WO2005025591A2 - CASPASE DERIVED CARDs - Google Patents

Abstract

The present invention relates to caspase derived caspase recruitment domains (CARDs). More specifically, the invention relates to CARDs derived from procaspase-1 and procaspase-2, and their use to induce nuclear factor of the Κ-enhancer in B-cells (NF-ΚB) activation and/or p38 MAP kinase activation.

Description

CASPASE DERIVED CARDs The present invention relates to caspase derived caspase recruitment domains (CARDs). More specifically, the invention relates to CARDs derived from caspase-1 and caspase-2, and their use to induce nuclear factor of the κ-enhancer in B-cells (NF-κB) activation and/or activation of p38 MAP kinase. Caspases, a family of cysteinyl aspartate-specific proteases, are central mediators of apoptotic and .inflammatory pathways. Caspases are synthesized as zymogens with a prodomain of variable length followed by a large subunit (p20) and small subunit (p10), called procaspases (Lamkanfi et al., 2003). The large prodomains of mammalian procaspases contain structural motifs that belong to the so-called 'death domain superfamily'. These structural motifs have emerged as the prime mediators of the interactions necessary for transducing inflammatory and cell death signals and can be found in a growing number of proteins involved in apoptosis, necrosis and inflammation. This superfamily consists of the death domain (DD), the death effector domain (DED), the procaspase recruiting domain (CARD) (Bouchier-Hayes and Martin, 2002; Lamkanfi et. al., 2003) and the recently identified PYRIN domain (Bert and DiStefano, 2000; Fairbrother et al., 2001; Martinon et al., 2001 ; Pawlowski et al., 2001). Each of these motifs interacts with other proteins through homotypic interactions. Among the four families (DD, DED, CARD, PYRIN), DDs are commonly found in upstream components of the apoptotic pathways, such as death receptors (e.g. CD95, TNF-R1) and the adaptor molecules that are recruited to these receptors (e.g. FADD, TRADD, RAIDD and RIP) (Lamkanfi et al., 2003). On the other hand, DEDs and CARDs are generally responsible for recruiting the initiator procaspases to death- or inflammation-inducing complexes through specific adaptor molecules (Bouchier-Hayes and Martin, 2002; Tibbetts et al., 2003). The PYRIN domain has only been found in zebrafish procaspases (Inohara and Nunez, 2000) and in the N-terminus of several proteins thought to function in apoptotic and inflammatory signaling pathways, such as the recently identified human CED-4 family members DEFCAP (Hlaing et al., 2001) and ASC (Masumoto et al., 2001). Caspases comprising a CARD are procaspase-1 , procaspase-2, procaspase-4, procaspase-5, procaspase-9, procaspase-11 and procaspase12. The procaspases are activated through proteolysis at specific Asp residues residing between the prodomain, p20 and p10 subunits. This results in the generation of mature tetrameric caspases consisting of two p20/p10 heterodimers aligning in a head-to-tail configuration, thereby positioning the two active sites at opposite ends of the molecule. The catalytic mechanism is exerted by a Cys-His catalytic diad (Lamkanfi et al., 2003). One powerful way to modulate assembly of procaspase-containing signaling complexes is through the presence of decoy molecules that attenuate the assembly process. The most dramatic examples of this are the virally encoded DEDs from a number of γ-herpesviruses including two human oncogenic viruses (Kaposi sarcoma associated-herpesvirus and molluscum contagiosum virus) that bind the DEDs of FADD and/or procaspase-8 and disrupt assembly of the Fas-receptor associated death inducing signaling complex. These viral proteins, termed v-FLIPs are closely related to c-FLIP, an endogenous cellular protein present as various splice forms, the longest of which (c-FLIP ) is a potent inhibitor of FADD-mediated procaspase-8 activation (Peter and Krammer, 2003). Both viral and cellular decoy molecules are capable of regulating assembly of signaling complexes held together by homotypic interactions involving six-helix-bundle interaction domains. In a similar vein, it was found that procaspase-1 activation and subsequent generation of lnterleukin-1β (IL-1β) is regulated by small CARD-containing decoy molecules termed ICEBERG and Pseudo-ICE/COP (Humke et al., 2000; Druilhe et. al., 2001; Lee et al., 2001). These decoy molecules have been shown to bind to the CARD motif present in the prodomain of procaspase-1.

A potential mechanism by which procaspase-1 is regulated became clear with the identification of a serine/threonine kinase RIP2/CARDIAK/RICK that binds procaspase-1 and promotes its processing (McCarthy et al., 1998; Thome et al., 1998). RIP2 engages procaspase-1 through a direct protein-protein interaction involving corresponding CARDs present at the C-terminus of RIP2 and within the prodomain of procaspase-1. Recently another potent procaspase-1 activating mechanism was discovered with the cloning of another CARD domain containing protein, designated lpaf-1 (Poyet et al., 2001). The CARD module mediates the interaction between a number of large prodomain procaspases and their corresponding upstream activator adaptors, the prototypical examples being procaspase-9 and Apaf-1. Structurally, the CARD motif resembles the DD and the DED. All possess six helices and have propensity to self associate. These homotypic interactions form the glue that binds the signaling machinery responsible for procaspase activation. Procaspase-2, initially described as Nedd2 in mice and lch-1 in human, shows a domain organization that is similar to procaspase-1, comprising a CARD, a p20 and a p10 domain. The CARD of procaspase-2 interacts with the CARD of RAIDD/CRADD (Duan and Dixit, 1997; Ahmad et. al., 1997). This latter molecule also contains a DD that can associate with the DD of the serine/threonine kinase RIP (Ahmad et al., 1997). RIP is interacting with the Tumor Necrosis Factor Receptor-1 (TNFR-1) via TRADD (Hsu etal., 1995).

NF-κB, classically a heterodimer composed of the p50 and p65 subunits, is a transcription factor whose activity is tightly regulated at multiple levels (Baldwin, 1996; Mayo and Baldwin, 2000; Zandi and Karin, 1999; Ghosh et al., 1998). NF-κB is normally sequestered in the cytoplasm as an inactive complex bound by an inhibitor known as lκB (Baldwin, 1996). Following cellular stimulation, lκB proteins become phosphorylated by the lκB kinase (IKK), which subsequently targets lκB for ubiquitination and degradation through the 26S proteasome (Zandi and Karin, 1999). The degradation of lκB proteins liberates NF-κB, allowing this transcription factor to translocate to the nucleus. In addition to regulation by lκB, NF-κB is also regulated by phosphorylation events that positively upregulate the transactivation potential of NF-κB subunits (May and Ghosh, 1998). The transactivation domains of NF-κB have been shown to be regulated by the catalytic domain of protein kinase A, casein kinase II, and by IKK itself (Zhong et al., 1997; Zhong et al., 1998; Wang et al., 2000; Mercurio et al., 1997; Sakurai et al., 1999). Although signals that regulate nuclear translocation of NF-κB have been regarded as the primary mechanism of NF-κB activation, alternative mechanisms involving the transactivation potential of p65 have been shown to be critical for NF-κB activation in vitro and in vivo (May and Ghosh, 1998;Hoeflich et al., 2000; Bonnard et al., 2000).

MAP kinases are an evolutionary conserved family of cytosolic serine/threonine kinases that modulate the activity of other intracellular proteins by adding phosphate groups to their serine/threonine residues. Activation of the MAP kinases themselves requires phosphorylation on both a threonine and tyrosine residue and thus needs the activity of dual specificity kinases, which are known as MEKs, or MAP kinase kinases. Three major groups of MAP kinase cascades are known: ERK1/2, JNK, and p38 MAP kinase. The p38 MAP kinase pathway is associated with inflammation, cell growth, cell differentiation, and cell death. Extracellular stimuli of the p38 MAP kinase pathway include a variety of cytokines (IL-1, IL-2, IL-7, IL-17, IL- 18, TGF-β, and TNF-α) and a number of pathogens that activate p38 through the different Toll receptors, including LPS, staphylococcal peptidoglycan, staphylococcal enterotoxin B, echovirus 1, and herpes simplex virus 1 (Ono and Han, 2000). Moreover, several growth factors (that is, granulocyte macrophage-colony stimulating factor, colony stimulating factor 1 , erythropoietin) (Foltz ef al., 1997) are capable of inducing p38 as well as environmental factors such as heat shock, changes in osmolarity, ultraviolet, oxygen radicals, and hypoxic states (Ono and Han, 2000). The downstream targets of p38 are either other kinases or transcription factors. The main biological response of p38 activation involves the production and activation of inflammatory mediators to initiate leucocyte recruitment and activation. Thus p38 MAP kinase probably plays a central role in the regulation of a wide range of immunological responses, as seen in inflammatory disorders.

Initially, CARD-CARD interactions were considered as mainly involved in the assembly of protein complexes that promote procaspase processing and activation in the context of apoptosis. However, as the family of CARD-containing proteins was growing, it became more and more apparent that many of the CARD comprising proteins are participating in NF-κB signalling pathways, rather than being involved in procaspase recruitment and activation. Their role in NF-κB activation can be both positive or negative. NOD1 and NOD2 are two examples of NF-κB-inducing CARD-containing proteins, while CARD6 inhibits RIP2-induced NF-κB activation (Bertin et al., 1999; Inohara et al., 1999; Stehlik et al., 2003). Up to now, no direct relation between the sequence of the CARD and its function has been found: although all CARDs are homologous, their role may be quite divergent. Although many CARDs are implicated in NF-κB signalling pathways, all CARDs derived form procaspases are thought to be solely implicated in procaspase regulation and proteolytic processing (Bouchier-Hayes and Martin, 2002). Surprisingly, we found that procaspase derived CARDs can be inducers of NF-κB activity and/or p38 MAP kinase phosphorylation. Depending on the procaspase, the derived CARD signals through a different pathway to NF-κB and p38 MAPK activation. More specifically, CARDs derived from procapase-1 and procaspase-2 are inducing NF-κB and/or p38 phosphorylation, while the CARDs of the closely related procaspase-11 and procapase-12 are inactive. Said activation of NF-κB and p38 MAP kinase is independent of the proteolytic processing of the procaspase. A first aspect of the invention is the use of a procaspase derived CARD to modulate NF-κB activation or p38 phosphorylation. Preferably, said modulation is an induction. Preferably, said CARD domain is derived from procaspase-1 or from procaspase-2. One preferred embodiment is the use of a procaspase derived CARD according to the invention, whereby said CARD comprises SEQ ID N°1. Preferably, said CARD comprises SEQ ID N°2. Another preferred embodiment is the use of a procaspase derived CARD according to the invention, whereby said CARD comprises SEQ ID N°3.

The use of a CARD, as used here, is not limited to the use of the domain as such, but covers also the use of proteins, comprising said CARD. Said proteins may be natural occurring proteins, like procaspase-1 and procaspase-2, or it may be mutants of said natural occurring proteins. As a non limiting example, said mutants may be caspase mutants without catalytic activity, such as a procaspase-1 C/A or a procaspase-2 C/A mutant, or may be deletion mutants such as CARD-p20 fusions of procaspase-1 or procaspase-2. Alternatively, the CARD may be fused to another, non-related polypeptide. The fusion may be aminoterminal, carboxyterminal or both. As the procaspases derived CARD exerts its inducing action by interacting with other proteins such as RIP2 and TRAF2, it is clear for the person skilled in the art that the induction of the NF-κB activity and/or p38 MAP kinase phosphorylation can be inhibited by inhibiting this interaction. Inhibition of said interaction can be realized in several ways. As non-limiting examples, antibodies may be generated against the CARD, or against the CARD binding domain of the interaction partner. Alternatively, CARD derived mutants or fragments, that interfere with the interaction without inducing the NF- B activity and/or p38 MAP kinase phosphorylation can be used. As the critical residues for the interaction have been defined, it is evident for the person skilled in the art how the interaction can be disrupted. Another aspect of the invention is the use of a procaspase derived CARD to screen for anti- inflammatory compounds. Preferably, said CARD domain is derived from procaspase-1 or from procaspase-2. One preferred embodiment is the use of a procaspase derived CARD according to the invention, whereby said CARD comprises SEQ ID N°1. Preferably, said CARD comprises SEQ ID N°2. Another preferred embodiment is the use of a procaspase derived CARD according to the invention, whereby said CARD comprises SEQ ID N°3. Indeed, as it is shown that the pro-inflammatory response is mediated by a CARD - protein interaction, compounds that disrupt this interaction will act as an anti-inflammatory compound. Methods to screen for compounds that disrupt protein-protein interactions are known to the person skilled in the art, and have been described in, as a non-limiting example, US5733726, WO9813502 and WO03004643.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Caspase-1 induces apoptosis, pro-IL-1β maturation and NF-κB activation. (A) 293T cells were transiently cotransfected with combinations of plasmids overexpressing nuclear localization signal-containing GFP, caspase-1 (CASP1), CrmA and empty vector (CTRL). Total DNA was maintained at 1 μg by the addition of control plasmid DNA. Microscopy fluorographs show that caspase-1 induces apoptotic cell death of transfected cells that can be blocked by CrmA. As a control, empty vector-transfected cells do not die. (B) 293T cells were transiently transfected with a plasmid encoding pro-IL-1β either alone or in combinations with caspase-1 (CASP1 ) and CrmA. Supernatant was analyzed for presence of biologically active IL-1β 24h after transfection. (C) 293T cells were transiently cotransfected with a NF-κB-dependent luciferase reporter and the indicated plasmids. Cells were lysed 24 h after transfection and NF- KB activity was measured as described in Materials and Methods. Figure 2: Caspase-1 -induced NF-κB activation is independent of pro-IL-1β maturation. (A) 293T cells were transiently cotransfected with an NF-κB-dependent luciferase reporter and the indicated amounts of plasmid encoding enzymatically inactive murine caspase-1 C284A. Total DNA was maintained at 0.5 μg by the addition of control plasmid DNA. 24 h after transfection, lysates were analyzed for NF-κB activation as described in Materials and Methods. (B) 293T cells were transiently transfected with the indicated plasmids. Supernatant was analyzed for presence of biologically active IL-1β 24 h after transfection. (C) 293T cells were transiently cotransfected with an NF-κB-dependent luciferase reporter, a plasmid encoding pro-IL-1β and the indicated μg of plasmid encoding murine caspase-1 2D/E. Total DNA was maintained at 0.5 μg by the addition of control plasmid DNA. 24 h after transfection, supernatants were analyzed for presence of mature IL-1β (upper panel right) and cell lysates for NF-κB activation (upper panel left) as described in Materials and Methods. Aliquots of the lysates were analyzed by SDS-PAGE/immunoblotting to confirm expression of caspase-1 2D/E (lower panel). Figure 3: Specificity of caspase-1 -induced NF-κB activation. (A) 293T cells were transiently cotransfected with an NF-κB-dependent luciferase reporter and 0.4 μg of the indicated plasmids. 24 h after transfection, lysates were analyzed for NF-κB activation as described in Materials and Methods. Due to death of transfected cells, activation of NF-κB by caspase-1 could not be measured. Wild type caspase-11 and -12 are much less cytotoxic. Caspase-1 C/A, but not caspase-11 C/A or caspase-12 C/A, leads to a dose-dependent induction of NF- KB activation. * Wild type caspase-1 leads to excessive cell death and leakage of caspase-1 into the supernatant. (B) 293T cells were transiently cotransfected with a NF-κB-dependent luciferase reporter and the indicated amounts of plasmid encoding enzymatically inactive human caspase-1 C285A. Total DNA was maintained at 0.5 μg by the addition of control plasmid DNA. 24 h after transfection, lysates were analyzed for NF-κB activation as described in Materials and Methods.

Figure 4: Caspase-1 CARD is necessary and sufficient for NF-κB activation. 293T cells were cotransfected with an NF-κB-dependent luciferase reporter and either empty vector, full-length caspase-1 C284A or the indicated deletion constructs. 24 h later, lysates were analyzed for NF-κB activation as described in Materials and Methods. Figure 5: Effect of dominant negative mutants and inhibitors of the signaling pathway on the procaspase-1 mediated induction of NF-κB activation. 293T cells were transiently cotransfected with an NF-κB-dependent luciferase reporter, caspase-1 C/A and plasmids encoding A20 or dominant negative molecules of either TRAF2, RIP1 , IKK-β or RIP2 CARD. The concentration of the plasmid used is as indicated in the figure. Lysates were analyzed for NF-κB activation as described in Materials and Methods (a) TRAF2 DN; (b) RIP DD; (c) IKK-β DN; (d) A20; (e) RIP2 CARD

Figure 6: (A) 293T cells were transiently cotransfected with an NF-κB-dependent luciferase reporter and plasmids encoding RIP2 or caspase-1 C/A in the presence or absence of RIP2 dominant negative. As a control, cells were treated with 500 lU/ml human TNF for induction of NF-κB activation. 24 h after transfection, lysates were analyzed for NF-κB activation as described in Materials and Methods. (B) Co-immunoprecipitation assays were performed using lysates (normalized for total protein content) from 293T cells that had been transiently transfected with plasmids encoding E-epitope tagged caspase-1 C285A and VSV-tagged RIP2 or RIP2 CARD. Immunoprecipitates were prepared using anti-VSV antibody adsorbed to protein G-sepharose and analyzed by SDS-PAGE/immunoblotting using anti-E-HRP antibody with ECL-based detection. Aliquots of the same lysates were also analyzed directly by SDS- PAGE/immunoblotting, as indicated. IP, immunoprecipitation; WB, Western blotting. Figure 7: NF-κB activation is independent of p38 MAPK activation. (A) 293T cells were transfected with plasmids encoding either EGFP or caspase-1 CARD. 24 h later, lysates were analyzed by SDS-PAGE/immunoblotting using antibodies against the indicated proteins. (B) 293T cells, untreated or pretreated with the p38 MAPK-specific inhibitor SB203508, were cotransfected with caspase-1 CARD and an NF-κB-dependent luciferase reporter. 24 h later, lysates were analyzed for NF-κB activation as described in Materials and Methods. Aliquots of the same lysates were also analyzed by SDS-PAGE/immunoblotting using antibodies against the indicated proteins to confirm the inhibition of p38 MAPK phosphorylation. Figure 8: Induction of NF-κB activation by wild type procaspase-2 (a) and by the catalytically inactive procaspase-2 C/A mutant (b). The experiment was essentially carried out as described for procaspase-1 in Figure 1 (wild type) and 2 (mutant), but using procaspase-2 instead of procaspase -1 Figure 9: Induction of NF-κB activation by the murine procaspase-2 CARD. The experiment was essentially carried out as described for procaspase-1 as indicated in the legend of Figure 4, but using procaspase-2 CARD instead of procaspase-1 CARD

Figure 10: Effect of dominant negative mutants and inhibitors of the signaling pathway on the procaspase-2 mediated induction of NF-κB activation, (a) TRAF2 DN; (b) IKK-β DN; (c) A20; (d) RIP2 CARD. The experiment was essentially carried out as described for procaspase-1 as indicated in the legend of Figure 5, but using procaspase-2 in stead of procaspase-1.

Figure 11: RIP1 is not a caspase-2 substrate and interacts with the caspase-2/TRAF2 complex in a TRAF2-dependent manner. (A) Co-immunoprecipitation assays were performed using lysates of 293T cells that had been transiently transfected with plasmids encoding E- tagged TRAF2, HA-tagged caspase-2 C320A and V5-HIS-tagged RIP1. Immunoprecipitates were prepared using an anti-HA antibody adsorbed to protein G-sepharose and analyzed by SDS-PAGE/immunoblotting using the indicated antibodies. Aliquots of the same lysates were also analyzed directly by SDS-PAGE/immunoblotting as indicated. IP, immunoprecipitation; WB, Western blotting. (B) In vitro ³⁵S-labeled caspase-2 and RIP1 were incubated with recombinant caspase-2 and -8 for 45 min at 37 °C in cell free system buffer and then analyzed by 15% SDS-PAGE and autoradiography. Arrowheads indicate the resulting cleavage fragments.

Figure 12: CASP2 CARD induces p38 phosphorylation. The experiment was essentially carried out as described for procaspase-1 as indicated in the legend of Figure 7, but using procaspase-2 in stead of procaspase-1 Figure 13: 293T cells were cotransfected with an NF-κB-dependent luciferase reporter and the indicated amounts of plasmids encoding full length caspase-1 C285A, COP/Pseudo-ICE or ICEBERG. Total DNA content of 0.5 μg was maintained by the addition of control empty vector. 24 h later, lysates were analyzed for NF-κB activation as described in Materials and Methods (upper panel). Aliquots of the same lysates were also analyzed by SDS- PAGE/immunoblotting using anti-E and anti-T7 epitope tag antibodies to confirm the appropriate expression of all constructs (lower panels). Figure 14: NF- B induction by four multiple mutants and one single mutant construct of mouse procaspase-1 CARD, in comparison with the wild type CARD domain of procaspase-1. Empty vector and RIP2 are used as control. The expression of wild type and mutant procaspase-1 CARDs, as checked by western blot, is shown below the graphical representation of the activity. Figure 15: NF-κB induction by 8 single mutant constructs of mouse procaspase-1 CARD, in comparison with the wild type CARD domain of procaspase-1. Empty vector is used as control. The expression of wild type and mutant procaspase-1 CARDs, as checked by western blot, is shown below the graphical representation of the activity.

EXAMPLES

Materials and methods to the examples

Plasmids and vectors - The cloning of cDNAs encoding murine caspases-1 , -11 and -12 have been described (Van de Craen et al., 1997). pCAGGS-caspase-1 C284A, coding for the enzymatically inactive mutant of murine caspase-1, was constructed by site-directed mutagenesis PCR. pCAGGS-caspase-12 C298A has been described elsewhere (Kalai et al., 2003). pCAGGS-caspase-11 C254A, encoding an inactive caspase-11 mutant, and pCAGGS- caspase-1 2D/E, in which the cleavage sites Asp103 and Asp122 were mutated to Glu, were kind gifts from Dr. P. Schotte (Ghent University, Ghent, Belgium). Caspase-1 deletion mutants were generated by PCR using modified complementary PCR adaptor primers. E-epitope tagging was done by cloning the PCR-generated cDNAs of the respective ORFs in frame into the pCAGGS-E vector. The following expression plasmids were obtained from the indicated sources: pNF-conLuc, encoding the luciferase reporter gene driven by a minimal NF-κB responsive promoter was a generous gift from Dr. A. Israel (Institut Pasteur, Paris, France). The plasmid pUT651 , encoding β-galactosidase, was obtained from Eurogentec (Seraing, Belgium). pCAGGS-pro-IL-1β has been described previously (Van de Craen et al., 1997). pEGFP-C3 was purchased from Clontech (Palo Alto, CA, USA). pCAGGS-CrmA has been described (Vercammen et al., 1998). pCR3-RIP2 and pCR3-RIP2-CARD were kindly provided by Dr. J. Tschopp (University of Lausanne, Epalinges, Switzerland) and have been described elsewhere (Thome et al., 1998). Plasmids encoding dominant negative forms of IKK-β and TRAF2 were generous gifts from Dr. J. Schmid (University of Vienna, Vienna, Austria) and Dr. D.V. Goeddel (Genentech, South San Fransisco, CA, USA), respectively. The plasmid encoding murine A20 has been described elsewhere (Klinkenberg et al., 2001) and was kindly provided by Dr. K. Heyninck (Ghent University, Ghent, Belgium). Plasmids encoding T7- epitope tagged COP/Pseudo-ICE and ICEBERG have been described previously (Druilhe et al., 2001) and were kindly provided by Dr. E. S. Alnemri (Thomas Jefferson University, Philadelphia, PA, USA). All the PCR products described above were checked by sequencing to ensure that no errors had been introduced by PCR.

Cloning of the human caspase-1 gene — THP-1 human macrophage cells were cultured in LPS-free RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FCS (Invitrogen, Carlsbad, CA, USA), 0.03% L-Gln (Merck, Darmstadt, Germany), 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μM β-mercaptoethanol at 37°C in a humidified 5% CO₂ atmosphere. THP-1 cells were seeded at 400 000 cells/ml medium. After seeding the cells were allowed to grow for another 24h during after which they were stimulated with human IFN- γ (1000 lU/ml). Total RNA was isolated from cells using the RNeasy isolation kit (Qiagen, Hilden, Germany). RT-PCR was performed according to instructions with the Superscript PreAmpIification system (Invitrogen, Carlsbad, CA, USA) using the Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). From this THP-1 cDNA library the cDNA encoding full-length human caspase-1 was amplified by PCR. The resulting PCR products were size-fractionated by electrophoresis in 1.0% agarose gels and cloned in frame with the N- terminal E-epitope tag into pCAGGS-E vector. The caspase-1 encoding sequence has been checked by sequencing to ensure that no errors were introduced by PCR.

Co-immunoprecipitations and immunoblotting assays — 293T is a human embryonal kidney carcinoma. 293T was routinely transfected using the calcium phosphate precipitation method (O'Mahoney and Adams, 1994). Cells were seeded the day before transfection at 2x10⁵ cells/ 6-well. Cells were transfected for 4 h, washed and incubated for another 24 h before lysates were prepared and/or supernatant was collected and tested in a biological assay for IL-1β. Lysates were prepared by harvesting the cells and lysing them in ice-cold NP-40 lysis buffer (10mM HEPES [pH 7.4], 142.5 mM KCI, 0.2% NP-40, 5 mM EGTA), supplemented with 1 mM DTT, 12.5 mM β-glycerophosphate, 1 μM Na₃VO , 1 mM PMSF, and 1x protease inhibitor mix (Roche, Basel, Switzerland). Cell lysates (0.5 ml) were clarified by centrifugation at 14,000xg for 5 minutes, and subjected to immunoprecipitation using specific antibodies, including anti- VSV (Sigma, MO, USA) and anti-HA antibodies (Babco, CA, USA) in combination with 15 μl Protein A-Sepharose. Immune-complexes were fractionated by sodium dodecyl sulfate- polyacrylamide gel electroforesis (SDS-PAGE) and transferred to nitrocellulose membranes. The blots were subsequently incubated with various antibodies, including anti-E antibodies (Amersham Biosciences, Freiburg, Germany), anti-Myc antibodies (Invitrogen, CA, USA), followed by horseradish peroxidase-conjugated secondary antibodies, and detection by an enhanced chemiluminescence (ECL) method. Alternatively, lysated were analyzed directly by immunoblotting after normalization for total protein content.

prolL-1 β-processing assay — Biologically active IL-1β was determined using growth factor- dependent D10(N4)M cells (Hopkins and Humphreys, 1989). Cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 2mM L-glutamine, 100 lU/ml penicillin G, 100 μg/ml streptomycin, 1mM sodium pyruvate, 5mM β-mercaptoethanol and 10% supernatant of phorbol ester-stimulated EL-4 cells as a source of IL-2, and 10% of phorbol ester-stimulated P388D1 cells as a source of IL-1. The day before the assay, D10(N4)M cells were washed and transferred to culture containing only 10% EL-4 supernatant. The next day, cells were washed again and added to serial dilutions of IL-1β-containing samples (10⁴ cells/96-weII), followed by incubation for 24 h at 37°C in a CO₂ incubator; proliferation was quantified by [³H]thymidine incorporation (0.5 μCi/well) for the last 6 h. Cells were harvested and incorporated [³H]thymidine was determined in a microplate scintillation counter (Packard Instrument Co., CT, USA). Samples were quantified according to a standard preparation of IL-1β with a specific biological activity of 10⁹ lU/mg (obtained from the National Institute for Biological Standards and Control, Potters Bar, UK).

Quantification of NF-κB activity — 293T cells were transfected with various expression vectors plus 100 ng NF-κB-luciferase and pUT651-LacZ reporter plasmids. Twenty-four hours after transfection the cells were collected, washed in phosphate buffer saline and lysed in NP-40 lysis buffer. In some experiments, cells were treated for 6h with 500 lU/ml of TNF-α prior to harvesting. NF-κB activity was assayed on 20 μl of cell lysates by chemiluminescence. To normalize for transfection efficiency, cell lysates were also subjected to β-galactosidase spectrophotometric assay. In brief, 20 μl of cell lysate were incubated for 5 min at room temperature with 200 μl of a solution containing 0.9 mg/ml o-nitrophenyl-β-D- galactopyranoside, 1 mM MgCI₂, 45 mM β-mercaptoethanol, 100 mM sodium phosphate, pH 7,5. The optical density was read at a wavelength of 595 nm. Results are expressed as relative luciferase units per second/optical density for β-galactosidase activity. Mutagenesis of caspase-1 CARD - Mutants used are summarized in Table 1 and Table 2. The mutagenesis was carried out using mutagenic primers (as listed in Table 3). For every mutation, two PCR fragments were synthesized: one upstream and one downstream from the mutant site. The fragments were fused by PCR using the mCasp-1 Notl fwr and the mCasp-1 Bglll rev primers. The Notl-Bglll fragment was cloned in the vector pCAGGS-E. The PCR mix consisted of 1 μl Pfx polymerase, 5 μl 10x Pfx amplication buffer, 2 μl enhancerbuffer, 2μl 50 mM MgSO₄, 1 μl dNTP's, 1 μl forward primer and 1 μl reverse primer, both at 50 pmol/μl, 4 μl template DNA (200 ng plasmid DNA) and sterile water up to 50 μl. The cycle was carried out for 2 min at 94°C, 1 min at 80°C, 15 sec at 94°C, 30 sec at 35, 45 or 50°C and 1 min 30 sec at 72. This cycle was repeated for 26 times, and stopped with 5 min at 72°C and cooling down to 10°C.

Example 1: procaspase-1 induces NF-κB activity Caspase-1 is a potent inducer of apoptotic cell death upon overexpression in 293T cells (Figure 1A). Co-expression of pro-IL-1 β and caspase-1 in these cells causes maturation of pro- IL-1 β to its biologically active form (Figure 1B). Cytokine response modifier A (CrmA) is a cowpox-derived serpin that covalently binds to the catalytic site of caspase-1 and -8, irreversibly inhibiting their enzymatic activity. Enzymatic activity of caspase-1 is essential for both cell death and pro-IL-1 β maturation, as inhibition of the enzymatic activity of caspase-1 by CrmA completely abrogates both effects in caspase-1 overexpressing cells (Figure 1A and B). However, when cells are protected from caspase-1 induced apoptosis by CrmA, we observed a significant activation of NF-κB (Figure 1C). This activation of NF-κB occurs in a caspase-1 - dependent manner and is not due to CrmA, as even high doses of CrmA do not significantly induce NF-κB activation (Figure 1C).

Example 2: procaspase-1 mediated NF-κB activation is due to CARD domain

To further assess that the observed activation of NF-κB is independent of catalytic activity, we generated an enzymatically inactive Cys/Ala mutant of caspase-1. Indeed, caspase-1 C/A is a potent inducer of NF-κB activation (Figure 2A) demonstrating that NF-κB activation occurs in a manner independent of enzymatic activity of caspase-1. As caspase-1 C/A is not capable of maturating pro-IL-1 β, we can also exclude the possibility that the observed activation of NF-κB is the result of an endogenous autocrine loop of IL-1β, a potent inducer of NF-κB, since transfection with wild type caspase-1 or caspase-1 C/A does not lead to detectable IL-1 β in the supernatant of transfected cells (Figure 2B). As the induction of NF-κB activity is independent of pro-IL-1 β maturation, we examined whether pro-IL-1 β processing and NF-κB activation are mutually exclusive or not. The strong proapoptotic activity of wild type caspase-1 masks the NF-κB read-out system. Therefore we made use of caspase-1 2D/E, a less cytotoxic variant of wild type caspase-1 in which the cleavage sites between the prodomain and the p20 domain are mutated. Both pro-IL-1 β maturation and a dose dependent induction of NF-κB activity could now be measured, suggesting that both the enzymatic activity and the non-enzymatic activity can occur simultaneously (Figure 2C). Conclusively, our data suggest induction of NF- KB activity is independent of the enzymatic activity of caspase-1 and therefore of the maturation of pro-inflammatory cytokines such as pro-IL-1 β and pro-IL-18. Moreover, caspase- 1 -mediated NF-κB activation and pro-IL-1 β maturation can occur simultaneously. Phylogenetically murine caspase-1 clusters with caspase-11 and caspase-12. This group is generally referred to as the inflammatory caspase subgroup (Lamkanfi et al., 2002). Therefore, we analyzed the specificity of the caspase-1 -dependent induction of NF-κB activation by comparing the ability of caspase-1 to activate NF-κB with that of its closest relatives caspase- 11 and -12. As already mentioned, wild type caspase-1 overexpression leads to quick apoptotic cell death of the transfected cells. This is reflected in the fact that caspase-1 is not detectable in cytosolic lysates by western blotting analysis (Figure 3A), but is probably present in the supernatant of dying cells (Denecker et al., 2001; Martinon et al., 2002). Apoptotic cell death is much less extensive when caspase-11 or -12 are overexpressed. This is in accordance with western blotting analysis showing clear expression of these proteins in cell lysates. In the case of caspase-11 , a 27 kDa fragment characteristic of its activation (Wang et al., 1996), is detected. However, neither caspase-11 nor caspase-12 induces NF-κB activation (Figure 3A). To exclude possible interference of cell death in the NF-κB assays, we also analyzed the enzymatic inactive C/A mutants of caspase-1 , -11 and -12. In contrast to the wild type caspases, none of the C/A mutants induces apoptosis. However, caspase-11 C/A and -12 C/A are completely unable of inducing NF-κB activity, while caspase-1 C/A strongly activated the NF-κB-dependent reporter (Figure 3A). Therefore, we can conclude that within the inflammatory subgroup of the murine caspases the ability to induce NF-κB activity is restricted to caspase-1. We next analyzed the NF-κB-inducing properties of human caspase-1. Therefore, human caspase-1 was cloned from THP-1 cells and the enzymatic inactive C/A mutant was generated to exclude interference with cell death during the assay. Potent dose- dependent induction of NF-κB activation was observed upon transfection of human caspase-1 C/A in 293T cells (Figure 3B), demonstrating that the NF-κB-inducing ability is evolutionary conserved from mouse to man.

In order to determine which domain of caspase-1 is responsible for the induction of the NF-κB activation, the CARD, p20 and p10 domain were cloned and tested separately, as well as the CARD-p20, and the p20-p10 combination. To make sure that the absence of induction is not due to insufficient expression, the expression of all the constructs was tested. The CARD domain is required and sufficient for the induction of the NF-κB activity, while the other domains of procaspase-1 do not induce NF-κB activity. In the CARD-p20 construct, the presence of the p20 domain seems to lead to some inhibition of the inducing capacity of the CARD (Figure 4).

Example 3: procaspase-1 mediated NF-κB activation occurs via IKK Several pathways are known to lead to NF-κB activation. Most of those pathways have the use of the IKK complex in common. Important pathways are starting from the Toll like receptor and using RIP2, or from the TNF receptor using TRAF2. In order to get an idea which pathway is implicated in procaspase-1 mediated NF-κB activation, the effect of several dominant negative (DN) mutants and inhibitors on procaspase-1 mediated activation of NF-κB activation was tested. TRAF2 DN has no effect on caspase-1 induced NF-κB activation, showing that TRAF2 is not involved downstream from caspase-1 (Figure 5a). However, TRAF2 DN is capable of inhibiting the TNF induced activation of NF-κB. As TRAF2 operates upstream in the TNF- pathway, it is likely that procaspase-1 is not involved in the TNF-pathway leading to activation of NF- B. RIP1 is a death domain containing kinase situated downstream of TRAF2. A dominant negative form of RIP1, lacking the kinase domain (RIP1 DD), blocks TNF mediated activation of NF-κB. However, RIP1 DD is not capable of inhibiting the procaspase-1 mediated induction of NF-κB activity (Figure 5b), indicating again that procaspase-1 is not involved in the TNF pathway.

As expected, IKK-β DN completely abolishes caspase-1 induced NF-κB activation, showing that procaspase-1 induces NF-κB through the IKK complex (Figure 5c). A20, a known inhibitor of many NF-κB pathways is also capable of inhibiting the caspase-1 mediated induction of the NF-κB activity. It indicates that A20 operates downstream of caspase-1. However, the precise role of A20 is not clear yet (Figure 5d).

As procaspase-1 is not involved in the TNF induction pathway, it may be involved in the Toll like receptor pathway. RIP2 is the central mediator of TLR-2, TLR-3, TLR-4 and of NOD1 and NOD2 induced NF-κB (Chin et al., 2002; Kobayashi et al., 2002). Indeed, a dominant negative form of RIP2, RIP2 lacking the kinase domain (RIP2-CARD) partially inhibits caspase-1 induced NF-κB activation (Figure 5e), supporting a role of RIP2 downstream of caspase-1 in its pathway. As shown in figure 6A, a deletion mutant of RIP2 lacking its kinase domain (RIP2 DN) functions as a dominant negative molecule on RIP2-induced activation of NF-κB (Figure 6A). This RIP2 DN inhibits caspase-1 -induced NF-κB activation to a similar extent (Figure 6A), suggesting a downstream function for RIP2 in the caspase-1 pathway. As a negative control, TNF-induced NF-κB activation is not blocked by RIP2 DN (Figure 6A). Co-immunoprecipitation experiments further confirmed that full-length caspase-1 physically interacts with RIP2 and with the isolated CARD domain of RIP2 (Figure 6B). Taken together, these results suggest a downstream role for RIP2 in caspase- -induced NF-κB activation.

Example 4: overexpression of procaspase-1 leads to p38 phosphorylation Beside the activation of NF-κB, phosphorylation of p38 MAP kinase is another downstream function of RIP2 (Chin et al., 2002; Kobayashi et al., 2002). Since caspase-1 -mediated activation of NF-κB occurs via a RIP2-dependent mechanism, we tested whether caspase-1 CARD is also capable of inducing p38 MAP kinase phosphorylation. Indeed, in caspase-1 CARD expressing cells a strong induction of p38 MAP kinase phosphorylation could be observed, while in GFP-transfected cells no active p38 MAP kinase was detectable (Figure 7A). As a control, both caspase-1 CARD as GFP-expressing cells contain basal levels of p38 MAP kinase (Figure 7A). This indicates that the phosphorylation of p38 MAP kinase by caspase-1 CARD is not due to general stress induced by the transfection procedure or by other aspecific stress-factors. Activation of both p38 MAP kinase and NF-κB occur in caspase- 1 CARD-expressing cells (Figure 7B). Therefore, we analyzed whether activation of p38 MAP kinase is involved in induction of NF-κB activation. As depicted in figure 7B, inhibition of p38 MAP kinase activation by the p38 MAP kinase-specific inhibitor SB203508 significantly decreases the levels of active p38 MAP kinase. However, in the same cells, the levels of NF- KB activation remained unchanged, suggesting that NF-κB activation by caspase-1 is independent of p38 MAP kinase activation and that activation of p38 MAP kinase and NF-κB are to separate pathways downstream of RIP2 recruitment.

Example 5: caspase-2 induces NF-κB activity

Overexpression of murine caspase-2 in 293T cells leads to cell death. This effect is depending upon the catalytic activity of caspase-2. The enzymatically inactive caspase-2 C/A mutant does not induce apoptosis. However, similar as for caspase-1 , both caspase-2 wild type, as well as the caspase-2 C/A mutant, are capable of inducing NF-κB activation in a dose dependent manner (Figure 8 a & b).

Example 6: caspase-2 mediated NF-κB activation is due to CARD domain

In order to determine which domain of caspase-2 is responsible for the induction of the NF-κB activation, the murine CARD, p20 and p10 domains were cloned and tested separately, as well as the CARD-p20, and the p20-p10 combination. To make sure that the absence of induction is not due to insufficient expression, the expression of all the constructs was tested. Comparable as for the results obtained with caspase-1 , the CARD domain of caspase-2 is required and sufficient for the induction of the NF-κB activity, while the other domains of procaspase-1 do not induce NF-κB activity. In the CARD-p20 construct, the presence of the p20 domain seems to lead to some inhibition of the inducing capacity of the CARD (Figure 9). The result was independently confirmed using human procaspase-2 derived CARD, which can also induce NF-κB activity in a dose dependent manner.

Example 7: procaspase-2 mediated NF-κB activation occurs via IKK In analogy to the experiments with procaspase-1 , the effect of several dominant negative (DN) mutants and inhibitors on the procaspase-2 NF-κB activation was tested to get an idea which pathway is used for the caspase-2 mediated NF-κB activation. Contrary to procaspase-1 , TRAF2 DN has a clear inhibiting effect on procaspase-2 induced activation of NF-κB, showing that TRAF2 is involved downstream of procaspase-2 (Figure 10a). TRAF2 is known to be involved in the TNF- and CD40- signaling pathways leading to NF-κB activation (Wajant et al., 2003; Hostager et al., 2003). This suggests that caspase-2 may be operating in aforementioned pathways to NF-κB activation. IKK-β DN completely abolishes procaspase-2 induced NF-κB activation, showing that procaspase-2 induces NF-κB through the IKK complex (Figure 10b). In a similar way, A20 is also capable of inhibiting the procaspase-2 mediated induction of the NF-κB activity (Figure 10c). It indicates that A20 operates downstream of procaspase-2, as is also the case for procaspase-1. As procaspase-2 is possibly involved in the TNF and CD40 induction pathways (Wajant et al., 2003), it may however not be involved in the Toll like receptor pathway. Indeed, RIP2 CARD does not inhibit procaspase-2 induced NF-κB activation (Figure 10d). RIP2 is the central mediator of TLR-2, TLR-3, TLR-4 and of NOD1 and NOD2 induced NF-κB (Chin et al., 2002; Kobayashi et al., 2002). The fact that RIP2 CARD cannot inhibit procaspase-2 mediated NF- KB activation strongly suggests that procaspase-2 probably is not involved in these pathways. Further confirmation that procaspase-2 may be operating in the TNF or CD40 pathways is obtained by immunoprecipitation of TRAF1 and TRAF2 with procaspase-2. Full length procaspase-2 (C/A mutant) physically interacts with both TRAF1 and TRAF2. These TRAF molecules are mediators of TNF- and CD40-induced NF-κB activation (Wajant et al., 2003; Hostager et al., 2003).

Example 8: RIP1 is recruited to the caspase-2/TRAF2 protein complex

The protein kinase RIP1 is another major regulator of NF-κB activation known to interact with

TRAF2 (Kelliher et al., 1998). Therefore, we studied the interaction between HA-tagged caspase-2 and RIP1 by immunoprecipitating caspase-2 and compared it to TRAF2 recruitment. In contrast to TRAF2, RIP1 was not present in HA-immunoprecipitates (Figure 11 A, left and middle panel). However, when both TRAF2 and RIP1 were co-expressed with HA-tagged caspase-2, RIP1 was clearly detectable in the HA-immunoprecipitates (Figure 11 A, right panel). The presence of TRAF2 in caspase-2 immunoprecipitates (Figure 11 A, middle panel) confirms our result that demonstrates the presence of caspase-2 in TRAF2- immunoprecipitates. These results demonstrate that RIP1 is recruited to caspase-2 only in the presence of TRAF2, suggesting the formation of a NF-κB signaling complex harboring caspase-2, TRAF2 and RIP1. Several reports have demonstrated that caspase-8 cleaves RIP1 in vitro, in TNF-stimulated HeLa cells and in FasL-stimuIated 293T and Jurkat T cells (Lin et al., 1999; Martinon et al., 2000). This cleavage at residue D324 leads to the release of a C- terminal RIP1 fragment that functions as a dominant negative molecule on NF-κB activation (Lin et al., 1999; Martinon et al., 2000). In this respect, caspase-8 promotes cell death by shutting down the NF-κB survival pathway. To rule out a similar function for caspase-2, in vitro translated ³⁵S-labeled RIP1 was incubated with purified recombinant caspase-2 and caspase-8 (Figure 11 B). In accordance to published results (Lin et al., 1999; Martinon et al., 2000), RIP1 was partially processed to fragments of 38 and 36 kDa by caspase-8 (Figure 11B). However, when incubated with caspase-2, RIP1 fragments were not detected and no weakening of the band representing full length RIP1 was observed (Figure 11B). In contrast to RIP1, ³⁵S-labeled caspase-2 was cleaved to a 38 kDa fragment by recombinant caspase-2, demonstrating that the enzyme was proteolytically active (Figure 11 B). These results suggest that RIP1 is not processed by caspase-2 to generate a dominant negative fragment when it is recruited to the caspase-2/TRAF2 complex.

Example 9: overexpression of procaspase-2 leads to p38 phosphorylation

Besides of the induction of NF-κB activation, similar to the CARD of procaspase-1 , the CARD of procaspase-2 is also able to induce phosphorylation of p38 (Figure 12). Also RIP1 is capable of inducing both NF-κB and p38 activation, which is in agreement with the role of RIP1 in the NF-κB and p38 MAPK activating effects of the CARD domain of procaspase-2. Furthermore, this suggests that both effects are downstream of the recruitment of RIP1 to the caspase-2 / TRAF2 complex. As a negative control, overexpression of GFP was unable to induce p38 MAP kinase phosphorylation.

Example 10: caspase mediated activation of NF-κB is specific for procaspase-1 and procaspase-2

Several other both CARD comprising and CARD non-comprising caspases were tested for their capacity to induce NF-κB activation. Although caspase-11 and caspase-12 are closely related to caspase-1 , neither the wild type molecule nor the catalytic inactive C/A mutants are capable of inducing NF-κB activation. The CARD-less caspase-3, caspase-7 and caspase-14 have also been tested, but didn't show any activation either. COP/Pseudo-ICE and ICEBERG are two human-specific CARD-only proteins that share a high degree of sequence homology to the prodomain of caspase-1 , reaching 93% and 73% respectively (Druilhe et al., 2001; Humke et al., 2000; Lee et al., 2001). Interestingly, the genes for both COP/Pseudo-ICE and ICEBERG are mapped to chromosome 11q22, beside caspase-1 and probably arose by a recent gene duplication event. Both COP/Pseudo-ICE and ICEBERG are capable of binding to caspase-1 and inhibit caspase-1 -mediated maturation of pro-IL-1 β (Druilhe et al., 2001 ; Humke et al., 2000; Lee et al., 2001). Since we observed that caspase-1 CARD is necessary and sufficient for NF-κB activation, we were interested to compare the NF-κB activating ability of human caspase-1 C/A with that of COP/Pseudo-ICE and ICEBERG. Human caspase-1 C/A and COP/Pseudo-ICE are comparable in their NF-κB- inducing capacity (Figure 13). ICEBERG, however, had very little effect on the basal NF-κB activity, though all proteins were appropriately expressed (Figure 13). This suggests that NF- KB activity is unlikely to be provoked by a non-specific cellular stress mediated by transient expression of caspase-1 and COP/Pseudo-ICE. The activation of NF-κB by COP/Pseudo-ICE but not by ICEBERG is in accordance with the results described by Druilhe et al (Druilhe et al.,

2001). As both caspase-1 CARD and COP/Pseudo-ICE, but not ICEBERG, can induce NF-κB activation, we can conclude that the NF-κB-activating capacity of caspase-1 CARD, is evolutionary conserved in COP/Pseudo-ICE, but is lost in the evolutionary more distant

ICEBERG. Furthermore, it is likely that caspase-1 CARD and COP/Pseudo-ICE share a common NF-κB-activating interface on their surface that has been lost in ICEBERG.

The caspase-1 -related CARD only molecules INCA has also been tested. However, notwithstanding its high homology, the INCA CARD, similar to the ICERBERG CARD, is not capable of inducing activation of NF-κB.

Comparison of the sequences of the active and non-active CARDs allows to pinpoint those amino acid residues in the caspase-1 CARD that are important in the NF-κB activation. The residues and their spacing are given in SEQ ID N°1. Said residues play probably an essential role in the caspase-1 CARD / RIP2 interaction.

Example 11: NF-κB activation by caspase-1 CARD mutants.

Several single and double mutations, and one triple mutation was introduced in the CARD domain of caspase-1 , as is indicated in Table 1 and Table 2. The NF-κB activity was tested as for the wild type CARD. The results are summarized in figure 14 and 15. As indicated in Figure 14, The mutant combinations D27G+E31G, K42G+L45D+I48D and K63G+Q67K were not longer capable of inducing NF-κB activity. To pinpoint which residues are essential in the NF-κB activation, the experiment was repeated with single mutations in the CARD domain. The aspartic acid residue at position 27 seems to play a crucial role, as the D27G mutation shows a dramatic effect on the activation. The mutations E31G and K63G do have a significant but less dramatic effect, whereas the mutations L45D, I48D, Q67K and Y82H have only a minor effect. No effect at all could be seen for the mutation K42G. However, from the study of the multiple mutations, it is clear that the combined mutations do have a synergetic effect, as the activation activity of the triple mutant K42G+L45D+I48D is strongly affected.

On the base of the structure, it can be concluded that the mutations affect the caspase-1 CARD RIP2 CARD interaction, stressing the importance of this interaction in the activation of NF-κB.

TABLES Table 1 : list of the positions of the single mutations

Table 2 : list of the combined mutations

Constructs with double and triple substitutions D27G + E31G R33E + N36S K42G + L45D + I48D K63G + Q67K Table 3: list of the primers used

REFERENCES - Ahmad, M., Srinivasula, S.M., Wang, L.J., Talanian, R.V., Litwack, G., Femandes- Alnemri, T. and Alnemri, E.S. (1997). Cradd, a novel human apoptotic adaptor molecule for caspase-2, and Fas/Tumor necrosis factor receptor-interacting protein Rip. Cancer Res. 57, 615-619. - Bertin, J., and DiStefano, P. S. (2000). The PYRIN domain: a novel motif found in apoptosis and inflammation proteins. Cell Death Differ 7, 1273-1274. - Bertin, 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., et al. (1999). Human CARD4 protein is a novel CED-4/Apaf-1 cell death family member that activates NF-kappaB. J Biol Chem 274, 12955-12958. - Bouchier-Hayes, L. and Martin, S.J. (2002) CARD games in apoptosis and immunity. EMBO reports, 3, 616-621.

- Baldwin, A. (1996). The NF-κB and lκB proteins: new discoveries and insights. Annu. Rev. Immunol. 14, 649-681.

- Bonnard, M., Mirtsos, C, Suzuki, S., Graham, K, Huang, J., Ng, M., Itie, A., Wakeham, A., Shahinian, A., Henzel, W. J., Elia, A. J., Shillinglaw, W., Mak, T. W., Cao, Z., and Yeh, W. C. (2000). Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-κB-dependent gene transcription. EMBO J. 19, 4976-4985. - Chin, A. I., Dempsey, P. W., Bruhn, K, Miller, J. F., Xu, Y., and Cheng, G. (2002). Involvement of receptor-interacting protein 2 in innate and adaptive immune responses. Nature 416, 190-194.

- Denecker, G., Vercammen, D., Steemans, M., Vanden Berghe, T., Brouckaert, G., Van Loo, G., Zhivotovsky, B., Fiers, W., Grooten, J., Declercq, W., and Vandenabeele, P. (2001). Death receptor-induced apoptotic and necrotic cell death: differential role of caspases and mitochondria. Cell Death Differ 8, 829-840.

- Druilhe, A., Srinivasula, S. M., Razmara, M., Ahmad, M., and Alnemri, E. S. (2001). Regulation of IL-1 beta generation by Pseudo-ICE and ICEBERG, two dominant negative caspase recruitment domain proteins. Cell Death Differ 8, 649-657. - Duan, H. and Dixit, V.M. (1997). Raidd is a new death adapter molecule. Nature, 385, 86-89.

- Fairbrother, W. J., Gordon, N. C, Humke, E. W., O'Rourke, K. M., Starovasnik, M. A., Yin, J. P., and Dixit, V. M. (2001 ). The PYRIN domain: a member of the death domain- fold superfamily. Protein Sci 10, 1911-1918. - Foltz, I. N., Lee, J. C, Young, P. R., and Schrader, J. W. (1997). Hemopoietic growth factors with the exception of interleukin-4 activate the p38 mitogen-activated protein kinase pathway. J Biol Chem 272, 3296-3301. - Ghosh, S., May, M., and Kopp, E. B. (1998). NF-κB and Rel proteins:evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16, 225-260. - Hlaing, T., Guo, R. F., Dilley, K. A., Loussia, J. M., Morrish, T. A., Shi, M. M., Vincenz, C, and Ward, P. A. (2001). Molecular cloning and characterization of DEFCAP-L and - S, two isoforms of a novel member of the mammalian Ced-4 family of apoptosis proteins. J Biol Chem 276, 9230-9238. - Hoeflich, K. P., Luo, J., Rubie, E. A., Tsao, M. S., Jin, O., and Woodgett, J. R. (2000). Requirement for glycogen synthase kinase-3beta in cell survival and NF-κB activation. Nature 406, 86-90. - Hopkins, S. J., and Humphreys, M. (1989). Simple, sensitive and specific bioassay of interleukin-1. J Immunol Methods 120, 271-276.

- Hostager, B. S., Haxhinasto, S. A., Rowland, S. L, and Bishop, G. A. (2003). TRAF2- deficient B lymphocytes reveal novel roles for TRAF2 in CD40 signaling. J Biol Chem.

- Hsu, H., Xiong, J, and Goeddel, D.V. (1995). The TNF receptor 1 -associated protein TRADD signals cell death and NF-κB activation. Cell, 81, 495- 504.

- Humke, E. W., Shriver, S. K., Starovasnik, M. A., Fairbrother, W. J., and Dixit, V. M. (2000). ICEBERG: a novel inhibitor of interleukin-1 beta generation. Cell 103, 99-111.

- Inohara, N., Koseki, T., del Peso, L., Hu, Y, Yee, C, Chen, S., Carrio, R., Merino, J., Liu, D., Ni, J., and Nunez, G. (1999). Nodi , an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J Biol Chem 274, 14560-14567.

- Inohara, N., and Nunez, G. (2000). Genes with homology to mammalian apoptosis regulators identified in zebrafish. Cell Death Differ 7, 509-510.

- Kalai, M., Lamkanfi, M., Denecker, G., Boogmans, M., Lippens, S., Meeus, A., Declercq, W., and Vandenabeele, P. (2003). Regulation of the expression and processing of caspase-12. J Cell Biol 162, 457-467.

- Kelliher, M.A., S. Grimm, Y. Ishida, F. Kuo, B.Z. Stanger, and P. Leder. (1998). The death domain kinase RIP mediates the TNF-induced NF-kappaB signal. Immunity. 8,297-303.

- Klinkenberg, M., Van Huffel, S., Heyninck, K., and Beyaert, R. (2001 ). Functional redundancy of the zinc fingers of A20 for inhibition of NF-kappaB activation and protein-protein interactions. FEBS Lett 498, 93-97.

- Kobayashi, K., Inohara, N., Hernandez, L. D., Galan, J. E., Nunez, G., Janeway, C. A., Medzhitov, R., and Flavell, R. A. (2002). RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems. Nature 416, 194-199. - Lamkanfi, M., Declercq, W., Kalai, M., Saelens, X., and Vandenabeele, P. (2002). Alice in caspase land. A phylogenetic analysis of caspases from worm to man. Cell Death Differ 9, 358-361. - Lamkanfi, M., Declercq, W., Depuydt, B., Kalai, M., Saelens, X., and Vandenabeele, P. (2003). The caspase family. In Caspases:Their Role in Cell Death and Cell Survival , Los, M., and Walczak, H., editors. Landes Bioscience, Kluwer Academic Press, Georgetown, TX. 1-40. - Lee, S. H., Stehlik, C, and Reed, J. C. (2001). Cop, a caspase recruitment domain- containing protein and inhibitor of caspase-1 activation processing. J Biol Chem 276, 34495.34500. - Lin, Y, A. Devin, Y Rodriguez, and Z.G. Liu. (1999). Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes Dev. 13,2514-26. - Martinon, F., N. Holler, C. Richard, and J. Tschopp. (2000). Activation of a pro- apoptotic amplification loop through inhibition of NF-kappaB-dependent survival signals by caspase-mediated inactivation of RIP. FEBS Lett. 468,134-6.

- Martinon, F., Hofmann, K., and Tschopp, J. (2001). The pyrin domain: a possible member of the death domain-fold family implicated in apoptosis and inflammation. Curr Biol 11, R118-120.

- Masumoto, J., Taniguchi, S., and Sagara, J. (2001). Pyrin N-terminal homology domain- and caspase recruitment domain-dependent oligomerization of ASC. Biochem Biophys Res Commun 280, 652-655.

- May, M. J., and Ghosh, S. (1998). Signal transduction though NF-κB. Immunol. Today 19, 80-88

- Mayo, M. W., and Baldwin, A. S. (2000). The transcription factor NF-κB: control of oncogenesis and cancer therapy resistance. Biochim. Biophys. Ada 1470, M55-M62

- McCarthy, J. V., Ni, J., and Dixit, V. M. (1998). RIP2 is a novel NF-kappaB-activating and cell death-inducing kinase. J Biol Chem 273, 16968-16975. - Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J., Young, D. B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997). IKK-1 and IKK-2: cytokine activated lκB kinases essential for NF-κB activation. Science 278, 860-866

- Ono, K, and Han, J. (2000). The p38 signal transduction pathway: activation and function. Cell Signal 12, 1-13. - O'Mahoney, J. V., and Adams, T. E. (1994). Optimization of experimental variables influencing reporter gene expression in hepatoma cells following calcium phosphate transfection. DNA Cell Biol 13, 1227-1232.

- Pawlowski, K, Pio, F., Chu, Z., Reed, J. C, and Godzik, A. (2001). PAAD a new protein domain associated with apoptosis, cancer and autoimmune diseases. Trends Biochem Sci 26, 85-87.

- Peter, M. E., and Krammer, P. H. (2003). The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ 10, 26-35. - Poyet, J. L., Srinivasula, S. M., Tnani, M., Razmara, M., Femandes-Alnemri, T., and Alnemri, E. S. (2001). Identification of Ipaf, a human caspase-1 -activating protein related to Apaf-1. J Biol Chem 276, 28309-28313. - Sakurai, H., Chiba, H., Miyoshi, H., Sugita, T, and Toriumi, W. (1999). IKB kinases phosphorylate NF-κB p65 subunit on serine 536 in the transactivation domain. J. Biol. Chem. 274, 30353-30356 - Stehlik, C, Hayashi, H., Pio, F., Godzik, A., and Reed, J. C. (2003). CARD6 is a modulator of NF-kappa B activation by Nodi- and Cardiak-mediated pathways. J Biol Chem 278, 31941-31949. - Thome, M., Hofmann, K, Burns, K., Martinon, F., Bodmer, J. L., Mattmann, C, and Tschopp, J. (1998). Identification of CARDIAK, a RIP-like kinase that associates with caspase-1. Curr Biol 8, 885-888.

- Tibbetts, M. D., Zheng, L., and Lenardo, M. J. (2003). The death effector domain protein family: regulators of cellular homeostasis. Nat Immunol 4, 404-409. - Van de Craen, M., Vandenabeele, P., Declercq, W., Van den Brande, I., Van Loo, G., Molemans, F., Schotte, P., Van Criekinge, W., Beyaert, R., and Fiers, W. (1997). Characterization of seven murine caspase family members. FEBS Lett 403, 61-69.

- Vercammen, D., Beyaert, R., Denecker, G., Goossens, V., Van Loo, G., Declercq, W., Grooten, J., Fiers, W., and Vandenabeele, P. (1998). Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med 187, 1477-1485.

- Wajant, H., Pfizenmaier, K., and Scheurich, P. (2003). Tumor necrosis factor signaling. Cell Death Differ 10, 45-65.

- Wang, S., Miura, M., Jung, Y, Zhu, H., Gagliardini, V., Shi, L, Greenberg, A. H., and Yuan, J. (1996). Identification and characterization of lch-3, a member of the interleukin-1 beta converting enzyme (ICE)/Ced-3 family and an upstream regulator of ICE. J Biol Chem 271, 20580-20587.

- Wang, D., Westerheide, S. D., Hanson, J. L., and Baldwin, A. S., Jr. (2000). Tumor necrosis factor alpha-induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II. J. Biol. Chem. 275, 32592-32597

- Zandi, E., and Karin, M. (1999). Bridging the gap: composition, regulation and physiological function of the IKB comples. Mol. Cell. Biol. 19, 4547-4551

- Zhong, H., SuYang, H., Erdjument-Bromage, H., Tempst, P., and Ghosh, S. (1997). Phosphorylation of NF-κB p65 by PKA stimulates transcritptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Cell 89, 413-424

- Zhong, H., Voll, R. E., and Ghosh, S. (1998) Mol. Cell Λ, 661-671

Claims

1. The use of a procaspase derived CARD to modulate NF-κB activation and/or p38 MAP kinase activation.

2. The use of a procaspase derived CARD, whereby said modulation is an induction of the activation.

3. The use of a procaspase derived CARD, to screen for anti-inflammatory compounds.

4. The use according to claim any of the claims 1-3, whereby said CARD comprises SEQ ID N°1.

5. The use according to claim 4, whereby said caspase is procaspase-1.

6. The use according to any of the claims 1-3, whereby said caspase is procaspase-2.

7. The use according to claim 5, whereby said CARD is essentially consisting of SEQ ID N°2.

8. The use according to claim 6, whereby said CARD is essentially consisting of SEQ ID N°3.