From the Institute for Molecular Virology, and Department of Biochemistry, Graduate School and College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706
Received for publication, November 8, 2000, and in revised form, February 22, 2001
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ABSTRACT |
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Activation of caspases by proteolytic processing
is a critical step during apoptosis in metazoans. Here we use high
resolution time lapse microscopy to show a tight link between caspase
activation and the morphological events delineating apoptosis in
cultured SF21 cells from the moth Spodoptera frugiperda, a
model insect system. The principal effector caspase,
Sf-caspase-1, is proteolytically activated during SF21
apoptosis. To define the potential role of initiator caspases in
vivo, we tested the effect of cell-permeable peptide inhibitors
on pro-Sf-caspase-1 processing. Anti-caspase peptide
analogues prevented apoptosis induced by diverse signals, including UV
radiation and baculovirus infection. IETD-fmk potently inhibited the
initial processing of pro-Sf-caspase-1 at the junction (TETD-G) of the large and small subunit, a cleavage that is blocked by
inhibitor of apoptosis Op-IAP but not pancaspase inhibitor P35. Because
Sf-caspase-1 was inhibited poorly by IETD-CHO, our data
indicated that the protease responsible for the first step in
pro-Sf-caspase-1 activation is a distinct apical caspase.
Thus, Sf-caspase-1 activation is mediated by a novel,
P35-resistant caspase. These findings support the hypothesis that
apoptosis in insects, like that in mammals, involves a cascade of
caspase activations.
Apoptosis is a dynamic process by which unwanted or diseased cells
are disassembled in a rapid but systematic manner. Apoptotic cells
undergo a series of dramatic and characteristic alterations in
morphology that include chromatin condensation, DNA fragmentation, cytoskeletal reorganization, cell shrinkage, and membrane blebbing (1,
2). These irreversible changes in cellular architecture are initiated
directly or indirectly by the proteolytic activity of the caspases, a
highly conserved family of cysteinyl aspartate-specific proteases that
play a major role in programmed cell death (3-7). Not surprisingly,
proper regulation of caspase activity is critical to apoptotic execution.
The caspases are activated from a latent proform (procaspase) by
proteolytic excision of the large and small subunits that interact to
generate the active enzyme. Procaspase processing occurs through
proximity-induced autoactivation or by the activity of other proteases,
including caspases (3, 4). In mammals, apoptotic signaling initiates a
caspase cascade wherein activated initiator caspases proteolytically
activate downstream effector caspases (6-8). Initiator caspases
possess long prodomains that interact with diverse proteins that
regulate protease activation. In contrast, effector caspases have short
prodomains. Initiator and effector caspases often exhibit different
substrate specificities in vitro (5, 9).
In invertebrates, programmed cell death plays a critical role in
development, control of DNA damage, and defense of pathogens, including viruses (10-12). Caspases are required for apoptosis in insects, like that in mammals. However, the mechanisms by which caspases are activated and the hierarchy of apical and effector caspases are still unclear (10, 13, 14). On the basis of sequence
similarity and biochemical activity, seven caspases have been
identified in Drosophila melanogaster (Order Diptera) (15). Drosophila DRONC and DCP-2/DREDD possess long prodomains and
by analogy to mammalian caspases are candidates as initiator caspases (16, 17). DCP-1, drICE, and DECAY contain short prodomains and
therefore are likely effector caspases (18-20). Caspases have also
been identified and characterized from lepidopteran insects (moths and
butterflies). In particular, Sf-caspase-1 is the principal effector caspase of SF21 cells (21, 22), an established cell line from
the nocturnal moth Spodoptera frugiperda (Order
Lepidoptera). These invertebrate cells have been used extensively for
studies on apoptosis because of their sensitivity to diverse death
stimuli, including baculovirus infection, UV radiation, and
overexpression of proapoptotic genes (i.e. Drosophila
reaper, hid, and grim) and their response to
known apoptotic regulators such as P35 and IAP (23-29).
Pro-Sf-caspase-1, which contains a short prodomain, is
activated by sequential proteolytic cleavages that are initiated only upon apoptotic signaling. The first cleavage occurs between the large
and small subunit at the caspase-recognition site TETD To define the mechanism by which Sf-caspase-1 is activated
upon apoptotic signaling and thereby gain insight into regulation of
invertebrate effector caspases, we characterized the protease activity
responsible for pro-Sf-caspase-1 activation. By using time
lapse video microscopy of SF21 cells, we observed an exact correlation
between the morphological hallmarks of apoptosis and caspase
activation. We report here that peptide-based fluoromethyl ketone
inhibitors potently blocked SF21 apoptosis induced by multiple signals.
In particular, zVAD-fmk and IETD-fmk prevented the initial proteolytic
processing of pro-Sf-caspase-1 at TETD Cells and Transfections--
S. frugiperda IPLB-SF21
(31) cells and Trichoplusia ni TN368 cells (32) were
propagated in TC100 growth medium (Life Technologies, Inc.)
supplemented with 10% heat-inactivated fetal bovine serum (HyClone
Laboratories) and 2.6 mg of tryptose broth/ml. SF21 cells were
transfected as described previously (22). In brief, plasmid DNA in
TC100 was mixed with N-[1-(2,
3-dioleoyloxy)propyl]-N,N,N- trimethylammonium methyl sulfate liposomes for 30 min at ambient temperature. The transfection mixture was added to cell monolayers previously washed with TC100. After 4 h of gentle rocking, the transfection mixture was replaced with supplemented TC100. Transfection efficiencies ranged from 60 to 80% as judged by lacZ
expression in control plates.
UV Irradiation and Virus Infection--
SF21 cell monolayers
were UV-B irradiated for 10 min at room temperature by using a Blak
Lamp (UVP, Upland, Calif) as described previously (33). For infection,
cell monolayers were inoculated with extracellular budded virus at the
indicated multiplicity of infection. Yields of infectious virus were
measured by standard plaque assay using apoptosis-resistant TN368
cells. Wild-type L-1 AcMNPV (p35+,
iap Time Lapse Video Microscopy--
SF21 cells were plated onto
glass coverslips mounted within 35-mm culture dishes. After cell
attachment, growth medium was replaced, and the cells were UV
irradiated or inoculated with virus as described. After a 2-h recovery
period at 27 °C, mineral oil was added to prevent evaporation. Cells
were viewed on a Nikon (Tokyo, Japan) Diaphot microscope using a 100×
oil emersion objective lens. Video images were obtained at the
indicated intervals with a Photometrics Series 300 or Micromax digital
camera. Images were background-subtracted and contrast-enhanced.
QuickTime movies were produced using Adobe Premiere 5.1 using Cinepak
compressor. Scale and time of compression are indicated.
Quantitation of Apoptosis--
Levels of apoptosis induced in
SF21 cell monolayers were determined by counting both apoptotic and
viable, nonapoptotic cells using a Zeiss Axiovert 135TV phase contrast
microscope (magnification, 200×) equipped with a digital camera and IP
Lab Spectrum P software. Cells undergoing plasma membrane blebbing
and/or cell body fragmentation were scored as apoptosis; both hallmarks
were readily distinguished from viable cells (see Fig. 1). The
mean ± standard deviation was calculated at the indicated times
from the percentage of apoptotic cells of at least six evenly
distributed fields of view and included from 1500 to 6000 cells.
Treatment with Peptide Analogues--
Irreversible fluoromethyl
ketone peptide inhibitors z-(benzyloxycarbonyl)-DEVD-fmk,
z-IETD-fmk, z-VAD-fmk, and z-FA-fmk
(Calbiochem, San Diego) dissolved in Me2SO were mixed in
supplemented TC100 and added to SF21 monolayers at the indicated
concentrations. Cells were irradiated in the presence of peptide
analogues and maintained at 27 °C. During infection, peptide
analogues were added 1 h after inoculation. Me2SO
vehicle was used as control.
Immunoblot Analysis--
Whole cell lysates or purified proteins
were subjected to SDS-polyacrylamide gel electrophoresis and
transferred to membranes. To detect Sf-caspase-1,
immunoblots were incubated with a 1:2,000 dilution of Protein Purification--
Escherichia coli strain
BL21 (DE3) cells were induced with IPTG
(isopropyl- Caspase Assays--
Sf-caspase-1 activity was
measured in reactions (20 µl) containing 25 mM HEPES, pH
7.5, 1 mM EDTA, 0.1%
CHAPS,1 10% sucrose, 10 mM dithiothreitol, and 10 µM tetrapeptide
substrates Ac-IETD-amc or Ac-DEVD-amc (Biomol Research). Accumulation
of fluorescent product (amc) was monitored by using a Molecular
Dynamics Biolumin 960 Kinetic Fluorescence/Absorbance microplate reader (excitation, 360 nm; emission, 465 nm) at 30-s intervals for 30 min.
Rate of product formation was obtained from the linear portion of the
reaction curves within the first 10% of substrate depletion and
averaged for triplicate assays. For inhibition assays, increasing concentrations of tetrapeptide aldehydes Ac-IETD-CHO or Ac-DEVD-CHO (Biomol Research) were incubated with 200 fmol of purified
Sf-caspase-1. After 1 h at ambient temperature,
substrate Ac-DEVD-amc (10 µM) was added, and residual
caspase activity was measured as described above.
Image Processing--
Stained gels and immunoblots were scanned
at a resolution of 300 dots per inch by using a Hewlett Packard
ScanJetIIcx. The resulting files were printed from Adobe Photoshop 3.0 and Illustrator 7.0 by using a Tektronics Phaser 450 dye sublimation printer.
UV Radiation-induced Apoptosis of Cultured SF21 Cells: Time Lapse
Microscopy--
Although many cellular components involved in
apoptosis have been identified, little is known about the kinetics and
morphological events of cell dismemberment. Cultured cells provide a
unique view into the morphology of programmed cell death. In
particular, SF21 cells from the moth S. frugiperda provide a
useful model system for studies on both biochemical and morphological
aspects of apoptosis (21, 23, 25, 27, 30, 33, 37-40). SF21 cells are
especially attractive because of their sensitivity to diverse apoptotic
stimuli and their classical apoptotic response, which includes
degradation of chromosomal DNA into nucleosomal-sized fragments and
vigorous membrane blebbing. Here, we used time lapse microscopy to
document SF21 morphological events during apoptosis as a means to link
them with intracellular biochemical processes.
SF21 cells rapidly succumb to UV radiation-induced apoptosis, which
consumes >90% of a culture within 9 h (25, 33). Because of the
large size (15-20 µm) and well defined nucleus of these cells,
nuclear and cytoplasmic events during apoptosis were readily discerned
by time lapse video microscopy (Fig. 1;
see also Video 1 in supplementary material). The first sign of UV
radiation-induced alterations was chromatin condensation, which
included formation of multiple opaque or dense bodies (2-3 µm in
diameter) within the nucleus (Fig. 1). The appearance of these
spheroidal inclusions coincided with the early activation of cellular
caspases 2-3 h after UV irradiation (33), which is consistent with
caspase-mediated detachment of chromatin from the nuclear envelope and
retraction into nuclear bodies (41-43). During this early period,
other transformations were observed within the nucleus, including rapid
migration of small particulate bodies and formation of vesicular-like
structures. Immediately thereafter, apoptotic blebbing was uniformly
initiated over the cell surface, upon which abundant microvilli-like
structures were still observed. Membrane blebs first appeared as small
rounded protrusions but grew rapidly to produce long extensions that
ultimately detached from the main cell body to form apoptotic bodies.
These vesicles were translucent or opaque, suggesting that their
contents varied. Blebbing lasted for 30-45 min and consumed the cell,
leaving behind a dense corpse. By 9-12 h after irradiation, only free floating apoptotic bodies and cell corpses remained (see Fig. 4A, panel ii).
Baculovirus-induced Apoptosis of SF21 Cells: Time Lapse
Microscopy--
SF21 cells are also highly sensitive to
baculovirus-induced apoptosis. AcMNPV mutants that lack
functional apoptotic suppressors (p35 or iap)
cause widespread apoptosis that severely restricts virus yields in part
because of premature host cell death (23, 26, 35, 44). Time lapse
microscopy of SF21 cells inoculated with the p35 deletion
mutant v Suppression of Apoptosis in AcMNPV p35+-infected Cells:
Time Lapse Microscopy--
During infection, baculovirus P35 prevents
premature host cell death by blocking apoptosis. The role that P35
plays in prolonging cell survival and contributing to virus
productivity is dramatically illustrated by time lapse microscopy of
wild-type AcMNPV-infected SF21 cells (Fig.
3; see also Video 3 in supplementary
material). The first visible signs of infection occurred 9-12 h after
inoculation, at which time the nucleus enlarged and the cell surface
became ruffled. Extracellular budded virus, the first of two
morphologically and temporally distinct forms of infectious virus (46),
is shed in abundance at 9-20 h and probably accounts for the
vesicular, nonapoptotic protrusions at the cell surface. During this
period, host caspases are activated and subsequently inhibited by newly synthesized P35 (22, 26, 36). The second and largest virus particle,
occluded virus (OV), appeared on the inside edge of the hypertrophied
nuclei beginning 22 h after infection (Fig. 3C).
Composed of nucleocapsids embedded within a matrix of the protein
polyhedrin (46), these polyhedral particles expanded to occupy the
entire nucleus (Fig. 3D). The number, size (1-3 µm dia),
and shape of OV particles varied between cells. OV are not produced
unless apoptosis is blocked (23, 35). Finally, in a dynamic process
that resembled necrosis, OV-containing cells expanded and ruptured
(Fig. 3; see also Video 3 in supplementary material). Thus, as
illustrated, the virus' apparent strategy is to prolong cell survival
long enough for progeny maturation, whereupon lysis facilitates virus
dissemination.
Caspase-targeted Peptide Inhibitors Block SF21 Apoptosis--
The
morphological events delineating apoptosis in SF21 cells coincided with
the early activation of caspases (21, 22, 26, 33). Current evidence
suggests that multiple caspases participate in SF21 apoptosis. To
define the in vivo role of initiator and effector caspases
in this invertebrate system, we first tested the anti-apoptotic
activity of peptide inhibitors that target the caspases, including the
membrane permeable peptides DEVD-fmk and IETD-fmk. Both tetrapeptides
were potent inhibitors of apoptosis induced by either UV radiation or
baculovirus infection (Fig. 4).
Incubation of UV irradiated cells with either tetrapeptide prevented
all morphological signs of apoptosis, which was widespread in untreated
cultures (Fig. 4A). Both peptides also blocked apoptosis induced by AcMNPV p35 deletion mutant v
In addition to promoting cell survival, the caspase inhibitory peptides
restored baculovirus multiplication. Upon treatment with DEVD- and
IETD-fmk, v Peptide Inhibitors Block Pro-Sf-caspase-1 Processing in
Vivo--
To identify the apoptotic step(s) affected by the permeable
peptide inhibitors, we determined their effect on proteolytic activation of the SF21 effector caspase, Sf-caspase-1.
Pro-Sf-caspase-1 is proteolytically activated in two steps
starting with cleavage at the caspase-like recognition site
TETD195 Differential Inhibition of Sf-caspase-1 by IETD- and DEVD-CHO in
Vitro--
Caspases exhibit distinct selectivities for peptide
substrates (9). The differential effects of IETD- and DEVD-fmk on
in vivo processing of pro-Sf-caspase-1 and the
differences in P4 to P1 residues at each
processing site (Fig. 6) suggested the participation of multiple
caspases during activation. To assess the involvement of
Sf-caspase-1 itself in this processing, we tested the
in vitro sensitivity of purified Sf-caspase-1 to
peptide aldehydes. As determined in dose response assays (Fig.
7), DEVD-CHO was ~100 times more
effective than IETD-CHO for inhibiting
Sf-caspase-1His (IC50 = 1.8 and 180 nM, respectively). Human caspase-3, the mammalian counterpart to Sf-caspase-1 (21), also has a greater
sensitivity to DEVD-CHO when compared with IETD-CHO
(Ki = 0.23 and 195 nM, respectively)
(9). Consistent with these results, purified Sf-caspase-1
hydrolyzed DEVD-amc efficiently, whereas even high levels of protease
cleaved IETD-amc poorly (data not shown). Thus, Sf-caspase-1
prefers DXXD- over IETD-containing substrates. These data
suggested that IETD-fmk inhibition of in vivo TETD Role of an Apical Caspase in Apoptosis of Invertebrate SF21
Cells--
Current evidence supports a cascade pathway for caspase
activation during execution of apoptosis in the insect S. frugiperda (Fig. 8). The principal
effector protease Sf-caspase-1 is activated by consecutive
proteolytic steps, each mediated by a distinct enzyme. The first step
involves cleavage of the large-small subunit junction
(TETD195
Our data indicate that Sf-caspase-X is an apical caspase
that is distinguished by its resistance to pancaspase inhibitor P35. Sf-caspase-X activity was inhibited in vivo by
the peptide analogues zVAD-, IETD-, and less well by DEVD-fmk (Fig. 6).
These inhibitors prevented cleavage of pro-Sf-caspase-1 at
TETD Mechanisms of Caspase Activation in Insects--
Our results that
Sf-caspase-X is an apical caspase provide evidence that
insects use a cascade of caspase-mediated cleavages to initiate
apoptosis. In Drosophila, the pro-caspase forms of DCP-1,
drICE, and DECAY contain short prodomains and undergo proteolytic activation at a TETD Role of Apoptosis in Baculovirus Replication--
Upon infection,
diverse viruses induce apoptosis (51-53). In some cases, apoptosis
facilitates virus multiplication and spread, as suggested by studies on
influenza virus and Sindbis virus (53). Conversely, apoptosis can
function as a host defense strategy that reduces virus yields and
thereby prohibits virus spread. In particular, infection with
apoptosis-causing baculovirus mutants reduce virus yields as much as
10,000-fold in SF21 cells (35). Moreover, the infectivity of these
mutants in insect larvae is 25-1000-fold lower than that of wild-type
apoptosis-suppressing virus, suggesting that apoptosis impedes
baculovirus multiplication in the host (44).
Here, we demonstrated that caspase-specific peptide inhibitors blocked
virus-induced apoptosis and restored multiplication of p35
and iap null mutants to wild-type levels (Figs. 4 and 5). These findings indicated that the primary function of
baculovirus-encoded apoptotic inhibitors is to negate caspase
activation or activity during infection and thereby block apoptosis.
Moreover, there were no obvious negative effects of these caspase
inhibitors on virus replication and maturation. Thus, host caspases are
not required for any phase of baculovirus replication in cultured cells. Collectively, these data argue that apoptosis is solely an
anti-virus defense mechanism employed by the host. Thus, for certain
viruses, suppression of the host suicide response provides a
significant selective advantage. Understanding the molecular mechanisms
by which such viruses circumvent this host defense strategy will
continue to provide insight into the regulation of apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G, which is
also conserved in Drosophila DCP-1 and drICE. This initial cleavage event is blocked by baculovirus Op-IAP but is insensitive to
the pancaspase inhibitor P35 (22). Op-IAP functions upstream from P35
to block apoptosis in Spodoptera (25, 30). Thus, it
has been hypothesized that the first step in
pro-Sf-caspase-1 activation is mediated by an apical caspase
that is distinguished by its novel resistance to P35 (22).
G. Because Sf-caspase-1 itself was inhibited poorly by IETD-CHO, our
data indicated that the protease responsible for the first step in pro-Sf-caspase-1 activation is a distinct caspase,
designated Sf-caspase-X. On the basis of these data, we
concluded that the P35-insensitive activity of Sf-caspase-X
is responsible for caspase activation in Spodoptera SF21
cells and that insects, like mammals, use a cascade of caspase-mediated
events to execute apoptosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) (34) and AcMNPV recombinants
wt/lacZ (p35+, iap
)
(35), v
p35 (p35
,
iap
) and v
p35/lacZ (p35
,
iap
) (36), and vOp-IAP (p35
,
iap+) (25) were described previously.
-Sfcasp1 (22)
and goat anti-rabbit immunoglobulin G (Pierce) conjugated to alkaline
phosphate. Color development was as described previously (36).
-D-thiogalactopyranoside) for overexpression
from Sf-caspase-1-encoding pET plasmids (22). C-terminal
His6-tagged proteins were purified by nickel
(Ni+2) affinity chromatography as described previously (22,
24). Isolated proteins were >90% homogenous as determined by
SDS-polyacrylamide gel electrophoresis and Colloidal Burst Coomassie G
Stain (Z axis). Protein concentrations were determined by
using the Bio-Rad Protein Assay (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Time lapse microscopy of UV radiation-induced
apoptosis. Monolayer SF21 cells were photographed every
30 s, beginning 2 h after UV irradiation by using a Nikon
Diaphot microscope equipped with a digital camera. Time of compression,
450×. Arrows depict condensed chromatin (white)
and apoptotic vesicles (black). Bar, 10 µm. See
also Video 1 in supplementary material .
p35 (Fig. 2; see also Video 2 in supplementary material) revealed that the morphological hallmarks of
virus-induced apoptosis were similar to those induced by UV radiation.
However, v
p35-induced apoptosis occurred later and was less
synchronous. The first signs of apoptosis included formation of dense,
refractile material within and around the inside edge of hypertrophied
nuclei (Fig. 2, B and C). The appearance of this intranuclear density coincided with caspase activation, which begins 9 and 12 h after infection (22, 26). Because nuclear aggregates were
absent in cells infected with wild-type virus that encodes caspase
inhibitor P35 (see below), it is likely that this material is condensed
chromatin resulting from caspase activity. Soon after these nuclear
events, apoptotic blebbing was initiated. Blebbing initiated 9-19 h
after infection (Fig. 2; see also Video 2 in supplementary material).
This asynchrony may be due to variations in the timing of virus-induced
apoptotic signaling. Blebbing was invariably followed by an unusual and
striking series of fusions in which apoptotic bodies of an individual
cell form a single spherical mass of vesicles. Although the mechanism
for this resurrection-like process is unknown, it may involve
virus-encoded surface proteins that mediate membrane fusion (45).
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Fig. 2.
Time lapse microscopy of
p35 baculovirus-induced apoptosis.
SF21 cells were inoculated with p35 deletion mutant v
p35
and photographed every 5 min, beginning 2 h after infection. Time
of compression, 3,000×. Bar, 10 µm. See also Video 2 in
supplementary material
.
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Fig. 3.
Time lapse microscopy of
p35+ baculovirus (AcMNPV)
infection. SF21 cells were inoculated with wild-type
p35+ AcMNPV and photographed every 5 min, beginning 2 h after infection. Time of compression, 3,000×.
Arrows depict early membrane ruffling and occluded virus
particles, respectively. Bar, 10 µm. See also Video 3 in
supplementary material .
p35
(Fig. 4B). At the highest extracellular dose tested (200 µM), DEVD- and IETD-fmk reduced apoptosis to less than
5%. At lower concentrations (30-50 µM), IETD-fmk was
two to three times more effective than DEVD-fmk. In contrast, the
control fluoromethyl ketone FA-fmk failed to affect apoptosis induced
by either death stimulus (data not shown). The intracellular
concentration of each peptide inhibitor was not determined.
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Fig. 4.
Prevention of UV- and virus-induced apoptosis
by peptide inhibitors. A, UV irradiation. SF21 cells
were UV irradiated in the absence (panel ii) or presence
(panels iii and iv) of 200 µM
DEVD-fmk or IETD-fmk and photographed 10 h later by using a
phase-contrast microscope (20× objective). Untreated cells
(panel i) were included. B, baculovirus
infection. SF21 cells were inoculated with p35 deletion
mutant v p35/lacZ in the presence of increasing concentrations of
DEVD- or IETD-fmk. The fraction of apoptotic cells was scored 24 h
later by computer-aided microscopy, counting from 1500 to 6000 cells in
at least six evenly distributed fields of view. Average numbers of
apoptotic cells ± standard deviation from triplicate assays are
reported.
p35-infected SF21 cells accumulated OV particles at
levels comparable with that of cells infected with wild-type virus that
encodes caspase-inhibitor P35 (Fig. 5A). In the absence of
tetrapeptide, OV was not produced. To compare the effects on infectious
virus production, we monitored yields of budded virus from
peptide-treated cells. DEVD-, IETD-, and zVAD-fmk increased virus
yields 1,000-fold to levels that were comparable with
p35+ viruses (Fig. 5B). Thus, the
peptide inhibitors did not interfere with virus replication. The
restoration of virus productivity ruled out the possibility that these
inhibitors blocked virus-induced apoptosis by preventing virus
replicative events that are required for apoptotic signaling (26).
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Fig. 5.
Restoration of virus replication by peptide
inhibitors. A, occluded virus. SF21 cells inoculated
with v p35 in the absence (panel ii) or presence of
DEVD-fmk (panel iii) or IETD-fmk (panel iv) were
photographed 48 h after infection as described in legend to Fig.
4. Cells inoculated with wild-type (wt)
p35+ AcMNPV in the absence of
inhibitors (panel i) were included. Arrows depict
OV particles. B, extracellular budded virus. Growth medium
was collected 48 h after infection with viruses v
p35/lacZ or
p35+ wt/lacZ (5 plaque-forming units/cell) in
the absence (control) or presence of 200 µM peptide
inhibitors (DEVD, IETD, and VAD). The yield of budded virus was
determined by plaque assay using TN368 cells and is reported as
plaque-forming units/ml ± standard deviation from triplicate
infections.
G (Fig.
6A). The resulting p25
fragment is then cleaved at DEGD28
A to remove the
prodomain and generate the mature enzyme complete with large (p19) and
small (p12) subunits (21, 22). Caspase processing was monitored by
immunoblot analyses using
-Sfcasp1 antiserum, which recognizes the
proform, p25, and p19 subunit of Sf-caspase-1. Upon
infection with p35 deletion mutant v
p35, p19 was the
predominant product (Fig. 6B, lane 2). DEVD-fmk
decreased accumulation of p19 (lane 3). However, at
equivalent concentrations, IETD-fmk and zVAD-fmk eliminated p19 and
reduced p25 to background levels (compare lanes 4 and
5 with lane 1). As shown previously (22), P35
fails to block cleavage of pro-Sf-caspase-1 at TETD
G but
inhibits subsequent DXXD cleavages. Thus, p25 is the major processing intermediate upon infection with wild-type
p35+ virus (lane 6). In the presence
of DEVD-fmk, p25 and p19 accumulation was reduced (lane 7).
However, in p35+ virus-infected cells, IETD- and
zVAD-fmk prevented the appearance of these intermediates (lanes
8 and 9). The effectiveness of IETD- and zVAD-fmk was
comparable with that of Op-iap (lane 10), which fully blocks the initial cleavage of pro-Sf-caspase-1 (22,
30). Thus, IETD- and zVAD-fmk inhibited the protease responsible for the first TETD
G activation cleavage of Sf-caspase-1 and
indicated that this upstream activity is mediated by a caspase.
Peptides IETD- and zVAD-fmk were more effective inhibitors of this
caspase activity than DEVD-fmk, which blocked the downstream
DXXD cleavages of pro-Sf-caspase-1.
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Fig. 6.
Effect of tetrapeptide inhibitors on
Sf-caspase-1 processing. A,
pro-Sf-caspase-1. The large (p19) and small (p12) subunits
are separated by an 11-residue linker and preceded by a 28-residue
prodomain. Proteolytic processing occurs first at
TETD195 G to generate p25, which is subsequently cleaved
at DEGD28
A to generate p19. B, immunoblot
analysis. SF21 cells were mock infected (lane 1) or infected
with either p35 deletion mutant v
p35 or wild-type
p35+ AcMNPV in the absence
(lanes 2 and 6) or presence of the indicated
peptide-inhibitors (lanes 3-5 and 7-9). Total
cell lysates (2.5 × 105 cell equivalents) prepared
24 h after infection were subjected to immunoblot analysis by
using
-Sfcasp1 serum specific to the p19 large subunit. Lysate from
cells infected with Op-iap expressing virus vOp-IAP
(lane 10) was included. vp39 is a virus-specific late
protein that is recognized by
-Sfcasp1. Molecular mass markers are
indicated on the left.
G
processing of pro-Sf-caspase-1 is principally due to
inhibition of an apical caspase and not Sf-caspase-1 itself.
This conclusion is consistent with the previous finding that in
vivo processing of pro-Sf-caspase-1 at TETD
G is
insensitive to caspase inhibitor P35, which potently inhibits active
Sf-caspase-1 (21, 22).
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Fig. 7.
Inhibition profiles of purified
Sf-caspase-1.
Sf-caspase-1His (0.2pmol) was mixed with
increasing concentrations of the reversible inhibitors DEVD- or
IETD-CHO. After 1 h, residual caspase activity was determined
using DEVD-amc as substrate. Values are the averages ± standard
deviation of triplicate assays and are expressed as a percentage of
total protease activity. Relative rates of product (amc)
formation were determined from the linear portion of the reaction
curves within the first 10% of substrate depletion.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G) by the apical caspase designated
Sf-caspase-X. The second step removes the
Sf-caspase-1 prodomain by a DXXD
G cleavage to generate the mature enzyme. This maturation step is blocked in vivo by caspase inhibitor P35, whereas the initial cleavage step by Sf-caspase-X is not (22). Despite its resistance to P35
inhibition, Sf-caspase-X activity is fully inhibited
in vivo by cell-permeable peptide analogues containing a
P1-aspartate residue. Furthermore, Sf-caspase-X-mediated activation of Sf-caspase-1
is blocked directly or indirectly by baculovirus Op-IAP and probably
its cellular homolog Sf-IAP (22, 47) (Fig. 8). The molecular
mechanism by which Sf-caspase-X is activated by diverse
apoptotic signals, including UV radiation and virus infection, remains
to be determined.
View larger version (23K):
[in a new window]
Fig. 8.
Model for activation of effector
Sf-caspase-1 by Sf-caspase-X.
Upon apoptotic signaling, activated Sf-caspase-X processes
pro-Sf-caspase-1 at the initial cleavage site
TETD195 G. Whereas P35 fails to affect
Sf-caspase-X cleavage of Sf-caspase-1, Op-IAP
functions upstream to block Sf-caspase-X activity or
activation. P35 blocks maturation cleavages (DXXD
(G/A)) of the
Sf-caspase-1 intermediate p25, most likely by direct
inhibition of active Sf-caspase-1. Peptides IETD-fmk and
VAD-fmk preferentially inhibit pro-Sf-caspase-1 cleavage at
TETD
G, whereas DEVD-fmk inhibits multiple cleavages and blocks
Sf-caspase-1 amplification.
G, an event required for commitment to apoptosis in SF21 cells.
Cytosolic extracts of SF21 cells also contain a P35-insensitive
Sf-caspase-X-like activity, which is inhibited by
iodoacetate, IETD-CHO, and DEVD-CHO, but not FA-fmk or
E64.2 Although resistant to
P35 (22), Sf-caspase-X activity is blocked in
vivo by baculovirus P49, a caspase-specific inhibitor that functions upstream from P35 and thus inhibits an apical
caspase.3 Consistent with the
involvement of an apical caspase in SF21 apoptosis, ectopic expression
of a cDNA encoding human apical caspase-8 induced widespread
apoptosis, whereas overexpression of effector caspases human caspase-3
and Sf-caspase-1 did not (data not shown and Refs. 22 and
30). Formal classification of Sf-caspase-X will require its
purification and sequence determination.
G site located at the large-small subunit junction analogous to that of pro-Sf-caspase-1 (22, 48).
Thus, the function of Sf-caspase-X as an apical caspase is
most likely conserved in Drosophila. Indeed, recent
identification of large prodomain-containing caspases, DREDD and DRONC,
suggests that apical caspases exist in Drosophila (13, 16).
DREDD and DRONC prodomains contain potential DED and CARD motifs that
are found in human caspases-8 and -9, respectively, and participate in
regulation of caspase activation (6). Interestingly, purified DRONC
failed to cleave P35, nor was it affected by this caspase inhibitor
(48, 49), suggesting that DRONC resembles P35-insensitive
Sf-caspase-X. Additionally, p35 failed to block
apoptosis induced by overexpression of dronc in transgenic
flies (48, 49). These findings were unexpected because downstream
effector caspases activated by a potential initiator caspase like DRONC
should have been inhibited by pancaspase inhibitor P35, thereby
preventing apoptosis. Subsequent studies have suggested that
ectopic expression of dronc in Drosophila eyes
can be blocked by p35 (50). Thus, additional experimentation is required to address the apical role of DRONC and DREDD in
Drosophila.
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ACKNOWLEDGEMENTS |
---|
We thank Alexander Verkhovsky, Thomas Keating, Vladimir Rodionov, and Gary Borisy (Laboratory of Molecular Biology, University of Wisconsin-Madison) for the use of time lapse microscopy instrumentation, assistance, and advice during this study. We thank Brooke Milde and Judit Jané-Valbuena for technical assistance and Brock Binkowski for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by Public Health Service Grants AI25557 and AI40482 from the NIAID, National Institutes of Health (to P. D. F.) and a Steenbock/Wharton Predoctoral Fellowship from the Department of Biochemistry (to G. A. M).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 on-line version of this article (available at
http://www.jbc.org) contains three QuickTime videos as
supplemental material.
Current address: Millennium Pharmaceuticals, Cambridge, MA 02139
§ To whom correspondence should be addressed: Inst. for Molecular Virology, R. M. Bock Laboratories, University of Wisconsin, 1525 Linden Dr., Madison, WI 53706-1596. Tel.: 608-262-7774; Fax: 608-262-7414; E-mail: pfriesen@facstaff.wisc.edu.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M010179200
2 B. Binkowski and P. Friesen, unpublished results.
3 S. Zoog, J. Schiller, J. Wetter, N. Chejanovsky, and P. Friesen, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-propane sulfonate; OV, occluded virus amc, 7-amino- 4-methylcoumarin.
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