PYPAF7, a Novel PYRIN-containing Apaf1-like Protein That Regulates Activation of NF-κB and Caspase-1-dependent Cytokine Processing
PYRIN-containing Apaf1-like proteins (PYPAFs) are members of the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signal transduction proteins. We report here that PYPAF7 is a novel PYPAF protein that activates inflammatory signaling pathways. The expression of PYPAF7 is highly restricted to immune cells, and its gene maps to chromosome 19q13.4, a locus that contains a cluster of genes encoding numerous PYPAF family members. Co-expression of PYPAF7 with ASC results in the recruitment of PYPAF7 to distinct cytoplasmic loci and a potent synergistic activation of NF-κB. To identify other proteins involved in PYPAF7 and ASC signaling pathways, we performed a mammalian two-hybrid screen and identified pro-caspase-1 as a binding partner of ASC. Co-expression of PYPAF7 and ASC results in the synergistic activation of caspase-1 and a corresponding increase in secretion of interleukin-1β. In addition, PYPAF1 induces caspase-1-dependent cytokine processing when co-expressed with ASC. These findings indicate that PYPAF family members participate in inflammatory signaling by regulating the activation of NF-κB and cytokine processing. PYRIN-containing Apaf1-like proteins (PYPAFs) are members of the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signal transduction proteins. We report here that PYPAF7 is a novel PYPAF protein that activates inflammatory signaling pathways. The expression of PYPAF7 is highly restricted to immune cells, and its gene maps to chromosome 19q13.4, a locus that contains a cluster of genes encoding numerous PYPAF family members. Co-expression of PYPAF7 with ASC results in the recruitment of PYPAF7 to distinct cytoplasmic loci and a potent synergistic activation of NF-κB. To identify other proteins involved in PYPAF7 and ASC signaling pathways, we performed a mammalian two-hybrid screen and identified pro-caspase-1 as a binding partner of ASC. Co-expression of PYPAF7 and ASC results in the synergistic activation of caspase-1 and a corresponding increase in secretion of interleukin-1β. In addition, PYPAF1 induces caspase-1-dependent cytokine processing when co-expressed with ASC. These findings indicate that PYPAF family members participate in inflammatory signaling by regulating the activation of NF-κB and cytokine processing. nucleotide-binding site leucine-rich repeat PYRIN-containing Apaf1-like protein interleukin-1β caspase recruitment domain hemagglutinin enzyme-linked immunosorbent assay The nucleotide-binding site (NBS)1 and leucine-rich repeat (NBS/LRR) family of proteins functions as a component of signaling pathways that regulate the activation of NF-κB, cytokine processing, and apoptosis. Each family member contains an N-terminal protein-protein interaction domain that mediates assembly with a downstream signaling partner, a central NBS domain that regulates activation of the signaling complex, and a C-terminal LRR domain that likely functions as a binding site for specific upstream regulators. CARD4 (Nod1), Nod2 (CARD15), and CARD12 (Ipaf/Clan) are NBS/LRR family members that contain an N-terminal caspase recruitment domain (CARD), a protein-protein interaction domain that belongs to the death domain-fold superfamily (1Bertin 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 (311) Google Scholar, 2Inohara N. Koseki T. del Peso L., Hu, Y. Yee C. Chen S. Carrio R. Merino J. Liu D., Ni, J. Nunez G. J. Biol. 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Inohara N. Hernandez L.D. Galan J.E. Nunez G. Janeway C.A. Medzhitov R. Flavell R.A. Nature. 2002; 416: 194-199Crossref PubMed Scopus (742) Google Scholar, 8Chin A.I. Dempsey P.W. Bruhn K. Miller J.F., Xu, Y. Cheng G. Nature. 2002; 416: 190-194Crossref PubMed Scopus (330) Google Scholar). Confirming the importance of NOD2 in inflammatory signaling, mutations within its gene confer susceptibility to Crohn's disease and Blau syndrome, two chronic immune disorders (9Hugot J.P. Chamaillard M. Zouali H. Lesage S. Cezard J.P. Belaiche J. Almer S. Tysk C. O'Morain C.A. Gassull M. Binder V. Finkel Y. Cortot A. Modigliani R. Laurent-Puig P. Gower-Rousseau C. Macry J. Colombel J.F. Sahbatou M. Thomas G. Nature. 2001; 411: 537-539Crossref PubMed Scopus (4652) Google Scholar, 10Ogura Y. Bonen D.K. Inohara N. Nicolae D.L. Chen F.F. Ramos R. Britton H. Moran T. Karaliuskas R. Duerr R.H. Achkar J.P. Brant S.R. Bayless T.M. Kirschner B.S. Hanauer S.B. Nunez G. Cho J.H. 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Intriguingly, the PYRIN domain is found at the N terminus of numerous NBS/LRR family members instead of a CARD domain. Because of the structural similarity to the apoptosis protein Apaf1, we have named these proteins PYPAFs forPYRIN-containing Apaf1-like proteins. Thus far, eight members of this NBS/LRR subfamily have been identified, including PYPAF1 (cryopyrin/NALP3), PYPAF2 (NBS1/NALP2), PYPAF3, PYPAF4 (PAN2/NALP4), PYPAF5, PYPAF6, MATER (PYPAF8), and CARD7 (NAC/DEFCAP/NALP1) (12Bertin J. DiStefano P.S. Cell Death Differ. 2000; 7: 1273-1274Crossref PubMed Scopus (159) Google Scholar, 14Staub E. Dahl E. Rosenthal A. Trends Biochem. Sci. 2000; 26: 83-85Abstract Full Text Full Text PDF Scopus (90) Google Scholar, 15Pawlowski K. Pio F. Chu Z. Reed J.C. Godzik A. Trends Biochem. Sci. 2001; 26: 85-87Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 16Hlaing T. Guo R.F. Dilley K.A. Loussia J.M. Morrish T.A. Shi M.M. Vincenz C. Ward P.A. J. Biol. 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The PYRIN domain is similar in structure to the CARD domain, death domain, and death effector domain, and it is therefore a new member of the death domain-fold superfamily (12Bertin J. DiStefano P.S. Cell Death Differ. 2000; 7: 1273-1274Crossref PubMed Scopus (159) Google Scholar, 13Martinon F. Hofmann H. Tschopp J. Curr. Biol. 2001; 11: 118-120Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 14Staub E. Dahl E. Rosenthal A. Trends Biochem. Sci. 2000; 26: 83-85Abstract Full Text Full Text PDF Scopus (90) Google Scholar, 21Fairbrother W.J. Gordon N.C. Humke E.W. O'Rourke K.M. Starovasnik M.A. Jian-Ping Y. Dixit V.M. Protein Sci. 2001; 10: 1911-1918Crossref PubMed Scopus (125) Google Scholar). This domain likely mediates homotypic interactions between PYRIN-containing proteins, resulting in the formation of a complex that participates in signal transduction. Indeed, PYPAF1 was shown recently to assemble with the PYRIN-containing protein ASC and activate NF-κB signaling (18Manji G. Wang L. Geddes B. Brown M. Merriam S., Al- Garawi A. Mak S. Lora J.M. Briskin M. Jurman M. Cao J. DiStefano P.S. Bertin J. J. Biol. Chem. 2001; 277: 11570-11575Abstract Full Text Full Text PDF Scopus (226) Google Scholar). A role for PYPAF family members in inflammatory signaling is indicated by the recent finding that mutations within PYPAF1 are associated with Muckle-Wells syndrome and familial cold auto-inflammatory syndrome, two genetically determined immune disorders that are similar to familial Mediterranean fever (19Hoffman H.M. Mueller J.L. Broide D.H. Wanderer A.A. Kolodner R.D. Nat. Genet. 2001; 29: 301-305Crossref PubMed Scopus (1279) Google Scholar). We report here that PYPAF7 is a novel PYRIN-containing NBS/LRR protein that is recruited by ASC to distinct cytoplasmic loci. Co-expression of PYPAF7 and ASC results in the synergistic activation of NF-κB and caspase-1-dependent cytokine processing. These findings identify PYPAF7 as a novel regulator of inflammatory signal transduction pathways. Plasmids expressing either full-length PYPAF7 (PYPAF7-FL, residues 1–1061, accession number AY095146) or a PYPAF7 truncation mutant lacking the PYRIN domain (PYPAF7ΔPYRIN, residues 89–1061) with a C-terminal FLAG epitope were constructed using pCI (Promega). The panel of CARD domains used for the mammalian two-hybrid screen was described previously (22Wang L. Guo Y. Huang W.J., Ke, X. Poyet J.L. Manji G.A. Merriam S. Glucksmann M.A. DiStefano P.S. Alnemri E.S. Bertin J. J. Biol. Chem. 2001; 276: 21405-21409Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Plasmid pCMV-CARD6-CARD/BD was constructed by inserting the CARD of CARD6 (residues 1–109, accession number AF356193) into pCMV/BD (Stratagene). Plasmid pCMV-ASC-PYRIN/AD was described previously (18Manji G. Wang L. Geddes B. Brown M. Merriam S., Al- Garawi A. Mak S. Lora J.M. Briskin M. Jurman M. Cao J. DiStefano P.S. Bertin J. J. Biol. Chem. 2001; 277: 11570-11575Abstract Full Text Full Text PDF Scopus (226) Google Scholar). Plasmids expressing mouse pro-IL-1β with a C-terminal FLAG epitope or human pro-caspase-1 with a C-terminal Myc epitope were constructed using pCI. Plasmids expressing either dominant negative IKKγ or inactive pro-caspase-1 (C285) were described previously (4Geddes B.J. Wang L. Huang W-J. Lavellee M. Manji G.A. Brown M. Jurman M. Cao J. Morgenstern J. Merriam S. Glucksmann M.A. DiStefano P.S. Bertin J. Biochem. Biophys. Res. Commun. 2001; 284: 77-82Crossref PubMed Scopus (96) Google Scholar, 18Manji G. Wang L. Geddes B. Brown M. Merriam S., Al- Garawi A. Mak S. Lora J.M. Briskin M. Jurman M. Cao J. DiStefano P.S. Bertin J. J. Biol. Chem. 2001; 277: 11570-11575Abstract Full Text Full Text PDF Scopus (226) Google Scholar). Total RNA from cells in culture was extracted (Qiagen), and expression profiles were determined by real time quantitative PCR analysis (Taqman™). In brief, an oligonucleotide probe (5′-TCCGCAGGAAATTCCGGCTCATG-3′) was designed to anneal to PYPAF7 between two PCR primers (forward, 5′-CCCAGGAAACCTACAGGGACTA-3′ and reverse, 5′-CGCGCATTGCGGTCTT-3′). The probe was then fluorescently labeled with FAM (reporter dye) on the 5′-end and TAMRA (quencher dye) on the 3′-end. Probe and PCR primers designed for β2microglobulin were described previously (18Manji G. Wang L. Geddes B. Brown M. Merriam S., Al- Garawi A. Mak S. Lora J.M. Briskin M. Jurman M. Cao J. DiStefano P.S. Bertin J. J. Biol. Chem. 2001; 277: 11570-11575Abstract Full Text Full Text PDF Scopus (226) Google Scholar). PCR reactions were performed on cDNAs from specific cell types using primers and probes for both PYPAF7 and β2 microglobulin genes. Fluorescent emissions generated during the PCR reaction were measured using a Sequence Detector 7700 (PerkinElmer Life Sciences). The expression level of PYPAF7 was normalized to the expression of β2 microglobulin for each sample. NF-κB and mammalian two-hybrid assays were described previously (22Wang L. Guo Y. Huang W.J., Ke, X. Poyet J.L. Manji G.A. Merriam S. Glucksmann M.A. DiStefano P.S. Alnemri E.S. Bertin J. J. Biol. Chem. 2001; 276: 21405-21409Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). For mammalian two-hybrid assays, 293T cells were transfected with pCMV/AD and pCMV/BD plasmids, pFR-Luc firefly reporter (Stratagene), and pRL-TK Renilla reporter (Promega). For NF-κB assays, 293T cells were transfected with pNF-κB luciferase reporter (Stratagene), pRL-TK Renillareporter, and indicated expression plasmids. Cells were harvested, and firefly and Renilla luciferase activities were determined using the Dual-Luciferase reporter assay system (Promega). 293T cells were transfected in poly(d-lysine)-coated glass chamber slides (BioCoat, BD Labware) with plasmids expressing HA-tagged ASC and either FLAG-tagged PYPAF7 or PYPAF7ΔPYRIN using SuperFect transfection reagent (Qiagen). Cells were fixed 24 h after transfection in 4% paraformaldehyde, permeabilized and blocked in a buffer containing 0.3% Triton X-100, and incubated with the following primary and secondary antibodies: rabbit anti-HA polyclonal Y-11 (Santa Cruz Biotechnology), mouse anti-FLAG monoclonal antibody M2 (Sigma), Alexa-350 goat anti-rabbit IgG and goat IgG were generated as described previously J. Wang L. Guo Y. Poyet Srinivasula S.M. Merriam S. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). cells were in using transfection reagent with plasmids encoding mouse pro-IL-1β and indicated expression plasmids were 24 h after transfection and to for mouse to the The caspase-1 was to cells following transfection at a of We the High Throughout Genome (HTG) data base of genomic using the and and identified a novel PYPAF family was PYPAF7 PYPAF7 maps to chromosome 19q13.4, a locus that contains a cluster of genes that encode PYPAF family members including PYPAF3, PYPAF6, and A corresponding to the predicted gene was and found to encode a protein of with a predicted of A of the protein data base a domain structure of an N-terminal PYRIN domain (residues a central NBS domain (residues and a C-terminal domain (residues of at least LRR The NBS domain belongs to the subfamily of and contains including the (residues L. Trends Biochem. Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The PYRIN domain of PYPAF7 shares similarity with the PYRIN domains of other PYRIN-containing proteins, including PYPAF1 and analysis using a expression PYPAF7 expression in not or expression was in other and cell a role for this protein in inflammatory To the cells that were into distinct cell time quantitative PCR analysis PYPAF7 to in and with expression in of PYPAF7 PYPAF7 expression in immune cells by real time quantitative The level of PYPAF7 expression in cell types was normalized to the expression of β2 indicate that ASC to PYRIN-containing proteins including and F. Hofmann H. Tschopp J. Curr. Biol. 2001; 11: 118-120Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 18Manji G. Wang L. Geddes B. Brown M. Merriam S., Al- Garawi A. Mak S. Lora J.M. Briskin M. Jurman M. Cao J. DiStefano P.S. Bertin J. J. Biol. Chem. 2001; 277: 11570-11575Abstract Full Text Full Text PDF Scopus (226) Google Scholar, N. P. A. J. E. A. D.L. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). We therefore performed to interactions between PYPAF7 and ASC. FLAG-tagged PYPAF7 and HA-tagged ASC were co-expressed in cells and using a of anti-HA and anti-FLAG PYPAF7 a cytoplasmic that the ASC to cytoplasmic when not J. S. K. H. T. N. E. T. T. J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). when the two proteins were was found to with ASC We also the of a PYPAF7 mutant lacking the N-terminal PYRIN domain to recruited to the ASC PYPAF7ΔPYRIN a cytoplasmic similar to that of PYPAF7ΔPYRIN to with the ASC that the PYRIN domain of PYPAF7 is for recruitment We have been to an interaction between PYPAF7 and ASC by when both proteins are in to an interaction of the of complexes that in the cell when these proteins are the interaction between PYPAF7 and ASC its by the of PYPAF7 with ASC to cytoplasmic loci is for the synergistic activation of NF-κB and caspase-1-dependent cytokine processing We PYPAF7 and ASC participate in NF-κB signaling pathways using a luciferase reporter of PYPAF7 in cells to activate NF-κB at protein and data not In NF-κB was when of ASC were in cells not (18Manji G. Wang L. Geddes B. Brown M. Merriam S., Al- Garawi A. Mak S. Lora J.M. Briskin M. Jurman M. Cao J. DiStefano P.S. Bertin J. J. Biol. Chem. 2001; 277: 11570-11575Abstract Full Text Full Text PDF Scopus (226) Google Scholar). when ASC was at protein that not activate NF-κB, with of PYPAF7 in a increase in NF-κB The N-terminal PYRIN domain of PYPAF7 was for NF-κB signaling of this domain the synergistic of NF-κB and analysis that ASC protein were not when co-expressed with that the activation of NF-κB was not by of ASC NF-κB signaling the complex of IKKγ and blocked the of PYPAF7 to activate NF-κB and These data that PYPAF7 is an of NF-κB signaling when co-expressed with ASC. ASC is of an N-terminal PYRIN domain and a C-terminal CARD domain J. S. K. H. T. N. E. T. T. J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The of a CARD domain in ASC that a CARD-containing protein involved in PYPAF7 and ASC To identify CARD domains that with the CARD of we performed a mammalian two-hybrid analysis and for binding to the CARD domains of family members The CARD domain of ASC was found to with the CARD domain of resulting in a activation of luciferase The CARD domain of CARD12 also with the CARD domain of ASC as previously (4Geddes B.J. Wang L. Huang W-J. Lavellee M. Manji G.A. Brown M. Jurman M. Cao J. Morgenstern J. Merriam S. Glucksmann M.A. DiStefano P.S. Bertin J. Biochem. Biophys. Res. Commun. 2001; 284: 77-82Crossref PubMed Scopus (96) Google Scholar). The PYRIN domain of ASC to with the CARD domain of that the CARD domain of ASC is both and for binding In addition, of HA-tagged ASC pro-caspase-1 The of pro-caspase-1 that with ASC is the level of ASC that is in the cell A in proteins that with ASC been in (18Manji G. Wang L. Geddes B. Brown M. Merriam S., Al- Garawi A. Mak S. Lora J.M. Briskin M. Jurman M. Cao J. DiStefano P.S. Bertin J. J. Biol. Chem. 2001; 277: 11570-11575Abstract Full Text Full Text PDF Scopus (226) Google Scholar, N. P. A. J. E. A. D.L. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). in cells, ASC and interactions and We that the of pro-caspase-1 found to with ASC are by the of complexes that in the cell when both proteins are the interaction between ASC and pro-caspase-1 the mammalian two-hybrid and interaction that ASC to pro-caspase-1 its C-terminal CARD domain. finding that ASC with pro-caspase-1 to PYPAF7 and ASC also a role in caspase caspase-1 resulting in the of from To the activation of a transfection assay was used in plasmids expressing pro-caspase-1 and mouse pro-IL-1β were transfected into cells with plasmids encoding ASC and In this the of into the culture after transfection with the of caspase-1 Stehlik C. Reed J.C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). We ASC functions as an of Co-expression of ASC and pro-caspase-1 in a increase in the of The of cytokine by ASC was caspase-1-dependent or was in the of encoding pro-caspase-1 ASC was to cytokine when co-expressed with a caspase-1 active site mutant that is to and J.L. Srinivasula S.M. Tnani M. Razmara M. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2001; 276: 28309-28313Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). We PYPAF7 plays a role in pro-caspase-1 ASC was at protein that in of caspase with PYPAF7 in a synergistic activation of pro-caspase-1 and a corresponding increase in and The N-terminal PYRIN domain of PYPAF7 was for pro-caspase-1 activation of this domain the synergistic of and analysis that PYPAF7ΔPYRIN was at similar to that of that of was not by protein not PYPAF7 and ASC were to cytokine expression when co-expressed with a caspase-1 active site mutant of a caspase-1 blocked the of PYPAF7 and ASC to the secretion of these data that PYPAF7 and ASC activate We also member of the PYPAF functions as an of Co-expression of PYPAF1 with ASC in the activation of pro-caspase-1 and a corresponding increase in The N-terminal PYRIN domain of PYPAF1 was for pro-caspase-1 activation of this domain the synergistic of and analysis that was at similar to that of that of was not by protein not The increase in was on pro-caspase-1 and PYPAF1 is also an of pro-caspase-1 when co-expressed with ASC. In we have identified PYPAF7 as a novel PYPAF family finding that PYPAF7 is recruited to ASC in the of cells that these two proteins assemble into a complex that mediates signal transduction. The PYRIN domain of PYPAF7 is for its that this domain mediates the assembly of a signaling complex of PYPAF7 and ASC. The restricted expression of PYPAF7 to a role for this PYPAF family member in inflammatory data that PYPAF7 and ASC participate in the activation of NF-κB and of ASC and pro-caspase-1 by PYPAF7 an to the activation of by Dixit V.M. Sci. S. 1999; PubMed Scopus Google Scholar). of this Srinivasula S.M. Poyet Razmara M. P. Alnemri E.S. J. Biol. Chem. 2002; a similar finding that ASC to and activates with the C-terminal CARD domain of ASC was found to interactions with both and pro-IL-1β and by caspase-1 to active G. C.A. J. 1999; PubMed Scopus Google Scholar). PYPAF7 therefore an role in innate and adaptive by regulating the of and by immune PYPAF1 was also found to activate pro-caspase-1 when co-expressed with an role for this protein in the of and Muckle-Wells syndrome and familial cold auto-inflammatory syndrome therefore associated with of NF-κB and caspase-1-dependent cytokine processing. In to PYPAF7 and CARD12 also been identified as an upstream regulator of caspase-1 (4Geddes B.J. Wang L. Huang W-J. Lavellee M. Manji G.A. Brown M. Jurman M. Cao J. Morgenstern J. Merriam S. Glucksmann M.A. DiStefano P.S. Bertin J. Biochem. Biophys. Res. Commun. 2001; 284: 77-82Crossref PubMed Scopus (96) Google Scholar, 5Poyet J.L. Srinivasula S.M. Tnani M. Razmara M. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2001; 276: 28309-28313Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 6Damiano J.S. Stehlik C. Pio F. Godzik A. Reed J.C. Genomics. 2001; 75: 77-83Crossref PubMed Scopus (62) Google Scholar). These NBS/LRR family members likely as that signal transduction in to distinct upstream these proteins to similar in distinct cell to determined other PYPAF family members also the activation of NF-κB and ASC recently been identified as a signaling partner of CARD7 and F. Hofmann H. Tschopp J. Curr. Biol. 2001; 11: 118-120Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, N. P. A. J. E. A. D.L. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google findings on PYPAF7 and PYPAF1 signaling that CARD7 and also regulate the activation of NF-κB and pro-caspase-1 binding to ASC. ASC therefore as a protein that PYRIN-containing proteins to downstream signaling pathways. The findings here a role for PYPAF7 in the transduction of signals to the activation of NF-κB and