From the Department of Biochemistry, Weill Medical
College of Cornell University, New York, New York 10021 and the
¶ Center for Biomolecular Interaction Analysis, School of
Medicine, University of Utah, Salt Lake City, Utah 84132
Received for publication, November 14, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apoptosis is a highly regulated multistep process
for programmed cellular destruction. It is centered on the activation
of a group of intracellular cysteine proteases known as caspases. The
baculoviral p35 protein effectively blocks apoptosis through its broad
spectrum caspase inhibition. It harbors a caspase recognition sequence
within a highly protruding reactive site loop (RSL), which gets cleaved
by a target caspase before the formation of a tight complex. The
crystal structure of the post-cleavage complex between p35 and
caspase-8 shows that p35 forms a thioester bond with the active site
cysteine of the caspase. The covalent bond is prevented from hydrolysis
by the N terminus of p35, which repositions into the active site of the
caspase to eliminate solvent accessibility of the catalytic residues.
Here, we report mutational analyses of the pre-cleavage and
post-cleavage p35/caspase interactions using surface plasmon resonance
biosensor measurements, pull-down assays and kinetic inhibition
experiments. The experiments identify important structural elements for
caspase inhibition by p35, including the strict requirement for a Cys
at the N terminus of p35 and the rigidity of the RSL. A bowstring
kinetic model for p35 function is derived in which the tension
generated in the bowstring system during the pre-cleavage interaction
is crucial for the fast post-cleavage conformational changes required
for inhibition.
The development and homeostasis of multicellular organisms depend
on a delicate balance of cell proliferation and programmed cell death
or apoptosis. Failure to control either of these processes can lead to
serious diseases that threaten the existence of the organism (1, 2).
For example, the down-regulation of apoptosis is often associated with
cancer, autoimmune disorders, and persistent viral infections. The
up-regulation of apoptosis is observed in many forms of degenerative
disorders such as Alzheimer's disease, ischemic injury from stroke,
and post-menopausal osteoporosis.
The central effectors of apoptotic cell death are caspases, a group of
cysteine proteases specific for aspartate residues (3). Caspases are
highly regulated at several different levels. First, they are
synthesized as inactive single-chain zymogens. Second, caspase
activation is achieved through controlled proteolytic cascades, with
upstream caspases (Group III, such as caspase-8 and caspase-9)
activated by signal-mediated oligomerization and autoprocessing and
downstream caspases (Group II, such as caspase-3 and casapse-7)
activated by upstream caspases. While caspases have a dominant
requirement for Asp at the P1 position, neighboring sequences at P5-P1'
(in particular P4-P2) influence the substrate specificity of each group
of caspases (4).
Active caspases are in addition subject to inhibition by specific viral
and cellular caspase inhibitors (5, 6). Most notably, the p35 protein
from baculoviruses is an effective and the only wide-spectrum caspase
inhibitor. It blocks apoptosis induced by numerous stimuli and in
diverse organisms (7-9). Transgenic expression of p35 shows immense
promise in controlling apoptosis and degenerative diseases (10-20).
Previous biochemical and structural studies showed that caspase
inhibition by p35 requires the cleavage of a caspase recognition
sequence (DQMD87) within a solvent exposed and highly
protruding reactive site loop
(RSL)1 (21), followed by the
formation of a tight post-cleavage complex with the caspase.
Previously, we reported the crystal structure of the post-cleavage
complex between p35 and caspase-8, a group III initiator caspase
involved in Fas-mediated apoptosis (22). The structure revealed that
the caspase is inhibited via a covalent thioester linkage between the
active site Cys360 of caspase-8 (Cys285
of caspase-1 numbering) and the cleavage residue Asp87 of
p35. During normal substrate cleavage, the thioester intermediate is
quickly hydrolyzed by an enzyme bound water molecule. In the p35-caspase-8 complex, the thioester bond is preserved by the N
terminus of p35, which interacts with the caspase active site and
excludes solvent from the catalytic His317 of caspase-8
(His237 of caspase-1 numbering). The interaction between
the p35 N terminus and the caspase is realized through a series of
dramatic post-cleavage conformational changes (22).
In the structure of the p35-caspase-8 post-cleavage complex, three
regions of p35 directly contact the caspase, the N terminus, the KL
region (including the KL loop and the K and L strands) and the
substrate sequence of p35 (Fig. 1). To further understand the
structural determinant of caspase inhibition by p35, we performed structure-based mutagenesis at these three regions of p35. The mutants
were extensively analyzed for their effects on the pre-cleavage association, as assessed by surface plasmon resonance biosensor measurements, and on the post-cleavage inhibition, as determined by
both qualitative pull-down assays and quantitative kinetic inhibition
experiments. These experiments not only identified functionally
important structural elements of p35 in caspase inhibition, but also
led to a novel bowstring kinetic model of caspase inhibition by p35. In
this model, the p35 may be considered as a bowstring system with the
RSL being the string. The tension produced in the string during the
pre-cleavage association with a target caspase appears to control the
efficiency of caspase inhibition by facilitating fast post-cleavage
conformational changes.
Protein Expression and Purification--
Wild-type and mutant
baculoviral p35 (residues 1-299, with a C-terminal His-tag), human
caspase-8 (residues Ser201-Asp463, with or
without a C-terminal His-tag), human caspase-3 (residues 1-277, with
or without a C-terminal His-tag), and human caspase-3 active site
mutant (C163A, with a C-terminal His-tag) were expressed in the pET
bacterial expression system using overnight
isopropyl-1-thio- His-tag Pull-down Assay--
Purified His-tagged wild-type or
mutant p35 proteins were mixed with cell pellets of non-tagged
caspase-8 or caspase-3 and lysed in 20 mM Tris at pH 7.4, 150 mM NaCl, 10 mM imidazole, and 10 mM Kinetic Analysis--
Caspase inhibition by p35 was assayed by
progress curve analysis as described earlier (9). The fluorogenic
caspase substrate Ac-DEVD-AFC was purchased from Enzyme Systems.
Fluorescence detection upon substrate cleavage by caspases was carried
out on a SpectraMax Gemini plate reader using an excitation
wavelength of 400 nm and an emission wavelength of 505 nm. The
Ac-DEVD-AFC substrate (200 µM) and various concentrations
of p35 mutants were first added to the 100-µl reaction wells in a
buffer containing 50 mM Hepes at pH 7.4, 100 mM
NaCl, 10 mM dithiothreitol, 0.1% (w/v) CHAPS, and
10% sucrose and equilibrated at 37 °C for 20 min. The reactions were initiated by adding pre-warmed caspase-8 or caspase-3 to the
mixtures and the substrate hydrolyzes, expressed as relative fluorescence units, were monitored at 20-s intervals for 60-90 min.
The final concentrations of caspase-8 and caspase-3 were 0.1 and 1 nM, respectively. The concentration series of p35 used for
caspase-8 were 0.01, 0.05, 0.07, 0.1, and 0.3 µM, while
those for caspase-3 were 0.01, 0.03, 0.05, 0.07, and 0.1 µM. If no inhibition was observed under these conditions,
the analyses were repeated with p35 concentration series of 0.1, 0.3, 0.5, 0.7, and 1.0 µM for either caspase-3 or
caspase-8.
As the p35 concentrations were kept at a large excess over caspases,
the reactions were rendered pseudo-first order for convenience of
analysis (9). The apparent inhibitory constant in the presence of
substrate, Ki(app), is given by the
linear regression as shown in the following equation,
Biosensor Measurement--
All surface plasmon resonance
biosensor measurements were performed using a BIACORE 2000 equipped
with a research-grade B1 sensor chip. Mature active-site mutant of
caspase-3 (C163A) was immobilized on one flow cell of the sensor chip
using amine-coupling chemistry to a surface density of ~200 resonance
units. Another activated flow cell was blocked with a 7-min
injection of 1 M ethanolamine, pH 8.0. To collect kinetic
binding data, concentration series of each p35 protein, in 10 mM Hepes, 300 mM NaCl, 3 mM EDTA, 5 mM dithiothreitol, and 0.05% P20 at pH 7.4, were
injected for 30 s over the flow cells at 25 °C using a flow
rate of 50 µl/min. No regeneration of the ligand surface was required
between injections.
All response data were double-referenced (23), thereby correcting for
bulk refractive index changes and nonspecific p35 binding to the sample
and reference surfaces. Data were fit globally to a simple interaction
model (A + B = AB) using CLAMP (24). For weak interactions,
response data sets were fit simultaneously with the wild-type p35 data
to obtain more accurate affinity constants.
Caspase inhibition by p35 presumably proceeds through two distinct
steps: a pre-cleavage association, which is the reversible mutual
recognition between p35 and the caspase, followed by the cleavage, and
the formation of an irreversible post-cleavage complex (Fig.
1A). To obtain a thorough
understanding on the mechanism of caspase inhibition by p35, we mutated
a series of p35 residues in contact with caspase-8 in the post-cleavage
complex, including the p35 N terminus, the KL region and the RSL (Fig.
1B). These mutants were characterized to derive an integral
understanding of the inhibitory process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-galactopyranoside induction
at 20 °C. The His-tagged proteins were purified by nickel (nickel-nitrilotriacetic acid) affinity and gel-filtration
chromatography. Both caspase-8 and caspase-3 were autoprocessed to
their mature forms during the protein expression and purification
procedures. To generate the mature form of the mutant caspase-3
(C163A), the cell pellets of His-tagged caspase-3 (C163A) were mixed
with those of non-tagged caspase-8. The mixed cells were lysed by
sonication and incubated for 30 min at room temperature to allow the
processing of caspase-3 (C163A) by the active caspase-8 in the lysate.
-mercaptoethanol. Pre-equilibrated
nickel-nitrilotriacetic acid resins were incubated with the mixtures at
4 °C overnight and washed three times with the same buffer. The
bound proteins were eluted using 250 mM imidazole and
analyzed on SDS-PAGE.
where
(Eq. 1)
0 and
i are the steady state rates of substrate
hydrolysis in the absence and presence of p35 concentration [I],
respectively. The Ki can be converted from
Ki(app) by the following equation,
where [S] is the substrate concentration, and
Km is the kinetic parameter of caspase-3 or
caspase-8 for the Ac-DEVD-AFC substrate (9).
(Eq. 2)
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (47K):
[in a new window]
Fig. 1.
p35 structures. A, the
potential pathway of caspase inhibition by p35 through the formation of
pre-cleavage and post-cleavage complexes. Only half of the complex (a
full complex contains a heterotetrameric caspase with two p35
molecules) is shown. B, the three regions of p35 in contact
with caspase-8 in the p35-caspase-8 post-cleavage structure.
Magenta and orange, large and small subunits of
caspase-8; cyan, p35. Segments of p35 in contact with
caspase-8 are shown in red for the N-terminal residues,
blue for the RSL residues, magenta for the KL
region residues, and green for location of the deletion
mutant. C, free p35 structure showing the buried N terminus
and the cleavage sites for caspases and for the caspase-like protease
gingipain-K.
Kinetic Characterization of the Pre-cleavage Association--
To
trap the pre-cleavage interaction, we used an active site mutant of
caspase-3 (C163A) that was processed to its active conformation through
trans-activation by its upstream caspase, caspase-8. Measurement by
surface plasmon resonance for the interaction between p35 and caspase-3
(C163A) gave rise to an association rate of 6.7 × 105
M1 s
1, similar to many
diffusion-controlled rigid body macromolecular associations (25, 26),
and a dissociation rate of 0.091s
1 (Table
I). This fast rate of association
suggests that the interaction between p35 and a caspase does not
involve complex conformational changes, which would otherwise slow down
the interaction. The calculated equilibrium dissociation constant is
0.13 µM, which is ~200-fold stronger than the
Km between caspase-3 and a peptide substrate (9).
Because the direct contact between p35 and a caspase during
pre-cleavage interaction is likely not much more extensive than
substrate recognition by a caspase (since the KL loop does not appear
to be energetically indispensable, see below), the stronger interaction
between p35 and a caspase is likely due to the decrease in entropic
loss often associated with the recognition of flexible peptide
substrates. These kinetic and thermodynamic observations are consistent
with the apparent rigidity of the highly solvent accessible RSL in the
free p35 structure (21) (Fig. 1C).
|
Strict Requirement of the Cys2 Residue and Importance
of the Structural Integrity of the N-terminal Segment in Free
p35--
In the crystal structure of the p35/caspase-8 complex, the N
terminus of p35 repositions into the active site of caspase-8 to block
thioester bond hydrolysis (22). Two p35 N-terminal residues,
Cys2 and Val3, directly contact the caspase and
both the C2G and V3G mutations have been shown to render p35 defective
in caspase inhibition (22). To further understand the structural
requirement of these two contacting residues, we performed both alanine
mutations and conservative substitutions on these residues (Table I,
Fig. 2, A and
B).
|
Similar to the phenotype of the C2G mutation, both C2A and the isosteric C2S mutants failed to pull-down with either caspase-3 or caspase-8, although both were cleaved efficiently by the caspases. As these mutants exhibited identical solution behavior with wild-type p35, as shown by gel-filtration profiles, the mutational effects are likely due to the direct deletion of interactions with the caspases. The drastic phenotype of the C2S mutant suggests that the interaction between residue Cys2 of p35 and a target caspase exhibits a strict specificity. This specificity may be explained by the structural observation that the side chain thiol of Cys2 may form a hydrogen bond with the imidazole ring of His317 in caspase-8 (22). None of these mutations affected the pre-cleavage association.
Conservative substitution V3I did not significantly change its inhibitory activity against caspases. In contrast, the V3A mutant failed to form a stable complex with either caspase-3 or caspase-8, although it could be readily cleaved by these caspases. Since Val3 is buried in the free p35 structure, this mutant phenotype may be explained by either a direct effect on a p35-caspase complex or an indirect effect through perturbation of the free p35 structure. Although it is not possible to distinguish these effects, the fact that the V3A mutant tends to aggregate in solution supports that structural perturbation may be at play in this mutant.
To determine whether residues at the N-terminal segment that do not directly contact the caspase (residues 4-13) can influence the ability of p35 to inhibit caspases through structural perturbation, we selectively mutated a few residues at the N terminus. Most N-terminal residues such as Ile4, Phe5, and Pro6 are buried in the free p35 structure, with the exception of Asp10 and Gln13. Interestingly, the I4A, F5A, and P6A mutations in p35 were detrimental to the inhibition, while the D10A and Q13G mutants behaved similarly as the wild-type p35 in their ability to inhibit either caspase-3 or caspase-8. The I4A, F5A, and P6A mutants showed aggregation in their gel-filtration profiles and therefore may have possible local structural perturbations. Accordingly, biosensor measurements showed that the pre-cleavage association of these p35 mutants to caspase-3 (C163A) exhibited complex behavior, indicating the presence of a mixture of stoichiometries in the interactions. In contrast, both the D10A and Q13A mutants behaved as the wild-type p35 in solution. These results support that structural perturbation of the buried N-terminal arm in the free p35 structure can abrogate the inhibitory activity of p35.
The KL Region Makes Modest and Variable Energetic Contribution to
Caspase Interaction but Supports a Crucial Contact with the
RSL--
In the structure of the p35-caspase-8 complex, residues in
the KL region make direct contact with residues 414-427 in the small
subunit of caspase-8 at the L4 loop and the 4 helix. However, these
residues in caspase-8 only show limited sequence conservation among
different caspases. Since there are significant conformational changes
in the KL loop region between the bound and the free p35, we had
earlier proposed that this flexibility of the KL loop might be
important for its ability to interact with different caspases (22).
We created a series of point mutations and deletions to determine the
functional role of the KL region (Table I and Fig. 2, B and
C). We generated single-site mutations on residues in direct
contact with caspase-8, S253A, W254A, K256A, and Y260A. We also created
triple alanine mutations on S253, W254, and Y260, the three residues
that show most extensive surface area burial in the complex. In
addition, we created deletion mutations that remove one
(Trp254), five (
253-257) and seven (
252-258)
residues from the KL loop and the adjoining
strands.
Surprisingly, the mutational data suggest that the KL loop does not
seem to play an indispensable role as would have been predicted from
the structure. The W254A, Y260A, Trp254, and
253-257
mutants exhibited only modest, but significant, effects against
caspase-8 and behaved essentially as wild-type against caspase-3, an
effector caspase. In addition, the pre-cleavage interactions between
the p35 mutants and caspase-3 (C163A) are also very similar to the
wild-type interaction. These results show that the KL region is not
crucial for caspase inhibition and suggest that there may be
significant differences in the role of the KL region against different
caspases. Interestingly, a low resolution structure of the complex
between p35 and an insect effector caspase showed that there is an
orientational difference between the p35 in complex with an initiator
caspase, caspase-8, and with the effector caspase (27). Although the KL
region appears to contact the target caspase in both complexes, their
energetic roles may be different.
The relative unimportance of the KL region also explains the ability of p35 to inhibit gingipain-K, a caspase-like bacterial cysteine protease that cleaves a different site on the RSL of p35 (28) (Fig. 1C). Because gingipain-K cleaves further downstream (91DSIK94) from the caspase recognition site (84DQMD87), it is unlikely that gingipain-K would be close enough to the KL loop for a direct interaction.
Interestingly, the KL loop deletion mutant 252-258 completely
abolished the inhibitory activity of p35 against both caspase-3 and
caspase-8, without drastically affecting the formation of the
pre-cleavage complex. We have previously shown that residue Tyr82 near the beginning of the RSL is essential for the
ability of p35 to inhibit caspases (22). We had postulated that
Tyr82 helps to glue the RSL with the neighboring KL
strands, in addition to its role in direct caspase contact. Because
mutant
253-257 does not exhibit a drastic decrease in caspase
inhibition, and the two additional residues deleted in
252-258 do
not directly contact the caspase, the defective phenotype of the
252-258 mutant may be best explained by the perturbation of the
local conformation of the K and L strands. Among other interactions,
the K and L strands harbor two large aromatic residues
Phe248 and Trp262 that interact with
Tyr82 of the RSL. As the C
distance between residues 251 and 259 is 5.2 Å, longer than a typical C
distance between two
adjacent residues, the deletion mutant
252-258 has to undergo
conformational changes to join these two residues, leading to
structural perturbations. Therefore, a crucial role of the KL region
appears to provide a proper "glue" patch for the RSL.
Substrate Region Residues; Pre-cleavage Association Directly Affects Inhibition, but May Not Be Rate-limiting-- The non-covalent interaction between p35 and caspase-8 centers around the tetrapeptide caspase recognition sequence (P4-D84QMD87-P1) in the reactive site loop of p35. It has been shown previously that the P1 Asp residue is essential for p35 function (21, 29), consistent with the absolute specificity of caspases to cleave after Asp residues. To elucidate the specific role of these residues in the function of p35, we generated a series of point mutations at the P2-P4 positions of the caspase recognition sequence.
None of the mutations on the P2-P4 positions completely abolished the caspase inhibitory activity of p35, but produced a range of different effects against caspase-3 and caspase-8 (Table I and Fig. 2) that are largely consistent with the substrate specificity of these caspases (30). For example, mutations on the P2 residue are relatively mild against both caspases, consistent with P2 being the most tolerable. In contrast, P4 mutations are more drastic, especially for caspase-3, which is consistent with the known preference of Asp in this position for the group II apoptotic effector caspases such as caspase-3. The most surprising is the mutational phenotype on the P3 residue, which generated drastic effects on caspase-8, but did not seem to harm its inhibition on caspase-3, even though both caspases appear to prefer Glu at this position based on combinatorial substrate analyses (3, 30).
How does the strength of pre-cleavage association affect the post-cleavage caspase inhibition? It appears that an efficient pre-cleavage interaction is required for inhibition because most of the p35 mutants that have a decreased pre-cleavage interaction are also less effective in caspase inhibition. However, substrate recognition or pre-cleavage interaction seems not to be the rate-limiting step in caspase inhibition by wild-type p35, as exemplified by the M86V mutant, which exhibits significantly stronger pre-cleavage association, but is wild-type in inhibition.
Bowstring Kinetic Model of p35 Inhibition-- The molecular event following p35 cleavage suggests the existence of two opposing forces in caspase inhibition by p35. As the post-cleavage strand of p35 departs the active site of the caspase, a series of cooperative conformational changes occur in p35 that allows the release of its N terminus from the core of p35 for interacting with the caspase active site. This has to occur before the caspase is able to hydrolyze the thioester intermediate formed between the caspase and p35. The existence of such a race between the catalytic power of the caspase and the rate of post-cleavage conformational changes may be exemplified by the observed leakage in caspase inhibition by p35, which often requires higher than stoichiometric quantity of p35 to completely inhibit a target caspase (9). In addition, since stronger pre-cleavage association does not necessarily translate into stronger inhibition, the rate-limiting step of the inhibition could rest on the rate of these post-cleavage conformational changes.
So what controls the rate of conformational changes and therefore the efficiency of caspase inhibition? A most conspicuous feature of the free p35 structure is the conformational rigidity of the entirely solvent exposed RSL, as shown by the visibility of the loop in the electron density map (21). In addition, our measurements on the association rate and the strength of the pre-cleavage association also suggest a nearly rigid-body interaction between p35 and the caspase.
During the pre-cleavage association in which the caspase recognition
sequence in RSL interacts with the active site of the caspase, this
rigidity is likely to transform into tension in the RSL, because RSL
residues after the cleavage site would have to be stretched by 4 Å due
to the pinching of the caspase recognition sequence (Fig.
3A). The C distance between
the cleavage residue Asp87 and Lys97 is 26.7 Å in the free p35 structure. Assuming that residue Lys97 does
not move significantly in the pre-cleavage complex, this distance may
be increased to 30.6 Å, which would very likely create tension in this
already rigid RSL.
|
The rigidity and tension in the RSL suggest that p35 might function
analogously as a bowstring system for shooting arrows (Fig.
3B). In this model, the RSL may be considered as a string and the remainder of p35, especially the long base helix 1, the bow.
During the pre-cleavage association, the string is pulled by the
caspase and the tension generated may distribute to the entire
bowstring system, possibly causing a bent in the base helix. Upon
cleavage of the RSL, similarly as in shooting arrows upon release of
the string, this stored energy in the bowstring system will pull the
part of the RSL after the cleavage site toward the bow and facilitate
fast conformational changes.
Based on this bowstring model, for p35 to function efficiently,
mutations that compromise the integrity of the bowstring system of p35
will be detrimental. Consistent with this prediction, mutations that
weaken the association between the RSL and the p35 bow (Y82A and
252-258), mutations that weaken the integrity of the base helix
(V71P and I67K) (21, 31), and an insertion mutation that lengthens the
string (insertion of Ala-Ser after residue 83) (29), all abolish
caspase inhibition by p35. This tension model is further supported by
the structural similarity between the cleaved structure of an
inhibition-defective p35 mutant V71P and the cleaved structure of
wild-type p35 as in complex with caspase-8 (22, 32). This similarity
suggests that the V71P mutant does not have a gross structural defect
but exhibits a tension defect due to a weaker base helix.
Mechanistic Similarities to Serpins-- Besides p35, the only other known case of covalent inhibition or suicide inhibition by protease inhibitors is mediated by serpins, a family of inhibitors mostly for serine proteases (33-35). Serpins harbor protease recognition sequences and upon cleavage stay covalently bound to the target proteases via acyl-enzyme intermediates. This covalent inhibition is achieved through dramatic post-cleavage strand insertion in serpins, which result in deformation and denaturation of the protease active sites and therefore the trapping of the acyl-enzyme intermediates from being hydrolyzed by the proteases (35).
While the exact structural basis of covalent protease inhibition is
different in the two cases, there are several remarkable mechanistic
similarities. First, both serpins and p35 undergo dramatic
conformational changes, or refolding, upon cleavage. Second, they both
exhibit wide spectrum protease inhibition, because the most important
requirement for inhibition appears to be the existence of a protease
recognition site. Third, the efficiency of protease inhibition both
appears to be driven by the rate of post-cleavage conformational
changes. In keeping with this observation, leakage in inhibition has
been observed for both serpins and p35 (9, 33). Therefore, in either
case, the inhibitory characteristic of the inhibitor is determined by
the kinetic property of the system.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Carl Nathan for the use of the SpectraMax Gemini plate reader.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant AI50872 (to H. W.).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.
§ Present address: Immunology Program, Sloan-Kettering Division, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021.
Pew scholar of biomedical sciences and a Rita Allen Scholar.
To whom correspondence should be addressed: Dept. of
Biochemistry, Weill Medical College of Cornell University, Whitney-2,
1300 York Ave., New York, NY 10021. Tel.: 212-746-6451; Fax:
212-746-4843; E-mail: haowu@med.cornell.edu.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M211607200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RSL, reactive site loop; Ac-DEVD-AFC, acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Thompson, C. B. (1995) Science 267, 1456-1461[Medline] [Order article via Infotrieve] |
2. | Johnstone, R. W., Ruefli, A. A., and Lowe, S. W. (2002) Cell 108, 153-164[Medline] [Order article via Infotrieve] |
3. | Nicholson, D. W. (1999) Cell Death Differ. 6, 1028-1042[CrossRef][Medline] [Order article via Infotrieve] |
4. | Stennicke, H. R., Renatus, M., Meldal, M., and Salvesen, G. S. (2000) Biochem. J. 350, 563-568[CrossRef][Medline] [Order article via Infotrieve] |
5. | Ekert, P. G., Silke, J., and Vaux, D. L. (1999) Cell Death Differ. 6, 1081-1086[CrossRef][Medline] [Order article via Infotrieve] |
6. | Stennicke, H. R., Ryan, C. A., and Salvesen, G. S. (2002) Trends Biochem. Sci. 27, 94-101[CrossRef][Medline] [Order article via Infotrieve] |
7. | Bump, N. J., Hackett, M., Hugunin, M., Seshagiri, S., Brady, K., Chen, P., Ferenz, C., Franklin, S., Ghayur, T., Li, P., Mankovich, J., Shi, L. F., Greenburg, A. H., Miller, L. K., and Wong, W. W. (1995) Science 269, 1885-1888[Medline] [Order article via Infotrieve] |
8. | Xue, D., and Horvitz, H. R. (1995) Nature 377, 248-251[CrossRef][Medline] [Order article via Infotrieve] |
9. | Zhou, Q., Krebs, J. F., Snipas, S. J., Price, A., Alnemri, E. S., Tomaselli, K. J., and Salvesen, G. S. (1998) Biochemistry 37, 10757-10765[CrossRef][Medline] [Order article via Infotrieve] |
10. | Araki, T., Shibata, M., Takano, R., Hisahara, S., Imamura, S., Fukuuchi, Y., Saruta, T., Okano, H., and Miura, M. (2000) Cell Death Differ. 7, 485-492[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Beidler, D. R.,
Tewari, M.,
Friesen, P. D.,
Poirier, G.,
and Dixit, V. M.
(1995)
J. Biol. Chem.
270,
16526-16528 |
12. | Clem, R. J., Fechheimer, M., and Miller, L. K. (1991) Science 254, 1388-1390[Medline] [Order article via Infotrieve] |
13. |
Datta, R.,
Kojima, H.,
Banach, D.,
Bump, N. J.,
Talanian, R. V.,
Alnemri, E. S.,
Weichselbaum, R. R.,
Wong, W. W.,
and Kufe, D. W.
(1997)
J. Biol. Chem.
272,
1965-1969 |
14. |
Hay, B. A.,
Wolff, T.,
and Rubin, G. M.
(1994)
Development (Camb.)
120,
2121-2129 |
15. |
Hisahara, S.,
Araki, T.,
Sugiyama, F.,
Yagami, K.,
Suzuki, M.,
Abe, K.,
Yamamura, K.,
Miyazaki, J.,
Momoi, T.,
Saruta, T.,
Bernard, C. C.,
Okano, H.,
and Miura, M.
(2000)
EMBO J.
19,
341-348 |
16. | Martinou, I., Fernandez, P. A., Missotten, M., White, E., Allet, B., Sadoul, R., and Martinou, J. C. (1995) J. Cell Biol. 128, 201-208[Abstract] |
17. | Morishima, N., Okano, K., Shibata, T., and Maeda, S. (1998) FEBS Lett. 427, 144-148[CrossRef][Medline] [Order article via Infotrieve] |
18. | Rabizadeh, S., LaCount, D. J., Friesen, P. D., and Bredesen, D. E. (1993) J. Neurochem. 61, 2318-2321[Medline] [Order article via Infotrieve] |
19. | Robertson, N. M., Zangrilli, J., Fernandes-Alnemri, T., Friesen, P. D., Litwack, G., and Alnemri, E. S. (1997) Cancer Res. 57, 43-47[Abstract] |
20. | Sugimoto, A., Friesen, P. D., and Rothman, J. H. (1994) EMBO J. 13, 2023-2028[Abstract] |
21. |
Fisher, A. J.,
Cruz, W.,
Zoog, S. J.,
Schneider, C. L.,
and Friesen, P. D.
(1999)
EMBO J.
18,
2031-2039 |
22. | Xu, G., Cirilli, M., Huang, Y., Rich, R. L., Myszka, D. G., and Wu, H. (2001) Nature 410, 494-497[CrossRef][Medline] [Order article via Infotrieve] |
23. | Myszka, D. G. (1999) J. Mol. Recognit. 12, 279-284[CrossRef][Medline] [Order article via Infotrieve] |
24. | Myszka, D. G., and Morton, T. A. (1998) Trends Biochem. Sci. 23, 149-150[CrossRef][Medline] [Order article via Infotrieve] |
25. | Park, Y. C., Ye, H., Hsia, C., Segal, D., Rich, R. L., Liou, H.-L., Myszka, D. G., and Wu, H. (2000) Cell 101, 777-787[Medline] [Order article via Infotrieve] |
26. | Cunningham, B. C., and Wells, J. A. (1993) J. Mol. Biol. 234, 554-563[CrossRef][Medline] [Order article via Infotrieve] |
27. | Eddins, M. J., Lemongello, D., Friesen, P. D., and Fisher, A. J. (2002) Acta Crystallogr. Sect. D Biol. Crystallogr. 58, 299-302[CrossRef][Medline] [Order article via Infotrieve] |
28. | Snipas, S. J., Stennicke, H. R., Riedl, S., Potempa, J., Travis, J., Barrett, A. J., and Salvesen, G. S. (2001) Biochem. J. 357, 575-580[CrossRef][Medline] [Order article via Infotrieve] |
29. | Bertin, J., Mendrysa, S. M., LaCount, D. J., Gaur, S., Krebs, J. F., Armstrong, R. C., Tomaselli, K. J., and Friesen, P. D. (1996) J. Virol. 70, 6251-6259[Abstract] |
30. |
Thornberry, N. A.,
Rano, T. A.,
Peterson, E. P.,
Rasper, D. M.,
Timkey, T.,
Garcia-Calvo, M.,
Houtzager, V. M.,
Nordstrom, P. A.,
Roy, S.,
Vaillancourt, J. P.,
Chapman, K. T.,
and Nicholson, D. W.
(1997)
J. Biol. Chem.
272,
17907-17911 |
31. |
Zoog, S. J.,
Bertin, J.,
and Friesen, P. D.
(1999)
J. Biol. Chem.
274,
25995-26002 |
32. |
dela Cruz, W. P.,
Friesen, P. D.,
and Fisher, A. J.
(2001)
J. Biol. Chem.
276,
32933-32939 |
33. | Wright, H. T., and Scarsdale, J. N. (1995) Proteins 22, 210-225[Medline] [Order article via Infotrieve] |
34. | Engh, R. A., Huber, R., Bode, W., and Schulze, A. J. (1995) Trends Biotechnol. 13, 503-510[CrossRef][Medline] [Order article via Infotrieve] |
35. | Huntington, J. A., Read, R. J., and Carrell, R. W. (2000) Nature 407, 923-926[CrossRef][Medline] [Order article via Infotrieve] |