title: Calcium-dependent activation and autolysis of Arabidopsis metacaspase 2d.
abstract: Metacaspases (MCPs) are members of a new family of cysteine proteases found in plants, fungi, and protozoa that are structurally related to metazoan caspases. Recent studies showed that plant MCPs are arginine/lysine-specific cysteine proteases with caspase-like processing activities in vitro and in vivo, and some of the plant type II MCPs exhibit Ca(2+) dependence for their endopeptidase activity in vitro. However, the mechanisms and biological relevance of Ca(2+) dependence and self-processing of plant MCPs remains unclear. Here we show that recombinant AtMCP2d, the most abundantly expressed member of Arabidopsis type II MCPs at the transcriptional level, exhibits a strict Ca(2+) dependence for its catalytic activation that is apparently mediated by intramolecular self-cleavage mechanism. However, rapid inactivation of AtMCP2d activity concomitant with Ca(2+)-induced self-processing at multiple internal sites was observed. Because active AtMCP2d can cleave its inactive form, intermolecular cleavage (autolysis) of AtMCP2d could also occur under our assay conditions. Ca(2+)-induced self-processing of recombinant AtMCP2d was found to correlate with the sequential appearance of at least six intermediates, including self-cleaved forms, during the proenzyme purification process. Six of these peptides were characterized, and the cleavage sites were mapped through N-terminal protein sequencing. Mutation analysis of AtMCP2d revealed that cleavage after Lys-225, which is a highly conserved residue among the six Arabidopsis type II MCPs, is critical for the catalytic activation by Ca(2+), and we demonstrate that this residue is essential for AtMCP2d activation of H(2)O(2)-induced cell death in yeast. Together, our results provide clues to understand the mode of regulation for this class of proteases.
authors.affiliation: Department of Plant Biology and Pathology, Rutgers University, New Brunswick, New Jersey 08901-8550, USA.
authors.forename: Naohide
authors.initials: N
authors.lastname: Watanabe
authors.forename: Eric
authors.initials: E
authors.lastname: Lam
mesh_terms: D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020169:Caspases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D020169:Caspases; D002118:Calcium