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

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 NP₀01218), 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 used A. thaliana and 1 mm and the was to ml with 1 mm mm 10% (w/v) sodium 1 mm and 1 mm The was to a with The was with 1 with of (w/v) sodium then with with mm metacaspases were with mm in and by (w/v) N-terminal of the Atmc9 and were on a with an and a was in two the was on a A in to the in two 1 and and of The was using a 100 A in The was as a at of B. of Metacaspase 4 and 9 and bacterial expression vectors and were into E. coli strain and was induced for with mm isopropyl were in mm mm 1 mm and a were by and the was with and at 4 were in the on a and was with with mm Metacaspase and and substrates were obtained from for and inhibitors from for the caspase and were in with in metacaspase For the of mm (pH mm 10% (w/v) (w/v) mm for the of mm (pH mm 10% 100 mm mm for caspase activity the of mm (pH mm and mm For the of the of metacaspases, the of mm mm and 100 mm J. F. PubMed Scopus Google Scholar, J. Z. W. Nucleic Acids Res. 1997; 25: PubMed Scopus Google Scholar). of was on a and activity was as the of in each and Cloning of Arabidopsis of the Arabidopsis metacaspases 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) a of Arabidopsis genes T. A. P. Scopus Google Scholar) identified one metacaspase leading to a of metacaspase genes in the of A. thaliana to The of the corresponding sequences is in 1 and the corresponding in analysis has revealed that the Arabidopsis of a large number of which be the of one or Arabidopsis 2000; PubMed Scopus Google Scholar, J. K. C. Van J. 2003; PubMed Scopus Google Scholar, C. K. Van M. Van S. A. 2002; PubMed Scopus Google Scholar). of the of Arabidopsis metacaspases and these that the is with genes and by an on not In these genes to are as a a of on I. By the and this this metacaspase to a of the that was by a in a of the of this with from to the corresponding open reading frames of one genes by reverse on pooled RNA from of several we not a cDNA corresponding to are in we that is a of the Arabidopsis metacaspase For of the the of 1 was to the De R. Google Scholar). On the side a of the of the Arabidopsis metacaspases is The is in the large is in the small is in and the and are in and and the are as at the to by the Arabidopsis Arabidopsis 2000; PubMed Scopus Google of the Arabidopsis metacaspase proteins and have been designated as N-terminal from to in 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 a that is in caspases. this be responsible for metacaspases other of 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, M. S. H. Salvesen G. S. Mol. Cell. 2003; 11: Full Text Full Text PDF PubMed Scopus Google Scholar). The Arabidopsis metacaspase are in and or and two to the a of the hypersensitive response with to 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, R. A. R. C. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The other metacaspases to a large the of a be and were designated type II metacaspases 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 a of with the of caspases (11Earnshaw W. C. Martins L. M. Kaufmann S. H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar). the is of the of caspases. In these and a is that type and type II this or is in type metacaspases, in type II metacaspases in from to to In to this structural the of metacaspases as caspase is based on the of a in the The of the in Arabidopsis metacaspases is an intermediate for the as in caspases (11Earnshaw W. C. Martins L. M. Kaufmann S. H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar). The cysteine in caspases is in a with the to the P1 The metacaspases of Arabidopsis have a that this is by an Bacterial of Arabidopsis II to in caspases in a cysteine-dependent to generate proteases (11Earnshaw W. C. Martins L. M. Kaufmann S. H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar). a biochemical analysis by N-terminal of the Arabidopsis type II metacaspases in E. coli with revealed that type II metacaspases be in that this to their in analogy to caspases. for was this a C-terminal from to was resulting in a in the of the For a detailed analysis of the type II metacaspases, we as a of the by the to with Atmc9. For these two forms in which the cysteine for and for was by an were with on bacterial and and Atmc9. of resulted in the of the and of an N-terminal of For to the of the and an N-terminal of These suggest that both metacaspases, are to the large and small For the cysteine such processing that is the of cysteine-dependent autocatalytic of the type II Atmc9 after an bacterial of type II metacaspases is for we were to the and of Atmc9 by N-terminal peptide and by His6 Atmc9 was by and with of and were at and were analysis revealed that the the of Atmc9 be with the the the the mature after of the His6 and possibly a the other be the be the and did not a His6 in that did we observed that processing and of the which after resulted in the of these The was purified and resulted in the peptide that the was by after by to of revealed the of Atmc9 a of that of on type II metacaspases an or a at this The of the autocatalytic of Atmc9 caspases and metacaspases are structural in with a at the P1 for the N-terminal of the revealed that was of the His6 at an by the cloning not A downstream a were in both type and type II is that the processing is by the and can be for on processing on the N-terminal side of the large and Atmc9 P1 the of and Atmc9 in the purified recombinant proteins and their cysteine were for their to cleave the synthetic oligopeptide P1 in a in both and Atmc9 have activity The corresponding cysteine were not at For was with activity in the range of to the for Atmc9 activity was (pH range to activity at the of the was P1 activities of purified and Atmc9 were oligopeptide substrates with an or at the P1 and P1 substrates are by both and at Atmc9 showed activity the P1 that type II metacaspases P1 we and Atmc9 cleave the caspase substrates and of the caspase substrates were by the metacaspases the that conditions the of caspase activity in we cloned and purified human caspase 7 as a fusion in the vector as the Arabidopsis metacaspases and potential to cleave caspase substrates. in recombinant human caspase 7 and at On the other human caspase 7 did not cleave of the substrates. These that caspase substrates be by Arabidopsis and Atmc9 and to be responsible for the caspase-like activities in plants reported in of on the of II the on and Atmc9 of several protease inhibitors with as we the caspase inhibitors and However, of these metacaspase activity at up to 100 the as at this of protease inhibitors on the activity of and Atmc9 with as Open in a the inhibitors a and cysteine protease and to a and metacaspase activity at 100 and both activities at the and 100 activity not that of the protease on metacaspase activity at up to 5 inhibitors with a at the P1 type II metacaspase activity and Atmc9 are by and two protease at as as 1 The oligopeptide and Atmc9 activities at the activity on Atmc9 was at 1 that both metacaspases in their In an of proteases, the activities of both and Atmc9. these the of and Atmc9 for P1 in their substrates. of Atmc9 a for caspases are by processing of the caspases or by autocatalytic in or cells G. S. Oncogene. 2004; 23: PubMed Scopus Google Scholar). Arabidopsis type II metacaspase activity and of the large and small subunits, we a of Atmc9 the P1 at the was by an this a was up in which both and Atmc9 were for at that the into a large and a small the that of Atmc9 at and that that for this residue. In we the of Atmc9 showed activity with Atmc9 and were added to and and of was of Atmc9 to a at corresponding to the of the proform In activity be with the with the type II metacaspases, the caspases in animals, on in to be Cell death in plants has been with caspase-like induced cell death in cells can be by the of caspase inhibitors Jong A. J. Hoeberichts F. A. Yakimova E. T. Maximova E. Woltering E. J. Planta. 2000; 211: 656-662Crossref PubMed Scopus (114) Google Scholar). plants with the protease activity as by a synthetic for caspase inhibitors with an the hypersensitive response in Pozo O. Lam E. Curr. Biol. 1998; 8: 1129-1132Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). cells caspase as with the H. A. Berecki G. Bruin W. van Duijn B. Wang M. FEBS Lett. 2000; 475: 139-144Crossref PubMed Scopus (92) Google Scholar). activity has been reported in cell death during (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). However, the genes the caspase-like proteases have not been identified reported by 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) on their in of and metacaspases, to plant metacaspases be responsible for these caspase-like cloned and characterized one of the reported Arabidopsis By of type II metacaspases in autocatalytic processing on a defined cysteine in the formation of and However, this processing was not observed with bacterial of type metacaspases not In animals, the caspases are in the of and their activation induced M. S. H. Salvesen G. S. Mol. Cell. 2003; 11: Full Text Full Text PDF PubMed Scopus Google Scholar, G. S. Dixit V. M. S. A. 1999; PubMed Scopus Google Scholar). type metacaspases, the to be a that we are type II metacaspases to the animal which are proteolytically by caspases. N-terminal peptide of the after bacterial Atmc9 that of plant metacaspases is not to aspartate at the P1 to and various synthetic oligopeptide substrates a P1 are by and the caspase substrates and are metacaspase activity is by and two and the P1 inhibitors and in the of the of and Atmc9 as a of the to of type II that metacaspases are arginine/lysine-specific from In P1 is by and in both the and subunits, which to the aspartate in the (11Earnshaw W. C. Martins L. M. Kaufmann S. H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar). on the of the Arabidopsis metacaspases and caspases and in analogy with A. R. A. J. J. W. J. 1999; 18: PubMed Scopus Google of which is metacaspases in other the P1 of the with two of the and in the is that these aspartate are in as that the be proteases not For we have that processing at is for the activation of the as in caspases of of the the and in of the and of the S. J. P. M. S. R. Salvesen G. S. W. S. A. 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Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar). cell death with of the and be by the caspase for the of caspase-like activity after metacaspase in be that caspase-like proteases are by the directly by or by of cellular be to the of P1 inhibitors on caspase activity in The of in which metacaspases are in plants be by or metacaspase of substrates and inhibitors into the of the biochemical characterization of plant on their and metacaspases can be as a family of proteases characterized by their R. A. A. J. FEBS Lett. 1998; PubMed Scopus Google Scholar, A. J. Nucleic Acids Res. 2002; PubMed Scopus Google which the and caspases and their for P1 metacaspases are to the other proteases and that type II metacaspases are to other proteases their cysteine-dependent autocatalytic In the of are to activity J. T. J. M. Salvesen G. S. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). is obtained after two autocatalytic processing D. C. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). metacaspase activity of on their structural and biochemical we suggest that metacaspases be as a family of the of proteases. De for in the