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Type II Metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana Cleave Substrates after Arginine and Lysine

2004; Elsevier BV; Volume: 279; Issue: 44 Linguagem: Inglês

10.1074/jbc.m406329200

1083-351X

Dominique Vercammen, Brigitte van de Cotte, Geert De Jaeger, Dominique Eeckhout, Peter Casteels, Klaas Vandepoele, Isabel Vandenberghe, Jozef Van Beeumen, Dirk Inzé, Frank Van Breusegem,

Insect Resistance and Genetics

Nine potential caspase counterparts, designated metacaspases, were identified in the Arabidopsis thaliana genome. Sequence analysis revealed two types of metacaspases, one with (type I) and one without (type II) a proline- or glutamine-rich N-terminal extension, possibly representing a prodomain. Production of recombinant Arabidopsis type II metacaspases in Escherichia coli resulted in cysteine-dependent autocatalytic processing of the proform into large and small subunits, in analogy to animal caspases. A detailed biochemical characterization with a broad range of synthetic oligopeptides and several protease inhibitors of purified recombinant proteins of both metacaspase 4 and 9 showed that both metacaspases are arginine/lysine-specific cysteine proteases and did not cleave caspase-specific synthetic substrates. These findings suggest that type II metacaspases are not directly responsible for earlier reported caspase-like activities in plants. Nine potential caspase counterparts, designated metacaspases, were identified in the Arabidopsis thaliana genome. Sequence analysis revealed two types of metacaspases, one with (type I) and one without (type II) a proline- or glutamine-rich N-terminal extension, possibly representing a prodomain. Production of recombinant Arabidopsis type II metacaspases in Escherichia coli resulted in cysteine-dependent autocatalytic processing of the proform into large and small subunits, in analogy to animal caspases. A detailed biochemical characterization with a broad range of synthetic oligopeptides and several protease inhibitors of purified recombinant proteins of both metacaspase 4 and 9 showed that both metacaspases are arginine/lysine-specific cysteine proteases and did not cleave caspase-specific synthetic substrates. These findings suggest that type II metacaspases are not directly responsible for earlier reported caspase-like activities in plants. Primarily based on morphological features, animal cell death is usually referred to as apoptosis or necrosis. Apoptosis is characterized by membrane blebbing, cytosolic condensation, cell shrinkage, nuclear condensation, breakdown of nuclear DNA, and finally, the formation of apoptotic bodies that can easily be taken up by other cells (1Fiers W. Beyaert R. Declercq W. Vandenabeele P. Oncogene. 1999; 18: 7719-7730Crossref PubMed Scopus (744) Google Scholar). Necrosis, as defined on a microscopic level, denotes cell death in which cells swell, round up, and then suddenly collapse, spilling their contents into the medium. However, in animals, other forms of cell death exist, such as autophagic and autolytic death and intermediate varieties (2Lockshin R.A. Zakeri Z. Curr. Opin. Cell Biol. 2002; 14: 727-733Crossref PubMed Scopus (205) Google Scholar). In plants “programmed cell death” usually denotes apoptosis-like cell death characterized by chromatin aggregation, cell shrinkage, cytoplasmic, and nuclear condensation, and DNA fragmentation (3Buckner B. Johal G.S. Janick-Buckner D. Physiol. Plant. 2000; 108: 231-239Crossref Scopus (49) Google Scholar, 4Jabs T. Biochem. Pharmacol. 1999; 57: 231-245Crossref PubMed Scopus (494) Google Scholar, 5O'Brien I.E.W. Murray B.G. Baguley B.C. Morris B.A. Ferguson I.B. Exp. Cell Res. 1998; 241: 46-54Crossref PubMed Scopus (40) Google Scholar). Apoptotic characteristics have been observed during hypersensitive response and after abiotic stress, such as exposure to ozone, UV irradiation, chilling, and salt stress (6Danon A. Gallois P. FEBS Lett. 1998; 437: 131-136Crossref PubMed Scopus (141) Google Scholar, 7Katsuhara M. Plant Cell Physiol. 1997; 38: 1091-1093Crossref Scopus (99) Google Scholar, 8Kratsch H.A. Wise R.R. Plant Cell Environ. 2000; 23: 337-350Crossref Scopus (365) Google Scholar, 9Dat J.F. Pellinen R. Beeckman T. Van De Cotte B. Langebartels C. Kangasjärvi J. Inzé D. Van Breusegem F. Plant J. 2003; 33: 621-632Crossref PubMed Scopus (259) Google Scholar, 10Pennell R.I. Lamb C. Plant Cell. 1997; 9: 1157-1168Crossref PubMed Scopus (657) Google Scholar). On a biochemical level, apoptosis in animals is characterized and commonly defined by the activation of a family of cysteine-dependent aspartate-specific proteases, or caspases (11Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar). Caspases can proteolytically activate downstream caspases or cut various cellular substrates, resulting in a plethora of structural and metabolic alterations, ultimately leading to cell death (11Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar, 12Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4084) Google Scholar, 13Utz P.J. Anderson P. Cell Death Differ. 2000; 7: 589-602Crossref PubMed Scopus (142) Google Scholar). Caspases hydrolyze peptide bonds at the C-terminal side of an aspartate, the so-called P1 residue. By using a variety of synthetic oligopeptide caspase substrates and inhibitors with an aspartate at this P1 position, caspase-like activity has recently been demonstrated in various plant cell death models (14Bozhkov P.V. Filonova L.H. Suarez M.F. Helmersson A. Smertenko A.P. Zhivotovsky B. von Arnold S. Cell Death Differ. 2004; 11: 175-182Crossref PubMed Scopus (124) Google Scholar, 15De Jong A.J. Hoeberichts F.A. Yakimova E.T. Maximova E. Woltering E.J. Planta. 2000; 211: 656-662Crossref PubMed Scopus (114) Google Scholar, 16del Pozo O. Lam E. Curr. Biol. 1998; 8: 1129-1132Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 17Korthout H.A. Berecki G. Bruin W. van Duijn B. Wang M. FEBS Lett. 2000; 475: 139-144Crossref PubMed Scopus (92) Google Scholar). However, the corresponding genes for these activities have never been identified. Previously, two families of distant caspase homologues in plants, fungi, protozoa, and animals have been reported; that is, paracaspases, restricted to the metazoa, and metacaspases, identified in plants, fungi, and protozoa (18Madeo F. Herker E. Maldener C. Wissing S. Lachelt S. Herlan M. Fehr M. Lauber K. Sigrist S.J. Wesselborg S. Frohlich K.U. Mol. Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 19Szallies A. Kubata B.K. Duszenko M. FEBS Lett. 2002; 517: 144-150Crossref PubMed Scopus (110) Google Scholar, 20Uren A.G. O'Rourke K. Aravind L.A. Pisabarro M.T. Seshagiri S. Koonin E.V. Dixit V.M. Mol. Cell. 2000; 6: 961-967Abstract Full Text Full Text PDF PubMed Google Scholar). Here, we report the identification, cloning, and biochemical characterization of two metacaspases of Arabidopsis thaliana as arginine/lysine-specific cysteine-dependent proteases. Cloning of Metacaspase Open Reading Frames in Arabidopsis— First-strand cDNA was synthesized from pooled RNA obtained from leaves, inflorescences, and roots of young and mature plants with the Superscript II RNase H– reverse transcriptase (Invitrogen) according to the manufacturer's instructions and used as template for PCR reactions with PLATINUM Pfx DNA polymerase (Invitrogen) and the forward and reverse primers: 5′-ATGTACCCGCCACCTCC-3′ and 5′-CTAGAGAGTGAAAGGCTTTGCATA-3′ for Atmc1; 5′-ATGTTGTTGCTGGTGGACTG-3′ and 5′-TTATAAAGAGAAGGGCTTCTCATATAC-3′ for Atmc2; 5′-ATGGCTAGTCGGAGAGAAG-3′ and 5′-TCAGAGTACAAACTTTGTCGCGT-3′ for Atmc3; 5′-ATGACGAAAAAGGCGGTGCTT-3′ and 5′-TCAACAGATGAAAGGAGCGTTGG-3′ for Atmc4; 5′-ATGGCGAAGAAAGCTGTGTTG-3′ and 5′-TTAACAAATAAACGGAGCATTCAC-3′ for Atmc5; 5′-ATGGCCAAGAAAGCTTTACTG-3′ and 5′-TCAACATATAAACCGAGCATTGAC-3′ for Atmc6; 5′-ATGGCAAAGAGAGCGTTGTTG-3′ and 5′-TTAGCATATAAACGGAGCATTCAC-3′ for Atmc7; 5′-ATGGCGAAGAAAGCACTTTTG-3′ and 5′-TTAGTAGCATATAAATGGTTTATCAAC-3′ for Atmc8; 5′-ATGGATCAACAAGGGATGGTC-3′ and 5′-TCAAGGTTGAGAAAGGAACGTC-3′ for Atmc9. For forward primers, 5′-AAAAAGCAGGCTCCACC-3′ was attached to the 5′ end to enable subsequent amplification with the attB1 primer 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′; for reverse primers, the 5′ extension was 5′-AGAAAGCTGGGTC-3′ to allow annealing with the attB2 primer 5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-3′. PCR products were cloned into pDONR201 (Invitrogen) to generate entry vectors for each metacaspase. For cloning of human caspase 7 (Ref. 21Denault J.-B. Salvesen G.S. J. Biol. Chem. 2003; 278: 34042-34050Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar; accession number NP_001218), the forward and reverse primers 5′-ATGGCAGATGATCAGGGCTGT-3′ and 5′-CTATTGACTGAAGTAGAGTTCC-3′ were used. Extensions were added to allow annealing of attB1 and attB2 primers as mentioned above for metacaspase cloning. GenBank™ accession numbers for the reported metacaspase sequences are AY219826-AY219834. Alignment of Metacaspase Sequences—Sequences were aligned with ClustalX (22Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35075) Google Scholar) and manually edited with BioEdit (23Hall T.A. Nucleic Acids Symp. Ser. 1999; 41: 95-98Google Scholar). Bacterial Production and N-terminal Peptide Sequencing of Atmc9 Fragments—The open reading frames were cloned into the bacterial expression vector pDEST17 (Invitrogen), resulting in the N-terminal fusion with a His6 epitope tag. Transformed cultures of Escherichia coli strain BL21(DE3) were induced with 1 mm isopropyl β-d-thiogalactopyranoside for 1–3 h. Cells were centrifuged and lysed under denaturing conditions (24Rogl H. Kosemund K. Kuhlbrandt W. Collinson I. FEBS Lett. 1998; 432: 21-26Crossref PubMed Scopus (160) Google Scholar). The bacterial cell pellet from a 500-ml culture was lysed with 5 ml of 100 mm Tris-HCl (pH 8.0), 20 ml of 8.0 m urea, and 2.7 ml of 10% (w/v) sodium N-lauroylsarcosinate completed with 1 mm phenylmethylsulfonyl fluoride (PMSF) 1The abbreviations used are: PMSF, phenylmethylsulfonyl fluoride; Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-amido-4-methylcoumarin (AMC); Atmc, A. thaliana metacaspase; C/A, cysteine-to-alanine mutation; Z-VAD-fmk, benzyloxycarbonyl (Z)-Val-Ala-Asp(O-methyl)fluoromethyl ketone (fmk); Boc-GRR-AMC, t-butyloxycarbonyl-Gly-Arg-Arg-amido-4-methylcoumarin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; tbmk, trimethylbenzoyloxymethyl ketone; TLCK, Nα-tosyl-l-lysine-chloromethyl ketone; cmk, chloromethyl ketone; E-64, l-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane; MES, 4-morpholineethanesulfonic acid; R/A, arginine-to-alanine mutation. and 1 mm oxidized glutathione. After sonication and centrifugation, the volume was brought to 50 ml with buffer 1 (20 mm Tris-HCl, pH 8.0, 200 mm NaCl, 10% (v/v) glycerol, 0.1% (w/v) sodium N-lauroylsarcosinate, 1 mm PMSF, and 1 mm oxidized glutathione). The lysate was applied to a 2-ml nickel nitrilotriacetic acid column (Qiagen, Hilden, Germany) equilibrated with buffer 1. The column was first washed with buffer 2 (buffer 1 with 0.1% (v/v) Triton-X100 instead of 0.1% (w/v) sodium N-lauroylsarcosinate), then with buffer 2 supplemented with 10 mm imidazole. Recombinant metacaspases were eluted with 300 mm imidazole in buffer 2 and checked by 12% (w/v) PAGE. Automated N-terminal Edman degradation of the immobilized Atmc9 fragments and mass determination were performed on a 476A pulsed-liquid sequencer equipped with an on-line phenylthiohydantoin-derivative analyzer and a 4700 proteomics analyzer, respectively (Applied Biosystems, Foster City, CA). Reversed-phase high performance liquid chromatography was done in two steps; the nickel nitrilotriacetic acid-purified bacterially produced protein was applied on a PLRP-S column (Polymer Laboratories, Amherst, MA; 4.6 × 200 mm; eluent A = 0.1% (v/v) trifluoroacetic acid, B = 90% isopropanol in 0.07% (v/v) trifluoroacetic acid) to separate the mixture in two fractions, fractions 1 and 2, between ∼80–85 and ∼85–95% of eluent B, respectively. The second fraction was separated using a μRPC column (Amersham Biosciences; 4.6 × 100 mm; eluent A = 0.1% (v/v) trifluoroacetic acid; eluent B = 90% (v/v) MeCN in 0.1% trifluoroacetic acid). The p10-like fragment was eluted as a single peak at ∼60% of eluent B. Native Preparation of Purified Recombinant Metacaspase 4 and 9 and Human Caspase 7—The bacterial expression vectors pDEST17Atmc4, pDEST17Atmc4C/A, pDEST17Atmc9, pDEST17Atmc9C/A, pDEST17Atmc9R/A, and pDEST17HsCasp7 were transformed into E. coli strain BL21(DE3)pLysE, and production was induced for 24 h with 0.2 mm isopropyl 1-thio-β-d-galactopyranoside. Cultures were harvested, resuspended in extraction buffer (50 mm phosphate buffer, pH 7.0, 300 mm NaCl, 1 mm oxidized glutathione), and passed through a French press. Cellular debris were removed by centrifugation, and the supernatant was mixed with TALON cobalt affinity resin (BD Biosciences) and incubated overnight at 4 °C. Beads were washed twice in the same buffer, loaded on a column, and washed twice again. Bound protein was eluted with extraction buffer supplemented with 150 mm imidazole. Metacaspase and Caspase Assays, Substrates, and Inhibitors—All tested fluorogenic substrates were obtained from Bachem (Bubendorf, Switzerland), except for d-VKKR-AMC (Enzyme Systems Products, Livermore, CA) and inhibitors from Sigma-Aldrich (St. Louis, MO), except for the caspase inhibitors, Z-FA-fmk and Z-FK-tbmk (Enzyme Systems Products). Assays were performed in 150 μl with 50 μm substrate in optimized metacaspase assay buffers. For Atmc9, the buffer consisted of 50 mm MES (pH 5.5), 150 mm NaCl, 10% (w/v) sucrose, 0.1% (w/v) CHAPS, 10 mm dithiothreitol; for Atmc4 the buffer consisted of 50 mm Hepes (pH 7.5), 150 mm NaCl, 10% (v/v) glycerol, 100 mm CaCl2, 10 mm dithiothreitol; for caspase activity assays, the buffer consisted of 50 mm Hepes (pH 7.5), 150 mm NaCl, and 10 mm 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane. For the determination of the pH profile of metacaspases, the buffer consisted of 50 mm acetic acid, 50 mm MES, and 100 mm Tris (25Ellis K.J. Morrison J.F. Methods Enzymol. 1982; 87: 405-426Crossref PubMed Scopus (642) Google Scholar, 26Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (58771) Google Scholar). Time-dependent release of free amido-4-methylcoumarin (AMC) was measured on a FLUOstar OPTIMA reader (BMG Labtechnologies, Offenburg, Germany), and activity was expressed as the increase per minute of fluorescence units in each well. Identification and Cloning of Arabidopsis Metacaspases—A sequence homology search (BLASTP; 26) of the eight published Arabidopsis metacaspases (20Uren A.G. O'Rourke K. Aravind L.A. Pisabarro M.T. Seshagiri S. Koonin E.V. Dixit V.M. Mol. Cell. 2000; 6: 961-967Abstract Full Text Full Text PDF PubMed Google Scholar) against a database of predicted Arabidopsis protein-encoding genes (EuGène; Ref. 27Schiex T. Moisan A. Rouzé P. Lecture Notes Comput. Sci. 2001; 2066: 111-125Crossref Scopus (74) Google Scholar) identified one extra putative metacaspase gene, leading to a total of nine metacaspase genes in the genome of A. thaliana (designated Atmc1 to Atcm9). The alignment of the corresponding protein sequences is shown in Fig. 1 and the corresponding phylogenetic tree in Fig. 2. Previous genomic analysis has revealed that the Arabidopsis genome consists of a large number of duplicated blocks, which might be the results of one or many complete genome duplications (28Initiative Arabidopsis Genome Nature. 2000; 408: 796-815Crossref PubMed Scopus (7006) Google Scholar, 29Raes J. Vandepoele K. Simillion C. Saeys Y. Van de Peer Y. J. Struct. Funct. Genomics. 2003; 3: 117-129Crossref PubMed Scopus (61) Google Scholar, 30Simillion C. Vandepoele K. Van Montagu M.C. Zabeau M. Van de Peer Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13627-13632Crossref PubMed Scopus (413) Google Scholar). Comparison of the genomic organization of all nine Arabidopsis metacaspases and these duplicated segments shows that the Atmc8 gene is linked with genes Atmc4, Atmc5, Atmc6, and Atmc7 by an internal duplication event on chromosome I (data not shown). In addition, these genes (Atmc4 to Atmc7) are present as a tandem within a region of 10.6 kilobases on chromosome I. By combining the phylogenetic tree topology (Fig. 2) and this genomic organization, this metacaspase cluster (genes Atmc4 to Atmc7) originated through a block duplication of the Atmc8 gene that was followed by a tandem duplication. As shown in Fig. 1, a high degree of similarity exists between the members of this cluster, with amino acid sequence identities ranging from 56% to 71%. We isolated the corresponding open reading frames of all but one genes by reverse transcription-PCR on pooled RNA isolated from different organs of Arabidopsis. Despite several attempts we could not isolate a full-length Atmc8 cDNA fragment. Because no expressed sequence tags corresponding to Atmc8 are found in public databases, we assume that it is probably a pseudogene.Fig. 2Unrooted phylogenetic tree of the Arabidopsis metacaspase family. For construction of the tree, the alignment of Fig. 1 was subjected to the TREECON software package (52Van de Peer Y. De Wachter R. Comput. Appl. Biosci. 1994; 10: 569-570PubMed Google Scholar). On the right side a tentative schematic representation of the structure of the nine Arabidopsis metacaspases is presented. The putative prodomain is in dark gray, the large subunit (p20) is in white, the small subunit (p10) is in black, and the linker/loop regions between p20 and p10 are in light gray. H and C designate His and Cys residues, respectively. Cysteine residues within the putative prodomain zinc fingers are shown as white stripes. Numbers given at the right correspond to those provided by the Arabidopsis Genome Initiative (28Initiative Arabidopsis Genome Nature. 2000; 408: 796-815Crossref PubMed Scopus (7006) Google Scholar).View Large Image Figure ViewerDownload (PPT) Three of the Arabidopsis metacaspase proteins (Atmc1, Atmc2, and Atmc3) have previously been designated as “type I” metacaspases. They possess N-terminal extensions ranging from ∼80 to 120 amino acids in length (Fig. 2; Ref. 20Uren A.G. O'Rourke K. Aravind L.A. Pisabarro M.T. Seshagiri S. Koonin E.V. Dixit V.M. Mol. Cell. 2000; 6: 961-967Abstract Full Text Full Text PDF PubMed Google Scholar) and could represent a prodomain that is also present in mammalian upstream “initiator” caspases. As such, this domain may be responsible for protein-protein interactions between metacaspases and/or other oligomerizing components of different signaling complexes, leading to subsequent metacaspase activation (11Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar, 31Boatright K.M. Renatus M. Scott F.L. Sperandio S. Shin H. Pedersen I.M. Ricci J.-E. Edris W.A. Sutherlin D.P. Green D.R. Salvesen G.S. Mol. Cell. 2003; 11: 529-541Abstract Full Text Full Text PDF PubMed Scopus (771) Google Scholar). The Arabidopsis metacaspase prodomains are rich in proline (Atmc1 and Atmc2) or glutamine (Atmc3), and all contain two putative CXXC-type zinc finger structures, similar to the lesion-simulating disease-1 protein, a negative regulator of the hypersensitive response with homology to GATA-type transcription factors (20Uren A.G. O'Rourke K. Aravind L.A. Pisabarro M.T. Seshagiri S. Koonin E.V. Dixit V.M. Mol. Cell. 2000; 6: 961-967Abstract Full Text Full Text PDF PubMed Google Scholar, 32Dietrich R.A. Richberg M.H. Schmidt R. Dean C. Dangl J.L. Cell. 1997; 88: 685-694Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). The other metacaspases (Atmc4 to Atmc9) lack a large prodomain, although the existence of a short prodomain cannot be excluded, and were designated type II metacaspases (20Uren A.G. O'Rourke K. Aravind L.A. Pisabarro M.T. Seshagiri S. Koonin E.V. Dixit V.M. Mol. Cell. 2000; 6: 961-967Abstract Full Text Full Text PDF PubMed Google Scholar). In both types, a conserved region of ∼150 amino acids could correspond with the p20 subunit of mammalian caspases (11Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar). At the C terminus, another conserved domain is reminiscent of the p10 of caspases. In between these putative p20 and p10 domains, a variable region is present that differs considerably between type I and type II metacaspases; this putative loop or linker region is ∼20 amino acids long in type I metacaspases, whereas in type II metacaspases it varies in size from 90 (for Atmc9) to 150 residues (Atmc4 to Atmc8). In addition to this structural domain homology, the designation of metacaspases as caspase homologs is based on the presence of a conserved histidine/cysteine dyad in the p20 domain. The sequence context of the catalytic histidine residue in Arabidopsis metacaspases is H(Y/F)SGHG, suggesting an intermediate stabilizing role for the adjacent glycine, as in mammalian caspases (11Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar). The catalytic cysteine in caspases is contained in a QAC(R/Q)G context, with the glutamine residue shown to coordinate the P1 aspartate. The metacaspases of Arabidopsis have a D(A/S)C(H/N)SG signature, suggesting that this role is now played by an aspartate. Bacterial Overproduction of Arabidopsis Type II Metacaspases Leads to Cysteine-dependent Autocatalytic Processing—Upon overproduction in bacteria, caspases autoprocess in a cysteine-dependent manner to generate fully active proteases (11Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar). We initiated a biochemical analysis by overproducing N-terminal His6-tagged versions of the Arabidopsis type II metacaspases in E. coli (Fig. 3a). Detection with anti-His6 antibody revealed that all type II metacaspases could be produced in bacteria and, more interestingly, that this overproduction led to their processing, in analogy to caspases. Only for Atmc6 was cleavage less clear. During this processing, a C-terminal region ranging from 15 to 30 kDa was removed, resulting in a concomitant decrease in the size of the detected His6-tagged protein. For a detailed analysis of the type II metacaspases, we chose Atmc4 as a representative of the closely related group encoded by the gene cluster Atmc4 to Atmc7, together with Atmc9. For these two proteins, mutant forms in which the presumed catalytic cysteine (Cys139 for Atmc4 and Cys147 for Atmc9) was replaced by an alanine (designated C/A mutation) were also produced. Fig. 3b shows immunoblots with anti-His6 antibodies on bacterial lysates overproducing wild-type and mutated Atmc4 and Atmc9. Overproduction of Atmc4 resulted in the presence of the His6-tagged full-length protein (apparent molecular mass 60 kDa) and of an N-terminal fragment of 33 kDa. For Atmc9, overproduction led to the detection of the His6-tagged full-length protein (apparent molecular mass 46 kDa) and an N-terminal fragment of 28 kDa. These patterns suggest that both metacaspases, like caspases, are able to autoprocess, thereby separating the putative large (p20) and small (p10) subunits. For the cysteine mutants, no such processing occurred, demonstrating that it is the result of cysteine-dependent autocatalytic action of the type II metacaspases. Atmc9 Autoprocesses after an Arginine Residue—Because bacterial overproduction of type II metacaspases is sufficient for autoprocessing, we were able to characterize the putative p20 and p10 subunits of Atmc9 by N-terminal peptide sequencing and by molecular mass determination via mass spectrometry. When His6 tag-purified Atmc9 was analyzed by PAGE and silver staining, major fragments with apparent molecular masses of 22 and 15 kDa were visible, whereas bands at 38 and 28 kDa were less clear (Fig. 4). Western blot analysis revealed that besides the full-length C/A mutant, only the 28-kDa fragment of wild-type Atmc9 could be detected with the anti-His6 antibody and, thus, represented the His6-tagged p20-like subunit. Therefore, the 22-kDa band could represent the mature p20 subunit after removal of the His6 tag and possibly a very short prodomain, whereas the other fragment (15 kDa) could be the p10-like subunit. It should be noted that, although the p20- and p10-like fragments did not carry a His6 tag, they probably co-purified in complexes that did so. Furthermore, we observed that processing and concomitant disappearance of the full-length zymogen, which occurred after purification, also resulted in the presence of these non-tagged fragments. The 15-kDa protein band was sufficiently purified and Edman degradation sequencing resulted in the peptide sequence ALPFKAV, indicating that the fragment was generated by cleavage after Arg183. Molecular mass determination by matrix-assisted laser absorption deionization coupled to tandem time of flight revealed the p10-like subunit of Atmc9 had a mass of 15,442 Da, confirming that it consists of amino acids 184–325. As seen on Fig. 1, all type II metacaspases possess either an arginine or a lysine at this position. The nature of the autocatalytic cleavage site of Atmc9 already suggests that, although mammalian caspases and metacaspases are structural homologs, they differ in substrate specificity, with a Lys/Arg specificity at the P1 position for the latter. N-terminal sequencing of the 22-kDa fragment revealed that it was generated through removal of the His6 tag at an artificial site, LYKK↓AGST, introduced by the Gateway cloning procedure (data not shown). A few residues downstream a similar sequence were present, conserved in both type I and type II metacaspases. Therefore, it is possible that the natural processing site is masked by the artificial one, and hence, no conclusions can be drawn for now on any additional processing on the N-terminal side of the large subunit. Atmc4 and Atmc9 Cleave P1 Arginine/Lysine Substrates—To study the substrate specificity of Atmc4 and Atmc9 in more detail, the purified recombinant proteins and their respective cysteine mutants were tested for their ability to cleave the synthetic fluorogenic oligopeptide P1 arginine substrate t-butyloxycarbonyl-Gly-Arg-Arg-amido-4-methylcoumarin (Boc-GRR-AMC) in a wide pH range. As shown in Fig. 5a, both Atmc4 and Atmc9 have prominent activity toward GRR. The corresponding catalytic cysteine mutants were not active at all (see also below). For Atmc4, optimal buffer pH was 7.5–8.0, with detectable activity in the pH range of 6.5 to 9.0. Interestingly, the pH optimum for Atmc9 activity was 5.0–5.5 (pH range 4.5 to 6.0), whereas activity at the physiological pH of the cytoplasm (7.0–7.5) was completely abolished. To explore substrate P1 preference, proteolytic activities of purified Atmc4 and Atmc9 were tested against additional oligopeptide substrates with an arginine or lysine at the P1 position; benzyloxycarbonyl-Phe-Arg-amido-4-methylcoumarin (Z-FR-AMC), Boc-GRR-AMC, t-butyloxycarbonyl-Gly-Lys-Arg-AMC (Boc-GKR-AMC), d-VKKR-AMC, and H-Ala-Phe-Lys-AMC (AFK-AMC). All tested P1 arginine substrates are indeed cleaved by both Atmc4 and Atmc9, albeit at different efficiencies (Fig. 5b). Noteworthy, Atmc9 also showed some activity toward the P1 lysine substrate H-AFK-AMC. To confirm that type II metacaspases prefer basic rather than acidic P1 residues, we tested whether Atmc4 and Atmc9 could cleave the caspase substrates Ac-DEVD-AMC, Ac-YVAD-AMC, and ZVAD-AMC. None of the caspase substrates were cleaved by the metacaspases (Fig. 5b). To exclude the possibility that specific experimental conditions would impair the measurement of caspase activity in our assays, we cloned and purified human caspase 7 as a His6-tag fusion in the same vector as the Arabidopsis metacaspases and assessed its potential to cleave caspase substrates. As shown in Fig. 5b, recombinant human caspase 7 efficiently hydrolyzed Ac-DEVD-AMC and also Ac-YVAD-AMC, although at hardly measurable efficiency. On the other hand, human caspase 7 did not cleave any of the arginine/lysine substrates. These results demonstrate that caspase substrates cannot be cleaved by Arabidopsis Atmc4 and Atmc9 and are, hence, very unlikely to be responsible for the caspase-like activities in plants reported in literature. Effect of Protease Inhibitors on the Activity of Type II Metacaspases—We assessed the effect on Atmc4 and Atmc9 of several protease inhibitors with Boc-GRR-AMC as substrate (Table I). First, we tested the caspase inhibitors Z-YVAD-chloromethyl ketone (cmk), Z-DEVD-cmk, and Z-VAD-cmk. However, none of these compounds could block metacaspase activity at concentrations up to 100 μm. Also, the cathepsin B inhibitor Z-FA-fmk had no effect as well at this concentration.Table IEffect of different protease inhibitors on the activity of Atmc4 and Atmc9 with Boc-GRR-AMC as substrateReagentConcentrationActivityAtmc4Atmc9%Z-YVAD-cmk100 μm97 ± 599 ± 9Z-DEVD-cmk100 μm94 ± 1494 ± 8Z-VAD-fmk100 μm90 ± 7103 ± 1Z-FA-fmk100 μm97 ± 492 ± 3Chymostatin100 μm14 ± 210 ± 1Soybean trypsin inhibitor100 μg/ml34 ± 050 ± 4Pepstatin100 μm66 ± 169 ± 0Benzamidine5 mm2 ± 15 ± 1Iodoacetamide10 mm0 ± 03 ± 0PMSF1 mm23 ± 098 ± 14E-64100 μm44 ± 293 ± 6Aprotinin5 μg/ml114 ± 11113 ± 7Leupeptin1 μm12 ± 15 ± 1Antipain1 μm6 ± 01 ± 1Z-FK-tbmk1 μm5 ± 012 ± 0TLCK250 μm41 ± 50 ± 0TLCK1 μm79 ± 50 ± 1TPCK250 μm72 ± 079 ± 6 Open table in a new tab Of the broad-spectrum inhibitors tested, only chymostatin, a reversible serine and cysteine protease inhibitor, and to a lesser extent soybean trypsin inhibitor and pepstatin could block metacaspase activity at 100 μm. Benzamidine and iodoacetamide inhibited both activities at the millimolar range.