Substrate and Inhibitor Specificity of Interleukin-1β-converting Enzyme and Related Caspases
1997; Elsevier BV; Volume: 272; Issue: 11 Linguagem: Inglês
10.1074/jbc.272.11.7223
ISSN1083-351X
AutoresNara Margolin, Scott A. Raybuck, Keith P. Wilson, Wenyong Chen, Ted Fox, Yong Gu, David J. Livingston,
Tópico(s)Trace Elements in Health
ResumoInterleukin-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)oxy]methyl 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 This gene is 29% identical to human ICE. Due to the central role of the CED-3 protein in C. elegans apoptosis, Yuan and colleagues deduced that ICE or ICE homologs might play a similar role in mammalian apoptosis. Overexpression of ICE in rat fibroblast, mammalian COS cells, and neuronal cell lines demonstrated that this protease can indeed induce apoptosis (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, 10Muira M. Zhu H. Rotello R. Hartweig E.A. Yuan J. Cell. 1993; 75: 653-660Abstract Full Text PDF PubMed Scopus (1325) Google Scholar, 11Gagliardi V. Fernandez P.A. Lee R.K. Drexier H.C. Rotello R.J. Fishman M.C. Yuan J. Science. 1994; 263: 826-828Crossref PubMed Scopus (604) Google Scholar). Subsequently, Kuida et al. (12Kuida K. Lippke J.A. Ku G. Harding M.W. Livingston D.J. Su M.S.-S. Flavell R.A. Science. 1995; 267: 2000-2003Crossref PubMed Scopus (1443) Google Scholar) confirmed an in vivo role for ICE in Fas-mediated apoptosis by disruption of the murine ICE gene.A family of ICE-related proteases (the caspases) was discovered by searching human cDNA libraries for sequences homologous to ICE or ced-3 (13Kumar S. Trends Biochem. Sci. 1995; 20: 198-202Abstract Full Text PDF PubMed Scopus (366) Google Scholar, 14Takahashi A. Earnshaw W.C. Curr. Opin. Genet. Dev. 1996; 6: 50-55Crossref PubMed Scopus (150) Google Scholar). At present, at least 10 human homologs of ICE possessing cysteine protease activity have been identified. These homologs can be grouped by sequence similarity into three subfamilies. The most closely related homologs to ICE are TX (caspase-4, also denoted ICH-2 or ICErelII) (15Faucheu C. Diu A. Chan A.W. Blanchet A.M. Miossec C. Herve F. Collard-Dutilleul V. Gu Y. Aldape R.A. Lippke J.A. Rochet C. Su M.S.-S. Livingston D.J. Hercend T. Lalanne J.-L. EMBO J. 1995; 14: 1914-1922Crossref PubMed Scopus (322) Google Scholar, 16Kamens J. Paskind M. Hugunin M. Talanian R.V. Allen H. Banach D. Bump N. Hackett M. Johnston C.G. Li P. Mankovich J.A. Terranova M. Ghayur T. J. Biol. Chem. 1995; 270: 15250-15256Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 17Munday N.A. Vaillancourt J.P. Ali A. Casano F.J. Miller D.K. Molineaux S.M. Yamin T.-T. Yu V.L. Nicholson D.W. J. Biol. Chem. 1995; 270: 15870-15876Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) and TY (caspase-5, also denoted ICErelIII) (17Munday N.A. Vaillancourt J.P. Ali A. Casano F.J. Miller D.K. Molineaux S.M. Yamin T.-T. Yu V.L. Nicholson D.W. J. Biol. Chem. 1995; 270: 15870-15876Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 18Faucheu C. Blanchet A.-M. Collard-Dutilleul V. Lalanne J.-L. Diu-Hercend A. Eur. J. Biochem. 1996; 236: 207-213Crossref PubMed Scopus (59) Google Scholar), which are 58 and 57% identical to ICE at the amino acid level. Another homolog, ICH-1 (caspase-2, corresponding to the murine Nedd-2 gene) (19Wang L. Muira M. Bergeron L. Zhu H. Yuan J. Cell. 1994; 78: 739-750Abstract Full Text PDF PubMed Scopus (800) Google Scholar, 20Kumar S. Kinoshita M. Noda M. Copelan N.G. Jenkins N.A. Genes Dev. 1994; 8: 1613-1626Crossref PubMed Scopus (585) Google Scholar), is 20% identical to ICE and belongs to a distinct subfamily. A third group of homologs comprises proteins that show a higher sequence similarity to CED-3 than to ICE. These include CPP32 (caspase-3) (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, 21Fernandes-Alnemri T. Litwack G. Alnemri E.S. J. Biol. Chem. 1994; 269: 30761-30764Abstract Full Text PDF PubMed Google Scholar), MCH-2 (caspase-6) (22Fernandes-Alnemri T. Litwack G. Alnemri E.S. Cancer Res. 1995; 55: 2737-2742PubMed Google Scholar), and CMH-1 (caspase-7, also called MCH-3) (23Lippke J.A. Gu Y. Sarnecki C. Caron P.R. Su M.S.-S. J. Biol. Chem. 1996; 271: 1825-1828Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 24Fernandes-Alnemri T. Takahashi A. Armstrong R. Krebs J. Fritz L. Tomaselli K.J. Wang L. Yu Z. Croces C.M. Salveson G. Earnshaw W.C. Litwack G. Alnemri E.S. Cancer Res. 1995; 55: 6045-6052PubMed Google Scholar). Based in part on sequence similarity to the C. elegans gene and inhibition by a tetrapeptide aldehyde based on the PARP cleavage sequence (DEVD-CHO), CPP32 was claimed to be the caspase responsible for apoptosis in mammalian cells (25Nicholson 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 (3777) Google Scholar). The dependence of apoptosis in vivo on the presence of CPP32 remains to be confirmed, however, and the inhibition of other caspases by DEVD-CHO has not been addressed. Subsequent work has shown that pro-CPP32 is activated by other caspases during apoptosis (26Slee E.A. Zhu H. Chow S.C. MacFarlane M. Nicholson D.W. Cohen G.M. Biochem J. 1996; 315: 21-24Crossref PubMed Scopus (396) Google Scholar, 27Liu X. Kim C.N. Pohl J. Wang X. J. Biol. Chem. 1996; 271: 13371-13376Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar).Although a thorough investigation of the kinetics of ICE has been reported (28Thornberry N.A. Methods Enzymol. 1994; 244: 615-631Crossref PubMed Scopus (202) Google Scholar), little kinetic information on ICE homologs exists in the literature. A rigorous kinetic analysis of these proteases is thus essential to evaluate the putative differential substrate specificity of these proteases (24Fernandes-Alnemri T. Takahashi A. Armstrong R. Krebs J. Fritz L. Tomaselli K.J. Wang L. Yu Z. Croces C.M. Salveson G. Earnshaw W.C. Litwack G. Alnemri E.S. Cancer Res. 1995; 55: 6045-6052PubMed Google Scholar, 25Nicholson 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 (3777) Google Scholar). For example, in contradistinction to the claim by Nicholson et al. (25Nicholson 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 (3777) Google Scholar) that ICE is unable to cleave poly(ADP-ribosyl) polymerase (PARP, a cellular enzyme cleaved during apoptosis), we recently reported that ICE is fully competent to cleave this substrate, albeit at enzyme concentrations higher than those required 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). Indeed, every ICE homolog identified is able to cleave PARP as a substrate (14Takahashi A. Earnshaw W.C. Curr. Opin. Genet. Dev. 1996; 6: 50-55Crossref PubMed Scopus (150) Google Scholar), and the MCH-3 homolog appears to be more active in PARP cleavage than CPP32 itself (24Fernandes-Alnemri T. Takahashi A. Armstrong R. Krebs J. Fritz L. Tomaselli K.J. Wang L. Yu Z. Croces C.M. Salveson G. Earnshaw W.C. Litwack G. Alnemri E.S. Cancer Res. 1995; 55: 6045-6052PubMed Google Scholar). Similarly, TX is able to cleave pre-IL-1β (16Kamens J. Paskind M. Hugunin M. Talanian R.V. Allen H. Banach D. Bump N. Hackett M. Johnston C.G. Li P. Mankovich J.A. Terranova M. Ghayur T. J. Biol. Chem. 1995; 270: 15250-15256Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), but does so inefficiently.We have selected for our kinetic studies two members from the ICE subfamily (ICE and TX) and two members of the CPP32 subfamily (CMH-1/MCH-3 and CPP32). We report a kinetic analysis of substrate hydrolysis by these proteases and the inhibition constants for both reversible and irreversible peptidyl inhibitors. Such compounds have been used widely to study the role of caspases in apoptosis across a variety of cell lines (29Enari M. Hug H. Nagata S. Nature. 1995; 375: 78-81Crossref PubMed Scopus (797) Google Scholar–32Cain K. Inayat-Hussain S.H. Couet C. Cohen G.M. Biochem. J. 1996; 314: 27-32Crossref PubMed Scopus (71) Google Scholar). This report is the first comprehensive comparison of the proteolysis kinetics by multiple caspases.EXPERIMENTAL PROCEDURESRecombinant ICE and ICE HomologsICERecombinant human interleukin-1β convertase was expressed from a p30 construct containing an N-terminal T7 tag in Trichoplusia ni insect cells using a baculovirus expression system. Active T7 ICE containing p20 and p10 subunits was purified from the medium by affinity chromatography using an immobilized T7 antibody column according to the manufacturer's protocol (Novagen). The expression, purification, and characterization of this protein has been published elsewhere (33Chen W. Raybuck S.A. Fulghum J.R. Petrillo R.A. Margolin N. Chambers S.P. Protein Exp. Purif. 1997; (in press)Google Scholar).Alternatively, ICE was expressed as a p32 construct in Escherichia coli and purified as the inactive p30 precursor from inclusion bodies using size-exclusion chromatography. Refolding and subsequent autoprocessing was performed at a protein concentration of 3 mg/ml in 25 mM Tris-HCl, 1 mM DTT, pH 7.5, at 25°C to give the active enzyme, which was immediately frozen in 10% glycerol at −78°C (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 parameters of the E. coli and baculovirus expressed ICE were similar, regardless of the purification method used.Quantification of active enzyme was performed with the irreversible inhibitor Z-Val-Ala-Asp-DCB under standard assay conditions using published methods (28Thornberry N.A. Methods Enzymol. 1994; 244: 615-631Crossref PubMed Scopus (202) Google Scholar). Kinetic parameters for the Suc-YVAD-pNA substrate under the assay conditions below are given in Table I.Table I.Comparison of kinetic parameters for pre-IL-1β and synthetic substrates for ICE and TXEnzymeSubstrateKmkcatkcat/KmμMs−1M−1s−1ICEPre-IL-1β (YVHDAPVR)4.01.23.0 × 105ICEAc-YVAD-AMC11.51.09.1 × 104ICESuc-YVAD-pNA21.51.67.7 × 104ICEDABCYL-YVADAPV-EDANSa4-(4′-Dimethylaminophenylazo)benzoic acid-YVADAPV-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid.13.40.413.1 × 104TXPre-IL-1βNDbND, not determined.NDbND, not determined.1.2 × 103cValue determined from the linear part of the rate vs concentration profile as described under “Experimental Procedures.”TXAc-YVAD-AMC350.51.4 × 104TXSuc-YVAD-pNA481.12.3 × 104a 4-(4′-Dimethylaminophenylazo)benzoic acid-YVADAPV-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid.b ND, not determined.c Value determined from the linear part of the rate vs concentration profile as described under “Experimental Procedures.” Open table in a new tab TXA cDNA encoding the p30 form of human TX was obtained from C. Faucheu (Roussel Uclaf, Romainville, France) (15Faucheu C. Diu A. Chan A.W. Blanchet A.M. Miossec C. Herve F. Collard-Dutilleul V. Gu Y. Aldape R.A. Lippke J.A. Rochet C. Su M.S.-S. Livingston D.J. Hercend T. Lalanne J.-L. EMBO J. 1995; 14: 1914-1922Crossref PubMed Scopus (322) Google Scholar). An N-terminal T7-tagged TX fusion described previously (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 similarly expressed in T. ni insect cells using the baculovirus expression system and purified from the medium using an immobilized T7 antibody column as described above. Active TX comprised two subunits of 24 and 10 kDa. Quantification of the active enzyme was performed with the irreversible inhibitor Z-Val-Ala-Asp-DCB. Kinetic parameters for the Suc-YVAD-pNA substrate under standard assay conditions are given in Table I.CPP32The expression plasmid for N-terminally (His)6-tagged wild type CPP32 lacking the prosequence was constructed by introducing XhoI sites at the 5′ and 3′ ends of CPP32 cDNAs by PCR using primers 5′-CGGCTGCAGCTCGAGTCTGGAATATCCCTGGACAACAGT and 5′-GGGAATTCTCGAGTTAGTGATAAAAATAGAGTTCTTTTGTGAGC and then ligating the resulting XhoI fragments into a XhoI-cut E. coli expression vector pET-15b (Novagen). The resulting plasmids direct the synthesis of polypeptides of 272 amino acids consisting of a 23-residue peptide (MGSSSSGHMLE, where LVPRGS represents a thrombin cleavage site) fused in frame to the N terminus of CPP32 at Ser29, as confirmed by DNA sequencing and by N-terminal sequencing of the expressed proteins. E. coli strain BL21(DE3) carrying the plasmid (4000 ml) was induced with 0.8 mM isopropyl-1-thio-β-D-galactopyranoside for 2 h at 30°C, harvested, and lysed by microfluidization in 150 Buffer A (20 mM sodium phosphate, pH 8.2, 300 mM NaCl, 2 mM dithiothreitol, 10% glycerol, 0.4 mM phenylmethylsulfonyl fluoride, and 2.5 μg/ml leupeptin). Lysates were cleared by centrifugation at 100,000 × g for 30 min, and the supernatant (S100) was loaded onto an ∼1.8-ml nickel-NTA-agarose column. After washing with Buffer A containing 25 mM imidazole, CPP32 protein was eluted with 100 mM imidazole in Buffer A. The eluate was desalted and applied onto a DEAE-Sepharose column in 20 mM Tris-HCl (pH 8.8), washed with the same buffer containing 50 mM NaCl, and eluted with the same buffer containing 100 mM NaCl. SDS-PAGE analysis indicated that the purified protein contained two major polypeptides of approximately 12 and 18 kDa, respectively, representing the two subunits of the protease, as confirmed by N-terminal sequencing.Quantification of the active enzyme (10 nM) was performed with the reversible inhibitor Ac-DEVD-CHO. Kinetic parameters for the Ac-DEVD-AMC substrate under standard assay conditions are: Km = 10 μM and kcat = 1.0 s−1.CMH-1Recombinant human N-terminal (His)6-tagged CMH-1 (Ala24-Gln303) was expressed in E. coli and purified by affinity chromatography using a nickel-NTA column as described previously (23Lippke J.A. Gu Y. Sarnecki C. Caron P.R. Su M.S.-S. J. Biol. Chem. 1996; 271: 1825-1828Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar).Quantification of the active enzyme (50 nM) was performed with the potent reversible inhibitor Ac-DEVD-CHO. Kinetic parameters for the Ac-DEVD-AMC substrate under standard assay conditions are: Km = 100 μM and kcat = 0.4 s−1.Purification of Pre-IL-1βRecombinant pre-IL-1β was cloned and expressed in E. coli Cell pellets (20 g) were resuspended in 100 ml of 10 mM Tris, pH 8.0, containing 0.05 M NaCl, 10% glycerol, 1 mM DTT, 1 mM PMSF, 2 mM EDTA and lysed by microfluidization. Cell extracts were prepared by centrifugation at 4°C, for 30 min at 35,000 × g Pre-IL-1β was extracted from the cell pellet with 100 ml of 10 mM Tris, pH 8.0, containing 0.05 M NaCl, 1 mM PMSF, 8 M urea and subsequently dialyzed against the lysis buffer without PMSF. The urea extract was loaded on a (3.5 × 26-mm) DEAE-Sephacel column, equilibrated in the same buffer, washed with five column volumes of buffer, and the bound protein was eluted with a linear gradient from 0.05 to 1 M NaCl in 10 mM Tris, pH 8.1, 10% glycerol. Pre-IL-1β was detected in these fractions by SDS-PAGE and by Western blotting using a monoclonal antibody generated against IL-1β (from M. DeCenzo, Vertex). Pre-IL-1β-containing fractions were concentrated by 40% ammonium sulfate precipitation. The subsequent pellet was solubilized in 10 mM Tris, pH 8.0, containing 2 mM EDTA, 2 mM DTT, 0.5 M NaCl, 5% glycerol and loaded on a Sephadex G-75 column (100 × 26 mm), calibrated, and equilibrated in the same buffer. Pre-IL-1β eluted at an apparent molecular mass of approximately 40 kDa. The sample was dialyzed against 10 mM Tris, pH 8.0, containing 0.1 M NaCl, 1 mM DTT and concentrated on a YM10 Amicon membrane. The yield of purified material was 70%, and on Coomassie-stained gel reduced pre-IL-1β migrated as a single band with a mobility corresponding to 33 kDa.Purification of Truncated Poly(ADP-ribose) PolymeraseThe DNA binding domain of PARP containing an N-terminal T7 tag was expressed in E. coli Cell pellets from 100-ml cultures were resuspended in lysis buffer containing 50 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaHSO3, 0.2 M NaCl, 10% glycerol, 5 mM DTT, 10 μg/ml pepstatin, 10 μg/ml leupeptin, 2 mM PMSF, and 1 mM benzamidine and disrupted in a microfluidizer. This suspension was centrifuged at 4°C, 30 min at 35,000 × g The cell pellet was solubilized in buffer (9 ml), containing 8 M urea, 50 mM Tris, pH 8.0, 50 mM NaHSO3, 0.2 M NaCl, 10% glycerol, and 5 mM DTT, and dialyzed stepwise against the same buffer without urea in 100-ml increments. The dialyzed sample was applied to a (1.8 × 26-mm) heparin-Sepharose column. The bound protein was eluted with a linear gradient from 0.2 to 1.2 M NaCl in the dialysis buffer at 0.35 M NaCl. Fractions were collected and analyzed by SDS-PAGE and Western blot, using a monoclonal antibody against the T7 tag. The protein migrated as a single band of apparent molecular mass 45 kDa. The total amount of pure PARP recovered was 3 mg, which corresponds to 30% yield of purification.In Vitro Cleavage of Pre-IL-1β and PARP by ICE and Homologs35S-Labeled pre-IL-1β and PARP proteins, which contain 12 and 11 methionine residues, respectively, were prepared by in vitro transcription-translation (IVTT) using the TNT T7-coupled reticulocyte lysate system (Promega) and [35S]methionine (500 Ci/mmol, Amersham Corp.).Cleavage experiments were performed using one or both of two methods. The first method, in which the amount of radioactivity per assay was kept constant, 35S-labeled substrate (40 nM/0.5 μCi) was incubated in reaction mixtures of 25 μl containing 10-40 nM enzyme and varying amounts (100 nM to 10 μM) of unlabeled protein substrate in 10 mM Tris-HCl buffer, pH 7.5, 0.1% CHAPS, 1 mM DTT, 37°C. Aliquots were removed, quenched by the addition of sample buffer, and applied to Novex Tris-glycine 4-20% gradient denaturing gels. After gel electrophoresis and autoradiography, the concentrations of cleavage products were determined by densitometry. Alternatively, the specific activity of the substrate was kept constant and a stock containing both 35S-labeled IVTT substrate and unlabeled protein substrate was prepared and used identically in reaction mixtures as described above. For both methods, calculation of kinetic parameters was performed by nonlinear least squares fitting of the rate versus concentration data using the commercial program Enzfitter (Biosoft).Spectrophotometric AssaysSynthetic peptidyl substrates were purchased from Bachem and corrected for purity by HPLC analysis on a Vydac C18 column (4.5 × 250 mm) using a water/acetonitrile gradient in 0.1% trifluoroacetic acid. Purity was also assessed by exhaustive enzymatic digestion, followed by quantification of the chromophoric leaving group by comparison to a standard curve of either p-nitroaniline or aminomethylcoumarin under identical assay conditions.Assays were conducted in 96-well microtiter plates as described previously and contained: 65 μl of assay buffer (10 mM Tris, 1 mM DTT, 0.1% CHAPS, pH 7.5), 10 μl of enzyme solution (final concentration 2-40 nM), 5 μl of Me2SO containing the inhibitor and 20 μl of substrate (34Mullican M.D. Lauffer D.J. Gillespie R.J. Matharu S.S. Kay D. Porritt G.M. Evans P.L. Golec J.M.C. Murcko M.A. Luong Y.-P. Raybuck S.A. Livingston D.J. Bioorg. & Med. Chem. Lett. 1994; 4: 2359-2364Crossref Scopus (28) Google Scholar). Production of p-nitroaniline (pNA) from reaction mixtures containing Suc-YVAD-pNA was measured by following the absorbance at 405 nm minus that at 650 nm using a Thermomax visible plate reader from Molecular Devices. Production of 7-amino-4-methylcoumarin (AMC) from enzyme catalyzed cleavage of Ac-YVAD-AMC or Ac-DEVD-AMC was measured fluorometrically on a Perkin Elmer LS50B equipped with microtiter plate capabilities and temperature control from a circulating water bath. The time course of AMC production was followed using an excitation wavelength of 360 nm and an emission wavelength of 480 nm, with slit widths of 5 and 15 nm, respectively. ICE-catalyzed cleavage of the resonance energy transfer substrate, 4-(4′-dimethylaminophenylazo)benzoic acid-YVADAPV-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (sodium salt), was monitored fluorometrically on the same instrument above using an excitation wavelength of 340 nM, an emission wavelength of 490 nM, with slit widths of 10 and 15 nm. Product was quantified with correction for the inner filter effect using standard curves from mixtures of 4-(4′-dimethylaminophenylazo)benzoic acid and 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (sodium salt).Peptidyl inhibitors used in this study were purchased from Bachem or synthesized in-house and had a purity of >95% as quantified by HPLC. Ac-YVAD-AMC or Suc-YVAD-pNA at a concentration of 2-4 times the Km value was used as a substrate for ICE or TX assays. Ac-DEVD-AMC at a concentration of 2-4 times the Km value was used as the substrate when CPP32 (Km = 10 μM) or CMH-1 (Km = 100 μM) were present.Assays containing reversible inhibitors were conducted as above in duplicate with the inhibitor and enzyme preincubated in the microtiter plate for 15 min at 30°C. The assay was started by the addition of substrate, and the initial rates were determined from progress curves at early reaction times. Ki values were calculated from rate versus inhibitor data with nonlinear least squares fitting to the tight binding equation of Morrison (35Morrison J.F. Biochim. Biophys. Acta. 1969; 185: 269-286Crossref PubMed Scopus (712) Google Scholar). A commercial program, KineTic, was used for this purpose. Reported values are from at least two replicate determinations, and standard errors are not larger than 20%.Second order rate constants (k) for irreversible inhibitors of ICE homologs were determined from assays where the reaction buffer and containing inhibitor and substrate were preincubated at 37°C and the reaction initiated by the addition of enzyme warmed to 37°C. Progress curves of product versus time w
Referência(s)