Substrate and Inhibitor Specificity of Interleukin-1β-converting Enzyme and Related Caspases

Interleukin-1β-converting enzyme (ICE) is a novel cysteine protease responsible for the cleavage of pre-interleukin-1β (pre-IL-1β) to the mature cytokine and a member of a family of related proteases (the caspases) that includes the Caenorhabditis elegans cell death gene product, CED-3. In addition to their sequence homology, these cysteine proteases display an unusual substrate specificity for peptidyl sequences with a P1 aspartate residue. We have examined the kinetics of processing pre-IL-1β to the mature form by ICE and three of its homologs, TX, CPP-32, and CMH-1. Of the ICE homologs, only TX processes pre-IL-1β, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADP-ribose) polymerase, a DNA repair enzyme that is cleaved within minutes of the onset of apoptosis. Every caspase examined cleaves PARP, with catalytic efficiencies ranging from 2.3 × 106M−1 s−1 for CPP32 to 1.0 × 103M−1 s−1 for TX. In addition, we report kinetic constants for several reversible inhibitors and irreversible inactivators, which have been used to implicate one or more caspases in the apoptotic proteolysis cascade. Ac-Asp-Glu-Val-Asp aldehyde (DEVD-CHO) is a potent inhibitor of CPP-32 with a Ki value of 0.5 nM, but is also potent as inhibitor of CMH-1 (Ki = 35 nM) and ICE (Ki = 15 nM). The x-ray crystal structure of DEVD-CHO complexed to ICE presented here reveals electrostatic interactions not present in the Ac-YVAD-CHO co-complex structure (Wilson, K. P., Black, J.-A. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-275), accounting for the surprising potency of this inhibitor against ICE. Interleukin-1β-converting enzyme (ICE) is a novel cysteine protease responsible for the cleavage of pre-interleukin-1β (pre-IL-1β) to the mature cytokine and a member of a family of related proteases (the caspases) that includes the Caenorhabditis elegans cell death gene product, CED-3. In addition to their sequence homology, these cysteine proteases display an unusual substrate specificity for peptidyl sequences with a P1 aspartate residue. We have examined the kinetics of processing pre-IL-1β to the mature form by ICE and three of its homologs, TX, CPP-32, and CMH-1. Of the ICE homologs, only TX processes pre-IL-1β, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADP-ribose) polymerase, a DNA repair enzyme that is cleaved within minutes of the onset of apoptosis. Every caspase examined cleaves PARP, with catalytic efficiencies ranging from 2.3 × 106M−1 s−1 for CPP32 to 1.0 × 103M−1 s−1 for TX. In addition, we report kinetic constants for several reversible inhibitors and irreversible inactivators, which have been used to implicate one or more caspases in the apoptotic proteolysis cascade. Ac-Asp-Glu-Val-Asp aldehyde (DEVD-CHO) is a potent inhibitor of CPP-32 with a Ki value of 0.5 nM, but is also potent as inhibitor of CMH-1 (Ki = 35 nM) and ICE (Ki = 15 nM). The x-ray crystal structure of DEVD-CHO complexed to ICE presented here reveals electrostatic interactions not present in the Ac-YVAD-CHO co-complex structure (Wilson, K. P., Black, J.-A. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-275), accounting for the surprising potency of this inhibitor against ICE. INTRODUCTIONICE 1The following abbreviations are used: ICEinterleukin 1β-converting enzymeIL-1βinterleukin 1βPARPpoly(ADP-ribose) polymerasePAGEpolyacrylamide gel electrophoresisDTTdithiothreitolpNAp-nitroanilidePMSFphenylmethylsulfonyl fluorideIVTTin vitro transcription translationCHAPS3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonateAc-YVAD-AMCAc-Tyr-Val-Ala-Asp-aminomethylcoumarinSuc-YVAD-pNASuc-Tyr-Val-Ala-Asp-p-nitroanilideAc-DEVD-AMCAc-Asp-Glu-Val-Asp-aminomethylcoumarinZ-Val-Ala-Asp-DCBcarbobenzoxy-Val-Ala-Asp-(2,6-dichlorobenzoyl)oxymethyl ketoneAc-DEVD-CHOAc-Asp-Glu-Val-Asp-aldehydeAc-YVAD-CHOAc-Tyr-Val-Ala-Asp-aldehydeHPLChigh performance liquid chromatography. is the prototypical member of a new family of mammalian cysteine proteases (the caspases) 2Caspase denotes the cysteine protease subfamily that includes ICE and its human homologs. For a discussion of the nomenclature of ICE and its homologs, please see 1Alnemri 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 (2129) Google Scholar. that is distinct from cysteine proteases in the papain superfamily (1Alnemri 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 (2129) Google Scholar–3Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kosture M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Ellison K.O. Ayala J.M. Casano F.J. Chin J. Ding J.-F.G. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.-T. Lee T.D. Shively J.E. MacCross M. Mumford R.A. Schmidt J.A. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2185) Google Scholar). The mutagenesis experiments and crystal structure reported by Wilson et al. (4Wilson K.P. Black J.-A.F. Thomson J.A. Kim E.E. Griffith J.P. Navia M.A. Murcko M.A. Chambers S.P. Aldape R.A. Raybuck S.A. Livingston D.J. Nature. 1994; 370: 270-275Crossref PubMed Scopus (752) Google Scholar) revealed a different active site geometry and catalytic mechanism for ICE than observed for papain. The structure of the ICE active site contains a Cys-His catalytic diad, and two Arg residues that confer high selectivity for peptidyl substrates with Asp residues at the P1 position (N-terminal to the scissile bond) (4Wilson K.P. Black J.-A.F. Thomson J.A. Kim E.E. Griffith J.P. Navia M.A. Murcko M.A. Chambers S.P. Aldape R.A. Raybuck S.A. Livingston D.J. Nature. 1994; 370: 270-275Crossref PubMed Scopus (752) Google Scholar, 5Walker N.P.C. Talanian R.V. Brady K.D. Dang L.C. Bump N.J. Ferenz C.R. Franklin S. Ghayur T. Hackett M.C. Hammill L.D. Cell. 1994; 78: 343-352Abstract Full Text PDF PubMed Scopus (526) Google Scholar). Although ICE has recently been reported to cleave other proteins in vitro (6Gu Y. Sarnecki C. Aldape R.A. Livingston D.J. Su M.S.-S. J. Biol. Chem. 1995; 270: 18715-18718Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 7Tewari M. Quan L.T. O'Rourke K. Desnoyers D. Zeng Z. Beidler D.R. Poirer G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar), it was identified from its essential role in processing the inactive 31-kDa precursor of interleukin-1β (pre-IL-1β) to the mature 17-kDa cytokine (8Black R.A. Kronheim S.R. Cantrell M. Deeley M.C. March C.J. Prickett K.S. Wignall J. Conlon P.J. Cosman D. Hopp T.P. Mochizuki D.Y. J. Biol. Chem. 1988; 263: 9437-9442Abstract Full Text PDF PubMed Google Scholar).In 1993, Yuan et al. (9Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2234) Google Scholar) reported the sequence of the Caenorhabditis elegans programmed cell death gene ced-3 gene is to human ICE. to the role of the in C. elegans Yuan and that ICE or ICE a role in mammalian apoptosis. of ICE in mammalian and cell that this protease (6Gu Y. Sarnecki C. Aldape R.A. Livingston D.J. Su M.S.-S. J. Biol. Chem. 1995; 270: 18715-18718Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, M. R. Yuan J. Cell. 1993; 75: Full Text PDF PubMed Scopus Google Scholar, Lee M.C. Yuan J. 1994; 263: PubMed Scopus Google Scholar). et al. K. J.A. G. Livingston D.J. Su M.S.-S. R.A. 1995; PubMed Scopus Google Scholar) an in role for ICE in by of the ICE family of proteases (the caspases) was by human for sequences to ICE or ced-3 S. 1995; Full Text PDF PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). at human of ICE cysteine protease have been by sequence three The related to ICE are TX also or C. A.M. C. Y. Aldape R.A. J.A. C. Su M.S.-S. Livingston D.J. T. J. 1995; PubMed Scopus Google Scholar, J. M. M. Talanian R.V. D. Bump Hackett M. J.A. M. Ghayur T. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, N.A. J.P. Casano F.J. Miller D.K. Molineaux S.M. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) and also N.A. J.P. Casano F.J. Miller D.K. Molineaux S.M. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. 1996; PubMed Scopus Google Scholar), which are and to ICE at the to the M. Yuan J. Cell. 1994; 78: Full Text PDF PubMed Scopus Google Scholar, S. M. M. N.A. 1994; PubMed Scopus Google Scholar), is to ICE and to a distinct of proteins that a sequence to than to ICE. CPP32 M. Quan L.T. O'Rourke K. Desnoyers D. Zeng Z. Beidler D.R. Poirer G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar, T. G. E.S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), T. G. E.S. 1995; Google Scholar), and CMH-1 also J.A. Y. Sarnecki C. Su M.S.-S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, T. R. J. Z. G. G. E.S. 1995; Google Scholar). in sequence to the C. elegans gene and by a aldehyde the cleavage sequence CPP32 was to the caspase responsible for in mammalian D.W. Thornberry N.A. J.P. Ding M. Y. M. N.A. Raju S.M. Yamin T.-T. Miller D.K. Nature. 1995; PubMed Scopus Google Scholar). The of in the of CPP32 to and the of other caspases by DEVD-CHO has not been has that is by other caspases M. Nicholson D.W. J. 1996; PubMed Scopus Google Scholar, Kim J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google a of the kinetics of ICE has been reported N.A. 1994; PubMed Scopus Google Scholar), kinetic ICE in the kinetic of these proteases is essential to the substrate specificity of these proteases T. R. J. Z. G. G. E.S. 1995; Google Scholar, D.W. Thornberry N.A. J.P. Ding M. Y. M. N.A. Raju S.M. Yamin T.-T. Miller D.K. Nature. 1995; PubMed Scopus Google Scholar). For in to the by Nicholson et al. D.W. Thornberry N.A. J.P. Ding M. Y. M. N.A. Raju S.M. Yamin T.-T. Miller D.K. Nature. 1995; PubMed Scopus Google Scholar) that ICE is to cleave a enzyme cleaved we recently reported that ICE is to cleave this albeit at enzyme than for cleavage of pre-IL-1β (6Gu Y. Sarnecki C. Aldape R.A. Livingston D.J. Su M.S.-S. J. Biol. Chem. 1995; 270: 18715-18718Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). ICE identified is to cleave as a substrate 1996; PubMed Scopus Google Scholar), and the to more active in cleavage than CPP32 T. R. J. Z. G. G. E.S. 1995; Google Scholar). TX is to cleave pre-IL-1β J. M. M. Talanian R.V. D. Bump Hackett M. J.A. M. Ghayur T. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), but have for kinetic two from the ICE subfamily and and two of the CPP32 subfamily and We report a kinetic of substrate by these proteases and the constants for reversible and irreversible peptidyl have been used to the role of caspases in a of cell M. S. Nature. 1995; PubMed Scopus Google K. C. J. 1996; PubMed Scopus Google Scholar). report is the of the proteolysis kinetics by ICE and ICE human interleukin-1β was from a an in a ICE and was from the by an to the The and of this has been Raybuck S.A. J.R. R.A. Chambers S.P. ICE was as a in and as the inactive precursor from chromatography. and was at a of in at to the active which was in at (4Wilson K.P. Black J.-A.F. Thomson J.A. Kim E.E. Griffith J.P. Navia M.A. Murcko M.A. Chambers S.P. Aldape R.A. Raybuck S.A. Livingston D.J. Nature. 1994; 370: 270-275Crossref PubMed Scopus (752) Google Scholar). The kinetic of the E. and ICE of the of active enzyme was with the irreversible inhibitor N.A. 1994; PubMed Scopus Google Scholar). for the substrate the are in of kinetic for pre-IL-1β and substrates for ICE and × × × × not not × from the of the as × × not from the of the as in a new the form of human TX was from C. C. A.M. C. Y. Aldape R.A. J.A. C. Su M.S.-S. Livingston D.J. T. J. 1995; PubMed Scopus Google Scholar). TX (6Gu Y. Sarnecki C. Aldape R.A. Livingston D.J. Su M.S.-S. J. Biol. Chem. 1995; 270: 18715-18718Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) was in T. the and from the an as TX two of and of the active enzyme was with the irreversible inhibitor for the substrate are in for CPP32 the was by at the and of CPP32 by and and the a E. The the of of of a a cleavage in to the of CPP32 at as by DNA and by of the E. the was with for at and by in and by at × for and the was an with CPP32 was with in The was and a in with the and with the that the two of and the two of the as by of the active enzyme nM) was with the reversible inhibitor for the substrate = and = 1.0 human CMH-1 was in E. and by a as J.A. Y. Sarnecki C. Su M.S.-S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google of the active enzyme nM) was with the potent reversible inhibitor for the substrate = and = of pre-IL-1β was and in E. in of and by by at for at × was from the cell with of and against the The was a × in the with of and the was with a from to in was in these by and by a against M. by The was in 0.5 and a × and in the at an of The was against and a The of was and gel pre-IL-1β as a with a to of DNA of an was in E. from in and and in a was at at × The cell was in and and against the in The was to a × The was with a from to in the at and by and a against the The as a of The of was which to of of and by ICE and pre-IL-1β and which and by in vitro the and experiments one or of two The in which the of was substrate was in of enzyme and to of substrate in by the addition of and to gel and the of cleavage by the of the substrate was and a substrate and substrate was and used in as For of kinetic was by of the the peptidyl substrates from and for by a × a in was also by by of the by to a of or in as and of of enzyme of the inhibitor and of substrate D.J. D. Murcko M.A. Raybuck S.A. Livingston D.J. Chem. 1994; Scopus Google Scholar). of from was by following the at that at a from of from enzyme cleavage of or was a with and from a The of was an of and an of with of and 15 cleavage of the was the an of nM, an of nM, with of and 15 was with for the from of and inhibitors used in this from or and a of as by or at a of the value was used as a substrate for ICE or TX at a of the value was used as the substrate CPP32 = or CMH-1 = reversible inhibitors as in with the inhibitor and enzyme in the for 15 at The was by the addition of and the from at Ki from inhibitor with to the of PubMed Scopus Google Scholar). was used for this are from at two and are not than constants for irreversible inhibitors of ICE from the and inhibitor and substrate at and the by the addition of enzyme to of the to the for Cell. constants from of to the = × for the of irreversible inhibitors of ICE have been by Thornberry et al. N.A. E.P. Howard A.D. Chapman K.T. 1994; PubMed Scopus Google and of the of ICE by in was with and to the at from the was used to x-ray at to The crystal to with cell a = = = structure of Ac-YVAD-CHO to ICE was used as a for of the but with the of the inhibitor and the of for the P1 and residues of the but for the Asp was less of the inhibitor and of and to The has an of for and with from and of and was used for and 1992; Google selectivity of ICE for pre-IL-1β is by the constants for the substrates in the and substrates are for the cleavage the for ICE cleavage of the substrates and pre-IL-1β are that of the to interactions in the selectivity of the ICE TX in pre-IL-1β is by of is with the that ICE and only ICE is responsible for the processing of to the mature we kinetic for ICE and TX substrates and inhibitors are that the peptidyl of are of TX from the ICE structure that residues the the in the C. A.M. C. Y. Aldape R.A. J.A. C. Su M.S.-S. Livingston D.J. T. J. 1995; PubMed Scopus Google has been as a substrate for proteases 1996; PubMed Scopus Google Scholar). is of as is a DNA repair enzyme that is cleaved two at the onset of apoptosis. The of as a substrate for proteases an an apoptotic and a member of the ced-3 cell death protease Of the ICE we CPP32 to the at ICE and TX cleave PARP, but at and more with the report of et al. (6Gu Y. Sarnecki C. Aldape R.A. Livingston D.J. Su M.S.-S. J. Biol. Chem. 1995; 270: 18715-18718Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The of this is at the present as the role of its in to it is cleaved the apoptotic sequence of Desnoyers S. Poirer G.G. Nature. 1994; PubMed Scopus Google Scholar). in the gene are and and that this gene not an essential role in or T. G.G. 1995; Full Text PDF PubMed Scopus Google Scholar, D. M. 1995; PubMed Scopus Google these two inhibitors have been as to and implicate the of ICE in the pre-IL-1β cleavage is an inhibitor of proteases with the to ICE with Ki of and against ICE and TX is not against CPP32 or CMH-1. In a was from the cleavage sequence to a potent inhibitor of CPP32 D.W. Thornberry N.A. J.P. Ding M. Y. M. N.A. Raju S.M. Yamin T.-T. Miller D.K. Nature. 1995; PubMed Scopus Google Scholar), is the as the the protease with Ki of 0.5 and is also against the CMH-1. surprising is the potency of this inhibitor against ICE and TX, this to one of the inhibitors to The x-ray crystal structure of complexed to ICE reveals an the of the inhibitor and an in the of ICE. of new the of and the enzyme are present which not observed in the structure of ICE with new as as the by the of the aldehyde the surprising potency of the inhibitor to the cleavage efficiency of ICE for the irreversible inhibitors the ICE cleavage sequence at selectivity for ICE and TX the CPP32 of The in potency for the the is for ICE and in this and to interactions of the the of the enzyme for of the ICE that the in for these irreversible inhibitors is for by the of the inhibitor or the at of or and for than as are used for cell of these irreversible to of the ICE family of also to of the in of by other caspases M. S. Nature. 1995; PubMed Scopus Google K. C. J. 1996; PubMed Scopus Google Scholar). is in reversible caspase inhibitors as a of the constants against caspases has not been the present not to and inhibitors of or have to INTRODUCTIONICE 1The following abbreviations are used: ICEinterleukin 1β-converting enzymeIL-1βinterleukin 1βPARPpoly(ADP-ribose) polymerasePAGEpolyacrylamide gel electrophoresisDTTdithiothreitolpNAp-nitroanilidePMSFphenylmethylsulfonyl fluorideIVTTin vitro transcription translationCHAPS3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonateAc-YVAD-AMCAc-Tyr-Val-Ala-Asp-aminomethylcoumarinSuc-YVAD-pNASuc-Tyr-Val-Ala-Asp-p-nitroanilideAc-DEVD-AMCAc-Asp-Glu-Val-Asp-aminomethylcoumarinZ-Val-Ala-Asp-DCBcarbobenzoxy-Val-Ala-Asp-(2,6-dichlorobenzoyl)oxymethyl ketoneAc-DEVD-CHOAc-Asp-Glu-Val-Asp-aldehydeAc-YVAD-CHOAc-Tyr-Val-Ala-Asp-aldehydeHPLChigh performance liquid chromatography. is the prototypical member of a new family of mammalian cysteine proteases (the caspases) 2Caspase denotes the cysteine protease subfamily that includes ICE and its human homologs. For a discussion of the nomenclature of ICE and its homologs, please see 1Alnemri 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 (2129) Google Scholar. that is distinct from cysteine proteases in the papain superfamily (1Alnemri 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 (2129) Google Scholar–3Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kosture M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Ellison K.O. Ayala J.M. Casano F.J. Chin J. Ding J.-F.G. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.-T. Lee T.D. Shively J.E. MacCross M. Mumford R.A. Schmidt J.A. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2185) Google Scholar). The mutagenesis experiments and crystal structure reported by Wilson et al. (4Wilson K.P. Black J.-A.F. Thomson J.A. Kim E.E. Griffith J.P. Navia M.A. Murcko M.A. Chambers S.P. Aldape R.A. Raybuck S.A. Livingston D.J. Nature. 1994; 370: 270-275Crossref PubMed Scopus (752) Google Scholar) revealed a different active site geometry and catalytic mechanism for ICE than observed for papain. The structure of the ICE active site contains a Cys-His catalytic diad, and two Arg residues that confer high selectivity for peptidyl substrates with Asp residues at the P1 position (N-terminal to the scissile bond) (4Wilson K.P. Black J.-A.F. Thomson J.A. Kim E.E. Griffith J.P. Navia M.A. Murcko M.A. Chambers S.P. Aldape R.A. Raybuck S.A. Livingston D.J. Nature. 1994; 370: 270-275Crossref PubMed Scopus (752) Google Scholar, 5Walker N.P.C. Talanian R.V. Brady K.D. Dang L.C. Bump N.J. Ferenz C.R. Franklin S. Ghayur T. Hackett M.C. Hammill L.D. Cell. 1994; 78: 343-352Abstract Full Text PDF PubMed Scopus (526) Google Scholar). Although ICE has recently been reported to cleave other proteins in vitro (6Gu Y. Sarnecki C. Aldape R.A. Livingston D.J. Su M.S.-S. J. Biol. Chem. 1995; 270: 18715-18718Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 7Tewari M. Quan L.T. O'Rourke K. Desnoyers D. Zeng Z. Beidler D.R. Poirer G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar), it was identified from its essential role in processing the inactive 31-kDa precursor of interleukin-1β (pre-IL-1β) to the mature 17-kDa cytokine (8Black R.A. Kronheim S.R. Cantrell M. Deeley M.C. March C.J. Prickett K.S. Wignall J. Conlon P.J. Cosman D. Hopp T.P. Mochizuki D.Y. J. Biol. Chem. 1988; 263: 9437-9442Abstract Full Text PDF PubMed Google Scholar).In 1993, Yuan et al. (9Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2234) Google Scholar) reported the sequence of the Caenorhabditis elegans programmed cell death gene ced-3 gene is to human ICE. to the role of the in C. elegans Yuan and that ICE or ICE a role in mammalian apoptosis. of ICE in mammalian and cell that this protease (6Gu Y. Sarnecki C. Aldape R.A. Livingston D.J. Su M.S.-S. J. Biol. Chem. 1995; 270: 18715-18718Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, M. R. Yuan J. Cell. 1993; 75: Full Text PDF PubMed Scopus Google Scholar, Lee M.C. Yuan J. 1994; 263: PubMed Scopus Google Scholar). et al. K. J.A. G. Livingston D.J. Su M.S.-S. R.A. 1995; PubMed Scopus Google Scholar) an in role for ICE in by of the ICE family of proteases (the caspases) was by human for sequences to ICE or ced-3 S. 1995; Full Text PDF PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). at human of ICE cysteine protease have been by sequence three The related to ICE are TX also or C. A.M. C. Y. Aldape R.A. J.A. C. Su M.S.-S. Livingston D.J. T. J. 1995; PubMed Scopus Google Scholar, J. M. M. Talanian R.V. D. Bump Hackett M. J.A. M. Ghayur T. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, N.A. J.P. Casano F.J. Miller D.K. Molineaux S.M. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) and also N.A. J.P. Casano F.J. Miller D.K. Molineaux S.M. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. 1996; PubMed Scopus Google Scholar), which are and to ICE at the to the M. Yuan J. Cell. 1994; 78: Full Text PDF PubMed Scopus Google Scholar, S. M. M. N.A. 1994; PubMed Scopus Google Scholar), is to ICE and to a distinct of proteins that a sequence to than to ICE. CPP32 M. Quan L.T. O'Rourke K. Desnoyers D. Zeng Z. Beidler D.R. Poirer G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2264) Google Scholar, T. G. E.S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), T. G. E.S. 1995; Google Scholar), and CMH-1 also J.A. Y. Sarnecki C. Su M.S.-S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, T. R. J. Z. G. G. E.S. 1995; Google Scholar). in sequence to the C. elegans gene and by a aldehyde the cleavage sequence CPP32 was to the caspase responsible for in mammalian D.W. Thornberry N.A. J.P. Ding M. Y. M. N.A. Raju S.M. Yamin T.-T. Miller D.K. Nature. 1995; PubMed Scopus Google Scholar). The of in the of CPP32 to and the of other caspases by DEVD-CHO has not been has that is by other caspases M. Nicholson D.W. J. 1996; PubMed Scopus Google Scholar, Kim J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google a of the kinetics of ICE has been reported N.A. 1994; PubMed Scopus Google Scholar), kinetic ICE in the kinetic of these proteases is essential to the substrate specificity of these proteases T. R. J. Z. G. G. E.S. 1995; Google Scholar, D.W. Thornberry N.A. J.P. Ding M. Y. M. N.A. Raju S.M. Yamin T.-T. Miller D.K. Nature. 1995; PubMed Scopus Google Scholar). For in to the by Nicholson et al. D.W. Thornberry N.A. J.P. Ding M. Y. M. N.A. Raju S.M. Yamin T.-T. Miller D.K. Nature. 1995; PubMed Scopus Google Scholar) that ICE is to cleave a enzyme cleaved we recently reported that ICE is to cleave this albeit at enzyme than for cleavage of pre-IL-1β (6Gu Y. Sarnecki C. Aldape R.A. Livingston D.J. Su M.S.-S. J. Biol. 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