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• W2014602273 abstract "The PYRIN and CARD domains are members of the six-helix bundle death domain-fold superfamily that mediates assembly of large signaling complexes in the apoptotic and inflammatory signaling pathways. Here we show that the PYRIN-CARD protein ASC functions as a caspase-1-activating adaptor. ASC interacted specifically with procaspase-1 via CARD-CARD interactions and induced its oligomerization. Consistent with these results ectopic expression of full-length ASC, but not its isolated CARD or PYRIN domain, with procaspase-1 induced activation of procaspase-1 and processing of pro-interleukin-1β in transfected cells. Substitution of the PYRIN domain of ASC with an inducible FKBP12 oligomerization domain produced a molecule that can induce caspase-1 activation in response to stimulation with the oligomerization drug AP20187, suggesting that the PYRIN domain functions as an oligomerization domain, whereas the CARD domain functions as the effector domain in the caspase-1 activation pathway. Furthermore stable expression of an isolated CARD of ASC in THP-1 cells diminished interleukin-1β generation in response to pro-inflammatory cytokines. These results indicate that ASC is involved in the caspase-1 signaling pathway by mediating the assembly of a caspase-1-inflammasome signaling complex in response to pro-inflammatory cytokine stimulation. The PYRIN and CARD domains are members of the six-helix bundle death domain-fold superfamily that mediates assembly of large signaling complexes in the apoptotic and inflammatory signaling pathways. Here we show that the PYRIN-CARD protein ASC functions as a caspase-1-activating adaptor. ASC interacted specifically with procaspase-1 via CARD-CARD interactions and induced its oligomerization. Consistent with these results ectopic expression of full-length ASC, but not its isolated CARD or PYRIN domain, with procaspase-1 induced activation of procaspase-1 and processing of pro-interleukin-1β in transfected cells. Substitution of the PYRIN domain of ASC with an inducible FKBP12 oligomerization domain produced a molecule that can induce caspase-1 activation in response to stimulation with the oligomerization drug AP20187, suggesting that the PYRIN domain functions as an oligomerization domain, whereas the CARD domain functions as the effector domain in the caspase-1 activation pathway. Furthermore stable expression of an isolated CARD of ASC in THP-1 cells diminished interleukin-1β generation in response to pro-inflammatory cytokines. These results indicate that ASC is involved in the caspase-1 signaling pathway by mediating the assembly of a caspase-1-inflammasome signaling complex in response to pro-inflammatory cytokine stimulation. caspase recruitment domain Fas-associated death domain apoptosis-associated speck-like protein containing a caspase recruitment domain interleukin lipopolysaccharide glutathione S-transferase IL-1β-converting enzyme interferon enzyme-linked immunosorbent assay hemagglutinin FK-506-binding protein The mechanism of activation of caspase-1 in response to pro-inflammatory cytokines is not yet fully understood. In particular, the signaling molecules that associate with caspase-1 and transduce the activation signals from the cell membrane to the cytoplasmic procaspase-1 are largely unknown or not fully characterized at present. Nevertheless since the mechanism of activation of the long prodomain caspases, such as caspase-1, caspase-8, or caspase-9, is likely to be conserved, it is expected that caspase recruitment domain (CARD)1-containing adaptor molecules structurally related to Apaf-1 or the death receptor adaptor protein FADD activate caspase-1. With the recent advances in sequencing of the entire human genome, several genes encoding novel N-terminal CARD-containing Apaf-1-related proteins have been identified. These include Nod1 (CARD4), Nod2 (CARD15), and Ipaf (CARD12/CLAN) (1Inohara 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 (628) Google Scholar, 2Bertin 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, 3Ogura 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 (1170) Google Scholar, 4Poyet 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 (348) Google Scholar, 5Geddes 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 (97) Google Scholar, 6Damiano J.S. Stehlik C. Pio F. Godzik A. Reed J.C. Genomics. 2001; 75: 77-83Crossref PubMed Scopus (62) Google Scholar). Each family member contains a CARD, a nucleotide-binding domain, and a leucine-rich repeat domain that likely functions as a binding region for upstream regulators. Nod1 and Nod2 have been identified as important components of the inflammatory signaling pathways. Nod1 and Nod2 appear to function in activating the NF-κB pathway via an interaction with RICK (also called RIP2 or CARDIAK), a serine-threonine kinase that contains a C-terminal CARD (1Inohara 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 (628) Google Scholar, 2Bertin 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, 3Ogura 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 (1170) Google Scholar, 7Inohara N. del Peso L. Koseki T. Chen S. Nunez G. J. Biol. Chem. 1998; 273: 12296-12300Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 8McCarthy J.V., Ni, J. Dixit V.M. J. Biol. Chem. 1998; 273: 16968-16975Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 9Thome 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). More recently Nod2 has been implicated in susceptibility to Crohn's disease and Blau syndrome, two chronic inflammatory disorders (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. Nature. 2001; 411: 603-606Crossref PubMed Scopus (4177) Google Scholar, 11Hugot 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: 599-603Crossref PubMed Scopus (4694) Google Scholar, 12Miceli-Richard C. Lesage S. Rybojad M. Prieur A.M. Manouvrier-Hanu S. Hafner R. Chamaillard M. Zouali H. Thomas G. Hugot J.P. Nat. Genet. 2001; 29: 19-20Crossref PubMed Scopus (791) Google Scholar). Ipaf was found recently to associate directly and specifically with the CARD of procaspase-1 through CARD-CARD interaction (4Poyet 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 (348) Google Scholar, 5Geddes 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 (97) Google Scholar, 6Damiano J.S. Stehlik C. Pio F. Godzik A. Reed J.C. Genomics. 2001; 75: 77-83Crossref PubMed Scopus (62) Google Scholar). Additional experiments revealed that Ipaf induces autocatalytic processing and activation of procaspase-1 and caspase-1-dependent apoptosis in transfected cells (4Poyet 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 (348) Google Scholar). Thus 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. Interestingly other Apaf-1-related family members such as DEFCAP (NAC/CARD7/NALP1) and cryopyrin/PYPAF1 contain an N-terminal PYRIN domain instead of the CARD (13Hlaing T. Guo R.F. Dilley K.A. Loussia J.M. Morrish T.A. Shi M.M. Vincenz C. Ward P.A. J. Biol. Chem. 2001; 276: 9230-9238Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 14Chu Z.L. Pio F. Xie Z. Welsh K. Krajewska M. Krajewski S. Godzik A. Reed J.C. J. Biol. Chem. 2001; 276: 9239-9245Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 15Hoffman H.M. Mueller J.L. Broide D.H. Wanderer A.A. Kolodner R.D. Nat. Genet. 2001; 29: 301-305Crossref PubMed Scopus (1301) Google Scholar, 16Manji G.A. Wang L. Geddes B.J. 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. 2002; 277: 11570-11575Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Secondary structure analysis revealed that the PYRIN domain is a member of the six-helix bundle death domain-fold superfamily that includes death domains, death effector domains, and CARDs (17Bertin J. DiStefano P.S. Cell Death Differ. 2000; 7: 1273-1274Crossref PubMed Scopus (159) Google Scholar, 18Fairbrother W.J. Gordon N.C. Humke E.W. O'Rourke K.M. Starovasnik M.A. Yin J.P. Dixit V.M. Protein Sci. 2001; 10: 1911-1918Crossref PubMed Scopus (125) Google Scholar, 19Martinon F. Hofmann K. Tschopp J. Curr. Biol. 2001; 11: R118-R120Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Similar to other members of the death domain-fold superfamily, the PYRIN domain likely mediates assembly of large signaling complexes via PYRIN-PYRIN interactions between different family members. The PYRIN domain is also found at the N terminus of the apoptosis proteins ASC and two zebrafish caspases related to mammalian caspase-1, suggesting that this domain might play a role in caspase activation (20Masumoto J. Taniguchi S. Ayukawa K. Sarvotham H. Kishino T. Niikawa N. Hidaka E. Katsuyama T. Higuchi T. Sagara J. J. Biol. Chem. 1999; 274: 33835-33838Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 21Inohara N. Nunez G. Cell Death Differ. 2000; 7: 509-510Crossref PubMed Scopus (96) Google Scholar). In addition to the PYRIN domain, ASC contains a C-terminal CARD, suggesting that this protein could function as an adaptor to link specific members of the PYRIN-containing proteins with CARD-containing proteins. In this report we show that ASC associates specifically with procaspase-1. This association is mediated by the CARDs of ASC and procaspase-1. Induced oligomerization of the CARD of ASC was sufficient to promote caspase-1 activation and secretion of IL-1β from transfected cells. Furthermore the isolated CARD of ASC was found to be a dominant negative inhibitor of IL-1β generation in response to LPS. Combined, our data suggest that ASC is a specific activating adaptor for caspase-1. Cells were cultured either in Dulbecco's modified Eagle's medium (293T and Phoenix) or RPMI 1640 medium (THP-1) supplemented with 10% fetal bovine serum, 200 μg/ml penicillin, and 100 μg/ml streptomycin sulfate. RPMI 1640 medium for THP-1 also contained 10 mm HEPES, 1 mm sodium pyruvate, and 55 μm β-mercaptoethanol. Constructs encoding full-length ASC, DEFCAP, cryopyrin, PYPAF3 or Pyrin, or truncated mutants (PYRINs, residues 1–100) were generated by PCR using modified complementary PCR adapter primers based on the ASC, DEFCAP, cryopyrin, PYPAF3, or Pyrin cDNA sequences (GenBankTM accession numbers AB023416, NM_033004, AF464765, AF410477, and AF018080). FLAG or T7 epitope tagging was done by cloning the PCR-generated cDNAs in-frame into pFLAG CMV-2 (IBI Kodak) or pcDNA3-T7 vectors, respectively. GST tagging was done by cloning the PCR generated cDNAs together with GST cDNA in-frame into pET-21 vector (Novagen). GST fusion proteins were overexpressed inEscherichia coli strain BL21 and purified on glutathione-Sepharose beads. The FKBP12-tagged PYRIN and CARD domains of ASC were constructed in a modified pcDNA3-T7 vector by fusing three tandem repeats of FKBP12 cDNA in-frame with the cDNA of ASC-CARD (residues 105–195) or ASC-PYRIN (residues 1–105). Pro-IL-1β cDNA was generated by PCR and cloned into pcDNA3. Ipaf, procaspase-1, and pseudo-ICE constructs have been described previously (4Poyet 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 (348) Google Scholar, 22Druilhe A. Srinivasula S.M. Razmara M. Ahmad M. Alnemri E.S. Cell Death Differ. 2001; 8: 649-657Crossref PubMed Scopus (147) Google Scholar, 23Razmara M. Srinivasula S.M. Wang L. Poyet J.L. Geddes B.J. DiStefano P.S. Bertin J. Alnemri E.S. J. Biol. Chem. 2002; 277: 13952-13958Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). In vitro GST pull-down assays were performed as described previously (23Razmara M. Srinivasula S.M. Wang L. Poyet J.L. Geddes B.J. DiStefano P.S. Bertin J. Alnemri E.S. J. Biol. Chem. 2002; 277: 13952-13958Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). 293T cells (5 × 106) in 100-mm dishes were transiently transfected with the expression plasmid(s) using the LipofectAMINETM reagent (Invitrogen). Cells were lysed, and the cellular lysates were immunoprecipitated and Western blotted as described before (23Razmara M. Srinivasula S.M. Wang L. Poyet J.L. Geddes B.J. DiStefano P.S. Bertin J. Alnemri E.S. J. Biol. Chem. 2002; 277: 13952-13958Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Western blotting with the anti-ASC monoclonal antibody was done according to the manufacturer's recommendation (Medical & Biological Laboratories, Nagoya, Japan). THP-1 cells stably expressing the isolated CARD or PYRIN domains of ASC were generated by retroviral bulk infection as described before (22Druilhe A. Srinivasula S.M. Razmara M. Ahmad M. Alnemri E.S. Cell Death Differ. 2001; 8: 649-657Crossref PubMed Scopus (147) Google Scholar, 23Razmara M. Srinivasula S.M. Wang L. Poyet J.L. Geddes B.J. DiStefano P.S. Bertin J. Alnemri E.S. J. Biol. Chem. 2002; 277: 13952-13958Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). To assay for IL-1β secretion, THP-1 cells (1 × 106 cells/ml) were incubated for 4 h with IFNγ and then 14 h with 1 μg/ml LPS. The culture media of the cells were then used to quantify IL-1β by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN). 293T cells were transfected with pro-IL-1β (0.15 μg) together with or without procaspase-1 (0.05 μg) and different amounts of ASC, Ipaf, or ASC truncated mutant expression constructs in different combinations. IL-1β secretion from transfected 293T cells was assayed 36 h after transfection as described for THP-1 cells above without stimulation with IFNγ and LPS. ASC has a dual PYRIN-CARD domain structure similar to the dual domain structures of the caspase adaptor proteins FADD and CRADD (Fig.1A). A BLAST analysis of the PYRIN domain of ASC revealed that this domain is also present at the N termini of two zebrafish caspases highly related to mammalian interleukin-1β-converting enzyme (ICE) or caspase-1 (Ref. 21Inohara N. Nunez G. Cell Death Differ. 2000; 7: 509-510Crossref PubMed Scopus (96) Google Scholar and GenBankTM accession numbers NP_571580 and AAG45230) and several Ipaf-related proteins (Refs. 13Hlaing T. Guo R.F. Dilley K.A. Loussia J.M. Morrish T.A. Shi M.M. Vincenz C. Ward P.A. J. Biol. Chem. 2001; 276: 9230-9238Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 14Chu Z.L. Pio F. Xie Z. Welsh K. Krajewska M. Krajewski S. Godzik A. Reed J.C. J. Biol. Chem. 2001; 276: 9239-9245Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 15Hoffman H.M. Mueller J.L. Broide D.H. Wanderer A.A. Kolodner R.D. Nat. Genet. 2001; 29: 301-305Crossref PubMed Scopus (1301) Google Scholar, 16Manji G.A. Wang L. Geddes B.J. 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. 2002; 277: 11570-11575Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar and 19Martinon F. Hofmann K. Tschopp J. Curr. Biol. 2001; 11: R118-R120Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar and GenBankTM accession numbers AAL12497, NP_127497, AAL87104, and AAL87105). This suggested that ASC and perhaps the Ipaf-related PYRIN-containing proteins could function as activation adaptors in the caspase-1 signaling pathway(s). To test this hypothesis we determined the ability of ectopically expressed ASC to induce activation of procaspase-1. Since IL-1β secretion is one of the consequences of caspase-1 activation, we measured the levels of IL-1β in the culture media of 293 cells that ectopically express increasing amounts of ASC together with fixed amounts of procaspase-1 and pro-IL-1β. As shown in Fig. 1B, full-length ASC was able to induce IL-1β secretion from the transfected 293 cells in a dose- and caspase-1-dependent manner. The increase in IL-1β generation was associated with a decrease in the proform of caspase-1 indicating that ASC-induced IL-1β secretion is a result of autocatalytic processing/activation of procaspase-1 (Fig.1C). Consistent with this result, ASC was unable to induce processing of the procaspase-1 C285A or IL-1β secretion in the presence of procaspase-1 C285A (Fig. 1, B and C). Interestingly the CARD-only protein pseudo-ICE/Cop (22Druilhe A. Srinivasula S.M. Razmara M. Ahmad M. Alnemri E.S. Cell Death Differ. 2001; 8: 649-657Crossref PubMed Scopus (147) Google Scholar, 24Lee S.H. Stehlik C. Reed J.C. J. Biol. Chem. 2001; 276: 34495-34500Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), which has been previously shown to diminish IL-1β generation from THP-1 cells in response to LPS, diminished ASC-induced IL-1β secretion from 293 cells (Fig. 1B). Combined, the above results suggest that ASC is a caspase-1 activator and could be the physiological target of pseudo-ICE in THP-1 cells. Beside ASC, we have shown recently that Ipaf is another activating adaptor for caspase-1 (4Poyet 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 (348) Google Scholar). To compare the activities of ASC and Ipaf, equal amounts of ASC or the constitutively active Ipaf-557 were transiently expressed together with procaspase-1 and pro-IL-1β in 293 cells (Fig. 1D). ASC was as efficient as the constitutively active Ipaf-557 in inducing the secretion of IL-1β from the transfected 293 cells. Interestingly, when Ipaf-557 was co-expressed with the isolated CARD or PYRIN domain of ASC, the CARD, but not the PYRIN domain of ASC, was able to diminish secretion of IL-1β by Ipaf-557 (Fig. 1D). The ability of the isolated CARD of ASC to interfere with Ipaf-induced activation of caspase-1 suggests that the CARD of ASC exerts its dominant negative effect by interacting directly with either the CARD of procaspase-1 or Ipaf or both. To test this hypothesis we performedin vitro GST pull-down assays to map the interactions of ASC with procaspase-1. As shown in Fig.2A, the GST-tagged CARD of caspase-1 was able to bind to in vitro translated ASC and its isolated CARD but not its PYRIN domain, indicating that the CARD of procaspase-1 interacts with ASC via CARD-CARD interactions (Fig.2A). Similar results were obtained when a GST-tagged CARD of ASC was incubated with in vitro translated procaspase-1; the GST-CARD of ASC was able to bind full-length procaspase-1 (Fig.2B), indicating that the interaction between ASC and procaspase-1 is mediated by the CARDs of the two proteins. These results were confirmed by immunoprecipitation experiments in 293 cells transfected with FLAG-tagged ASC, ASC-CARD, or ASC-PYRIN and T7-tagged procaspase-1. ASC and ASC-CARD, but not ASC-PYRIN, were able to immunoprecipitate procaspase-1 (Fig. 2C), thus confirming that ASC can indeed interact via CARD-CARD interactions with procaspase-1. The GST-CARD of procaspase-1 was also able to bind to the endogenous ASC protein of the THP-1 monocytic cell line, providing additional evidence that ASC interacts specifically with procaspase-1 (Fig. 2D). No interactions were observed between ASC and the CARDs of the caspase-1-related caspase-4 and -5 (not shown), indicating that the interactions between the CARD of caspase-1 and ASC is very specific. GST pull-down experiments with a GST-tagged CARD of Ipaf revealed that ASC could also interact with the CARD of Ipaf (Fig.2E). This and the above observations that ASC interacts via CARD-CARD interactions with procaspase-1 explain the dominant negative effect of the isolated CARD of ASC on Ipaf-mediated caspase-1 activation. Furthermore, consistent with the ability of pseudo-ICE to inhibit ASC-induced caspase-1 activation (Fig. 1B), GST-tagged pseudo-ICE was able to interact strongly with full-length ASC and its CARD (Fig.3E). The initiator caspases, such as caspase-8 or caspase-9, are activated by an induced proximity mechanism involving oligomerization by an adaptor molecule such as FADD or Apaf-1, respectively (25Salvesen G.S. Dixit V.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10964-10967Crossref PubMed Scopus (774) Google Scholar). In the case of FADD, its N-terminal death effector domain interacts with procaspase-8 and acts as the effector domain, while its C-terminal death domain, which interacts with death receptors, functions as the oligomerization domain. The dual domain structure of ASC and its ability to interact through the CARD with procaspase-1 suggests that ASC, like FADD, functions as an adaptor molecule. The CARD of ASC functions as the effector domain, while the PYRIN domain functions as the oligomerization domain. To test this hypothesis we fused the isolated PYRIN and CARD of ASC to an inducible FKBP12 oligomerization cassette (Fig. 3A). We then expressed the fusion proteins in 293T cells together with procaspase-1 and pro-IL-1β and treated the cells with the oligomerization drug AP20187 (ARIAD Pharmaceuticals). Treatment of the FKp3-CARD-expressing cells with AP20187 resulted in an 8-fold increase in IL-1β secretion compared with the untreated control (Fig. 3B). This increase in IL-1β secretion was diminished ∼50% by the caspase-1 dominant negative protein pseudo-ICE, a result consistent with the findings that pseudo-ICE binds to the CARD of ASC and interferes with its ability to activate procaspase-1. No IL-1β secretion was observed in FKp3-PYRIN-expressing cells after treatment with AP20187. Taken together these results indicate that the CARD is the effector domain of ASC and that the role of the PYRIN domain is to regulate the oligomerization status of the CARD by upstream signaling molecules in this caspase-1 activation pathway. Finally to test whether ASC is a critical component in the caspase-1 signaling pathway in THP-1 cells, we generated stable THP-1 cell lines that express the isolated CARD or PYRIN domains of ASC. To determine the effect of these domains on the caspase-1 signaling pathway we assayed IL-1β generation in response to IFNγ and LPS stimulation. As expected, the CARD of ASC reduced IL-1β secretion by ∼50% compared with control vector-transfected cells. The dominant negative effect of the CARD of ASC is likely due to its ability to bind to caspase-1 and interfere with its activation and/or activity. Consistent with this, the stably transfected ASC-CARD, but not the PYRIN domain, was able to immunoprecipitate the endogenous caspase-1 from the THP-1 cells (Fig. 3D). Interestingly the PYRIN domain of ASC increased IL-1β secretion by 2-fold. This effect might be attributed to PYRIN-PYRIN interactions between the isolated PYRIN domain of ASC and endogenous ASC, which may enhance oligomerization of endogenous ASC and activation of caspase-1. Pyrin, cryopyrin/PYPAF1, PYPAF3, and DEFCAP/NAC contain N-terminal PYRIN domains (13Hlaing T. Guo R.F. Dilley K.A. Loussia J.M. Morrish T.A. Shi M.M. Vincenz C. Ward P.A. J. Biol. Chem. 2001; 276: 9230-9238Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 14Chu Z.L. Pio F. Xie Z. Welsh K. Krajewska M. Krajewski S. Godzik A. Reed J.C. J. Biol. Chem. 2001; 276: 9239-9245Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 15Hoffman H.M. Mueller J.L. Broide D.H. Wanderer A.A. Kolodner R.D. Nat. Genet. 2001; 29: 301-305Crossref PubMed Scopus (1301) Google Scholar, 16Manji G.A. Wang L. Geddes B.J. 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. 2002; 277: 11570-11575Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 26The International FMF ConsortiumCell. 1997; 90: 797-807Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar). Pyrin and cryopyrin/PYPAF1 have been shown to form cytosolic speck-like structures with ASC when overexpressed together in transfected HeLa or 293 cells (16Manji G.A. Wang L. Geddes B.J. 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. 2002; 277: 11570-11575Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 27Richards N. Schaner P. Diaz A. Stuckey J. Shelden E. Wadhwa A. Gumucio D.L. J. Biol. Chem. 2001; 276: 39320-39329Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). The formation of these structures is thought to be mediated by PYRIN-PYRIN interactions. To test whether the PYRIN domain of these proteins interacts with ASC we performed GST pull-down experiments (Fig. 4, A and B). Unlike the strong interaction observed between in vitro translated full-length ASC and the GST-CARD of procaspase-1, no significant interactions were observed between full-length ASC and the GST-tagged PYRIN domains of Pyrin, cryopyrin/PYPAF1, PYPAF3, or DEFCAP/NAC under these conditions (Fig. 4A). However, a weak interaction was seen between full-length ASC and the GST-tagged PYRIN domain of ASC. Similarly, under the same conditions in which a detectable interaction can be seen between 35S-labeled procaspase-1 and GST-ASC, no significant interactions were seen betweenin vitro translated full-length Pyrin, cryopyrin/PYPAF1, PYPAF3, or DEFCAP/NAC and GST-tagged ASC or ASC-PYRIN (Fig.4B). Furthermore no significant interactions were detected by co-immunoprecipitation experiments with overexpressed full-length proteins in transfected 293 cells, although the overexpressed proteins were readily detectable in the total extracts of transfected cells (data not shown). These observations indicate that ASC does not form stable complexes with Pyrin, cryopyrin/PYPAF1, PYPAF3, or DEFCAP/NAC via PYRIN-PYRIN interactions. However, we cannot rule out the possibility that ASC might interact transiently with these proteins in a stimulus-dependent manner under physiological circumstances. In conclusion, we have demonstrated that ASC is a specific procaspase-1 binding partner. Interaction of ASC with procaspase-1 in transfected cells induces its activation and results in processing of pro-IL-1β and secretion of mature IL-1β. The CARD of ASC represents its effector domain, which recruits procaspase-1 and induces its activation by an induced proximity mechanism. Consistent with this, enforced oligomerization of the CARD of ASC causes activation of procaspase-1 and secretion of IL-1β. Furthermore the isolated CARD of ASC is a dominant negative inhibitor of IL-1β secretion in response to LPS stimulation of THP-1 cells. Finally we hypothesize that the PYRIN domain of ASC represents its oligomerization domain, which likely interacts with upstream effectors of the caspase-1 signaling pathway in response to stimulation with proinflammatory cytokines. However, the upstream interactors that bind to the PYRIN domain of ASC remain unknown at present." @default.
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