From the The Burnham Institute, La
Jolla, California 97037,
Department of Hematology and
Oncology, Charité, Virchow-Clinic, Berlin, Germany, and
The University of Texas M. D. Anderson Cancer Center, Houston,
Texas 77030
Received for publication, October 3, 2002, and in revised form, January 16, 2003
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ABSTRACT |
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Here we report on the identification of peptides
targeting the X-inhibitor of apoptosis protein (XIAP). XIAP functions
as a caspase inhibitor and is a member of the inhibitors of apoptosis (IAP) family of proteins. IAPs are often overexpressed in cancers and
leukemias and are associated with an unfavorable clinical prognosis. We
have selected peptides from a phage library by using recombinant
full-length human XIAP or a fragment containing only the baculovirus
IAP repeat 2 (BIR2) domain. A consensus motif, C(D/E/P)(W/F/Y)-acid/basic-XC, was recovered from
two independent screenings by using different libraries.
Phage-displaying variations of the consensus sequence bound
specifically to the BIR2 domain of XIAP but not to other IAPs. The
interaction was specific as it could be blocked by the cognate
synthetic peptides in a dose-dependent manner. Phage
displaying the XIAP-binding motif CEFESC bound to the BIR2 domain of
XIAP with an estimated dissociation constant of 1.8 nM as
determined by surface plasmon resonance. Protein-protein interaction
assays revealed that caspase-3 and caspase-7 (but not caspase-8)
blocked the binding of the CEFESC phage to XIAP, indicating that this
peptide targets a domain within XIAP that is related to the
caspase-binding site. In fact, the sequence EFES is homologous to a
loop unique to the executioner caspase-3 and caspase-7 that are
targeted by XIAP. Finally, we demonstrated that an internalizing
version of the XIAP-binding peptide identified in our screenings (PFKQ)
can induce programmed cell death in leukemia cells. Peptides
interacting with XIAP could serve as prototypes for the design of low
molecular weight modulators of apoptosis.
The inhibitor of apoptosis proteins
(IAPs)1 represent a
family of anti-apoptotic proteins found in both vertebrates and
invertebrates (reviewed in Ref. 1). All of the human IAP homologs have
been shown to inhibit programmed cell death (1, 2). The human IAP
family members, XIAP, c-IAP1, and c-IAP2, bind to caspase-3 and
caspase-7 with inhibitory constant values (Ki) of 0.2-10 nM (3-5). XIAP also binds to and suppresses
specifically caspase-9, an initiator caspase, that is at the apical
protease in the cytochrome c/mitochondrial pathway for
apoptosis (6-9).
All of the IAP family members have at least one and up to three copies
of an We identified XIAP ligands containing the motif CEFESC. This motif
appears to mimic the XIAP-binding site within specific caspases because
the binding of this peptide to XIAP is inhibited by preincubation with
caspase-3 and caspase-7 but not by caspase 9. Binding assays using
individual phage displaying this motif and a panel of purified targets
confirmed that the phage interacts specifically with the BIR2 domain in
a pattern consistent with caspase-type ligands. No binding was observed
when BIR1, BIR3, or the RING finger domain of XIAP were tested nor when
other IAP family members such as cIAP1, cIAP2, NAIP, and Survivin were
evaluated under the same experimental conditions. We also show that
XIAP-binding peptides can affect cell viability. Taken together, our
results contribute to the understanding of the structural requirements and functional domains that are important in the regulation of programmed cell death.
Plasmids, Protein Expression, and Purification--
Cloning and
synthesis of full-length XIAP and the XIAP fragments BIR1, BIR2, BIR3,
BIR1-2, BIR2-3, and RING as well as full-length cIAP1, cIAP2, NAIP,
and Survivin have been described previously (4, 10, 12). Recombinant
caspase proteins containing His6 tags were prepared as
described previously (8) and were a gift from Dr. G. Salvesen (The
Burnham Institute, La Jolla, CA).
Identification of XIAP-binding Peptides by Phage
Display--
fUSE5-based phage peptide libraries displaying either
cyclic or linear random peptides (CX4C or
X6; C = cysteine and X = random amino acid) were made and screened as described
previously (13, 14). Polystyrene 96-well plates were coated with 50 µl/well of 1 mg/ml GST-XIAP fusion protein or GST fusion proteins of
various fragments of XIAP, cIAP1, cIAP2, NAIP, Survivin, BSA, or GST in PBS overnight at 4 °C. The wells were washed with PBS, blocked with
3% BSA (first round of panning), casein (second round), or Peptide Synthesis--
Peptides were synthesized with an
ACT-350 multiple peptide synthesizer by using Fmoc synthesis on
Rink amide MBHA resin or Fmoc-Lys(-LC-D-Biotin)-Rink amide-MBHA resin for
C-terminally biotinylated peptides. The peptides were cleaved with
92.5% trifluoroacetic acid, 2.5% water, 2.5% EDTA, and 2.5%
TIS and precipitated with cold ethyl ether. Quantitative
cyclization was achieved in 20% Me2SO,
H2O, pH 4.0-7.0 in 1 day as described previously (16). Crude peptides were purified by high pressure liquid chromatography on
a reverse phase C-18 column with a gradient of
water/acetonitrile containing 0.1% trifluoroacetic acid. The
purified peptides showed one peak by analytical high pressure liquid
chromatography, and the molecular weight was confirmed by
matrix-assisted laser desorption ionization time-of-flight mass spectroscopy.
Caspase Competition Assays--
Inhibition of phage binding to
XIAP by recombinant caspases was assayed by coating polystyrene 96-well
plates with 1 µg/well of recombinant XIAP protein (50 µl of a 1 mg/ml solution), various fragments of XIAP, or control proteins (as
indicated) in PBS overnight at 4 °C. The wells were washed once with
PBS, blocked with 3% BSA for 1 h, washed again, and incubated
with various amounts of recombinant active caspases-3, -7, or -8 for 30 min at room temperature. The wells were then washed once with PBS, and
XIAP-binding or control phage was added (108 TU/well) and
incubated for 30 min. Wells were then washed nine times with PBS,
0.01% Tween 20, and washed once more in PBS. Bound phage were eluted
by adding 200 µl of a log-phase K91Kan terrific broth culture. The
number of bound phage was determined as described above.
Surface Plasmon Resonance--
The interaction of XIAP-binding
phage and control phage with the BIR2 domain of XIAP was investigated
using the BIAcore 3000 system (BIAcore, Uppsala, Sweden). GST-BIR2 was
covalently attached by its primary amine residues to CM5 sensor chips
(17). Binding was detected in resonance units after injecting phage in
a range of concentrations in HBS buffer (10 mM HEPES, pH
7.4, with 0.15 M NaCl, 3.0 mM EDTA, and 0.05%
surfactant P20). After each run, the chip surfaces were regenerated
with 10 mM glycine, pH 4.5. Non-linear regression analysis
was used to determine equilibrium-binding constants that fit to a
single site-binding model (18-20).
Peptide Internalization and Cell Viability Assays--
Uptake of
penetratin-linked peptides into OCI/AML-4 cells (an acute myeloblastic
leukemia cell line, for review see Ref. 21) was monitored as described
previously (22). Cells growing in 24-well plates were incubated with
increasing doses (1-20 µM) of penetratin alone,
synthetic peptides alone (CEFESC, CPFKQC, or ARGKER), or
penetratin-linked versions of each peptide. Cell viability was assessed
at different time points (12, 24, and 72 h).
Identification of XIAP-binding Peptides--
XIAP-binding peptides
were identified by selecting phage display peptide libraries on
immobilized human GST-XIAP. The libraries contained six amino acid
inserts in which either all six residues were randomized or the first
and last positions were fixed as cysteines to promote cyclization by
disulfide bonding (X6 or
CX4C libraries, respectively). The enrichment of
phage on XIAP was monitored by counting the number of TU recovered from
the XIAP-coated wells versus the number recovered from wells
coated with the control protein. We observed a pronounced enrichment
for phage binding to GST-XIAP (Fig. 1).
The DNA inserts of 36 randomly chosen phage clones (18 from the
X6 library and 18 from the
CX4C library) recovered from the third round of
biopanning on XIAP were sequenced. A total of 32 phage clones derived
from both libraries displayed the consensus motif C-(E/D/P)-(aromatic
amino acid = W/Y/F)-charged amino acid-(X = random)-C (Table I). In addition, marked
enrichment was observed for phage displaying the motif (8 of 18 in the
second round and 16 of 18 in the third round by using the
X6 library). In contrast, biopanning experiments
using the same libraries on other IAPs such as Survivin or on GST
protein alone failed to select for phage with the above consensus
motif, demonstrating the specificity of the selection process.
Phage Specificity and Mapping of Binding Sites on XIAP--
The
specificity of XIAP-binding phage was tested in individual
phage-binding assays on immobilized GST-XIAP or GST fusion proteins of
other members of the IAP family such as Survivin, cIAP1, cIAP2, and
NAIP. Representative data are shown for two of the XIAP-binding phage
displaying the CEFESC- or the CPFKQC-peptide. Controls were insertless
fd-tet phage and phage-displaying ARGKER, an unrelated control insert.
In these assays, both the CEFESC and CPFKQC phage bound to XIAP but not
to the other IAP family members, BSA, or GST. Control phage did not
bind to GST-XIAP, indicating specificity. These data confirm the
specificity of phage clones displaying the consensus motif for XIAP
binding (Fig. 2).
We next mapped the domain within XIAP that mediates the binding of the
isolated phage clones using a panel of XIAP deletion mutants expressed
as GST fusion proteins. XIAP-binding phage bound to GST fusion proteins
containing the BIR2 domain of XIAP alone or the BIR2 domain in
combination with flanking domains. In contrast, fragments of XIAP
containing only the BIR1, BIR3, or RING domains failed to adsorb
XIAP-binding phage. The insertless fd-tet phage as well as the control
ARGKER-phage did not bind to either GST-XIAP or GST-BIR2, indicating
that the target proteins do not bind recombinant phage nonspecifically
(Fig. 3).
Synthetic Peptides Representing Phage-derived Sequences Bind
XIAP--
To further confirm that the peptides displayed on
XIAP-binding phage interact with XIAP independently of other phage
components, experiments were performed using synthetic peptides. Cyclic
peptides corresponding to the sequences displayed by XIAP-binding phage clones were synthesized and tested for their ability to specifically inhibit phage binding to GST-XIAP and GST-BIR2. The cyclic peptides but
not the irrelevant control peptides inhibited phage binding of the
corresponding phage clones in a concentration-dependent manner (Fig. 4). Non-cyclic versions of
the same peptides failed to prevent phage binding.
Binding Properties of XIAP-binding Phage--
To further
characterize the interaction of XIAP-binding phage with the BIR2
domain, we used surface plasmon resonance. The BIR2 domain of XIAP was
conjugated to the chip surface (solid phase) and phage displaying a
XIAP-binding peptide were used as an analyte (mobile phase). A variety
of concentrations of phage displaying CEFESC, CPFKQC, or CDFKAC were
injected, and the CEFESC-phage clone was the strongest binder of the
three phages analyzed (Fig. 5). This
phage bound to BIR2 with an estimated ka of 4.9 × 105 M XIAP-binding Phage and Caspases Compete for Binding to
XIAP--
Because the BIR2 domain of XIAP has been shown to be
necessary and sufficient for binding caspases-3 and -7 (10), we asked whether these caspases compete with XIAP-binding phage for binding to
XIAP. GST-BIR2 or various GST control proteins were immobilized on
glutathione-Sepharose and incubated with XIAP-binding CEFESC-phage in
the presence or absence of recombinant caspase-3 or -7. Recombinant caspase-8 was used as a control because it does not bind XIAP (3). The
binding of CEFESC-phage to BIR2 was inhibited in a concentration-dependent manner upon the addition of caspase-7 or caspase-3. In contrast, neither BSA nor caspase-8 blocked the binding of CEFESC-phage to XIAP (Fig. 6).
These data suggest that phage displaying the CEFESC-peptide occupy
sites on the BIR2 domain that overlap with or are adjacent to sites
involved in caspase binding.
To determine whether the XIAP-targeting peptides could affect cell
viability, we designed and synthesized internalizing versions of the
CEFESC-, CPFKQC-, or ARGKER-peptides (negative control) by using the
penetratin system for intracellular delivery. Penetratin (Pen) is a
peptide containing 16 amino acids that is part of the third helix of
the antennapedia protein homeodomain (22). Because penetratin has
translocating properties, it is capable of carrying hydrophilic
compounds across the plasma membrane and delivering them directly to
the cytoplasm without degradation (22). We fused the XIAP-binding
peptides or a control unrelated peptide to penetratin and added a
biotin moiety to visualize internalization. We used leukemia cells
(OCI/AML-4) as a model system to test the effect of the internalizing
peptides because these cells (i) express XIAP and caspases and (ii)
respond to regulators of apoptosis (21). Pen-linked peptides were
internalized and remained in the cytoplasm (data not shown). Penetratin
alone was also internalized and uniformly distributed in the cytoplasm.
Peptides lacking penetratin were not internalized (data not shown). We
evaluated cell survival in the cells exposed, internalizing versions of
the XIAP-binding peptides. After a 72-h incubation, reduced cell
viability was observed in cells treated with Pen-CPFKQC (Fig.
7). No cell death was observed when cells
were untreated, treated with penetratin alone, or treated with an
unrelated control peptide fused to penetratin. Moreover, incubation
with non-internalizing versions of each synthetic peptide had no effect
(Fig. 7).
IAPs are overexpressed in a variety of cancers and leukemias, and
antagonistic small molecules could be useful for modifying the
antiapoptotic activity of XIAP in vivo. In this study, we screened phage-display peptide libraries in search of short peptide motifs capable of binding this caspase-inhibitory protein. An analysis
of the peptides displayed by phage binding to XIAP revealed a consensus
sequence in which position 1 is invariably a cysteine, position 2 is
either an acidic residue (aspartic acid or glutamic acid) or a proline,
and position 3 is usually an aromatic amino acid (tryptophan,
phenylalanine, or tyrosine) followed by two variable amino acids and
finally a cysteine. Although position 4 was variable among the
XIAP-binding phage randomly sequenced, the most highly represented
types of amino acids at position 4 were charged (lysine, arginine,
histidine, aspartic acid, or glutamic acid in 13 of 32 cases). The
binding of these phage to XIAP was highly selective with no evidence of
significant interactions with other members of the IAP family of
proteins such as cIAP1, cIAP2, NAIP, or Survivin. Thus, the
XIAP-binding phage recognize a binding site unique to XIAP, which may
be exploitable for applications aimed either at detecting XIAP or
selectively inhibiting XIAP function.
We showed that the isolated peptides bind to the BIR2 domain of XIAP.
The specificity of the motif for BIR2 was confirmed independently by
repeating library screenings using GST-BIR2 as a target, resulting in
the selection of phage whose inserts shared the same consensus
sequence, which was obtained originally using GST full-length XIAP.
Interactions of these peptide motifs with BIR2 are biologically
relevant, because our previous studies indicate that the BIR2 domain is
necessary and sufficient for inhibiting caspases-3 and -7 (10).
Functional studies using the cyclic XIAP-binding peptides show that
these probes do not act as repressors of XIAP-mediated caspase
inhibition. We also show that an internalizing version of the
XIAP-binding peptide identified in our screenings (PFKQ) can induce
programmed cell death in leukemia cells in a specific,
dose-dependent, and time-dependent manner.
These findings suggest that development of peptidomimetics following
affinity maturation strategies to increase binding affinity may lead to tools that can be used as modulators of programmed cell death.
During programmed cell death, effector caspase zymogens are cleaved at
conserved aspartic acid residues, generating large and small subunits,
which together constitute the active protease. The activation of
effector caspases such as caspases-3 and -7 is a nearly universal event
associated with apoptosis induced by multiple stimuli. Our previous
observation that XIAP and other IAP family proteins directly bind
active executioner caspases-3 and -7, resulting in their potent
suppression in vitro and in cultured cells (3, 4), suggests
a general mechanism for IAP-mediated apoptosis inhibition. Recently, a
naturally occurring IAP inhibitor termed Smac/DIABLO has been
identified (23, 24). Smac performs a critical function in
apoptosis by eliminating the inhibitory effect of IAPs on
caspases. Smac promotes not only the proteolytic activation of
procaspase-3 but also the enzymatic activity of mature caspase-3, both
of which depend upon the ability of Smac to interact physically with
IAPs. A seven-residue peptide derived from the N terminus of Smac
promotes procaspase-3 activation in vitro (23). Although the
amino acid sequence of this peptide is different from our sequences,
identification of Smac and Omi provides further evidence that
inhibition of IAPs by peptides represents a viable approach to
induction of apoptosis in mammalian cells.
Interestingly, the sequence EFES, which is embedded in one of the
XIAP-binding phage in a cyclic context (CEFESC), occurs in caspase-3, a
protease that binds to BIR2 of XIAP with a KD Although XIAP is widely expressed, abnormally high levels of XIAP have
been observed in certain types of leukemias and solid tumors (28).
Overexpression of XIAP has also been shown to render tumor cells more
resistant to apoptosis induction by anti-cancer drugs in
vitro (2). Thus, agents that interfere with XIAP activity may be
useful to treat cancers. The phage-derived peptides described here
could provide a starting point for the generation of small molecule
compounds that bind to and inhibit XIAP, thereby providing a new
approach to the treatment of malignancies. Alternatively, cell
membrane-penetrating versions of XIAP-binding peptides or tumor-targeted delivery of genes expressing XIAP-binding peptides fused
to ubiquitin ligases could potentially be exploited as mechanism-based strategies for improved cancer treatment.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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70-amino acid zinc-binding domain termed the baculovirus IAP
repeat (BIR) (1). We have performed mutagenesis-based studies to show
that in the context of XIAP, the second of the three BIR domains (BIR2)
is necessary and sufficient to inhibit the effector proteases caspase-3
and caspase-7 (10). In contrast, for XIAP to suppress caspase-9, the
third BIR domain is required (11). These data suggest that a single BIR
domain can mediate anti-apoptotic activity. To map functionally
relevant interacting sites within molecules associating with XIAP, we
screened peptide libraries using the full-length protein and on the
isolated BIR2 domain.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globulins (third round) for 1 h, and incubated with
1010 transducing units (TU) of the primary library for
1 h at room temperature. The wells were washed nine times in PBS,
0.01% Tween 20 to remove unbound phage and once with PBS. Bound phages
were eluted by adding 200 µl of a log-phase K91Kan terrific broth
culture and amplified overnight in 10 ml of LB medium containing 40 µg/ml tetracycline. Randomly selected phage clones from the third
round of panning were sequenced as described previously (15).
Individual phage clones displaying similar peptide motifs were tested
for specific binding to GST fusion proteins of XIAP, fragments of XIAP,
cIAP1, cIAP2, NAIP, Survivin, BSA, or GST alone. These binding assays
were performed following the protocol of the initial screening. The
number of bound phage was determined by plating serial dilutions of the
recovered phage-bacteria mixture on LB agar plates containing 40 µg/ml tetracycline.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Fig. 1.
Enrichment of XIAP-binding phage on
recombinant XIAP. A CX4C random
phage-display peptide library was selected on immobilized GST-XIAP as
described under "Experimental Procedures." Aliquots of the
recovered phage-bacteria mixture were plated on LB plates containing
tetracycline to determine the number of TU recovered. Data represent
the number of TU recovered from wells coated either with GST-XIAP
(black bars) or a control protein (white bars;
casein for screening round II, bovine -globulins for screening round
III).
Sequences of XIAP-binding peptides
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Fig. 2.
CEFESC and CPFKQC selectively bind to XIAP
but not to other IAP family members. Phage displaying no peptide
insert (fd-tet), the XIAP-binding sequences CEFESC or CPFKQC, or a
control sequence ARGKER were incubated in microtiter plate wells coated
with 2 µg of various GST fusion proteins (XIAP, Survivin, cIAP1,
cIAP2, and NAIP), GST alone, or BSA. Bound phage were recovered by
bacterial infection. Transduced bacteria were grown overnight on LB
plates containing tetracycline to determine the number of TU recovered.
Data are expressed as the percentage relative to the number of TU
recovered from wells coated with GST-XIAP (set to 100%). Data
represent the means from triplicate platings ± S.D.
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Fig. 3.
XIAP-binding phage interact with the BIR2
domain of XIAP. Phage were incubated in microtiter plate wells
coated with 2 µg of immobilized GST fusion proteins representing
full-length XIAP, various fragments of XIAP, or BSA (control). Bound
phage were rescued and quantified by colony counting as in Fig.
2.
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Fig. 4.
Synthetic peptides displaying phage-derived
sequences bind XIAP. Binding of CEFESC phage to XIAP is inhibited
by the cognate peptide. Binding of phage displaying the XIAP-binding
peptide-CEFESC or a control peptide (ARKGER) to GST-XIAP or GST-BIR2
was measured as in Fig. 2 in the presence or absence of cyclic
CEFESC-synthetic peptide. Data represent the means from triplicate
platings ± S.D. and are expressed as the percentage relative to
the number of TU obtained in the absence of synthetic peptides.
1s
1. Upon
switching to phage-free flow solution, a very slow dissociation of
phage from the chip surface was observed with an approximate kd of 8.6 × 10
4. A
KD of 1.8 nM was estimated for binding
of the CEFESC-phage to BIR2 as compared with a KD of
>10 µM for either insertless fd-tet phage or phage
displaying a control peptide-ARGKER.
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Fig. 5.
Surface plasmon resonance characterizes the
binding of the CEFESC-phage to the BIR2 domain of XIAP.
Recombinant BIR2 of XIAP was immobilized on a sensor chip, and a range
of concentrations of CEFESC phage was injected over the chip surface.
Data represent resonance units plotted as a function of time. The
arrow indicates the time at which the mobile phase was
switched to the buffer lacking phage. The binding constant
(KD) was estimated from association and dissociation
rate constants using sensograms analyzed by Bioevaluation 3.0 (BIAcore)
software.
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Fig. 6.
Caspase-7 but not caspase-8 inhibits binding
of phage to XIAP. GST-XIAP or GST alone were immobilized on
microtiter plate wells (100 ng/well) and incubated with recombinant
caspase-7 or caspase-8 (control) in a total volume of 50 µl at the
indicated concentrations. XIAP-binding CEFESC-phage were added at
107 TU/well and incubated for 1 h, and bound phage
were recovered after washing. Recovered phage were quantified as in
Fig. 2.
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Fig. 7.
An internalizing version of the XIAP-binding
peptide triggers cell death. Internalization of Pen-CPFKQC leads
to growth arrest and induces programmed cell death in OCI/AML-4 cells.
Cell viability (%) was evaluated at 72 h after no treatment
(No peptide) or incubation with multiple peptides as
indicated. Pen-CPFKQC decreased cell viability. *, p < 0.01. Shown are mean ± S.E. obtained from triplicate wells.
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
5 nM (10). This sequence is located in the 381-loop of
caspase-3 in a region only found in caspases-3 and -7. Because this
loop is important in the binding of BIR2 to caspases-3 and -7 (5, 25,
26) (for review see Ref. 27), one might speculate that the
EFES-containing peptide corresponds to an important region within the
caspase-3-XIAP interacting site. In fact, the recently reported x-ray
crystallographic structure of the XIAP-BIR2 domain/caspases-3 and -7 complex shows that the substrate-binding pocket within these caspases
is formed by four surface loops, L1, L2, L3, and L4 (5, 25, 26). The
sequence EFES is exposed for binding within the 381-loop, and the
second Glu residue in the sequence corresponds to an interaction site
between caspase-3 and BIR2 (5). This observation underscores the power
of phage display technology for the mapping for biologically relevant
protein-interacting sites.
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ACKNOWLEDGEMENTS |
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We thank Drs. Guy S. Salvesen, Frank C. Marini III, and Luiz V. Rizzo for critical reading of this paper and helpful insights.
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FOOTNOTES |
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* This work was supported in parts by National Institutes of Health Grants CA55164 and AG15402 (to J. R.), CA90270 and CA8297601 (to R. P.), and CA90270 and CA9081001 (to W. A.) and by a Gilson-Longenbaugh Foundation Award (to R. P. and W. 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.
§ Both authors contributed equally to this work.
¶ Supported by a fellowship from the Mildred-Scheel-Stiftung fuer Krebsforschung and a grant from the José Carreras Leukämie-Stiftung.
** Supported by fellowships from the Deutsche Forschungsgemeinschaft and the Susan G. Komen Breast Cancer Foundation. Present address: Dept. of Hematology and Oncology and Institute for Molecular Medicine and Cell Research, University of Freiburg Medical Center, 79106 Freiburg, Germany.
§§ To whom correspondence should be addressed: rpasqual{at}notes.mdacc.tmc.edu, jreed{at}burnham-inst.org.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M210133200
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ABBREVIATIONS |
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The abbreviations used are: IAP, inhibitor of apoptosis; XIAP, X-inhibitor of apoptosis protein; BIR, baculovirus IAP repeat; GST, glutathione S-transferase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TU, transducing units; Fmoc, N-(9-fluorenyl) methoxycarbonyl; Pen, penetratin.
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