Caspase-2 Induces Apoptosis by Releasing Proapoptotic Proteins from Mitochondria
2002; Elsevier BV; Volume: 277; Issue: 16 Linguagem: Inglês
10.1074/jbc.m108029200
ISSN1083-351X
AutoresYin Guo, Srinivasa M. Srinivasula, Anne Druilhe, Teresa Fernandes‐Alnemri, Emad S. Alnemri,
Tópico(s)Autophagy in Disease and Therapy
ResumoCaspase-2 is one of the earliest identified caspases, but the mechanism of caspase-2-induced apoptosis remains unknown. We show here that caspase-2 engages the mitochondria-dependent apoptotic pathway by inducing the release of cytochrome c (Cyt c) and other mitochondrial apoptogenic factors into the cell cytoplasm. In support of these observations we found that Bcl-2 and Bcl-xL can block caspase-2- and CRADD (caspase and RIP adaptor with death domain)-induced cell death. Unlike caspase-8, which can process all known caspase zymogens directly, caspase-2 is completely inactive toward other caspase zymogens. However, like caspase-8, physiological levels of purified caspase-2 can cleave cytosolic Bid protein, which in turn can trigger the release of Cyt c from isolated mitochondria. Interestingly, caspase-2 can also induce directly the release of Cyt c, AIF (apoptosis-inducing factor), and Smac (second mitochondria-derived activator of caspases protein) from isolated mitochondria independent of Bid or other cytosolic factors. The caspase-2-released Cytc is sufficient to activate the Apaf-caspase-9 apoptosomein vitro. In combination, our data suggest that caspase-2 is a direct effector of the mitochondrial apoptotic pathway. Caspase-2 is one of the earliest identified caspases, but the mechanism of caspase-2-induced apoptosis remains unknown. We show here that caspase-2 engages the mitochondria-dependent apoptotic pathway by inducing the release of cytochrome c (Cyt c) and other mitochondrial apoptogenic factors into the cell cytoplasm. In support of these observations we found that Bcl-2 and Bcl-xL can block caspase-2- and CRADD (caspase and RIP adaptor with death domain)-induced cell death. Unlike caspase-8, which can process all known caspase zymogens directly, caspase-2 is completely inactive toward other caspase zymogens. However, like caspase-8, physiological levels of purified caspase-2 can cleave cytosolic Bid protein, which in turn can trigger the release of Cyt c from isolated mitochondria. Interestingly, caspase-2 can also induce directly the release of Cyt c, AIF (apoptosis-inducing factor), and Smac (second mitochondria-derived activator of caspases protein) from isolated mitochondria independent of Bid or other cytosolic factors. The caspase-2-released Cytc is sufficient to activate the Apaf-caspase-9 apoptosomein vitro. In combination, our data suggest that caspase-2 is a direct effector of the mitochondrial apoptotic pathway. Apoptosis is a form of cellular suicide that is essential for development and tissue homeostasis of all metazoan organisms. Caspases, a family of cysteine-dependent aspartate-directed proteases, play critical roles in initiation and execution of apoptosis (1.Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2133) Google Scholar). Determining the cellular processes, which lead to activation of caspases during apoptosis, and identifying the relevant intracellular caspase substrates and regulators have been the subjects of intensive investigation in the last several years. Two relatively well-characterized apoptotic pathways have been identified. The first pathway is mediated by death receptors, such as Fas or Tumor Necrosis Factor (TNF) 1The abbreviations used are: TNFtumor necrosis factorDNdominant negativeDMdouble mutantAIFapoptosis-inducing factorSmac/Diablosecond mitochondria-derived activator of caspases proteinApaf-1apoptotic protease activating factor-1Coxcytochrome oxidaseCyt ccytochromecIAPinhibitor of apoptosis proteinCRADDcaspase and RIP adaptor with death domainz-VAD-fmkbenzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketoneVDVAD-afcVal-Asp-Val-Ala-Asp-7-amino-4-trifluoromethyl coumarinCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidcaspase-associated recruitment domainCARD, caspase-associated recruitment domainPTpermeability transitionVDACvoltage-dependent anion channel1The abbreviations used are: TNFtumor necrosis factorDNdominant negativeDMdouble mutantAIFapoptosis-inducing factorSmac/Diablosecond mitochondria-derived activator of caspases proteinApaf-1apoptotic protease activating factor-1Coxcytochrome oxidaseCyt ccytochromecIAPinhibitor of apoptosis proteinCRADDcaspase and RIP adaptor with death domainz-VAD-fmkbenzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketoneVDVAD-afcVal-Asp-Val-Ala-Asp-7-amino-4-trifluoromethyl coumarinCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidcaspase-associated recruitment domainCARD, caspase-associated recruitment domainPTpermeability transitionVDACvoltage-dependent anion channelreceptor (2.Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4542) Google Scholar). Specific adaptor proteins such as FADD (also known as MORT1) (3.Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2153) Google Scholar, 4.Boldin M.P. Varfolomeev E.E. Pancer Z. Mett I.L. Camonis J.H. Wallach D. J. Biol. Chem. 1995; 270: 7795-7798Abstract Full Text Full Text PDF PubMed Scopus (938) Google Scholar) or CRADD (also known as RAIDD) (5.Ahmad M. Srinivasula S.M. Wang L. Talanian R.V. Litwack G. Fernandes-Alnemri T. Alnemri E.S. Cancer Res. 1997; 57: 615-619PubMed Google Scholar, 6.Duan H. Dixit V.M. Nature. 1997; 385: 86-89Crossref PubMed Scopus (469) Google Scholar) bind to the ligand-bound receptor complexes. Interaction between the adaptor molecules and the prodomain of initiator caspases 2, 8, or 10 triggers sequestration-mediated autoactivation of these caspases (7.Yang X. Chang H.Y. Baltimore D. 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In the second pathway, Cyt c is released from mitochondria to the cytoplasm in cells exposed to chemotherapeutic drugs, UV irradiation, growth factor withdrawal, or ligation of Fas and TNF receptors (12.Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4265) Google Scholar, 13.Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4394) Google Scholar, 14.Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1106) Google Scholar, 15.Kharbanda S. Pandey P. Schofield L. Israels S. Roncinske R. Yoshida K. Bharti A. Yuan Z.M. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (368) Google Scholar, 16.Adachi S. Cross A.R. Babior B.M. Gottlieb R.A. J. Biol. Chem. 1997; 272: 21878-21882Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Released Cyt c binds to Apaf-1 (apoptotic protease-activating factor-1) and promotes its oligomerization (17.Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4447) Google Scholar,18.Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (962) Google Scholar). Recruitment of procaspase-9 to this active apoptosome results in its autoactivation and subsequent activation of caspase-3 by the active caspase-9·Apaf-1 complex (18.Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (962) Google Scholar, 19.Li 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 (6197) Google Scholar, 20.Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2736) Google Scholar). In both pathways activation of effector caspases by initiator caspases amplifies the apoptotic signal to ensure fast and irreversible cell death. In addition, there appears to be cross-talk between the death receptor and the mitochondrial apoptotic pathways. Caspase-8, an initiator caspase in the death receptor pathway, can connect death receptors to the core apoptotic machinery by directly cleaving downstream executioner caspases (11.Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (481) Google Scholar). Alternatively, it may activate the caspase cascade indirectly by cleaving Bid, a proapoptotic Bcl-2 family member. The cleaved Bid fragment translocates from the cytosol to the outer mitochondrial membrane resulting in disruption of the outer mitochondrial membrane and release of Cyt c from the mitochondrial intermembrane space (21.Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3067) Google Scholar, 22.Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3771) Google Scholar). Engagement of the mitochondria-dependent pathway is more efficient than the mitochondria-independent pathway, as it only requires a small amount of active caspase-8 (23.Kuwana T. Smith J.J. Muzio M. Dixit V. Newmeyer D.D. Kornbluth S. J. Biol. Chem. 1998; 273: 16589-16594Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Recent reports suggest that induction of Cytc release from mitochondria is not only restricted to caspase-8. Effector caspases, caspase-3, -6, and -7, can also facilitate rapid Cyt c release from the mitochondria when other cytosolic factors are present (24.Bossy-Wetzel E. Green D.R. J. Biol. Chem. 1999; 274: 17484-17490Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). tumor necrosis factor dominant negative double mutant apoptosis-inducing factor second mitochondria-derived activator of caspases protein apoptotic protease activating factor-1 cytochrome oxidase cytochromec inhibitor of apoptosis protein caspase and RIP adaptor with death domain benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone Val-Asp-Val-Ala-Asp-7-amino-4-trifluoromethyl coumarin 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid CARD, caspase-associated recruitment domain permeability transition voltage-dependent anion channel tumor necrosis factor dominant negative double mutant apoptosis-inducing factor second mitochondria-derived activator of caspases protein apoptotic protease activating factor-1 cytochrome oxidase cytochromec inhibitor of apoptosis protein caspase and RIP adaptor with death domain benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone Val-Asp-Val-Ala-Asp-7-amino-4-trifluoromethyl coumarin 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid CARD, caspase-associated recruitment domain permeability transition voltage-dependent anion channel Because of the critical role of mitochondria in apoptosis, it has been the subject of intensive research recently. Proapoptotic factors such as Cyt c (13.Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4394) Google Scholar, 17.Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4447) Google Scholar), procaspases 2, 3, 9 (25.Mancini M. Nicholson D.W. Roy S. Thornberry N.A. Peterson E.P. Casciola-Rosen L.A. Rosen A. J. Cell Biol. 1998; 140: 1485-1495Crossref PubMed Scopus (372) Google Scholar, 26.Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Brenner C. Larochette N. Prevost M.C. Alzari P.M. Kroemer G. J. Exp. Med. 1999; 189: 381-394Crossref PubMed Scopus (637) Google Scholar), apoptosis-inducing factor (AIF) (27.Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. 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Upon apoptotic challenge, however, a rapid release of these factors through the outer mitochondrial membrane into the cytoplasm signals the initiation of the apoptotic process. The mechanism of release of these mitochondrial apoptotic factors into the cytoplasm remains unclear. Caspase-2, initially described as Nedd-2/Ich-1, has been identified as a protein related to the Caenorhabditis elegans cell death protein CED-3 and mammalian interleukin-1β-converting enzyme (caspase-1) (32.Wang L. Miura M. Bergeron L. Zhu H. Yuan J. Cell. 1994; 78: 739-750Abstract Full Text PDF PubMed Scopus (801) Google Scholar). Two distinct caspase-2 mRNA species derived from alternative splicing encode two proteins, caspase-2L and caspase-2S. Overexpression of caspase-2L induces cell death, whereas overexpression of caspase-2S can antagonize cell death. Caspase-2L is the dominant isoform that is expressed in most tissues (32.Wang L. Miura M. Bergeron L. Zhu H. Yuan J. 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Genes Dev. 1998; 12: 1304-1314Crossref PubMed Scopus (600) Google Scholar). Thus, for caspase-2 knockout mice, defects in apoptosis are cell-type and stimulus-dependent. So far the mechanism by which caspase-2 can induce activation of the effector caspases is not fully understood. CRADD, an adaptor molecule for TNF receptor, recruits pro-caspase-2 to the death receptor pathway (5.Ahmad M. Srinivasula S.M. Wang L. Talanian R.V. Litwack G. Fernandes-Alnemri T. Alnemri E.S. Cancer Res. 1997; 57: 615-619PubMed Google Scholar, 6.Duan H. Dixit V.M. Nature. 1997; 385: 86-89Crossref PubMed Scopus (469) Google Scholar). It has been suggested that the prodomain of caspase-2 interacts with the CARD domain of CRADD (5.Ahmad M. Srinivasula S.M. Wang L. Talanian R.V. Litwack G. Fernandes-Alnemri T. Alnemri E.S. Cancer Res. 1997; 57: 615-619PubMed Google Scholar, 6.Duan H. Dixit V.M. Nature. 1997; 385: 86-89Crossref PubMed Scopus (469) Google Scholar), allowing the dimerization and autoactivation of procaspase-2. However, events subsequent to caspase-2 activation remain largely unknown. To understand how caspase-2 triggers the apoptotic pathway, we examined the possibility that caspase-2 employs the mitochondrial pathway to activate the caspase cascade. Our results indicate that physiological amounts of recombinant caspase-2 protein could directly provoke release of Cyt c and other proapoptotic factors from the mitochondria independent of any cytosolic protein and also through cleavage of Bid. The released proapoptotic factors trigger activation of the downstream caspases, thus setting into motion the apoptotic cascade. Anti-Cyt c antibody (7H8.2 c12, 6H2.B4) was obtained from BD PharMingen (San Diego, CA). Anti-caspase-2 antibody (directed toward amino acids 225–401) was purchased from Transduction Laboratories (Lexington, KY). Anti-AIF and anti-Bid were purchased from Santa Cruz Biotechnology Inc. Anti-caspase-3 antibody was purchased from BD PharMingen. Anti-human cytochrome oxidase subunit II (A-6404) was purchased from Molecular Probes. Anti-Smac monoclonal antibody was raised against mature Smac. VDVAD-afc was purchased from Enzyme Systems. Anti-citrate synthase polyclonal antibody was a gift from Yuri Lazebnik. Protease inhibitors were purchased from Roche Molecular Biochemicals. Protein concentrations were determined by the Bio-Rad assay kit (Hercules, CA). For apoptosis assays, we used the mammalian double-expression vector pRSC-LacZ, which allows expression of lacZ under the Rous sarcoma virus promoter, and the recombinant proteins (caspase-2 or CRADD) under the cytomegalovirus promoter. Constructs encoding CRADD, caspase-2, caspase-9-DN, Bcl-2, Bcl-xL, p35, and X-linked inhibitor of apoptosis protein have been described before (5.Ahmad M. Srinivasula S.M. Wang L. Talanian R.V. Litwack G. Fernandes-Alnemri T. Alnemri E.S. Cancer Res. 1997; 57: 615-619PubMed Google Scholar, 19.Li 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 (6197) Google Scholar, 35.MacFarlane M. Ahmad M. Srinivasula S.M. Fernandes-Alnemri T. Cohen G.M. Alnemri E.S. J. Biol. Chem. 1997; 272: 25417-25420Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 36.Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Bid wild type, cleavage site Asp-59 to Glu mutant, and double mutant (Asp-59 and Asp-75 to Glu) genes were excised from constructs generously given by Dr. J. Yuan (Harvard Medical School, Boston, MA) and then subcloned into a T7pcDNA3 vector. For bacterial expression, cDNA was cloned in pET 21 or pET 28 (Invitrogen). Recombinant proteins with C-terminal or N-terminal His6 tags were expressed in BL-21(DE3) bacteria and purified on a resin of Ni2+affinity by standard affinity purification procedures as described previously (18.Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (962) Google Scholar). MCF-7 cells were transiently cotransfected with pRSC-lacZ constructs in the presence or absence of different apoptosis inhibitors. The cells were stained with β-galactosidase 30–36 h after transfection or 20 μm z-VAD-fmk treatment and examined for morphological signs of apoptosis (36.Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Normal and apoptotic blue cells were counted by phase-contrast microscopy. The graphs depict the mean percentage of stained apoptotic cells as a fraction of the total number of blue cells after subtracting the percentage of apoptotic cell in the empty vector-transfected control cells (means ± S.D.). Data provided represent the average of at least three individual experiments (n ≥ 3 and S.D. ± 1–4%). Wild type caspases and Bid and Bid mutants were in vitro translated in the presence of [35S]methionine in rabbit reticulocyte lysate with a T7-RNA polymerase-coupled TnT kit (Promega), using the pRSC-LacZ, T7pcDNA3, or pET 28 constructs as templates according to the manufacturer's recommendations. Equal amounts of the translation reactions were diluted in 10 μl of interleukin-1β-converting enzyme buffer (25 mm HEPES, 1 mm EDTA, 5 mm dithiothreitol, and 0.1% CHAPS (pH 7.5)) and incubated with purified recombinant caspase-2 and caspase-8, respectively, at 30 °C for 1 h. The reactions were fractionated by 10% SDS-PAGE and analyzed by autoradiography. These were done as previously described (36.Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). HeLa cells were collected by centrifugation at 600 × g for 10 min. The cell pellets were washed twice with ice-cold phosphate-buffered saline (pH 7.4) and resuspended with five volumes of buffer A (250 mm sucrose, 20 mm HEPES, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mmdithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, pH 7.5). The cells were homogenized in a glass Dounce homogenizer until ∼60% of the cells became Trypan blue-positive (60 strokes for HeLa cells). The homogenates were centrifuged twice at 750 ×g for 10 min at 4 °C. Supernatants were centrifuged at 10,000 × g for 15 min at 4 °C, and the resulting mitochondrial pellets were re-suspended in buffer A (13.Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4394) Google Scholar). In some cases mitochondria were further purified on Percol gradient (Amersham Biosciences, Inc.) as per the manufacturer's recommendations. The supernatants of the 10,000 × g spin were further centrifuged at 100,000 × g for 1 h at 4 °C, and the resulting supernatants (designated S-100) were frozen as aliquots at −80 °C for subsequent experiments. In some experiments Percol gradient-purified mouse liver mitochondria were used (37.Ellerby H.M. Martin S.J. Ellerby L.M. Naiem S.S. Rabizadeh S. Salvesen G.S. Casiano C.A. Cashman N.R. Green D.R. Bredesen D.E. J. Neurosci. 1997; 17: 6165-6178Crossref PubMed Google Scholar). A 5-μl aliquot of mitochondria (2 μg/μl) was incubated with 10 μg of cytosolic extract (S-100) and recombinant caspase-2 or caspase-8, in a final volume of 25 μl of buffer A at 37 °C for 1 h. The reaction mixture was then centrifuged at 12,000 × g for 10 min at 4 °C to pellet the mitochondria. Supernatants and the pellets were subjected to SDS-PAGE and immunoblotting. Similar procedures were used to assay the release of Smac/Diablo and AIF. To gain a better understanding of how caspase-2 and its adaptor molecule CRADD engage the death pathway, we investigated their apoptotic and procaspase-3 processing ability in the presence of various apoptosis inhibitors. Among the inhibitors used, Bcl-2 and Bcl-xL inhibit apoptosis by blocking release of Cyt c and other proapoptotic factors from the mitochondria, whereas caspase-9-DN (active site cysteine 287 to alanine) interferes with formation of a functional Apaf-1·caspase-9 complex by a dominant negative mechanism. As shown in Fig. 1A, CRADD- and caspase-2-induced apoptosis in MCF7-Fas cells was efficiently blocked by Bcl-2, Bcl-xL, and caspase-9-DN (Fig. 1A), suggesting that they require the participation of the mitochondria and the Apaf-1·caspase-9 complex to trigger activation of the apoptotic pathway. To determine whether overexpression of caspase-2 and CRADD can induce processing of procaspase-3, 293T cells were transiently transfected with CRADD and caspase-2 expression plasmids together with or without expression plasmids encoding Bcl-xL or caspase-9-DN. The cell lysates were immunoblotted with antibody against the processed caspase-3-p20 subunit. As expected, overexpression of caspase-2 and CRADD induced processing/activation of procaspase-3 (Fig. 1B, lanes 2 and 3). This processing was completely blocked when 4-fold excess of caspase-9-DN or Bcl-xL was coexpressed together with CRADD or caspase-2 (Fig. 1B, lanes 5,6 and lanes 8, 9). These observations are consistent with the result in Fig. 1A, confirming the importance of the mitochondrial pathway in activation of the effector caspases by CRADD and caspase-2. Caspase-8 can process and activate most known caspase zymogens (procaspases) (11.Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (481) Google Scholar). To compare the relative activities of caspase-2 and caspase-8 toward other known caspase zymogens, recombinant caspase-2 and caspase-8, were expressed in bacteria and then affinity-purified. Expression of casapase-2 and -8 zymogens in bacteria allowed autoactivation of the two zymogens to generate the two processed subunits of the mature caspase-2 and caspase-8 (here referred to as caspase-2 and caspase-8) (Fig. 2A). Equal amounts of caspase-2 and caspase-8 were incubated with a panel of in vitro-translated [35S]methionine-labeled caspase zymogens, and the reactions were then analyzed by SDS-PAGE and autoradiography. Unlike caspase-8, caspase-2 was not able to process zymogens of effector caspases 3, 6, or 7, or initiator caspases 8, 9, or 10. However, caspase-2 was able to process its own zymogen and the baculovirus IAP inhibitor, p35 (Fig. 2, B and C). This suggests that caspase-2 could execute the apoptotic program independent of other effector or initiator caspases, or by activating another unknown caspase. Alternatively, caspase-2 could activate the caspase cascade indirectly via the mitochondrial pathway by causing release of Cyt c and other apoptotic factors into the cytoplasm. To examine whether caspase-2 induces Cyt c release from the mitochondria, purified mitochondria from HeLa cells were incubated with purified caspase-2 in vitro with or without cytosolic extracts. Caspase-8 was used at the same time as a control. When mitochondria were incubated with cytosol alone, almost no Cytc release was observed in the supernatant, indicating that the mitochondrial preparation was not releasing Cyt cnonspecifically (Fig. 3A,lane 2). However, when mitochondria were incubated with cytosol and increasing amounts of caspase-2, an increase in the amount of Cyt c in the supernatant was detected by immunoblotting (Fig. 3A, lanes 3–7). The same results were obtained with caspase-8 (Fig. 3A, lanes 8–12). The amount of Cyt c released into the supernatant by a fixed amount of caspase-2 or caspase-8 was also increased by increasing the amount of the S-100 cytosol (Fig. 3B). This suggests that both caspases are capable of inducing Cyt c release, presumably by proteolytically activating one or more cytosolic factors. Surprisingly, when the S-100 cytosol was omitted from the reaction mixture, caspase-2, but not caspase-8, was still able to induce Cytc release (Fig. 3, A and B, comparelanes 13 and 14), suggesting that one or more of the presumable cytosolic factors are dispensable for stimulation of mitochondria by caspase-2 but not by caspase-8. Bid is a proapoptotic Bcl-2 family member that triggers Cyt c release from mitochondria after proteolytic cleavage by caspase-8 (21.Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3067) Google Scholar, 22.Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3771) Google Scholar). Careful examination of the amino acid sequence of Bid identified two potential caspase cleavage sites that match the preferred cleavage sites for caspase-2 (38.Thornberry N.A. Rosen A. Nicholson D.W. Adv. Pharmacol. 1997; 41: 155-177Crossref PubMed Scopus (76) Google Scholar). We examined the ability of caspase-2, compared with caspase-8, to cleave Bid. In vitro translated 35S-labeled wild type Bid (WT), Bid D59E mutant (D59E), or Bid D59E/D75E double mutant (DM) were incubated with recombinant caspase-2 or caspase-8. As shown in Fig. 4A, both caspase-2 and caspase-8 were able to cleave WT Bid, but not the D59E or DM mutants, into two small fragments almost to the same extent. Based on these results, caspase-2 can efficiently cleave Bid at Asp-59, suggesting that caspase-2 induces Cyt c release by proteolytically activating Bid. To determine whether Bid is necessary for caspase-2- or caspase-8-induced Cyt c release from the mitochondria, endogenous Bid was immunodepleted from HeLa cell S-100 extracts before incubating with caspase-2 or -8 and mitochondria. As expected, depletion of Bid from the S100 extracts totally abolished the Cytc-releasing ability of caspase-8 (Fig. 4B, compare lane 3 with lane 7). However, depletion of Bid decreased, but did not totally inhibit the Cyt creleasing ability of caspase-2 (Fig. 4B, compare lane 4 with lane 8). This suggests that Bid may not be critical for caspase-2-induced Cyt c release but its presence could enhance this caspase-2 activity. Cyt c, AIF, and Smac are normally localized in the intermembrane space of the mitochondria (27.Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. C
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