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J. Biol. Chem., Vol. 275, Issue 46, 36152-36157, November 17, 2000
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,,From the Center for Apoptosis Research and the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, August 8, 2000, and in revised form, August 18, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Smac/DIABLO is a mitochondrial protein that is
released along with cytochrome c during apoptosis and
promotes cytochrome c-dependent caspase
activation by neutralizing inhibitor of apoptosis proteins (IAPs). We
provide evidence that Smac/DIABLO functions at the levels of both the
Apaf-1-caspase-9 apoptosome and effector caspases. The N terminus of
Smac/DIABLO is absolutely required for its ability to interact with
the baculovirus IAP repeat (BIR3) of XIAP and to promote
cytochrome c-dependent caspase activation.
However, it is less critical for its ability to interact with BIR1/BIR2 of XIAP and to promote the activity of the effector caspases. Consistent with the ability of Smac/DIABLO to function at the level of the effector caspases, expression of a cytosolic Smac/DIABLO in Type II cells allowed TRAIL to bypass Bcl-xL inhibition of death receptor-induced apoptosis. Combined, these data suggest that
Smac/DIABLO plays a critical role in neutralizing IAP inhibition of the
effector caspases in the death receptor pathway of Type II cells.
Apoptosis is a highly conserved cell suicide program essential for
development and tissue homeostasis of all metazoan organisms (1-3).
Key to the apoptotic program is a family of cysteine proteases termed
caspases that cleave their substrates after specific aspartic acid
residues (reviewed in Refs. 4 and 5). Caspases reside in cells as
inactive zymogens, known as procaspases, and can be activated by
proteolytic cleavage after specific aspartate residues present between
what will become the large and small subunits of the active caspase (4,
5). During apoptosis, the initiator caspase zymogens are activated by
autocatalytic cleavage, which then activate the effector caspases by
cleaving their inactive zymogens (6, 7). Active caspases can then
induce the characteristic apoptotic changes through their ability to
cleave certain key protein substrates in the cell (4, 5).
The initiator caspase zymogens are activated by adaptor proteins such
as FADD and Apaf-1, which associate in a
stimulus-dependent manner with the prodomains of these
zymogens and promote their activation via oligomerization (6, 7). For
example, ligation of the cell surface death receptors triggers binding
of procaspase-8 to FADD and its subsequent activation and release from
the death receptor complex (reviewed in Refs. 6 and 8). Likewise, release of cytochrome c from the mitochondria in response to
apoptotic stimuli such as serum starvation, ionization radiation, DNA
damaging agents, etc. triggers oligomerization of Apaf-1 in an ATP- or dATP-dependent manner (7, 9-11). The oligomeric Apaf-1
apoptosome then recruits and activates procaspase-9.
Given the potentially irreversible caspase cascade triggered by
activation of the upstream initiator caspases, it is crucial that
activation of caspases in the cell be tightly regulated. A number of
cellular proteins have been shown to modulate caspase activation and
activity. One of these, FLAME/FLIP, inhibits death receptor-mediated activation of caspase-8 by binding to FADD (12, 13).
Others, such as the antiapoptotic members of the Bcl-2 family, inhibit
Apaf-1-mediated activation of caspase-9 by blocking cytochrome
c release from the mitochondria (reviewed in Refs. 14 and
15). Heat shock proteins Hsp70 and Hsp90 also interfere with the
mitochondrial apoptotic pathway by modulating the formation of a
functional Apaf-1 apoptosome (16-18). Finally, members of the
IAP1 family, such as XIAP,
c-IAP-1, and c-IAP-2, block both the death receptor and mitochondrial
pathways by inhibiting the activity of the effector caspase-3 and -7 and the initiator caspase-9 (reviewed in Ref. 19).
Recently, Smac/DIABLO, a mitochondrial protein that is released
together with cytochrome c from the mitochondria in response to apoptotic stimuli, was found to promote caspase activation by
binding and neutralizing the IAPs (20, 21). In this report, we
investigated in detail the caspase-promoting activity of Smac/DIABLO and its interaction with XIAP. We demonstrate that Smac/DIABLO promotes
the caspase activity of the initiator caspase-9 and the effector
caspase-3 and -7, by neutralizing the inhibitory effect of IAPs. Like
the Drosophila Reaper, Grim, and Hid proteins (19, 22, 23),
Smac/DIABLO requires its N terminus to interact with IAPs and promote
caspase activation. Moreover, Smac/DIABLO
can potentiate death receptor-induced apoptosis in the presence of Bcl-xL, suggesting that it could play an important role in this pathway.
cDNA Cloning and Expression Constructs--
The entire open
reading frames of human Smac/DIABLO-L and Smac/DIABLO-S cDNAs were
cloned from Jurkat mRNA by RT-PCR using complementary PCR adaptor
primers spanning the initiation and stop codons of these cDNAs. The
PCR primers were designed based on the sequence of mouse
DIABLO2 and human GenBankTM
clones AW16150 and AK001399. All deletion mutants were also
generated by PCR using modified complementary PCR adaptor primers. All
Smac/DIABLO constructs were cloned in pET 28(a) in the
NcoI/XhoI site with C-terminal His6
tag or GST tag. All XIAP constructs were cloned in the
EcoRI/XhoI site of pGEX-4T or
MYC-pcDNA3. The MYC-pcDNA3 constructs were used for in vitro translations.
In Vitro Interaction Assay--
All in vitro
interactions were performed by incubating 10 µl of TNT®
(Promega) translation reaction at 4 °C for 2 h with
equal amounts of proteins (200 ng) bound to 50 µl of
glutathione-Sepharose (Amersham Pharmacia Biotech) or
TALONTM-agarose (CLONTECH) beads in a 100-µl
reaction. The beads in all the samples were washed at least 4 times and
then boiled in 30 µl of sample buffer. 15 µl of each sample was
loaded onto a 10% SDS polyacrylamide gel and separated by
electrophoresis. The gels were dried and then exposed to x-ray films.
Smac/DIABLO and XIAP Purification--
His6- or
GST-tagged Smac/DIABLO or XIAP were purified using TALONTM resin or
glutathione-Sepharose by standard affinity-purification procedures as
described previously (7).
Caspase-3 Activation and DEVD Cleavage
Assays--
Cytochrome c-dependent activation
of caspase-9 and caspase-3 was done in 293T S100 extracts as described
previously (7, 20). Caspase-3 and -7 enzymatic assays were performed in
a 20-µl volume in buffer A (20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM
EDTA, 1 mM EGTA, 1 mM dithiothreitol) at
37 °C. Pure GST-XIAP was incubated with 5 nM of pure
caspase-3 (at 20 nM concentration) or caspase-7 (at 5 nM concentration) with various amounts of Smac/DIABLO and
50 µM Asp-Glu-Val-Asp-7-Amino-Y-methyl coumarin
(DEVD-AMC) substrate for 30 min. The release of AMC from the
DEVD-AMC substrate was measured using a PerkinElmer Life
Sciences luminescence spectrometer. The results were expressed
as percent of DEVD cleavage from at least three experiments.
Apoptosis Assay--
MCF-7 cells (0.5 × 105
cells/well) in 12-well plates were transfected with 0.5 µg of
pEGFP-N1 reporter plasmid (CLONTECH) or GFP-Smac/DIABLO expression construct together with 0.5 µg of empty vector plasmids or plasmids encoding Bcl-xL using the LipofectAMINETM method. 24 h after transfection cells were treated with TRAIL (0.5 or 2 µg/ml) for 10 h and then the normal (flat and attached) and
apoptotic (round and detached) GFP-expressing cells were counted using
fluorescence microscopy. The percentage of apoptotic cells in each
experiment was expressed as the mean percentage of apoptotic cells as a
fraction of the total number of GFP-expressing cells.
Identification of a Cytosolic Isoform of Smac/DIABLO--
The
Smac/DIABLO precursor contains a 55-residue mitochondrial targeting
sequence (MTS) at its N terminus that is cleaved in the mitochondria to
generate the mature mitochondrial Smac/DIABLO. Genomic analysis of the
Smac/DIABLO gene on chromosome 12q (GenBankTM accession number
AC048338) revealed that the MTS is encoded by the first two
exons. We found that the exons encoding the MTS are spliced out in two
human expressed sequence tag clones (AA156765 and AA305624)
and one full-length clone (AK001399) in the data base. The three clones
contain an open reading frame that generates Smac/DIABLO-S
lacking the MTS (Fig. 1a). The
alternatively spliced Smac/DIABLO-S mRNA is expressed in all cell
lines tested by RT-PCR using isoform-specific primers (Fig.
1b), although at a lower level than that of Smac/DIABLO-L.
Transient transfection of constructs encoding GFP fusion proteins of
the two isoforms into MCF-7 cells revealed that Smac/DIABLO-S is
targeted to the cytosol, whereas Smac/DIABLO-L is targeted to the
mitochondria (Fig. 1c).
Smac/DIABLO Functions at the Initiation and Execution Steps of the
Caspase Cascade--
Smac/DIABLO has been shown to enhance cytochrome
c-dependent activation of caspase-3 by
neutralizing the inhibitory effect of IAPs in S100 extracts. To compare
the activity of the two isoforms, we expressed Smac/DIABLO-S and
Smac/DIABLO-L without its MTS (mature Smac/DIABLO) in bacteria,
purified them, and examined their ability to enhance cytochrome
c-dependent activation of caspase-9 and -3 in
XIAP-containing S100 extracts. Mature Smac/DIABLO was able to relieve
the inhibitory effect of XIAP and enhance caspase-9 and -3 processing
in a dose-dependent manner (Fig. 2a).
Interestingly, Smac/DIABLO-S was almost completely inactive in this
assay (Fig. 2a). Because the
only difference between the two proteins is the substitution of the
N-terminal AVPIA sequence in mature Smac/DIABLO with MKSDFYF in
Smac/DIABLO-S, this indicated that the first 5 N-terminal residues of
mature Smac/DIABLO are critical for its activity (see below).
XIAP has been shown to inhibit the enzymatic activity of the effector
caspases, caspase-3 and -7 (24). To examine whether Smac/DIABLO could
also relieve the XIAP-inhibitory effect on the enzymatic activity of
mature caspase-3 and -7, we tested the effect of Smac/DIABLO and
Smac/DIABLO-S on the activity of caspase-3 and -7 in the presence of
XIAP. Smac/DIABLO was able to promote the enzymatic activity of both
caspase-3 and -7 (Fig. 2b). Interestingly, compared with its
very low activity with caspase-9, Smac/DIABLO-S had ~30% of the
activity of wild type Smac/DIABLO with caspase-3 and -7, suggesting
that Smac/DIABLO-S may play a role in regulating caspase-3 and -7 activity in vivo. Combined, these data suggest that
Smac/DIABLO has a dual role in the caspase cascade, to promote the
activities of the initiator caspase-9 and the effector caspases.
The ability of Smac/DIABLO to promote the enzymatic activity of
caspases depends on its interaction with IAPs (20, 21). To determine
whether the weak activity of Smac/DIABLO-S is due to altered
interaction with XIAP, we performed in vitro interaction assays with 35S-labeled full-length XIAP or isolated BIR
domains of XIAP (Fig. 2c). Consistent with its weak
activity, Smac/DIABLO-S was not able to interact with the BIR3 domain
(XIAP-BIR3/RING) of XIAP but was still able to interact with the
BIR1/BIR2 domains (XIAP-BIR1/2) of XIAP. Because Smac/DIABLO-S can
interact with the isolated BIR1/BIR2 domains (XIAP-BIR1/2) of XIAP, it
was not surprising that it was also able to interact with full-length
XIAP, although to a lesser extent than the wild type Smac/DIABLO.
The Caspase-promoting Activity of Smac/DIABLO Resides in Its N
Terminus--
The low activity of Smac/DIABLO-S compared with that of
mature Smac/DIABLO suggests that the N terminus of mature Smac/DIABLO is very important for its activity. To understand the molecular basis
for this difference we generated a series of Smac/DIABLO N-terminal
deletion mutants (Fig. 3a) and
expressed them in bacteria. The recombinant proteins were purified to
homogeneity and assayed for their ability to promote cytochrome
c-dependent caspase-3 activation in
XIAP-containing S100 extracts (Fig. 3b, upper
panel). Interestingly, deletion of only the first 4 residues of
mature Smac/DIABLO (
The results with the N-terminal deletion mutants suggest that the N
terminus harbors the caspase-promoting activity of Smac/DIABLO. To test
this hypothesis, we generated three C-terminal deletion mutants fused
at their C termini to GST. These mutants, which contain the first 7, 30, or 39 N-terminal residues of mature Smac/DIABLO (N7, N30, and N39,
respectively) (Fig. 3a), were expressed in bacteria and
purified to homogeneity. As shown in Fig. 3b, lower panel, N30 and N39 were able to promote caspase-3 activation in S100 extracts containing XIAP, although at a higher concentration than
the wild type Smac/DIABLO. The smallest mutant, N7, was the least
effective among the mutants. N30 and N39 were also able to relieve the
XIAP inhibition of caspase-7 and caspase-3 (Fig. 3c),
although they were slightly more effective with caspase-7 than
caspase-3. However, within the range of concentrations used in this
experiment N7 had no detectable activity with caspase-3 and -7. Taken
together, these results suggest that the caspase-promoting activity of
Smac/DIABLO resides within an approximately 30-residue-long domain at
its N terminus.
Like Smac/DIABLO-S, the N-terminal deletion mutants
Interestingly, all the three C-terminal deletion mutants (N7, N30, and
N39) were able to interact with full-length XIAP, as well as the
isolated BIR domains of XIAP (Fig. 3d, right
panel). However, the interaction of N7 with XIAP and its isolated
BIR domains was weaker than that observed with N30 and N39. The weaker activity of N30 and N39 compared with wild type Smac/DIABLO (Fig. 3,
b and c) suggests that the sequences C-terminal
to residue 39 are still required to achieve optimal activity, probably
because these sequences are necessary to maintain an overall structure capable of disrupting the interaction of XIAP with the active site of
caspases at lower concentrations of Smac/DIABLO. Indeed, according to
the crystal structure of Smac/DIABLO (26), the residues that are
involved in dimerization of Smac/DIABLO are located within a
hydrophobic interface C-terminal to residue 25. Mutations that disrupt
Smac/DIABLO dimerization were found to diminish its caspase-promoting
activity (26). Thus we believe that the weak activity of the short
N-terminal peptides, although they can still interact with XIAP, is
perhaps due to removal of the dimerization domain C-terminal to these peptides.
Chemically Synthesized Smac/DIABLO N-terminal Peptides Can Promote
Caspase Activation--
The finding that short sequences derived from
the N terminus of Smac/DIABLO can still interact with XIAP, cIAP-1, and
cIAP-2 (Fig. 3d and data not shown) and promote the caspase
activity of caspase-9, -3, and -7 (Figs. 2 and 3) suggests that these
sequences can be used to make peptides that could enhance the ability
of chemotherapeutic agents to induce apoptosis in cancer cells with elevated levels of IAPs. To test this hypothesis, we synthesized four
peptides based on the N-terminal sequences of mature Smac/DIABLO and
Smac/DIABLO-S and tested their ability to promote cytochrome c-dependent activation of caspase-3 in S100
extracts containing XIAP (Fig.
3e). The activity of caspase-3
in the S100 extracts was measured using the peptide substrate DEVD-AMC.
The peptides containing the first 7 or 35 residues (Smac/DIABLO-N7
(pept-1) and Smac/DIABLO-N35 (pept-4),
respectively) of mature Smac/DIABLO were very effective in promoting
caspase-3 activation in the XIAP containing extracts at 100-500
µM concentrations. The longer peptide (Smac/DIABLO-N35)
was noticeably better than the short peptide (Smac/DIABLO-N7) in
promoting caspase-3 activation. The peptide derived from the N terminus
of Smac/DIABLO-S (Smac/DIABLO-S-N9 (pept-2)) or derived from
an internal Smac/DIABLO sequence (Smac/DIABLO 27-35
(pept-3)) were almost completely inactive in this assay. These results provide a proof of principle that short peptides derived
from the N terminus of mature Smac/DIABLO could be used in the future
as effective activators of caspases at attainable concentrations to
kill cancer cells that overexpress IAPs.
Expression of a Cytosolic Smac/DIABLO Can Convert Type II Cells to
Type I Cells--
In Type II cells (e.g. breast
adenocarcinoma MCF-7 cells) death receptor-induced apoptosis can be
blocked by expression of Bcl-2 or Bcl-xL, but in Type I cells
(e.g. B lymphoblastoid cell line SKW6.4) it can not
(27, 28). In Type II cells it is possible that direct activation of the
effector caspases by caspase-8 is blocked at the level of the effector
caspases by IAPs, such as XIAP (29). In this scenario, the cleavage of
BID by caspase-8 might be required to release Smac/DIABLO to
neutralize the IAPs and allow direct activation of the effector
caspases by caspase-8 (30, 31). If this hypothesis is correct, then
Type II cells, which are not sensitive to death receptor-induced
apoptosis, can be made sensitive to death receptor-induced apoptosis
(Type I cells) by expression of a cytosolic form of Smac/DIABLO. To
test this hypothesis, we constructed a mammalian GFP-Smac/DIABLO
expression construct, which allows the expression of a cytosolic
GFP-Smac/DIABLO fusion protein that can be cleaved by caspase-8 to
generate mature Smac/DIABLO. This was achieved by introduction of an
IETD-A cleavage site between the N-terminal GFP and the C-terminal
Smac/DIABLO that lacks its MTS (Fig.
4a). Treatment of MCF-7 cells
with TRAIL induced apoptosis in ~28-40% of the cells.
Overexpression of Bcl-xL inhibited TRAIL-induced apoptosis of MCF-7
cells confirming previous observations that these cells require the
mitochondrial pathway for death receptor signaling (7, 32).
Interestingly, transfection of GFP-Smac/DIABLO into the
Bcl-xL-expressing MCF-7 cells bypassed the Bcl-xL inhibition and
sensitized these cells to TRAIL-induced apoptosis to a level almost
similar to that observed in the absence of Bcl-xL (~22-30%
apoptosis). Moreover, transfection of GFP-Smac/DIABLO into MCF-7 cells
in the absence of overexpressed Bcl-xL potentiated TRAIL-induced
apoptosis and resulted in ~65-80% cell death. The ability of
GFP-Smac/DIABLO to potentiate TRAIL-induced apoptosis in the absence of
overexpressed Bcl-xL is consistent with the presence of an IAP block in
these cells. Indeed, we found that MCF-7 cells express high levels of
XIAP (data not shown). These results suggest that the inability of
death receptor ligands to induce apoptosis in Type II cells in the
presence of overexpressed Bcl-xL is most likely attributed to
inhibition of the effector caspases by IAPs. This inhibition can only
be bypassed by release of Smac/DIABLO from the mitochondria, a function
that is performed by BID in Type II cells after caspase-8 cleavage
(Fig. 4b). Therefore, if Smac/DIABLO gene inactivation is
not proven to be lethal in the future, we expect that the phenotype of
Smac/DIABLO-deficient mice would be similar to that of the
BID-deficient mice (33) with respect to sensitivity of normal
hepatocytes to FAS-induced apoptosis. Because normal hepatocytes are
Type II cells, we predict that Smac/DIABLO gene inactivation should
make these cells resistant to Fas-induced apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Expression and subcellular localization
of the short isoform of Smac/DIABLO. a, a schematic
representation of the N termini of the Smac/DIABLO precursor
(Smac/DIABLO-L) and the alternatively spliced short isoform
of Smac/DIABLO (Smac/DIABLO-S). Smac/DIABLO-S begins with
the MKSDFYF sequence, which replaces the MTS and residues 55-60
(AVPIA) of the Smac/DIABLO-L. Smac/DIABLO-L and
Smac/DIABLO-S are 100% identical after residue 60. The
arrow indicates the cleavage site, which removes the MTS to
generate mature Smac/DIABLO. b, RT-PCR analysis of the
expression of Smac/DIABLO-L (lane 1) and Smac/DIABLO-S
(lane 2) in different cell lines (Jurkat, 293, THP1, MCF-7,
A431, and 697). The cloned Smac/DIABLO-L or Smac/DIABLO-S cDNAs
were used as control templates (cDNA lane).
c, MCF-7 cells were transfected with constructs encoding GFP
(A) or C-terminal GFP-tagged Smac/DIABLO-S (B) or
Smac/DIABLO-L (C). 24 h after transfection, cells were
visualized by confocal microscopy and photographed. Note the uniform
cytoplasmic fluorescence in panels A and B and
the punctate perinuclear (mitochondrial) fluorescence in panel
C.
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Fig. 2.
Smac/DIABLO relieves XIAP inhibition of the
initiator and effector caspases. a, 293T S100 extracts
were mixed with purified XIAP (20 nM) and then stimulated
with cytochrome c plus dATP in the presence of the indicated
amounts of purified mature Smac/DIABLO or Smac/DIABLO-S. S100 extracts
without XIAP (-) were used as controls. The reactions were
carried out in the presence of 35S-labeled procaspase-9
(upper panel) or procaspase-3 (lower panel).
After a 1-h incubation, the samples were analyzed by SDS polyacrylamide
gel electrophoresis and autoradiography. b, purified
caspase-3 or caspase-7 was mixed with XIAP (20 nM in the
case of caspase-3, and 5 nM in the case of caspase-7), and
the mixtures were then incubated with increasing amounts of purified
mature Smac/DIABLO or Smac/DIABLO-S (100, 200, 500, or 1000 nM, respectively). The reactions were carried out in the
presence of the peptide substrate DEVD-AMC (50 µM) for 30 min, and the substrate cleavage was measured by luminescence
spectrometry. The caspase activity in all the samples is plotted as a
percentage of the activity of caspase-3 or -7 in the absence of XIAP
(100%). c, interaction of XIAP and its isolated BIR domains
with mature Smac/DIABLO and Smac/DIABLO-S. Mature Smac/DIABLO
(lanes 2, 5, and 8) or Smac/DIABLO-S
(lanes 3, 6, and 9) was expressed in
bacteria with His6 tag and then immobilized onto
TALONTM-affinity resin. The resin was incubated with in
vitro-translated 35S-labeled XIAP or BIR1/BIR2 or
BIR3/RING domains of XIAP, washed extensively, and then analyzed by SDS
polyacrylamide gel electrophoresis and autoradiography. A
His6-tagged GST was used as a negative control (lanes
1, 4, and 7). XIAP and its isolated BIR
domains used in this experiment are represented as bar
diagrams (upper panel).
4) dramatically reduced Smac/DIABLO activity to a level similar to that seen with Smac/DIABLO-S. Deletion of the first
21 residues (21) further reduced Smac/DIABLO activity to undetectable levels. Other larger N-terminal deletions produced insoluble mutant proteins, which could not be used in this assay. However, one N-terminal deletion mutant lacking the first 139 residues
(139) was soluble but found to be completely inactive. The
N-terminal deletion mutants were then assayed for their ability to
enhance caspase-3 and -7 activity in the presence of XIAP (Fig. 3c). Like Smac/DIABLO-S, the N-terminal deletion mutants
4 and 21 had ~30-40% of the activity of wild type Smac/DIABLO
with caspase-3 and -7. However, the N-terminal deletion mutant 139 was also completely inactive in this assay.
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Fig. 3.
The N terminus of Smac/DIABLO is important
for its activity and interaction with the BIR3 of XIAP.
a, schematic diagram of mature Smac/DIABLO and the
N-terminal and C-terminal deletion mutants of Smac/DIABLO. Mature
Smac/DIABLO and the N-terminal deletion mutants were expressed in
bacteria with C-terminal His6 tags. The C-terminal deletion
mutants were expressed with C-terminal GST tags. b, effect
of the N-terminal deletion mutants (upper panel) or the
C-terminal deletion mutants (lower panel) on cytochrome
c-mediated caspase-3 activation. The reactions were carried
out with 293T S100 extracts and the indicated amounts of purified
mature Smac/DIABLO or mutants as in Fig. 2a. c,
effect of the N-terminal deletion mutants (upper panels) or
the C-terminal deletion mutants (lower panels) on the
enzymatic activity of caspase-3 (left panels) and -7 (right panels). The reactions were carried out as in Fig.
2b with increasing amounts of purified mature Smac/DIABLO or
mutants (100, 200, 500, or 1000 nM, respectively). The data
with mature Smac/DIABLO are essentially very similar to those in Fig.
2b, and thus are not shown here. d, interaction
of XIAP and its isolated BIR domains with the N-terminal and C-terminal
deletion mutants. The reactions were carried out as in Fig.
2c. The GST control contains a 47-residue-long
T7-His6 tag at its N terminus. e, effect of
chemically synthesized Smac/DIABLO N-terminal peptides on cytochrome
c-mediated caspase-3 activation. 293T S100 extracts were
mixed with purified XIAP (20 nM) and then stimulated with
cytochrome c plus dATP in the presence of increasing amounts
of mature Smac/DIABLO (25, 100, and 500 nM) or the
indicated purified N-terminal Smac/DIABLO or Smac/DIABLO-S peptides
(25, 100, and 500 µM). The reactions were carried
out in the presence of DEVD-AMC as a substrate. pept-1,
AVPIAQK; pept-2, MKSDFYFQK; pept-3, TDSTSTFL;
pept-4, AVPIAQKSEPHSLSSEALMRRAVSLVTDSTSTFL.
4 and 21 were
not able to interact with the BIR3 domain (XIAP-BIR3/RING) of XIAP but
were still able to interact with the BIR1/BIR2 domains (XIAP-BIR1/2) of
XIAP and to a slightly lesser extent with full-length XIAP (Fig.
3d, left panel). Based on these observations, we
suggest that the first 4 residues of mature Smac/DIABLO are essential for its ability to interact with the BIR3 domain of XIAP. Because the
BIR3 domain of XIAP is the domain that binds and inhibits caspase-9
(25), this could explain the very weak activity of Smac/DIABLO-S and
the N-terminal deletion mutants 4 and 21 in the caspase-3
activation assay, which measures caspase-9 activity. Thus, deletion or
substitution of the first 4 residues impairs the ability of Smac/DIABLO
to interact with the BIR3 domain of XIAP and consequently its ability
to promote caspase-3 activation by the caspase-9 apoptosome in the
presence of XIAP. Nevertheless, because Smac/DIABLO-S and the
N-terminal deletion mutants 4 and 21 can still interact with the
BIR1/BIR2 domains of XIAP, which is important for caspase-3 and -7 inhibition (25), they had better caspase-promoting activity with
caspase-3 and -7 (Figs. 2b and 3c) compared with
that with caspase-9 in the presence of XIAP.
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Fig. 4.
Smac/DIABLO is required to relieve the IAP
inhibition of the death receptor pathway in MCF-7 cells.
a, schematic diagram of the GFP-Smac/DIABLO fusion protein
and its cleavage by caspase-8 to generate mature Smac/DIABLO.
b, effect of expression of cytosolic Smac/DIABLO on
TRAIL-induced apoptosis. MCF-7 cells were transfected with GFP or
GFP-Smac/DIABLO expression constructs together with equal amounts of
empty vector or a construct encoding Bcl-xL. 24 h after
transfection the cells were left untreated (0 µg/ml) or treated with
TRAIL (0.5 or 2 µg/ml). The percentage of round apoptotic cells was
determined as described under "Experimental Procedures."
c, a model of cross-talk between the death receptor pathway
and the mitochondrial pathway and the role of Smac/DIABLO in
neutralizing the inhibitory effect of XIAPs on the initiator and
effector caspases.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. Vaux for the mouse DIABLO cDNA and for communicating results before publication. We also thank Y. Shi for communicating results before publication. We thank J.-L. Poyet for help in preparing the figures.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AG14357, AG13487, and CA78890 (to E. S. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF298770.
These authors contributed equally to this work.
§ To whom correspondence and requests for materials should be addressed: Thomas Jefferson University, Kimmel Cancer Inst., Bluemle Life Sciences Bldg., 904, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4632; Fax: 215-923-1098; E-mail: E_Alnemri@ lac.jci.tju.edu.
Published, JBC Papers in Press, August 18, 2000, DOI 10.1074/jbc.C000533200
2 D. Vaux, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: IAP(s), inhibitor of apoptosis protein(s); Smac/DIABLO-S, short Smac/DIABLO isoform; Smac/DIABLO-L, long Smac/DIABLO isoform; RT, reverse transcriptase; PCR, polymerase chain reaction; GST, glutathione S-transferase; GFP, green fluorescent protein; MTS, mitochondrial targeting sequence; DEVD-AMC, Asp-Glu-Val-Asp-7-Amino-Y-methyl coumarin; DEVD, Asp-Glu-Val-Asp; AMC, 7-Amino-4-methyl coumarin; BIR, baculovirus IAP repeat.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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| 1. | Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972) Br. J. Cancer 26, 239-257 |
| 2. | Vaux, D. L., Haecker, G., and Strasser, A. (1994) Cell 76, 777-779 |
| 3. | Vaux, D. L., and Korsmeyer, S. J. (1999) Cell 96, 245-254 |
| 4. | Salvesen, G. S., and Dixit, V. M. (1997) Cell 91, 443-446 |
| 5. | Thornberry, N. A., and Lazebnik, Y. (1998) Science 281, 1312-1316 |
| 6. | Salvesen, G. S., and Dixit, V. M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 10964-10967 |
| 7. | Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T., and Alnemri, E. S. (1998) Mol Cell 1, 949-957 |
| 8. | Ashkenazi, A., and Dixit, V. M. (1998) Science 281, 1305-1308 |
| 9. | Reed, J. C. (1997) Cell 91, 559-562 |
| 10. | Saleh, A., Srinivasula, S. M., Acharya, S., Fishel, R., and Alnemri, E. S. (1999) J. Biol. Chem. 274, 17941-17945 |
| 11. | Goldstein, J. C., Waterhouse, N. J., Juin, P., Evan, G. I., and Green, D. R. (2000) Nat. Cell Biol. 2, 156-162 |
| 12. | Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J. L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L. E., and Tschopp, J. (1997) Nature 388, 190-195 |
| 13. | Srinivasula, S. M., Ahmad, M., Ottilie, S., Bullrich, F., Banks, S., Wang, Y., Fernandes-Alnemri, T., Croce, C. M., Litwack, G., Tomaselli, K. J., Armstrong, R. C., and Alnemri, E. S. (1997) J. Biol. Chem. 272, 18542-18545 |
| 14. | Adams, J. M., and Cory, S. (1998) Science 281, 1322-1326 |
| 15. | Green, D. R., and Reed, J. C. (1998) Science 281, 1309-1312 |
| 16. | Saleh, A., Srinivasula, S. M., Balkir, L., Robbins, P. D., and Alnemri, E. S. (2000) Nat. Cell Biol. 2, 476-483 |
| 17. | Beere, H. M., Wolf, B. B., Cain, K., Mosser, D. D., Mahboubi, A., Kuwana, T., Tailor, P., Morimoto, R. I., Cohen, G. M., and Green, D. R. (2000) Nat. Cell Biol. 2, 469-475 |
| 18. | Pandey, P., Saleh, A., Nakazawa, A., Kumar, S., Srinivasula, S. M., Kumar, V., Weichselbaum, R., Nalin, C., Alnemri, E. S., Kufe, D., and Kharbanda, S. (2000) EMBO J. 19, 4310-4322 |
| 19. | Deveraux, Q. L., and Reed, J. C. (1999) Genes Dev. 13, 239-252 |
| 20. | Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Cell 102, 33-42 |
| 21. | Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G. E., Moritz, R. L., Simpson, R. J., and Vaux, D. L. (2000) Cell 102, 43-53 |
| 22. | Wang, S. L., Hawkins, C. J., Yoo, S. J., Muller, H. A., and Hay, B. A. (1999) Cell 98, 453-463 |
| 23. | Goyal, L., McCall, K., Agapite, J., Hartwieg, E., and Steller, H. (2000) EMBO J. 19, 589-597 |
| 24. | Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. (1997) Nature 388, 300-304 |
| 25. | Deveraux, Q. L., Leo, E., Stennicke, H. R., Welsh, K., Salvesen, G. S., and Reed, J. C. (1999) EMBO J. 18, 5242-5251 |
| 26. | Chai, J., Du, C., Wu, J.-W., Kyin, S., Wang, X., and Shi, Y. (2000) Nature 406, 855-862 |
| 27. | Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K. J., Debatin, K. M., Krammer, P. H., and Peter, M. E. (1998) EMBO J. 17, 1675-1687 |
| 28. | Medema, J. P., Scaffidi, C., Krammer, P. H., and Peter, M. E. (1998) J. Biol. Chem. 273, 3388-3393 |
| 29. | Green, D. G. (2000) Cell 102, 1-4 |
| 30. | Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998) Cell 94, 491-501 |
| 31. | Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998) Cell 94, 481-490 |
| 32. | Li, F., Srinivasan, A., Wang, Y., Armstrong, R. C., Tomaselli, K. J., and Fritz, L. C. (1997) J. Biol. Chem. 272, 30299-30305 |
| 33. | Yin, X. M., Wang, K., Gross, A., Zhao, Y., Zinkel, S., Klocke, B., Roth, K. A., and Korsmeyer, S. J. (1999) Nature 400, 886-891 |
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