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INTRODUCTION |
Apoptosis, the ordered dismantling of animal cells that results in
their death and removal from the organism, requires specific proteolysis of a subset of cellular proteins, mediated by caspases. Many apoptotic responses are initiated by activation of the apical caspases-8 or -9; the former by recruitment to ligated cell surface receptors belonging to tumor necrosis factor receptor-1 family (1, 2),
and the latter by recruitment to Apaf-1, in the presence of ATP or
dATP, following delivery of cytochrome c
(cyto-c)1 from
mitochondria (3). Activation of either of these two initiator caspases
can lead to activation of the executioner caspase-3 (casp-3), by direct
proteolysis of the casp-3 precursor (4, 5). Indeed all caspase zymogens
are thought to be activated by limited proteolysis within a linker
segment which results in generation of the characteristic large and
small subunits of the catalytically active enzyme. Thus it has been
specifically demonstrated that casp-1, -3, -7, and -8 gain marked
increases in activity after proteolytic processing (6-9). This is
typical of proteolytic enzymes from all families and catalytic classes.
As pointed out by Neurath (10), the zymogens are stored in an inactive
form to prevent adventitious proteolysis before it is needed at the
required site of action. Frequently protease zymogens are activated by
other proteases, in a cascade mechanism that results in either
amplification or localization to specific sites. There are few
exceptions to the rule that zymogen activation requires proteolysis,
and these exceptional zymogens, such as tissue plasminogen activator
(tPA), usually have specific allosteric modulators that allow them to
become active at the required site, without proteolysis.
The initiator casp-8 has some of these exceptional properties. Casp-8
initiates apoptotic signaling induced by specific ligation of death
receptors (reviewed in Refs. 11 and 12). Recruitment of procasp-8 to
the cytosolic face of ligated Fas or tumor necrosis factor receptor-1
results in its autoactivation by a mechanism involving clustering of
zymogen molecules (9, 13). The zymogen possesses ~1% of the activity
of the activated enzyme, and it is hypothesized that this small amount
of activity of the zymogen is sufficient to drive cleavage of clustered
casp-8 zymogen molecules to the active form. Thus casp-8 has a
zymogenicity ratio of 100 (defined as the ratio of the activity of the
enzyme to the zymogen). Nevertheless, proteolysis of the casp-8 zymogen
is thought to be required for its activation (9, 13).
In light of the casp-8 activation studies, it has been hypothesized
that the initiator caspases, including caspases-8 and -9 (14) and
Caenorhabditis elegans CED-3, share a common mechanism of
activation (15). This would require clustering zymogens that possess a
small amount of activity in their single chain forms, with production
of the active protease dependent on proteolytic processing of the
zymogen. In turn, this would mean that preventing proteolysis of the
zymogen in the interdomain linker should inactivate apoptotic signal
transmission. We tested this hypothesis for casp-9 by utilizing a
cell-free system (16) that allows dissection of the requirements for
zymogen activation.
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EXPERIMENTAL PROCEDURES |
Materials--
Acetyl-DEVD-p-nitroanilide
(Ac-DEVD-pNA) and carbobenzoxy-VAD-fluoromethyl ketone (Z-VAD-FMK) were
from Bachem. Flourogenic 7-amino-4-trifluoromethyl coumarin (AFC)
caspase substrates (Ac-DEVD-AFC and Ac-LEHD-AFC) were from Enzyme
System Products. Dithiothreitol was from Diagnostic Chemicals Ltd.
Sucrose was from Mallickrodt. All other chemicals were from Sigma.
Rabbit anti-caspase-9 was kindly provided by Dr. Douglas R. Green, La
Jolla Institute for Allergy and Immunology (8). Rabbit antisera to
caspases-3 and -7 were from in-house collections (17). The casp-3
zymogen was expressed and purified from Escherichia coli as
described previously (8). The protein concentration of purified
zymogens was determined from the absorbance at 280 nm based on the
molar absorption coefficients calculated from the Edelhoch relationship
(18).
Methods--
The specific mutants casp-9 (D315A), casp-9
(D330A), and casp-9 (D315A/D330A) were constructed using overlap
polymerase chain reaction techniques. A CARD domain deleted form
(
CARD casp-9) was constructed by truncating the cDNA and
introducing an initiator codon substituting Val139. All
constructs were expressed in E. coli and isolated by virtue of an engineered C-terminal His6 purification tag as
described previously (19, 20). The sizes and composition of purified
proteins and protein fragments were analyzed by SDS-PAGE and/or
MALDI-TOF.
Cytosolic extracts of 293 cells, and casp-9-depleted extracts were
prepared as described (8). The extracts were stored in appropriate
portions at
80 °C. Activation of casp-9 in cytosolic extracts was
initiated by addition of 1 mM dATP and 10 µM
cyto-c followed by incubation at 37 °C for 30 min (3).
These conditions routinely result in 100% activation of the endogenous
executioner casp-3 as judged by comparison with recombinant, fully
active casp-3. The caspase activity in 2.5 µl of cytosolic extract
was determined by measuring the cleavage of Ac-DEVD-AFC as described previously (8, 20, 21). Alternatively, the generation of activity can
be followed by release of pNA from Ac-DEVD-pNA, reflecting the
instantaneous generation of executioner caspases, which in 293 cells is
predominantly due to casp-3 (8).
SDS-PAGE was performed using 8-18% acrylamide gels in the
2amino-2-methyl-1,3-propandiol/glycine discontinuous buffer system (22). After electrophoresis the gels were either stained using Gel-Code
(Pierce) according to the manufacturer's protocol or blotted to
Immobilon-P (Millipore) (23). Western blot analysis was performed with
the ECL kit (Amersham Pharmacia Biotech) using rabbit anti-casp-3 and
anti-casp-9.
Cell death assays were performed as described previously (24). Briefly,
2.5 × 105 293T cells were transfected with 1 µg of
the mutant or wild-type plasmids plus 0.1 µg of
cytomegalovirus-
-galactosidase in six-well tissue culture dishes
using calcium phosphate precipitation. Approximately 36 h after
transfection, the cells were fixed with 0.5% glutaraldehyde and then
stained with
5-bromo-4-chloro-3-indoxyl-
-D-galactopyranoside and
examined by phase contrast microscopy. At least 300
-galactosidase-positive cells were counted for each transfection and
identified as apoptotic or nonapoptotic based on morphological
alterations typical of adherent cells undergoing apoptosis, including
becoming rounded, condensed, and detached from the dish (25). Percent
apoptosis represents the mean value from two independent experiments
(mean ± S.D.).
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RESULTS AND DISCUSSION |
Purified Recombinant Casp-9 Is Maximally Active Only in the
Presence of Cytosolic Components--
Recombinant casp-9 was obtained
in the fully processed form, consisting of two chains caused by
autolytic cleavage at Asp315, as determined by N-terminal
Edman degradation and MALDI-TOF analysis of the isolated material (Fig.
1, A and B). The
CARD domain-deleted form of casp-9 was obtained in the same way, with the same internal cleavage site. Both purified proteins had very low
activity when measured with Ac-LEHD-AFC and contained only 1-5%
active sites as judged Z-VAD-FMK titration. Thus the recombinant material is largely inactive compared with other caspases such as 3, 6, 7, and 8, which normally demonstrate 50-100% active sites following
expression and purification by the same procedure (5). Moreover,
activation of the natural substrate procasp-3 was extremely slow,
leading us to speculate that the recombinant casp-9 forms purified from
E. coli are not equivalent to those operating in vivo. To test this we immuno-depleted endogenous casp-9 from a cytosolic extract made from 293 cells, and reconstituted with the
recombinant forms.

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Fig. 1.
Reconstitution of casp-9 in cytosolic
extracts. A, recombinant proteins were analyzed for
purity by SDS-PAGE. The wild-type, CARD, and D330A versions were
obtained in the two-chain form due to autolytic processing at
Asp315 (white asterisk), whereas D315A and
D315A/D330A were largely unprocessed, but contained 5.1% of material
cleaved at Ser307 (black asterisk).
B, representation of the casp-9 precursor. The start codon
for the CARD version replaces Val139. The position of
the catalytic cysteine (Cys287), the autolytic cleavage
sites observed at Ser307 and Asp315, and the
Asp330 site cleaved by casp-3 are denoted by
arrows, and asterisks corresponding to the
cleavage products identified in A. C, naive
cytosolic extracts were immunodepleted of casp-9 and reconstituted with
the indicated concentrations of casp-9 recombinants (nanomolar).
Reconstitution of the depleted extracts recapitulated caspase
activation, measured by DEVD-AFC substrate hydrolysis, only in the
presence of cyto-c/dATP and only when casp-9 contained an
intact CARD domain.
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Quantitative immunoblot analyses revealed that the concentration of
casp-9 in the extract was approximately 20 nM (not shown). Addition of 200 nM (10-fold excess of the endogenous
concentration) of the interdomain cleaved forms of
CARD or
full-length casp-9 to the depleted extract resulted only in limited
induction of DEVD-AFC cleaving activity (Fig. 1C). In this
assay, the cleavage of DEVD-AFC measures the activity of protease(s)
that are activated by casp-9 in cytosolic extracts and thereby acts as
an indirect measure of the activity of casp-9 itself. The rate is
greatly amplified upon addition of cyto-c/dATP, which
activates casp-9 in cytosolic extracts (3, 26). While the activity of
the
CARD casp-9 decreases slightly upon addition of
cyto-c/dATP, full-length casp-9 recapitulated the activation
rate of the endogenous material, even when added at 20 nM,
equivalent to the endogenous casp-9 concentration. This is equivalent
to a 2000-fold amplification of activity relative to the action of
fully processed casp-9 on purified procasp-3 and demonstrates that
binding to a cytosolic factor, probably the reported casp-9 activator
Apaf-1 (26), is essential for the activity of casp-9 and not just for
its activation. This is consistent with the demonstration that cleavage
of casp-9 in cytosolic extracts devoid of cyto-c failed to
accelerate caspase activation (8).
Proteolytic Processing of Casp-9 Is Not Required for
Activation--
Based on the consensus view that procaspase activation
requires proteolysis in the interdomain linker segment (reviewed in Ref. 4), we expected mutants that prevent processing would prevent
activation. Moreover, since casp-9 is recruited to Apaf-1 for
activation, the noncleavable mutants may displace endogenous casp-9
from its activation complex and thus prevent its activation. Thus we
constructed the processing site mutants casp-9 (D315A), casp-9 (D330A),
and casp-9 (D315A/D330A), as well as a catalytically incompetent mutant
casp-9 (C287A), see Fig. 1, A and B, for details, and tested them in the in vitro cytosolic reconstitution
system. Substitution of endogenous casp-9 by any of the cleavage site mutants recapitulated caspase activation. The single mutants were only
slightly less effective than endogenous casp-9, while the double mutant
(D315A/D330A) was the least effective, but still reconstituted 40% of
the wild-type level (Fig.
2A).

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Fig. 2.
Activation of casp-9 mutants. Purified
casp-9 mutants were added to immunodepleted extracts at 20 nM, followed by addition of cyto-c/dATP. Caspase
activity was measured either after 30 min at 37 °C by using
Ac-DEVD-AFC (A) or followed continuously at 37 °C in the
presence of 0.4 mM Ac-DEVD-pNA (B). In
A, additional controls were performed with 1 nM
processed casp-9 (designated 5% wt) and a mixture of 2 nM processed casp-9 with 18 nM catalytic mutant
(designated 10% wt + C287A). C, cytosolic
extracts immunodepleted of endogenous casp-9, to which 20 nM recombinant proteins were added, were Western blotted
with an anti-casp-9 antiserum before ( ) or after addition of
cyto-c/dATP for 30 min at 37 °C (+). Note that the
portion of material cleaved at Ser307 does not increase
during activation by cyto-c/dATP (black
asterisks). The extra band seen following activation of the D315A
extract is due to cleavage at Asp330 by casp-3 (14).
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Interestingly, the linker site mutants contained a small amount of
aberrantly processed material that was determined by N-terminal sequence analysis and MALDI-TOF to be due to cleavage between Ser307 and Pro308 (Fig. 1A). The
amount of this material relative to the D315A/D330A single chain
zymogen was determined to be 5.1 (±1.2)% based on quantitative image
analysis of Coomassie-stained gels. The origin of this cleaved material
is a mystery, but appears to require a competent catalytic site in
casp-9, since it was absent from the C287A mutant. The presence of this
aberrantly processed material raises two possibilities which pertain
directly to whether proteolytic processing of casp-9 is required for
activation. Is the small amount of aberrant material the activator, not
the residual 95% of unprocessed material in the D315A/D330A cleavage
site mutant? This is unlikely since an amount of wild-type processed
casp-9 equal to the aberrantly processed material was only able to
generate 9% maximal activity, as opposed to the 40% attained by the
D315A/D330A cleavage site mutant (Fig. 2A). Moreover, if
casp-9 requires cleavage for activation, it should compete with
unprocessed material (D315A/D330A) for the activator complex. Indeed,
when we simulated this by adding 2 nM wild-type processed
casp-9 to 18 nM catalytic mutant (C287A), activation was
abolished, presumably because the catalytic mutant prevents recruitment
of processed casp-9 to the activator complex. Therefore we are left
with a second possibility: the unprocessed D315A/D330A mutant is
directly responsible for generating activity. To test whether the
aberrant material increased during the incubation, Western blot
analysis was conducted on cytosolic extracts that had been activated by
cyto-c/dATP (Fig. 2C). No increase in the amount
of the aberrant material, quantitated by image analysis, could be
observed. This rules out adventitious proteolytic activation of the
D315A/D330A mutant during the course of the assay and confirms that
casp-9 can become active without proteolytic processing.
The generation of DEVD-pNA cleavage as a function of time provides a
measure of instantaneous casp-3 activation. Casp-9 mutants that can be
cleaved at the Asp315 autoprocessing site (wild-type casp-9
and D330A) demonstrate kinetics very similar to endogenous casp-9, but
mutants that cannot be cleaved at Asp315 (D315A and
D315A/D330A) cause DEVD-pNA cleavage more slowly (Fig. 2B).
However, in the D315A mutant there is a significant beneficial effect
of being able to form a two chain enzyme by cleavage at Asp330. Thus, the rate of caspase activation increases
significantly as an increasing amount of caspase activity is generated,
presumably due to a feedback of casp-3 onto the Asp330 site
of casp-9 as proposed by Srinivasula et al. (14).
Because the activation of procasp-3 by casp-9 is a complex process, it
is difficult to derive an exact quantitation of the activity of casp-9.
However, the cumulative caspase activity caused by endogenous casp-9 is
2.5-fold that of the double mutant, and this occurs about four times
faster for the endogenous casp-9 than for the double mutant (Fig. 2,
A and B). The product of these two measures of
activation indicates that the double mutant has at least 10% of the
activity of endogenous casp-9 when both are allowed to activate under
identical conditions in cyto-c-programmed cytosols. Thus,
since the casp-9 D315A/D330A mutant can be considered a zymogen, the
zymogenicity ratio, which describes the activity of an active enzyme
relative to its latent precursor, is at most 10.
Cleavage Site Mutants Do Not Act as Dominant Negative Inhibitors of
Endogenous Casp-9--
These results combine to suggest that the
cleavage site mutants would not act as dominant negative inhibitors and
that they indeed should support apoptosis. Only the catalytic mutant
should act as a dominant negative. To test this hypothesis, the
constructs were subcloned into pcDNA3 and transiently expressed in
293T cells. As demonstrated in Fig. 3,
both D315A and D315A/D330A induced apoptosis to the level observed for
the wild-type and D330A, whereas the catalytic mutant casp-9 (C287A)
failed to induce more apoptosis than the empty vector control. Only the
catalytic mutant acted to suppress casp-9 activation in
vivo, presumably by competing with endogenous casp-9 for binding
to the cytosolic activators.

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Fig. 3.
The processing site mutants are
proapoptotic. Human 293T cells were transfected with plasmids
encoding the indicated constructs, or an empty vector control, and
cells were quantitated for apoptosis. The cleavage site mutants are
proapoptotic, and only the catalytic mutant (C287A) acts as a dominant
negative inhibitor of caspase activation.
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Zymogenicity and Casp-9--
Several caspases undergo proteolytic
processing when expressed in E. coli, probably due to
intrinsic catalytic properties of the zymogens which, though low, are
sufficient to allow autolysis under the high concentrations obtained in
expression (4, 5). However, unlike other caspases which demonstrate
substantial activity when purified from E. coli, casp-9
appears to be largely inactive until it is delivered to the correct
cellular environment. It is not clear whether the endogenous cytosolic
factors (presumably Apaf-1 in the presence of cyto-c/dATP)
bind casp-9 and then release it in an active conformation or whether
casp-9 must remain attached to retain activity. Experiments with
purified cytosolic factors should answer this question. Nevertheless,
proteolysis of casp-9 does not seem to be required for its activation.
Certainly, processing in the interdomain linker, characteristic for
other caspases, enhances casp-9 activity, but only by 10-fold, thus the
zymogenicity ratio of casp-9 is about 10. On the other hand, binding to
cytosolic factors enhances activity 2000-fold to give a robust enzyme
capable of activating available procasp-3 in a few minutes. In the
natural cellular environment it is clear that casp-9 is processed
during apoptosis, as demonstrated in (14) for example, and this can occur either due to an autoprocessing cleavage at
Asp315 or by trans-processing by casp-3 at
Asp330. However, in light of our data it would now be
necessary to distinguish between processing (which may be adventitious)
and activation, which does not appear to derive its driving force
simply from proteolytic processing.
These observations on the relation of proteolytic processing to zymogen
activation are reminiscent of tPA, which demonstrates a very small
zymogenicity ratio, 2-10 (27). However, upon binding to fibrin, the
ability of tPA to activate its physiologic substrate plasminogen
increases several thousandfold (28). Presumably enzymes such as tPA and
casp-9 have abolished the requirement for proteolysis as a mechanism of
substantially increasing their activities, because allosteric
regulators substitute this function, fibrin for tPA and Apaf-1 for
casp-9. In the case of tPA, specific side chain interactions, absent in
other members of the chymotrypsin family, allow activity of the
zymogen. However, in the absence of a molecular structure of the casp-9
zymogen, few clues are available to explain the high activity of the
unprocessed protein. One clue is suggested by the structure of active
casp-1 and casp-3, each composed of two catalytic units thought to
arise from dimerization of monomeric zymogens (reviewed in Ref. 5). If
activation of zymogens of the initiator caspases-8 and -9 and CED3
operates by clustering, then the clustering phenomenon may be explained by adapter-driven homodimerization of monomers. In the case of casp-9,
the putative dimerization would be envisaged to activate the protease,
and from a mechanistic point of view, though not necessarily from a
biologic one, proteolysis is an epiphenomenon. It will be interesting
to determine whether similar processing-independent mechanisms for
activation apply to the other initiator caspases.