From the Department of Pathology and Comprehensive
Cancer Center, The University of Michigan Medical School and
¶ Department of Biology, The University of Michigan, Ann Arbor,
Michigan 48109, the Departments of § Laboratory Medicine and
Molecular Oncology and Angiology, Research Center on Aging and
Adaptation, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, Nagano 390-8621, Japan, and the ** Institute of
Neuroscience, University of Oregon, Eugene, Oregon 97403
Received for publication, April 23, 2002, and in revised form, November 22, 2002
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ABSTRACT |
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The pyrin domain was identified recently
in multiple proteins that are associated with apoptosis and/or
inflammation, but the physiological and molecular function of these
proteins remain poorly understood. We have identified Caspy and Caspy2,
two zebrafish caspases containing N-terminal pyrin domains. Expression
of Caspy and Caspy2 induced apoptosis in mammalian cells that were
inhibited by general caspase inhibitors. Biochemical analysis revealed
that both Caspy and Caspy2 are active caspases, but they exhibit
different substrate specificity. Caspy, but not Caspy2, interacted with the zebrafish orthologue of ASC (zAsc), a pyrin- and caspase
recruitment domain-containing protein identified previously in mammals.
The pyrin domains of both Caspy and zAsc were required for their
interaction. Furthermore, zAsc and Caspy co-localized to the
"speck" when co-transfected into mammalian cells. Enforced
oligomerization of zAsc, but not simple interaction with zAsc, induced
specific proteolytic activation of Caspy and enhanced
Caspy-dependent apoptosis. Injection of zebrafish
embryos with a morpholino antisense oligonucleotide corresponding to
caspy resulted in an "open mouth" phenotype
associated with defective formation of the cartilaginous pharyngeal
skeleton. These studies suggest that zAsc mediates the activation of
Caspy, a caspase that plays an important role in the morphogenesis of the jaw and gill-bearing arches.
Apoptosis, a morphologically distinguished form of programmed cell
death, is critical during development and tissue homeostasis and plays
a role in the pathogenesis of a variety of diseases. The morphological
features of apoptosis and demise of the cell are because of the
cleavage of structural and non-structural proteins by a family of
proteases called caspases (for review, see Ref. 1). Upon stimulation by
apoptotic stimuli, proximal caspases are activated and cleave
downstream effector caspases leading to an amplification of proteolytic
activity within the dying cells. Activation of proximal caspases
requires upstream regulators that have domains homologous to those
present in the N-terminal prodomains of proximal caspases, namely death
effector domains (DEDs)1 and
caspase recruitment domains (CARDs). Homophilic interactions between
the CARDs or DEDs of upstream regulators and proximal caspases mediate
caspase activation (1). For example, homophilic interactions between the DEDs of Fas-associated death
domain protein and procaspase-8 and between the CARDs of Apaf-1 and
procaspase-9 are essential for caspase activation (2). Activation of
proximal caspases is thought to be induced via proximity of their
catalytic subunits, a process that is mediated through oligomerization
of their upstream regulators. For example, Fas-associated death domain protein and Apaf-1 contain a death domain (DD) and nucleotide-binding domain, respectively, that mediate their oligomerization in a ligand-dependent manner (1).
Bioinformatic and protein structure analyses have revealed that the
CARD, DED, and DD share similar structural and functional features
(3-6). These domains are found not only in apoptosis regulators but
also in proteins that are associated with inflammation, cell cycle
regulation, and cytoskeletal organization (4, 7). Thus, these
The pyrin domain (also called DAPIN, PYRIN, and PAAD) was originally
found in pyrin, the product of the familial Mediterranean fever-associated gene (12), and in ASC, a component of the "speck," a granular structure induced in the cytosol of certain apoptotic cells
(13). Subsequently, pyrin domains were also identified in multiple
proteins whose molecular functions are largely unknown (14-19).
Computer modeling has predicted that the pyrin domain is The vertebrate zebrafish (Danio rerio) is a model organism
whose genome encodes the great majority of the components that mediate
apoptosis and inflammation in mammals including humans (10, 21). Thus,
analysis of zebrafish components should provide insight into the
mechanisms that mediate apoptosis and inflammation in mammalian
systems. Here we report the identification and initial characterization
of the zebrafish orthologue of ASC (zAsc) and two zebrafish
pyrin-containing caspases, Caspy and Caspy2. We provide evidence that
zAsc associates with and activates Caspy through a homophilic
pyrin-pyrin domain interaction. In addition, we show that Caspy is
required for normal development of the jaw and pharyngeal arches.
Identification of Caspy, Caspy2, and zAsc cDNAs and
Preparation of Expression Plasmids--
Zebrafish genes with homology
to the pyrin domain of human ASC were searched in public data bases of
ESTs, using the program TBLASTN (National Center for Biotechnology
Information). The nucleotide sequences of the EST clones AW174631,
AI384922 (zAsc), AI331460 (Caspy), and BF156256 (Caspy2) were
determined by dideoxy sequencing. The entire open reading frames of
zebrafish Caspy, Caspy2, and zAsc were amplified by polymerase chain
reaction from a cDNA pool prepared from the whole body of an adult
zebrafish. The EST clones AW174631 and BF156256, respectively, were
cloned into pEGFP-C2, pDsRed-N1 (Clontech),
pFLAG-CMV-4 (Sigma), pcDNA3, pcDNA3-Myc, pcDNA3-HA (22),
and pcDNA3-Fpk3-Myc (23) to generate pEGFP-Caspy, pEGFP-zAsc, pDsRed-zAsc, pFLAG-Caspy, pcDNA3- Caspy,
pcDNA3-Caspy2, pcDNA3-Caspy-Myc, pcDNA3-Caspy2-Myc,
pcDNA3-zAsc-HA, pcDNA3-zAsc-Myc, and pcDNA3-zAsc-Fpk3-Myc.
pcDNA3-Caspy-(1-100)-Myc and pcDNA3-Caspy-(101-383)-Myc were
generated from the DNA fragments corresponding to the regions between
residue 1 and 100 and between residue 101 and 383, respectively, in
Caspy subcloned in pcDNA3-Myc. pcDNA3-zAsc-(1-103)-Myc and pcDNA3-zAsc-(104-203)-Myc were generated from the DNA fragments corresponding to the regions between residue 1 and 103 and between 104 and 203, respectively, in zAsc subcloned in pFLAG-CMV4. The plasmids
pcDNA3-Caspase-9-FLAG, pcDNA3-Apaf-1XL-Myc, and pEF1BOS- Genetic Mapping and in Situ Hybridization--
Genetic mapping
was performed on the heat shock doubled haploid meiotic mapping panel
by single strand conformation polymorphism as described (25), using the
following mapping primers: caspy, 5'-GAAGTAAAATGGGCACAAGTCACCTA-3' and 5'-AGGGTCCCCAATTCGCTAAA-3'; caspy2 Set1, 5'-TCAGAGGGAAACAGCACAGG-3' and
5'-TAAAGCCCAAGCAATACAAATAAA-3'; Set2, 5'-CAAAGCGGCTCGAGTCCAATCT-3' and
5'-GAGCCGGACAGTTCACAGAGTTCA-3'; Set3, 5'-CAAAGCGGCTCGAGTCCAATCT-3' and
5'-TCCGCGTTCTCCTCGTCTCTGT-3'.
For in situ hybridization, digoxigenin-labeled antisense RNA
probes were synthesized from full-length cDNAs using an in
vitro transcription kit (Promega). As a control, sense RNA-labeled
probes were synthesized and used for hybridization as above. In
situ hybridization and development of whole-mount zebrafish
embryos were performed as described (26).
Transfection, Expression, Immunoprecipitation, and
Immunodetection of Tagged Proteins--
Cells were transfected, and
immunoprecipitation assay was performed as described with slight
modifications (27). The total amount of transfected plasmid DNA was
adjusted with pcDNA3 to always be the same within individual
experiments. 1 × 106 COS-7 cells were transfected
with 2.4 µg of expression plasmids using LipofectAMINE-PLUS reagent
(Invitrogen) according to the manufacturer's instructions. The
supernatant was immunoprecipitated with 20 µl of protein G-Sepharose
4B (Amersham Biosciences) conjugated with anti-Myc polyclonal antibody
(Santa Cruz Biotechnology, Inc.). Immunoprecipitated proteins were
subjected to 16% SDS-polyacrylamide electrophoresis and detected by
Western blotting using anti-FLAG monoclonal antibody M2 (Sigma). For
cellular localization assay, COS-7 cells were transiently cotransfected
with pEGFP-zAsc and pEGFP-Caspy. For coexpression assay, COS-7 cells
were transiently cotransfected with pDsRed-zAsc and pEGFP-Caspy. After
24 h, cells were fixed with 70% ethanol. Signals were detected by
immunofluorescence microscopy.
Cell Death Assays and Caspase Enzymatic Assay--
Cell death
assays with 293T cells were performed as described (28). For Fig.
4B, transfected cells were incubated with 20 µM zVAD-fmk
(benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone) or left
untreated. FLAG-Caspy and Caspy2-Myc were purified by immunoprecipitation from 293T cells transfected by
pcDNA3-FLAG-Caspy and pcDNA3-Caspy2-Myc, respectively, as
described above, and caspase enzymatic assays were performed as
described (29) using caspase-type-specific substrates (Calbiochem, La
Jolla, CA) indicated in the figure legends. The concentration of the
cleavage products was estimated from comparison with the fluorescence
of AMC (7-amino-4-methyl-coumarin) or AFC
(7-amino-4-(trimethyl-fluoromethyl) coumarin) standards.
Microinjection of Morpholino Oligonucleotides and Phenotypic
Analyses--
Each morpholino (Gene Tools, LLC) was resuspended in
sterile water to a concentration of 1 mM. For injections,
the stock solution was diluted to 250 nM in Danieau buffer.
A 3-nl volume of the solution was injected into one to four cell-stage
embryos at the yolk and cytoplasm interface. The sequences of
caspy and control morpholino oligonucleotides were
5'-GCCATGTTTAGCTCAGGGCGCTGAC-3' and 5'-CCTCTTACCTCAGTTACAATTTATA-3',
respectively. For the morphological assay, larvae were euthanized in
tricane, fixed in 4% paraformaldehyde, and washed for 3 min in
acid-alcohol (0.37% HCl, 70% ethanol). The fixed larvae were stained
for 1 h with 0.1% Alcian blue. After rinsing in ethanol, animals
were suspended in 50% glycerol, 0.25% KOH overnight. Stained
preparations were clarified with 1% H2O2 and
mounted in glycerol-KOH.
Identification of a Novel Class of Caspases with Pyrin
Domain--
To identify novel pyrin domain-containing proteins in
zebrafish, we screened the zebrafish GenBankTM EST data
base for cDNAs encoding amino acid sequences with homology to the
pyrin domain (amino acids 1-91) of human ASC by the program TBLASTN.
We identified the zebrafish orthologue of human ASC (referred to here
as zAsc) and two zebrafish caspases (designated here as Caspy and
Caspy2). Sequence analysis of zAsc cDNA revealed that it encodes a
protein of 203 amino acid residues composed of a N-terminal pyrin
domain linked to a C-terminal CARD as reported for its mammalian
orthologues (GenBankTM accession numbers were as
follows: human ASC, AB023416; mouse ASC, AB032249; bovine ASC,
AB050006; rat ASC, AB053165). Caspy and Caspy2 contained N-terminal
pyrin domains and C-terminal caspase catalytic domains (Fig.
1A). The N-terminal pyrin
domain of Caspy was most homologous to that present in zAsc (87%
similarity) whereas that of Caspy2 was most homologous to that of human
Cryopyrin/PYPAF1 (46% similarity), a pyrin-domain containing
Nod-family protein (30, 31). Comparison of these zebrafish and human
caspases showed that the catalytic domains of Caspy and Caspy2 share
highest homology with those of human caspase-1 and caspase-5,
respectively (54 and 57% similarity, respectively). Caspy and Caspy2
contained conserved His and Cys amino acid residues, which are
essential for proteolytic catalysis (depicted by arrows in
Fig. 1A), and also conserved residues, which are essential
for interaction with critical core residues of substrates, suggesting
that both Caspy and Caspy2 are enzymatically active. Notably, Caspy,
like human caspase-1, has His at position 318, corresponding to the
residue at position 342, which binds the specific P3 residue of
the substrate (Ala of YVAD) in human caspase-1 (32), whereas Caspy2 and
human caspase-5, which can not digest peptides carrying Ala residue at
P3 (33), have Asn and Asp residues, respectively, at the corresponding
positions (arrowhead; see Fig. 1A).
Genomic Mapping of asc, caspy, and
caspy2--
Genomic mapping experiments using the heat shock
doubled haploid meiotic mapping panel revealed that caspy
and caspy2 are located on linkage groups 16 and 1, respectively (Fig. 2). Human CASP1 and CASP5, which are homologues of
caspy and caspy2, are located as tandem genes on
human chromosome 11. We did not find any conclusive evidence supporting
conserved syntenies between the zebrafish and human regions containing
the zebrafish and human caspase genes (Fig. 2). Notably,
caspy was adjacent to asc on linkage group 16 (z06s003336 at www.ensembl.org/Danio_rerio/blastview), whereas the human orthologue of asc is located on chromosome
16. Sequence analysis also showed that the pyrin domain of Caspy2 exhibited significant homology not only to pyrin domains but also to
the CARDs of Xenopus caspase-1 (49% similarity) and bovine caspase-13 (45% similarity, previously known as human caspase-13) (Fig. 1B). Thus, both the pyrin domain and CARD are
predicted to be zAsc, Caspy, and Caspy2 mRNA Exhibit a Similar Pattern of
Expression in the Zebrafish Embryo--
We performed in
situ hybridization to assess the expression of zAsc, Caspy, and
Caspy2 mRNA in zebrafish embryos. At embryonic development of 48 and 72 h post-fertilization, zAsc (Fig.
3, A-C), Caspy
(Fig. 3, E-G), and Caspy2 (Fig. 3,
I-K) mRNA exhibited a similar labeling
pattern with expression being detected primarily in epidermis (arrows),
mouth (single arrowhead), and pharyngeal arches (white
arrowheads) shown in Fig. 3. Significantly, the expression of
asc and caspy2 appeared stronger than that
observed for caspy. Similar results were observed in embryos
at 36 h
post-fertilization.2 The
expression pattern of asc (Fig. 3, A and
B) is consistent with that of human ASC, which was reported
to be expressed in epithelial cells including squamous epithelium of
skin and mouth (34).
Caspy and Caspy2 Induce Apoptosis in Mammalian Cells--
To begin
to assess the molecular function of Caspy and Caspy2, we constructed
expression plasmids of tagged and non-tagged Caspy and Caspy2 and
transiently transfected the plasmids into 293T cells, respectively. A
significant percentage of Caspy- and Caspy2-transfected cells displayed
morphological features of adherent cells undergoing apoptosis such as
rounding, membrane blebbing, and detachment from the dish (Fig.
4A). At 24 h
post-transfection, about 20-40 and 95% of the cells transfected with
the highest dose of both tagged and non-tagged Caspy and Caspy2
constructs, respectively, displayed morphological features of apoptosis
compared with less than 1% of the cells transfected with control
plasmid (Fig. 4A). To determine whether the proapoptotic
activity of Caspy and Caspy2 is because of the their enzymatic
activities, the ability of these proteins to induce apoptosis was
tested in the presence of the caspase inhibitors, p35, CrmA, and
zVAD-fmk. Expression of baculoviral p35 attenuated apoptosis induced by
both Caspy and Caspy2 whereas incubation with zVAD-fmk greatly
inhibited apoptosis (Fig. 4B). Interestingly, CrmA, an
inhibitor of human caspase-1 and certain caspases (35), inhibited
apoptosis induced by Caspy but not by Caspy2 (Fig. 4B).
These data were also similar compared with the results using non-tagged
Caspy and non-tagged Caspy2 (Fig. 4B). These results suggest
that apoptosis induced by Caspy and Caspy2 is dependent on caspase
activity and that the proapoptotic activity of Caspy and Caspy2 can be
dissociated based on the inhibition with CrmA.
Caspy and Caspy2 Are Active Caspases with Different Substrate
Specificity--
To test more directly whether Caspy and Caspy2 are
active enzymes, a panel of fluorogenic peptide substrates of mammalian caspases were incubated with Caspy or Caspy2 immunoprecipitated from
extracts of cells transfected with plasmids producing the relevant
caspases. Both caspases cleaved several caspase peptide substrates,
demonstrating that Caspy and Caspy2 are active caspases (Fig. 4,
C and D). Notably, Caspy preferentially cleaved
AcYVAD-AMC, a caspase-1 substrate, whereas Caspy2 was more active on
AcWEHD-AFC, a preferred substrate of caspase-5, and it did not cleave
AcYVAD-AMC (Fig. 4, C and D). These results
suggest that Caspy and Caspy2 are enzymatically active caspases that
exhibit different substrate specificity.
Zebrafish ASC Interacts with Caspy but Not with
Caspy2--
Because Caspy and Caspy2 contain a pyrin domain highly
homologous to that of zAsc, the ability of both caspases to interact with zAsc was tested using a co-immunoprecipitation assay. zAsc was
co-immunoprecipitated with Caspy but not with Caspy2 (Fig. 5). Mutational analysis revealed that a
zAsc mutant containing the pyrin domain but lacking the CARD was
co-immunoprecipitated with Caspy (Fig. 5, FLAG-zAsc-PD),
suggesting that the pyrin domain of zAsc mediates the binding to Caspy.
Furthermore, a zAsc mutant-lacking pyrin domain did not
co-immunoprecipitate with Caspy (Fig. 5, FLAG-zAsc- Enforced Oligomerization of zAsc Induces
Caspy-dependent Apoptosis--
To determine the functional
relevance of the interaction between Caspy and zAsc, we first tested
whether co-expression of zAsc enhances apoptosis induced by Caspy and
Caspy2. In these experiments we expressed low amounts of plasmid so
that we could assess whether zAsc could enhance tagged and non-tagged
Caspy- and Caspy2-induced apoptosis. Under these experimental
conditions, expression of zAsc did not augment apoptosis induced by
either Caspy or Caspy2 (Fig. 7,
A and B). This result suggested that the
interaction between Caspy and zAsc was not sufficient to induce caspase
activation. Enforced oligomerization of the CARD of Apaf-1 and that of
the DD of Fas, which are protein modules structurally related to the
pyrin domain, is known to induce the activation of target caspases (36,
37). Therefore, we hypothesized that zAsc might similarly promote Caspy
activation through zAsc oligomerization. To test this hypothesis, we
constructed a plasmid to express a zAsc-Fpk3 fusion protein that can be
oligomerized with the ligand AP1510 (14). Co-expression of zAsc-Fpk3,
like that of zAsc, did not enhance apoptosis induced by Caspy in the
absence of AP1510 (Fig. 7, A and B). In contrast,
greater than 50% of the cells underwent apoptosis when both zAsc-Fpk3
and Caspy were co-expressed in the presence of AP1510 (Fig. 7,
A and B). In control experiments, co-expression
of zAsc-Fpk3 did not enhance the ability of Caspy2 or caspase-9 to
induce apoptosis in the presence of AP1510 (Fig. 7B).
In reciprocal experiments, co-expression of Apaf-1, a regulator of
caspase-9, enhanced apoptosis induced by caspase-9, but it did not
augment Caspy- or Caspy2-mediated apoptosis (Fig. 7B). These
results suggest that oligomerization of zAsc is required for
enhancement of Caspy-dependent apoptosis. Oligomerization of zAsc is presumably induced through an interaction between the C-terminal CARD of zAsc and a putative upstream regulator that needs to
be identified. Because Caspy2 did not interact with zAsc, we suggest
that Caspy2, which possesses caspase-5 like enzymatic activity,
mediates apoptosis and/or inflammation in a zAsc-independent manner.
Enforced Oligomerization of zAsc Induces Proteolytic Processing of
Caspy--
To examine whether the activation of Caspy is enhanced by
zAsc, we determined whether oligomerization of zAsc induces the processing of Caspy. Analysis of cell extracts by immunoblotting revealed that a processed form of Caspy was immunodetected in extracts
from cells co-expressing zAsc-Fpk3 and Caspy in the presence but not in
the absence of dimerizer AP1510 (Fig. 7C). When Caspy was
immunoprecipitated with antibody, processing of Caspy was observed in
extracts from cells co-expressing Caspy and zAsc even in the absence of
the dimerizer (Fig. 7C). A possible explanation for the
latter finding is that Caspy activation can be induced through
antibody-mediated aggregation of zAsc-Caspy complexes in
vitro resulting from the immunoprecipitation procedure.
Significantly, zAsc was still required for processing of Caspy in
vitro, suggesting that the interaction of zAsc with Caspy is
important for caspase processing and activation.
Morpholino Antisense Oligonucleotide-based Knocked-down of
Caspy Affects Development of the Jaw and Posterior Pharyngeal
Arches--
To assess whether Caspy was required for normal
development of zebrafish, we designed a morpholino antisense
oligonucleotide to interfere with caspy translation.
Microinjection of the blocking antisense oligonucleotide into one- to
four-cell stage embryos resulted in an open mouth phenotype as
observed by simple morphological examination of larvae at 6 days
post-injection. This phenotype was observed at least 4 days
post-injection. To assess the phenotype in more detail, we injected
caspy and control morpholino oligonucleotides into early
cleavage embryos and stained the head cartilage of the resulting larvae
with Alcian blue. The analysis revealed that 12/19 larvae injected with
the caspy antisense morpholino exhibited several
abnormalities in the development of the cartilaginous pharyngeal
skeleton. The abnormal phenotype included deformed and thinned
Meckel's and palatoquadrate cartilages, which form the jaw, deformed
ceratohyal cartilages, which are pointing posteriorly, and disorganized
branchial cartilages (Fig. 8). The larvae
were still alive approximately 1 week post-fertilization but then
started to die as yolk disappeared. Of the seven remaining larvae
derived from early embryos injected with the caspy antisense
oligonucleotide, 5/19 showed a similar but less marked phenotype,
whereas 2/19 larvae appeared normal. The phenotype is consistent with
the expression of caspy in the pharyngeal arches (Fig. 3).
No other abnormalities were identified in any other part of the body
including the brain, eyes, or pectoral fins of the larvae injected with
the caspy antisense oligonucleotide. The latter is
consistent with the observation that caspy is not expressed
in the brain, eyes, or pectoral fins during embryonic development (Fig.
3). None of the embryos injected with control morpholino
oligonucleotide showed any alteration in the cartilaginous structures
of the jaw or gills.
In this report we provide evidence that the zebrafish ASC mediates
the activation of Caspy, a pyrin domain-containing caspase. Although
mammalian orthologues of zebrafish Caspy have not been yet identified,
ASC is highly conserved in vertebrates. It is possible that mammals
have Caspy-like molecules with typical pyrin domains, but they need to
be identified. Because the CARD is highly related to the pyrin domain,
it is also possible that one of the mammalian CARD-containing caspases
is the Caspy orthologue. Caspy exhibits the highest homology to human
caspase-1 and preferentially cleaved caspase-1 substrates whereas
Caspy2 showed the highest homology to human caspase-5 and preference
for caspase-5-like substrates. Apoptosis induced by Caspy was blocked
by CrmA, a caspase inhibitor that inhibits human caspase-1 at low
concentrations (35) (Fig. 4B). Both caspase-1 and caspase-5
belong to the same subfamily of CARD-containing caspases (33), but
their activator(s) are unknown or are poorly understood. Thus, it will
be important to test for interactions between these known
CARD-containing caspases and mammalian ASC.
zAsc, like its mammalian orthologues, is composed of an N-terminal
pyrin domain linked to a C-terminal CARD. Recent studies of the
three-dimensional structure of several apoptosis regulators have
suggested that the stoichiometry of the homophilic interaction between
the CARD and related domains is one by one (38, 39). Therefore, it is
unlikely that the pyrin domain of zAsc binds to other pyrin or
CARD-containing factors in the Caspy/zAsc signaling complex. Enforced
oligomerization of zAsc enhanced Caspy-induced apoptosis. These
findings suggest that the C-terminal CARD of zAsc and that of its
mammalian orthologues might be important for oligomerization. In this
model, zAsc acts as an adaptor molecule linking a putative upstream
factor to caspases such as Caspy. Potential upstream factors include
Defcap/NAC/CARD7/NALP1 and Ipaf-1/CARD12/CLAN, Cryopyrin/PYPAF1, CARD-,
or pyrin domain-containing proteins with structural homology to a
family of Apaf-1-like molecules that include Nod1 and Nod2 (9, 15-16,
30-31, 40-45). Such a factor may interact via its CARD with Caspy, an
event that may lead to zAsc oligomerization and association with Caspy,
leading to caspase activation. Alternatively, self-oligomerization of
zAsc might be mediated via its CARD, which might be induced by upstream
molecules such as protein kinases or other factors. Consistent with the latter, it has been shown that a mutant form of human ASC carrying the
CARD alone can self-associate (20). Further studies are required to
understand the mechanism by which caspases are activated by zAsc and
its mammalian orthologues.
Analysis of animals in which caspy activity was knocked down
by morpholino antisense oligonucleotide revealed abnormal development of the pharyngeal skeleton containing the jaw and branchial arches. The
pharyngeal skeleton in vertebrates derives from migrating neural crest
cells, and its development involves a complex program of interactions
among neural crest cells, mesodermal mesenchyme, and surrounding
epithelia (46). The abnormal phenotype involving the anterior and
posterior arch cartilages indicates that Caspy is required for proper
development of neural crest derivatives. It is possible that Caspy is
involved in the execution of a program of neural crest cell death
required for proper development and assembly of the cartilages. For
example, cell death may be required to release the ceratohyal from
ventral attachment to the basibranchials allowing it to assume its
normal anterior projection. Terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL) analysis did
not reveal any differences in the level of apoptosis between
caspy knocked-down and control morphants.2
However, we cannot rule out a subtle defect in apoptosis that was not
revealed by terminal deoxynucleotidyl transferase-mediated dUTP nick
end labeling analysis. Another possibility is that Caspy is involved in
processing a zebrafish homologue of interleukin-1/interleukin-18 and/or another factor that is required for normal development of
pharyngeal arch cartilages. For example, mutant mice deficient in the
endothelin-1 converting enzyme, which processes the endothelin-1 precursor to biologically active endothelin-1, exhibit defects in
branchial arch-derived craniofacial tissues (47). Furthermore, endothelin-1 is essential for development of the ventral aspect of
zebrafish pharyngeal arches (48). Genetic screens in the zebrafish have
revealed a large number of loci required for pharyngeal arch
development (49, 50). Some of the mutant fish exhibit phenotypes that
are similar but not identical to that observed in caspy
morpholino knocked-down animals. Thus, it is possible that Caspy is a
component of a signaling pathway that includes some of the genes
identified in the genetic screens. Additional studies are needed to
understand the precise role of Caspy in the genetic program involved in
the development of the jaw and branchial arch cartilages.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix-rich domains mediate homophilic interactions between
signaling components of diverse signaling pathways, most notably those
involved in apoptosis and inflammation (8-11).
-helix-rich
and structurally related to the CARD, DED, and DD. Furthermore, the
pyrin domain mediates homophilic interactions between pyrin
domain-containing proteins (16, 20). However, the functional
significance of these pyrin-pyrin homophilic interactions is presently
poorly understood.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal have been described (9, 24).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structure of Caspy and Caspy2.
A, amino acid sequences of zebrafish Caspy and
Caspy2. The pyrin and caspase domains are indicated by a bar
and box, respectively. The conserved catalytic histidine and
cysteine residues of caspases and the histidine residue corresponding
to that of caspase-1, which binds to P3 of substrates, are indicated by
arrows and an arrowhead, respectively.
B, homology between pyrin domains and CARDs. The putative
-helices (H1a to H5) are shown according to
the three-dimensional structure of the CARD of Apaf-1 (6).
-helix rich and to share a similar organization in
their predicted secondary structure (Fig. 1B).
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Fig. 2.
Genomic mapping of asc,
caspy, and caspy2. The
chromosomal locations of asc, caspy, and
caspy2 were determined as described under "Experimental
Procedures." The localization of asc, caspy,
and caspy2 is indicated by arrows. The
chromosomal localization of human orthologues was determined by
NCBI-BLAST programs (www.ncbi.nlm.nih.gov/BLAST) and are given in
parentheses.
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Fig. 3.
In Situ hybridization analysis of
zebrafish ASC, Caspy, and Caspy2 mRNA expression in zebrafish
embryos. Panels A and D, E and
H, and I and L depict lateral views of
the head of whole-mounted embryos at 48 h post-fertilization, and
panels C, G, and K depict ventral
views of the head of whole-mounted embryos at 72 h
post-fertilization, analyzed by in situ hybridization with
the indicated antisense and sense probes of asc,
caspy, and caspy2, respectively. Labeling of the
epidermis (black arrows), mouth (black
arrowhead), and pharyngeal arches (white arrowheads) is
indicated. Panels B, F, and J depict
high power views to show labeling of individual cells in epidermis
(black arrows). Scale bars are 40 µm
(A, C-E, G-I,
K, and L) and 20 µm (B,
F, and J).
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Fig. 4.
Both Caspy and Caspy2 induce apoptosis in
mammalian cells and exhibit differential substrate specificity.
A, dose-dependent induction of apoptosis by
Caspy and Caspy2 in 293T cells. 5 × 104 293T cells
were cotransfected with the indicated amount of empty vector ( ),
pcDNA3-FLAG-Caspy (Caspy, tagged),
pcDNA3-Caspy2-Myc (Caspy2, tagged),
pcDNA3-Caspy (Caspy, non-tagged), or
pcDNA3-Caspy2 (Caspy2, non-tagged) and 73 ng
of pcDNA3-
-gal. 24 h post-transfection, the percent of
apoptotic cells ± S.D. was calculated in triplicate cultures.
B, suppression of Caspy- and Caspy2-induced apoptosis by
caspase inhibitors. 5 × 104 293T cells were
co-transfected with 667 ng of pcDNA3-FLAG-Caspy (Caspy,
tagged), 333 ng of pcDNA3-Caspy2-Myc (Caspy2,
tagged), 667 ng of pcDNA3-Caspy (Caspy,
non-tagged), 333 ng of pcDNA3-Caspy2 (Caspy2,
non-tagged), and 167 ng of empty vector (
), pcDNA3-p35
(p35), or pcDNA3-CrmA (CrmA) in the
presence of pcDNA3-
-gal. 8 h post-transfection,
transfected cell were incubated with 20 µM zVAD-fmk
(zVAD-fmk) or medium alone (other lanes). 24 h post-transfection, the percent of apoptotic cells ± S.D. was
calculated in triplicate cultures. C and D, Caspy
and Caspy2 exhibit differential substrate specificity. 3 × 106 293T cells were co-transfected with 10 µg of
pcDNA3-FLAG-Caspy (A) or pcDNA3-Caspy2-Myc
(B). 24 h post-transfection, the caspases were
immunopurified by anti-Myc and anti-FLAG Abs, and 1/10 of the
aliquot was incubated with the indicated fluorogenic peptide substrates
for 60 min.
PD),
indicating the pyrin domain of zAsc is necessary and sufficient for the
interaction with Caspy. In addition, a Caspy mutant lacking the pyrin
domain (Fig. 5, Myc-Caspy-
PD) failed to interact with a
zAsc mutant containing a pyrin domain (Fig. 5, FLAG-zAsc-PD)
suggesting that zAsc and Caspy associate through a homophilic
pyrin-pyrin domain interaction. To further assess the association
between zAsc and Caspy, we constructed plasmids to express GFP-Caspy,
GFP-zAsc, and DsRed-zAsc fusion proteins to facilitate the analysis of
their subcellular localization. Expression of GFP-Caspy and GFP-zAsc
induced the formation of specks (Fig.
6B, GFP-Caspy and
GFP-zAsc, respectively), and double labeling analysis of the
same cells revealed that both GFP-Caspy (Fig. 6A,
Green) and DsRed-zAsc (Fig. 6A, Red)
co-localized to the same speck visualized in yellow (Fig.
6A, Merge). These observation demonstrates that
zAsc interacts with Caspy in the same speck.
View larger version (25K):
[in a new window]
Fig. 5.
Interaction between zAsc and Caspy. zAsc
interacts with Caspy but not with Caspy2. 1 × 106
COS-7 cells were transfected with 2.4 µg of pcDNA3-Myc (vector),
pcDNA3-Caspy-Myc (Caspy-WT),
pcDNA3-Caspy-(101-383)-Myc (Caspy- PD), or
pcDNA3-Caspy2-Myc and pFLAG-zAsc (zAsc-WT),
pFLAG-zAsc-(1-103) (zAsc-PD), or pFLAG-zAsc-(104-203)
(zAsc-
PD). 24 h post-transfection cells were lysed,
and proteins were immunoprecipitated by anti-Myc polyclonal Ab.
Co-immunoprecipitated proteins were detected by anti-FLAG M2 Ab
(upper panel). As controls, co-immunoprecipitated proteins
were detected by anti-Myc monoclonal Ab (middle panel).
Proteins in total lysate were detected by anti-FLAG Ab (bottom
panels).
View larger version (23K):
[in a new window]
Fig. 6.
Co-localization of Caspy and zAsc.
A, co-expression of both the GFP-Caspy and the DsRed-zAsc in
COS-7 cells. 1 × 103 COS-7 cells were transiently
co-transfected with 4.8 ng of pEGFP-Caspy (Green) and
pDsRed-zAsc (Red). After 24 h, cells were fixed with
70% ethanol. Blue nuclei were stained by DAPI
(4',6-diamidino-2-phenylindole dihydrochloride)
(Blue). Signals were detected by immunofluorescence
microscopy. Green fluorescence, red fluorescence, and blue fluorescence
were visualized in the same field (Merge). GFP-Caspy signal
(Green) and DsRed-zAsc signal (Red) accumulate in
the same speck (arrowheads). B, as controls,
cells were transiently transfected with pEGFP or pDsRed as a vector
control. Diffuse green or red florescent signals were detected in the
cells (GFP and DsRed, respectively). Cells were
transiently transfected with pEGFP-zAsc alone or pEGFP-Caspy alone
(GFP-zAsc and GFP-Caspy, respectively). Green
fluorescence signals were detected in the cells
(arrowheads).
View larger version (40K):
[in a new window]
Fig. 7.
The induced proximity of zAsc enhanced
apoptosis induced by Caspy and processing of Caspy. 5 × 104 293T cells were co-transfected with 17 ng of
pcDNA3, pcDNA3-Caspase-9-FLAG, pcDNA3-FLAG-Caspy,
pcDNA3-Caspy2-Myc, pcDNA3-Caspy, or pcDNA3-Caspy2 and 33 ng
of pcDNA3-zAsc-Myc, pcDNA3-zAsc-Fpk3-Myc, or
pcDNA3-Apaf-1XL-Myc in the presence of pEF1BOS- -gal. 8 h
post-transfection cells were treated with 200 nM AP1510 or
left alone. 24 h post-transfection the cells were fixed and
stained with X-gal. A, the proximity of zAsc enhanced
apoptosis induced by Caspy but not that of Caspase-9. Apoptotic cells
are indicated by arrows. B, the proximity of zAsc
enhanced apoptosis induced by Caspy but not those of Caspy2 and
Caspase-9. The percentage of apoptotic cells was measured in triplicate
and is shown with mean ± S.D. C, 5 × 105 293T cells were co-transfected with 1 µg of
pcDNA3 (lane 1), pcDNA3-FLAG-Caspy
(Caspy) (lanes 1 and 2), and 1 µg of
pcDNA3 (lanes 1 and 2) and
pcDNA3-zAsc-Fpk3-Myc (zAsc) (lane 3). 8 h post-transfection cells were treated with 200 nM AP1510
(lane 5) or left untreated (lanes 1 and
4). 24 h post-transfection cells were lysed, and
proteins were precipitated with anti-FLAG M2 monoclonal Ab and eluted
by FLAG peptide and detected by anti-FLAG M2 monoclonal Ab. The
asterisk indicates the nonspecific band.
View larger version (110K):
[in a new window]
Fig. 8.
Targeted knocked-down of caspy
resulted in malformation of jaw and branchial arches.
A-C and D-F depict
Alcian blue-stained pictures of the head of larvae at 6 days
post-injection with control morpholino or antisense caspy
oligonucleotides. A and D, lateral view showing
wide open mouth in larvae injected with caspy antisense
oligonucleotide compared with larvae injected with control
oligonucleotide. B and E, ventral view showing
malformation of the cartilages of the jaw and brachial arches in larvae
injected with caspy antisense oligonucleotide compared with
larvae injected with control oligonucleotide. Meckel's and
palatoquadrate cartilages, which form the jaw, are deformed, and the
ceratohyal cartilages are also deformed and inverted in larvae injected
with caspy antisense oligonucleotide. The deformation of
these anterior cartilages is also observed in lateral view
(D). C and F, high power of ventral
view showing disorganized and ill defined branchial cartilages
(arrows) in larvae injected with caspy antisense
oligonucleotide. m, Meckel's cartilage; pq,
palatoquadrate cartilage; ch, ceratohyal cartilage;
co, coracoid of pectoral fin; cb, ceratobranchial
cartilage. Scale bars are 100 µm (A,
B, D, and E) and 40 µm (C
and F).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank V. Rivera (Ariad Pharmaceuticals) for Fpk3 plasmids and dimerization agent AP1510, L. McAllister-Lucas for critical review of the manuscript, and C. Wilson and B. Issa for technical assistance.
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FOOTNOTES |
---|
* The research was supported in part by National Institutes of Health Grants GM-60421 (to N. I.), CA-64556 and CA-64421 (to G. N.), and R01 RR10715 and P01HD22486 (to J. H. P.) and by the Japan Clinical Pathology Foundation for International Exchange and the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to J. 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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF233434, AF327410, AF231013, AB050006, and AB053165.
To whom correspondence should be addressed: Dept. of Pathology,
University of Michigan Medical School, Ann Arbor, MI 48109. Tel.:
734-764-8514; Fax: 734-647-9654; E-mail: ino@umich.edu.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M203944200
2 W. Zhou and J. Y. Kuwada, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: DED, death effector domain; CARD, caspase recruitment domain; DD, death domain; EST, expressed sequence tag; GFP, green fluorescent protein; Ab, antibody; zAsc, zebrafish orthologue of ASC.
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