From the Research Center for Experimental Biology,
the § Department of Bioengineering, and ¶ Frontier
Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
Received for publication, April 29, 2002, and in revised form, November 26, 2002
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ABSTRACT |
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Upon engagement with Fas ligand (FasL), Fas
rapidly induces recruitment and self-processing of caspase-8 via the
adaptor protein Fas-associated death domain (FADD), and
activated caspase-8 cleaves downstream substrates such as caspase-3. We
have found that penicillic acid (PCA) inhibits FasL-induced apoptosis
and concomitant loss of cell viability in Burkitt's lymphoma Raji
cells. PCA prevented activation of caspase-8 and caspase-3 upon
treatment with FasL. However, PCA did not affect active caspase-3 in
FasL-treated cells, suggesting that PCA primarily blocks early
signaling events upstream of caspase-8 activation. FasL-induced
processing of caspase-8 was severely impaired in the death-inducing
signaling complex, although FasL-induced recruitment of FADD and
caspase-8 occurred normally in PCA-treated cells. Although PCA
inhibited the enzymatic activities of active recombinant caspase-3,
caspase-8, and caspase-9 at similar concentrations, PCA exerted weak
inhibitory effects on activation of caspase-9 and caspase-3 in
staurosporine-treated cells but strongly inhibited caspase-8 activation
in FasL-treated cells. Glutathione and cysteine neutralized an
inhibitory effect of PCA on caspase-8, and PCA bound directly to the
active center cysteine in the large subunit of caspase-8. Thus, our
present results demonstrate that PCA inhibits FasL-induced apoptosis by targeting self-processing of caspase-8.
The death receptor Fas is widely expressed in diverse cell types
and plays an essential role in the killing of harmful cells by
cytotoxic T lymphocytes, as well as regulation of the immune system
such as lymphocyte homeostasis (1-4). Defective apoptotic signals
in the Fas/FasL system result in accumulation of abnormal T cells in
the peripheral lymphoid organs and develop autoimmune diseases in human
and mice (1-4). In addition to death-inducing function, the Fas/FasL
system is also involved in T cell proliferation, as evidenced by
reports from us and other groups (5-7) showing that Fas provides
costimulatory signals for human T cells, and caspase inhibitors block
CD3-induced proliferation and interleukin-2 production by human T
cells. Accelerated apoptotic signals via the Fas/FasL system are
involved in the pathogenesis of fulminant hepatitis and autoimmune
diseases such as experimental autoimmune encephalomyelitis, because
virus-infected hepatocytes and oligodendrocytes are killed by cytotoxic
T lymphocytes in a FasL-dependent fashion (8-10).
Therefore, suppression of Fas-mediated signals might have therapeutic
potential in the treatment of autoimmune diseases and fulminant
hepatitis by blocking T cell-mediated killing and T cell proliferation.
Fas contains a cytoplasmic sequence known as the death domain that is
utilized to interact with the adaptor protein Fas-associated death domain (FADD),1 which
contains both a death domain and a death effector domain (1-4).
Membrane-bound Fas ligand (FasL) induces Fas oligomerization that
allows FADD recruitment to the death domain (1-4). Through the
interaction of their mutual death effector domains, FADD binds to
caspase-8, which contains two death effector domains and a caspase
domain, allowing the formation of death-inducing signaling complex
(DISC) (11, 12). In the DISC, caspase-8 zymogens are placed in
proximity, which facilitates self-processing to generate the active
form (13). Activated caspase-8 cleaves downstream substrates such as
caspase-3, essential for apoptosis execution (14, 15).
The early signaling pathway of Fas-mediated apoptosis is a complex
process modulated at various steps by cellular and viral inhibitors
(16, 17). Functional and structural analyses of these inhibitors have
contributed to clarify the molecular basis of Fas-mediated apoptosis.
Moreover, membrane-permeable small molecules that modulate Fas-mediated
apoptosis seem to be valuable reagents to clarify the molecular basis
of initial signaling events (18, 19), as well as to lead compounds for
pharmaceutical intervention. To search for specific inhibitors that
block Fas-mediated apoptosis, we have screened natural products such as
microbial metabolites, and we have identified penicillic acid (PCA)
(Fig. 1) as an inhibitor of Fas-induced cell death. In the present
paper, we show that PCA targets self-processing of caspase-8.
Cells--
Murine B lymphoma A20 cells and human Burkitt's
lymphoma Raji cells were maintained in RPMI 1640 medium (Invitrogen)
supplemented with 10% (v/v) heat-inactivated fetal calf serum (JRH
Bioscience, Lenexa, KS), and penicillin/streptomycin/neomycin
antibiotic mixture (Invitrogen) in the presence or the absence of 50 µM 2-mercaptoethanol, respectively.
Reagents--
PCA and staurosporine were purchased from Sigma
and Wako Pure Chemical Industries Ltd. (Osaka, Japan), respectively.
Z-VAD-fluoromethyl ketone (Z-VAD-fmk) was obtained from the Peptide
Institute Inc. (Osaka, Japan).
Assays for Apoptosis and Cell Viability--
A20 cells were
labeled with 37 kBq of [3H]thymidine (TdR) (ICN
Biomedicals, Costa Mesa, CA) for 16 h and washed three times with
RPMI 1640 medium. A20 cells (5 × 104 cells, 100 µl)
were cultured with hamster anti-mouse Fas antibody Jo2 (Pharmingen) for
4 h in 96-well microtiter plates and then lysed by pipetting in
the presence of 1% Triton X-100. The plates were centrifuged (600 × g, 5 min), and supernatants were collected. DNA
fragmentation (%) was calculated using the following formula: (experimental release Screening System--
Five thousand extracts of microbial
culture broths and a hundred extracts of herbal medicines were screened
to look for specific inhibitors that block DNA fragmentation induced by
anti-Fas antibody Jo2 as monitored by [3H]TdR-labeled A20
cells. The first screening led to an identification of 10 positive
candidates. One sample did not suppress reduced cell viability of
Jo2-treated A20 cells as analyzed by MTT assay. Two samples were
excluded because of lack of reproducibility when prepared from new
microbial culture broths. The bleb-forming assay using the human
chronic myeloid leukemia K562 cells was utilized to detect activators
of protein kinase C (21), because activators of protein kinase C block
apoptosis induced by anti-Fas antibodies (22, 23). Six samples induced
a significant blebbing on K562 cells. Therefore, one crude extract
derived from an unidentified fungus was further subjected to HPLC and
UV spectrometric analysis. The active component was recovered as a
single peak and identified as PCA. The anti-apoptotic activity of PCA
was confirmed by the standard compound obtained from the commercial supplier.
Clonogenic Assay--
Raji cells (106 cells/ml) were
pretreated with various concentrations of PCA for the indicated times
and washed with the RPMI 1640 medium to remove PCA. The cells were then
treated with or without cross-linked FasL (500 ng/ml) for 2 h and
washed with the medium to remove FasL. The cells were diluted with the
medium (10 cells/ml) and cultured in 96-well microtiter plates (1 cell/well, 100 µl) for 10 days. The number of colonies formed was counted.
Western Blotting--
Cells were washed with PBS and lysed in
the lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1%
Triton X-100, 1 mM DTT, 0.5 mM EDTA, and
protease inhibitor mixture (Complete; Roche Diagnostics). After
centrifugation (10,000 × g, 5 min), supernatants were
collected. Postnuclear lysates (30 µg/lane) were separated by
SDS-PAGE and analyzed by Western blotting using ECL detection reagents
(Amersham Biosciences). Antibodies for caspase-3 (Santa Cruz
Biotechnology), caspase-8 (Medical and Biological Laboratories Co.,
Ltd. (MBL), Nagoya, Japan), caspase-9 (MBL), and FADD (BD Biosciences)
were used.
Assay for Caspase Activity--
Ac-DEVD-4-methylcoumaryl-7-amide
(MCA) for the caspase-3 substrate, Ac-IETD-MCA for the caspase-8
substrate, and Ac-LEHD-MCA for the caspase-9 substrate were obtained
from the Peptide Institute Inc. Postnuclear lysates (40 µg) were
mixed with the caspase substrates in the reaction buffer (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.1% CHAPS,
10% sucrose, 1 mM DTT) for 1 h. To block
caspase-8-independent cleavage of Ac-IETD-MCA, the proteasome inhibitor
MG-132 (Peptide Institute Inc.) was included in the reaction mixture at
the final concentration of 2.5 µM (24). Active
recombinant human caspase-3 (MBL; 2 units/ml), active recombinant human
caspase-8 (MBL; 2 units/ml), and active recombinant human caspase-9
(MBL; 2 units/ml) were mixed with Ac-DEVD-MCA, Ac-IETD-MCA, and
Ac-LEHD-MCA in the reaction buffer (20 mM PIPES (pH 7.5),
100 mM NaCl, 0.1% CHAPS, 10% sucrose, 1 mM
EDTA) for 1 h, respectively. The release of 7-amino-4-methylcoumarin (AMC) was measured with an excitation at
360 nm and an emission at 460 nm by CytoFluor multiwell plate reader
series 4000 (Applied Biosystems, Foster City, CA).
FACS Analysis--
Raji cells were treated with or without mouse
anti-human Fas antibody DX2 (Calbiochem) for 1 h on ice. The cells
were repeatedly washed and then stained with fluorescein
isothiocyanate-conjugated rabbit anti-mouse IgG (H + L) (Jackson
ImmunoResearch, West Grove, PA) for 45 min on ice. After washing, the
stained cells were analyzed under flow cytometry (FACSCalibur; BD Biosciences).
DISC Analysis--
Raji cells were treated with 2 µg/ml FasL
in the presence of 2 µg/ml anti-FLAG antibody M2 (Sigma) for 15 min
and quickly cooled down by adding 5 volumes of ice-cold PBS. Cells were
washed twice with PBS and lysed with 0.2% Nonidet P-40, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM sodium vanadate, 10% glycerol, and the protease
inhibitor mixture. Postnuclear lysates were precleared with Sepharose
6B (Sigma) for 90 min and then incubated with protein A-Sepharose CL-4B
(Amersham Biosciences) for 3 h. Sepharose beads were washed four
times with the lysis buffer. Proteins were separated by SDS-PAGE and
analyzed by Western blotting using anti-FADD and anti-caspase-8 antibodies.
Construction and Purification of Caspase-8 Large Subunit
(p20)--
DNA fragment corresponding to caspase-8 p20 was amplified
by PCR from mouse full-length caspase-8 with 5'-forward primer
containing BamHI site (5'-AAAGGATCCGAAGTGAGTCACGGACTTC-3')
and 3'-reverse primer containing EcoRI site
(5'-GATGAATTCCTAATCCACTTCTAAAGTG-3') and cloned into pET-32c(+)
(Novagen Inc., Madison, WI) containing N-terminal His tag. Cell lysates
were prepared from Escherichia coli AD494 (DE3) strain
harboring caspase-8 p20/pET32c(+) after induction with
isopropyl-thio- Mass Spectrometry--
Caspase-8 p20 (30 µg) was treated with
100 µM PCA in Tris-HCl (pH 8.5), 100 mM NaCl,
5% sucrose for 3 h. Caspase-8 p20 was precipitated by acetone and
then digested with 2 µg of lysyl endoprotease (Wako Pure Chemical
Industries, Ltd., Osaka, Japan) in 25 mM Tris-HCl (pH 9.0),
1 mM DTT in the presence of 2 M urea at
37 °C for 14 h. The resulting peptide mixture was subjected to
liquid chromatography and tandem mass spectrometry (MS/MS). The
digested peptides were separated by a reversed-phase column (Symmetry
C18 5 µm, 0.32 × 150 mm, Waters Associates, Milford, MA) using
a capillary HPLC system (CapLC system, Waters Associates). The column
was eluted with a linear gradient of 5-50% acetonitrile in 0.1%
formic acid at a flow rate of 5 µl/min. The isolated peptides were
identified by mass measurements with quadrupole time-of-flight
mass spectrometer (Micromass Ltd., Altrincham, UK) equipped with an
electrospray interface that was connected directly to the above HPLC system.
PCA Blocks Apoptosis Induced by Agonistic Anti-Fas Antibody or
Cross-linked FasL--
Fas-mediated apoptosis is induced by agonistic
Fas antibodies or physiological FasL. Murine B lymphoma A20 cells are
highly sensitive to anti-Fas antibody Jo2 (19). To search for specific inhibitors that block Fas-mediated apoptosis, we screened microbial secondary metabolites and herbal medicines, and we identified PCA as an
active component in the culture broth of an unidentified fungus. The
rationale of the screening system was described under "Experimental
Procedures." A20 cells underwent DNA fragmentation after treatment
with Jo2 in a dose-dependent manner for 16 h (Fig. 1B). Four hours of incubation
with 200 ng/ml Jo2 was sufficient to induce nearly maximal DNA
fragmentation (data not shown). PCA inhibited DNA fragmentation induced
by Jo2 in a dose-dependent manner, whereas PCA exhibited
apoptotic effects marginally (Fig. 1C). PCA markedly
reversed a reduction in cell viability of A20 cells treated with Jo2
(Fig. 1D). In addition to A20 cells, human Burkitt's
lymphoma Raji cells were killed by cross-linked FasL in a
dose-dependent manner, and most of the cells underwent
apoptosis by 500 ng/ml FasL within 4 h (Fig.
1E). PCA also inhibited FasL-induced apoptosis in Raji
cells at 100-200 µM (Fig. 1F). Although PCA alone slightly affected cell viability, PCA at 100-200
µM effectively blocked FasL-induced loss of cell
viability (Fig. 1G). To determine the limits at which PCA
does not affect long term viability, clonogenic assays were performed
with a range of doses and incubation times with PCA (Fig. 1,
H and I). One hour of treatment with 200 µM PCA reduced the long term viability by about 20%
(Fig. 1H). However, cloning efficiency was markedly
decreased when Raji cells were treated with 100-200 µM
PCA for 2 h (Fig. 1I). Therefore, the 1-h treatment
with 200 µM PCA was used for assessment of its inhibitory effects on FasL-induced apoptosis by using clonogenic assay. Under this
condition, PCA inhibited FasL-induced apoptosis in Raji cells and
increased their survival rate slightly but significantly (Fig. 1J).
PCA Inhibits Caspase-3 Activity, but Not Active Caspase-3, in
FasL-treated Cells--
Upon Fas oligomerization, caspase-8 is
activated by self-cleavage, and active caspase-8 subsequently processes
caspase-3 to an active form (1-4). Consistent with this model,
caspase-3 activity was undetectable during the first 30 min after FasL
addition and increased rapidly thereafter in FasL-treated Raji cells
(Fig. 2A). PCA inhibited
caspase-3 activity at 100-200 µM in cells treated with
FasL for 2 h (Fig. 2B). To examine whether PCA inhibits
active caspase-3 directly in FasL-treated cells, PCA was added to Raji cells 0 or 60 min (sufficient time to induce active caspase-3) after
treatment with FasL. In contrast to the caspase inhibitor Z-VAD-fmk,
PCA did not prevent caspase-3 activity when PCA was added 60 min after
FasL treatment (Fig. 2C). These results suggest that PCA
does not inhibit active caspase-3 in FasL-treated cells.
PCA Blocks Activation of Caspase-8 in FasL-treated Cells--
In
FasL-treated cells, PCA prevented processing of caspase-3 into active
subunits (Fig. 3A), suggesting
that PCA blocks the signaling pathway upstream of caspase-3 activation.
Cross-linked FasL induced cleavage of two caspase-8 isoforms (8/a and
8/b) (25) into fragments corresponding to p43 and p41, respectively, and these intermediate forms were subsequently processed into a p18
active large subunit (Fig. 3B). FasL-induced processing of
caspase-8 was markedly inhibited at 100-200 µM in
PCA-treated cells (Fig. 3B). PCA still effectively prevented
caspase-8 processing in cells treated with higher concentrations of
FasL (2 µg/ml) (Fig. 3C). Similar to PCA, Z-VAD-fmk
inhibited processing of caspase-8 induced by cross-linked FasL (Fig.
3D).
PCA Blocks Self-processing of Caspase-8 in the DISC--
Upon FasL
engagement, Fas binds to FADD via the death domain interaction, and in
turn recruits caspase-8 via the homotypic interaction of their death
effector domain, allowing the formation of the DISC (1-4, 11, 12).
Immediately after recruitment, caspase-8 molecules are placed in close
proximity facilitating self-processing (13). PCA affected neither the
cell-surface expression of Fas nor the cellular level of FADD and
caspase-8 up to 5 h (Fig. 4,
A and B). After treatment with FasL, PCA did not
affect the recruitment of FADD and caspase-8 into the DISC (Fig.
4C). However, in cells treated with 100-200
µM PCA, only full-length caspase-8, but not processed
caspase-8 (p43 and p41), was detected in the DISC (Fig.
4C). Caspase-8 processing in the DISC was significantly
increased for 30-60 min after treatment with FasL, and PCA still
effectively blocked caspase-8 processing in the DISC (Fig.
4D). Similar to PCA, Z-VAD-fmk inhibited the processing of
caspase-8 in the DISC without affecting the recruitment of FADD and
caspase-8 (Fig. 4E).
Caspase-8 Is the Preferential Target of PCA in Living
Cells--
The above observations suggest the possibility that
caspase-8 is a direct target of PCA. Consistent with this hypothesis, PCA inhibited the enzyme activity of active recombinant caspase-8 in a
dose-dependent manner (Fig.
5A). However, the enzymatic
activities of active recombinant caspase-3 and active recombinant
caspase-9 were also inhibited by PCA at similar concentrations (Fig. 5, B and C). To address whether PCA inhibits
caspase-8 preferentially at the enzyme level, competition assay between
caspase-3 and caspase-8 was performed (Fig. 5D). Even in the
presence of excess caspase-8 large subunit (p20) where the consensus
active center QACXG is located (15, 26), the inhibitory effect of PCA
on caspase-3 activity was not suppressed (Fig. 5D). Thus,
these observations suggest that PCA does not selectively inhibit active
caspase-8 at the enzyme level. To determine whether PCA inhibits
caspase-9 at the cellular level, Raji cells were pretreated with PCA
for 1 h and then incubated for 6 h with staurosporine which
induces apoptosis mediated by caspase-9 (Fig. 5E).
Caspase-9 is processed into p35 subunit by self-cleavage, whereas
active caspase-3 cleaves caspase-9 into p37 subunit (27). Caspase-9
processing was slightly inhibited by PCA in staurosporine-treated
cells, although PCA profoundly inhibited caspase-8 processing in
FasL-treated cells (Fig. 5E). Because, unlike caspase-3 and
caspase-8, the cellular level of caspase-9 was markedly reduced when
Raji cells were treated with 200 µM PCA for over 7 h
(Fig. 5F), a marginal reduction of caspase-9 processing
might be due to the PCA-induced reduction of pro-caspase-9 rather than
the inhibitory effect of PCA on caspse-9 activation. To avoid the
extensive reduction of caspase-9 by PCA, Raji cells were first
preincubated with staurosporine for 3 h and then treated with PCA
or Z-VAD-fmk when caspase-9 activity was increasing (Fig.
5G). In contrast to Z-VAD-fmk, PCA exerted weak inhibitory
effects on activation of caspase-9 and caspase-3 in
staurosporine-treated cells. Moreover, a similar experiment was
performed using FasL-treated Raji cells (Fig. 5H). Z-VAD-fmk prevented activities of caspase-8 and caspase-3 to the background level. By contrast, PCA strongly inhibited newly activated caspase-8 and caspase-3 but partially prevented already activated caspase-8 and
caspase-3 in FasL-treated cells. Thus, these observations indicate that
PCA preferentially targets activation of caspase-8 in living cells.
PCA Binds Directly to the Active Center Cysteine of
Caspase-8--
PCA has a molecular structure of Caspases play a central role in apoptotic cell death (14, 15).
Active caspases are modulated directly by cellular and viral proteins
that interact with these proteases (14, 16, 17). Cell-permeable caspase
inhibitors are important research tools for addressing the molecular
basis of apoptosis, as well as being therapeutic agents for various
diseases (28-33). In contrast to cell-free screening for inhibitors of
recombinant proteins such as caspases, we adopted cell-based models of
apoptosis to overcome disadvantages such as the poor membrane
permeability of inhibitors. We screened for specific inhibitors that
block Fas-mediated apoptosis and identified PCA. In the present paper, we demonstrate that PCA primarily blocks self-processing of caspase-8 in the DISC by binding the active center cysteine.
PCA inhibited the enzymatic activities of active recombinant caspase-3,
caspase-8, and caspase-9 at similar concentrations. The non-selective
inhibitory effects of PCA on active recombinant caspases at the enzyme
level might be explained by the fact that PCA binds to cysteine
residues of proteins via an In contrast to caspase-3 and caspase-8, the cellular amount of
caspase-9 was selectively reduced in PCA-treated cells. The caspase-9
reduction by PCA was prevented by the proteasome inhibitor MG-132,2 suggesting that
caspase-9 is proteolytically degraded by the ubiquitin-proteasome
system in PCA-treated cells. The molecular mechanism of caspase-9
degradation induced by PCA remains to be elucidated.
Among peptide-based caspase inhibitors so far developed, Z-VAD-fmk is
an irreversible inhibitor that targets many (but not all) caspases (33,
37) and is most frequently used to block apoptosis. It was reported
that a series of non-peptide inhibitors of caspase-3 and caspase-7
(isatin sulfonamides) prevent apoptosis and maintain cell functionality
(38). Unlike PCA, the isatin sulfonamides do not possess an
Caspase-8 is the initiator caspase that functions as a key molecule
critical for apoptosis induced by death receptors (1-4, 39, 40).
Signaling by death receptors plays an essential role in homeostasis of
various types of cells in the immune system and autoimmunity (41-43).
Recently, our group and others (6, 7) reported that caspase activation
is required for T cell proliferation. In agreement with this notion, we
have shown that the cellular caspase-8 inhibitor c-FLIP is essential
for the regulation of T cell proliferation (44, 45). Caspase inhibitors
might thus be applicable to block T cell proliferation and prevent
autoimmune diseases. Our present results demonstrate that PCA blocks
self-processing of caspase-8 in the DISC. However, PCA exhibited a
limited selectivity and induced unfavorable cytotoxic effects in the
long term culture, possibly due to binding to sulfhydryl groups of
other proteins. Thus, further design of synthetic analogues of PCA
might lead to highly selective inhibitors for caspase-8.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
spontaneous release)/(maximum
release
spontaneous release) × 100. Cross-linked FasL was
used as described previously (20). Raji cells were cultured with
cross-linked FasL for 4 h and fixed with 4% paraformaldehyde/PBS,
followed by staining with 300 µM Hoechst 33342 (Calbiochem). The number of normal and condensed nuclei was counted
under fluorescent microscopy. Apoptotic cells (%) were calculated as
(condensed nuclei/total nuclei) × 100. A20 cells and Raji cells
were cultured with Jo2 and cross-linked FasL for 4 h,
respectively, and pulsed with 500 µg/ml
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT;
Sigma) for 4 h, and MTT-formazan was then solubilized with 5% SDS
overnight. Absorbance at 595 nm was measured. Cell viability (%) was
calculated as (experimental absorbance
background
absorbance)/(control absorbance
background absorbance) × 100.
-D-galactopyranoside. His-tagged caspase-8 p20 was purified with HiTrap chelating column (Amersham Biosciences) and desalted with PD-10 column (Amersham Biosciences) according to the manufacturer's instruction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PCA blocks apoptosis induced by anti-Fas
antibody or physiological FasL. A, structure of
PCA. B, [3H]TdR-labeled A20 cells were
incubated with various concentrations of anti-Fas antibody Jo2 for
16 h. Radioactivity of fragmented DNA was measured. Data points
represent the mean ± S.D. of triplicate determinations.
C, [3H]TdR-labeled A20 cells were
pretreated with serial dilutions of PCA for 2 h and then incubated
with (filled circles) or without (open circles)
Jo2 (200 ng/ml) for 4 h. Radioactivity of fragmented DNA was
measured. Data points represent the mean ± S.D. of triplicate
determinations. D, A20 cells were pretreated with
serial dilutions of PCA for 1 h and then incubated with
(filled circles) or without (open circles) Jo2
(200 ng/ml) for 4 h. Cell viability (%) was measured by MTT
assay. Data points represent the mean ± S.D. of triplicate
cultures. E, Raji cells were incubated with various
concentrations of cross-linked FasL for 4 h. Apoptotic cells (%)
were measured by Hoechst 33342 staining. Data points represent the
mean ± S.D. of triplicate determinations. F, Raji
cells were pretreated with various concentrations of PCA for 1 h,
and then incubated with (filled circles) or without
(open circles) cross-linked FasL (500 ng/ml) for 4 h.
Apoptotic cells (%) were measured by Hoechst 33342 staining. The
results represent the mean ± S.D. of triplicate determinations.
G, Raji cells were pretreated with serial dilutions of
PCA for 1 h and then incubated with (filled circles) or
without (open circles) cross-linked FasL (500 ng/ml) for
4 h. Cell viability (%) was measured by MTT assay. Data points
represent the mean ± S.D. of triplicate cultures.
H, Raji cells were incubated with 200 µM
PCA for indicated times and washed with the medium to remove PCA. The
cells were diluted and cultured in 96-well microtiter plates (1 cell/well) for 10 days. The number of colonies formed was counted. The
results represent the mean ± S.D. of triplicate determinations.
I, Raji cells were treated with various concentrations
of PCA for 2 h and washed with the medium to remove PCA. The cells
were diluted and cultured in 96-well microtiter plates (1 cell/well)
for 10 days. The number of colony formed was counted. The results
represent the mean ± S.D. of triplicate determinations.
J, Raji cells were pretreated with or without 200 µM PCA for 1 h and washed with the medium to remove
PCA. The cells were then incubated in the presence or the absence of
cross-linked FasL (500 ng/ml) for 2 h and washed with the medium
to remove FasL. The cells were diluted and cultured in 96-well
microtiter plates (1 cell/well) for 10 days. The number of colonies
formed was counted. The results represent the mean ± S.D. of
triplicate determinations. Student's t test was used to
determine the statistical significance. Asterisk,
p < 0.001 relative to FasL treatment.
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Fig. 2.
PCA inhibits activation of caspase-3 in
FasL-treated cells. A, Raji cells were incubated
with cross-linked FasL (500 ng/ml) for the indicated times. Caspase-3
activity was measured by hydrolysis of Ac-DEVD-MCA. Data points
represent the mean ± S.D. of triplicate determinations.
B, Raji cells were pretreated with various
concentrations of PCA for 1 h and then incubated with cross-linked
FasL (500 ng/ml) for 2 h. Caspase-3 activity was measured by
hydrolysis of Ac-DEVD-MCA. The results represent the mean ± S.D.
of triplicate determinations. C, Raji cells were
incubated with cross-linked FasL (500 ng/ml). Two hundred
µM PCA or 10 µM Z-VAD-fmk was included at
the same time with addition of FasL 0 or 60 min later. After 120 min of
incubation, postnuclear lysates were prepared and measured for
caspase-3 activity by hydrolysis of Ac-DEVD-MCA. The results represent
the mean ± S.D. of triplicate determinations.
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Fig. 3.
PCA blocks activation of caspase-8 in
FasL-treated cells. A, Raji cells were pretreated
with various concentrations of PCA for 1 h and then incubated with
cross-linked FasL (500 ng/ml) for 2 h. Caspase-3 processing was
analyzed by Western blotting. B, Raji cells were
treated with various concentrations of PCA for 1 h and then
treated with cross-linked FasL (500 ng/ml) for 2 h. Caspase-8
processing was analyzed by Western blotting. C, Raji
cells were treated with or without 200 µM PCA for 1 h and then incubated with cross-linked FasL (2 µg/ml) for 30 min.
Caspase-8 processing was analyzed by Western blotting.
D, Raji cells were treated with various concentrations
of Z-VAD-fmk for 30 min and then incubated with cross-linked FasL (2 µg/ml) for 30 min. Caspase-8 processing was analyzed by Western
blotting.
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Fig. 4.
PCA blocks self-processing of caspase-8 in
the DISC. A, Raji cells were treated with or
without 200 µM PCA for 5 h. Surface expression of
Fas (solid lines) and background staining (dotted
lines) were detected by FACS analysis. B, Raji
cells were incubated with various concentrations of PCA for 5 h.
Expression of FADD and caspase-8 was analyzed by Western blotting.
C, Raji cells were pretreated with various
concentrations of PCA for 30 min and then incubated with cross-linked
FasL (2 µg/ml) for 15 min. The DISC and cell lysates were analyzed by
Western blotting. Nonspecific band is indicated as ns.
D, Raji cells were pretreated with or without 200 µM PCA for 30 min and then incubated with cross-linked
FasL (2 µg/ml) for 15-60 min. The DISC and cell lysates were
analyzed by Western blotting. Nonspecific band is indicated as
ns. E, Raji cells were pretreated with or
without 100 µM Z-VAD-fmk for 1 h and then incubated
with cross-linked FasL (2 µg/ml) for 15 min. The DISC and cell
lysates were analyzed by Western blotting.
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Fig. 5.
Caspase-8 is the preferential target of PCA
in living cells. A, active recombinant caspase-8
was pretreated with serial dilutions of PCA (filled circles)
or Z-VAD-fmk (open circles) for 30 min and incubated with 50 µM Ac-IETD-MCA for 60 min. Fluorescent intensity of AMC
release was measured. Data points represent the mean ± S.D. of
triplicate determinations. B, active recombinant
caspase-3 was pretreated with serial dilutions of PCA (filled
circles) or Z-VAD-fmk (open circles) for 30 min and
incubated with 50 µM Ac-DEVD-MCA for 60 min. Fluorescent
intensity of AMC release was measured. Data points represent the
mean ± S.D. of triplicate determinations. C,
active recombinant caspase-9 was pretreated with serial dilutions of
PCA (filled circles) or Z-VAD-fmk (open circles)
for 10 min and incubated with 50 µM Ac-LEHD-MCA for 60 min. Fluorescent intensity of AMC release was measured. Data points
represent the mean ± S.D. of triplicate determinations.
D, active recombinant caspase-3 (600 ng/ml)
was pretreated with various concentrations of PCA in the presence
(filled circles) or the absence (open circles) of
caspase-8 p20 (30 µg/ml) for 10 min and incubated with 50 µM Ac-DEVD-MCA for 60 min. Fluorescent intensity of AMC
release was measured. Data points represent the mean ± S.D. of
triplicate determinations. E, Raji cells were
pretreated with various concentrations of PCA for 1 h and then
incubated with 5 µM staurosporine (upper
panel) or 500 ng/ml cross-linked FasL (lower panel) for
6 h. The processing of caspase-8 and caspase-9 was analyzed by
Western blotting. F, Raji cells were treated with 200 µM PCA for indicated times. The cellular amount of
caspase-3, caspase-8, and caspase-9 was analyzed by Western blotting.
G, Raji cells were treated with 5 µM
staurosporine, and 200 µM PCA (filled circles)
or 20 µM Z-VAD-fmk (filled squares) was added
3 h later. The cells were incubated for indicated times, and
postnuclear lysates were prepared. The activity of caspase-9 and
caspase-3 was measured by hydrolysis of Ac-LEHD-MCA and Ac-DEVD-MCA,
respectively. Data points represent the mean ± S.D. of triplicate
determinations. H, Raji cells were treated with
cross-linked FasL (100 ng/ml), and 200 µM PCA
(filled circles) or 20 µM Z-VAD-fmk
(filled squares) was added 60 min later. The cells were
incubated for the indicated times, and postnuclear lysates were
prepared. The activity of caspase-8 and caspase-3 was measured by
hydrolysis of Ac-IETD-MCA and Ac-DEVD-MCA, respectively. Data points
represent the mean ± S.D. of triplicate determinations.
,
-unsaturated
lactone moiety capable of binding cysteine sulfhydryl groups. In
agreement with this, an inhibitory effect of PCA on the enzyme activity of caspase-8 was neutralized by addition of cysteine or glutathione but
not serine (Fig. 6A).
Glutathione also suppressed the inhibitory effect of PCA on caspase-8
processing in the DISC (Fig. 6B). To address whether PCA
binds to the active site cysteine, the recombinant caspase-8 p20 was
mixed with PCA, and direct binding of PCA was assessed by liquid
chromatography-MS/MS (Fig. 6, C and D). Compared with an unmodified peptide, the profiles of fragment ions of the peptide spanning the active center clearly revealed that PCA binds to
the active center cysteine of caspase-8 catalytic domain.
View larger version (30K):
[in a new window]
Fig. 6.
PCA binds directly to the active site
cysteine of caspase-8. A, active caspase-8 was
pretreated with (filled circles) or without (open
circles) 30 µM PCA in the presence of glutathione
(left panel), cysteine (middle panel), or serine
(right panel) for 30 min, and then incubated with 50 µM Ac-IETD-MCA. Fluorescent intensity of AMC release was
measured. Data points represent the mean ± S.D. of triplicate
determinations. B, Raji cells were pretreated with or
without 1 mM glutathione for 30 min. The cells were treated
with or without 200 µM PCA for 30 min and then incubated
with cross-linked FasL (2 µg/ml) for 15 min. The DISC and cell
lysates were analyzed by Western blotting. Nonspecific band is
indicated as ns. C and D,
caspase-8 p20 was treated with 100 µM PCA. An unmodified
peptide covering active site cysteine (IFFIQACQGSNFQK) (C)
and the same peptide covalently coupled to PCA (D) were
isolated by the HPLC system. The doubly charged ion of the unmodified
peptide (m/z 815.97) and the PCA-bound peptide
(m/z 900.98) was subjected to MS/MS analysis.
Observed fragment ions of b and y series are
indicated above and below the peptide sequence,
respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
-unsaturated lactone moiety.
Nevertheless, PCA strongly inhibited self-activation of caspase-8 but
weakly inhibited activation of caspase-3 and caspase-9, in cells
treated with FasL or staurosporine. These observations suggest that
caspase-8 should be the preferential target of PCA in intact cells. The
overall structure of caspase-8 including the substrate-binding subsite
(S1) resembles the caspase-3 structure, with the exception
of subsites (S3 and S4) essential for substrate
specificity (34-36). The similarity and difference in the binding
cleft of caspase-3 and caspase-8 does not explain the preferential
blockade of caspase-8 by PCA in intact cells. Although the
three-dimensional structure of the caspase-8 zymogen is currently
unknown, it seems likely that the substrate-binding pocket differs
between the fully active caspase-8 heterodimer and the caspse-8
zymogen. As the likely explanation, we speculate that PCA might have a
higher affinity to the caspase-8 zymogen and inactivate its intrinsic
proteolytic activity.
,
-unsaturated lactone moiety that is capable of binding cysteine
residues. Instead, these inhibitors primarily interact with the
substrate binding cleft (S2) unique to caspase-3 and
caspase-7 (38). Most of the isatin sulfonamide derivatives do not
inhibit caspase-8 (38). Thus, PCA seems to be the non-peptide inhibitor
that blocks self-processing of caspase-8 in cell-based models of apoptosis.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Ralph C. Budd and Jürg Tschopp for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant-in-aid for Scientific Research (C) from the Japan Society for the Promotion of Science and research grants by Mitsubishi Chemical Corporation Fund and Kato Memorial Bioscience Foundation.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: Dept. of Biological Chemistry, Chubu
University, 1200 Matsumoto-cho, Kasugai 487-8501, Japan.
** To whom correspondence should be addressed: Research Center for Experimental Biology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Tel.: 81-45-924-5830; Fax: 81-45-924-5832; E-mail: tkataoka@bio.titech.ac.jp.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M204178200
2 M. Bando, K. Nagai, and T. Kataoka, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: FADD, Fas-associated death domain; FasL, Fas ligand; DISC, death-inducing signaling complex; AMC, 7- amino-4-methylcoumarin; MCA, 4-methylcoumaryl-7-amide; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltretazolium bromide; PCA, penicillic acid; TdR, thymidine; Z-VAD-fmk, benzyloxycarbonyl-VAD-fluoromethyl ketone; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; HPLC, high pressure liquid chromatography; FACS, fluorescence-activated cell sorter; MS, mass spectrometry.
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