Biochemical and Bioinformatic Characterization of Type II Metacaspase Protein (TaeMCAII) from Wheat
E. Piszczek
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M. Dudkiewicz
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M. Mielecki
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M. Mielecki Department of Protein Biosynthesis Institute of Biochemistry and Biophysics, Polish Academy of Sciences
, Pawinskiego 5a, 02106, Warsaw,
Poland
1
M. Dudkiewicz Department of Experimental Design and Bioinformatics, Warsaw University of Life Sciences
, Nowoursynowska 159, 02776, Warsaw,
Poland
2
) Department of Biochemistry, Warsaw University of Life Sciences
, Nowoursynowska 159, 02776, Warsaw,
Poland
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.
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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. Plants and animals
share many similarities in the morphological features and
enzymatic machinery of PCD (Sun et al. 2012; Sanmartin et
al. 2005). 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 crystal structures of caspases show that the
active enzymes are heterotetramers that contain two small and
two large subunits. 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. Two
residues, cysteine and histidine, are present in the active sites
and participate in catalysis (Ho and Hawkins 2005; Cohen
1997). 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).
The existence of distant caspase relatives named
caspaselike proteases has been demonstrated previously in plant cells
undergoing PCD (Sanmartin et al. 2005). Metacaspases, which
are caspase-like enzymes, were discovered in silico in the
Arabidopsis genome more than a decade ago, and they are
present in protozoa, fungi and plants (Uren et al. 2000).
Phylogenetic analysis has revealed that metacaspases are distant
homologs and ancestors of animal caspases (Vercammen et al.
2007). These proteases, together with eukaryotic caspases,
metazoan paracaspases, legumains, separases and the bacterial
clostripains and gingipains, are classified as members of clan
CD cysteine proteases (Bonneau et al. 2008). All proteins from
this clan share a common structural feature, the presence of the
caspasehemoglobinase fold (Bonneau et al. 2008).
On the basis of metacaspase structure, they can be
subdivided into two groups: type I and type II. Type I metacaspases
possess an N-terminal prodomain with a Zn finger motif,
which is absent in type II. The distinguishable feature of type
II metacaspases is the presence of a linker region between the
large and the small subunit (Piszczek and Gutman 2007).
Until now, it has been shown for Arabidopsis thaliana and
Picea abies type II metacaspases that they are synthesized as
inactive zymogens, and that they are activated by
autoprocessing, similar to effector caspases from mammals (Vercammen
et al. 2004; Bozhkov et al. 2005). In contrast, type I
metacaspases from Arabidopsis do not autoprocess, and most likely
similar to initiator mammalian caspases, they require
oligomerization for activity (Vercammen et al. 2004). Metacaspases
and animal caspases contain a conserved catalytic His/Cys
dyad in their active site, with the Cys residue acting as a
nucleophile for substrate peptide bond hydrolysis (Piszczek
and Gutman 2007). The striking difference between all
discovered metacaspases and caspases is the formers preference
for Arg or Lys residues in their substrates (Vercammen et al.
2004; Bozhkov et al. 2005). Metacaspase activities can be
modified by post-tr (...truncated)