Purification and Characterization of an Interleukin-1β-converting Enzyme Family Protease That Activates Cysteine Protease P32 (CPP32)
1996; Elsevier BV; Volume: 271; Issue: 23 Linguagem: Inglês
10.1074/jbc.271.23.13371
ISSN1083-351X
AutoresXuesong Liu, Caryn Naekyung Kim, Jan Pohl, Xiaodong Wang,
Tópico(s)Signaling Pathways in Disease
ResumoCPP32, a member of the interleukin-1β-converting enzyme (ICE) family of cysteine proteases, cleaves poly(ADP-ribose) polymerase and sterol regulatory element binding proteins during apoptosis. CPP32 normally exists in the cytosol as a 32-kDa inactive precursor and only becomes activated when cells are undergoing apoptosis. The activation is a proteolytic event that generates a p20/p11 heterodimer. We report here the identification, purification, and characterization of a hamster CPP32-activating protease (CAP) that cleaves and activates CPP32. The biochemical properties of CAP suggest that it is another member of the ICE family of proteases. Purified CAP consists of two prominent polypeptides of 19 and 13 kDa. Protein sequencing revealed that CAP is derived from the hamster homolog of Mch2α, a member of the ICE family recently identified based on the sequence conservation among the ICE family members. CAP activity is inhibited by CrmA, a cowpox virus protein that prevents host cell apoptosis. CAP itself is also activated through proteolytic cleavage. These data are consistent with the idea that the activation of the ICE family of proteases during apoptosis proceeds through a cascade of proteolytic events. CPP32, a member of the interleukin-1β-converting enzyme (ICE) family of cysteine proteases, cleaves poly(ADP-ribose) polymerase and sterol regulatory element binding proteins during apoptosis. CPP32 normally exists in the cytosol as a 32-kDa inactive precursor and only becomes activated when cells are undergoing apoptosis. The activation is a proteolytic event that generates a p20/p11 heterodimer. We report here the identification, purification, and characterization of a hamster CPP32-activating protease (CAP) that cleaves and activates CPP32. The biochemical properties of CAP suggest that it is another member of the ICE family of proteases. Purified CAP consists of two prominent polypeptides of 19 and 13 kDa. Protein sequencing revealed that CAP is derived from the hamster homolog of Mch2α, a member of the ICE family recently identified based on the sequence conservation among the ICE family members. CAP activity is inhibited by CrmA, a cowpox virus protein that prevents host cell apoptosis. CAP itself is also activated through proteolytic cleavage. These data are consistent with the idea that the activation of the ICE family of proteases during apoptosis proceeds through a cascade of proteolytic events. INTRODUCTIONCPP32, 1The abbreviations used are: CAPCPP32-activating proteaseICEinterleukin-1β-converting enzymeCPP32cysteine protease P32PARPpoly(ADP-ribose) polymeraseSREBPsterol regulatory element binding proteinNEMN-ethylmaleimideIAAiodoacetamideDTTdithiothreitolPAGEpolyacrylamide gel electrophoresisAc-DEAD-CHOAc-Asp-Glu-Ala-Asp-aldehydeAc-YVAD-CHOAc-Tyr-Val-Ala-Asp-aldehydePCRpolymerase chain reactionPMSFphenylmethylsulfonyl fluoride. an interleukin-1β-converting enzyme (ICE)-like cysteine protease, has been implicated in the pathway of apoptosis in mammalian cells based on several observations. First, CPP32 is closely related to an apoptosis promoting gene (ced-3 of Caenorhabditis elegans) in terms of both sequence similarity and substrate specificity (1Fernandes-Alnemri T. Litwack G. Alnemri E.S. J. Biol. Chem. 1994; 269: 30761-30764Abstract Full Text PDF PubMed Google Scholar, 2Xue D. Horvitz H.R. Nature. 1995; 377: 248-251Crossref PubMed Scopus (436) Google Scholar). Second, CPP32 activity is markedly elevated in cells undergoing apoptosis induced by a variety of reagents (3Nicholson W.D. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulsom M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3777) Google Scholar, 4Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar, 5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). Third, a cowpox virus protein CrmA and a baculovirus protein p35, both of which prevent cells from undergoing apoptosis, inhibit the activity of CPP32 (2Xue D. Horvitz H.R. Nature. 1995; 377: 248-251Crossref PubMed Scopus (436) Google Scholar, 4Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar, 6Bump N.J. Hackett M. Hugunin M. Seshagiri S. Brady K. Chen P. Ferenz C. Franklin S. Ghayur T. Li P. Licari J. Mankovich L. Shi L. Greenberg A.H. Miller L.K. Wong W.W. Science. 1995; 269: 1885-1888Crossref PubMed Scopus (600) Google Scholar). Finally, a tetrapeptide aldehyde inhibitor that specifically inhibits CPP32 activity also blocks the ability of cytosol from apoptotic cells to induce apoptosis-like changes in normal nuclei in vitro. (3Nicholson W.D. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulsom M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3777) Google Scholar).When activated, CPP32 specifically cleaves poly(ADP-ribose) polymerase (PARP) (3Nicholson W.D. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulsom M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3777) Google Scholar, 4Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar) and sterol regulatory element binding proteins (SREBPs) (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar, 7Wang X. Pai J. Wiedenfeld E.A. Medina J.C. Slaughter C.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). PARP is an enzyme implicated in DNA repair and genome surveillance and integrity. The proteolytic cleavage of PARP during the onset of apoptosis by CPP32 results in the separation of its DNA binding and catalytic domains. This cleavage prevents the catalytic domain of PARP from being recruited to sites of DNA damage and presumably disables the ability of PARP to coordinate subsequent repair and genome maintenance events (3Nicholson W.D. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulsom M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3777) Google Scholar). Furthermore, the Ca2+/Mg2+- dependent endonuclease implicated in the internucleosomal DNA cleavage, a hallmark of apoptosis, is negatively regulated by polyADP-ribosylation, and this inhibition may be retrieved when PARP is cleaved (8Tanaka Y. Yoshihara K. Itaya A. Kamiya T. Koide S.S. J. Biol. Chem. 1984; 259: 6579-6585Abstract Full Text PDF PubMed Google Scholar). SREBPs are a family of transcription factors that stimulate transcription of genes involved in cholesterol and fatty acid metabolism, including the low density lipoprotein receptor, 3-hydroxy-3-methylglutaryl-CoA synthase, and fatty acid synthase genes (9Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (783) Google Scholar, 10Hua X. Yokoyama C. Wu J. Briggs M.R. Brown M.S. Goldstein J.L. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11603-11607Crossref PubMed Scopus (499) Google Scholar, 11Bennett M.K. Lopez J.M. Sanchez H.B. Osborne T.F. J. Biol. Chem. 1995; 270: 25578-25583Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 12Kim J.B. Spotts G.D. Halvorsen Y.-D. Shih H.-M. Ellenberger T. Towle H.C. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 2582-2588Crossref PubMed Scopus (295) Google Scholar). The amino-terminal halves of SREBPs are bona fide basic helix-loop-helix zipper transcription factors. Unlike any other transcription factors, they are linked to extended carboxyl-terminal halves by two trans-membrane domains that anchor the proteins to the membranes of the endoplasmic reticulum and nuclear envelope. In cells starved for cholesterol or undergoing apoptosis, a proteolytic cleavage event between the leucine zipper and the membrane attachment region frees the amino-terminal fragment which enters the nucleus and activates its target genes (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar, 13Wang X. Sato R. Brown S.M. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (851) Google Scholar). The CPP32-mediated cleavage of SREBPs during apoptosis is not regulated by cellular cholesterol content and occurs at a different site compared with that of sterol-regulated proteolysis (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). The physiological function of activated SREBPs in apoptotic cells is still obscure. Nevertheless, since the activated SREBPs should have a profound impact on cellular lipid metabolism, it has been speculated that cleavage of SREBPs by CPP32 during apoptosis is involved in preserving the cytoplasmic membrane integrity of apoptotic cells, and/or preparing the membrane for phagocytosis (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar).The ability of activated CPP32 to trigger apoptosis implies that cells must have a tight mechanism to control this activation to prevent unwanted cell death. CPP32 is activated by multiple proteolytic cleavages at aspartic acid residues. The eventual result is cleavage of the 32-kDa precursor into the 20/11-kDa active form (3Nicholson W.D. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulsom M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3777) Google Scholar, 5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). The mechanism that triggers CPP32 activation is not known. Partially purified active CPP32 from HeLa cell extracts was able to cleave the CPP32 precursor in vitro (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). This reaction was partially, but not completely, inhibited by a CPP32-specific tetrapeptide inhibitor, suggesting autocatalytic activation as well as the existence of another activating enzyme (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). ICE has also been shown to be able to cleave and activate CPP32 in vitro (4Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar). However, since ICE knockout mice have no general defects in apoptosis (14Li P. Allen H. Benerjee S. Franklin S. Herzog L. Johnston C. McDowell J. Paskind M. Rodman L. Salfeld J. Towne E. Tracey D. Wardwell S. Wei F.-Y. Wong W. Kamen R. Seshadri T. Cell. 1995; 80: 401-411Abstract Full Text PDF PubMed Scopus (1299) Google Scholar, 15Kuida K. Lippke J.A. Ku G. Harding M.W. Livingston D.J. Su M.S.-S. Flavell R.A. Science. 1995; 276: 2000-2003Crossref Scopus (1443) Google Scholar), ICE does not appear to be a general mediator of apoptosis. Accordingly, since CPP32 is activated by cleavage at aspartic acid residues, a hallmark of ICE-like proteases (16Thornberry N.A. Molineaux S.M. Protein Sci. 1995; 4: 3-12Crossref PubMed Scopus (152) Google Scholar), a cascade of ICE-like proteolytic cleavages leading to apoptosis has been proposed (4Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar, 5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). Such a protease cascade would provide both the regulation and signal amplification necessary for a highly controlled yet rapid and irreversible process of apoptosis.In this paper, we report the identification and purification of a CPP32-activating protease (CAP) from hamster liver extract. This enzyme specifically cleaves and activates CPP32. The biochemical properties of this protease suggest that it is an ICE-like cysteine protease distinct from ICE and CPP32, the two enzymes that have previously been implicated in the activation of CPP32 (4Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar, 5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). The protein sequence of purified CAP revealed that CAP is derived from the hamster homolog of Mch2α, a member of the ICE family recently cloned by PCR based on sequence conservation of the ICE family (17Fernandes-Alnemri T. Litwack G. Alnemri E.S. Cancer Res. 1995; 55: 2737-2742PubMed Google Scholar). Mch2α may represent the upstream protease acting on CPP32 and may initiate the ICE-like protease cascade leading to apoptosis. We also find that CAP activity is more sensitive to inhibition by CrmA than is CPP32, defining a new and more efficient target for CrmA blockage of the onset of apoptosis.EXPERIMENTAL PROCEDURESGeneral Methods and MaterialsWe obtained male Golden Syrian hamsters (~150 g) from Sasco (Omaha, NE); [35S]methionine was from Amersham Corp.; N-ethylmaleimide (NEM), iodoacetamide (IAA), phenylmethylsulfonyl fluoride (PMSF), imidazole, and aprotinin were from Sigma; Ac-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) were from Bachem Bioscience, Inc.; Ac-Asp-Glu-Ala-Asp-aldehyde (Ac-DEAD-CHO) was from Julio C. Medina of Tularik, Inc. (7Wang X. Pai J. Wiedenfeld E.A. Medina J.C. Slaughter C.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar); molecular weight standards for SDS-PAGE and gel-filtration chromatography were from Bio-Rad. cDNA clones of human SREBP-2 and hamster CPP32 were described in the indicated references. HeLa cell cytosol was prepared as described (13Wang X. Sato R. Brown S.M. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (851) Google Scholar). Protein concentration was determined by the Bradford method. Silver staining was carried out using a Silver Stain Plus kit from Bio-Rad. Plasmids were purified using a Megaprep kit from Qiagen.In Vitro Translation of SREBP-2, CPP32, and CrmASREBP-2 was translated in a TNT SP6 transcription/translation kit from Promega as described (7Wang X. Pai J. Wiedenfeld E.A. Medina J.C. Slaughter C.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). A PCR fragment encoding amino acids 29-277 of hamster CPP32 (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar) was cloned into NdeI and BamHI sites of pET 15b vector (Novagen). The resulting fusion protein of six histidines with hamster CPP32 (amino acids 29-277) was translated in a TNT T7 transcription/translation kit in the presence of [35S]methionine according to the manufacturer's instruction. The translated protein was passed through a 1-ml nickel affinity column (Qiagen) equilibrated with buffer A (20 mM Hepes-KOH, pH 6.8, 10 mM KCl, 1.5 mM MgCl2, 1 mM Na-EDTA, 1 mM Na-EGTA, 1 mM dithiothreitol (DTT), and 0.1 mM PMSF). After washing the column with 10 ml of buffer A, the translated CPP32 was eluted with buffer A containing 250 mM imidazole. CrmA cDNA was cloned into the EcoRI site of pBK-CMV vector (Stratagene) and translated in a TNT T3 transcription/translation kit in the presence of [35S]methionine. The translated CrmA (200 µl) was purified by passing the translation mixture through a 10-ml Sephadex G-25 gel-filtration column equilibrated with buffer A. The translated proteins contained within the exclusion volume of the column were collected.Assay for CPP32-activating Protease10 µl of purified, 35S-labeled, in vitro translated hamster CPP32 was incubated at 37°C for various times with the indicated enzyme fractions in a final volume of 25 µl of buffer A. At the end of the incubation, SDS sample buffer was added to each tube. After boiling for 3 min, the samples were subjected to 15% SDS-PAGE. The gel was subsequently transferred to a nitrocellulose filter and exposed to a Kodak X-Omat AR x-ray film.Purification of CAP from Hamster LiverAll purification steps were carried out at 4°C. All the chromatography steps except the SP-Sepharose column were carried out using an automatic fast protein liquid chromatography station (Pharmacia Biotech Inc.).Step 1: Preparation of S-100 FractionLivers from 25 hamsters were rinsed twice with cold buffer A and homogenized for 45 s in the same buffer (0.5 g/ml) in a Waring blender followed by three strokes of a motor-driven homogenizer. The homogenates were centrifuged at 105× g for 1 h in a SW 28 rotor (Beckman). The resulting supernatant (S-100) fraction was dialyzed overnight against three changes of 4 liters of buffer A.Step 2: SP-Sepharose ChromatographyThe S-100 fraction from step 1 was applied to a SP-Sepharose column (200 ml) equilibrated with buffer A. The flow-through fraction was supplemented with DTT to a final concentration of 10 mM and incubated at 30°C for 2 h before being loaded onto a fresh SP-Sepharose column (100 ml) equilibrated with buffer A. After washing with 4-column volumes of buffer A, the column was eluted with buffer A containing 400 mM NaCl.Step 3: Ammonium Sulfate PrecipitationSolid ammonium sulfate was added to the SP-Sepharose column eluate (150 ml) to 40% saturation. After stirring for 1 h, the precipitate was collected by centrifigation at 15,000 rpm in a JA 20 rotor (Beckman) for 20 min. The pellet was resuspended in 10 ml of buffer A.Step 4: Superdex-200 Gel-filtration ChromatographyThe resuspended ammonium sulfate pellet was loaded onto a Superdex-200 gel filtration column (Phamacia, 300 ml) equilibrated with buffer A. The column was eluted with the same buffer, and fractions of 10 ml were collected and assayed for CAP activity.Step 5: Mono Q ChromatographyThe active fractions from Superdex 200 column were pooled and loaded onto a Mono Q 5/5 column after adjusting the pH to 7.6 with 1 M KOH. The column was equilibrated with buffer B (buffer A adjusted to pH 7.6) and eluted with buffer B containing 400 mM NaCl. The flow-through and the bound materials were assayed for CAP activity.Step 6: Heat TreatmentThe flow-through fraction from the Mono Q column was adjusted to pH 6.8 with 1 N HCl followed by incubation at 55°C for 15 min. The denatured proteins were pelleted by centrifigation at 15,000 rpm in a JA 20 rotor for 20 min.Step 7: Mono S ChromatographyThe supernatant after heat treatment was loaded onto a Mono S 5/5 column equilibrated with buffer A. The column was eluted with a 20-ml linear salt gradient of buffer A to buffer A containing 440 mM NaCl. Fractions of 1 ml were collected and assayed for CAP activity.NH2-terminal Sequencing Analysis of CAPThe CAP peak fractions from Mono S column (step 7) were subjected to electrophoresis in a 15% SDS gel and then transferred onto a piece of poly(vinylidene difluoride) membrane (Millipore). The 19- and 13-kDa subunits were visualized by Coomassie Blue staining and excised for direct NH2-terminal sequencing on a sequencer (Biosystem).Western Blot AnalysisA polyclonal antibody against hamster CPP32 was produced as described (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). A monoclonal antibody against human CPP32 was purchased from Transduction Laboratories. Immunoblot analysis of CPP32 was performed with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G using Enhanced Chemiluminescence Western blotting detection reagents (Amersham Corp.) as described previously (13Wang X. Sato R. Brown S.M. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (851) Google Scholar).Expression and Purification of Recombinant His6-tagged CPP32 and CrmAThe plasmid containing fusion protein of six histidine and hamster CPP32 (amino acid 29-277) was the same as used for the in vitro translation described above. The plasmid was transformed into DE3 competent cells (Novagen). The entire coding sequence of CrmA was PCR-amplified from the plasmid p996 containing cowpox virus crmA gene and cloned into the SalI and HindIII sites of pQE 30 vector (Qiagen). The plasmid was transformed into the M15 competent cells (Qiagen). The bacteria cultures (1 liter for each plasmid) were grown at 37°C until the density reach A600 reading of 0.6. Isopropyl-1-thio-β-D-galactopyranoside was then added to the final concentration of 2 mM. After 1-h induction, the bacteria were pelleted and lysed in buffer A through sonication. After centrifigation, the supernatants were loaded onto two 3-ml nickel-Sepharose (Qiagen) columns equilibrated with buffer A. The columns were washed with 10 ml of buffer A, followed by 10 ml of buffer A containing 500 mM NaCl, and again with 10 ml of buffer A. The fusion proteins were eluted with buffer A containing 250 mM imidazole. The peak protein fractions of the nickel column eluates were further purified through a FPLC Superdex-200 16/30 column equilibrated with buffer A, and 1-ml fractions were collected. The column fractions of the recombinant CPP32 were assayed for its enzymatic activity by incubating with 35S-labeled SREBP-2 as described previously (7Wang X. Pai J. Wiedenfeld E.A. Medina J.C. Slaughter C.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The purity of recombinant CPP32 enzyme and CrmA were analyzed by SDS-PAGE followed by silver staining. INTRODUCTIONCPP32, 1The abbreviations used are: CAPCPP32-activating proteaseICEinterleukin-1β-converting enzymeCPP32cysteine protease P32PARPpoly(ADP-ribose) polymeraseSREBPsterol regulatory element binding proteinNEMN-ethylmaleimideIAAiodoacetamideDTTdithiothreitolPAGEpolyacrylamide gel electrophoresisAc-DEAD-CHOAc-Asp-Glu-Ala-Asp-aldehydeAc-YVAD-CHOAc-Tyr-Val-Ala-Asp-aldehydePCRpolymerase chain reactionPMSFphenylmethylsulfonyl fluoride. an interleukin-1β-converting enzyme (ICE)-like cysteine protease, has been implicated in the pathway of apoptosis in mammalian cells based on several observations. First, CPP32 is closely related to an apoptosis promoting gene (ced-3 of Caenorhabditis elegans) in terms of both sequence similarity and substrate specificity (1Fernandes-Alnemri T. Litwack G. Alnemri E.S. J. Biol. Chem. 1994; 269: 30761-30764Abstract Full Text PDF PubMed Google Scholar, 2Xue D. Horvitz H.R. Nature. 1995; 377: 248-251Crossref PubMed Scopus (436) Google Scholar). Second, CPP32 activity is markedly elevated in cells undergoing apoptosis induced by a variety of reagents (3Nicholson W.D. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulsom M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3777) Google Scholar, 4Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar, 5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). Third, a cowpox virus protein CrmA and a baculovirus protein p35, both of which prevent cells from undergoing apoptosis, inhibit the activity of CPP32 (2Xue D. Horvitz H.R. Nature. 1995; 377: 248-251Crossref PubMed Scopus (436) Google Scholar, 4Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar, 6Bump N.J. Hackett M. Hugunin M. Seshagiri S. Brady K. Chen P. Ferenz C. Franklin S. Ghayur T. Li P. Licari J. Mankovich L. Shi L. Greenberg A.H. Miller L.K. Wong W.W. Science. 1995; 269: 1885-1888Crossref PubMed Scopus (600) Google Scholar). Finally, a tetrapeptide aldehyde inhibitor that specifically inhibits CPP32 activity also blocks the ability of cytosol from apoptotic cells to induce apoptosis-like changes in normal nuclei in vitro. (3Nicholson W.D. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulsom M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3777) Google Scholar).When activated, CPP32 specifically cleaves poly(ADP-ribose) polymerase (PARP) (3Nicholson W.D. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulsom M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3777) Google Scholar, 4Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar) and sterol regulatory element binding proteins (SREBPs) (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar, 7Wang X. Pai J. Wiedenfeld E.A. Medina J.C. Slaughter C.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). PARP is an enzyme implicated in DNA repair and genome surveillance and integrity. The proteolytic cleavage of PARP during the onset of apoptosis by CPP32 results in the separation of its DNA binding and catalytic domains. This cleavage prevents the catalytic domain of PARP from being recruited to sites of DNA damage and presumably disables the ability of PARP to coordinate subsequent repair and genome maintenance events (3Nicholson W.D. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulsom M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3777) Google Scholar). Furthermore, the Ca2+/Mg2+- dependent endonuclease implicated in the internucleosomal DNA cleavage, a hallmark of apoptosis, is negatively regulated by polyADP-ribosylation, and this inhibition may be retrieved when PARP is cleaved (8Tanaka Y. Yoshihara K. Itaya A. Kamiya T. Koide S.S. J. Biol. Chem. 1984; 259: 6579-6585Abstract Full Text PDF PubMed Google Scholar). 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The amino-terminal halves of SREBPs are bona fide basic helix-loop-helix zipper transcription factors. Unlike any other transcription factors, they are linked to extended carboxyl-terminal halves by two trans-membrane domains that anchor the proteins to the membranes of the endoplasmic reticulum and nuclear envelope. In cells starved for cholesterol or undergoing apoptosis, a proteolytic cleavage event between the leucine zipper and the membrane attachment region frees the amino-terminal fragment which enters the nucleus and activates its target genes (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar, 13Wang X. Sato R. Brown S.M. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (851) Google Scholar). The CPP32-mediated cleavage of SREBPs during apoptosis is not regulated by cellular cholesterol content and occurs at a different site compared with that of sterol-regulated proteolysis (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). The physiological function of activated SREBPs in apoptotic cells is still obscure. Nevertheless, since the activated SREBPs should have a profound impact on cellular lipid metabolism, it has been speculated that cleavage of SREBPs by CPP32 during apoptosis is involved in preserving the cytoplasmic membrane integrity of apoptotic cells, and/or preparing the membrane for phagocytosis (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar).The ability of activated CPP32 to trigger apoptosis implies that cells must have a tight mechanism to control this activation to prevent unwanted cell death. CPP32 is activated by multiple proteolytic cleavages at aspartic acid residues. The eventual result is cleavage of the 32-kDa precursor into the 20/11-kDa active form (3Nicholson W.D. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulsom M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3777) Google Scholar, 5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). The mechanism that triggers CPP32 activation is not known. Partially purified active CPP32 from HeLa cell extracts was able to cleave the CPP32 precursor in vitro (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). This reaction was partially, but not completely, inhibited by a CPP32-specific tetrapeptide inhibitor, suggesting autocatalytic activation as well as the existence of another activating enzyme (5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). ICE has also been shown to be able to cleave and activate CPP32 in vitro (4Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar). However, since ICE knockout mice have no general defects in apoptosis (14Li P. Allen H. Benerjee S. Franklin S. Herzog L. Johnston C. McDowell J. Paskind M. Rodman L. Salfeld J. Towne E. Tracey D. Wardwell S. Wei F.-Y. Wong W. Kamen R. Seshadri T. 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Such a protease cascade would provide both the regulation and signal amplification necessary for a highly controlled yet rapid and irreversible process of apoptosis.In this paper, we report the identification and purification of a CPP32-activating protease (CAP) from hamster liver extract. This enzyme specifically cleaves and activates CPP32. The biochemical properties of this protease suggest that it is an ICE-like cysteine protease distinct from ICE and CPP32, the two enzymes that have previously been implicated in the activation of CPP32 (4Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar, 5Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO. J. 1996; 15: 1012-1020Crossref PubMed Scopus (295) Google Scholar). The protein sequence of purified CAP revealed that CAP is derived from the hamster homolog of Mch2α, a member of the ICE family recently cloned by PCR based on sequence conservation of the ICE family (17Fernandes-Alnemri T. Litwack G. Alnemri E.S. Cancer Res. 1995; 55: 2737-2742PubMed Google Scholar). Mch2α may represent the upstream protease acting on CPP32 and may initiate the ICE-like protease cascade leading to apoptosis. We also find that CAP activity is more sensitive to inhibition by CrmA than is CPP32, defining a new and more efficient target for CrmA blockage of the onset of apoptosis.
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