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While fungal and protozoan metacaspases have been impli-
cated in a range of biological processes [ 2 , 4 ], the physiological
role of plant metacaspases has so far been only linked to cell death
regulation. In Arabidopsis , type I metacaspases AtMC1 and AtMC2
antagonistically control hypersensitive response-associated cell
death activated by intracellular immune receptors [ 5 ], whereas
type II metacaspases are positive regulators of cell death induced
by abiotic (AtMC4 and AtMC8) and biotic stress (AtMC4) [ 6 , 7 ].
mcII-Pa type II metacaspase of Norway spruce is required for vac-
uolar cell death in the embryo-suspensor, which is essential for cor-
rect embryonic patterning [ 8 ].
Considering a growing evidence for metacaspases to represent
bona fi de plant cell-death proteases functionally equivalent to
metazoan caspases, and assuming potential involvement of plant
metacaspases in other, cell death unrelated, processes (by analogy
with fungal and protozoan metacaspases [ 9 ], there is a need for a
set of robust methods to detect activation and to measure proteo-
lytic activity of recombinant and endogenous plant metacaspases.
These methods are also required to advance our understanding of
molecular mechanisms regulating metacaspase activation.
Similar to caspases, maturation of metacaspase enzyme involves
autocatalytic processing of the zymogen [ 2 ]; thus autoprocessing
can be used as a marker of activated metacaspase. Detection of pro-
cessed form of metacaspase in cell lysates or in situ should however
be interpreted with caution, since inhibitor binding (e.g., serpin
[ 10 ]) can antagonize autoprocessing-mediated activation. Active
metacaspase is a monomer [ 11 , 12 ] for type I metacaspases (PV
Bozhkov and AP Smertenko, unpublished results, for type II meta-
caspase mcII-Pa), a feature diverging metacaspases and caspases,
because the latter require dimerization as a part of activation mecha-
nism [ 13 ]. Another feature contrasting metacaspases and caspases is
a requirement of Ca 2+ for activation. While caspases are Ca 2+ -
independent proteases, a vast majority of metacaspases studied so far
require millimolar concentrations of Ca 2+ to become active in vitro
[ 14 – 16 ]. All above-described biochemical characteristics should be
taken into consideration when setting up assays for detecting active
forms of metacaspases and measuring their activity.
However, the chief property of a protease that must be known
for correct measurement of its catalytic activity is substrate cleavage
specifi city. In contrast to Asp-specifi c caspases, metacaspases cleave
peptide bonds C-terminal to Arg or Lys residues [ 2 , 14 ]. We have
previously pointed to inapplicability of caspase substrates for meta-
caspase activity assays and urged to use peptidic substrates with Arg
or Lys at the P1 position [ 2 ] ( see Note 1 ).
There are two further points that require special attention when
measuring metacaspase activity in the total or fractionated plant cell
extracts. First, it remains unknown how much the extended sub-
strate specifi city (i.e., P2–P4 and P1′–P4′) differs between type I
and type II metacaspases, as well as between different metacaspasesElena A. Minina et al.http://www.ebook3000.com