From the Burnham Institute, La Jolla, California 92037
Received for publication, July 17, 2000, and in revised form, December 8, 2000
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ABSTRACT |
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Apaf1/CED4 family members play central roles in
apoptosis regulation as activators of caspase family cell death
proteases. These proteins contain a nucleotide-binding (NB)
self-oligomerization domain and a caspase recruitment domain (CARD). A
novel human protein was identified, NAC, that contains an
NB domain and CARD. The CARD of NAC
interacts selectively with the CARD domain of Apaf1, a
caspase-activating protein that couples mitochondria-released cytochrome c (cyt-c) to activation of cytosolic caspases.
Cyt-c-mediated activation of caspases in cytosolic extracts and in
cells is enhanced by overexpressing NAC and inhibited by reducing NAC
using antisense/DNAzymes. Furthermore, association of NAC with Apaf1 is
cyt c-inducible, resulting in a mega-complex (>1 MDa) containing both
NAC and Apaf1 and correlating with enhanced recruitment and proteolytic
processing of pro-caspase-9. NAC also collaborates with Apaf1 in
inducing caspase activation and apoptosis in intact cells, whereas
fragments of NAC representing only the CARD or NB domain suppress
Apaf1-dependent apoptosis induction. NAC expression
in vivo is associated with terminal differentiation of
short lived cells in epithelia and some other tissues. The ability of
NAC to enhance Apaf1-apoptosome function reveals a novel paradigm for
apoptosis regulation.
CED4 family proteins constitute a unique family of
caspase-activating molecules. The founding member of this family, CED4, was discovered in the nematode Caenorhabditis elegans in
screens for genes that are essential for developmental programmed cell death (1). CED4 contains an N-terminal
CARD1 followed by an NB
domain, the later containing classical Walker A and B box motifs
recognized as important in binding nucleotide triphosphates. CED4
functions as an activator of the caspase, CED3, in vitro and
in vivo (2, 3). The NB domain of CED4 oligomerizes in an
ATP-dependent manner (4, 5), whereas the CARD binds a
complementary N-terminal CARD found in the zymogen proform of CED3 (6).
Protease activation is thought to result from the induced proximity of
CED3 zymogens bound to oligomerized CED4, where the weak intrinsic
protease activity of the proenzymes is sufficient for trans-proteolysis
of closely juxtaposed pro-caspases (4, 7). Proteolytic cleavage of
pro-CED3 then produces the large and small subunits of the
heterotetrameric, autonomously active enzyme.
The closest homologue of CED4 identified in humans and other mammals
thus far is Apaf1 (apoptosis protease-activating factor-1) (8). Similar
to CED4, the Apaf1 protein contains a CARD, followed by an NB domain
that shares significant amino acid sequence identity with the NB
domains of CED4 and a family of ATPases associated with pathogen
resistance (R genes) in plants (3, 5, 9), thus constituting the
NB-ARC (Apaf-1/R gene/CED4) domain family (also known as NACHT
domain). Unlike CED4, however, the NB-ARC domain of Apaf1 is followed
by multiple WD repeats. These WD domains participate in auto-repression
of Apaf1, locking it into an inactive, unoligomerized state until bound
by cyt-c. In response to multiple cell death stimuli, changes in
mitochondrial membrane permeability result in release of cyt-c into the
cytosol, where it binds and activates Apaf-1, thus coupling
mitochondrial damage to a mechanism for caspase activation (10). Cyt-c,
in conjunction with dATP or ATP, induces formation of a large Apaf1
oligomer (estimated to be an octamer), via its NB-ARC domain, and
exposes the CARD of Apaf1 for interactions with a complementary CARD
found in the N-terminal prodomain of pro-caspase-9 (11-14). By the
induced proximity method, juxtaposed pro-caspase-9 (pro-Casp9) zymogens
then trans-proteolyze each other, generating the characteristic large
and small subunits typical of activated caspases. Active caspase-9
bound to oligomerized Apaf1 then directly cleaves and activates
pro-caspase-3, an effector caspase that is responsible both for
cleavage and activation of additional downstream caspases and for
direct cleavage of a variety of substrate proteins that commit the cell
to an apoptotic demise. A close homologue of Apaf1 has recently been
identified in the fly, Drosophila melanogaster, apparently
operating as a caspase-activator via a similar cyt-c-inducible
mechanism (15-17). Gene ablation studies in mice and flies indicate
that Apaf1 plays a critical role in programmed cell death in certain
tissues and in response to many types of cell death stimuli in
vivo (18).
In this report, we describe the identification and initial functional
characterization of a novel regulator of Apaf1, which we have termed NAC.
cDNA Cloning and Plasmid Construction--
The NAC cDNA
sequence was found using PSI-BLAST and the CARD sequence of CARD4/Nod1
as query. This search revealed homology with a predicted protein of EST
clone KIAA0926 in the Kazusa DNA Research Institute brain
genomic data base. Jurkat total RNA was reverse-transcribed to
cDNAs with Moloney murine leukemia virus reverse transcriptase
(Stratagene) and random hexanucleotide primers. Three overlapping
cDNA fragments of NAC were amplified using Turbo Pfu DNA
polymerase (Stratagene) and three sets of oligonucleotide primers as follows: set 1, 5'-CCGAATTCACCATGGCTGGCGGAGCCTGGGGC-3' (forward) and 5'-CCGCTCGAGTCAACAGAGGGTTGTGGTGGTCTTG-3' (reverse); set
2, 5'-CCCGAATTCGAACCTCGCATAGTCATACTGC-3' (forward) and
5'-GTCCCACAACAGAATTCAATCTCAACGGTC-3' (reverse);, and set 3, 5'-TGTGATGAGAGAAGCGGTGAC-3' (forward) and 5'-CCGCTCGAGCAAAGAAGGGTCAGCCAAAGC-3' (reverse). The resultant cDNA
fragments were ligated into mammalian expression vector pcDNA3-Myc. From these overlapping cDNA fragments, full-length NAC cDNA
was assembled in pcDNA3-Myc and pcDNA3-HA at EcoRI
and XhoI cloning sites. The nucleotide sequence of the
assembled full-length NAC was confirmed by DNA sequencing analysis. The
regions encoding the CARD or the NB domain (amino acids 329-547) were
polymerase chain reaction-amplified from Jurkat cDNA using primer
set 3 and the primers 5'- CCCGAATTCGAACCTCGCATAGTCATACTGC-3' (forward)
and CCGCTCGAGTCAACAGAGGGTTGTGGTGGTCTTG-3' (reverse),
respectively. The resultant polymerase chain reaction fragments were
digested with EcoRI and XhoI and ligated into
pcDNA3-Myc and into vector pGEX-4T1 for GST fusion protein production.
Antibodies--
Polyclonal antisera were generated in rabbits
using keyhole limpet hemocyanin- and ovalbumin-conjugated
(Pierce) synthetic peptides with sequences corresponding to residues aa
161-180 (Bur241) or aa 1058-1077 (Bur242) of NAC. Mouse monoclonal
antibody recognizing human APAF1 was purchased from R & D
Systems (Minneapolis, MN). Epitope-specific antibodies for FLAG, HA, or
Myc tag were obtained from Sigma, Roche Molecular Biochemicals, and
Santa Cruz Biotechnology, respectively.
DNAzymes--
An anti-NAC DNAzyme oligonucleotide was designed
by the method of Joyce (19), targeting the translation initiation
region of NAC mRNA and containing 2-O-methylnucleosides
at the 5'-end and an inverted thymidine at the 3'-end for nuclease
resistance. The sequences of the catalytic (AS) and control (C)
noncatalytic oligonucleotides are as follows: AS,
5'-(2-O-MeC2-O-MeCAGCCAGGCTAGCTA-CAACGACTCTGTCC-InvT)-3', and (C,
5'-(2-O-MeC2-O-MeCAGCCAGGCTACCTACAACGACTCTGTCC-InvT)-3', respectively (Operon Technologies).
Immunohistochemistry--
Normal human tissues for
immunohistochemistry analysis were obtained from biopsy and autopsy
specimens, fixed in Bouin's solution (Sigma), and embedded in
paraffin. Tissue sections were immunostained using a
diaminobenzidine-based detection method employing the Envision-Plus-horseradish peroxide system (Dako). Nuclei were counterstained with hematoxylin.
Coimmunoprecipitation and Immunoblotting Assays--
For
immunoprecipitation and immunoblotting analyses, cells were lysed in
either buffer A (142.4 mM KCl, 5 mM
MgCl2, 10 mM HEPES (pH 7.4), 0.5 mM
EGTA, 1 mM EDTA, and 0.2% Nonidet P-40 for cytoplasmic
extracts), buffer B (20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, and 1 mM EDTA, for hypotonic lysis), or ELB (50 mM
HEPES (pH 7.4), 250 mM NaCl, 5 mM EDTA, and
0.4% Nonidet P-40, for whole cell extracts), all supplemented with 1 mM dithiothreitol, 12.5 mM In Vitro Protein Interaction Assays--
GST fusion proteins
were expressed from pGEX-4T1 in XL-1-blue Escherichia coli
cells (Stratagene) and affinity-purified using GSH-Sepharose. Purified
GST fusion proteins (0.1-0.5 µg) immobilized on 10-15 µl of
GSH-Sepharose beads were incubated with 1 mg/ml bovine serum albumin in
100 µl of buffer A for 30 min at 25 °C. The beads were then
incubated overnight at 4 °C with 1 µl of rabbit reticulocyte
lysates (TnT-lysate; Promega) containing 35S-labeled,
IVT proteins in 100 µl of buffer A supplemented with 0.5 mg/ml
bovine serum albumin. Proteins on beads were washed four times in 500 µl of buffer A, followed by boiling in 20 µl of Laemmli-SDS sample
buffer, SDS-PAGE and detection by fluorography.
Gel-sieve Chromatography Analysis--
Cytosolic extracts were
prepared using buffer B (above) as described (21) and incubated (1.5 mg) with cyt-c (10 µM) and dATP (1 mM) for 10 min at 30 °C, and then 100 µM ZVAD-fmk was added. The
treated protein lysates were immediately fractionated by using a
Superose-6 HR 10/30 gel filtration column in elution buffer containing
50 mM Tris (pH 7.4), 100 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and 1 mM dithiothreitol. Column fractions (0.5 ml) were analyzed
for NAC and Apaf1 by SDS-PAGE, followed by immunoblotting.
Caspase Assays--
Cytosolic extracts were prepared in
hypotonic buffer B as described (21) and incubated (10 µg) with
various concentrations of cyt-c and 1 mM dATP in Caspase
buffer (21) for 30 min at 30 °C. Caspase substrate Ac-DEVD-AFC (100 µM) (Calbiochem) was then added, and protease activity
was measured continuously by monitoring the release of fluorigenic AFC
at 37 °C. Alternatively, transfected cells were directly lysed in
Caspase Lysis buffer (10 mM HEPES (pH 7.4), 25 mM NaCl, 0.25% Triton X-100, and 1 mM EDTA),
normalized for protein content, and monitored for cleavage of
Ac-DEVD-AFC as described (21). Processing of IVT
35S-labeled pro-caspase-9 in cytosolic extracts was
monitored by SDS-PAGE (20).
Apoptosis Assays--
Cells were transfected with pEGFP
(CLONTECH) and effector plasmids using SuperFect
transfection reagents (Qiagen) as indicated. After culturing 1.5 days
in media containing reduced serum (0.1% fetal bovine serum), floating
and adherent cells (recovered by trypsinization) were pooled,
and cells were fixed in 3.7% formaldehyde/PBS, stained with 1 µg/ml
4',6-diamidino-2-phenylindole (DAPI), and the percentage of
GFP-positive cells with apoptotic morphology (nuclear fragmentation,
chromatin condensation) was determined by fluorescence microscopy (20,
21).
Molecular Modeling--
A three-dimensional model of the CARD
domain of NAC was generated using the MODELLER program, essentially as
described (22), based on the structures of the CARDs of Apaf1,
pro-Casp9, and Raidd (23, 24).
NAC-encoding cDNAs were obtained by reverse
transcriptase-polymerase chain reaction, revealing a continuous open
reading frame encoding a 1473-amino acid protein (Fig.
1). The predicted NAC protein contains an
NB domain, followed by leucine-rich repeats (LRR), and a CARD domain.
Thus, unlike Apaf1/CED4 family proteins that also contain CARD and NB
domains, the CARD domain of NAC is located at its C rather than N
terminus. The NB domain of NAC contains classical Walker A and B boxes
indicative of ATP-binding proteins and is most similar in amino acid
sequence to the NB domain of Nod1/CARD4 (29%) (25, 26), followed by
human APAF1 (17%), the Drosophila Apaf1 homologue
(12%), and the C. elegans CED4 protein (12%) (Fig.
1C). Moreover, recombinant NAC NB domain was observed to
bind ATP and to self-associate in an ATP-dependent manner
in vitro (not shown). The CARD domain of NAC shares 21, 19, and 8% amino acid identity with the CARD domains of Nod1/CARD4, huApaf1, and CED4, respectively (Fig. 1D). The NAC CARD
sequence was readily threaded onto the structures of other CARDs using the MODELLER program (Fig. 1E), suggesting conservation of
the 6
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-glycerol
phosphate, 1 µM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1× protease
inhibitor mix (Roche Molecular Biochemicals). Cell lysates were
clarified by centrifugation and subjected to immunoprecipitation using
specific antibodies and protein G or A beads. Immune complexes were
resolved in SDS-PAGE gels, transferred to nitrocellulose membranes, and
immunoblotted with antibodies followed by detection using ECL (Amersham
Pharmacia Biotech) (20).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-helical fold typical of these domains (23). The LRRs of NAC
are reminiscent of Nod1/CARD4 and plant stress-response (R) proteins,
which also contain LRRs. In NB-containing plant R proteins, the LRRs
function as interaction motifs for pathogen responses (9), suggesting a
possible role of these structures in linking NAC to specific signaling
pathways. Additional NAC cDNAs were obtained that presumably
represent alternative mRNA splicing products that encode shorter
proteins lacking 31- or 45-amino acid segments (or both) located
between the LLR and CARD (Fig. 1).
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Fig. 1.
Sequence analysis of NAC. A,
schematic representation showing domain structure of human NAC. The NB
domain (aa 329-547, filled box), the leucine-rich repeats
(aa 808-947, filled bars), and the CARD (aa 1373-1473,
dotted box) are depicted. Hatched boxes indicate
sequences derived from two alternatively spliced exons. B,
predicted amino acid sequences of human NAC. The positions for the
P-loop (Walker A) and Walker B of NB domain are indicated. The amino
acids sequences of LRR repeats and CARD are underlined and
in bold letters, respectively. Italic letters
indicate sequences for the alternatively spliced exons. C,
alignment of the NB domain of NAC (aa 329-547) with NB domains of
Nod1/CARD4 (aa 197-408), Apaf1 (aa 138-355), and C. elegans CED4 (aa 154-374). Alignment was conducted using
ClustalW. Identical and similar residues are shown in black
and gray shades, respectively. Positions of
P-loop and Walker B sequences are indicated. D, alignment of
CARDs of NAC (aa 1373-1465), Nod1/CARD4 (aa 15-104), Apaf1 (aa
1-89), and CED4 (aa 2-89). Identical and similar residues are shown
in black and gray shading,
respectively. E, three-dimensional model of NAC CARD domain,
showing predicted 6 -helixes (labeled H1-H6).
NAC mRNAs were widely expressed in human tissues, with highest
levels found in blood leukocytes, thymus, spleen, and heart (Fig.
2A). Antisera were raised
against synthetic NAC peptides and confirmed to bind specifically the
NAC protein by immunoblotting and immunoprecipitation assays (Fig.
2B, and not shown). NAC protein was detected in several
adult human tissues, with highest levels in kidney, brain, and
epidermis among the tissues examined (Fig. 2C). A smaller
anti-NAC immunoreactive band was detected in thymus lysates, which
remains to be characterized (Fig. 2C). Differences in NAC
mRNA and protein expression in some tissues suggest the possibility
of post-transcriptional regulation (Fig. 2, A and C).
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Immunohistochemical analysis of the in vivo patterns of NAC expression in adult human tissues demonstrated association with differentiation in stratified epithelia of the skin, esophagus, intestine, and cervix, as well as in the prostate gland where differentiated luminal secretory cells were NAC-immunopositive and undifferentiated basal cells were immunonegative (Fig. 2D and not shown). Differentiated macrophages and granulocytes were also strongly NAC-immunopositive (3-4 intensity/on 0-4 scale), whereas their bone marrow precursors were immunonegative (not shown). In the testis, NAC immunointensity also increased in concert with the differentiation program from spermatocytes (negative), to spermatids (0-1 intensity/on 0-4 scale), to spermatozoa (1-2 intensity), reaching highest intensity in the residual bodies that represent cellular remnants (2-3 intensity) (not shown). Thus, NAC expression is associated with differentiation of some types of short lived cells in vivo.
In vitro binding experiments were performed using a glutathione S-transferase (GST) fusion protein containing the CARD of NAC. The CARD of NAC bound efficiently to itself and also interacted selectively in vitro with the CARDs of Apaf1, Nod1, and CED4 but not with the CARDs of Bcl10, pro-Casp9, pro-caspase-1, pro-caspase-2, pro-caspase-11, Raidd, or Cardiak (RIP2) (Fig. 2A and not shown). The ability of NAC to interact with itself, Apaf1, Nod1, and CED4 in cells was confirmed by coimmunoprecipitation of epitope-tagged proteins from transiently transfected HEK293 cells (Fig. 2B and not shown). The endogenous ~160-kDa NAC protein could also be coimmunoprecipitated with endogenous Apaf1 (but not Nod1) from cells that intrinsically express NAC, using anti-NAC antisera (Fig. 2D).
Interestingly, compared with full-length Apaf1, NAC
coimmunoprecipitated more efficiently with a truncation mutant of Apaf1 which lacks the WD repeats that normally maintain this protein in an
auto-repressed state (14), suggesting that "activated" Apaf1
interacts preferentially with NAC (Fig. 2B). Consistent with
this observation, association of full-length Apaf1 with NAC was
inducible by stimuli such as staurosporine, which trigger cyt-c release
and result in Apaf1 activation (27). Before exposure to death stimuli,
relatively little NAC was coimmunoprecipitated with Apaf1, but within
10 min NAC/Apaf1 complexes were readily detected (Fig.
3E).
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Since Apaf1 is known to form complexes with pro-Casp9 (10), we examined the effects of NAC on Apaf1/pro-Casp9 interactions by coimmunoprecipitation. Overexpression of NAC by transient transfection in HEK293 cells increased the relative amount of Apaf1 that coimmunoprecipitated with pro-Casp9 (Fig. 3C), suggesting that NAC enhances rather than inhibits interactions of these proteins. These coimmunoprecipitation experiments also hinted that NAC may exist in a complex simultaneously with Apaf1 and pro-Casp9, since NAC was recovered in anti-Casp9 immunoprecipitates when Apaf1 was coexpressed (Fig. 3C), even though NAC cannot directly bind pro-Casp9 (Fig. 3A and not shown) and was not recovered in anti-Casp9 immune complexes when Apaf1 was not coexpressed (Fig. 3B).
Cyt-c induces formation of a large holoenzyme complex (apoptosome) containing multiple Apaf1 and Casp9 molecules in an estimated 8:8 stoichiometry (12). The apoptosome can be monitored by gel-sieve chromatography of cyt-c-stimulated cytosolic extracts and typically migrates at ~700 kDa (28). However, a larger apoptosome of >1 MDa has been reported in some types of cells, suggesting that other proteins may associate with the Apaf1-Casp9 complex (29). In cyt-c-stimulated extracts from HEK293 cells, which contain relative little endogenous NAC, as determined by immunoblotting (not shown), a single Apaf1-containing apoptosome of ~700 kDa was observed by gel-sieve chromatography (Fig. 2F). However, in extracts from NAC-transfected HEK293 cells, two Apaf1-containing apoptosomes were evident, including a large >1 MDa complex that contained both Apaf1 and NAC (as determined by coimmunoprecipitation analysis of the column fractions in which Apaf1 and NAC coeluted) (Fig. 2F). Similarly, in cells that contain relative high levels of endogenous NAC, such as Jurkat T-cells, the endogenous NAC and Apaf1 molecules coeluted in gel-sieve experiments, revealing two apoptosomes where greater amounts of NAC were present in the larger of these multiprotein complexes (Fig. 2F, top). In extracts lacking cyt-c treatment, Apaf1 eluted as a monomer, whereas NAC was spread over multiple fractions without a clear elution peak (not shown). Also, NAC did not coelute in gel-sieve chromatography experiments with Nod1 using untreated or cyt-c-stimulated extracts,2 confirming that NAC associates selectively with the Apaf1 apoptosome.
To explore whether the Apaf1-containing apoptosome with which NAC
associates displays caspase activity, endogenous NAC was immunoprecipitated from control- and cyt-c-stimulated Jurkat cell extracts, and associated caspase activity was measured based on cleavage of the fluorigenic caspase-substrate Ac-DEVD-AFC (21). These
experiments revealed that NAC is associated with active caspases after
but not before cyt-c stimulation (Fig.
4A). Control immunoprecipitates lacked significant caspase activity, confirming the
specificity of these results. Although activities of different size
apoptosomes can vary depending on salt concentrations, use of
detergents, and the presence of endogenous inhibitors
(X-chromosone-linked inhibitor of apoptosis protein and second
mitochondrial activator of caspases) (29),2 these findings
argue that the larger apoptosome containing NAC is active.
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In HEK293 cells, which contain little endogenous NAC, overexpression of NAC by transient transfection promotes formation predominantly of the larger >1-MDa apoptosome upon cyt-c stimulation, with relatively little of the smaller ~700-kDa apoptosome present (Fig. 3F). To contrast the function of Apaf1 in the presence and absence of NAC, therefore, we compared cyt-c-induced proteolytic processing of pro-Casp9 and activation of downstream caspases (Ac-DEVD-AFC cleavage) in extracts prepared from control- and NAC-transfected HEK293 cells (Fig. 4B). Extracts containing elevated NAC displayed increased processing of pro-Casp9 and greater cyt-c-induced activation of downstream caspases, suggesting that NAC enhances Apaf1 activity. Conversely, reducing NAC protein levels using antisense expression plasmids (not shown) or antisense/DNAzyme oligonucleotides decreased the ability of cyt-c to activate caspases in cell extracts in vitro. Fig. 4C, for example, shows experiments performed using Jurkat cells, which contain relatively higher levels of endogenous NAC, demonstrating DNAzyme-mediated ablation of NAC protein without concomitant changes in the levels of Apaf1. Extracts prepared from anti-NAC DNAzyme-treated Jurkat cells were less sensitive to cyt-c compared with control oligonucleotide-treated cells, in terms of caspase activation. In contrast, sensitivity to granzyme B-mediated caspase activation was not affected, confirming a specific defect in the cyt-c pathway which depends on Apaf1 for caspase activation. Further evidence that NAC regulates the cyt-c/Apaf1-dependent activation of caspases in cell extracts was obtained by affinity preabsorption of NAC from extracts using a GST fusion protein containing the CARD of NAC and by expression in cells of a dominant-negative fragment of NAC consisting of only the CARD domain (not shown).
Consistent with experiments involving cell extracts, NAC also collaborated with Apaf1 in inducing caspase activation and apoptosis in intact cells. In transient transfection experiments using HEK293 (Fig. 4, D and E) or other cell lines (not shown), overexpression of NAC by itself (not shown) or in combination with pro-Casp9 had little effect on caspase activation or apoptosis. In contrast, overexpressing NAC together with Apaf1 and pro-Casp9 resulted in synergistic increases in activation of caspases and induction of apoptosis, as determined from cotransfections that employed suboptimal amounts of Apaf1-encoding plasmid (Fig. 4, D and E). Overexpression of NAC also sensitized cells to suboptimal concentrations of apoptosis inducers such as staurosporine, which triggers apoptosis through an Apaf1-dependent mechanism (30), but not by anti-Fas antibody which utilizes an Apaf1-independent pathway (Fig. 4E and not shown). Immunoblotting experiments demonstrated that the enhanced sensitivity of NAC-transfected cells to staurosporine was not due to differences in the levels of Apaf1 or pro-Casp9 proteins produced in cells (not shown).
Whereas full-length NAC enhanced apoptosis and caspase activation induced by overexpressing Apaf1 or by treatment of cells with Apaf1-dependent apoptotic stimuli (Fig. 4, D and E), fragments of NAC containing only the CARD or NB domain had the opposite effect, interfering with apoptosis induced by coexpression of Apaf1/pro-Casp9 and by Apaf1-dependent stimuli such as staurosporine without affecting Apaf1-independent pathways activated by Fas (Fig. 4F). Again, immunoblotting experiments demonstrated that these reductions in Apaf1-dependent apoptosis caused by these dominant-negative fragments of NAC were not secondary to effects on levels of the Apaf1 or pro-Casp9 proteins (not shown).
Although some CARD-containing proteins, including Nod1/CARD-4 (25, 26)
and Bcl10/mE10 (31, 32), reportedly induce NFB activation, NAC did
not induce NF-
B, when overexpressed in cells (data not shown).
Cells of various tissues vary in their sensitivity to cyt-c-induced
activation of caspases, a finding that cannot be accounted for by
differences in the levels of Apaf1 protein or downstream caspases (33,
34). The discovery of NAC suggests a mechanism for fine-tuning Apaf1
function, based on whether NAC is expressed and perhaps on whether NAC
interacts with unidentified proteins via its LRR or other domains.
Apaf1/CED4 family proteins directly bind CARD-containing caspases,
promoting protease activation upon oligomerization by bringing the
suboptimally active pro-enzymes into close proximity, allowing them to
trans-process each other (4, 12-14). Although NAC enhances
cyt-c-mediated pro-Casp9 processing, it does not directly bind this
caspase (nor caspases-1, -2, -6, -7, -8, -10, or -11).2
Rather, interactions of NAC with Apaf1 facilitate Apaf1-mediated activation of pro-Casp9, thus revealing a new paradigm for apoptosis regulation. The precise mechanism by which NAC enhances Apaf1 function
remains to be elucidated.
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ACKNOWLEDGEMENTS |
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We thank E. Alnemri for caspase-9 and Apaf1 cDNAs; G. Joyce for DNAzyme design; H. Bettendorf for technical assistance; S. Matsuzawa, H. Chan, H. Zhang, and S. Kitada for discussions; and R. Cornell for manuscript preparation.
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FOOTNOTES |
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* This work was supported by CaPCURE, National Institutes of Health Grants GM61694 and NS36821, and the United States Department of Defense.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.
Current address: Arena Pharmaceuticals, 6166 Nancy Ridge Dr., San
Diego, CA 92121.
§ To whom correspondence should be addressed: Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3140; Fax: 858-646-3194, E-mail: jreed@burnham.org.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M006309200
2 Z.-L. Chu, F. Pio, Z. Xie, K. Welsh, M. Krajewska, S. Krajewski, A. Godzik, and J. C. Reed, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CARD, caspase recruitment domain; LRR, leucine-rich repeat; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; IP, immunoprecipitation; IVT, in vitro translated; PAGE, polyacrylamide gel electrophoresis; aa, amino acids; HA, hemagglutinin; cyt-c, cytochrome c; pro-Casp9, pro-caspase-9; ZVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone; NB, nucleotide binding; Ac-DEVD-AFC, acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl-coumarin; DAPI, 4',6-diamidino-2-phenylindole; GST, glutathione S-transferase.
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