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J. Biol. Chem., Vol. 277, Issue 24, 21119-21122, June 14, 2002

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ACCELERATED PUBLICATION
The PYRIN-CARD Protein ASC Is an Activating Adaptor for Caspase-1^*

Srinivasa M. Srinivasula^§, Jean-Luc Poyet^§, Marjaneh Razmara^§, Pinaki Datta^§, ZhiJia Zhang, and Emad S. Alnemri^¶

From the Center for Apoptosis Research and the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Received for publication, March 26, 2002, and in revised form, April 18, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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-1 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-1 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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) (1-6). 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-B pathway via an interaction with RICK (also called RIP2 or CARDIAK), a serine-threonine kinase that contains a C-terminal CARD (1-3, 7-9). More recently Nod2 has been implicated in susceptibility to Crohn's disease and Blau syndrome, two chronic inflammatory disorders (10-12).

Ipaf was found recently to associate directly and specifically with the CARD of procaspase-1 through CARD-CARD interaction (4-6). Additional experiments revealed that Ipaf induces autocatalytic processing and activation of procaspase-1 and caspase-1-dependent apoptosis in transfected cells (4). 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 (13-16). 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 (17-19). 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 (20, 21). 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-1 from transfected cells. Furthermore the isolated CARD of ASC was found to be a dominant negative inhibitor of IL-1 generation in response to LPS. Combined, our data suggest that ASC is a specific activating adaptor for caspase-1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cell Culture-- 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 -mercaptoethanol.

Expression Vectors-- 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 (GenBank^TM 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 in Escherichia 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-1 cDNA was generated by PCR and cloned into pcDNA3. Ipaf, procaspase-1, and pseudo-ICE constructs have been described previously (4, 22, 23).

In Vitro Binding Assays-- In vitro GST pull-down assays were performed as described previously (23).

Transfection, Immunoprecipitation, and Immunoblot Analysis-- 293T cells (5 × 10⁶) in 100-mm dishes were transiently transfected with the expression plasmid(s) using the LipofectAMINE^TM reagent (Invitrogen). Cells were lysed, and the cellular lysates were immunoprecipitated and Western blotted as described before (23). Western blotting with the anti-ASC monoclonal antibody was done according to the manufacturer's recommendation (Medical & Biological Laboratories, Nagoya, Japan).

Assay of IL-1 Secretion from THP-1 and Transfected 293T Cells-- THP-1 cells stably expressing the isolated CARD or PYRIN domains of ASC were generated by retroviral bulk infection as described before (22, 23). To assay for IL-1 secretion, THP-1 cells (1 × 10⁶ cells/ml) were incubated for 4 h with IFN and then 14 h with 1 µg/ml LPS. The culture media of the cells were then used to quantify IL-1 by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN). 293T cells were transfected with pro-IL-1 (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-1 secretion from transfected 293T cells was assayed 36 h after transfection as described for THP-1 cells above without stimulation with IFN and LPS.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

ASC Is a Procaspase-1 Activator-- 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-1-converting enzyme (ICE) or caspase-1 (Ref. 21 and GenBank^TM accession numbers NP_571580 and AAG45230) and several Ipaf-related proteins (Refs. 13-16 and 19 and GenBank^TM 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-1 secretion is one of the consequences of caspase-1 activation, we measured the levels of IL-1 in the culture media of 293 cells that ectopically express increasing amounts of ASC together with fixed amounts of procaspase-1 and pro-IL-1. As shown in Fig. 1B, full-length ASC was able to induce IL-1 secretion from the transfected 293 cells in a dose- and caspase-1-dependent manner. The increase in IL-1 generation was associated with a decrease in the proform of caspase-1 indicating that ASC-induced IL-1 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-1 secretion in the presence of procaspase-1 C285A (Fig. 1, B and C). Interestingly the CARD-only protein pseudo-ICE/Cop (22, 24), which has been previously shown to diminish IL-1 generation from THP-1 cells in response to LPS, diminished ASC-induced IL-1 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.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   ASC induces caspase-1-dependent IL-1 generation in transfected 293 cells. A, a bar diagram representing the dual domain structure of ASC. B, 293T cells were co-transfected with pro-IL-1 together with empty vector (Vector), ASC (0.6 µg), empty vector plus procaspase-1, ASC (0.2, 0.4, and 0.8 µg) plus procaspase-1, ASC (0.2, 0.4, and 0.6 µg) and pseudo-ICE (0.5 µg) plus procaspase-1, or ASC (0.2, 0.4, and 0.6 µg) plus procaspase-1 C285A expression constructs as indicated. The total amount of DNA in all transfection reactions was kept constant by inclusion of empty vector plasmid. 36 h after transfection, the culture media were assayed for IL-1 by ELISA. C, total cellular lysates of 293T cells co-transfected with increasing amounts of HA-ASC expression construct (0.2, 0.4, and 0.6 µg) and a fixed amount of FLAG-tagged wild type procaspase-1, procaspase-1 plus pseudo-ICE, or procaspase-1 C285A expression construct were fractionated by SDS-PAGE and detected by Western blot analysis using anti-FLAG-M2/M5 (for FLAG-caspase-1) or anti-HA (for HA-ASC) antibodies. The processed caspase-1 cannot be detected because of cleavage of the FLAG tag during processing of procaspase-1. D, 293T cells were co-transfected with pro-IL-1 together with empty vector (Vector), Ipaf-(1-557), ASC, empty vector plus procaspase-1, Ipaf-(1-557) plus procaspase-1, ASC plus procaspase-1, Ipaf-(1-557) and ASC-PYRIN plus procaspase-1, or Ipaf-(1-557) and ASC-CARD plus procaspase-1 expression constructs as indicated. 36 h after transfection, the culture media were assayed for IL-1 by ELISA. pcasp-1, procaspase-1; Casp-1, caspase-1; Ps-ICE, pseudo-ICE.

Beside ASC, we have shown recently that Ipaf is another activating adaptor for caspase-1 (4). 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-1 in 293 cells (Fig. 1D). ASC was as efficient as the constitutively active Ipaf-557 in inducing the secretion of IL-1 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-1 by Ipaf-557 (Fig. 1D).

ASC Interacts with Procaspase-1 via CARD-CARD Interaction-- 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 performed in 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.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   ASC interacts with procaspase-1. A and E, GST or GST-tagged caspase-1-CARD, Ipaf-CARD, or pseudo-ICE bound to glutathione-Sepharose beads were incubated with in vitro translated 35S-labeled ASC, ASC-CARD, or ASC-PYRIN as indicated. The interactions were then analyzed by SDS-PAGE and autoradiography. The GST fusion proteins were stained with Coomassie (lower panels). B, GST or GST-tagged ASC, ASC-CARD, or ASC-PYRIN were incubated with in vitro translated 35S-labeled procaspase-1 as indicated and analyzed as in A. C, 293T cells were co-transfected with expression constructs encoding T7-procaspase-1 C285A and FLAG-tagged ASC, ASC-CARD, or ASC-PYRIN or with empty vector. 36 h after transfection, cells were lysed, and the lysates were immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were immunoblotted with anti-T7 antibody as indicated. D, GST or GST-tagged caspase-1-CARD bound to glutathione-Sepharose beads were incubated with THP-1 cellular lysate. After incubation the beads (Pellet) and the supernatants (Supe) were fractionated by SDS-PAGE and then immunoblotted with an anti-ASC monoclonal antibody. The Lysate lane represents total THP-1 lysate. The amounts of GST fusion proteins used in this experiment are equal to those shown in the lower panel of A. IP, immunoprecipitation; WB, Western blot; Casp-1, caspase-1; pcasp-1, procaspase-1.

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).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   A and B, induced oligomerization of ASC-CARD promotes procaspase-1 activation. 293T cells were co-transfected with pro-IL-1 and FKp3-ASC-CARD or FKp3-ASC-PYRIN together with or without procaspase-1 as indicated. Pseudo-ICE was also included in some transfections as indicated. 36 h after transfection, cells were incubated with or without the oligomerization drug AP20187 (0.1 µg/ml) for 5 h as indicated. After incubation, the culture media were harvested and assayed for IL-1 by ELISA. The FKBP constructs used in this experiment are represented schematically in A. C, the CARD of ASC is a dominant negative inhibitor of LPS-induced IL-1 generation in THP-1 cells. Stable THP-1 cells generated by retroviral bulk infection with control retroviral vector (Vector) or retroviral vector encoding T7-ASC-CARD (ASC-CARD), HA-ASC-PYRIN (ASC-PYRIN) were treated with IFN and LPS for 18h. IL-1 secretion in the culture media was quantified by ELISA. Inset, expression levels of the stably expressed ASC-CARD (CARD) and ASC-PYRIN (PYRIN) in the THP-1 cells used in this experiment as determined by Western blotting with anti-T7 and -HA antibodies. D, the CARD of ASC interacts with endogenous procaspase-1. THP-1 cells stably infected with control retroviral vector (Vector) or retroviral vector encoding T7-ASC-CARD (ACS-CARD) or HA-ASC-PYRIN (ASC-PYRIN) were lysed and incubated with a mixture of anti-HA.II and -T7 monoclonal antibodies. The complexes were bound to protein G-Sepharose and then eluted by boiling in SDS sample buffer. The eluted proteins were resolved by SDS-PAGE and detected by Western analysis with an anti-caspase-1 antibody (upper panel) or anti-HA plus -T7 antibodies (lower panels). Procasp-1, procaspase-1; WB, Western blot; Casp-1, caspase-1.

ASC Promotes Caspase-1 Activation by an Induced Proximity Mechanism-- 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 (25). 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-1 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-1 secretion compared with the untreated control (Fig. 3B). This increase in IL-1 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-1 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-1 generation in response to IFN and LPS stimulation. As expected, the CARD of ASC reduced IL-1 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-1 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.

ASC Does Not Form Stable Complexes with Pyrin, Cryopyrin/PYPAF1, PYPAF3, or DEFCAP/NAC-- Pyrin, cryopyrin/PYPAF1, PYPAF3, and DEFCAP/NAC contain N-terminal PYRIN domains (13-16, 26). Pyrin and cryopyrin/PYPAF1 have been shown to form cytosolic speck-like structures with ASC when overexpressed together in transfected HeLa or 293 cells (16, 27). 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 between in 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.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   ASC does not form stable complexes with PYRIN proteins. A, GST or GST-tagged ASC-PYRIN, cryopyrin-PYRIN, PYPAF3-PYRIN, Pyrin-PYRIN, DEFCAP-PYRIN, or caspase-1-CARD were bound to in vitro translated 35S-labeled ASC as indicated and then analyzed by SDS-PAGE and autoradiography. B, GST or GST-tagged ASC or ASC-PYRIN were bound to in vitro translated 35S-labeled cryopyrin, PYPAF3, Pyrin, DEFCAP, or procaspase-1 as indicated and then analyzed by SDS-PAGE and autoradiography. The GST fusion proteins were stained with Coomassie (lower panels). Casp-1, caspase-1; pcasp-1; procaspase-1.

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-1 and secretion of mature IL-1. 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-1. Furthermore the isolated CARD of ASC is a dominant negative inhibitor of IL-1 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.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants CA85421 and AG14357 (to E. S. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A special fellow of the Leukemia and Lymphoma Society.

^§ These authors contributed equally to this work.

^¶ To whom correspondence should be addressed: Kimmel Cancer Inst., Thomas Jefferson University, Bluemle Life Sciences Bldg., Rm. 904, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4632; Fax: 215-923-1098; E-mail: E_Alnemri@lac.jci.tju.edu.

Published, JBC Papers in Press, April 19, 2002, DOI 10.1074/jbc.C200179200

	ABBREVIATIONS

The abbreviations used are: CARD, caspase recruitment domain; FADD, Fas-associated death domain; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; IL, interleukin; LPS, lipopolysaccharide; GST, glutathione S-transferase; ICE, IL-1-converting enzyme; IFN, interferon; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; FKBP, FK-506-binding protein.

	REFERENCES

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