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Planta

, Volume 239, Issue 1, pp 147–160 | Cite as

Two aspartate residues at the putative p10 subunit of a type II metacaspase from Nicotiana tabacum L. may contribute to the substrate-binding pocket

  • Alexis Acosta-Maspons
  • Edgar Sepúlveda-García
  • Laura Sánchez-Baldoquín
  • Junier Marrero-Gutiérrez
  • Tirso Pons
  • Mario Rocha-Sosa
  • Lien González
Original Article

Abstract

Metacaspases are cysteine proteases present in plants, fungi, prokaryotes, and early branching eukaryotes, although a detailed description of their cellular function remains unclear. Currently, three-dimensional (3D) structures are only available for two metacaspases: Trypanosoma brucei (MCA2) and Saccharomyces cerevisiae (Yca1). Furthermore, metacaspases diverged from animal caspases of known structure, which limits straightforward homology-based interpretation of functional data. We report for the first time the identification and initial characterization of a metacaspase of Nicotiana tabacum L., NtMC1. By combining domain search, multiple sequence alignment (MSA), and protein fold-recognition studies, we provide compelling evidences that NtMC1 is a plant metacaspase type II, and predict its 3D structure using the crystal structure of two type I metacaspases (MCA2 and Yca1) and Gsu0716 protein from Geobacter sulfurreducens as template. Analysis of the predicted 3D structure allows us to propose Asp353, at the putative p10 subunit, as a new member of the aspartic acid triad that coordinates the P1 arginine/lysine residue of the substrate. Nevertheless, site-directed mutagenesis and expression analysis in bacteria and Nicotiana benthamiana indicate the functionality of both Asp348 and Asp353. Through the co-expression of mutant and wild-type proteins by transient expression in N. benthamiana leaves we found that polypeptide processing seems to be intramolecular. Our results provide the first evidence in plant metacaspases concerning the functionality of the putative p10 subunit.

Keywords

Autoprocessing Cell death Protein modeling Transient expression 

Abbreviations

AMC

7-Amino-4-methylcoumarin

IPTG

Isopropyl β-d-1-thiogalactopyranoside

MSA

Multiple sequence alignment

PCD

Programmed cell death

RACE

Rapid amplification of cDNA ends

Notes

Acknowledgments

We thank Patricia Rueda for technical assistance, Eugenio López-Bustos and Paul Gaytán for oligonucleotide synthesis and Jorge Yáñez for sequencing. This work was funded by Consejo Nacional de Ciencia y Tecnología (CONACYT, Grant No. 58761) and the International Foundation for Science (Grant C/4702-1). A A-M, E S-G and L S-B were supported by CONACYT fellowships. A A-M wants to acknowledge to Programa de Posgrado en Ciencias Biológicas, UNAM. The present work is as a part of the requirements for obtaining his PhD degree.

Supplementary material

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Supplementary material 1 (DOCX 113 kb)
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Supplementary material 2 (PDF 80 kb)
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Supplementary material 7 (TIFF 10034 kb)
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Supplementary material 8 (PDF 29 kb)

References

  1. Belenghi B, Romero-Puertas MC, Vercammen D, Brackenier A, Inzé D, Delledonne M, Van Breusegem F (2007) Metacaspase activity of Arabidopsis thaliana is regulated by S-nitrosylation of a critical cysteine residue. J Biol Chem 282:1352–1358PubMedCrossRefGoogle Scholar
  2. Bowie JU, Lüthy R, Eisenberg DA (1991) A method to identify protein sequences that fold into a known three-dimensional structure. Science 253:164–170PubMedCrossRefGoogle Scholar
  3. Bozhkov PV, Suarez MF, Filonova LH, Daniel G, Zamyatnin AA Jr, Rodriguez-Nieto S, Zhivotovsky B, Smertenko A (2005) Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis. Proc Natl Acad Sci USA 102:14463–14468PubMedCrossRefGoogle Scholar
  4. Cambra I, Garcia FJ, Martinez M (2010) Clan CD of cysteine peptidases as an example of evolutionary divergences in related protein families across plant clades. Gene 449:59–69PubMedCrossRefGoogle Scholar
  5. Castillo-Olamendi L, Bravo-García A, Morán J, Rocha-Sosa M, Porta H (2007) AtMCP1b, a chloroplast-localised metacaspase, is induced in vascular tissue after wounding or pathogen infection. Funct Plant Biol 34:1061–1071CrossRefGoogle Scholar
  6. Choi CJ, Berges JA (2013) New types of metacaspases in phytoplankton reveal diverse origins of cell death proteases. Cell Death Differ 4:e490. doi: 10.1038/cddis.2013.21 Google Scholar
  7. Coll NS, Vercammen D, Smidler A, Clover C, Van Breusegem F, Dangl JL, Epple P (2010) Arabidopsis type I metacaspases control cell death. Science 330:1393–1397PubMedCrossRefGoogle Scholar
  8. Curtis M, Grossniklaus U (2003) A Gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469PubMedCentralPubMedCrossRefGoogle Scholar
  9. Dudkiewicz MZ, Piszczek E (2012) Bacterial putative metacaspase structure from Geobacter sulfureducens as a template for homology modeling of type II Triticum aestivum metacaspase (TaeMCAII). Acta Biochim Pol 59:401–406PubMedGoogle Scholar
  10. Frangioni JV, Neel BG (1993) Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem 210:179–187PubMedCrossRefGoogle Scholar
  11. Fuentes-Prior P, Salvesen GS (2004) The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem J 384:201–232PubMedCrossRefGoogle Scholar
  12. Hao L, Goodwin PH, Hsiang T (2007) Expression of a metacaspase gene of Nicotiana benthamiana after inoculation with Colletotrichum destructivum or Pseudomonas syringae pv. tomato, and the effect of silencing the gene on the host response. Plant Cell Rep 26:1879–1888PubMedCrossRefGoogle Scholar
  13. He R, Drury GE, Rotari VI, Gordon A, Willer M, Farzaneh T, Woltering EJ, Gallois P (2008) Metacaspase-8 modulates programmed cell death induced by ultraviolet light and H2O2 in Arabidopsis. J Biol Chem 283:774–783PubMedCrossRefGoogle Scholar
  14. Helmersson A, von Arnold S, Bozhkov PV (2008) The level of free intracellular zinc mediates programmed cell death/cell survival decisions in plant embryos. Plant Physiol 147:1158–1167PubMedCentralPubMedCrossRefGoogle Scholar
  15. Hoeberichts FA, ten Have A, Woltering EJ (2003) A tomato metacaspase gene is unregulated during programmed cell death in Botrytis cinerea infected leaves. Planta 217:517–522PubMedCrossRefGoogle Scholar
  16. Hooft RW, Vriend G, Sander C, Abola EE (1996) Errors in protein structures. Nature 381:272PubMedCrossRefGoogle Scholar
  17. Kelley LA, Sternberg MJE (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363–371PubMedCrossRefGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  19. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948PubMedCrossRefGoogle Scholar
  20. Laskowski RA, MacArthur MW, Moss D, Thornton JM (1993) Procheck-a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291CrossRefGoogle Scholar
  21. McLuskey K, Rudolf J, Proto WR, Isaacs NW, Coombs GH, Moss CX, Mottram JC (2012) Crystal structure of a Trypanosoma brucei metacaspase. Proc Natl Acad Sci USA 109:7469–7474PubMedCrossRefGoogle Scholar
  22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  23. Piszczek E, Dudkiewicz M, Mielecki M (2012) Biochemical and bioinformatic characterization of type II metacaspase protein (TaeMCAII) from wheat. Plant Mol Biol Rep 30:1338–1347CrossRefGoogle Scholar
  24. Reape TJ, McCabe PF (2008) Apoptotic-like programmed cell death in plants. New Phytol 180:13–26PubMedCrossRefGoogle Scholar
  25. Sambrook JE, Fritsch F, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  26. Scheer JM, Ryan CA (2001) A method for the quantitative recovery of proteins from polyacrylamide gels. Anal Biochem 298:130–132PubMedCrossRefGoogle Scholar
  27. Shi Y (2002) Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 9:459–470PubMedCrossRefGoogle Scholar
  28. Suarez MF, Filonova LH, Smertenko A, Savenkov EI, Clapham DH, von Arnold S, Zhivotovsky B, Bozhkov PV (2004) Metacaspase dependent programmed cell death is essential for plant embryogenesis. Curr Biol 14:R339–R340PubMedCrossRefGoogle Scholar
  29. Sundström JF, Vaculova A, Smertenko AP, Savenkov EI, Golovko A, Minina E, Tiwari BS, Rodriguez-Nieto S, Zamyatnin AA Jr, Välineva T, Saarikettu J, Frilander MJ, Suarez MF, Zavialov A, Ståhl U, Hussey PJ, Silvennoinen O, Sundberg E, Zhivotovsky B, Bozhkov PV (2009) Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome. Nat Cell Biol 11:1347–1354PubMedCrossRefGoogle Scholar
  30. Tsiatsiani L, Van Breusegem F, Gallois P, Zavialov A, Lam E, Bozhkov PV (2011) Metacaspases. Cell Death Differ 18:1279–1288PubMedCrossRefGoogle Scholar
  31. Uren AG, O’Rourke K, Aravind L, Pisabarro MT, Seshagiri S, Koonin EV, Dixit VM (2000) Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. Mol Cell 6:961–967PubMedGoogle Scholar
  32. van Doorn WG, Beers EP, Dangl JL, Franklin-Tong VE, Gallois P, Hara-Nishimura I, Jones AM, Kawai-Yamada M, Lam E, Mundy J, Mur LAJ, Petersen M, Smertenko A, Taliansky M, Van Breusegem F, Wolpert T, Woltering E, Zhivotovsky B, Bozhkov PV (2011) Morphological classification of plant cell deaths. Cell Death Differ 18:1241–1246PubMedCrossRefGoogle Scholar
  33. Vercammen D, van de Cotte B, De Jaeger G, Eeckhout D, Castells P, Vandepoele K, Vandenberghe I, Van Beeumen J, Inze D, Van Breusegem F (2004) Type II metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana cleave substrates after arginine and lysine. J Biol Chem 279:45329–45336PubMedCrossRefGoogle Scholar
  34. Vercammen D, Belenghi B, van de Cotte B, Beunens T, Gavigan J-A, De Rycke R, Brackenier A, Inzé D, Harris JL, Breusegem F (2006) Serpin1 of Arabidopsis thaliana is a suicide inhibitor for metacaspase 9. J Mol Biol 364:625–636PubMedCrossRefGoogle Scholar
  35. Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956PubMedCrossRefGoogle Scholar
  36. Watanabe N, Lam E (2005) Two Arabidopsis metacaspases AtMCP1b and AtMCP2b are arginine/lysine-specific cysteine proteases and activate apoptosis-like cell death in yeast. J Biol Chem 280:14691–14699PubMedCrossRefGoogle Scholar
  37. Watanabe N, Lam E (2011a) Arabidopsis metacaspase 2d is a positive mediator of cell death induced during biotic and abiotic stresses. Plant J 66:969–982PubMedCrossRefGoogle Scholar
  38. Watanabe N, Lam E (2011b) Calcium-dependent activation and autolysis of Arabidopsis metacaspase 2d. J Biol Chem 286:10027–10040PubMedCrossRefGoogle Scholar
  39. Wong AHH, Yan C, Shi Y (2012) Crystal structure of the yeast metacaspase Yca1. J Biol Chem 287:29251–29259PubMedCrossRefGoogle Scholar
  40. Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinforma 9:40CrossRefGoogle Scholar
  41. Zhang Y, Lam E (2011) Sheathing the swords of death. Post-translational modulation of plant metacaspases. Plant Signal Behav 6:2051–2056PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Alexis Acosta-Maspons
    • 1
  • Edgar Sepúlveda-García
    • 1
  • Laura Sánchez-Baldoquín
    • 1
    • 2
  • Junier Marrero-Gutiérrez
    • 2
  • Tirso Pons
    • 3
  • Mario Rocha-Sosa
    • 1
  • Lien González
    • 4
  1. 1.Departamento de Biología Molecular de Plantas, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico
  2. 2.Departamento de Biología Vegetal, Facultad de BiologíaUniversidad de La HabanaHavanaCuba
  3. 3.Programa de Biología Estructural y BiocomputaciónCentro Nacional de Investigaciones Oncológicas-Carlos III (CNIO)MadridSpain
  4. 4.Facultad de Ingenierías y Ciencias AgropecuariasUniversidad de Las Américas (UDLA)QuitoEcuador

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