Identification of Ipaf, a Human Caspase-1-activating Protein Related to Apaf-1
Procaspase-9 contains an NH2-terminal caspase-associated recruitment domain (CARD), which is essential for direct association with Apaf-1 and activation. Procaspase-1 also contains an NH2-terminal CARD domain, suggesting that its mechanism of activation, like that of procaspase-9, involves association with an Apaf-1-related molecule. Here we describe the identification of a human Apaf-1-related protein, named Ipaf that contains an NH2-terminal CARD domain, a central nucleotide-binding domain, and a COOH-terminal regulatory leucine-rich repeat domain (LRR). Ipaf associates directly and specifically with the CARD domain of procaspase-1 through CARD-CARD interaction. A constitutively active Ipaf lacking its COOH-terminal LRR domain can induce autocatalytic processing and activation of procaspase-1 and caspase-1-dependent apoptosis in transfected cells. Our results suggest that Ipaf is a specific and direct activator of procaspase-1 and could be involved in activation of caspase-1 in response to pro-inflammatory and apoptotic stimuli. Procaspase-9 contains an NH2-terminal caspase-associated recruitment domain (CARD), which is essential for direct association with Apaf-1 and activation. Procaspase-1 also contains an NH2-terminal CARD domain, suggesting that its mechanism of activation, like that of procaspase-9, involves association with an Apaf-1-related molecule. Here we describe the identification of a human Apaf-1-related protein, named Ipaf that contains an NH2-terminal CARD domain, a central nucleotide-binding domain, and a COOH-terminal regulatory leucine-rich repeat domain (LRR). Ipaf associates directly and specifically with the CARD domain of procaspase-1 through CARD-CARD interaction. A constitutively active Ipaf lacking its COOH-terminal LRR domain can induce autocatalytic processing and activation of procaspase-1 and caspase-1-dependent apoptosis in transfected cells. Our results suggest that Ipaf is a specific and direct activator of procaspase-1 and could be involved in activation of caspase-1 in response to pro-inflammatory and apoptotic stimuli. caspase recruitment domain nucleotide-binding domain leucine-rich repeats reverse transcriptase-polymerase chain reaction green fluorescent protein glutathioneS-transferase Dulbecco's modified Eagle's medium polyacrylamide gel electrophoresis peripheral blood leukocytes nuclear factor κB death effector filament lipopolysaccharide Apoptosis is a physiological cellular suicide process, which is essential for normal development and homeostasis of multicellular organisms and has been highly conserved throughout evolution (1Vaux D.L. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2239-2244Crossref PubMed Scopus (907) Google Scholar). Genetic studies in Caenorhabditis elegans have identified four genes, which encode four proteins that are required for programmed cell death (2Horvitz H.R. Cancer Res. 1999; 59: 1701-1706PubMed Google Scholar, 3Hengartner M.O. Curr. Opin. Genet. Dev. 1996; 6: 34-38Crossref PubMed Scopus (91) Google Scholar, 4Metzstein M.M. Stanfield G.M. Horvitz H.R. Trends Genet. 1998; 14: 410-416Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Among these proteins, CED-3 is the apoptotic initiator caspase, which is activated by binding to the adaptor protein CED-4. This activation involves mutual recognition of the respective caspase-associated recruitment domains (CARDs)1 of CED-3 and CED-4 and oligomerization of CED-4 (5Yang X. Chang H.Y. Baltimore D. Science. 1998; 281: 1355-1357Crossref PubMed Scopus (234) Google Scholar). So far, three proteins, Apaf-1, Nod1 (also called CARD 4), and Nod2 have been identified as mammalian homologues of CED-4 (6Zou 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, 7Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Nunez G. J. Biol. Chem. 1999; 274: 14560-14567Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, 8Bertin J. Nir W.J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 9Ogura Y. Inohara N. Benito A. Chen F.F. Yamaoka S. Nunez G. J. Biol. Chem. 2001; 276: 4812-4818Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar). Like CED-4, Apaf-1, Nod1, and Nod2 contain one, or two in the case of Nod2, NH2-terminal CARD domains followed by a centrally located nucleotide-binding domain (NBD). Apaf-1, Nod1, and Nod2 differ from CED-4 by having a long COOH-terminal regulatory domain, containing WD-40 repeats in Apaf-1 or leucine-rich repeats (LRRs) in Nod1 and Nod2. Binding of cytochrome c to the WD-40 repeats of Apaf-1 leads to ATP/dATP-dependent oligomerization of Apaf-1 and subsequent recruitment, processing, and activation of caspase-9 (5Yang X. Chang H.Y. Baltimore D. Science. 1998; 281: 1355-1357Crossref PubMed Scopus (234) Google Scholar, 10Srinivasula 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, 11Saleh A. Srinivasula S.M. Acharya S. Fishel R. Alnemri E.S. J. Biol. Chem. 1999; 274: 17941-17945Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). Nod1 and Nod2 on the other hand appear to function in activating the NF-κB pathway via an interaction with RICK (also called RIP2, CARDIAK), a serine-threonine kinase that contains a COOH-terminal CARD (7Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Nunez G. J. Biol. Chem. 1999; 274: 14560-14567Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, 8Bertin J. Nir W.J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 9Ogura Y. Inohara N. Benito A. Chen F.F. Yamaoka S. Nunez G. J. Biol. Chem. 2001; 276: 4812-4818Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar, 12Inohara N. del Peso L. Koseki T. Chen S. Nunez G. J. Biol. Chem. 1998; 273: 12296-12300Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 13McCarthy J.V. Ni J. Dixit V.M. J. Biol. Chem. 1998; 273: 16968-16975Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 14Thome M. Hofmann K. Burns K. Martinon F. Bodmer J.L. Mattmann C. Tschopp J. Curr. Biol. 1998; 8: 885-888Abstract Full Text Full Text PDF PubMed Google Scholar). Nod1 has also been shown to bind and activate caspase-9 and to enhance caspase-9-induced apoptosis (7Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Nunez G. J. Biol. Chem. 1999; 274: 14560-14567Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, 8Bertin J. Nir W.J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Because humans, unlike C. elegans, contain multiple initiator caspases with CARD domains (15Hofmann K. Bucher P. Tschopp J. Trends Biochem. Sci. 1997; 22: 155-156Abstract Full Text PDF PubMed Scopus (447) Google Scholar), it can be speculated that other novel CED-4/Apaf-1 family members are likely to be found in humans. In this study, we identified and characterized Ipaf (ICE-protease-activating factor), a novel human CED-4/Apaf-1 family member that associates specifically with and activates caspase-1/ICE. Ipaf can also induce apoptosis in human cells in a caspase-1-dependent manner. The nucleotide sequence encoding a CARD-containing peptide (GenBankTM accession number AL121653) with homology to the CARD of procaspase-1 was found in the NCBI public genomic data base using the TBLASTN program. The complete cDNA of this new CARD-containing protein (designated Ipaf) was isolated from a human peripheral blood leukocytes (PBL) cDNA library by the polymerase chain reaction (PCR) using Ipaf-specific primers. The cDNA sequence of Ipaf was verified by nucleotide sequencing. Constructs encoding full-length Ipaf or truncated mutants were generated by PCR using modified complementary PCR adapter-primers. FLAG- and T7-epitope tagging was done by cloning the PCR generated cDNAs of the respective genes in-frame into pFLAG CMV-2 (IBI Kodak) and pcDNA3-T7 (Invitrogen) vectors, respectively. Plasmids encoding GFP fusions were constructed using pEGFP-N1 (CLONTECH). The Ipaf-CARD-GST construct was made by cloning residues 1–88 of Ipaf with COOH-terminal GST in pET-28a (Novagen). Procaspase-1 and RICK constructs have been described previously (12Inohara N. del Peso L. Koseki T. Chen S. Nunez G. J. Biol. Chem. 1998; 273: 12296-12300Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 16Alnemri E.S. Fernandes-Alnemri T. Litwack G. J. Biol. Chem. 1995; 270: 4312-4317Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Cells were cultivated either in Dulbecco's modified Eagle's medium (DMEM) (MCF7 cells) or DMEM/F-12 (293T cells) (Life Technologies, Inc.), supplemented with 10% fetal bovine serum, 200 µg·ml−1 penicillin and 100 µg·ml−1 streptomycin sulfate. MCF7 cells were grown on coverslips and then transfected with constructs encoding GFP-tagged Ipaf. 24 h after transfection, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 30 min. The coverslips were mounted on a glass slide, and the fluorescence of GFP was detected by confocal microscopy using excitation wavelength of 488 nm and detection wavelength of 522 nm. Images were Kalman-averaged. 293T cells (5 × 106 cells) in 100-mm dishes were transiently transfected with the expression plasmids using the LipofectAMINETM (Life Technologies, Inc.) method as per the manufacturer's instructions. 24 h after transfection, cells were lysed in 50 mm Tris/HCl, pH 7.6, 150 mm NaCl containing 0.5% Nonidet P-40, 10 µg·ml−1 leupeptin and aprotinin, and 0.1 mm phenylmethylsulfonyl fluoride and clarified by centrifugation at 15,000 × g for 15 min. The clarified lysates were preabsorbed on protein G-Sepharose (Amersham Pharmacia Biotech) and then incubated with anti-FLAG-M5 monoclonal antibody (Eastman Kodak Co.) for 2 h, followed by protein G-Sepharose-agarose IgG beads. Immune complexes were washed extensively in the lysis buffer and eluted by boiling in SDS sample buffer. The eluted proteins were resolved by SDS-PAGE and detected by Western blot analysis with a horseradish peroxidase-conjugate T7 antibody (Novagen). GST pull down assays were performed as described previously (17Ahmad M. Srinivasula S.M. Wang L. Talanian R.V. Litwack G. Fernandes-Alnemri T. Alnemri E.S. Cancer Res. 1997; 57: 615-619PubMed Google Scholar). Total lysates from 293T cells (from 100-mm plates) transfected with 1 µg of NH2-terminally FLAG-tagged procaspase-1 in the presence of either 5 µg of RICK or different Ipaf constructs were prepared 18 h post-transfection and subjected to 15% SDS-PAGE. Tagged caspase-1 was detected by anti-FLAG antibody. 293T cells (0.5 × 105cells/well) in 12-well plates were transfected with 0.1 µg of LacZ reporter plasmid, 0.2 µg of the caspase-1 plasmid, and 0.4 µg of various Ipaf expression plasmids using the LipofectAMINETMmethod as per the manufacturer's instructions. Cells were stained for β-galactosidase activity 20 h after transfection. Normal and apoptotic blue cells were counted. The percentage of apoptotic cells in each experiment was expressed as the mean percentage of stained apoptotic cells as a fraction of the total number of blue cells (n = 3). To identify novel members of the CED-4/Apaf-1 family, we searched the NCBI public data bases for genes having sequence similarity to the CARD of procaspase-1. We identified a genomic sequence in human chromosome 2 (GenBankTM accession number AL121653) that encodes a CARD-containing protein with high similarity to the CARD of procaspase-1. Based on the predicted cDNA sequence of this gene, we cloned the entire open reading frame by PCR from a human PBL library with two sets of primers corresponding to overlapping sequences of the coding region of the gene. The deduced amino acid sequence encodes a protein of 1024 residues with a predicted molecular mass of 116.1 kDa (Fig. 1A). Because of its activity, this protein was termed Ipaf forICE-protease activatingfactor (see below). A BLAST search of protein data bases indicated that Ipaf is a novel protein composed of three putative functional domains (Fig. 1 B), including an NH2-terminal CARD (residues 1–88), followed directly by a putative nucleotide binding domain (residues 163–457) and a COOH-terminal region (residues 656–1024) containing multiple leucine-rich repeats. The CARD domain of Ipaf is mostly related to the CARD domains of procaspase-1 and other caspase-1-related procaspases (Fig. 1 C). The CARD/NBD/LRR domain structure of Ipaf is similar to that of CED-4/Apaf-1/Nod1, thus establishing Ipaf as a new member of the CED4/Apaf-1/Nod1 family of proteins. RT-PCR analysis of the expression of the mRNA of Ipaf in multiple human tissues and cell lines revealed that Ipaf is highly expressed in bone marrow and to a lesser extent in lymph node, placenta, and spleen. Ipaf mRNA was also detected in the brain (Fig. 1 D). Among the different cell lines tested, Ipaf mRNA was found only in the THP-1 monocytic cell line. Subcellular localization is often important for regulating signaling molecules (18Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (830) Google Scholar). To gain insight into the intracellular location of Ipaf, a COOH-terminal GFP-tagged construct was created and transfected in MCF7 cells. Interestingly, the fusion protein exhibited a cytoplasmic filament pattern (Fig.2A) similar to the death effector filaments (DEF) formed by CRADD/RAIDD, FADD, or the death effector domain of procaspase-8 (19Siegel R.M. Martin D.A. Zheng L. Ng S.Y. Bertin J. Cohen J. Lenardo M.J. J. Cell Biol. 1998; 141: 1243-1253Crossref PubMed Scopus (214) Google Scholar). A similar construct in which the CARD domain of Ipaf has been removed showed mostly diffuse cytoplasmic subcellular localization (Fig. 2 A). The formation of DEF-like structures by the full-length Ipaf but not the CARD-truncated mutant indicates that Ipaf may dimerize or oligomerize in a CARD-mediated manner. To test if the Ipaf molecules are able to interact with each other, 293T cells were transfected with expression vectors encoding wild type or truncated FLAG-tagged Ipaf and T7-tagged Ipaf. As shown in Fig.2 B, the FLAG-tagged Ipaf was able to bind to the T7-tagged Ipaf, indicating that the Ipaf molecules can indeed associate with each other. This association was dependent on the NH2-terminal CARD as deletion of this domain prevented the CARD-truncated Ipaf mutant from interacting with the full-length Ipaf (Fig.2 B). Moreover, the epitope-tagged CARD of Ipaf was able to coimmunoprecipitate the full-length protein. This CARD-mediated interaction was also demonstrated in vitro as35S-labeled Ipaf was able to associate with an Ipaf-CARD-GST fusion protein but not the GST control (Fig.2 C). Taken together, the above results suggest that the CARD domain of Ipaf mediates its oligomerization. Because Ipaf shows significant structural homology with Nod1 and Nod2, and the latter proteins activate NF-κB (7Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Nunez G. J. Biol. Chem. 1999; 274: 14560-14567Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, 8Bertin J. Nir W.J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 9Ogura Y. Inohara N. Benito A. Chen F.F. Yamaoka S. Nunez G. J. Biol. Chem. 2001; 276: 4812-4818Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar), we tested whether Ipaf can activate NF-κB signaling using a reporter luciferase construct. Surprisingly, no NF-κB activation was detected after expression of Ipaf in 293T cells (data not shown), which indicates that Ipaf is not involved in the NF-κB pathway but could play a role in another signaling pathway. The CARD and its structurally related death effector domain have been shown to function in diverse signaling pathways that mediate apoptosis (20Nunez G. Benedict M.A. Hu Y. Inohara N. Oncogene. 1998; 17: 3237-3245Crossref PubMed Scopus (942) Google Scholar). Because the CARD of Ipaf shows high similarity to the CARD of procaspase-1, we tested the ability of Ipaf to associate with procaspase-1 in 293T cells. Interestingly, immunoprecipitation of epitope-tagged Ipaf transiently expressed in 293T cells quantitatively coprecipitated procaspase-1 (Fig.3A). The interaction was mediated by the CARD motifs in the two molecules as truncated Ipaf or procaspase-1 containing only the CARD motifs could interact with each other, whereas a truncated version of Ipaf lacking its CARD (ΔCARD) could not precipitate procaspase-1 (Fig. 3 A). To rule out the possibility that other proteins were necessary for the Ipaf-procaspase-1 interaction, we analyzed the ability of the Ipaf-CARD-GST fusion protein to associate with in vitro 35S-labeled procaspase-1. In agreement with a direct interaction, 35S-labeled procaspase-1 to the Ipaf-CARD-GST fusion protein but not to the GST control B). no significant interaction was detected and other procaspases as as the CARD-containing proteins Apaf-1 and (data not results suggest that Ipaf associates with procaspase-1 through a CARD-CARD interaction. This Ipaf-procaspase-1 interaction to whether Ipaf be able to procaspase-1 activation. is as a which is to be activated through J.R. M.J. S.M. J.R. J. PubMed Scopus Google Scholar, Talanian R.V. S. T. Full Text PDF PubMed Scopus Google Scholar, M.A. M.A. PubMed Scopus Google Scholar, T. S. M. D. L. A. L. L. Talanian R. M. Wong R. D. H. 1997; PubMed Scopus Google Scholar). data suggest that Ipaf is able to oligomerize (Fig. A and B). Moreover, Ipaf a COOH-terminal LRR domain, which could be to the long WD-40 repeats of Apaf-1, thus regulating Ipaf To whether Ipaf procaspase-1 processing, we transfected 293T cells with expression constructs encoding NH2-terminally FLAG-tagged procaspase-1 with or constructs encoding T7-tagged full-length Ipaf or a As a we an experiment in which procaspase-1 was with which is to procaspase-1 processing M. Hofmann K. Burns K. Martinon F. Bodmer J.L. Mattmann C. Tschopp J. Curr. Biol. 1998; 8: 885-888Abstract Full Text Full Text PDF PubMed Google Scholar, M.A. W.J. Dixit V.M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, of the Ipaf but not the full-length protein, was able to induce activation of procaspase-1 C). Moreover, but not the full-length Ipaf, was able to enhance apoptosis in 293T cells (Fig. of full-length Ipaf or Ipaf no on cell expressed in 293T cells (Fig. A). results that Ipaf is a activator of procaspase-1 and that the COOH-terminal LRR domain of Ipaf as a of Ipaf activity, as of this domain results in a activation of Ipaf. Ipaf to apoptotic cell death by or (data not shown), suggesting that the activity was specific for 293T cells were with a constructs encoding Ipaf wild type procaspase-1, and a procaspase-1 active mutant the procaspase-1 mutant was able to Ipaf thus indicating that Ipaf activates specifically procaspase-1 (Fig. A). To the sequence of Ipaf that is able to activation of procaspase-1, we mutant of Ipaf and tested for ability to activate procaspase-1. As shown in of Ipaf mutants containing the CARD and the Ipaf Ipaf and lacking the LRR with procaspase-1, in apoptotic cell in the of Ipaf but not its activity (Fig. A). of the CARD of Ipaf with procaspase-1 was to induce a in apoptotic cell death by caspase-1 (Fig. A). deletion of the CARD of Ipaf Ipaf (Fig. A). In agreement with these only the constitutively active which contain the CARD and entire of Ipaf, were able to induce procaspase-1 processing (Fig. B). The CARD-truncated Ipaf was not able to induce processing of procaspase-1. The or LRR were (data not results that the CARD and of Ipaf are necessary and to bind and activate procaspase-1. To whether processing of procaspase-1 by we the constitutively active Ipaf with a T7-tagged procaspase-1 active mutant in 293T cells and if this mutant could be As shown in no processing of the procaspase-1 mutant could be detected in the presence of Ipaf of is likely that Ipaf the of procaspase-1 by its in with the In this the identification and the of Ipaf, a new member of the CED-4/Apaf-1 Ipaf is able to specifically associate with procaspase-1 via CARD-CARD interaction and to procaspase-1 activation. Interestingly, the of Ipaf mRNA is similar to that of caspase-1 M.A. W.J. Dixit V.M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, S. and S. Cell in Scholar). Ipaf mRNA was detected in the THP-1 monocytic cell which is to high of procaspase-1 M.A. W.J. Dixit V.M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, S. and S. Cell in Scholar). Like Apaf-1, Ipaf is composed of an NH2-terminal CARD followed by a Apaf-1 has been shown to induce autocatalytic processing of Ipaf can also suggesting that Apaf-1 and Ipaf may a mechanism for activation, with the with this Ipaf was to induce activation of an active mutant of procaspase-1, suggesting that activation of procaspase-1 involves autocatalytic activation of procaspase-1. the role of the COOH-terminal this domain is likely to function in a to the Apaf-1 WD-40 domain, Ipaf activation by of the Ipaf constitutively active and of procaspase-1 processing, of other stimuli. of the WD-40 repeats of Apaf-1 has also been shown to Apaf-1 constitutively active 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, M.A. Hu Y. Inohara N. Nunez G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Ipaf, RICK is the only activator of caspase-1 M. Hofmann K. Burns K. Martinon F. Bodmer J.L. Mattmann C. Tschopp J. Curr. Biol. 1998; 8: 885-888Abstract Full Text Full Text PDF PubMed Google Scholar, M.A. W.J. Dixit V.M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the role of caspase-1 in apoptosis is it has been demonstrated that this an important role in programmed cell death as caspase-1 a in apoptosis K. G. Science. 1995; PubMed Scopus Google Scholar, P. H. S. S. L. C. J. M. L. J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). Moreover, these are highly to lipopolysaccharide P. H. S. S. L. C. J. M. L. J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). Interestingly, the LRR domain is to be involved in and in binding to proteins J. Curr. Opin. Biol. 1995; PubMed Scopus Google Scholar, A. A. M. A. 1998; PubMed Scopus Google Scholar). Ipaf may be an intracellular for other that to the activation of procaspase-1. The of Ipaf the of the of Ipaf in the activation of