Biochemical and Bioinformatic Characterization of Type II Metacaspase Protein (TaeMCAII) from Wheat

Abstract

The biochemical analysis and homology modeling of a tertiary structure of a cereal type II metacaspase protein from wheat (Triticum aestivum), TaeMCAII, are presented. The biochemical characterization of synthetic oligopeptides and protease inhibitors of Escherichia coli-produced and purified recombinant TaeMCAII revealed that this metacaspase protein, similar to other known plant metacaspases, is an arginine/lysine-specific cysteine protease. Thus, a model of a plant type II metacaspase structure based on newly identified putative metacaspase-like template was proposed. Homology modeling of the TaeMCAII active site tertiary structure showed two cysteine residues, Cys140 and 23, in close proximity to the catalytic histidine, most likely participating in proton exchange during the catalytic process. The autoprocessing that leads to activation of TaeMCAII was highly dependent on Cys140. TaeMCAII required high levels of calcium ions for activity, which could indicate its involvement in stress signaling pathways connected to programmed cell death.

Type III metacaspases: calcium‐dependent activity proposes new function for the p10 domain

“…The activity of the majority of type I and type II metacaspases characterized to date has been shown to be affected by the presence of calcium ions. Although TbMC2 is the only type I metacaspase requiring high micromolar concentrations of CaCl 2 for activity (Machado et al ., ), yeast type I metacaspase Yca1, as well as type II metacaspases and the type III metacaspases reported here, require millimolar concentrations for full proteolytic activity (Watanabe & Lam, ; Zhang & Lam, ; Piszczek et al ., ). The only exception is the A. thaliana type II metacaspase AtMC9, whose activity has been reported to be calcium independent (Zhang & Lam, ).…”

“…Increasing biochemical evidence has shown that the catalytic activity of type I and type II metacaspases depends on the presence of calcium ions, with the only exception of an Arabidopsis thaliana type II metacaspase, AtMC9, whose activity is calcium independent (Zhang & Lam, ). In type II metacaspases, the presence of CaCl 2 in millimolar concentrations induces specific cleavage in the linker region between p20 and p10 domains, similar to the activation observed in caspases (Vercammen et al ., ; Lam & Zhang, ; Piszczek et al ., ). Although cleavage between the two domains in type I metacaspases does not seem to be essential for activation, the presence of calcium is a fundamental requirement for their catalytic activity (Moss et al ., ; Wong et al ., ).…”

Two-dimensional zymography in detection of proteolytic enzymes in wheat leaves

“…Authors have mentioned two isoforms of 35 and 27 kDa isolated by affinity chromatography in denaturing conditions. Type II metacaspase from wheat leaves (TaeMCAII) was modeled in silico, cloned and characterized (Piszczek et al 2011;Piszczek et al 2012). Studies of Arabidopsis thaliana dwarf mutant, impaired in carbohydrate metabolism, enabled characterization of NANA, chloroplastlocated, aspartic proteinase (Paparelli et al 2012).…”

Bacterial putative metacaspase structure from Geobacter sulfureducens as a template for homology modeling of type II Triticum aestivum metacaspase (TaeMCAII).

“…Animal caspases are cysteine endopeptidases cleaving their substrates on the carboxyl side of aspartate residues (Kaufmann & Hengartner, 2001). Studies with recombinant plant metacaspases, including TaeMCAII, showed that metacaspases are unable to cleave caspase substrates and that their preferred cleavage site was after Arg or Lys residues (Vercammen et al, 2004;Bozhkov et al, 2005;Vercammen et al, 2006;Piszczek et al, 2012). Arginine and lysine have positively charged side-chains; thus, negatively charged residues surrounding the active site can facilitate ligand binding.…”