132
and apoptosis. The protocol below is adapted from Nutt et al.
(2005) [ 13 ]. In this study, we found that glucose-6-phosphate
(G6P) was capable of inhibiting caspase-3/7 activity, using the
assay described in Subheading 3.4. The initiating signal for induc-
tion of the apoptotic pathway was believed to be upstream of mito-
chondria, and G6P was believed to be imposing a block on this
pre-mitochondrial signal. To test this hypothesis, we tested the
activation of caspase-3/7 in the presence of G6P and tBID. If G6P
was inhibiting apoptosis upstream of mitochondria, then tBID
would be able to override and bypass this inhibition and induce
cytochrome c release and subsequent caspase-3/7 activation, which
is what we observed. This indicated that there was a signal upstream
of mitochondria and BCL-2 family proteins that was responsible
for initiating apoptosis.- Supplement egg extract from Subheading 3.1 with 1:20 dilu-
tion of 20× EM. Divide this extract into two separate aliquots.
Treat one of these samples with 20 mM G6P, and treat the
other sample with 20 mM G6P and 25 nM tBID. - Incubate the extract at room temperature. Every 30–60 min,
take a 3 μL aliquot to perform the caspase-3/7 assay from
Subheading 3.3 and 20 μL to perform the cytochrome c
release assay from Subheading 3.6.
An important anti-apoptotic BCL-2 family member is BCL-xL.
This protein belongs to a subfamily of anti-apoptotic BCL-2 family
proteins that prevent proteins like tBID and other members of the
BH3-only protein family from inducing MOMP, cytochrome c
release, and caspase activation. This property allows it to be used in
a similar fashion to tBID in Subheading 3.7.1 , in determining if the
signals initiating apoptosis are upstream of mitochondria and BCL-2
family proteins. The protocol below is also adapted from Nutt et al.
(2005) [ 13 ]. We hypothesized that the pre- mitochondrial signal
responsible for inducing apoptosis was caspase-2. Caspase-2 was
known to be important in regulating apoptosis in oocytes, and it had
been shown to be capable of cleaving BID to its active tBID form.
To test this hypothesis, we assessed the processing of caspase- 2 in X.
laevis egg extract, using the protocol described in Subheading 3.5 ,
in the absence and presence of BCL-xL. We also examined cyto-
chrome c release in these same extracts, in the absence and presence
of BCL-xL, using the protocol described in Subheading 3.6. If cas-
pase-2 activation was the pre-mitochondrial signal initiating apop-
tosis, then the addition of BCL-xL would inhibit cytochrome c
release but not affect caspase-2 activation. We observed that in
the presence of BCL-xL, caspase-2 was still processed, while
cytochrome c release was completely inhibited. This indicated that
caspase-2 was indeed an upstream of cytochrome c release.3.7.2 Using Recombinant
BCL-xL to Determine if
Inhibition of Caspase
Activation Lies Upstream
of Mitochondria and BCL-2
Family Proteins
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