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m308576200
ISSN1083-351X
AutoresFrank Altznauer, Sébastien Conus, Andrea Cavalli, Gerd Folkers, Hans‐Uwe Simon,
Tópico(s)Mosquito-borne diseases and control
ResumoIn the absence and in the resolution of inflammatory responses, neutrophils rapidly undergo spontaneous apoptosis. Here we report about a new apoptosis pathway in these cells that requires calpain-1 activation and is essential for the enzymatic activation of the critical effector caspase-3. Decreased levels of calpastatin, a highly specific intrinsic inhibitor of calpain, resulted in activation of calpain-1, but not calpain-2, in neutrophils undergoing apoptosis, a process that was blocked by a specific calpain-1 inhibitor or by intracellular delivery of a calpastatin peptide. Further support for the importance of the calpastatin-calpain system was obtained by analyzing neutrophils from patients with cystic fibrosis that exhibited delayed apoptosis, associated with markedly increased calpastatin and decreased calpain-1 protein levels compared with neutrophils from control individuals. Additional studies were designed to place calpain-1 into the hierarchy of biochemical events leading to neutrophil apoptosis. Pharmacological calpain inhibition during spontaneous and Fas receptor-induced neutrophil apoptosis prevented cleavage of Bax into an 18-kDa fragment unable to interact with Bcl-xL. Moreover, calpain blocking prevented the mitochondrial release of cytochrome c and Smac, which was indispensable for caspase-3 processing and enzymatic activation, both in the presence and absence of agonistic anti-Fas receptor antibodies. Taken together, calpastatin and calpain-1 represent critical proximal elements in a cascade of pro-apoptotic events leading to Bax, mitochondria, and caspase-3 activation, and their altered expression appears to influence the life span of neutrophils under pathologic conditions. In the absence and in the resolution of inflammatory responses, neutrophils rapidly undergo spontaneous apoptosis. Here we report about a new apoptosis pathway in these cells that requires calpain-1 activation and is essential for the enzymatic activation of the critical effector caspase-3. Decreased levels of calpastatin, a highly specific intrinsic inhibitor of calpain, resulted in activation of calpain-1, but not calpain-2, in neutrophils undergoing apoptosis, a process that was blocked by a specific calpain-1 inhibitor or by intracellular delivery of a calpastatin peptide. Further support for the importance of the calpastatin-calpain system was obtained by analyzing neutrophils from patients with cystic fibrosis that exhibited delayed apoptosis, associated with markedly increased calpastatin and decreased calpain-1 protein levels compared with neutrophils from control individuals. Additional studies were designed to place calpain-1 into the hierarchy of biochemical events leading to neutrophil apoptosis. Pharmacological calpain inhibition during spontaneous and Fas receptor-induced neutrophil apoptosis prevented cleavage of Bax into an 18-kDa fragment unable to interact with Bcl-xL. Moreover, calpain blocking prevented the mitochondrial release of cytochrome c and Smac, which was indispensable for caspase-3 processing and enzymatic activation, both in the presence and absence of agonistic anti-Fas receptor antibodies. Taken together, calpastatin and calpain-1 represent critical proximal elements in a cascade of pro-apoptotic events leading to Bax, mitochondria, and caspase-3 activation, and their altered expression appears to influence the life span of neutrophils under pathologic conditions. Neutrophils are important players within the innate immune system. Neutrophil products are often toxic, inasmuch as they are made for killing microorganisms. As targeting of the toxic neutrophil products is not specific, inflammatory responses are associated with more or less tissue damage (1Weiss S. N. Engl. J. Med. 1989; 320: 365-376Crossref PubMed Scopus (3872) Google Scholar). To reduce neutrophil numbers, for instance after successful removal of the antigenic initiators of an inflammatory response, a safe removal of the cells is required. Such removal occurs through apoptosis, a form of cell death that prevents the release of inflammatory mediators from the dying cell and, therefore, limits tissue damage (2Savill J. J. Leukocyte Biol. 1997; 61: 375-380Crossref PubMed Scopus (566) Google Scholar, 3Simon H.-U. Immunol. Rev. 2003; 193: 101-110Crossref PubMed Scopus (299) Google Scholar). Clearly, any failure in the process of neutrophil apoptosis would result in additional and/or persistent inflammation. Delayed neutrophil apoptosis has been associated with several acute and chronic inflammatory diseases and appears to largely be mediated by overexpression of survival factors for these cells (4Dibbert B. Weber M. Nikolaizik W.H. Vogt P. Schöni M.H. Blaser K. Simon H.-U. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13330-13335Crossref PubMed Scopus (265) Google Scholar, 5Coxon A. Tang T. Mayadas T.N. J. Exp. Med. 1999; 190: 923-933Crossref PubMed Scopus (142) Google Scholar). The induction of neutrophil apoptosis during the resolution of a neutrophilic inflammatory response can be mimicked in vitro by culturing the cells in the absence of sufficient amounts of survival factors, a process that is called spontaneous apoptosis. Spontaneous neutrophil apoptosis can be enhanced by Fas receptor stimulation (6Iwai K. Miyawaki T. Takizawa T. Konno A. Ohta K. Yachie A. Taniguchi N. Blood. 1994; 84: 1201-1208Crossref PubMed Google Scholar, 7Watson R.W.G. O'Neill A. Brannigen A.E. Coffey R. Marshall J.C. Brady H.R. Fitzpatrick J.M. FEBS Lett. 1999; 453: 67-71Crossref PubMed Scopus (91) Google Scholar). Both spontaneous and Fas receptor-mediated neutrophil apoptosis involves the activation of a family of cysteine proteases, which cut cellular substrates at an obligatory aspartic acid within a preferred sequence (8Daigle I. Simon H.-U. Int. Arch. Allergy Immunol. 2001; 126: 147-156Crossref PubMed Scopus (77) Google Scholar). Members of this family are the so-called caspases. Caspases have been shown to cut cellular substrates by limited proteolysis that results in either activation or inactivation, but not degradation, of proteins involved in RNA splicing, DNA repair, maintenance of cell structure, and others (9Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6191) Google Scholar). Their action in apoptosis appears to be critical. Inhibitor studies, however, have provided functional evidence for the importance of other proteases in the execution of apoptosis as well (10Knepper-Nicolai B. Savill J. Brown S.B. J. Biol. Chem. 1998; 273: 30530-30536Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Recently a significant focus has been directed toward calpain, a non-caspase cysteine protease with several isoforms (11Saido T.C. Sorimachi H. Suzuki K. FASEB J. 1994; 8: 814-822Crossref PubMed Scopus (617) Google Scholar, 12Squier M.K.T. Cohen J.J. Cell Death Differ. 1996; 3: 275-283PubMed Google Scholar). The ubiquitous calpain isoforms are calpain-1 (calpain I, μ-calpain) and calpain-2 (calpain II, m-calpain), distinguished by their in vitro calcium requirements. Both calpain-1 and calpain-2 are heterodimers consisting of a calcium-binding catalytic 80-kDa subunit and a regulatory 30-kDa subunit, which is functionally essential (13Arthur J.S.C. Elce J.S. Hegadorn C. Williams K. Greer P.A. Mol. Cell. Biol. 2000; 20: 4474-4481Crossref PubMed Scopus (297) Google Scholar). There is accumulating evidence for the involvement of calpain in several physiological processes such as cell-cycle regulation, activation of transcription factors, differentiation, and apoptosis (11Saido T.C. Sorimachi H. Suzuki K. FASEB J. 1994; 8: 814-822Crossref PubMed Scopus (617) Google Scholar, 12Squier M.K.T. Cohen J.J. Cell Death Differ. 1996; 3: 275-283PubMed Google Scholar). Calpain has been described as an upstream regulator of apoptosis based on the observation that dexamethasone-induced thymocyte apoptosis was prevented by calpain inhibitors, whereas DNA fragmentation in isolated thymocyte nuclei was not (14Squier M.K.T. Cohen J.J. J. Immunol. 1997; 158: 3690-3697PubMed Google Scholar). Recently, Bax, a pro-apoptotic member of the Bcl-2 family involved in triggering apoptosis via mitochondria, has been identified as a target of calpain in HL-60 (15Wood D.E. Thomas A. Devi L.A. Berman Y. Beavis R.C. Reed J.C. Newcomb E.W. Oncogene. 1998; 17: 1069-1078Crossref PubMed Scopus (308) Google Scholar) and Jurkat cells (16Gao G. Dou Q.P. J. Cell. Biochem. 2000; 80: 53-72Crossref PubMed Scopus (267) Google Scholar). Whereas in HL-60 cells Bax cleavage into an 18-kDa fragment occurred several hours after cleavage of poly(ADP-ribose) polymerase and retinoblastoma protein (15Wood D.E. Thomas A. Devi L.A. Berman Y. Beavis R.C. Reed J.C. Newcomb E.W. Oncogene. 1998; 17: 1069-1078Crossref PubMed Scopus (308) Google Scholar), the results reported in Jurkat cells suggested that the generation of the 18-kDa Bax fragment is an early event required for the induction of apoptosis via mitochondria (16Gao G. Dou Q.P. J. Cell. Biochem. 2000; 80: 53-72Crossref PubMed Scopus (267) Google Scholar). Because Bax appears to be a critical apoptosis-regulating molecule in neutrophils (4Dibbert B. Weber M. Nikolaizik W.H. Vogt P. Schöni M.H. Blaser K. Simon H.-U. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13330-13335Crossref PubMed Scopus (265) Google Scholar, 17Weinmann P. Gaehtgens P. Walzog B. Blood. 1999; 93: 3106-3115Crossref PubMed Google Scholar, 18Maianski N.A. Mul F.P.J. van Buul J.D. Kuijpers T.W. Blood. 2002; 99: 672-679Crossref PubMed Scopus (153) Google Scholar), we studied the calpastatin-calpain-system and its importance for Bax cleavage and apoptosis induction in this particular type of granulocytes. Here, we demonstrate that calpain-1, but not calpain-2, is activated in spontaneous and Fas receptor-mediated apoptosis of neutrophils. We show that calpain-1 activation involves caspase-dependent proteolysis of calpastatin, generates an 18-kDa cleavage product of Bax unable to interact with anti-apoptotic Bcl-xL, and is required for mitochondrial release of cytochrome c and Smac as well as subsequent enzymatic caspase-3 activation and apoptosis. Moreover, neutrophils purified from patients with a systemic inflammatory disease (cystic fibrosis) provide evidence for decreased activity of the calpain-calpastatin system associated with delayed apoptosis. Reagents—The caspase inhibitors N-benzyloxycarbonyl (z) 1The abbreviations used are: zN-benzyloxycarbonylfmkfluoromethylketoneFITCfluorescein isothiocyanateTRITCtetramethylrhodamine isothiocyanatePBSphosphate-buffered salineILinterleukinVDACvoltage-dependent anion channelIAPinhibitor of apoptosisChaps3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidGM-CSFgranulocyte/macrophage colony-stimulating factorHEKhuman embryonic kidneyE64-d(l)-3-carboxy-trans-2,3-epoxypropionyl-Leu-amido-(4-guanidino)butane ethylesterr.m.s.d.root mean square displacementBHBcl-2-homology regionTBSTris-buffered salineODoptical densityMDmolecular dynamics.-Val-Ala-Asp (VAD)-fluoromethylketone (fmk) and z-Ile-Glu-Thr-Asp (IETD)-fmk were purchased from Alexis Corporation (Läufelfingen, Switzerland). Bongkrekic acid, calpastatin-derived and scrambled control peptide, antennapedia-coupled Smac-N7, antennapedia-coupled control peptide (Ant-BH3-A78), as well as purified human calpain-1 and calpain-2 were obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA). Isoform-specific inhibitors of calpains were generous gifts from Dr. J. C. Powers (Georgia Institute of Technology, Atlanta, GA) and had the following chemical structures: Z-Leu-Abu-CONH-CH2-CH(OH)-C6H4-3-OC6H4(3-CF3) (compound 28 for calpain-1) and Z-Leu-Abu-CONH-CH2-2-pyridyl (compound 35 for calpain-2) (19Li Z. Ortega-Vilain A.C. Patil G.S. Chu D.L. Foreman J.E. Eveleth D.D. Powers J.C. J. Med. Chem. 1996; 39: 4089-4098Crossref PubMed Scopus (159) Google Scholar). GM-CSF was purchased from Novartis Pharma GmbH (Nürnberg, Germany). HEK 293T cells overexpressing Smac (20Adrain C. Creagh E.M. Martin S.J. EMBO J. 2001; 20: 6627-6636Crossref PubMed Scopus (363) Google Scholar) were obtained from Dr. S. Martin (Trinity College, University of Dublin, Dublin, Ireland). The general calpain inhibitor (l)-3-carboxy-trans-2,3-epoxypropionyl-Leu-amido-(4-guanidino)butane ethylester (E64-d) and all other chemicals were, unless stated otherwise, from Sigma (Buchs, Switzerland). N-benzyloxycarbonyl fluoromethylketone fluorescein isothiocyanate tetramethylrhodamine isothiocyanate phosphate-buffered saline interleukin voltage-dependent anion channel inhibitor of apoptosis 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid granulocyte/macrophage colony-stimulating factor human embryonic kidney (l)-3-carboxy-trans-2,3-epoxypropionyl-Leu-amido-(4-guanidino)butane ethylester root mean square displacement Bcl-2-homology region Tris-buffered saline optical density molecular dynamics. Cells—Peripheral blood neutrophils were purified from healthy normal individuals or patients suffering from cystic fibrosis by Ficoll-Hypaque centrifugation (4Dibbert B. Weber M. Nikolaizik W.H. Vogt P. Schöni M.H. Blaser K. Simon H.-U. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13330-13335Crossref PubMed Scopus (265) Google Scholar, 21Yousefi S. Green D.R. Blaser K. Simon H.-U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10868-10872Crossref PubMed Scopus (216) Google Scholar). The resulting cell populations contained more than 95% neutrophils. Isolation of eosinophils (22Yousefi S. Hoessli D. Blaser K. Mills G.B. Simon H.-U. J. Exp. Med. 1996; 183: 1407-1414Crossref PubMed Scopus (217) Google Scholar, 23Hebestreit H. Dibbert B. Balatti I. Braun D. Schapowal A. Blaser K. Simon H.-U. J. Exp. Med. 1998; 187: 415-425Crossref PubMed Scopus (162) Google Scholar) and generation of T lymphoblasts (24Schmandt R. Hill M. Amendola A. Mills G.B. Hogg D. J. Immunol. 1994; 152: 96-105PubMed Google Scholar) were performed as previously described. H9, SKW6.4, J16, and CEM cells (25Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2638) Google Scholar) were grown in RPMI 1640 with 10% heat-inactivated fetal calf serum (complete culture medium). 32D cells were cultured in the presence of IL-3 (26Daigle I. Yousefi S. Colonna M. Green D.R. Simon H.-U. Nat. Med. 2002; 8: 61-67Crossref PubMed Scopus (161) Google Scholar). Cell Cultures—Cells were cultured at 1 × 106 cells/ml in complete culture medium, and, where indicated, treated with E64-d, calpain-1, or calpain-2 inhibitor (19Li Z. Ortega-Vilain A.C. Patil G.S. Chu D.L. Foreman J.E. Eveleth D.D. Powers J.C. J. Med. Chem. 1996; 39: 4089-4098Crossref PubMed Scopus (159) Google Scholar), calpastatin (27Maki M. Bagci H. Hamaguchi K. Ueda M. Murachi T. Hatanaka M. J. Biol. Chem. 1989; 264: 18866-18869Abstract Full Text PDF PubMed Google Scholar, 28Kawasaki H. Emori Y. Imajoh-Ohmi S. Minami Y. Suzuki K. J. Biochem. (Tokyo). 1989; 106: 274-281Crossref PubMed Scopus (72) Google Scholar), Smac (29Srinivasula S.M. Hegde R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R. Robbins P.D. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (867) Google Scholar), or control peptides at the indicated concentrations. In other experiments cells were incubated with the caspase-8 inhibitor z-IETD-fmk (50 μm) or the pan-caspase inhibitor z-VAD-fmk (50 μm). Me2SO was always used as a solvent control. In the experiments in which agonistic anti-Fas receptor monoclonal antibodies (1 μg/ml CH11, Beckman Coulter International S.A., Nyon, Switzerland) were added, the inhibitors were given 1 h before Fas receptor stimulation. The duration of CH11 or GM-CSF (50 ng/ml) stimulation is indicated in each presented experiment. Determination of Cell Death and Apoptosis—Neutrophil death was assessed by uptake of 1 μm ethidium bromide and flow cytometric analysis (FACSCalibur, Becton Dickinson, Heidelberg, Germany) as previously described (4Dibbert B. Weber M. Nikolaizik W.H. Vogt P. Schöni M.H. Blaser K. Simon H.-U. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13330-13335Crossref PubMed Scopus (265) Google Scholar, 26Daigle I. Yousefi S. Colonna M. Green D.R. Simon H.-U. Nat. Med. 2002; 8: 61-67Crossref PubMed Scopus (161) Google Scholar, 30Heinisch I.V.W.M. Daigle I. Knöpfli B. Simon H.-U. Eur. J. Immunol. 2000; 30: 3441-3446Crossref PubMed Scopus (71) Google Scholar). To determine whether cell death was apoptosis, redistribution of phosphatidylserine (23Hebestreit H. Dibbert B. Balatti I. Braun D. Schapowal A. Blaser K. Simon H.-U. J. Exp. Med. 1998; 187: 415-425Crossref PubMed Scopus (162) Google Scholar, 30Heinisch I.V.W.M. Daigle I. Knöpfli B. Simon H.-U. Eur. J. Immunol. 2000; 30: 3441-3446Crossref PubMed Scopus (71) Google Scholar), oligonucleosomal DNA fragmentation (4Dibbert B. Weber M. Nikolaizik W.H. Vogt P. Schöni M.H. Blaser K. Simon H.-U. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13330-13335Crossref PubMed Scopus (265) Google Scholar, 23Hebestreit H. Dibbert B. Balatti I. Braun D. Schapowal A. Blaser K. Simon H.-U. J. Exp. Med. 1998; 187: 415-425Crossref PubMed Scopus (162) Google Scholar), and morphologic analysis were performed. In Vitro Autolytic Digestion of Purified Calpain-1—Purified procalpain-1 was dissolved in digestion buffer and incubated at 37 °C for 30 min, according to the instructions from the manufacturer (Calbiochem). The digestions were stopped by adding Tris-glycine loading buffer, and aliquots were subjected to gel electrophoresis and immunoblotting. Immunoprecipitation—Neutrophils (5 × 107) were washed with PBS and buffer A (50 mm Hepes, pH 7.2, 143 mm KCl, 5 mm MgCl2, 1 mm EGTA, phosphatases, and proteases inhibitors), and then lysed in buffer A plus 0.2% Nonidet P-40 on ice for 30 min. Cell lysates were precleared with 100 μl of 50% protein A-Sepharose (Sigma) at 4 °C for 1 h. Immunoprecipitation was performed using 5 μl of affinity-purified rabbit anti-human Bcl-xL S18 (Santa Cruz Biotechnology, Inc., LabForce, Nunningen, Switzerland) in the presence of 2.5 mg/ml ovalbumin at 4 °C for 2h. Fifty μl of a 50% protein A-Sepharose suspension were added and the immunocomplexes captured on a rotating wheel at 4 °C for 90 min. Immunocomplexes were washed three times in buffer A and boiled at 95 °C for 5 min before loading on 12% NuPAGE gels (Invitrogen, Groningen, Netherlands). Gel Electrophoresis and Immunoblotting—Neutrophils (5 × 106) were washed with PBS, lysed with radioimmune precipitation assay buffer (0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS in PBS) supplemented with a protease inhibitor mixture with frequent vortexing on ice for 40 min, including two freeze/thaw cycles using a methanol/dry ice bath. After a 10-min centrifugation step to remove insoluble particles, equal amounts of the cell lysates were loaded on Tris-glycine gels or NuPAGE gels (Invitrogen). Separated proteins were electrotransferred onto polyvinylidene difluoride membranes (Immobilion-P, Millipore, Bedford, MA). The filters were incubated with primary antibodies at room temperature for 2 h or at 4 °C (overnight) in TBS, 0.1% Tween 20, 3% nonfat dry milk. The primary antibodies were polyclonal anti-Bax, detecting amino acids 43-61 (15Wood D.E. Thomas A. Devi L.A. Berman Y. Beavis R.C. Reed J.C. Newcomb E.W. Oncogene. 1998; 17: 1069-1078Crossref PubMed Scopus (308) Google Scholar); polyclonal anti-caspase-3 (both 1/1000 and from BD Biosciences, Pharmingen); monoclonal anti-calpain-1 antibody (1/1000; Chemicon, Temecula, CA); monoclonal anti-calpain-1 antibody (1/200; detecting latent and activated forms (Ref. 31Saido T.C. Nagao S. Shiramine M. Tsukaguchi M. Sorimachi H. Murofushi H. Tsuchiya T. Ito H. Suzuki K. J. Biochem. (Tokyo). 1992; 111: 81-86Crossref PubMed Scopus (131) Google Scholar); kind gift of J. S. Elce, Queens University, Kingston, Ontario, Canada); monoclonal anti-calpain-2 antibody (1/500; Sigma); and monoclonal anti-calpastatin antibody (1/400; Chemicon). For loading controls, stripped filters were incubated with a monoclonal anti-β-actin antibody (1/10,000; Sigma). Filters were washed in TBS, 0.1% Tween 20 for 30 min and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Dübendorf, Switzerland) in TBS, 0.1% Tween 20, 5% nonfat dry milk for 1 h. Filters were developed by an ECL technique (ECL Kit, Amersham Biosciences) according to instructions from the manufacturer. Densitometry Analysis—Cleavage of calpastatin was analyzed by densitometry using nonsaturated chemiluminescence exposed films and NIH Image 1.9 software. OD values of calpastatin bands divided by the OD of the corresponding β-actin band are expressed as percentage of OD(calpastatin)/OD(β-actin) of freshly isolated neutrophils, which was defined as 100%. Bax/Bcl-xLin Vitro Binding Studies—One μg of purified plasmids containing sequence verified cDNAs encoding Bax (pCRII) or Bcl-xL (pBSK-, kind gift of L. Boise) was in vitro transcribed and translated in the presence of l-[35S]methionine using a coupled transcription/translation TnT kit (Promega, Catalys AG, Wallisellen, Switzerland) according to instructions from the manufacturer. 35S-Labeled truncated Bax was prepared by in vitro digestion of 35S-labeled Bax with purified calpain-1 (Calbiochem) in assay buffer (10 mm HEPES, pH 7.4, 2 mm CaCl2) at 37 °C for 5 h. The reaction was stopped by adding E64-d (50 μm). Potential residual full-length Bax was immunodepleted by adding 10 μl of anti-Bax antisera (N20, Santa Cruz) at 4 °C for 2 h (15Wood D.E. Thomas A. Devi L.A. Berman Y. Beavis R.C. Reed J.C. Newcomb E.W. Oncogene. 1998; 17: 1069-1078Crossref PubMed Scopus (308) Google Scholar), followed by incubation with 25 μl of protein A/G plus-agarose (Santa Cruz). The supernatant was subjected to two further cycles of immunodepletion and finally saved as 35S-labeled truncated Bax. 35S-Labeled Bax intended for co-immunoprecipitation experiments was treated in exactly the same way except that addition of purified calpain-1 and antisera (N20) was omitted. Ten μl of full-length or truncated Bax was incubated with 4 μl of translated Bcl-xL in 100 μl of buffer (10 mm Tris-HCl, pH 7.4, 142.5 mm KCl, 5 mm MgCl2, 1 mm EGTA, 0.5% Nonidet P-40) at 4 °C for 2 h. One hundred μl of buffer containing 2 μg of polyclonal Bax antisera (BD Biosciences, Pharmingen) was added at 4 °C for 2 h, followed by an additional 2-h incubation with 25 μl of protein A/G plus-agarose (Santa Cruz) at 4 °C. After three washing steps with buffer and two PBS washing steps, the bound proteins were eluted by adding loading buffer and heated at 80 °C for 10 min. An aliquot of the obtained protein solution was loaded on 12% NuPAGE gels (Invitrogen), followed by autoradiography. Subcellular Fractionation—Freshly purified neutrophils and neutrophils cultured in the presence or absence of E64-d (50 μm) and CH11 antibody for 8 h, were washed with cold PBS and digitonin-permeabilized essentially as described elsewhere (20Adrain C. Creagh E.M. Martin S.J. EMBO J. 2001; 20: 6627-6636Crossref PubMed Scopus (363) Google Scholar). Equal volumes of the NuPAGE loading buffer-supplemented fractions were loaded on 12% NuPAGE gels and subsequently transferred to polyvinylidene difluoride membranes. Filters were incubated with anti-cytochrome c monoclonal antibody (1/500; BD Biosciences, Pharmingen) or anti-Smac polyclonal antisera (1/500; Alexis), and binding was detected by using horseradish peroxidase-conjugated secondary antibodies and ECL. Stripped filters were incubated with anti-voltage-dependent anion channel (VDAC) monoclonal antibody (1/500; Calbiochem). Enzymatic Caspase Assay—Neutrophils were cultured in the presence or absence of E64-d (50 μm), CH11 antibody, and Smac peptide (50 μm) for 10 h, washed with cold PBS, and subsequently lysed in cell lysis buffer (50 mm HEPES, pH 7.4, 0.1% Chaps, 5 mm dithiothreitol, 0.1 mm EDTA) using a Teflon glass homogenizer (VWR International, Ismaning, Germany) on ice for 10 min. After a 10-min centrifugation step at 10,000 × g at 4 °C, caspase-3-like activity was measured in 10 μl of supernatants as enzymatic conversion of the colorimetric substrate Ac-DEVD-pNA at 405 nm according to the instructions from the manufacturer (QuantiZyme caspase-3 cellular activity assay kit; Biomol, Plymouth Meeting, PA). For controls, we also analyzed caspase-3-like activity of recombinant caspase-3 (Calbiochem) in the presence and absence of the indicated concentrations of E64-d and 50 μm Ac-DEVD. Confocal Laser Scanning Microscopy—Cytospins were made from freshly purified neutrophils and neutrophils cultured in the presence or absence of E64-d (50 μm) and CH11 antibody for 12 h. Cells were fixed in 4% paraformaldehyde at room temperature for 15 min and washed four times in PBS, pH 7.4. Permeabilization of cells was performed with 0.05% saponin in blocking buffer (3% bovine serum albumin in PBS) at room temperature for 5 min and with acetone at -20 °C for 15 min. To prevent nonspecific binding, slides were incubated in blocking buffer at room temperature for 1 h. Indirect immunostainings for cytochrome c, Smac, and VDAC were performed at room temperature for 1 h by using the following primary antibodies: anti-cytochrome c monoclonal antibody (1/100; diluted in blocking buffer), anti-Smac polyclonal antisera (1/100), and anti-VDAC monoclonal antibody (1/200). Incubation with appropriate TRITC- and FITC-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, Milan Analytica, La Roche, Switzerland) was performed in the dark at room temperature for 1 h. The anti-fading agent Slowfade (Molecular Probes) was added, and the cells were covered by coverslips. The slides were analyzed by confocal laser scanning microscopy (LSM 510, Carl Zeiss, Heidelberg, Germany) equipped with argon and helium-neon lasers. Molecular Protein Modeling—The models were based on the NMR structure of Bax (Protein Data Bank code 1F16) (32Suzuki M. Youle R.J. Tjandra N. Cell. 2000; 103: 645-654Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). The NH2-deleted Bax was built by simply removing the first 32 amino acids from the three-dimensional structure of the conformer number 1. All calculations were carried out with the AMBER 6 program (33Case D.A. Pearlman D.A. Caldwell J.W. Cheatham T.E. Ross III, W.S. Simmerling C.L. Darden T.A. Merz K.M. Stanton R.V. Cheng A.L. Vincent J.J. Crowley M. Tsui V. Radmer R.J. Duan Y. Pitera J. I. Massova G.L. Seibel U.C. Singh P. K. Weiner Kollman P.A. AMBER 6. University of California, San Francisco1999Google Scholar). The all-atom AMBER force field (34Cornell W.D. Cieplack P. Bayly C.I. Gould I.R. Merz K.M. Ferguson D.M. Spellmeyer D.C. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11687) Google Scholar) and the TIP3P model (35Jorgensen W.L. Chandrasekhar J. Madura J.D. Impey R.W. Klein M.L. J. Chem. Phys. 1983; 79: 926-935Crossref Scopus (30918) Google Scholar) were used for the proteins and the water molecules, respectively. The dielectric constant was set equal to 1. Long range electrostatic interactions were estimated by means of the particle mesh Ewald method (36Darden T.A. York D. Pedersen L. J. Chem. Phys. 1993; 98: 10089-10092Crossref Scopus (21714) Google Scholar), whereas the short range electrostatic and van der Waals interactions were truncated within a 12-Å cut-off. Constant temperature pressure was achieved by coupling the system with a Berendsen thermostat (T = 300 K; coupling constant = 1.0 ps) and barostat (P = 1 atm; coupling constant = 1.0 ps) (37Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. DiNola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (24191) Google Scholar). Full-length and truncated Bax were immersed in a 69 × 58 × 51-Å3 box containing ∼6000 water molecules, to account for the effects of the explicit solvent. The positions of the water molecules were equilibrated by running 5000 geometry optimization steps and 30-ps molecular dynamics (MD) simulations. The two systems were further studied by carrying out 2-ns MD simulations. Because a stable conformation was not achieved even after 2 ns of MD simulations, the NH2-deleted model was simulated up to 3.2 ns. Spontaneous and Fas Receptor-mediated Neutrophil Apoptosis Require Calpain-1 but Not Calpain-2—To determine whether calpain has a functional role within apoptotic pathways in neutrophils, we used the previously characterized pan-calpain inhibitor E64-d (14Squier M.K.T. Cohen J.J. J. Immunol. 1997; 158: 3690-3697PubMed Google Scholar, 38Mehdi S. Trends Biochem. Sci. 1991; 16: 150-155Abstract Full Text PDF PubMed Scopus (196) Google Scholar) in an in vitro system of neutrophil apoptosis. As shown in Fig. 1A, E64-d delayed spontaneous and Fas receptor (CH11)-mediated neutrophil death in a dose-dependent manner. The data represent cell death numbers of 24-h cultures, but the effect of E64-d was also observed in 12-, 36-, and 48-h cultures (data not shown). E64-d also inhibited phosphatidylserine redistribution (Fig. 1B), DNA fragmentation (Fig. 1C), and morphological changes associated with apoptosis (Fig. 1D), suggesting that the type of neutrophil death blocked by E64-d was apoptosis. Several lysosomal (ammonium chloride, chloroquine) and cathepsin inhibitors (compound 28 (Ref. 19Li Z. Ortega-Vilain A.C. Patil G.S. Chu D.L. Foreman J.E. Eveleth D.D. Powers J.C. J. Med. Chem. 1996; 39: 4089-4098Crossref PubMed Scopus (159) Google Scholar)) had no effect in this system (data not shown). To confirm the potential role of calpain in the regulation of neutrophil apoptosis, we applied cell-permeable inhibitors that block specific calpain isoforms. Whereas a specific calpain-1 inhibitor (19Li Z. Ortega-Vilain A.C. Patil G.S. Chu D.L. Foreman J.E. E
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