239of the same type. Yet extended specifi city of individual plant meta-
caspases appears to be rather loose, as follows from screening of
combinatorial peptide library with recombinant AtMC9 [ 10 ],
AtMC9 degradome [ 17 ] and analysis of four mcII- Pa cleavage sites
present in its natural target protein Tudor staphylococcal nuclease
(TSN) [ 18 ]. This implies that a given substrate could be the target
of two or several metacaspases and also provides explanation for a
high degree of redundancy among metacaspases. Second, synthetic
peptides with Arg(P1) or Lys(P1) could be cleaved by proteases
other than metacaspases, e.g., separases, cathepsins, or subtilisins.
Genetic knockouts or knockdowns of metacaspase gene(s) are far
and away the best tools to assess which fraction of Arg- and/or Lys-
specifi c proteolytic activity is attributed to metacaspases [ 15 ]. The
bottom line is that no truly specifi c synthetic substrates (and for the
same reasons, inhibitors) are available and that strict caution must
be exercised in ascribing observed activity to metacaspases in gen-
eral, to their specifi c type (I or II) or to individual metacaspase.
Accordingly, quantitative data obtained in the assays measuring
proteolysis of Arg(P1)- or Lys(P1)-containing synthetic peptides
refl ect “metacaspase- like activity”, unless there is an experimental
evidence for the absence of other types of Arg- and/or Lys-specifi c
proteases in the given biological system. In general it is preferably
to assess metacaspase activity by using a combination of different
methods as described in this chapter.
Two main approaches are currently used to detect metacaspase
activation and measure its proteolytic activity: (1) detection of pro-
cessed metacaspase and/or reduced zymogen forms by immunob-
lotting and (2) substrate cleavage assays using synthetic peptidic
substrates conjugated with fl uorescent moiety. New evidences
about in vivo substrates of metacaspases are appearing [ 17 , 18 ]. In
case of Norway spruce embryos where type II metacaspase mcII-
Pa is known to cleave TSN protein, immunoblotting analysis of
full-length TSN and its fragments is an additional tool to evaluate
mcII-Pa activity. In the following sections, we describe above-
mentioned methods step by step and how they are used in our
laboratories.2 Materials
- Sterile conical fl asks (50–500 mL).
- Shaker at 37 and 28 °C.
- Spectrophotometer to measure OD at 600 nm.
- Sonicator or French press.
- Nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen, 30210 or
similar). - Isopropyl β- D -thiogalactoside (IPTG) (Thermo Scientifi c,
R1171 or similar).
2.1 Production
of Recombinant
Metacaspases
Plant Metacaspases