Establishing a Blueprint for CED-3-dependent Killing through Identification of Multiple Substrates for This Protease

Abstract

Genetic studies have established that the cysteine protease CED-3 plays a central role in coordinating programmed cell death in Caenorhabditis elegans. However, it remains unclear how CED-3 activation results in cell death because few substrates for this protease have been described. We have used a global proteomics approach to seek substrates for CED-3 and have identified 22 worm proteins that undergo CED-3-dependent proteolysis. Proteins that were found to be substrates for CED-3 included the cytoskeleton proteins actin, myosin light chain, and tubulin, as well as proteins involved in ATP synthesis, cellular metabolism, and chaperone function. We estimate that ∼3% of the C. elegans proteome is susceptible to CED-3-dependent proteolysis. Notably, the endoplasmic reticulum chaperone calreticulin, which has been implicated in the recognition of apoptotic cells by phagocytes, was cleaved by CED-3 and was also cleaved by human caspases during apoptosis. Inhibitors of caspase activity blocked the appearance of calreticulin on the surface of apoptotic cells, suggesting a mechanism for the surface display of calreticulin during apoptosis. Further analysis of these substrates is likely to yield important insights into the mechanism of killing by CED-3 and its human caspase counterparts. Genetic studies have established that the cysteine protease CED-3 plays a central role in coordinating programmed cell death in Caenorhabditis elegans. However, it remains unclear how CED-3 activation results in cell death because few substrates for this protease have been described. We have used a global proteomics approach to seek substrates for CED-3 and have identified 22 worm proteins that undergo CED-3-dependent proteolysis. Proteins that were found to be substrates for CED-3 included the cytoskeleton proteins actin, myosin light chain, and tubulin, as well as proteins involved in ATP synthesis, cellular metabolism, and chaperone function. We estimate that ∼3% of the C. elegans proteome is susceptible to CED-3-dependent proteolysis. Notably, the endoplasmic reticulum chaperone calreticulin, which has been implicated in the recognition of apoptotic cells by phagocytes, was cleaved by CED-3 and was also cleaved by human caspases during apoptosis. Inhibitors of caspase activity blocked the appearance of calreticulin on the surface of apoptotic cells, suggesting a mechanism for the surface display of calreticulin during apoptosis. Further analysis of these substrates is likely to yield important insights into the mechanism of killing by CED-3 and its human caspase counterparts. The caspase family of cysteine proteases have been implicated in the regulation of programmed cell death in numerous organisms, from simple invertebrates to man (1Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar). Caspases are present in most cells as inactive proenzymes and become activated during apoptosis through diverse signaling pathways (2Martin S.J. Green D.R. Cell. 1995; 82: 349-352Abstract Full Text PDF PubMed Scopus (1256) Google Scholar, 3Stennicke H.R. Salvesen G.S. Cell Death Differ. 1999; 6: 1054-1059Crossref PubMed Scopus (152) Google Scholar). Upon activation, caspases promote programmed cell death by restricted proteolysis of numerous proteins within the dying cell (4Fischer U. Janicke R.U. Schulze-Osthoff K. Cell Death Differ. 2003; 10: 76-100Crossref PubMed Scopus (875) Google Scholar). Caspase-dependent proteolytic events produce dramatic rearrangements of the cellular architecture and engulfment of the dying cell by phagocytes (5Savill J. Fadok V. Nature. 2000; 407: 784-788Crossref PubMed Scopus (1266) Google Scholar). The nematode Caenorhabditis elegans has contributed greatly to our understanding of the regulation of cell death in metazoans through discovery of a series of cell death defective (ced) genes (6Ellis R.E. Horvitz H.R. Cell. 1986; 44: 817-829Abstract Full Text PDF PubMed Scopus (1332) Google Scholar, 7Lettre G. Hengartner M.O. Nat. Rev. Mol. Cell. Biol. 2006; 7: 97-108Crossref PubMed Scopus (221) Google Scholar). In several cases, this led directly to the discovery of a role for their mammalian counterparts in the regulation of apoptosis, the caspases being a notable example (8Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2229) Google Scholar, 9Miura M. Zhu H. Rotello R. Hartwieg E.A. Yuan J. Cell. 1993; 175: 653-660Abstract Full Text PDF Scopus (1325) Google Scholar, 10Nicholson D.W. 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. Smulson M.E. Yamin T.T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3768) Google Scholar). The worm caspase, CED-3, has been established as essential for the great majority of developmentally related programmed cell deaths in the worm (6Ellis R.E. Horvitz H.R. Cell. 1986; 44: 817-829Abstract Full Text PDF PubMed Scopus (1332) Google Scholar, 11Shaham S. Reddien P.W. Davies B. Horvitz H.R. Genetics. 1999; 153: 1655-1671Crossref PubMed Google Scholar). Similar to mammalian caspases, CED-3 is probably present in most cells as an inactive precursor but becomes activated in cells fated to die during development. CED-3 activation is achieved through recruitment of the inactive CED-3 precursor to the scaffold protein CED-4, which assembles into a tetrameric complex (12Yan N. Chai J. Lee E.S. Gu L. Liu Q. He J. Wu J.W. Kokel D. Li H. Hao Q. Xue D. Shi Y. Nature. 2005; 437: 831-837Crossref PubMed Scopus (175) Google Scholar). The latter event is normally prevented by CED-9, which represses CED-4 tetramerization and CED-3 recruitment (12Yan N. Chai J. Lee E.S. Gu L. Liu Q. He J. Wu J.W. Kokel D. Li H. Hao Q. Xue D. Shi Y. Nature. 2005; 437: 831-837Crossref PubMed Scopus (175) Google Scholar, 13Hengartner M.O. Ellis R.E. Horvitz H.R. Nature. 1992; 356: 494-499Crossref PubMed Scopus (707) Google Scholar). However, although CED-3 is firmly established as a central participant in the molecular control of programmed cell death in the worm, how activation of this protease results in cell death is obscure because few substrates for CED-3 have been identified thus far. Understanding how CED-3 kills cells in the worm is of considerable interest, not only because this is likely to shed further light upon how programmed cell death is coordinated in this organism, but because this is also likely to generate insight into how human caspases promote apoptosis. The observation that a single caspase is sufficient to coordinate apoptosis in the worm suggests that CED-3 harbors all of the activities that have subsequently become allocated to several different caspases in higher organisms. Here we have used two-dimensional gel-based proteomics to seek substrates for CED-3. This approach yielded 22 substrates and included proteins involved in protein and DNA synthesis, chaperone function, cellular metabolism, and the cytoskeleton. We estimate that ∼3% of the worm proteome is susceptible to CED-3-dependent proteolysis. These data suggest that CED-3 promotes cell death through simultaneously targeting diverse cellular functions. Detailed analysis of these substrates is likely to yield important insights into the mechanism of killing by CED-3. Reagents—The following antibodies were used for immunoblotting: anti-CED-3 C-20 (Santa Cruz), anti-GST (BD Pharmingen), anti-β-tubulin (ICN), anti-calreticulin polyclonal antibody (Affinity Bioreagents), and anti-calreticulin monoclonal antibody (Transduction Laboratories). Anti-calreticulin polyclonal antibody was also used for surface immunostaining in conjunction with anti-rabbit Alexafluor 488 as a secondary antibody (Molecular Probes). Rabbit polyclonal antibodies were generated against recombinant CED-3, as follows. Briefly, His-CED-3221-503 was expressed in Escherichia coli and purified over nickel-nitrilotriacetic acid-agarose, and rabbits were immunized with the purified protein. Polyclonal antibodies against p17 and p13 domain peptides of CED-3 were also generated and affinity-purified using the immunization peptides. The peptides z-VAD-fmk, 2The abbreviations used are: z, benzyloxycarbonyl; fmk, fluoromethyl ketone; DCI, 3,4-dichloroisocoumarin; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; FACS, fluorescence-activated cell sorter. Ac-DEVD-AFC, Ac-LEHD-AFC, Ac-IETD-AFC, and Ac-YVAD-AFC were purchased from Bachem. 17-cm immobilized pH gradient strips (pH 3-6 and pH 5-8), easy melt agarose and Bio-Lyte ampholytes™ were obtained from Bio-Rad. The serine protease inhibitors, tosylphenylalanylchloromethane, and 3,4-dichloroisocoumarin (DCI) were purchased from Merck. Unless otherwise indicated, all other reagents were purchased from Sigma. Plasmids and Site-directed Mutagenesis—The CED-3221-503 open reading frame was cloned from a pQE30-CED-3221-503 template (kindly provided by Dr. Shai Shaham) into the pGEX4TK2 vector to generate GST-tagged CED-33221-503. Crt-1, Tbb-2, and Ftt-2 open reading frames were amplified by PCR from a C. elegans cDNA library (Origene) and cloned into the pET45b vector. The human calreticulin open reading frame was amplified by PCR from an IMAGE clone (MRC) and inserted into pcDNA3. Site-directed mutagenesis was carried out using a QuikChange kit (Stratagene). All of the plasmids were verified by DNA sequencing. Expression and Purification of Recombinant CED-3—E. coli DH5α cells were transformed with pGEX4TK2-CED-3221-503. CED-3 protein expression was induced by the addition of 100 μm isopropyl β-d-thiogalactopyranoside to culture medium followed by a 3-h incubation at 25 °C. Proteins were affinity-purified on glutathione-Sepharose, as previously described, and eluted in protease reaction buffer (50 mm HEPES, pH 7.4, 75 mm NaC1, 0.1% CHAPS, 2 mm dithiothreitol) with 10 mm glutathione. CED-3 was then active site titrated against Ac-DEVD-AFC, using z-VAD-fmk as an inhibitor. Fluorimetry Assays—The Reactions containing recombinant GST-CED-3221-503 were typically assembled in a final volume of 100 μl, containing 50 μm Ac-DEVD-AFC, Ac-LEHD-AFC, Ac-IETD-AFC, or Ac-YVAD-AFC in protease reaction buffer. Fluorescence was then measured in an automated fluorimeter (Spectrafluor Plus; TECAN) at wavelengths of 430 nm (excitation) and 535 nm (emission). Coupled in Vitro Transcription/Translation—[35S]Methionine-labeled proteins were generated using the TnT kit (Promega) as described previously (35Martin S.J. Amarante-Mendes G.P. Shi L. Chuang T.H. Casiano C.A. O'Brien G.A. Fitzgerald P. Tan E.M. Bokoch G.M. Greenberg A.H. Green D.R. EMBO J. 1996; 15: 2407-2416Crossref PubMed Scopus (272) Google Scholar). Typically, 25-μl were assembled containing of template DNA and 2 of C. elegans were from a culture by at for were in 50 of 100 mm to of and for at were from the and in 100 mm a final at were in 2 of buffer mm pH 10 mm mm mm mm mm 100 μm 10 2 were by and was for at CED-3 was to C. elegans at nm by active site with otherwise The were for 2 at to CED-3-dependent proteolysis. were then by or to two-dimensional as described. of protein was to a final volume of in buffer CHAPS, 100 mm Bio-Lyte pH gradient strips were in the of protein The were then on a cell the following a to over at a to to over and at a V. immobilized pH gradient strips were for by a incubation in buffer in mm pH followed by a incubation in buffer in mm pH were then on using of easy melt agarose and at in a cell of were using a as previously described C. E.M. S.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). were using a and were for CED-3-dependent in and by protein were from two-dimensional with and identified by as previously described C. E.M. S.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Cell and of cells were in was induced by incubation in of tosylphenylalanylchloromethane, or apoptosis, the cells were with at and at 10 in buffer mm pH 7.4, mm mm mm mm Cell was measured on a cells were with containing and followed by a incubation in the buffer. were at and for a further secondary antibody was at for The cells were and for in with and then for of Cell cells were at and at 2 in buffer 50 mm pH The were by and of were from human cells, and caspases were activated by the addition of and to these as described previously C. E.M. S.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, E.A. Casiano C.A. D.W. E.S. Green D.R. S.J. J. Cell Biol. 1999; PubMed Scopus Google Scholar). CED-3 of and cell death in the nematode in cells during that of a of cells in the C. elegans of these die in a programmed Horvitz H.R. Biol. PubMed Scopus Google Scholar). studies established that it was not to coordinate programmed cell death in worm or in the cells of using or of protein or C. and S. J. The latter apoptosis in and mammalian cells, and their to in the worm suggests that a programmed cell death is in the majority of cells in the it was not to of dying cells from worm or we a approach to CED-3 This involved from worm to which recombinant active CED-3 was recombinant CED-3 we generated a of expression CED-3, a CED-3 the and the and However, most of these to produce and several and not Recombinant active CED-3 was by the domain of the protease and with and protein proteolytic activity against several caspase peptides and In CED-3 not a that is by caspases in CED-3 a for which is of mammalian caspases as but was also of and an CED-3 also activity substrates suggesting that the addition of the not the of CED-3 CED-3 to be the caspase in which is with the role of CED-3 as the only essential as well as caspase in the We also the activity of recombinant CED-3 using protein substrates that were by in CED-3 has previously been to its with its to D. Shaham S. Horvitz H.R. 1996; 10: PubMed Scopus Google Scholar). CED-3 is also to the cell death in D. Horvitz H.R. Nature. PubMed Scopus Google Scholar). we titrated recombinant and to the of CED-3 to these proteins CED-3 cleaved its precursor to of and with of the and through proteolysis at and However, the addition of C. elegans the activity of substrates and proteolysis of and was also by the z-VAD-fmk CED-3-dependent in C. elegans for CED-3 substrates within the C. elegans we generated worm as follows. of C. elegans were in were because this the of coli from the worm were then several in buffer followed by over of worm were generated by and the protein were by at to and These were then for 2 at in the or of recombinant CED-3. The of CED-3 was upon of recombinant CED-3 against in the and of worm in Proteins were by in incubation of C. elegans with active to to the worm suggesting that CED-3 is a protease that few at at this protease its precursor We worm using two-dimensional and these were in to the two-dimensional protein and and protein were then from the and the proteins were identified by in and 22 proteins were identified as CED-3-dependent proteolysis these These included proteins involved in a diverse of cellular functions. several of human caspase substrates were the proteins actin, and A. M. D. A. L. A. M. U. S. A. PubMed Scopus Google Scholar, C. G. P. S.J. J. Biol. 2006; Full Text Full Text PDF Scopus Google a protein J. M. D. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google and a P. L. J. K. A. J. K. Nat. 2005; PubMed Scopus Google Scholar). several of the substrates actin, tubulin, and calreticulin, have previously been implicated in events in mammalian C. G. P. S.J. J. 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PubMed Google the present a in our understanding of how CED-3 cell The substrates identified be in proteins that important during CED-3-dependent cell death in the Further analysis of the of these substrates also yield insights into the of apoptosis in organisms. is important to that the approach we have used to CED-3 substrates has several because of the of worm in our of proteins have been and this proteins to proteolysis in we have used recombinant at that well be higher in worm cells programmed cell of the of antibodies to the of CED-3 expressed in worm cells remains the of two-dimensional restricted to only the most proteins within the worm is a that numerous C. elegans of are also to proteolysis by CED-3 during cell These the of we the approach in this as to CED-3-dependent proteolysis in dying worm cells for The to this is of the of cells in the worm, with the that cell deaths are it was to CED-3-dependent to the worm proteome using or not as numerous to coordinate cell death in worm or protein and inhibitors, and were it is likely that the of these have contributed to the of the mechanism of of CED-3 during worm CED-3 plays a role in worm as an as well as an caspase in this We also the of CED-3 to human in of CED-3 a for its to the human the of CED-3 in are the caspases and However, CED-3 a higher of in its to the substrates and its role in C. elegans This was in the CED-3 identified in protein substrates of CED-3 a the other the was for or were were the most cleaved not the site was in all but by a suggesting that CED-3 site further the as for several of the human caspases H.R. 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This suggests that the dying cells from of the In substrates identified are involved in cellular This a of in apoptotic also to the of the apoptotic because cells are to essential are However, the of proteins identified in this analysis is to the proteomics used because two-dimensional the most proteins within the which proteins with are events to cell proteins as in several pathways and have been identified as substrates of human caspases J. M. D. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). proteins also be during apoptosis to to the chaperone calreticulin is for in C. elegans K. N. M. Full Text Full Text PDF PubMed Scopus (221) Google Scholar). studies also suggests that calreticulin is on the cell surface during apoptosis and as a and cell engulfment S.J. A. P. M. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. G.M. L. M. G. N. D. N. P. M. L. G. Nat. PubMed Scopus Google Scholar). of calreticulin in to to the of M. A. G.M. L. M. G. 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PubMed Scopus Google by CED-3 proteases to that cells not The observation that of the 22 substrates identified are for in C. elegans a to of apoptotic cells into few substrates for CED-3 have previously been described, substrates for the mammalian caspases have been identified S.J. Cell Death Differ. PubMed Scopus Google Scholar). However, the majority of these substrates have been at over a of 10 or for caspase to the have been to used a with to for human caspase substrates in cells apoptosis in to P. L. J. K. A. J. K. Nat. 2005; PubMed Scopus Google Scholar). This approach yielded caspase of which were as substrates for these The latter a and for the of generated protein P. L. J. K. A. J. K. Nat. 2005; PubMed Scopus Google Scholar). However, with as by P. L. J. K. A. J. K. Nat. 2005; PubMed Scopus Google it is that not the because several caspase substrates were not by this approach S.J. Cell Death Differ. PubMed Scopus Google Scholar). several the of proteolysis of the of the used for the analysis have an on the of the proteins on two-dimensional because of and peptides not well in the of proteolysis of and other of the it is likely that substrates for CED-3 be found by using In this the analysis of the proteins that undergo CED-3-dependent proteolysis in the nematode C. elegans and that CED-3 a restricted of proteins to coordinate cell of which are caspase substrates in We Dr. and Dr. Shai Shaham for the of expression C. elegans were provided by the C. elegans with