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INTRODUCTION |
Caspases are a unique class of cysteine proteases that function as
effectors of apoptosis, or programmed cell death (1, 2). Caspases are
expressed in virtually all metazoan cells as catalytically inactive
zymogens known as procaspases. Two mechanisms have been described for
activating caspases: noncovalent association with caspase activating
proteins (e.g. Fadd, Apaf-1) leading to autocatalytic
cleavage of the procaspase polypeptide at specific aspartic acid
residues, or cleavage at specific aspartic acid residues within the
zymogen by other activated caspases. Caspases have been divided into
initiators (caspases-2, -8, -9, and -10) and effectors (caspases-3, -6, and -7) based on their relative position within a caspase cascade
(3-7). Initiator caspases are activated by the former mechanism,
effector caspases by the latter. The combined proteolytic activities of
initiator and effector caspases cleave a variety of vital protein
substrates, including DFF45/ICAD, lamin B, gelsolin, Bid, and PAK2,
leading to the morphological and biochemical characteristics of
apoptosis (8-11).
Caspase-9 is the initiator caspase in the "intrinsic" or
mitochrondrial caspase pathway. Interaction of caspase-9 with Apaf-1, a
human homologue of the Caenorhabditis elegans CED4 protein, to form the apoptosome occurs in response to cytochrome c
release from the mitochondria of pre-apoptotic cells (12). The
apoptosome is a multiprotein complex comprised of Apaf-1, cytochrome
c, and caspase-9 in a 1:1:1 molar ratio (12, 13). The
function of the apoptosome is to cleave and activate the apoptosis
effector caspases-3, -6, and -7 (14, 29). The apoptosome is assembled when seven Apaf-1:cytochrome c heterodimers oligomerize to
form a symmetrical "wheel" and procaspase-9 molecules become
associated noncovalently to Apaf-1 via caspase-9 CARD/Apaf-1 CARD
heterophilic interaction (15, 16). Binding of procaspase-9 to Apaf-1 is important for two reasons. First, it increases the intrinsic catalytic activity of the caspase-9 protease leading to the autolytic cleavage of
procaspase-9 at Asp315 to yield a large (p35) and a small
(p12) subunit (14, 17, 18). And second, cleavage exposes a neo-epitope
comprising the NH2-terminal four amino acids (ATPF) of the
small p12 subunit that has been shown to be both necessary and
sufficient for binding to the BIR3 domain of XIAP, leading to
inhibition of caspase-9 (19). Once activated in the apoptosome,
caspase-9 cleaves procaspase-3 at Asp175 and activates
caspase-3. Studies in vitro (14, 29) and in intact cells
(20) have shown that caspase-3 is capable of feedback cleavage of
caspase-9 at Asp330, and that this cleavage is associated
with an increase in apoptosome activity. Caspase-3-directed feedback
cleavage of caspase-9 p35/p12 at Asp330 would remove the
BIR3 recognition motif of caspase-9 (19), creating a caspase-9 species,
p35/p10, which may be insensitive to XIAP inhibition.
Using purified, recombinant Apaf-1, caspase-9, and caspase-3 the
present study has addressed the regulation of the apoptosome at three
levels. 1) Does caspase-9 cleavage affect apoptosome activity? 2) What
is the impact of caspase-3-mediated feedback cleavage of caspase-9 on
apoptosome activity? And 3) following feedback cleavage of caspase-9,
what impact does the loss of the BIR3 binding motif from the linker
region have on XIAP inhibition. Our results show that recombinant
proteins can combine in a dATP- and cytochrome c-dependent
manner to yield a catalytically active apoptosome capable of cleaving
and activating recombinant procaspase-3. The data further demonstrate
that association of procaspase-9 with Apaf-1 leads to a partial active
apoptosome containing p35/p12 caspase-9. Activation of caspase-3 by the
p35/p12-containing apoptosomes leads to the initiation of a feedback
loop whereby caspase-3 cleaves caspase-9 at Asp330. Our
data further demonstrate that p10-containing apoptosomes have enhanced
catalytic properties relative to p12-containing species. Finally, our
study identifies a novel motif at the NH2 terminus of the
p10 subunit capable of mediating XIAP inhibition. Thus, whereas
feedback cleavage of caspase-9 by caspase-3 significantly increases the
proteolytic activity of the apoptosome, it does little to attenuate its
sensitivity to inhibition by the endogenous caspase-9 inhibitor, XIAP.
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EXPERIMENTAL PROCEDURES |
General Methods and Materials--
Tris glycine gels, molecular
weight standards for SDS-PAGE, and 10× Tris glycine gel running buffer
were from Invitrogen, dATP was obtained from Amersham
Biosciences. Horse heart cytochrome c was purchased
from Sigma and further purified by passing through the ion exchange
column (Mono S). The fluorogenic tetrapeptide substrates DEVD-AMC and
LEHD-AMC were synthesized at Idun Pharmaceuticals as previously
described (21). General molecular biology methods were used as
described in Sambrook et al. (22).
Production of Recombinant Proteins--
Recombinant Apaf-1,
procaspase-9, and procaspase-3 proteins were produced in a baculovirus
expression system. Expression plasmids for Apaf-1 and wild-type
procaspase-9 were constructed as described previously (12) and kindly
provided by Dr. Xiaodong Wang (University of Texas, Dallas, TX).
Caspase-3 with an in-frame His9 coding sequence at the
3'-end was subcloned into pFastBacI vector at BamHI and
NotI sites. Caspase-9 active site (C287A) mutant, cleavage site mutants (D315A, D330A, and D315A/D330A), and the p12 and p10
NH2-terminal mutants were generated by the PCR-SOEing
method (23). These mutants, engineered to express a His9
tag at the COOH termini, were subcloned into pFastBacI vector at
BamHI and EcoRI sites. Expression plasmids were
transformed into DH10Bac Escherichia coli cells
(Invitrogen), recombinant bacmids were purified as recommended
by the manufacturer (Invitrogen), and their identity were confirmed by
PCR amplification analysis. The DNA was then used to transfect
Sf21 cells, and virus was amplified as described (12). The virus
stocks were amplified to 200 ml and used to infect 1 liter of
Sf21 cells at a density of 1 × 106 cells/ml.
The infected cells were harvested after 38 h for Apaf-1, 20 h
for procaspase-9 and procaspase-3, and 24 h for the procaspase-9 mutants and processed caspase-9 (p35/p12). Recombinant proteins were
purified by nickel affinity chromatography as described (12), followed
by ion exchange chromotography (Mono Q). The eluted protein was
dialyzed with buffer A (20 mM Hepes-KOH, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM NaEDTA, 1 mM NaEGTA, 1 mM
dithiothreitol, and 0.1 mM phenylmethylsulfonyl
fluoride) and stored in multiple aliquots with 20% glycerol at
80 °C. To obtain the p35/p10 and p37/p10 forms of caspase-9,
2 × 104 units of recombinant human caspase-3 was
incubated with 17 mg of either caspase-9 p35/p12 or the D315A mutant of
caspase-9 at 30 °C for 60 min in a total of 5 ml, pH 7.5, ICE
buffer. The caspase-3 was separated from the caspase-9 by ion exchange
chromatography (Mono Q). The active site mutant of procaspase-3 (C163A)
was expressed from pET 15b in BL21(DE3) cells (Novagen), and purified
through the nickel affinity column. Active caspase-3 was expressed and purified as described previously (24). Recombinant full-length XIAP
with a COOH-terminal His6 tag was cloned into the pFastBacI vector and expression was achieved as described above in Sf21 cells. The BIR3 domain (amino acids 241-356) of XIAP was expressed and
purified as previously described (30).
Western Blot Analysis--
Polyclonal anti-caspase-9 and
anti-caspase-3 (CM1) antibodies were produced as described previously
(12, 25). The monoclonal anti-caspase-3 antibody was from Transduction
Labs. Following SDS-PAGE, samples were transferred to nylon membrane
and probed with either the polyclonal CM1 (1:2000) or caspase-9
(1:1500), or the mouse monoclonal for caspase-3 (1:2000). Washed blots
were incubated with horseradish peroxidase-conjugated goat anti-rabbit (Apaf-1, caspase-9) or goat anti-mouse (caspase-3) IgG, and reactive bands were visualized using the ECL Plus Western blotting detection system and analyzed with a Storm (Amersham Biosciences).
Time Course Analysis of Procaspase-9 and Procaspase-3
Cleavage--
Aliquots of 2.5 µg of Apaf-1, 0.75 µg of
procaspase-9, and 0.375 µg of wild-type procaspase-3 or active site
mutant procaspase-3 (C163A) were combined, brought to a final volume of
300 µl in buffer A with 200 µM dATP, 600 nM
cytochrome c, and incubated at 30 °C. Following
incubation for the indicated times, aliquots of 20 µl (each
containing 50 ng of caspase-9 and 25 ng of caspase-3) were mixed with
SDS loading buffer, boiled, and subjected to 16% SDS-PAGE. Detection
of immunoreactive caspases was achieved as described above.
Procaspase-3 Cleavage Analysis--
60 ng of wild-type
procaspase-3, or its active site mutant (C163A) were combined with
increasing amounts of a 1:1 molar ratio of Apaf-1 and caspase-9
(p35/p12), 200 µM dATP, and 600 nM cytochrome c in buffer A to a total volume of 40 µl. After incubation
at 30 °C for 30 min, 15-µl aliquots of each reaction mixture
(containing 22.5 ng of caspase-3) were mixed with SDS loading buffer,
boiled, and subjected to 16% SDS-PAGE. Detection of immunoreactive
caspases was performed as described above.
Detection of Caspase-9 and Caspase-3 Activity with Fluorogenic
Tetrapeptide Substrates--
Typically, Apaf-1 was mixed with
procaspase-9 and procaspase-3 at the desired concentration in a 30- or
40-µl reaction mixture in buffer A containing both dATP and
cytochrome c. At the end of 30 min, the reaction was stopped
by 5-fold dilution with ICE buffer (25 mM Hepes, 1 mM EDTA, 0.1%
Chaps,1 and 10% sucrose)
containing either DEVD-AMC (50 µM, pH 7.5, for caspase-3)
or LEHD-AMC (100 µM, pH 6.5, for caspase-9). Caspase activity was monitored as the release of the AMC product over 60 min at
room temperature using a Cytofluor fluorescence photometer. Caspase
activity was expressed as the change in fluorescence over time derived
from the linear phase of the reaction.
Active Site Titration of Caspase-9--
To determine accurately
the relative kinetic properties of the various caspase-9 species, two
irreversible caspase-9 inhibitors were used to determine the
concentration of caspase-9 active sites after association with Apaf-1.
Briefly, serial dilutions of the inhibitors were incubated with Apaf-1,
dATP, cytochrome c, and the various caspase-9 species for
2 h at room temperature to allow complete inactivation. Aliquots
were removed, mixed with 60 nM procaspase-3, and
subsequently incubated for an additional 30-min prior to the addition
of 50 µM DEVD-AMC. Caspase-3 activity assay was monitored
as above. The active enzyme concentration was defined as the minimal
inhibitor concentration that completely depleted the caspase-9
activities. Titration with either the two inhibitors resulted in
identical results. Caspase-9 and caspase-9 mutant concentrations used
in Figs. 3 and 4 and Tables I and II, refer to active site concentrations.
Determination of Caspase-9 Kinetic Parameters--
The
Km for LEHD-AMC was determined for each form of
Apaf-1/caspase-9 apoptosome. Aliquots containing 100 nM
Apaf-1 and 100 nM caspase-9 (concentration determined by
active site titration) were incubated at 30 °C for 30 min in the
presence of 200 µM dATP and 600 nM cytochrome
c in a total volume of 10 µl in buffer A to allow the
formation of Apaf-1/caspase-9 apoptosome. The reaction mixture was
subsequently assayed at various LEHD-AMC concentrations in a total
volume of 100 µl. Rate of catalysis was calculated using the initial
slope. Km and Vmax were determined using a nonlinear regression method to fit the
Michaelis-Menten equation: V = Vmax[S]n/([S]n + Kmn), where V = initial
catalytic rate, in nanomole of AMC/h, [S] = concentration of the
substrate in micromolar; Vmax = a limiting value
of V at sufficiently high or saturating [S];
n = the hill coefficient.
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RESULTS |
Recombinant Apaf-1, Procaspase-9, and Procaspase-3
Reconstitute dATP/Cytochrome c-regulated Caspase-3
Activation--
To characterize the biochemical events leading to
Apaf-1/caspase-9 apoptosome-mediated cleavage of procaspase-3, we
expressed recombinant Apaf-1, procaspase-9, and procaspase-3 using a
baculovirus expression system as described under "Experimental
Procedures." Each protein was purified to apparent homogeneity using
nickel affinity chromotography followed by ion exchange chromatography (Fig. 1B). To demonstrate that
these recombinant proteins could support dATP/cytochrome
c-dependent caspase activation, we monitored the
cleavage of procaspase-9 and -3 after incubation with Apaf-1, dATP, and
cytochrome c. As expected, procaspase-9 and procaspase-3 were processed in a time-dependent manner yielding products
that were consistent with previously published reports (Fig. 1,
A and C) (26, 27). To confirm that the cleaved
caspase-3 products were catalytically active, we incubated aliquots
with DEVD-AMC. The time-dependent appearance of DEVD-AMC
cleaving activity parallels the degree of caspase-3 processing in each
sample (Fig. 1C, lower panel).

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Fig. 1.
Recombinant Apaf-1, procaspase-9,
and procaspase-3 reconstitute dATP/cytochrome
c-regulated caspase-3 activation. A,
schematic diagram showing procaspase-9 and products obtained from
caspase-9 cleavage by caspase-9 at Asp315, caspase-3
cleavage of caspase-9 at Asp330, or both. B,
recombinant Apaf-1, procaspase-9, and procaspase-3 (5 µg each) were
subjected to 16% SDS-PAGE and detected with Coomassie Blue staining.
C, apaf-1 (4.7 nM), procaspase-9 (28.1 nM), and procaspase-3, (25.5 nM) were incubated
with cytochrome c (300 nM) and dATP (200 µM) at 30 °C. After 0, 10, 20, or 30 min of
incubation, 20 µl of the reaction mixture was subjected to 16%
SDS-PAGE followed by immunoblotting for caspase-9 (top) or
caspase-3 (middle). Duplicate aliquots were analyzed for
caspase-3 activity using the fluorogenic tetrapeptide substrate
DEVD-AMC (bottom panel). Fu, fluorescence
units.
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Cleavage Requirements for Procaspase-9 in the
Apoptosome--
Several publications have addressed the relationship
between caspase-9 cleavage status and the activity of the apoptosome (17, 19). These studies, employing cell-free extracts with and without
immunodepletion of endogenous caspase-9, disagree on the impact of the
cleavage state of caspase-9 on apoptosome activity. The establishment
of a completely recombinant system to study Apaf-1-mediated caspase
activation allowed us to address this issue under more defined
conditions. To determine the relationship between sites of procaspase-9
cleavage and apoptosome activity, we first expressed and purified
several procaspase-9 proteins containing point mutations, including
D315A, D330A, D315A/D330A, and C287A (Fig.
2A). Each of the mutant
proteins, and wild-type procaspase-9, were tested for their ability to
be cleaved, in vitro, by recombinant active caspase-3.
Consistent with previous reports (14), procaspase-9 mutants D330A and
D315A/D330A were not processed by caspase-3, whereas the cleavage site
mutant (D315A) and the procaspase-9 active site mutant (C287A) yielded
the expected cleavage product, p37. Incubation of wild-type caspase-9
with caspase-3 led to the formation of a p35 product, suggesting that caspase-3 directed cleavage of caspase-9 yields an active caspase-9 (p37/p10) capable of cleaving p37 subunits at Asp315 to
generate p35. The lack of p35 production in reactions containing procaspase-9 (C287A) supports this proposal (Fig. 2A).

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Fig. 2.
Defining the cleavage site requirements
within procaspase-9 for apoptosome activation. Recombinant
wild-type procaspase-9 and its cleavage sites mutants (D315A, D330A,
and D315A/D330A) and active site mutant (C287A) were purified as
described under "Experimental Procedures." A, 5 µg of
procaspase-9 or procaspase-9 mutants were directly subjected to 16%
SDS-PAGE (left), or incubated with 10 ng of caspase-3 at
30 °C for 30 min and then subjected to 16% SDS-PAGE
(right). Caspase-9 species were detected with Coomassie
Blue. B, procaspase-9 or procaspase-9 mutants (35.2 nM) were incubated with Apaf-1 (4.7 nM), dATP
(200 µM), and cytochrome c (300 nM) at 30 °C for 30 min. The reaction mixtures were
subjected to 16% SDS-PAGE followed by immunoblotting for caspase-9.
C, procaspase-3 (46 nM), dATP (200 µM), and cytochrome c (300 nM)
were incubated with different amounts of Apaf-1 and procaspase-9 or
procaspase-9 mutants at 30 °C for 30 min. The molar ratio of Apaf-1
to procaspase-9 was maintained at 1:1. Caspase-3 activity was
analyzed using the fluorogenic substrate DEVD-AMC. Fu,
fluorescence units.
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To identify which cleavage events occur upon association of
procaspase-9 with Apaf-1, we incubated either wild-type procaspase-9 or
its cleavage-site mutants with Apaf-1, dATP, and cytochrome c and monitored procaspase-9 cleavage. Incubation of Apaf-1
with wild-type procaspase-9 or the D330A mutant yielded a p35 cleavage product (Fig. 2B), whereas reactions with cleavage-site
mutants D315A or D315A/D330A yielded no products. These results confirm that Apaf-1 induces cleavage of procaspase-9 at Asp315
preferentially to Asp330 (14).
To determine the relative impact of each cleavage event on caspase-9
activity within the apoptosome, we preincubated procaspase-9 or the
procaspase-9 mutants with Apaf-1 and procaspase-3 in the presence of
dATP and cytochrome c. The reactions were then tested for
caspase-3 activity using the fluorogeneic tetrapeptide substrate DEVD-AMC. Addition of increasing amounts of wild-type procaspase-9 leads to an increase in caspase-3 activation as expected (Fig. 2C). In contrast to published work (17), reactions with the procaspase-9 mutant (D315A) generated nearly the same amount of caspase-3 activity as reactions containing the wild-type procaspase-9, whereas reactions with the procaspase-9 mutant (D330A) yielded almost
no activation of caspase-3 (Fig. 2C). These data show that efficient activation of procaspase-3 by the apoptosome requires cleavage of caspase-9 at Asp330. Thus, feedback cleavage of
procaspase-9 or caspase-9 p35/p12 by caspase-3 may lead to full
activation of the apoptosome.
Caspase-3-directed Cleavage of Caspase-9 Is Required for Full
Activation of the Apoptosome--
To directly demonstrate that full
activation of the apoptosome requires caspase-3 feedback cleavage of
caspase-9, we compared the processing of procaspase-3 in two separate
reconstituted reactions. One reaction contained wild-type procaspase-3,
whereas the other contained procaspase-3 with an active-site cysteine
mutation (C163A). As shown in Fig.
3A, the presence of a
catalytically active procaspase-3 in these reactions leads to a change
in both the procaspase-9 cleavage products and the rate of procaspase-3
cleavage. In the reactions containing wild-type procaspase-3, caspase-9
cleavage products of p37, p35 (note the p35/p37 doublet at 5 min), p12, and p10 were generated, whereas the majority of procaspase-3 was cleaved within 5 min (Fig. 3A). In the procaspase-3 (C163A)
mutant reaction, the caspase-9 cleavage products were exclusively p35 and p12, and no processing of procaspase-3 was observed within the
first 5 min (Fig. 3A, lower right panel). To rule
out the possibility that the difference in procaspase-3 cleavage
observed in Fig. 3A was because of cleavage of procaspase-3
by activated caspase-3 in the wild-type caspase-3 reactions, we
incubated procaspase-3 with increasing amounts of active caspase-3.
Fig. 3B demonstrates that up to 10 units of caspase-3 is
insufficient to cleave procaspase-3, whereas as little as 2 units
efficiently cleaves procaspase-9, indicating that all the procaspase-3
cleavage observed in Fig. 3A is apoptosome-mediated. In
addition to the differences in caspase-3 cleavage rate, the rates for
caspase-9 cleavage in each reaction were also different. In reactions
containing wild-type procaspase-3, procaspase-9 cleavage was observed
within 2 min and its cleavage was nearly complete by 10 min. In the
reactions with mutant procaspase-3 the rate of procaspase-9 cleavage
was slower, such that no cleavage was observed at 2 min and the
reaction had not reached completion by 20 min.

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Fig. 3.
Caspase-3 feedback cleavage of caspase-9 is
critical for full activation of Apaf-1/caspase-9 apoptosome.
A, time course analysis of procaspase-9 and procaspase-3
cleavage in the Apaf-1 reconstituted system determined by Western blot
analysis. Wild-type procaspase-3 (left panels) or its active
site mutant (C163A, right panels) were used as substrates in
the reactions. B, Coomassie Blue-stained gel of procaspase-9
and procaspase-3 following incubation with varying amounts of active
caspase-3. In C, Western blot analysis was used to follow
the processing of wild-type procaspase-3 (upper panel) or
its active site mutant (C163A) (lower panel) with increasing
amounts of the Apaf-1/caspase-9 (p35/p12) apoptosome. The molar ratio
of Apaf-1 to caspase-9 in the apoptosome was maintained at 1:1 in each
reaction and the concentration ranged from 0.4 to 50 nM in
2-fold increments. WT, wild type.
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To further establish that caspase-9 cleaved at Asp330 to
generate a p10 subunit is critical for full activation, we added
increasing amounts of caspase-9 p35/p12 apoptosomes (at a constant 1:1
molar ratio of Apaf-1:caspase-9) to either wild-type procaspase-3 or the C163A mutant. If caspase-3-mediated feedback can enhance the ability of caspase-9 to cleave procaspase-3, then we would expect procaspase-3 cleavage to be detected at lower apoptosome concentrations in reactions containing catalytically competent caspase-3. As show in
Fig. 3C, cleavage of wild-type procaspase-3 is observed when
the apoptosome concentration reaches 3.1 nM (Fig.
3C, upper panel), with substantial cleavage
occurring at 50 nM. In reactions with the C163A
procaspase-3 mutant, cleavage is not observed until the apoptosome
concentration reaches 25 nM with only marginal cleavage at
50 nM (Fig. 3C, lower panel). The
data presented in Fig. 3 therefore demonstrate that cleavage of
caspase-9 at Asp330 by caspase-3 can significantly enhance
the activity of the apoptosome. The magnitude of the enhancement was
calculated to be up to 8-fold.
Activities of Different Forms of Caspase-9 in the
Apoptosome--
To directly demonstrate that apoptosomes containing
fully processed caspase-9 are more active than those containing
partially processed caspase-9, we produced the three distinct processed forms of caspase-9. Each of the three purified proteins, p35/p10, p37/p10, and p35/p12, migrated as predicted in SDS-PAGE (Fig. 4A), and the molecular masses
of each individual subunit were confirmed using mass spectrometry (not
shown). We first compared the ability of apoptosomes containing either
p35/p12 or p37/p10 to process recombinant procaspase-3 (C163A) by
titration of the apoptosome added to the reaction. Caspase-9 p37/p10
apoptosomes were able to cleave procaspase-3 at concentrations of 0.4 to 0.8 nM, whereas caspase-9 p35/p12 apoptosome required
3.1 to 6.3 nM to cleave procaspase-3 during this period
(Fig. 4B). To assess the differences between apoptosomes in
a quantitative manner, we used the caspase-9 tetrapeptide substrate,
LEHD-AMC. After preincubation of Apaf-1 with p35/p12, p37/p10, or
p35/p10 to allow the formation of Apaf-1/csp-9 apoptosomes, LEHD-AMC
was added to the reactions and apoptosome activity was assessed by
following the liberation of AMC. The activities of p35/p10- and
p37/p10-containing apoptosomes were significantly greater than that of
p35/p12 over the entire range of apoptosome concentration (Fig.
4C). Together these results suggest that removal of the
NH2-terminal linker region from the p12 small subunit of
caspase-9 yields an apoptosome with greater specific activity.

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Fig. 4.
Activities of different forms of caspase-9 in
the apoptosome. A, Coomassie Blue-stained gel of the
various forms of recombinant caspase-9. In B, Western blot
analysis was used to follow the processing of procaspase-3 (C163A) when
increasing amounts of Apaf-1/caspase-9 (p35/p12) apoptosome
(upper panel), or Apaf-1/caspase-9 (p37/p10) apoptosome
(lower panel) were added to the reaction. The molar ratio of
Apaf-1 to caspase-9 in the apoptosome was maintained at 1:1 in each
reaction and the concentration ranged from 0.4 to 50 nM in
2-fold increments. C, increasing amounts of caspase-9
p35/p12 (circles), p35/p10 (squares), or p37/p10
(triangles) were incubated with Apaf-1 at a constant molar
ratio in the presence of dATP and cytochrome c at 30 °C
for 30 min. The activity of the apoptosomes formed was detected with
LEHD-AMC. D, caspase-9 p35/p12 (circles), p35/p10
(squares), or p37/p10 (triangles) were incubated
with Apaf-1 in the presence of dATP and cytochrome c at
30 °C for 30 min. Procaspase-3 and DEVD-AMC were then added to the
reactions and the liberation of AMC was continuously monitored over 60 min. Control reactions lacking Apaf-1 yielded no activity
(open circles). Fu, fluorescence units.
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To establish that the observed effects on LEHD-AMC cleavage were
because of changes in the intrinsic catalytic properties of the
apoptosomes, we determine the Km and
kcat for each of the apoptosome species. We
found that processing of procaspase-9 at Asp330, to form
the p10 subunit, was associated with a modest decrease in
Km for LEHD-AMC (Table
I). This effect was most pronounced with
the p37/p10 species yielding a Km 2-fold lower than that for p35/p12. The first-order rate constants,
kcat, of Apaf-1/caspase-9 (p35/p10) and
Apaf-1/caspase-9 (p37/p10) were 2.8- and 3.6-fold greater than that of
Apaf-1/(p35/p12), respectively (Table I). An apparent catalytic rate
can be derived from kcat/Km, and is a measure of the ability of the enzyme to turn over substrate. The combined effects of processing at Asp330 in
procaspase-9 to yield a p10 subunit is reflected in a nearly 8-fold
increase in the catalytic rate for apoptosomes containing the p37/p10
form of caspase-9.
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Table I
Kinetic parameters of different forms of caspase-9 in the apoptosome
The Km and kcat for LEHDamc was
determined for each form of Apaf-1/csp-9 apoptosome, and each value was
calculated using a nonlinear regression method to fit Michaelis-Menten
equation as described under "Experimental Procedures."
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The form of caspase-9 within the apoptosome can clearly impact cleavage
of the synthetic substrate LEHD-AMC. To ask whether this was also true
of full-length endogenous substrates, we assessed the ability of each
of the distinctly processed caspase-9 species, in association with
Apaf-1, to cleave and activate recombinant procaspase-3. Apaf-1 was
preincubated with p35/p12, p37/p10, or p35/p10 in the presence of dATP
and cytochrome c to allow the formation of apoptosomes.
Following preincubation, wild-type procaspase-3 and DEVD-AMC were added
simultaneously and the time-dependent activation of
caspase-3 was monitored. Apoptosomes containing either p35/p10 or
p37/p10 demonstrated similar activity in this experiment, reaching a
steady state level of caspase-3 activity within 10-15 min, with a rate
of DEVD-AMC cleavage during this phase of ~1500 fluorescence
units/h (Fig. 4D). In contrast, the activity of the
reaction containing p35/p12 during this time period was ~10-fold
lower at 150 fluorescence units/h (Fig. 4D). None of
these caspase-9 species demonstrated appreciable activity in the
absence of Apaf-1 under these conditions (Fig. 4D). At later time points in the p35/p12 reaction the rate of DEVD-AMC cleavage begins to approach that of the other two reactions. This change in the
rate of DEVD-AMC cleavage in the Apaf-1/p35/p12 containing reaction
(between 30 and 50 min) is a result of feedback cleavage of p12 by
active caspase-3 to yield a p10 subunit, and thus p35/p10-containing apoptosomes (not shown). The lag time required for this change in rate
to occur is inversely proportional to the amount of input procaspase-3 (not shown).
Fully Active Caspase-9 Is a Target for XIAP-mediated Inhibition
through the BIR3 Domain--
Srinivasula et al. (19) have
reported that the 316ATPF319 motif at the
NH2 terminus of the p12 domain, a motif similar to the AVPI
motif responsible for SMAC-mediated BIR3 binding, is critical for
inhibition of caspase-9 by the BIR3 domain of XIAP (19). Because
feedback cleavage of caspase-9 by caspase-3 removes the ATPF motif as
part of the linker, we were interested in determining whether p35/p10
or p37/p10 caspase-9 were less sensitive to inhibition by XIAP.
Accordingly, we reconstituted the apoptosome with various forms of
caspase-9 and tested the ability of BIR3 to inhibit caspase-9-mediated activation of procaspase-3. As expected, BIR3 inhibited caspase-3 activation by apoptosomes containing
procaspase-9 (not shown) or caspase-9 p35/p12 (Table II, procaspase-3
as substrate, Fig. 5A).
Surprisingly, apoptosomes containing the p35/p10 form of caspase-9
were inhibited with nearly the same IC50 values as
procaspase-9- and p35/p12-containing apoptosomes (Table II and Fig.
5A). These data implied that other regions within the
caspase-9 protein are able to mediate inhibition by BIR3. The
NH2 terminus of the fully cleaved p10 small subunit of
caspase-9 also contains a tetrapeptide motif,
331AISS334, with similarity to the SMAC and
caspase-9 p12 motifs (Fig. 5B). Although Srinivasula
et al. (19) found little binding of BIR3 to this motif, we
expressed point mutants targeting both of the potential BIR3 binding
motifs within caspase-9 (Fig. 5, C and D) to test
whether the p10 NH2-terminal motif is responsible for BIR3-mediated inhibition of fully processed caspase-9. Apoptosomes containing caspase-9 p35/p10 A331G/I332G (M4 in Table II and
Fig. 5C), or caspase-9 p35/p12 A316G/T317G/A331G/I332G
(M3) were no longer sensitive to BIR3-mediated inhibition in
the fully reconstituted system (Fig. 5A). This result is
consistent with a model where the AISS motif present at the
NH2 terminus of p10 is responsible for the inhibition by
BIR3 of caspase-9 p35/p10 observed in Fig. 5A.
Interestingly, caspase-9 p35/p12 A331G/I332G (M2) was also insensitive
to inhibition by BIR3, even though the input caspase-9 had a p12
subunit with the ATPF BIR3 recognition motif. These data can be
accommodated by at least two models, either ATPF-mediated inhibition by
BIR3 is reversible by removal of the linker region by caspase-3
cleavage of Asp330, or caspase-3 cleavage of
Asp330 occurs more rapidly than binding of BIR3 to the
NH2 terminus of p12. Finally, caspase-9 p35/p12 A316G/T317G
(M1) was inhibited by BIR3 with nearly the same IC50 as
caspase-9 p35/p10, suggesting this the M1 mutant was inhibited
following cleavage at Asp330 and exposure of the AISS
motif.
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Table II
IC50 (nM) of XIAP or BIR3 mediated inhibition of
caspase-9 and caspase-9 mutants in apoptosome
XIAP- or BIR3-mediated inhibition of wild type and mutant
Apaf-1/caspase-9 apoptosome was measured in Fig. 5. The IC50 of
the inhibition was determined by fitting the equation, %I = 100[I]n/(IC50n + [I]n) using
SigmaPlot program.
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Fig. 5.
Either of the two conserved XIAP binding
motifs within caspase-9, ATPF or AISS, are sufficient for inhibition by
XIAP or its BIR3 domain. A, caspase-9 or its mutant
were incubated with Apaf-1, procaspase-3, dATP, cytochrome
c, and different amounts of BIR3. The activity of processed
caspase-3 was measured by the cleavage of DEVD-AMC. Open
circles, p35/p12 (WT); open diamonds,
p35/p10 (WT); open squares, p35/p12
(M1); filled diamonds, p35/p12 (M2);
filled triangle, P35/p12 (M3); filled
square, P35/p10 (M4). Symbols are the same for
A, E, and F. B, alignment
of the NH2-terminal amino acids from mouse,
Xenopus, and human caspase-9 p10, along with the
NH2 terminus of human caspase-p12 and human SMAC/Diablo.
C, details of caspase-9 p35/p12 p35/p10 point mutants used
to assess amino acid requirements for BIR mediated inhibition.
D, Coomassie Blue-stained gel of caspase-9 small subunit
NH2-terminal mutants. In E and F,
caspase-9 or the caspase-9 mutants were incubated with Apaf-1 dATP,
cytochrome c, and either BIR3 or full-length XIAP. The
activity of Apaf-1/caspase-9 holoenzyme was directly measured by the
cleavage of LEHD-AMC.
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To demonstrate more directly that each of the NH2 termini
of the small subunits of caspase-9 can mediate BIR3 inhibition, we
tested the various apoptosomes in the absence of caspase-3 (and thus
without feedback cleavage at Asp330) using the LEHD-AMC
substrate. We observed potent BIR3 inhibition of p35/p12 WT and the
p35/p12 M2 mutant, whereas the p35/p12 M1 mutant was not sensitive to
BIR3 (Table II, LEHD as substrate, and Fig. 5E). As expected
from Fig. 5A, we also observed inhibition by BIR3 of
caspase-9 p35/p10 WT but no inhibition of the p35/p10 M4 mutant or the
p35/p12 M3 mutant. Finally, to demonstrate that the above observations
are relevant to BIR3-mediated inhibition in the context of full-length
XIAP, we repeated the experiment using purified full-length XIAP. As
shown in Fig. 5F, the ability of full-length XIAP to inhibit
the apoptosome is dependent on the presence of either ATPF or AISS
exposed at the NH2 terminus of the small subunit. The
IC50 values for each of these reactions is shown in Table
II (XIAP inhibitor, LEHD as substrate). These data support a model
where XIAP is able to inhibit caspase-9 at all levels of activation by
virtue of a conserved motif at the NH2 terminus of the less
active p12 subunit as well as the more active p10 subunit.
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DISCUSSION |
In the present study, we have addressed the potential
physiological role(s) of caspase-3-mediated feedback cleavage of
caspase-9 using purified, recombinant Apaf-1, caspase-9, and caspase-3
proteins in a reconstituted apoptosome in vitro. Because
caspase-9 can be cleaved by itself (Asp315) or by caspase-3
(Asp330), the combined action of the two proteases on
procaspase-9 can produce three different caspase-9 enzymes: p35/p12,
p37/p10, or p35/p10 (Fig. 1). We attempted to discriminate the role of
each cleavage site by generating various recombinant caspase-9 species. First we generated procaspase-9 species in which each cleavage site had
been eliminated alone or in combination and assessed their ability to
activate procaspase-3. Second we produced all three processed forms of
caspase-9, p35/p12, p37/p10, and p35/p10, and reconstituted them
separately into the apoptosome to compare their relative catalytic
activities and sensitivities to inhibition by the BIR3 domain of XIAP.
Experiments in which procaspase-9 with cleavage mutations were used
suggest that cleavage at Asp330 is required for full
activation of the apoptosome (Fig. 2C). This observation is
inconsistent with previously published reports (17-19) in which
little, if any, difference was observed in the ability of unprocessed
and processed caspase-9 to cleave and activate procaspase-3. Although
no definitive explanation for this discrepancy can be presented, there
were significant differences in methodology between these previous
reports and the present work. In general, the previous reports employed
cellular extracts as the context for assessing relative activities of
caspase-9, whereas the present work utilized recombinant proteins at
defined concentrations and molar ratios. The use of cellular extracts
may introduce additional cytosolic factors, including but not limited
to endogenous procaspase-9 that may modulate apoptosome activity and
obscure the potential impact of cleavage at Asp330.
Cleavage of caspase-9 at Asp330 by caspase-3 increases the
catalytic activity of caspase-9 in the apoptosome up to 8-fold. This is
because of an increase in kcat and a decrease in
Km of p10-containing forms of the apoptosome as
measured by cleavage of the caspase-9 substrate LEHD-AMC (Table I).
Thus, caspase-3 feedback cleavage of either p35/p12 (to generate
p35/p10) or uncleaved procaspase-9 (to generate p37/p10) has the
potential to amplify the proteolytic activity of the apoptosome. If
operational in cells, this would create a feed-forward mechanism to
accelerate the apoptotic destruction of the cell once the downstream
effector caspase-3 has been activated. At least two recent studies
support this possibility. Fujita et al. (20), using
caspase-9 cleavage site-specific antibodies, demonstrated in living
mouse cells a sequential cleavage of procaspase-9, first by caspase-9
and then by caspase-3. These authors also noted a correlation between
cleavage at Asp330 and increased cellular caspase-9
activity. In a second study, Slee et al. (29) using
cell-free extracts, have demonstrated a requirement for caspase-3
activity to achieve maximum apoptosome activity. The optimum activity
in this study also correlated with the presence of an
Asp330 cleavage product. Our data are consistent with these
observations and provide biochemical support for the potential impact
of such a feedback loop.
Recent work by Srinivasula et al. (19) would predict that
feedback cleavage of caspase-9 by caspase-3, and loss of the BIR3 binding motif, would be accompanied by a loss of sensitivity to inhibition by XIAP, representing a potential point of no return for the
cell. Thus, we were surprised to observe that either full-length XIAP
or the isolated BIR3 domain is equally effective at inhibiting p10-containing forms of caspase-9 (p35/p10, p37/p10) as the
p12-containing caspase-9 (p35/p12, Table II and Fig. 5). Cleavage by
caspase-3 at Asp330 exposes a tetrapeptide motif AISS that
appears to be responsible for XIAP inhibition (Fig. 5). Two lines of
evidence support this conclusion: 1) the similarity of the AISS
sequence to the SMAC/reaper tetrapeptide motif (Fig. 5B),
and 2) the nearly complete loss of function when the
NH2-terminal alanine of p10 is mutated to glycine (Fig. 5,
A, E, and F). This p10 NH2
terminus is well conserved (Fig. 5B), indicating that it is
important that caspase-3 feedback-cleaved caspase-9 still be subject to
regulation by IAPs.
The catalytic activity of caspase-9 in the apoptosome is regulated at
multiple levels. First, noncovalent association with Apaf-1 via
CARD/CARD interaction causes an increase in protease activity (18, 28).
The precise activation mechanism is unknown but may involve allosteric
changes in procaspase-9 induced upon binding to Apaf-1. Second,
autolytic cleavage at Asp315, perhaps because of the
induced proximity (14) of caspase-9 molecules in the multivalent
apoptosome, further increases caspase-9 protease activity. Third,
feedback cleavage at Asp330 by caspase-3 increases
caspase-9 activity still further, providing for an amplification of the
cascade. How the loss of the 15-amino acid linker region leads to
enhancement of the catalytic activity is not clear. In the recently
published structure of caspase-9 the NH2 terminus of the
p12 subunit was not resolved (28). However, the NH2
terminus of the p12 subunit was adjacent to the catalytic site,
suggesting that removal of the NH2 terminus could affect either substrate recognition or catalysis by caspase-9. Finally, all
forms of activated caspase-9 are subject to inhibition by XIAP. Thus,
even after full activation of the apoptosome, it appears that XIAP may
be capable of setting a threshold over which sufficient active
caspase-9 must be generated before a cell can complete the apoptotic program.