27caspases and to understand the impact of specific cleavage events
on caspase activity and specificity. Indeed, although it was known
for years that the initiator caspase-9 does not require cleavage to
display full enzymatic activity [ 37 ], other initiators are more stable
when cleaved [ 41 ]. Cleavage may also increase the activity of initia-
tor caspases without affecting their substrate specificity in vitro
[ 29 ]. Examples of caspase-7 and caspase-9 cleavage-site mutants
purified as described in this chapter are presented in Fig. 6.
Expression of wild-type zymogen forms of executioner cas-
pase- 3 and caspase-7 can be useful to study activation of these pep-
tidases by initiator caspases [ 35 , 38 , 42 , 43 ]. Because of the
intrinsic activity of initiator caspases and their propensity to dimer-
ize and become active, it is impossible to prepare uncleaved zymo-
gen forms of these caspases without resorting to cleavage-site
mutants (Fig. 6a).
Several other caspase mutants that result in the production of
particular molecular forms have been described in the literature.
These include mutations that prevent dimerization of caspase-8
[ 30 ], force caspase-9 dimerization [ 44 ], allow the activity of the
zymogen form of executioner caspases [ 45 ], and produce caspase
chimeras [ 31 , 32 ]. However, it must be stressed that although
these mutations were made to affect specific properties of caspases,
they may also alter the kinetic properties of the enzyme in vitro and
in cells.Fig. 6 Various molecular forms of caspases. (a) Zymogen (inactive) form of caspase-7 cleavage-site mutants.
The zymogen forms of wild-type (wt) caspase-7 or cleavage-site mutants were expressed for 30 min and
purified using the protocol described in Subheading 3.1.1. FL full-length, ΔN no N-terminal peptide (residues
1–23), LS large subunit, SS small subunit. These results were originally published in Molecular Cell [ 35 ] ©
Elsevier B.V. B) Cleavage site mutants of full-length caspase-9. Active wild-type caspase-9 (WT) or caspase-9
proteins cleaved at site 1 (C9AISS) or site 2 (C9ATPF) in the linker or a double cleavage-site mutant (DD → AA) were
expressed as described in Subheading 3.1.1. Gels were stained using Coomassie Blue. These results were
originally published in Biochemical Journal [ 60 ] © The Biochemical Society
Apoptotic Caspases Assays