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

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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.

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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

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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.

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Authors and Affiliations

  1. Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, 62250, Cuernavaca, Morelos, Mexico

    Alexis Acosta-Maspons, Edgar Sepúlveda-García, Laura Sánchez-Baldoquín & Mario Rocha-Sosa

  2. Departamento de Biología Vegetal, Facultad de Biología, Universidad de La Habana, 10400, Havana, Cuba

    Laura Sánchez-Baldoquín & Junier Marrero-Gutiérrez

  3. Programa de Biología Estructural y Biocomputación, Centro Nacional de Investigaciones Oncológicas-Carlos III (CNIO), 28029, Madrid, Spain

    Tirso Pons

  4. Facultad de Ingenierías y Ciencias Agropecuarias, Universidad de Las Américas (UDLA), Quito, Ecuador

    Lien González

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  1. Alexis Acosta-Maspons
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  2. Edgar Sepúlveda-García
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  3. Laura Sánchez-Baldoquín
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  6. Mario Rocha-Sosa
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  7. Lien González
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Correspondence to Alexis Acosta-Maspons or Lien González.

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M. Rocha-Sosa: Deceased, 8 September 2013.

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Acosta-Maspons, A., Sepúlveda-García, E., Sánchez-Baldoquín, L. et al. Two aspartate residues at the putative p10 subunit of a type II metacaspase from Nicotiana tabacum L. may contribute to the substrate-binding pocket. Planta 239, 147–160 (2014). https://doi.org/10.1007/s00425-013-1975-0

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