From the Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609, Republic of Singapore
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ABSTRACT |
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Caspase-10/a (Mch4) and caspase-10/b (FLICE2) are
related death effector domain-containing cysteine aspartases presumed
to be at or near the apex of apoptotic signaling pathways. We report the cloning and characterization of two novel proteins that are splice
isoforms of the caspase-10 family. Caspase-10/c is a truncated protein
that is essentially a prodomain-only form of the caspase that lacks
proteolytic activity in vitro but efficiently induces the
formation of perinuclear filamentous structures and cell death in
vivo. Caspase-10/c mRNA is specifically up-regulated upon TNF stimulation, suggesting a potential role of this isoform in amplifying the apoptotic response to extracellular stimuli such as cytokines. Caspase-10/d is a hybrid of the known caspases Mch4 and FLICE2, as it
is identical to FLICE2 except for the small (p12) catalytic subunit,
which is identical to Mch4. Caspase-10/d is proteolytically active
in vitro and also induces cell death in vivo,
although it is less active than Mch4. The mRNAs for all known
isoforms of caspase-10 are abundantly expressed in fetal lung, kidney, and skeletal muscle but are very poorly expressed or absent in these
tissues in the adult, implying a possible role for the caspase-10 family in fetal development.
Apoptosis is a process of regulated cell suicide crucial for the
development and homeostasis of multicellular organisms that is
characterized by chromatin condensation, DNA fragmentation, cell
shrinkage, and plasma membrane blebbing (1). The gene products of
ced-3 and ced-4 regulate the apoptotic death of
cells during normal development of the nematode Caenorhabditis
elegans (2). Subsequently, CED-3 was shown to have a mammalian
homologue named interleukin 1 Caspases are present as inactive proenzymes comprising a prodomain and
a catalytic protease domain that can be further processed to give a
large and a small subunit. Crystallographic data (6, 7) has revealed
(for caspases 1 and 3 and by extrapolation to the other caspase family
members) that the activated caspase exists as a heterodimeric tetramer
of two large and two small subunits, with each substrate binding pocket
spanning both subunits. Evidence for the sequential activation of
caspases has led to the concept of a caspase cascade (8-11), with
initiator caspases at the apex, transmitting signals to executor
caspases that cleave a wide variety of substrates. In this scheme,
caspases 2, 8, and 10 (12-16) have been demonstrated to associate with
the death receptors Fas and TNF1 receptor 1 and are
presumably initiator caspases.
In terms of their substrate specificities, the caspases can be
classified into three groups (17, 18), all of which have an absolute
requirement for an aspartate residue in the P1 position but otherwise
show varying degrees of flexibility. Group I caspases (caspases 1, 4, and 5) prefer the tetrapeptide sequence WEHD; group II caspases
(caspases 2, 3, and 7) have a DEXD specificity, whereas
group III caspases (caspases 6, 8, 9, and 10) cleave after (I/L/V)EXD. It should be noted, however, that these are only
the preferred substrate motifs and are not absolute, being dependent on
factors such as concentration, reaction time, and accessibility. Multiple redundancies are common among the caspases, with many caspases
having the ability to cleave the same substrates, at least in
vitro (17, 18). There is an additional level of complexity with
the discovery that multiple isoforms are present for many caspases,
including caspases 1, 2, 6, and 8 (19). These isoforms are most likely
splice variants (20) or variants derived from post-translational
modifications, but the biological significance of these variants is
mostly unclear.
In this paper we describe the identification and characterization of
two novel isoforms of caspase-10 (caspase-10/c and caspase-10/d) that
incorporate features of both Mch4 (caspase-10/a) (14) and FLICE2
(caspase-10/b) (15). We describe their tissue expression and activity
both in vitro and in vivo and examine the effect of exogenous stimulation by TNF on their mRNA expression.
Cell Lines and Reagents--
Expression vectors coding for PARP
or caspase-3/Yama and the 293T cell line were generous gifts from Dr.
V. Dixit. Antibodies against the large and small catalytic subunits of
caspase-10 were obtained from Research Diagnostics, Inc. The polyclonal
serum directed against a caspase-10/c-specific peptide sequence
(EGSCVQDESEPQRPL) was produced by Genosys, Inc. Chemiluminescent
Western blots were developed using the Immun-Star kit from Bio-Rad.
Unless otherwise mentioned, all other reagents were purchased from Sigma.
Cloning and Identification of Caspase-10 Variants--
PCR was
performed on human spleen, thymus, and peripheral blood leukocytes
cDNA libraries (CLONTECH) using primers 5MCH4
(AAA GGA TCC GCT AGC ATG AAA TCT CAA GGT CAA CAT TGG TAT TCC) and 3MCH4 (AAA GAA TTC CTA TAT TGA AAG TGC ATC CAG GGG CAC AGG) to obtain the
coding regions of Mch4, caspase-10/c, and caspase-10/d. Advantage-HF polymerase (CLONTECH) was used with cycling
conditions recommended by the manufacturer. The final clones were from
spleen (caspase-10/c) and thymus (caspase-10/d). The PCR products were
inserted into pCITE-4a(+) (Novagen) for in vitro expression
as S-Tag fusion proteins or pCI-neo (Promega) for in vivo
eukaryotic expression.
mRNA Expression Analysis--
The primers C614 (CCG AGT CGT
ATC AAG GAG AGG AAG AAC) and 3BRIDGE (TAT ATG CAC TGT GAA CCC AAG CCA)
were used in PCR and RT-PCR experiments to obtain short DNA fragments
corresponding to Mch4, caspase-10/c, and caspase-10/d. RNA from various
cell lines was purified using the Qiagen RNAeasy kit, and
reverse-transcription was performed using SuperScript II enzyme with
oligo(dT) primers from Life Technologies, Inc. PCR was then performed
for 30 cycles on the resulting cDNA at an annealing/extension
temperature of 68 °C using Advantage-HF polymerase or on cDNA
from adult and fetal multiple tissue cDNA panels
(CLONTECH), and the products were analyzed in
ethidium bromide-stained 2.5% agarose gels.
Transfections, Killing Assays, and Solubility Studies--
For
the killing assays, MCF7 cells were seeded at a density of 1 × 105 cells/60-mm dish in 5 ml of RPMI 1640 supplemented with
10% fetal bovine serum and 1% penicillin/streptomycin. At ~60-80%
confluence, each dish of cells was transfected with 4 µg of pCI-neo
expression vector with or without the CASP insert and 1 µg
of pCMV-
For the solubility studies, 293T or MCF7 cells were seeded at a density
of 5 × 106 cells/100-mm dish in 10 ml of growth
medium. At approximately 80% confluence, the cells were transfected
with 12 µg of plasmid DNA, with the addition of Ac-DEVD-CHO inhibitor
at a final concentration of 20 µM to delay cell death. At
24 h post-transfection, the cells were scraped and pelleted by
centrifugation. Total lysates were obtained by boiling the pellets in 1 ml of Laemmli sample buffer. In experiments separating soluble from
insoluble fractions, pellets were first incubated in lysis buffer
(0.1% Nonidet P-40, 250 mM NaCl, 50 mM Hepes
pH 7.4, 5 mM EDTA, Complete Protease Inhibitor mix
(Boehringer Mannheim)) for 30 min at 4 °C, then centrifuged for 20 min at 10,000 × g. The supernatant was harvested as
the soluble fraction, whereas the pellet was washed three times with lysis buffer, then boiled in 300 µl of Laemmli sample buffer and used
as the insoluble fraction. Immunoblotting was carried out using either
commercial goat anti-Mch4 p12 subunit antibody at 1:500 dilution or
custom anti-caspase-10/c polyclonal antiserum at 1:1000 dilution in
blocking buffer (5% milk/PBS, 0.05% Tween 20).
Confocal Microscopic Visualization--
Constructs of Mch4,
caspase-10/c, and caspase-10/d fused to the N terminus of EGFP were
made by cloning the respective cDNAs into pEGFP-N3
(CLONTECH). MCF7 cells were transfected in 60-mm dishes using 2 µg of pEGFP-N3-derived plasmids in the presence of 50 µg of Ac-DEVD-CHO. Approximately 16 h post-transfection, the
cells were fixed in 4% paraformaldehyde, PBS for 15 min at 25 °C,
then equilibrated in 2× SSC buffer (15 mM sodium citrate, 150 mM NaCl) and incubated for 30 min at 37 °C with 100 µg/ml DNase-free RNase in 2× SSC. After rinsing with 2× SSC, the
cells were stained for 2 min in propidium iodide for nuclear
visualization, mounted in Vectashield (Vector Laboratories), and
visualized under confocal laser scanning microscopy (Bio-Rad MRC 1024).
TNF Stimulation and mRNA Expression--
MCF7 cells were
seeded at 5 × 106 cells/100-mm dish in 10 ml of
growth medium, then grown to high density, usually 48 h
post-seeding. Recombinant human TNF Cytotoxicity Assay--
MCF7 cells were seeded at a density of
3 × 104 cells/well in 96-well plates. Approximately
48 h post-seeding, recombinant human TNF In Vitro Cleavage Analysis--
The coding regions of Mch4,
caspase-10/c, and caspase-10/d starting from Ile (194) (14) were each
inserted into the pGEX-2TK bacterial expression plasmid (Amersham
Pharmacia Biotech), enabling their expression as GST fusion proteins in
Escherichia coli JM109. The pellets from 200 ml of 1 mM
isopropyl- Cloning and Identification of Caspase-10 Variants--
Previous
reports had indicated the possible existence of multiple isoforms of
caspase-10 (15, 21). Our initial cloning by PCR of what appeared to be
a single band of approximately 1500 bp emerged on sequencing to consist
of three individual DNA fragments, wild type Mch4 (1440 bp),
caspase-10/c (1477 bp), and caspase-10/d (1569 bp). It was possible to
distinguish electrophoretically between these three DNAs by designing
PCR primers (5MCH4/3BRIDGE) spanning a mid-insert region (comprising
exons 6 and 7) common to caspase-10/d and FLICE2 (Fig.
1). Bands of 230, 260, and 350 bp in size
corresponded to Mch4, caspase-10/c, and caspase-10/d, respectively (see
below).
In the recent paper on the characterization of Usurpin (22), the
authors described the sequencing of the death effector domain
(DED)-caspase gene cluster on human chromosome 2 band q33-34, which
contains the genes for Usurpin, caspase-10/a/Mch4, and
caspase-8/a/FLICE. Based on the genomic map, the mid-insert region
mentioned above consists of exons 6 and 7 (Fig. 1). Hence Mch4 lacks
exons 6, 7, and 11. In contrast, FLICE2 contains exons 6 and 7 but
skips exon 10 and contains part of exon 11. Caspase-10/c contains exon 6 but not 7, whereas caspase-10/d contains both exons 6 and 7 (summarized in Fig. 1).
The predicted amino acid sequences show that caspase-10/d is a hybrid
of FLICE2 and Mch4 (Fig. 2).
Interestingly, the insertion of exon 6 in caspase-10/c leads to a
frameshift at the protein level, resulting in a protein that is
truncated shortly after the DED-containing prodomain. Thus,
caspase-10/c is essentially a prodomain-only protein (Fig. 2). We
confirmed the authenticity of the predicted protein sequences by
translation in vitro and by performing immunoblotting on the
products. As predicted, in vitro translated caspase-10/d was
slightly larger in size than Mch4 and was recognized by antibodies
against both the large and small protease subunits of Mch4 (Fig.
3). In vitro translated caspase-10/c was a much smaller protein of approximately 40 kDa on
SDS-polyacrylamide gel electrophoresis (Fig. 3, upper panel) that was not recognized by either of the anti-protease subunit antibodies (Fig. 3, lower panel).
Tissue- and Cell Line-specific mRNA Expression of
Caspase-10 Isoforms--
To determine the constitutive mRNA
expression profiles of the caspase-10 variants, PCR and RT-PCR was
performed on RNA extracted from various cell lines and on commercially
available cDNA panels. Because the CLONTECH
multiple tissue cDNA panels are prenormalized against several
housekeeping genes, we were able to directly compare the levels of
mRNA expression between samples. The bands representing Mch4,
caspase-10/c and caspase-10/d were 230, 260, and 350 bp in size,
respectively (Fig. 4). Their identity was
confirmed by subcloning and sequencing two independent clones of each
DNA fragment from spleen, thymus, and peripheral blood leukocytes
cDNA libraries. Although this method did not distinguish between
FLICE2 and caspase-10/d expression (because they both contain the same
exon 6 and 7 insert), it is clear that expression levels of
caspase-10-related mRNAs are generally higher in fetal than in
adult tissues (Fig. 4A). In particular, Mch4, caspase-10/c,
and FLICE2/caspase-10/d are highly expressed in fetal skeletal muscle,
lung, and kidney, whereas their expression in these tissues is
virtually undetectable in the adult.
Expression of the caspase-10 isoforms was highly variable among various
cell lines (Fig. 4B). There was no obvious pattern in their
distribution. The 350-bp FLICE2/caspase-10/d band was consistently
highly expressed in all cell lines tested, whereas caspase-10/c and
Mch4 were present at very low levels in the MCF7 breast carcinoma and
SW480T adenocarcinoma cell lines (Fig. 4B). Caspase-10/c was
not absent, as increasing the number of PCR cycles resulted in easier
visualization (not shown).
Caspase-10/d but Not Caspase-10/c Is Proteolytically Active in
Vitro--
Crude sonicated lysates of bacterial cultures expressing
recombinant Mch4 and caspase-10/d were proteolytically active.
Immunoblotting of the bacterial sonicates using antibodies against the
Mch4 p12 small subunit revealed that autoprocessing had occurred in the case of Mch4 and caspase-10/d with the release of the p12 subunit but
not with caspase-10/c (Fig. 5,
panel A).
The bacterial sonicates containing Mch4 and caspase-10/d cleaved
in vitro translated full-length Mch4 and caspase-10/d
proteins, demonstrating the possibility of intermolecular processing of caspase-10 isoforms (Fig. 5, panel B) and also cleaved
in vitro translated PARP and caspase-3 (Fig. 5, panels
C and D). Again, caspase-10/c was not proteolytically
active in any of these experiments. The activity of Mch4 or
caspase-10/d was completely abrogated by the addition of 1 µM specific caspase inhibitor Ac-DEVD-CHO but not
Ac-YVAD-CHO (Fig. 5, panels C and D). The
cleavage activity (and the inhibition thereof) is because of the
expressed recombinant caspase and not to the activation of any other
endogenous proteases because (i) bacteria do not have endogenous
caspase activity (Ref. 23 and vector controls in Fig. 5, panels
B-D), (ii) affinity-purified recombinant caspases are
proteolytically active (14,
23),2 and (iii) the in
vitro translation products used as substrates in some of our
experiments were first purified from any contaminating reticulocyte components.
In addition, we consistently observed that caspase-10/d was less active
than Mch4 after normalization of the starting quantities of bacterial
sonicates used based on the amount of processed p12 subunit. We
quantitated this activity using the caspase substrate DEVD-pNA and
confirmed that Mch4 was approximately twice as active as caspase-10/d
(not shown).
Caspases 10/c and 10/d Are Highly Toxic to MCF7 Cells--
Despite
the lack of a protease domain, caspase-10/c was highly efficient in
causing apoptosis when transiently overexpressed in the MCF7 cell line.
Caspase-10/d was also pro-apoptotic, as was Mch4 used as a control
(Fig. 6). This killing by all three isoforms was caspase-dependent, as shown first by the
dose-dependent inhibitory effect of the tetrapeptide
inhibitor Ac-DEVD-CHO (Fig. 6, panel A) and second by the
observation that PARP was cleaved in cells transfected with Mch4,
caspase-10/c, or caspase-10/d (Fig. 6, panel B, lanes
3-5). Furthermore, the fact that Ac-DEVD-CHO but not Ac-YVAD-CHO
inhibited killing indicated that apoptosis was transduced by downstream
caspase-3-like enzymes, and caspase-1-like proteases were probably not
involved (17, 18).
Mch4 and Caspase-10/d Are Soluble Proteins, Whereas Caspase-10/c Is
Insoluble and Localizes to Filamentous Perinuclear
Structures--
Lysates of mammalian cells transiently overexpressing
Mch4, caspase-10/c, or caspase-10/d were prepared as detergent-soluble and -insoluble fractions, and immunoblotting was performed using either
anti-Mch4 p12 antibodies to detect Mch4 and caspase-10/d or custom
anti-caspase-10/c polyclonal antiserum. In both MCF7 and 293T cell
lines, Mch4 and caspase-10/d were completely soluble, whereas
caspase-10/c was predominantly insoluble (Fig.
7). These results are consistent with
recent evidence that DED-only proteins might form insoluble filamentous
structures (death effector filaments, DEF) either on their own or in
association with as yet unidentified cytoskeletal elements (24). To
determine whether DEFs were formed by caspase-10 isoforms, Mch4,
caspase-10/c and caspase-10/d were ectopically expressed as EGFP fusion
proteins in MCF7 and 293T cells and visualized using confocal laser
scanning microscopy. Distinct perinuclear filamentous structures were
seen in all cells expressing caspase-10/c-EGFP (Fig.
8, panel A). In most cases these filaments were closely bundled together, but more haphazard arrangements were sometimes also observed. EGFP alone was present in
both the nucleus and diffused in the cytoplasm (Fig. 8, panels N1 and N2). Mch4-EGFP and caspase-10/d-EGFP were mostly
diffused throughout the cytoplasm (Fig. 8, panels
WT1 and B), but filaments could be seen in some
cells (Fig. 8, panels WT2 and B),
although these were never as distinct as those seen in
caspase-10/c-transfected cells. Killing assays performed on MCF7 cells
transiently overexpressing EGFP-caspase-10 fusion proteins confirmed
that these EGFP fusion proteins were functionally active and induced
apoptosis (data not shown).
Caspase-10/c mRNA Is Up-regulated by TNF--
It has been
shown that Mch4 and FLICE2 can associate with TNF receptor 1 via
homotypic DED interactions with Fas-associated death domain protein
(FADD) (15). It was of interest to know whether the expression of the
mRNAs of any of the caspase-10 isoforms was altered by TNF
stimulation. The addition of TNF resulted in a specific up-regulation
of caspase-10/c mRNA. This up-regulation was transient and
typically observed at 18 h after TNF stimulation (Fig.
9, panel A), although it was
sometimes observed at other times between 12 and 24 h
post-stimulation (data not shown). The peak of mRNA induction
occurred before or during the majority of apoptotic cell death (Fig. 9,
panel B) and was not observed in parallel control
experiments in which TNF was omitted (Fig. 9, panel A).
Previous work on the DED-containing protein MRIT
(c-FLIP/CASH/CLARP/I-FLICE/CASPER/FLAME-1/Usurpin) (22, 25-31) has
been controversial, because in some studies, MRIT had an inhibitory effect on apoptosis, whereas in others, MRIT induced cell death. Interestingly, this protein was reported to exist as two
splice-isoforms, with the shorter variant essentially comprising just
two DED domains, analogous in structure to the pro-apoptotic FLICE
isoform MACH How might caspase-10/c induce apoptosis after the possible formation of
the DEF? We showed that cell death induced by caspase-10/c was
inhibitable by Ac-DEVD-CHO, suggesting the requirement for caspase-3-like proteases. Yet, in MCF7 cells, caspase-3 is absent (32),
and we were unable to detect specific caspase-10/c-induced activation
of the two other known DEVD-inhibitable caspases 2 and 7,2
which is similar to the situation in TNF-induced apoptosis (33). Thus,
the caspases that are activated by the overexpression of caspase-10/c
in MCF7 cells remain to be identified.
It is known that caspase transcripts are constitutively present in many
cells, albeit at different levels of expression, and because apoptosis
can occur in the presence of protein synthesis inhibitors such as
cycloheximide (21), pro-caspases must already be present at sufficient
levels to allow the apoptotic response to proceed. Nonetheless, it has
been shown that caspase transcripts are up- and down-regulated in
development. For example, caspase-2 (Ich-1/Nedd2) mRNA is more
highly expressed in the mouse during embryonic development than in the
adult, particularly in the brain, liver, lung and kidney (34). Our
observations that the expression of Mch4 and caspases-10/c and
-10/d/FLICE2 is considerably higher in fetal than in adult tissues,
particularly in skeletal muscle, lung, and kidney, suggests a role for
the isoforms of caspase-10 during development.
Caspase mRNAs may also be up-regulated by extracellular stimuli as
well as in a genetic program during development. Caspase-1, -3, -4, -7, -8, and -10 transcripts are up-regulated within 16 h of
interferon- Accordingly, we propose a hypothetical model (Fig.
10) wherein the binding of TNF to its
receptors (most likely TNF receptor 1 (15)) results in the recruitment
and activation of caspase-8 and/or -10 at the plasma membrane, and
simultaneously, an as yet undefined signaling pathway stimulates
expression of caspase-10/c mRNA. A concomitant increase in
caspase-10/c protein may be sufficient for DEF scaffold formation and
subsequent intracellular caspase recruitment and activation, thereby
amplifying the apoptotic response. However, the existence of such an
amplification model in vivo remains to be proven, as we have
been unable to detect any increase in caspase-10/c protein levels by
Western blotting. First, the increase in caspase-10/c mRNA is
transient, occurring at any time from 12 to 24 h after TNF
treatment, and so the caspase-10/c protein synthesis may also be
transient. Second, it is possible that any TNF-induced increase in
caspase-10/c protein, although below the threshold of detection with
our antibody, is nonetheless sufficient to induce cell death.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-converting enzyme
(ICE1; caspase-1) (3), which
was the first of a series of proteases to be identified with an
active-site cysteine and a unique aspartate-Xaa substrate specificity.
In recognition of these properties, this family of apoptotic proteases
was renamed the caspases, for cysteine-aspartic acid proteases (4).
With the recent discovery of MICE (5), the number of caspases now
stands at 14, including murine proteases.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gal reporter plasmid (CLONTECH) using
Tfx-20 reagent (Promega) as directed by the manufacturer, except that
the cells were washed once with PBS before incubation with the
DNA-transfection reagent complex, and incubation was for 45 min at
37 °C. Where required, tetrapeptide inhibitors Ac-YVAD-CHO or
Ac-DEVD-CHO (Biomol) were added after this incubation. Approximately
16-24 h post-transfection, the cells were fixed in 0.25%
glutaraldehyde and stained with 0.2% 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal),
2 mM MgCl2, 5 mM
K4Fe(CN)6·3H2O, 5 mM
K3Fe(CN)6 for 2 to 10 h at 37 °C.
Blue-stained cells were scored as apoptotic on the basis of morphology.
Four random microscopic fields per dish were counted with a minimum of
400 blue cells per data set. For the concurrent determination of PARP
cleavage, transfected cells were boiled in 500 µl of Laemmli sample
buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 7.5% Ficoll
Type 400, 5%
-mercaptoethanol, 0.001% bromphenol blue), and the
extracts were immunoblotted using polyclonal anti-PARP antiserum
(Upstate Biotechnology, Inc.).
(BIOSOURCE)
was added directly to the conditioned medium at a final concentration
of 50 ng/ml where required. Treated cells were harvested, and RT-PCR
was performed essentially as described above.
was added at a final
concentration of 50 ng/ml. At various time points, the medium was
aspirated, and the cells were washed with 100 µl of PBS, fixed in 5%
formaldehyde, PBS for 5 min, then stained for 20 min at room
temperature in a solution of 0.5% crystal violet, 20% methanol. After
thorough washing to remove unincorporated dye, the plates were dried
completely at room temperature. The stained cells were then lysed using
50 µl of 33% acetic acid per well, and the absorbance at 550 nm was
measured in a microplate reader.
-D-thiogalactopyranoside-induced culture were
resuspended in 20 ml of ICE cleavage buffer (25 mM Hepes,
pH 7.4, 5 mM EDTA, 2 mM dithiothreitol, 0.1%
CHAPS) and sonicated for 3 × 20 s. After centrifugation for
30 min at 12,000 × g, the supernatants were collected
and used as the source of active caspase. Between 2 and 5 µl of
induced sonicate (depending on the amount of processed p12 small
subunit, as described under "Results") was added to 3 µl of
[35S]methionine-labeled substrates expressed in
vitro using the T7 Quick TnT system (Promega). ICE cleavage buffer
was then added to a final volume of 30 µl. Where used, the
tetrapeptide inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO were added to a
final concentration of 1 µM. In some experiments, the
radiolabeled in vitro translated proteins were first
affinity-purified by mixing the entire translation product with 200 µl of S-protein agarose slurry (Novagen) and using 10 µl of the
washed, protein-bound matrix as substrates.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Genomic organization of caspase-10 (adapted
from Ref. 22) and the predicted amino acid sequence alignments of the
four known isoforms on the basis of this organization. Exons are
numbered 1 to 12 at the top, with their lengths (in base pairs) shown
below. The amino acid sequence alignments are shown with translated
regions variously shaded as defined above. Unshaded regions
of exons are transcribed but do not code for protein. The translation
start (ATG) and stop (TAA, TAG) sites and caspase-processing points
(SQTD-V, IEAD-A) are shown.
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Fig. 2.
Amino acid sequence alignments of Mch4,
FLICE2, caspase-10/c, and caspase-10/d. Death effector domain
homology regions are underlined with thick horizontal
bars. The additional amino acids common to FLICE2, caspase-10/c,
and caspase-10/d coded for by exons 6-7 are in boldface
type, whereas the frameshifted sequence in caspase-10/c is
italicized. An open box is drawn around the amino
acids in caspase-10/d coded for by exon 11. The active-site cysteine
residue is highlighted by an asterisk. 10/c, caspase-10/c;
10/d, caspase-10/d.
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Fig. 3.
Antibodies against either the small or large
protease subunits of Mch4 recognize caspase-10/d but not
caspase-10/c. 5 µl of radiolabeled in vitro
translated Mch4, caspase-10/c, or caspase-10/d were electrophoresed in
12% SDS-polyacrylamide gels, transferred to nitrocellulose membranes,
and either autoradiographed (A) or immunoblotted (Western
blot) (B) with antibodies directed against the p12 subunit
of Mch4 (lanes 1-4) or the p17 subunit of Mch4 (lanes
5-8). , vector only; WT, Mch4; 10/c,
caspase-10/c; 10/d, caspase-10/d.
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Fig. 4.
Tissue and cell line distribution
of Mch4, caspase-10/c, and caspase-10/d/FLICE2 mRNAs.
A, first-strand cDNA (CLONTECH
multiple tissue cDNA (MTC)) prepared from various human adult or
fetal tissues was amplified by PCR using the primer pair C614/3BRIDGE
to distinguish between Mch4, caspase-10/c, and caspase-10/d/FLICE2
transcripts, as described in the text. The 200-bp fragment seen in the
adult pancreas lane is a spurious product not reproducible
in other experiments. The slight variation in
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels is
because of the multiple housekeeping gene normalization method employed
by the supplier. SK. MUSC., skeletal muscle; SMALL
INT., small intestine; PBL, peripheral blood leukocytes.
B, RT-PCR was performed on RNA prepared from various cell
lines. WT, 10/c, 10/d, control PCR
reactions using plasmids coding for Mch4, caspase-10/c, or
caspase-10/d, respectively. CTRLS, controls;
GAPDH, control RT-PCR using primers specific for
glyceraldehyde-3-phosphate dehydrogenase.
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Fig. 5.
Mch4 and caspase-10/d, but not caspase-10/c,
are autoprocessed and proteolytically active in
vitro. This activity is inhibited by Ac-DEVD-CHO but
not by Ac-YVAD-CHO. A, Mch4 and caspase-10/d undergo
autoprocessing. 20 µl of crude bacterial sonicates from 200-ml
bacterial cultures expressing GST fusion Mch4, caspase-10/c,
or cas pase-10/d were electrophoresed, transferred to
nitrocellulose, and immunoblotted with antibody against the Mch4 p12
small subunit. B, sonicates of GST-Mch4 or GST-caspase-10/d
cleave radiolabeled in vitro translated Mch4 and
caspase-10/d. C and D, cleavage of PARP and
caspase-3 by Mch4 or caspase-10/d is inhibitable by Ac-DEVD-CHO. 3 µl
of radiolabeled in vitro translated PARP (C) or
caspase-3 (D) were mixed with sonicates of GST-Mch4
(lanes 2, 6, and 10; 2 µl),
caspase-10/c (lanes 3, 7, 11; 5 µl),
or caspase-10/d (lanes 4, 8, 12; 5 µl). Lanes 1, 5, and 9 are controls
with 5 µl of pGEX-2TK vector sonicate added; , radiolabeled
in vitro translated product only. Lanes 5-8, 1 µM Ac-DEVD-CHO inhibitor added; lanes
9-12, 1 µM Ac-YVADCHO inhibitor added.
P1/P2, cleaved products of PARP;
Y1/Y2, cleaved products of caspase-3;
WT, 10/c, 10/d, Mch4, caspase-10/c,
and caspase-10/d, respectively; GWT, G10/c,
G10/b, GST-Ile194-Mch4,
GST-Ile194-caspase-10/c, and
GST-Ile194-caspase-10/d, respectively. TnT, T7
in vitro transcription and translation system
(Promega).
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Fig. 6.
Apoptosis induced by the transient
overexpression of Mch4, caspase-10/c, or caspase-10/d in MCF7 cells
involves PARP cleavage and is inhibited by Ac-DEVD-CHO but not by
Ac-YVAD-CHO. A, solid black bars represent
killing in the absence of inhibitors. Gray shaded bars
represent killing in the presence of Ac-DEVD-CHO. White bars
represent killing in the presence of Ac-YVAD-CHO. The expression levels
of the transfected proteins are shown in Fig. 7, panel B.
B, immunoblot showing PARP cleavage in transfected MCF7
cells. NIL, no DNA added. CI ( ), vector only;
WT, 10/c, 10/d, Mch4, caspase-10/c,
and caspase-10/d, respectively.
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Fig. 7.
Mch4 and caspase-10/d are soluble proteins,
whereas caspase-10/c is present mainly in the insoluble fraction of
cell lysates. 293T (A) or MCF7 (B) cells
transiently overexpressing Mch4, caspase-10/c, or caspase-10/d
(WT, 10/c, 10/d, respectively) were
lysed and separated into detergent-soluble and -insoluble fractions as
described in the text. Lysates were immunoblotted with anti-Mch4 p12
polyclonal antibody to detect Mch4 or caspase-10/d protein (open
arrows, upper blots) or with custom antiserum against
caspase-10/c protein (open arrows, lower blots).
+, total protein lysates of cells overexpressing caspase-10/c; ,
vector-only transfection.
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Fig. 8.
Caspase-10/c localizes to distinct
perinuclear filaments. MCF7 cells transiently overexpressing EGFP
(N1 and N2), Mch4-EGFP (WT1 and
WT2), caspase-10/c-EGFP (A), or caspase-10/d-EGFP
(B) were stained with propidium iodide to allow nuclear
visualization then observed under confocal laser scanning microscopy.
N2 is a single-channel output of N1 and shows the
nuclear and cytoplasmic distribution of EGFP.
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Fig. 9.
Caspase-10/c mRNA is specifically
up-regulated by TNF and correlates with TNF-induced cell death.
A, MCF7 cells were stimulated with 50 ng/ml TNF (lanes
4-7) and harvested at various times as indicated. RT-PCR was
performed on RNA prepared from these cells as detailed in the text, and
the products were electrophoresed in 2.5% agarose gels. Lanes
8-11, parallel experiment without TNF. The figure shows one set
of results that are representative of at least three similar
experiments. WT, 10/c, 10/d, control
PCR reactions using plasmids coding for Mch4, caspase-10/c, or
caspase-10/d, respectively. GAPDH, control RT-PCR using
primers specific for glyceraldehyde-3-phosphate dehydrogenase.
B, TNF was added to MCF7 cells grown in 96-well plates, and
the extent of killing at various time points was measured as described
under "Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (12) and our caspase-10/c. The recent work on DEF (24)
sheds some light on the reason for this controversy, suggesting that apoptotic activity is a concentration-dependent phenomenon.
Expression of only the DED of FLICE (but not the full-length protein)
above a certain concentration threshold resulted in the formation of an
insoluble filamentous perinuclear structure that apparently acted as a
scaffold to recruit and facilitate the autoactivation of cytoplasmic
DED-containing pro-caspases (24). We also showed that pro-apoptotic
caspase-10/c is present mainly in the insoluble fraction of cell
lysates (in contrast to the predominantly soluble full-length Mch4 and
caspase-10/d proteins) and that ectopically expressed caspase-10/c-EGFP
fusion protein localizes intracellularly as distinct perinuclear
structures identical to the DEFs described previously. These
observations provide a plausible explanation for the mechanism of
action of caspase variants containing only the DED and suggest that
caspase-10/c and the
-isoforms of FLICE and MRIT may play a role in
amplifying the apoptotic response through the formation of similar
"activating scaffolds."
stimulation (35, 36), and the CASP-2 and -3 genes are also up-regulated in response to etoposide treatment (37). We
showed TNF induces the expression of caspase-10/c mRNA but not
caspase-10/d or Mch4 transcripts, the first demonstration that the
mRNA of a protein encoding only the DED is up-regulated by a
specific stimulus. Overall, these studies suggest that cytokine-induced cytotoxicity could potentially involve the up-regulation of specific caspase isoforms.
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Fig. 10.
Hypothetical model for the involvement of
caspase-10/c and other DED-only proteins in the amplification of
apoptotic signals via TNF receptor 1 (TNFR 1).
Binding of TNF to its receptors results in the recruitment and
activation of caspases-8 and/or -10 at the plasma membrane
(A). Simultaneously, an as yet undefined signaling pathway
stimulates cell type-specific expression of caspase-10/c mRNA
(B), resulting in a concomitant increase in caspase-10/c
protein (C), perinuclear DEF scaffold formation
(D), and subsequent intracellular caspase recruitment and
activation (E), thereby amplifying the apoptotic response.
TRADD, TNF receptor-associated death domain protein.
It appears that within each family of the DED-containing proteins
(caspase-8, -10, and MRIT) there exists a cohort of isoforms (12, 25)
with variations in the composition of the protease domains depending on
variations in splicing. These isoforms appear to be analogous in
structure between the families, suggesting a common pattern of
functionality. For instance, expression of DED-only isoforms is
pro-apoptotic, whereas expression of longer isoforms (lacking catalytic
residues) inhibits apoptosis (12). Together with the results presented
here and elsewhere showing that caspases and caspase isoforms can be
regulated spatially (depending on tissue type) and temporally
(depending on stage of development), a picture emerges in which cells
are allowed a high degree of control over the apoptotic process.
Multiple caspases and caspase isoforms could potentially enable
specialization in response to external stimuli in organ development and
in cellular substrate cleavage, depending on local protein
concentrations and subcellular localization. Multiple isoforms of the
same caspase could also fine-tune caspase activity through
hetero-oligomerization or differential expression, resulting in either
an amplification or inhibition of the apoptotic response.
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ACKNOWLEDGEMENTS |
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We thank Dr. V. Dixit for the 293T cell line and the plasmids encoding PARP and caspase-3/Yama. We are grateful to Dr. V. Yu and Dr. P. Li for reviewing this manuscript and to Dr. M. Choi for useful discussions.
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FOOTNOTES |
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* This work was supported by the Institute of Molecular and Cell Biology, Singapore.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF111344 (caspase-10/c) and AF111345 (caspase-10/d).
To whom correspondence should be addressed: TNF and Cell Death
Group, Institute of Molecular and Cell Biology, 30 Medical Dr.,
Singapore 117609, Republic of Singapore. Tel.: 65-874-3761; Fax:
65-779-1117; E-mail: mcbagp{at}imcb.nus.edu.sg.
2 P. W. P. Ng, A. G. Porter, and R. U. Jänicke, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
ICE, interleukin
1-converting enzyme;
TNF, tumor necrosis factor;
DED, death effector
domain;
DEF, death effector filaments;
EGFP, enhanced green fluorescent
protein;
GST, glutathione S-transferase;
PARP, poly(ADP-ribose) polymerase;
PCR, polymerase chain reaction;
RT-PCR, reverse transcription-PCR;
CHAPS, 3-[3-cholamidopropyldimethylamino]-1-propanesulfonate;
PBS, phosphate-buffered saline;
bp, base pair(s).
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REFERENCES |
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