26
insoluble, and the alternative protocol described in Subheading 3.1.2
must be used. Alternatively, F122Y and L123S mutations in the
second DED of caspase-8 may be used to prevent aggregation and
render the protein soluble [ 36 ], but it is important to keep in mind
that it is no longer the wild-type enzyme.
Caspase-9
Caspase-9 is the easiest initiator caspase to produce, either as a
truncated form or as a full-length protein. Complete processing
occurs within 5 h of expression. Indeed, it is impossible to produce
uncleaved caspase-9 without the use of cleavage-site mutants [ 37 ].
Full-length and CARD-less caspase-9 have the same substrate pref-
erence [ 38 ], at least on small peptidic substrates, and for reasons
that are still poorly understood, the complete enzyme is less active
than the truncated caspase-9 [ 38 ].
Caspase-10
Caspase-10 expresses similarly to caspase-8. However, the large
subunit and small subunit of the catalytic domain bind each other
with much less strength so that some of the large subunit is lost
during purification. Consequently, care must be taken to keep frac-
tions that, once pooled, result in a 1:1 subunit ratio. It is assumed
that caspase-10, similarly to caspase-8, will be essentially insoluble
as a full-length form.Over the years, many variants of caspases were produced so that
their mechanisms could be studied. The most prominent classes of
those variants are either inactive forms or forms that carry cleavage-
site mutations. For example, catalytic mutants of caspase-3 and
caspase- 7 can be used as substrates in cleavage assays by initiator
caspases. Catalytic mutants of caspases are easily expressed (much
easier than the corresponding zymogen forms), with high yields
using the protocol described in Subheading 3.1.1. The use of cata-
lytic mutants of caspases can also be appropriate as a negative con-
trol. Inactive caspases are usually generated by mutating the
catalytic cysteine into an alanine residue [C285A mutation, cas-
pase- 1 structural nomenclature [ 8 ]], but some researchers have
replaced the catalytic cysteine with a serine residue [ 39 ]. Mutation
of histidine 237 to an alanine (H237A) has the same effect as the
cysteine mutation. This is because those two amino acids, Cys285
and His237, form the caspase catalytic dyad of the peptidase, and
mutation of either one of them is sufficient to completely abrogate
enzymatic activity [ 8 , 40 ].
Caspases participate in a proteolytic cascade and often cleave
themselves during apoptosis. Those cleavage events may activate
the caspases, regulate their association with activation platforms,
alter their substrate specificity or provide a way to regulate their
interactions with endogenous inhibitors and molecular partners.
Mutation of the aspartate residue at the cleavage site abrogates pro-
teolysis. These mutations are quite useful to study the activation of3.5 Useful Molecular
Forms of Caspases
Dave Boucher et al.http://www.ebook3000.com