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.
Highlights
Programmed cell death (PCD) is a process of elimination of unwanted cells during the ontogenesis of organisms and in response to environmental stresses
The overproduction of TaeMCAII in bacterial cells led to its autoactivation, which consisted of the removal of a 66-aminoacid fragment with a molecular weight of 7.45 kDa from the middle of the zymogen
When His6 tag-purified wild-type TaeMCAII and mutant form TaeMCAIIC23A were analyzed by PAGE and immunoblotted with anti-His antibodies, the appropriate fragments corresponding to those two subunits and zymogen were visible (Fig. 2a,b)
Summary
Programmed cell death (PCD) is a process of elimination of unwanted cells during the ontogenesis of organisms and in response to environmental stresses. It is common to all eukaryotic cells, including animal and plant cells. The initiators and executors of animal PCD are caspases, a family of cysteine-dependent proteases that cleave their substrates at the carboxyl-terminal side of aspartate residues. They are synthesized as inactive proenzymes that comprise an N-terminal prodomain together with one large and one small subunit. The enzymes have two active sites that are found at opposite ends of the molecules. Both the small and large subunits participate in the formation of active site. The activation of caspases during PCD processes such as apoptosis and autophagy results in the cleavage of important cellular proteins, including poly(ADP-ribose) polymerase and lamins, leading to the demise of the cell (Earnshaw et al 1999)
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