A Novel Enhancer of the Apaf1 Apoptosome Involved in Cytochrome c-dependent Caspase Activation and Apoptosis

Abstract

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 anNB 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 expressionin 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. 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 anNB 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 expressionin 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. caspase recruitment domain leucine-rich repeat enhanced green fluorescent protein phosphate-buffered saline immunoprecipitation in vitro translated polyacrylamide gel electrophoresis amino acids hemagglutinin cytochrome c pro-caspase-9 benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone nucleotide binding acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl-coumarin 4′,6-diamidino-2-phenylindole glutathioneS-transferase 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 (1Yuan J.Y. Horvitz H.R. Dev. Biol. 1990; 138: 33-41Crossref PubMed Scopus (441) Google Scholar). 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 andin vivo (2Seshagiri S. Miller L. Curr. Biol. 1997; 7: 455-460Abstract Full Text Full Text PDF PubMed Google Scholar, 3Chinnaiyan A. Chaudhary D. O'Rourke K. Koonin E. Dixit V. Nature. 1997; 388: 728-729Crossref PubMed Scopus (163) Google Scholar). The NB domain of CED4 oligomerizes in an ATP-dependent manner (4Yang X. Chang H. Baltimore D. Science. 1998; 281: 1355-1357Crossref PubMed Scopus (234) Google Scholar, 5Jaroszewski L. Rychlewski L. Reed J.C. Godzik A. Proteins. 2000; 39: 197-203Crossref PubMed Scopus (31) Google Scholar), whereas the CARD binds a complementary N-terminal CARD found in the zymogen proform of CED3 (6Chinnaiyan A.M. O'Rourke K. Lane B.R. Dixit V.M. Science. 1997; 275: 1122-1126Crossref PubMed Scopus (552) Google Scholar). 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 (4Yang X. Chang H. Baltimore D. Science. 1998; 281: 1355-1357Crossref PubMed Scopus (234) Google Scholar, 7Salvesen G.S. Dixit V.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10964-10967Crossref PubMed Scopus (762) Google Scholar). 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) (8Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2716) Google Scholar). 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 (3Chinnaiyan A. Chaudhary D. O'Rourke K. Koonin E. Dixit V. Nature. 1997; 388: 728-729Crossref PubMed Scopus (163) Google Scholar, 5Jaroszewski L. Rychlewski L. Reed J.C. Godzik A. Proteins. 2000; 39: 197-203Crossref PubMed Scopus (31) Google Scholar, 9Kobe B. Deisenhofer J. Curr. Opin. Struct. Biol. 1995; 5: 409-416Crossref PubMed Scopus (321) Google Scholar), 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 (10Li P. Nijhawan D. Budihardjo I. Srinivasula S. Ahmad M. Alnemri E. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6157) Google Scholar). 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 (11Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (958) Google Scholar, 12Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1771) Google Scholar, 13Saleh A. Srinivasula S. Acharya S. Fishel R. Alnemri E. J. Biol. Chem. 1999; 274: 17941-17945Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 14Adrain C. Slee E.A. Harte M.T. Martin S.J. J. Biol. Chem. 1999; 274: 20855-20860Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). 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 (15Rodriguez A. Oliver H. Zou H. Chen P. Wang X. Abrams J. Nat. Cell Biol. 1999; 1: 272-279Crossref PubMed Scopus (292) Google Scholar, 16Zhou L. Song Z. Tittel J. Steller H. Mol. Cell. 1999; 4: 745-755Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 17Kanuka H. Sawamoto K. Inohara N. Matsuno K. Okano H. Miura M. Mol. Cell. 1999; 4: 757-769Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). 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 (18Cecconi F. Alvarez-Bolado G. Meyer B.I. Roth K.A. Gruss P. Cell. 1998; 94: 727-737Abstract Full Text Full Text PDF PubMed Scopus (810) Google Scholar). In this report, we describe the identification and initial functional characterization of a novel regulator of Apaf1, which we have termed NAC. 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. 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. An anti-NAC DNAzyme oligonucleotide was designed by the method of Joyce (19Santoro S.W. Joyce G.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4262-4266Crossref PubMed Scopus (1237) Google Scholar), 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). 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. For immunoprecipitation and immunoblotting analyses, cells were lysed in either buffer A (142.4 mm KCl, 5 mmMgCl2, 10 mm HEPES (pH 7.4), 0.5 mmEGTA, 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 mmHEPES (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 β-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) (20Cardone M. Roy N. Stennicke H. Salvesen G. Franke T. Stanbridge E. Frisch S. Reed J. Science. 1998; 282: 1318-1321Crossref PubMed Scopus Google Scholar). GST fusion proteins were from pGEX-4T1 in (Stratagene) and using GST fusion proteins of were with 1 in of buffer A for at The were then at with 1 of lysates containing proteins in of buffer A supplemented with 0.5 were in of buffer followed by in of SDS-PAGE and detection by extracts were using buffer B as R. Salvesen G.S. Reed J.C. Nature. 1997; 388: PubMed Scopus Google and with cyt-c and dATP for 10 at and then μm was The protein lysates were by using a gel in buffer containing mm (pH 7.4), mm KCl, 1.5 mm MgCl2, 1 mm EDTA, and 1 mm were for NAC and Apaf1 by followed by extracts were in hypotonic buffer B as R. Salvesen G.S. Reed J.C. Nature. 1997; 388: PubMed Scopus Google and with of cyt-c and 1 mm dATP in buffer R. Salvesen G.S. Reed J.C. Nature. 1997; 388: PubMed Scopus Google for at substrate was then and protease activity was by the release of at cells were directly lysed in buffer mm HEPES (pH 7.4), mm NaCl, and 1 mm for protein and for cleavage of as R. Salvesen G.S. Reed J.C. Nature. 1997; 388: PubMed Scopus Google Scholar). of pro-caspase-9 in cytosolic extracts was by SDS-PAGE (20Cardone M. Roy N. Stennicke H. Salvesen G. Franke T. Stanbridge E. Frisch S. Reed J. Science. 1998; 282: 1318-1321Crossref PubMed Scopus Google Scholar). were with and effector using as 1.5 in containing and cells by were and cells were fixed in with 1 4′,6-diamidino-2-phenylindole and the of cells with apoptotic was by (20Cardone M. Roy N. Stennicke H. Salvesen G. Franke T. Stanbridge E. Frisch S. Reed J. Science. 1998; 282: 1318-1321Crossref PubMed Scopus Google R. Salvesen G.S. Reed J.C. Nature. 1997; 388: PubMed Scopus Google Scholar). A of the CARD domain of NAC was generated using the as S. R. K. Godzik A. B. Reed J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), the of the of Apaf1, and J. H. H. G. Cell. 1998; 94: Full Text Full Text PDF PubMed Scopus Google Scholar, H. Srinivasula S. G. Fernandes-Alnemri T. Alnemri E. Y. Nature. 1999; PubMed Scopus Google Scholar). cDNAs were obtained by reverse chain a encoding a acid protein The predicted NAC protein contains an NB domain, followed by leucine-rich and a CARD Apaf1/CED4 family proteins that also contain CARD and NB the CARD domain of NAC is at its The NB domain of NAC contains classical Walker A and B of proteins and is similar in amino acid sequence to the NB domain of J. C. P. J. J. M. K. G. M. P. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, N. T. L. Y. C. Chen S. R. J. Liu D. J. G. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), followed by human APAF1 the Drosophila Apaf1 homologue and the C. elegans CED4 protein NAC NB domain was to and to in an ATP-dependent vitro The CARD domain of NAC shares and amino acid identity with the CARD domains of and CED4, respectively 1 The NAC CARD sequence was the of other using the 1 of the typical of these domains J. H. H. G. Cell. 1998; 94: Full Text Full Text PDF PubMed Scopus Google Scholar). The of NAC are of and which also contain In R the function as motifs for pathogen B. Deisenhofer J. Curr. Opin. Struct. Biol. 1995; 5: 409-416Crossref PubMed Scopus (321) Google Scholar), a role of these in NAC to specific NAC cDNAs were obtained that mRNA that proteins or acid the and CARD NAC were in human with found in and were synthetic NAC peptides and confirmed to the NAC protein by immunoblotting and immunoprecipitation and NAC protein was in human with in and the tissues A anti-NAC was in which to be in NAC mRNA and protein expression in some tissues the of regulation 2, A analysis of the in vivo of NAC expression in human tissues association with differentiation in epithelia of the and as as in the where cells were and cells were and were also whereas were In the NAC also in with the differentiation from to to in the that NAC expression is associated with differentiation of some types of short lived In vitro binding were using a fusion protein containing the CARD of NAC. The CARD of NAC bound to and also selectively in vitro with the of Apaf1, and CED4 with the of or A and The ability of NAC to with Apaf1, and CED4 in cells was confirmed by of proteins from cells B and The NAC protein also be with Apaf1 from cells that NAC, using anti-NAC antisera with full-length Apaf1, NAC with a of Apaf1 which the WD that this protein in an state C. Slee E.A. Harte M.T. Martin S.J. J. Biol. Chem. 1999; 274: 20855-20860Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), that Apaf1 interacts with NAC with this association of full-length Apaf1 with NAC was by stimuli as which cyt-c release and result in Apaf1 activation J. Liu X. K. A.M. J. Wang X. Science. 1997; 275: PubMed Scopus Google Scholar). to death stimuli, NAC was with Apaf1, 10 complexes were Apaf1 is known to complexes with (10Li P. Nijhawan D. Budihardjo I. Srinivasula S. Ahmad M. Alnemri E. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6157) Google Scholar), we the of NAC interactions by of NAC by in cells the of Apaf1 that with 3 that NAC interactions of these These also that NAC in a with Apaf1 and NAC was in Apaf1 was 3 NAC directly 3 A and and was in complexes Apaf1 was induces formation of a large containing multiple Apaf1 and in an H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1771) Google Scholar). The be by of cytosolic extracts and at K. C. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). a of has been in some types of cells, that other proteins with the K. C. Walker G. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In extracts from cells, which contain NAC, as by immunoblotting a of was by in extracts from cells, were a large that both Apaf1 and NAC by analysis of the in which Apaf1 and NAC in cells that contain of NAC, as Jurkat the NAC and Apaf1 in where of NAC were in the of these complexes In extracts cyt-c Apaf1 as a whereas NAC was multiple a NAC in with using or F. Z. K. M. S. A. and J. C. that NAC selectively with the Apaf1 the with which NAC caspase NAC was from and Jurkat cell and associated caspase activity was cleavage of the R. Salvesen G.S. Reed J.C. Nature. 1997; 388: PubMed Scopus Google Scholar). These revealed that NAC is associated with active caspases cyt-c significant caspase the of these of of and the of inhibitor of apoptosis protein and mitochondrial activator of K. C. Walker G. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google these that the containing NAC is In cells, which contain NAC, of NAC by formation of the cyt-c with of the the function of Apaf1 in the and of NAC, we proteolytic processing of and activation of downstream caspases in extracts from and cells containing NAC processing of and activation of downstream that NAC Apaf1 reducing NAC protein using expression or oligonucleotides the ability of cyt-c to caspases in cell extracts in for using Jurkat cells, which contain of NAC, ablation of NAC protein changes in the of from anti-NAC Jurkat cells were to cyt-c with control cells, in of caspase In to caspase activation was a specific in the cyt-c which Apaf1 for caspase that NAC the activation of caspases in cell extracts was obtained by of NAC from extracts using a GST fusion protein containing the CARD of NAC and by expression in cells of a of NAC of only the CARD domain with cell NAC also with Apaf1 in inducing caspase activation and apoptosis in intact In using D and or other cell of NAC by or in with caspase activation or In overexpressing NAC with Apaf1 and in in activation of caspases and of as from that of D of NAC also cells to of apoptosis as which apoptosis an Apaf1-dependent mechanism H. R. A. R. Cell. 1998; 94: Full Text Full Text PDF PubMed Scopus Google Scholar), by antibody which an and that the enhanced of cells to was to in the of Apaf1 or proteins in cells full-length NAC enhanced apoptosis and caspase activation induced by overexpressing Apaf1 or by of cells with Apaf1-dependent apoptotic stimuli D fragments of NAC containing only the CARD or NB domain the with apoptosis induced by of and by Apaf1-dependent stimuli as activated by immunoblotting that these in Apaf1-dependent apoptosis by these fragments of NAC were to of the Apaf1 or proteins some J. C. P. J. J. M. K. G. M. P. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, N. T. L. Y. C. Chen S. R. J. Liu D. J. G. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google and T. D. H. A. M. H. L. I. L. T. R. P. M. Cell. 1999; 96: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. S. A. Dixit V. J. Biol. Chem. 1999; 275: Full Text Full Text PDF Scopus (98) Google Scholar), NAC in cells of tissues in to activation of a that be for by in the of Apaf1 protein or downstream caspases D. M. T. K. F. S. I. 1999; M. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The of NAC a mechanism for Apaf1 NAC is and NAC interacts with proteins via its or other Apaf1/CED4 family proteins directly protease activation by the active into close to each other (4Yang X. Chang H. Baltimore D. Science. 1998; 281: 1355-1357Crossref PubMed Scopus (234) Google Scholar, 12Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1771) Google Scholar, 13Saleh A. Srinivasula S. Acharya S. Fishel R. Alnemri E. J. Biol. Chem. 1999; 274: 17941-17945Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 14Adrain C. Slee E.A. Harte M.T. Martin S.J. J. Biol. Chem. 1999; 274: 20855-20860Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). NAC it directly this caspase or interactions of NAC with Apaf1 activation of thus a paradigm for apoptosis regulation. The mechanism by which NAC Apaf1 function to be E. Alnemri for caspase-9 and Apaf1 G. Joyce for DNAzyme H. for S. H. H. and S. for and R. for