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Proteome-wide Substrate Analysis Indicates Substrate Exclusion as a Mechanism to Generate Caspase-7 Versus Caspase-3 Specificity

2009; Elsevier BV; Volume: 8; Issue: 12 Linguagem: Inglês

10.1074/mcp.m900310-mcp200

ISSN

1535-9484

Autores

Dieter Demon, Petra Van Damme, Tom Vanden Berghe, Annelies Deceuninck, Joost Van Durme, Jelle Verspurten, Kenny Helsens, Francis Impens, Magdalena Wejda, Joost Schymkowitz, Frédéric Rousseau, Annemieke Madder, Joël Vandekerckhove, Wim Declercq, Kris Gevaert, Peter Vandenabeele,

Tópico(s)

Machine Learning in Bioinformatics

Resumo

Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6–P5′ undecapeptide retained complete specificity for caspase-7. The corresponding P6–P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P′ residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4–P1 residues constitute the core cleavage site but that P6, P5, P2′, and P3′ residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7. Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6–P5′ undecapeptide retained complete specificity for caspase-7. The corresponding P6–P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P′ residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4–P1 residues constitute the core cleavage site but that P6, P5, P2′, and P3′ residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7. Caspases, a family of evolutionarily conserved proteases, mediate apoptosis, inflammation, proliferation, and differentiation by cleaving many cellular substrates (1Lamkanfi M. Festjens N. Declercq W. Vanden Berghe T. Vandenabeele P. Caspases in cell survival, proliferation and differentiation.Cell Death Differ. 2007; 14: 44-55Crossref PubMed Scopus (459) Google Scholar, 2Timmer J.C. Salvesen G.S. Caspase substrates.Cell Death Differ. 2007; 14: 66-72Crossref PubMed Scopus (333) Google Scholar, 3Lüthi A.U. Martin S.J. The CASBAH: a searchable database of caspase substrates.Cell Death Differ. 2007; 14: 641-650Crossref PubMed Scopus (345) Google Scholar). The apoptotic initiator caspases (caspase-8, -9, and -10) are activated in large signaling platforms and propagate the death signal by cleavage-induced activation of executioner caspase-3 and -7 (4Boatright K.M. Salvesen G.S. Mechanisms of caspase activation.Curr. Opin. Cell Biol. 2003; 15: 725-731Crossref PubMed Scopus (1084) Google Scholar, 5Riedl S.J. Salvesen G.S. The apoptosome: signalling platform of cell death.Nat. Rev. Mol. Cell Biol. 2007; 8: 405-413Crossref PubMed Scopus (861) Google Scholar). 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The substrate degradomes of the two main executioner caspases have not been determined but their identification is important to gaining greater insight in their cleavage specificity and biological functions. The specificity of caspases was rigorously profiled by using combinatorial tetrapeptide libraries (8Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1852) Google Scholar), proteome-derived peptide libraries (9Schilling O. Overall C.M. Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites.Nat. Biotechnol. 2008; 26: 685-694Crossref PubMed Scopus (320) Google Scholar), and sets of individual peptide substrates (10Talanian R.V. Quinlan C. Trautz S. Hackett M.C. Mankovich J.A. Banach D. Ghayur T. Brady K.D. Wong W.W. Substrate specificities of caspase family proteases.J. Biol. Chem. 1997; 272: 9677-9682Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar, 11Stennicke H.R. Renatus M. Meldal M. Salvesen G.S. Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8.Biochem. J. 2000; 350: 563-568Crossref PubMed Scopus (264) Google Scholar). The results of these studies indicate that specificity motifs for caspase-3 and -7 are nearly indistinguishable with the canonical peptide substrate, DEVD, used to monitor the enzymatic activity of both caspase-3 and -7 in biological samples. This overlap in cleavage specificity is manifested in their generation of similar cleavage fragments from a variety of apoptosis-related substrates such as inhibitor of caspase-activated DNase, keratin 18, PARP, 1The abbreviations used are:PARPpoly(ADP-ribose) polymeraseamc7-amino-4-methylcoumarineAbz2-amino-benzoic acidCFScell-free systemCOFRADICcombined fractional diagonal chromatographyIAAiodoacetamidekcatcatalytic constantKmMichaelis-Menten constantY(NO2)3-nitrotyrosineRPreverse phaseSILACstable isotope labeling by amino acids in cell culturezVAD-fmkbenzyloxycarbonyl-valine-alanine-aspartic acid(OMe)fluoromethyl ketonepNAp-nitroanilideFmocN-(9-fluorenyl)methoxycarbonylwtwild typeC3caspase-3C7caspase-7HDGFhepatoma-derived growth factorOtButert-butyl ester. protein-disulfide isomerase, and Rho kinase I (for reviews, see Refs. 2Timmer J.C. Salvesen G.S. Caspase substrates.Cell Death Differ. 2007; 14: 66-72Crossref PubMed Scopus (333) Google Scholar, 3Lüthi A.U. Martin S.J. The CASBAH: a searchable database of caspase substrates.Cell Death Differ. 2007; 14: 641-650Crossref PubMed Scopus (345) Google Scholar, and 7Fischer U. Jänicke R.U. Schulze-Osthoff K. Many cuts to ruin: a comprehensive update of caspase substrates.Cell Death Differ. 2003; 10: 76-100Crossref PubMed Scopus (891) Google Scholar). This propagated the view that these two caspases have completely redundant functions during apoptosis. Surprisingly, mice deficient in one of these caspases (as well as mice deficient in both) have distinct phenotypes. Depending on the genetic background of the mice, caspase-3-deficient mice either die before birth (129/SvJ) or develop almost normally (C57BL/6J) (12Lakhani S.A. Masud A. Kuida K. Porter Jr., G.A. Booth C.J. Mehal W.Z. Inayat I. Flavell R.A. Caspases 3 and 7: key mediators of mitochondrial events of apoptosis.Science. 2006; 311: 847-851Crossref PubMed Scopus (923) Google Scholar, 13Kuida K. Zheng T.S. Na S. Kuan C. Yang D. Karasuyama H. Rakic P. Flavell R.A. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.Nature. 1996; 384: 368-372Crossref PubMed Scopus (1713) Google Scholar, 14Woo M. Hakem R. Soengas M.S. Duncan G.S. Shahinian A. Kägi D. Hakem A. McCurrach M. Khoo W. Kaufman S.A. Senaldi G. Howard T. Lowe S.W. Mak T.W. Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes.Genes Dev. 1998; 12: 806-819Crossref PubMed Scopus (768) Google Scholar). This suggests that dynamics in the genetic background, such as increased caspase-7 expression, compensate for the functional loss of caspase-3 (15Houde C. Banks K.G. Coulombe N. Rasper D. Grimm E. Roy S. Simpson E.M. Nicholson D.W. Caspase-7 expanded function and intrinsic expression level underlies strain-specific brain phenotype of caspase-3-null mice.J. Neurosci. 2004; 24: 9977-9984Crossref PubMed Scopus (81) Google Scholar). In the C57BL/6J background, caspase-7 single deficient mice are also viable, whereas caspase-3 and -7 double deficient mice die as embryos, further suggesting redundancy (12Lakhani S.A. Masud A. Kuida K. Porter Jr., G.A. Booth C.J. Mehal W.Z. Inayat I. Flavell R.A. Caspases 3 and 7: key mediators of mitochondrial events of apoptosis.Science. 2006; 311: 847-851Crossref PubMed Scopus (923) Google Scholar, 13Kuida K. Zheng T.S. Na S. Kuan C. Yang D. Karasuyama H. Rakic P. Flavell R.A. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.Nature. 1996; 384: 368-372Crossref PubMed Scopus (1713) Google Scholar, 14Woo M. Hakem R. Soengas M.S. Duncan G.S. Shahinian A. Kägi D. Hakem A. McCurrach M. Khoo W. Kaufman S.A. Senaldi G. Howard T. Lowe S.W. Mak T.W. Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes.Genes Dev. 1998; 12: 806-819Crossref PubMed Scopus (768) Google Scholar). However, because caspase-3 and -7 probably arose from gene duplication between the Cephalochordata-Vertebrata diversion (16Bayascas J.R. Yuste V.J. Benito E. Garcia-Fernàndez J. Comella J.X. Isolation of AmphiCASP-3/7, an ancestral caspase from amphioxus (Branchiostoma floridae). Evolutionary considerations for vertebrate caspases.Cell Death Differ. 2002; 9: 1078-1089Crossref PubMed Scopus (36) Google Scholar), they might have acquired different substrate specificities during evolution. Caspase-3 and -7 do exhibit different activities on a few arbitrarily identified natural substrates, including BID, X-linked inhibitor of apoptosis protein, gelsolin, caspase-6, ataxin-7, and co-chaperone p23 (17Walsh J.G. Cullen S.P. Sheridan C. Lüthi A.U. Gerner C. Martin S.J. Executioner caspase-3 and caspase-7 are functionally distinct proteases.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12815-12819Crossref PubMed Scopus (415) Google Scholar, 18Young J.E. Gouw L. Propp S. Sopher B.L. Taylor J. Lin A. Hermel E. Logvinova A. Chen S.F. Chen S. Bredesen D.E. Truant R. Ptacek L.J. La Spada A.R. Ellerby L.M. Proteolytic cleavage of ataxin-7 by caspase-7 modulates cellular toxicity and transcriptional dysregulation.J. Biol. Chem. 2007; 282: 30150-30160Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 19Ju W. Valencia C.A. Pang H. Ke Y. Gao W. Dong B. Liu R. Proteome-wide identification of family member-specific natural substrate repertoire of caspases.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 14294-14299Crossref PubMed Scopus (45) Google Scholar, 20Song J.J. Lee Y.J. Differential cleavage of Mst1 by caspase-7/-3 is responsible for TRAIL-induced activation of the MAPK superfamily.Cell. Signal. 2008; 20: 892-906Crossref PubMed Scopus (59) Google Scholar). In addition, caspase-3 generally cleaves more substrates during apoptosis than caspase-7 and therefore appears to be the major executioner caspase. Moreover, a recent report describing caspase-1-dependent activation of caspase-7, but not of caspase-3, in macrophages in response to microbial stimuli supports the idea of a non-redundant function for caspase-7 downstream of caspase-1 (21Lamkanfi M. Kanneganti T.D. Van Damme P. Vanden Berghe T. Vanoverberghe I. Vandekerckhove J. Vandenabeele P. Gevaert K. Núñez G. Targeted peptidecentric proteomics reveals caspase-7 as a substrate of the caspase-1 inflammasomes.Mol. Cell. Proteomics. 2008; 7: 2350-2363Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). poly(ADP-ribose) polymerase 7-amino-4-methylcoumarine 2-amino-benzoic acid cell-free system combined fractional diagonal chromatography iodoacetamide catalytic constant Michaelis-Menten constant 3-nitrotyrosine reverse phase stable isotope labeling by amino acids in cell culture benzyloxycarbonyl-valine-alanine-aspartic acid(OMe)fluoromethyl ketone p-nitroanilide N-(9-fluorenyl)methoxycarbonyl wild type caspase-3 caspase-7 hepatoma-derived growth factor tert-butyl ester. Commercially available "caspase-specific" tetrapeptide substrates are widely used for specific caspase detection, but they display substantial promiscuity and cannot be used to monitor individual caspases in cells (22Agniswamy J. Fang B. Weber I.T. Plasticity of S2-S4 specificity pockets of executioner caspase-7 revealed by structural and kinetic analysis.FEBS J. 2007; 274: 4752-4765Crossref PubMed Scopus (47) Google Scholar, 23McStay G.P. Salvesen G.S. Green D.R. Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways.Cell Death Differ. 2008; 15: 322-331Crossref PubMed Scopus (258) Google Scholar). Detecting proteolysis by measuring the release of C-terminal fluorophores, such as 7-amino-4-methylcoumarin (amc), restricts the specificity of these peptide substrates to non-prime cleavage site residues, which may have hampered the identification of specific cleavage events. To address this limitation, a recently developed proteomics technique, called proteomic identification of protease cleavage sites, was used to map both non-prime and prime preferences for caspase-3 and -7 on a tryptic peptide library (9Schilling O. Overall C.M. Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites.Nat. Biotechnol. 2008; 26: 685-694Crossref PubMed Scopus (320) Google Scholar). However, no clear distinction in peptide recognition motifs between caspase-3 and -7 could be observed (9Schilling O. Overall C.M. Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites.Nat. Biotechnol. 2008; 26: 685-694Crossref PubMed Scopus (320) Google Scholar). Because not all classical caspase cleavage sites are processed (7Fischer U. Jänicke R.U. Schulze-Osthoff K. Many cuts to ruin: a comprehensive update of caspase substrates.Cell Death Differ. 2003; 10: 76-100Crossref PubMed Scopus (891) Google Scholar), structural or post-translational higher order constraints are likely involved in steering the cleavage site selectivity. Peptide-based approaches generally overlook such aspects. We made use of the COFRADIC N-terminal peptide sorting methodology (24Gevaert K. Goethals M. Martens L. Van Damme J. Staes A. Thomas G.R. Vandekerckhove J. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.Nat. Biotechnol. 2003; 21: 566-569Crossref PubMed Scopus (508) Google Scholar, 25Staes A. Van Damme P. Helsens K. Demol H. Vandekerckhove J. Gevaert K. Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC).Proteomics. 2008; 8: 1362-1370Crossref PubMed Scopus (129) Google Scholar, 26Van Damme P. Maurer-Stroh S. Plasman K. Van Durme J. Colaert N. Timmerman E. De Bock P.J. Goethals M. Rousseau F. Schymkowitz J. Vandekerckhove J. Gevaert K. Analysis of protein processing by N-terminal proteomics reveals novel species-specific substrate determinants of granzyme B orthologs.Mol. Cell. Proteomics. 2009; 8: 258-272Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) to profile proteolytic events of caspase-3 and -7 in a macrophage proteome labeled by triple stable isotope labeling by amino acids in cell culture (SILAC), which allowed direct comparison of peak intensities in peptide MS spectra and consequent quantification of N termini that are equally, preferably, or exclusively generated by the action of caspase-3 or -7 (26Van Damme P. Maurer-Stroh S. Plasman K. Van Durme J. Colaert N. Timmerman E. De Bock P.J. Goethals M. Rousseau F. Schymkowitz J. Vandekerckhove J. Gevaert K. Analysis of protein processing by N-terminal proteomics reveals novel species-specific substrate determinants of granzyme B orthologs.Mol. Cell. Proteomics. 2009; 8: 258-272Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 27Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4625) Google Scholar). We identified 55 cleavage sites in 48 protein substrates, encompassing mutual, preferred, and unique caspase-3 and -7 cleavage sites. Expression and purification of recombinant mouse caspase-3 and -7 have been described (28Van de Craen M. Declercq W. Van den brande I. Fiers W. Vandenabeele P. The proteolytic procaspase activation network: an in vitro analysis.Cell Death Differ. 1999; 6: 1117-1124Crossref PubMed Scopus (175) Google Scholar). Activity of both caspases was determined by active site titration using a serial dilution of zVAD-fmk (25 min at 37 °C) in cell-free system (CFS) buffer containing 220 mm mannitol, 170 mm sucrose, 5 mm NaCl, 5 mm MgCl2, 10 mm HEPES, pH 7.5, 2.5 mm KH2PO4, 2.1 µm leupeptin, 0.15 µm aprotinin, 100 µm PMSF, and 10 mm DTT. Proteolytic activity was quantified on Ac-DEVD-amc as described below (data not shown). Activity was also quantified on proendothelial monocyte-activating polypeptide II, the p43 component of the aminoacyl-tRNA complex as described previously (29Behrensdorf H.A. van de Craen M. Knies U.E. Vandenabeele P. Clauss M. The endothelial monocyte-activating polypeptide II (EMAP II) is a substrate for caspase-7.FEBS Lett. 2000; 466: 143-147Crossref PubMed Scopus (67) Google Scholar) (data not shown). Recombinant mouse caspase-3 or -7 was incubated with 100 µm fluorogenic Ac-DEVD-amc peptide (Peptide Institute, Osaka, Japan) in 150 µl of CFS buffer. The generation of free amc was continuously monitored for 50 min in a fluorometer (CytoFluor, PerSeptive Biosystems) at 360/460-nm excitation/emission wavelengths. The linear rate of fluorophore generation was used to quantify caspase activity. Fmoc-Asp(OtBu)-OH was converted to the corresponding amide using amc. After side chain deprotection, the Fmoc-Asp-amc residue was attached to 2-chlorotrityl resin followed by automated peptide synthesis using an O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate/diisopropylethylamine protocol. Subsequently, following N-terminal acetylation, acidic cleavage was done from the solid support. Finally, purification by precipitation and lyophilization yielded the C-terminal labeled Ac-peptide-amc. Identical kinetic values were obtained with commercial or in-house synthesized Ac-DEVD-amc, illustrating sufficiently high quality of in-house made peptides (Table I).Table IKinetic parameters of DEVD- and DVKD-based peptide cleavage by caspase-7 and -3SubstratekcatKmkcat/KmC7C3C7C3C7C3s−1µmm−1 s−1DEVDaCommercial source.0.17 ± 0.008.32 ± 0.329.0 ± 0.6108 ± 1418,900 ± 1,28076,500 ± 10,800DEVDbSynthesized in house.0.14 ± 0.008.39 ± 0.477.9 ± 0.6112 ± 1117,800 ± 1,38074,700 ± 8,700DVKDbSynthesized in house.0.23 ± 0.010.74 ± 0.0353 ± 6267 ± 274,330 ± 5902,790 ± 312ADVKDbSynthesized in house.0.11 ± 0.000.77 ± 0.0335 ± 4243 ± 273,080 ± 4373,180 ± 389KDVKDbSynthesized in house.0.59 ± 0.020.79 ± 0.03133 ± 13407 ± 364,390 ± 4941,940 ± 188AKDVKDbSynthesized in house.0.63 ± 0.020.48 ± 0.01129 ± 11439 ± 274,840 ± 4441,100 ± 77QKDVKDbSynthesized in house.0.43 ± 0.020.19 ± 0.0192 ± 12356 ± 394,690 ± 657560 ± 69"Quencher"cAbz-QKDVKDGKYSQY(NO2).0.25 ± 0.020.005 ± 0.001173 ± 27192 ± 681,450 ± 25326 ± 10a Commercial source.b Synthesized in house.c Abz-QKDVKDGKYSQY(NO2). Open table in a new tab 50 nm recombinant caspase-3 or -7 was incubated with a serial dilution of DEVD- or DVKD-based fluorogenic amc-linked substrates (concentration ranging between 1 and 1200 µm) in CFS buffer in a final volume of 80 µl. Similarly, 100 nm recombinant caspase-3 or -7 was incubated with a serial dilution of Abz-QKDVKDGKYSQY(NO2) (synthesized using the Fmoc chemistry on an Applied Biosystems 433A Peptide Synthesizer; Y(NO2) denotes a 3-nitrotyrosine residue). Generation of free amc or Abz was monitored as described above. The initial linear rates of fluorescence at all concentrations of the substrate were used to obtain plots of activity versus substrate concentration. The Michaelis-Menten (Km) and the catalytic constant (kcat) were determined from these plots. Absolute Km and kcat values were calculated using a standard curve determined with free amc. pCMV-SPORT6-GSTP1, pCMV- SPORT6-HDGF, pCMV-SPORT6-MYBBP1a, pCMV-SPORT6-NUCKS1, pCMV-SPORT6-PKM2, pCMV-SPORT6-RPL28, pCMV-SPORT6.1-MKI67ip, pYX-ASC-FAM21 (all from the German Resource Center for Genome Research (RZPD), Berlin, Germany), pFLCI-ACTA2 (from Geneservice Ltd.), and the four pCR3-based plasmids described under "Plasmids" were used as templates for [35S]methionine (PerkinElmer Life Sciences)-radiolabeled in vitro coupled transcription/translation in a rabbit reticulocyte lysate system according to the manufacturer's instructions (Promega). Before caspase treatment, the translated protein samples were alkylated with 5 mm iodoacetamide (IAA) for 30 min at 30 °C in the dark. Excess IAA was removed by making use of protein desalting spin columns (Perbio Science). The desalted translated proteins (2 µl) were subsequently incubated for 1.5 h at 37 °C in CFS buffer with the indicated concentrations of recombinant caspases in a total volume of 30 µl. Next, samples were boiled for 10 min after addition of Laemmli buffer, separated by 10, 15, or 20% SDS-PAGE, and transferred to a nitrocellulose membrane (Schleicher & Schuell) by semidry blotting. The blotted membranes were sealed to keep them humid and exposed to a film (Amersham Biosciences Hyperfilm™ ECL) for radiography. The cDNA encoding RPS18 was amplified by PCR from the pYX-ASC-RPS18 vector (RZPD) using the following primer pair: RPS18-forward, 5′-CTGAATTCGCCATGTCTCTAGTGATCCC-3′, and RPS18-reverse, 5′-ATATGCGGCCGCTCATTTCTTCTTGGATACACCC-3′. The amplified product was digested with EcoRI and NotI and cloned in a multiple cloning site-modified pCR3 vector (Invitrogen). The cDNA encoding the "ADVKD" mutant of RPS18 was generated by overlap PCR technology using the following primer pair: RPS18-K88A-forward, 5′-AACAGACAGGCGGATGTGAAGGATGGGAAG-3′, and RPS18-K88A-reverse, 5′-TTCACATCCGCCTGTCTGTTCAGGAACCAG-3′. The cDNA encoding PARP was amplified by PCR from the pGEM-PARP1 plasmid using the following primer pair: PARP-forward, 5′-ATCTGCGGCCGCTCATGGCGGAGTCTTCGGATAAGC-3′, and PARP-reverse, 5′-TATAGCGGCCGCTCCCAATTACCACAGGGAGGTC-3′. The amplified product was digested with NotI and cloned into a multiple cloning site-modified pCR3 vector (Invitrogen). The cDNA encoding the "KDEVD" mutant of PARP was generated by overlap PCR technology using the following primer pair: PARP-G210K-forward, 5′-AGAGAAAAAAGGATGAGGTGGATGGAGTGG-3′, and PARP-G210K-reverse, 5′-ACCTCATCCTTTTTTCTCTTTCCTTCACTCTTGC-3′. The cDNAs encoding caspase-3 and -7 devoid of their prodomain (i.e. caspase-p30) were cloned in the pLT10TH vector downstream of an N-terminal His6 tag as described (28Van de Craen M. Declercq W. Van den brande I. Fiers W. Vandenabeele P. The proteolytic procaspase activation network: an in vitro analysis.Cell Death Differ. 1999; 6: 1117-1124Crossref PubMed Scopus (175) Google Scholar). All plasmids were sequence-verified. Mf4/4 (30Desmedt M. Rottiers P. Dooms H. Fiers W. Grooten J. Macrophages induce cellular immunity by activating Th1 cell responses and suppressing Th2 cell responses.J. Immunol. 1998; 160: 5300-5308PubMed Google Scholar) mouse macrophages were grown in lipopolysaccharide-free RPMI 1640 medium (Invitrogen) containing either l-[12C6]arginine, l-[13C6]arginine, or l-[13C6,15N4]arginine (Cambridge Isotope Laboratories, Andover, MA) at a concentration of 287 µm (i.e. 25% of the normal concentration in RPMI 1640 medium at which l-arginine to proline conversion was not observed). Cell populations were cultured at 37 °C in a humidified 5% CO2 atmosphere for at least five doublings for complete incorporation of the labeled l-arginine. Mf4/4 cells were detached using enzyme-free cell dissociation buffer (Invitrogen), washed in D-PBS (Dulbecco's PBS), resuspended in D-PBS supplemented with 2.1 µm leupeptin, 0.15 µm aprotinin, 100 µm PMSF, and 1 mm oxidized glutathione, and freeze-thawed three times. Lysates were cleared by centrifugation for 15 min at 20,000 × g. Proteins were alkylated with 5 mm iodoacetamide for 30 min at 30 °C in the dark, and to remove excess IAA they were separately desalted on NAP-10 columns in D-PBS supplemented with leupeptin, aprotinin, PMSF, and oxidized glutathione. Subsequently, the separate samples were supplemented with DTT (10 mm final concentration) and incubated with either 130 nm recombinant caspase-7 (l-[12C6]Arg) or -3 (l-[13C6]Arg) or left untreated (l-[13C6,15N4]Arg) for 1.5 h at 37 °C. Solid guanidinium hydrochloride was then added to a final concentration of 4 m followed by downstream analysis as described previously (24Gevaert K. Goethals M. Martens L. Van Damme J. Staes A. Thomas G.R. Vandekerckhove J. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.Nat. Biotechnol. 2003; 21: 566-569Crossref PubMed Scopus (508) Google Scholar, 25Staes A. Van Damme P. Helsens K. Demol H. Vandekerckhove J. Gevaert K. Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC).Proteomics. 2008; 8: 1362-1370Crossref PubMed Scopus (129) Google Scholar, 26Van Damme P. Maurer-Stroh S. Plasman K. Van Durme J. Colaert N. Timmerman E. De Bock P.J. Goethals M. Rousseau F. Schymkowitz J. Vandekerckhove J. Gevaert K. Analysis of protein processing by N-terminal proteomics reveals novel species-specific substrate determinants of granzyme B orthologs.Mol. Cell. Proteomics. 2009; 8: 258-272Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). ESI LC-MS/MS analysis was performed as described before (26Van Damme P. Maurer-Stroh S. Plasman K. Van Durme J. Colaert N. Timmerman E. De Bock P.J. Goethals M. Rousseau F. Schymkowitz J. Vandekerckhove J. Gevaert K. Analysis of protein processing by N-terminal proteomics reveals novel species-specific substrate determinants of granzyme B orthologs.Mol. Cell. Proteomics. 2009; 8: 258-272Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). ESI-Q-TOF MS/MS peptide fragmentation spectra were converted to pkl files using the MassLynx® software (version 4.1, Waters Corp.), and ESI ion trap MS/MS spectra were converted to mgf files using the Automation Engine software (version 3.2, Bruker). Peptides were identified using a locally installed version of the Mascot database search engine version 2.1 (Matrix Science) and the Swiss-Prot (version 53.2 of UniProtKB/Swiss-Prot protein database, containing 269,293 sequence entries of which 13,316 originated from mouse protein sequences) and TrEMBL databases (version 35.0 of UniProtKB/TrEMBL protein database, containing 3,874,166 sequence entries comprising 52,403 mouse protein entries) were searched with restriction to mouse proteins. Truncated peptide databases made by DBToolkit (31Martens L. Vandekerckhove J. Gevaert K. DBToolkit: processing protein databases for peptide-centric proteomics.Bioinformatics. 2005; 21: 3584-3585Crossref PubMed Scopus (121) Google Scholar) were searched in parallel to pick up protein processing events more efficiently (e.g. Refs. 6Van Damme P. Martens L. Van Damme J. Hugelier K. Staes A. Vandekerckhove J. Gevaert K. Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis.Nat. Methods. 2005; 2: 771-777Crossref PubMed Scopus (206) Google Scholar and 32Vande Walle L. Van Damme P. Lamkanfi M. Saelens X. Vandekerckhove J. Gevaert K. Vandenabeele P. Proteome-wide identification of HtrA2/Omi substrates.J. Proteome Res. 2007; 6: 1006-1015Crossref PubMed Scopus (103) Google Scholar). The following search parameters were used. Peptide mass tolerance was set at 0.2 Da, and peptide fragment mass tolerance was set at 0.1 Da with the "ESI-QUAD-TOF" as selected instrument for peptide fragmentation rules for the Q-TOF Premier data. For ion trap data, peptide mass tolerance was set at 0.5 Da, and peptide fragment mass tolerance was set at 0.5 Da with the "ESI-IT" as selected instrument for peptide fragmentation rules. Endoproteinase Arg-C/P was set as the enzyme with a maximum number of one missed cleavage. Peptide charge was set to 1+, 2+, and 3+. Variable modifications were set to methionine oxidation (to methionine sulfoxide), pyroglutamate formation of N-terminal glutamine, pyrocarbamidomethyl formation of N-terminal alkylated cysteine, deamidation of asparagine, ace

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