256 Research Article

IntroductionToll-like receptors (TLRs) are on the frontline of innate immunity,ensuring a fast and efficient response against invading pathogens.At present, ten human TLRs are described, all defined byextracellular leucine-rich repeats (LRRs) and an intracellular Toll-IL-1 receptor (TIR) interaction domain. TLRs recognize a wholespectrum of both extracellular and intracellular pathogen-associatedmolecular patterns (PAMPs), such as lipopolysaccharide (LPS;TLR4) (Poltorak et al., 1998), bacterial lipoproteins (TLR2)(Yoshimura et al., 1999) or double-stranded RNA (TLR3)(Alexopoulou et al., 2001). Upon ligand binding, various adaptorproteins are recruited to the activated TLRs. This event is mediatedby TIR-TIR domain interactions. Four of these TIR-containingadaptor molecules have been identified so far. Myeloiddifferentiation factor 88 (MyD88) is considered to be the universaladaptor and is recruited by every TLR, except TLR3 (Wesche etal., 1997). MyD88 adaptor-like (Mal) is used by TLR2 and TLR4,and acts as a bridging adaptor, linking MyD88 to the activatedreceptor (Fitzgerald et al., 2001; Horng et al., 2001; Horng et al.,2002; Kagan and Medzhitov, 2006; Ulrichts et al., 2007; Yamamotoet al., 2002). TIR-domain-containing adaptor-inducing IFN- (Trif)is involved in TLR3 and TLR4 signaling (Oshiumi et al., 2003a).Finally, Trif-related adaptor molecule (Tram) is solely recruited toTLR4 where it couples Trif to the receptor (Fitzgerald et al., 2003;Kagan et al., 2008; Oshiumi et al., 2003b). TLR signaling is furtherpropagated by a cascade of phosphorylation and ubiquitinylationevents, ultimately resulting in the activation of transcription factorssuch as NF-B, AP-1 and various interferon-regulatory factors(IRFs) (Kawai and Akira, 2006). This leads to the induction of pro-inflammatory cytokines and type I interferons, which initiate apotent immune reaction. However, uncontrolled TLR signaling and

the concomitant excessive inflammation are extremely harmful forthe host. Mammals have therefore evolved several mechanisms toprevent inordinate TLR-induced pro-inflammatory cytokineproduction. This attenuation can be achieved at multiple levels,either by interfering with ligand binding, by inhibiting receptorexpression or by targeting intracellular signaling components (Langand Mansell, 2007).

Recently, the family of cysteine-aspartic acid proteases (caspases)was shown to target intracellular components of TLR signaling.Activation of caspases by various pro-apoptotic signals or upon viralinfection results in the cleavage and subsequent inactivation of theTLR adaptor Trif, and hence inhibits NF-B and IRF activation(Rebsamen et al., 2008). However, which caspase is involved inthis attenuation and the exact modalities of its activation remain tobe clarified. In addition to its cardinal role in inflammasome-mediated cleavage of pro-IL-1 and pro-IL-18, caspase-1 seemsalso to be involved in the regulation of TLR signaling, becauseTLR2 and TLR4 signaling in caspase-1–/– macrophages areabrogated (Miggin et al., 2007). Miggin and co-workersdemonstrated a direct interaction of Mal with caspase-1, and Malis cleaved by caspase-1. Mass spectrometry analysis identified D198of Mal as the caspase-1 cleavage site. A Mal D198A mutant is nolonger cleaved by Mal, shows no NF-B activation and acts as adominant-negative inhibitor of LPS- and Pam3Cys-inducedsignaling. These data suggest that Mal cleavage at D198 bycaspase-1 is indispensable for Mal-mediated signaling.

In this report, we further investigate the structural and functionalimplications of Mal cleavage by caspase-1. We investigate how theinteraction of Mal with TLR4 or MyD88 is affected by mutationsthat inhibit or mimic caspase-1 cleavage. For the study of theseinteractions, we use the mammalian two-hybrid system MAPPIT

Caspase-1 targets the TLR adaptor Mal at a crucialTIR-domain interaction sitePeter Ulrichts1,2, Celia Bovijn1,2, Sam Lievens1,2, Rudi Beyaert3,4, Jan Tavernier1,2,* and Frank Peelman1,2

1Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium2Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, B-9000 Ghent, Belgium3Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research, VIB, B-9052 Ghent, Belgium4Department of Biomedical Molecular Biology, Faculty of Sciences, Ghent University, B-9052 Ghent, Belgium*Author for correspondence (jan.tavernier@ugent.be)

Accepted 22 OctoberJournal of Cell Science 123, 256-265 Published by The Company of Biologists 2010doi:10.1242/jcs.056002

SummaryToll-like receptors (TLRs) are crucial components of innate immunity, ensuring efficient responses against invading pathogens. Afterligand binding, TLR signaling is initiated by recruitment of adaptor molecules, a step mediated by homotypic Toll-IL-1 receptor (TIR)domain interactions. Four TIR-containing TLR adaptor molecules are described, all of which are susceptible to modification and strictregulation. For example, caspase-1 is reported to cleave the TLR adaptor Mal at position D198, an event that is indispensible for Malfunction. In this report, we use the mammalian two-hybrid technique MAPPIT to study the implications of Mal cleavage. We showthat a Mal mutant, which mimics caspase-1 cleavage and a caspase-1-uncleavable MalD198A mutant, are abrogated in their bridgingfunction and lose the ability to activate NF-B. A MalD198E mutant is still fully functional, suggesting that caspase-1 cleavage ofMal is not necessary for Mal-mediated signaling. D198 of Mal is conserved in MyD88 and TLR4 TIR domains and the negativelycharged amino acid at this position is crucial for the interactions and function of Mal, MyD88 and TLR4 TIR. Our data suggest aninhibitory, rather than an activating role for caspase-1 in Mal regulation, and show that the caspase-1 cleavage site in Mal is part of aTIR-domain interaction site.

Key words: Caspase-1, Mal, TLR, TIR domain

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(mammalian protein-protein interaction trap) (Eyckerman et al.,2001). Our data show that caspase-1-mediated Mal cleavage is notrequired for Mal interaction with TLR4 or MyD88, nor for Malinduced NF-B activation. A mutant that mimics caspase-1-cleavedMal shows no interaction with TLR4 and MyD88. Our data suggestthat the role of caspase-1 in TLR2 and TLR4 signaling is notdependent on Mal cleavage by caspase-1. We show that D198 isclose to a very conserved surface area in Mal, which is likely to beinvolved in Mal function. Mutations of D198 and mutations of thecorresponding residue in the TIR domain of TLR4 and MyD88disrupt their TIR-TIR interactions. The TIR surface areacorresponding to D198 in Mal is important for TIR-TIR interactionsin Mal, MyD88 and TLR4.

ResultsMutation of the caspase-1 cleavage site in Mal prevents itsinteraction with MyD88 and TLR4Caspase-1-dependent cleavage was previously shown to be anessential event for Mal activation and downstream TLR2 and TLR4signal transduction (Miggin et al., 2007). This processing occurs atthe C-terminal portion of its ‘TIR’ domain at the aspartic acid atposition 198. Based on a model of Mal (Núñez et al., 2007), theremoval of the E helix in the TIR domain of Mal by caspase-1 wasargued to be essential for the generation of a steep groove involvedin MyD88 recruitment. Mutation of aspartic acid at position 198of Mal to alanine completely blocks TLR2 and TLR4 signaling.

This effect was attributed to a failing caspase-1 cleavage of thismutant form of Mal (Miggin et al., 2007). Using MAPPIT (Fig. 1),we investigated whether this MalD198A mutant indeed fails torecruit MyD88. As expected, a clearly reduced interaction of thismutant form of Mal with MyD88 could be detected (Fig. 2A) andthe mutant could not bridge MyD88 to TLR4 (Fig. 2B).

Cleaved Mal does not interact with MyD88 and TLR4Caspase-1-mediated cleavage of Mal was proposed to be essentialfor Mal-MyD88 binding (Núñez et al., 2007). We therefore clonedthe caspase-cleaved variant of Mal (containing only amino acids 1-198; further referred to as Malcasp), as both MAPPIT bait and prey,and tested its association with MyD88 and TLR4. Interaction oftruncated Mal with full-length MyD88 or its TIR domain(MyD88TIR) was completely abrogated (Fig. 3A). In addition, thereciprocal MAPPIT set-up also showed a clear reduction ofassociation (Fig. 3B). These MAPPIT results were confirmed by co-immunoprecipitation (Fig. 3C). FLAG-tagged MyD88 or

Fig. 1. The MAPPIT concept. A ‘bait’ is C-terminally fused with thetransmembrane and intracellular part of a leptin receptor that is deficient inSTAT3 recruitment. The extracellular domain of either the leptin receptor (LR)or of the erythropoietin (EpoR) can be used. The ‘prey’ protein is linked to aseries of six functional STAT3 recruitment sites of the gp130 chain.Association of bait and prey and ligand (L) stimulation leads to STAT3activation and induction of a STAT3-responsive luciferase reporter (rat PAPI-Luci).

Fig. 2. MalD198A mutant loses interaction with MyD88 and TLR4.(A)Heterodimerization of MalTIR or MalTIRD198A and MyD88. HEK293Tcells were transiently co-transfected with the MAPPIT bait vector pCLL-MyD88, together with prey plasmids pMG2-MalTIR, pMG2-MalTIRD198Aand the rat PAPI-luci reporter. After transfection, cells were left untreated (NS)or were stimulated with Leptin for 24 hours. Data are expressed as mean foldluciferase induction (ratio stimulated/non-stimulated) and s.d. of triplicatemeasurements are plotted. Expression of the FLAG-tagged prey proteins wasverified on lysates using anti-FLAG antibody. (B)MalD198A loses itsbridging function. HEK293T cells were transiently co-transfected with pCLL(control bait) or pCLL-TLR4ic, the MyD88TIR prey, a Mal expression vector(pCAGGSE-Mal/pCAGGSE-MalD198A) and the rat PAPI-luci reporter.Experimental set-up was as in A.

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MyD88TIR could be precipitated using wild-type E-tagged Mal, butnot by E-tagged Malcasp. Next, we investigated the interaction ofMal with TLR4 (Fig. 3D). As previously described, using theintracellular part of TLR4 as bait and Mal as prey, a clear MAPPITsignal could be monitored. Again, this interaction was clearlynegatively affected by Mal truncation. Finally, we also demonstratedthat Malcasp loses its ability to bridge MyD88 to TLR4 (Fig. 3E).

Caspase-1 targets Mal at an acidic amino acid that iscrucial for interactionTo find out whether the altered interaction profile of MalD198Aoccurs because of a loss of caspase-1 cleavage or to a loss of‘negative charge’ at position 198, we repeated the same experiments

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with a Mal mutant in which aspartic acid 198 was mutated toglutamic acid (MalD198E). Since it was shown that substitution ofthe aspartic acid to glutamic acid in the P1 position of caspase-1cleavage sites reduces selectivity over 1000-fold, the MalD198Emutant is expected to be resistant to caspase-1 cleavage (Stennickeet al., 2000). To unequivocally demonstrate the impossibility ofMalD198E cleavage by caspase-1, both overexpression assays (Fig.4A), as well as in vitro caspase-cleavage assays with purifiedMal(mutant) protein (Fig. 4B) were performed. A modest caspase-1-dependent cleavage could be observed using wild-type Mal. Thisphenomenon was completely absent when performing the assayswith MalD198E, indicating that this mutant indeed is not cleavedby caspase-1.

Fig. 3. Mal cleavage disrupts its interaction with MyD88 and TLR4. (A,B)Heterodimerization of Mal or Malcasp and MyD88. HEK293T cells were transientlyco-transfected with various combinations of MAPPIT bait vectors pCLL-Mal, pCLL-Malcasp, pCLL-MyD88, together with prey plasmids pMG2-MalTIR, pMG2-MalTIRcasp, pMG2-MyD88, pMG2-MyD88TIR and the rat PAPI-luci reporter. Experimental set-up was as in Fig. 2. (C)Co-immunoprecipitation analysis.HEK293T cells were transfected with combinations of pMet7-MyD88-FLAG, pMET7-MyD88TIR-FLAG, pCAGGSE-Mal and pCAGGSE-Malcasp. Cell lysateswere immunoprecipitated with an anti-E-tag antibody and subsequently immunoblotted (IB) with anti-FLAG or anti-E-tag. (D)TLR4 interaction profile. HEK293Tcells were transiently co-transfected with pCLL-TLR4ic, the rat PAPI-luci promoter and either Mal or Malcasp prey. (E)Cleavage of Mal results in a loss of itsbridging function. HEK293T cells were transiently co-transfected with pCLL or pCLL-TLR4ic, the MyD88TIR prey, a Mal expression vector (pCAGGSE-Mal orpCAGGSE-Malcasp) and the rat PAPI-luci reporter. Experimental set-up was as in Fig. 2.

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Strikingly, and in contrast to the MalD198A mutant, theMalD198E mutant still interacted with MyD88 and exerted abridging function for the MyD88-TLR4 interaction in a MAPPITcontext (Fig. 5A,B). These data indicate that an acidic amino acidat position 198, rather than caspase-1-mediated cleavage at this site,is indispensible for Mal-TIR interactions.

Mal cleavage is not essential for NF-B activationNext, we investigated the potency of Mal, Malcasp, MalD198Aand MalD198E to activate NF-B signaling. We therefore clonedthese Mal forms into a doxycyclin-inducible expression vector(pTre-tight, Clontech). In accordance with previous reports(Fitzgerald et al., 2001), we saw clear NF-B activation upon Malexpression (Fig. 6A). This effect seemed to be dependent on a full-length form of Mal, because Malcasp had no effect. The MalD198Amutant also failed to induce NF-B signaling, a feature that hasalready been described (Miggin et al., 2007). Strikingly, mutatingthe aspartic acid at position 198 into a glutamic acid only partiallyaffected NF-B induction. This reduction can partially be attributedto a reduced expression of the MalD198E mutant, as shown bywestern blot analysis (Fig. 6A). In addition, pre-incubation withthe specific caspase-1 inhibitor z-WEHD-fmk (Fig. 6B) or the

broad-range caspase inhibitor z-VAD-fmk (data not shown), did notdrastically affect Mal-induced NF-B activation.

Additionally, we tested the role of caspase-1 cleavage of Mal onTLR-induced NF-B activation in a Mal-deficient background. Inanalogy with a recent report (Nagpal et al., 2009), immortalizedMal-knockout macrophages were transduced with retrovirusesencoding Mal, Malcasp or MalD198E and an internal ribosomalentry site (IRES)-encoded green fluorescence protein (GFP). GFP-positive cells were sorted in bulk, and Mal(mutant) expression wasassayed (Fig. 7A). Mal-deficient macrophages were severelyimpaired in TNF- production in response to lipoteichoic acid (LTA)(Fig. 7B) and LPS (Fig. 7C), as measured by ELISA. Transductionof these Mal-deficient cells with wild-type Mal led to restored LTA-and LPS-induced TNF- production. This functionalcomplementation was not observed when transducing the Mal-knockout cells with the Malcasp mutant. MalD198E-transducedcells, on the other hand, displayed a comparable response to LTAand LPS as seen in wild-type Mal-transduced cells. Together withthe interaction experiments (see above), these data contradict thehypothesis that Mal cleavage is crucial for Mal function.

Mutations in TLR4 and MyD88 corresponding to D198Adisrupt TIR-TIR interactionsThe D198A mutation in Mal clearly disrupts its interaction withMyD88 and TLR4, and our data indicate that this might result from

Fig. 4. MalD198E mutant is not cleaved by caspase-1. (A)HEK293T cellswere co-transfected with different combinations of expression vectors coding forE-tagged Mal, MalD198E, caspase-1 p10 subunit and caspase-1 p20 subunit. Sixhours before lysis, cells were incubated with the proteasomal inhibitor MG132.Mal and caspase-1 were detected by immunoblotting with anti-E-tag. Totalprotein mass was severely reduced upon p10 and p20 co-expression (lane 3),most likely as a result of caspase-1-induced apoptosis. Therefore, lane 3 wasscanned with a higher intensity (lane 4, indicated with an asterisk). The caspase-1 cleaved form of Mal is indicated between two asterisks. (B)Purified His-tagged Mal or MalD198E was incubated for 2 hours with active recombinanthuman caspase-1 according to the manufacturer’s instructions. The caspase-1-cleaved form of Mal is indicated between two asterisks. Mal is detected byimmunoblotting with anti-Mal and anti-His antibodies.

Fig. 5. MalD198E mutant exhibits similar heterodimerization andbridging properties as wild-type Mal. (A) Hek293T cells were transientlyco-transfected with the MAPPIT bait vector pCLL-MyD88, the rat PAPI-lucipromoter and either MalTIR or MalTIRD198E prey. (B)Hek293T cells weretransiently co-transfected with pCLL or pCLL-TLR4ic, the MyD88TIR-prey,a Mal expression vector (pCAGGSE-Mal/ pCAGGSE-MalD198E) and the ratPAPI-luci reporter. Experimental set-up was as for Fig. 2.Jo

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the loss of an acidic amino acid at this site, rather than loss of acaspase-1 cleavage site. A sequence alignment of TIR domainsshows that D198 in Mal corresponds to E796 in TLR4 and D275in MyD88 (see later). Interestingly, it was previously shown thatmutation of the 796EWE798 motif to AAA in TLR4 leads to astrong inhibition of NF-B, C/EBP and AP-1 signaling (Ronni etal., 2003). Moreover, a D275A mutation in MyD88 was shown toinhibit MyD88-MyD88 interaction (Li et al., 2005). In view of ourfinding that D198A mutation in Mal disrupts its interaction withMyD88 and TLR4, we therefore investigated whether mutations ofE796 or D275 also affected the TIR-TIR interactions of TLR4 andMyD88, respectively. We carried out MAPPIT experiments usingeither the intracellular portion of wild-type TLR4 (TLR4ic) or itsmutant form (TLR4icEWE-AWA) as a bait and tested receptordimerization (using the TLR4ic prey) or adaptor recruitment (usingthe Tram prey) (Fig. 8A). We also assayed the homo- andheterodimerization properties of the TIR domain of MyD88 or its

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mutant form (MyD88D275A) in a MAPPIT context (Fig. 8B).Analogous to our experiments with Mal, a marked decrease wasdetected for all tested associations, indicating the importance of thisacidic amino acid for TIR-TIR interactions.

D198 is located close to a possible conserved binding sitein MalFig. 9 shows the structures and models of TLR4, MyD88 and Mal.We mapped the sequence conservation in orthologs of TLR4,MyD88 and Mal on the surface of these models, assuming thatresidues with an important role in TIR-TIR interactions would beconserved. E796 is part of a very conserved surface patch in TLR4,which consists mainly of residues 789-796 of the DE loop. Severalmutations in this area strongly affect TLR4 signal transduction(Ronni et al., 2003). This area is therefore probably involved ininteractions of TLR4. We found a corresponding conserved area inMal. Residues Y187, R192, F193, M194 and Y195 of the DE loopof Mal seemed to be central in this conserved surface patch.Residues 192-198 of Mal aligned with the residues of the conservedsurface patch in TLR4 (Fig. 9A). We used the prediction programWHISCY (de Vries et al., 2006) to predict possible protein-proteininteraction sites on the surface of the Mal model. WHISCYcombines residue conservation with structural information, such assurface neighbors and interface propensity, to predict the protein-protein interface residues. WHISCY prediction suggested that theconserved surface patch in Mal is part of a protein interaction site(Fig. 9E).

DiscussionThe recognition of pathogens by TLRs results in the recruitmentof one or more adaptor molecules to the activated receptorcomplex, which in part accounts for receptor-specific responses.This TLR-adaptor association is mediated by homotypic TIR-TIRdomain interactions. TIR domain crystal structures reveal a centralfive-stranded parallel -sheet, enclosed by five -helices (Khan etal., 2004; Nyman et al., 2008; Xu et al., 2000). Various regions inthe TIR domain seem to be involved in TIR-TIR association. Theimportance of the BB loop, linking the second -strand with thesecond -helix, is demonstrated in several studies (Jiang et al.,2006; Poltorak et al., 1998; Ulrichts et al., 2007). The TLR adaptorMal is cleaved by caspase-1, an event that is hypothesized to beessential for TLR2 and TLR4 signaling (Miggin et al., 2007).Núñez and colleagues presented a model of Mal, where the E-helixof Mal is removed after caspase-1 cleavage (Núñez et al., 2007).Removal of the E-helix leads to formation of a deep groove, whichis proposed to be involved in MyD88 recruitment. In this model,MalS180, is buried in this cleft and exposed after removal of theE-helix. A common Mal S180L polymorphism in humans clearlyattenuates TLR2 and TLR4 signaling, indicating that this residueis important for signaling (Khor et al., 2007). In analogy with aprevious study (Ulrichts et al., 2007), we tried to gain better insightin the effects of caspase-1 cleavage of Mal for its TIR domaininteractions using the MAPPIT two-hybrid technique. A D198Amutation severely affects the interaction of Mal with the TLR4and MyD88 TIR domains. The D198A mutant also loses thebridging function of Mal in our MAPPIT assay. We thereforeinvestigated the behavior of a Malcasp mutant corresponding toresidues 1-198 of Mal. This truncated Mal mutant mimics thecaspase-1-cleaved Mal. A clear reduction in the MAPPIT signalcompared with wild-type Mal was observed when assayingheterodimerization properties of Malcasp and MyD88 (Fig. 3A,B).

Fig. 6. Functional implications of Mal cleavage. (A)HEK293T cells weretransiently co-transfected with a NF-B-inducible luciferase reporter (pNF-conluc), the expression vector for the transactivator (rtTA-advanced) anddoxycyclin-inducible expression vectors pTRE-tight-Mal, pTRE-tight-Malcasp, pTRE-tight-MalD198A, pTRE-tight-MalD198E or empty vector. 24hours after transfection, cells were stimulated with doxycyclin (1g/ml) foranother 24 hours. Luciferase measurements were performed in triplicate. Dataare expressed as relative increase over control and s.d. of triplicatemeasurements are plotted. Protein expression was assayed on the remainder ofthe transfected cells stimulated for 24 hours with 1g/ml doxycyclin.(B)HEK293T cells were transiently co-transfected with pNF-conluc, rtTA-advanced and pTRE-tight-Mal. 24 hours after transfection, cells were pre-incubated for 2 hours with 50M caspase-1 inhibitor (ac-WEHD-fmk).DMSO was used as solvent control. The transfected cells were stimulated withdoxycyclin (1g/ml) for another 24 hours or were left untreated. Luciferasemeasurements were performed in triplicate and mean ± s.d. was plotted.

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These MAPPIT data were confirmed by co-immunoprecipitation(Fig. 3C). We also found that the recruitment of Mal to TLR4 wasaffected in the Malcasp mutant (Fig. 3D). In line with above-mentioned observations, we also demonstrated that the bridgingfunction of Mal was abolished when using the Malcasp mutant(Fig. 3E). The effects of the Malcasp mutation suggest thatcaspase-1 cleavage would inhibit, rather than promote proteininteractions of Mal. This hypothesis is further supported by theobservation that reconstitution of a Mal-deficient cell line with theMalcasp mutant does not lead to functional complementation (Fig.7B,C).

In an attempt to further assess the importance of caspase-1cleavage for the interactions of Mal, we tested the effects of a MalD198E mutation. The Mal D198E mutant conserves the negativecharge at position 198, but is not cleaved by caspase-1 (Fig. 4). Incontrast to the MalD198A mutant, the MalD198E mutation had

minimal effects on MAPPIT interaction with MyD88 (Fig. 5A) andbridging of MyD88 to TLR4 in MAPPIT (Fig. 5B). Overexpressionof the D198E mutant strongly induces NF-B activation, indicatingthat this mutant is fully functional (Fig. 6A).

Our data demonstrate that a conserved acidic amino acid atposition 198, rather than cleavage by caspase-1, is crucial for TIR-TIR domain interactions and NF-B signaling induced by Mal. Thisis supported by the effects of a caspase-1 inhibitor on NF-Bactivation, which is induced by Mal overexpression. We did notobserve a strong effect of a specific caspase-1 inhibitor on NF-Bactivation induced by overexpression of Mal (Fig. 6B). Moreover,TLR2 and TLR4 responses in MalD198E-transduced Mal-deficientmacrophages were comparable with that in cells transduced withwild-type Mal (Fig. 7B,C). These data indicate that Mal-inducedNF-B activation does not require a catalytically active caspase-1protein.

Fig. 7. Reconstitution of Mal-knockoutmacrophages does not support a criticalrole for caspase-1 in Mal activation.(A)Immortalized Mal-knockout macrophageswere transduced with retroviruses expressingwild-type Mal, Malcasp or MalD198E and anIRES-translated GFP. Transduced cells weresorted for GFP expression and Mal(mutant)expression was examined by western blotusing an anti-Mal antibody. TheseMal(mutant) transduced cells were tested forresponsiveness to TLR2 and TLR4 ligands.Following 6 hours of stimulation with variousconcentrations of LTA (B) and LPS (C), theTNF- levels in the supernatant weredetermined.

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D198 in Mal corresponds with negatively charged residues inthe sequence of TLR1, TLR2, TLR4 and MyD88. E796 in TLR4and D275 in MyD88 align with the caspase-1 cleavage site D198of Mal (Fig. 9A). In an alanine scan of TLR4, mutation of residues796-798 leads to loss of all downstream signaling (Ronni et al.,2003). We performed a mutagenesis study on the TIR domain ofTLR4. Using TLR4ic as a bait, we could clearly demonstratehomodimerization of TLR4ic and association of Tram and TLR4ic(Fig. 8A). These interactions were abrogated when two glutamicacid residues E796 and E798 in the E strand were mutated toalanine, indicating that these residues are crucial for TLR4interactions. Mutation of the corresponding D275 in MyD88 toalanine was reported to disrupt MyD88 homo-oligomerization,whereas a MyD88 TIR domain with the D275A mutation loses itsdominant-negative effect on IL-1 receptor signaling (Li et al., 2005).In our MAPPIT assays, the MyD88 D275A mutant lost itsinteraction with Mal and MyD88 (Fig. 8B). Together, the datasuggest that the conserved negative charge is important for TIR-TIR interaction and possibly part of a binding site in different TIRdomains.

In a homology model of TLR4, E796 is part of a stronglyconserved surface patch, which is likely to be important forprotein interactions of TLR4 (Fig. 9B). The conserved surface

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patch consists mainly of residues of the DE loop. In an alaninescan of TLR4, mutations in this surface patch lead to loss ofdownstream activation (Ronni et al., 2003). In our homologymodel for Mal (Fig. 9C,D), D198 was found next to a highlyconserved surface patch, which consists mainly of residues ofthe DE loop at a similar location as in TLR4. Mal phosphorylationby Bruton’s tyrosine kinase is required for TLR2 and TLR4 signaltransduction. The conserved surface patch contains the possibleBruton’s tyrosine kinase target Y187. Mutation of Y187 in Malto phenylalanine reduces Mal phosphorylation and LPS signaling,whereas NF-B activation by overexpression of this Mal mutantis reduced (Gray et al., 2006). WHISCY analysis of the modelconfirms that the surface patch is a good candidate for a protein-protein interaction site (Fig. 9E). The D198A and Malcaspmutations and caspase-1 cleavage might disrupt this interaction

Fig. 8. Mutations in TLR4 and MyD88 corresponding to D198A in Maldisrupt the TIR-TIR interactions. (A)Comparison of MAPPIT interactionprofile of wild-type TLR4ic-bait and TLR4icEWE-AWA-bait. (B)Homo- andheterodimerization properties of the MyD88TIRD275A-prey.

Fig. 9. Sequence alignment and residue conservation in models for TLR4and Mal. (A)sequence alignment of TLR1, TLR2, TLR4, Mal and MyD88.The position of the secondary structure elements in Mal are indicated underthe alignment. The position of D198 is indicated by an asterisk. Residues of aconserved surface patch are underlined in red. A reported TRAF6-bindingmotif of Mal (Mansell et al., 2004) is underlined in black. (B)Sequenceconservation in TLR4 orthologs mapped on the TLR4 homology model.Residues are colored according to their conservation on a linear scale from red(highly conserved) to blue (unconserved). (C)Sequence conservation in Malorthologs mapped on the Mal homology model, as in B. (D)Ribbonpresentation of the Mal model, oriented as in C. The -D, -E strands, the DEloop and residues S180 and D198 are indicated. (E)WHISCY prediction ofprotein-protein interaction sites on the surface of Mal. Residues are coloredaccording to their propensity of being in a protein-protein interaction interface.(Red, high propensity; blue, low propensity.) Extended patches of red coloredresidues indicate a possible protein-protein interaction site. Residues Y187,R192, F193, M194 and Y195 are part of such a surface patch.

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site, whereas phosphorylation of Y187 by Bruton’s tyrosinekinase might be required for interactions via this site.Alternatively, mutation of Y187 in the conserved surface patchmight directly inhibit the protein interactions of Mal, leading todecreased tyrosine phosphorylation of Mal. Interestingly, theposition of the conserved surface patch partially superposes witha strongly conserved patch in TLR4, whereas residues R192, F193and D198 are identical in MyD88. It is therefore possible thatthe conserved surface patch has an important role in proteininteractions of Mal, MyD88 and TLR4. The effect of the D198Amutation in Mal might be a consequence of disruption ofinteractions via this surface patch. Interestingly, R192 and F193are part of a proposed TRAF6-binding-site motif 188PPELRF193of Mal (Mansell et al., 2004). Overexpression of a Mal E190Amutant does not induce any of the signal transduction pathwaysnormally induced by Mal overexpression. However, the E190Amutant retains wild-type levels of TRAF6 binding, making itquestionable that the motif is an actual TRAF6 binding site. Therole of the conserved surface patch in Mal, MyD88 and TLR4 iscurrently under investigation.

Our data and homology model for Mal do not agree with themodel of Núñez and colleagues (Núñez et al., 2007). We found thatremoval of the E-helix by caspase-1 is not necessary for theinteractions of Mal with MyD88 or TLR4, whereas a mutant wherethe E-helix is removed loses this interaction. In our homology model,S180 was found at the C-terminal end of beta strand D, where it issurface exposed (Fig. 8D). D198 is found at the C-terminal end of-strand E, next to S180, but the side chains of S180 and D198protrude towards opposite sides of the central -sheet.

In summary, our data show that caspase-1 cleavage is notrequired for interaction of Mal with MyD88 or TLR4, or for Mal-induced NF-B activation. Mutations of D198 might affect a nearbybinding site. Two mechanisms have been proposed for therequirement of Mal cleavage by caspase-1. In one model, the C-terminal fragment is proposed to be inhibitory, and its release allowsinteractions of Mal. In the second model, the released C-terminalfragment itself has a signal transduction function. Our data excludea role for either mechanism in NF-B activation induced by Mal,and suggest that caspase-1 cleavage of Mal is inhibitory, ratherthan required. This would parallel the recent finding that the TLRadaptor Trif and the antiviral adaptor Cardif are cleaved andinactivated by caspases (Rebsamen et al., 2008). Our data clearlyindicate that the inhibitory effect of the Mal D198A mutation isinsufficient evidence for a requirement of caspase-1 cleavage ofMal in TLR2 and TLR4 signaling. However, we cannot excludea role for caspase-1 in TLR2 and TLR4 pathways downstream ofMal. Caspase-1 itself can mediate NF-B activation, and thisproperty does not rely on its enzymatic activity (Lamkanfi et al.,2004; Sarkar et al., 2006). Sarkar and co-workers demonstratedthat overexpression of a catalytically inactive mutant of caspase-1 in caspase-1–/– macrophages corrected the decreased NF-Bactivation upon LPS stimulation (Sarkar et al., 2006). It was shownthat caspase-1 associates with receptor interacting protein 2 (RIP2),which led to the proposition that this interaction is crucial for RIP2-IKK association and hence for NF-B activation. Moreover, RIP2-deficient cells are severely impaired in TLR2-, TLR4- or TLR3-induced cytokine production (Kobayashi et al., 2002), furtherunderscoring this hypothesis. Our data are in accordance with thesereports, because no apparent negative effect of a specific caspase-1 inhibitor on Mal induced NF-B activation could bedemonstrated.

Materials and MethodsPlasmidsGeneration of the pMG2-SVT, pMG2-Mal, pMG2-MalTIR, pMG2-MyD88, pMG2-MyD88TIR prey vectors and of the human LepR bait constructs pCLL, pCLL-MyD88,pCLL-Mal and pCLL-TLR4ic was described previously (Ulrichts et al., 2007). ThepMG2-Malcasp and pMG2-MalTIRcasp plasmids were generated by amplificationof Malcasp cDNA (corresponding to residues 1-198) or the caspase-1-cleaved formof the Mal TIR domain (residues 78-198) from the pMG2-Mal vector with primers1-2 and 3-2 (supplementary material Table S1), respectively. After EcoRI-XbaIdigestion, or BstEII-XbaI digestion, the fragment was cloned in the pMG2 vector,resulting in pMG2-MalCasp or pMG2-MalTIRcasp.

The pMET7-MyD88TIR-FLAG and pMet7-MyD88-FLAG vectors weregenerated by amplification of MyD88TIR or MyD88 and using primer pairs 4,5 and6,7, respectively, on the pMG2-MyD88 vector. The fragments were digested withBstEII-XbaI or EcoRI-XbaI and cloned in the pMet7 or pMet7-FLAG vector(Lemmens et al., 2003). The gene encoding human full-length Mal was amplifiedusing primers 8 and 9 on the pMG2-Mal plasmid. After NotI-XhoI digestion, thefragment was cloned in the pCAGGSE vector, resulting in pCAGGSE-Mal. In ananalogous manner, pCAGGSE-Malcasp was constructed, using primers 8 and 10.The MalD198A and MalD198E mutant vectors were generated by PCR-basedmutagenesis using primer pairs 11,12 and 13,14, respectively (Quikchange site-directed mutagenesis method, Stratagene). The vectors were cleaved with XhoI,followed by a Klenow fill-in reaction and EcoRI digestion. Subsequently, E-taggedMal(mutant) cDNA fragments were cloned in the pTre-tight vector (Clontech)linearized by a EcoRI-EcoRV digest.

pCLL-TLR4EWE-AWA and pMG2-MyD88TIRD275A were generated by PCR-based mutagenesis using primer pairs 15,16 and 17,18. Genes encoding the caspase-1subunits p10 and p20 were amplified with primer pairs 19,20 or 21,22 on cDNAfrom the human monocytic THP-1 cell line. Following a NotI-XhoI digestion, thefragments were ligated in the pCAGGSE vector, generating pCAGGSE-p10 orpCAGGSE-p20. The pET30HIS vector is a modified pET30a bacterial expressionvector (Novagen), in which the S-tag was removed. Mal and MalD198E cDNA wereobtained by BamHI-NotI digestion on the MAPPIT bait vectors and cloned in thepET30HIS-vector.

The pXP2d2-rPAPI-luciferase reporter, originating from the rat PAP1 (pancreatitisassociated protein I) promoter was used as previously described (Eyckerman et al.,2001). The pNFconluc reporter was a gift from Alain Israel (Institut Pasteur, Paris,France). The pMX-Mal(mutant)-IRES-GFP vectors were generated by BamHI-NotIdigestion of the MAPPIT bait vectors, followed by ligation of the inserts into thepMX-IRES-GFP vector (a gift from Stefan Constantinescu, Université Catholiquede Louvain, Brussels, Belgium).

MAPPIT conceptThroughout this study, we make use of the MAPPIT two-hybrid system, which isfounded on the basic principles of JAK-STAT signaling. In MAPPIT, a given ‘bait’protein is fused to the intracellular and transmembrane domain of the leptin receptor(LR) that is deficient in STAT3 recruitment. In this bait, the extracellular portion ofthe LR can be replaced by the extracellular domain of the erythropoietin receptor(EpoR). The ‘prey’ protein is coupled to a string of six functional STAT3 recruitmentsites of the gp130 chain. Association of bait and prey and ligand stimulation (leptinor Epo) results in STAT3 activation and induction of a STAT3-inducible luciferasereporter (Fig. 1). This MAPPIT set-up easily allows detection of both TLR adaptorsinteraction, as well as receptor-adaptor recruitment (Ulrichts et al., 2007; Ulrichtsand Tavernier, 2008).

Cell culture, transfection, luciferase reporter assays and expressioncontrolsCell culture conditions, transfection procedures and luciferase assays for HEK293Tcells were previously described (Ulrichts et al., 2007). Caspase-1 inhibitor z-WEHD-fmk was from R&D Systems, E. coli lipopolysaccharide (LPS) and S. aureuslipoteichoic acid (LTA) were from InvivoGen, and recombinant caspase-1 was fromBiovision.

For a typical luciferase experiment, 4�105 cells were seeded in six-well plates 24hours before transfecting them overnight with the desired constructs together withthe luciferase reporter gene. Cells were left untreated (negative control) or werestimulated overnight with 100 ng/ml leptin, followed by measurement of luciferaseactivity in cell lysates by chemiluminescence.

Prey expression was examined by western blot using anti-FLAG mouse monoclonalantibody (Sigma) on lysates of transfected cells. Expression of Mal(mutants) couldbe monitored using an anti-E monoclonal antibody (GE Healthcare). Functionalinteraction of all baits with a JAK2-interacting prey can be detected, ruling out someintrinsic inhibition of the MAPPIT signal (data not shown). All results arerepresentative for at least three independent transfection experiments.

Co-immunoprecipitationApproximately 2�106 HEK293T cells were transfected with different combinationsof expression vectors pMet7-FLAG-MyD88, pMet7-FLAG-MyD88TIR, pCAGGSE-Mal and pCAGGSE-Malcasp. Cleared lysates [modified RIPA lysis buffer: 200 mMNaCl, 50 mM Tris-HCl, pH 8, 0.05% SDS, 2 mM EDTA, 1% NP40, 0.5% sodium

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deoxycholic acid, Complete Protease Inhibitor Cocktail (Roche)] were incubated withprotein G-Sepharose (GE Healthcare). After immunoprecipitation, SDS-PAGE andwestern blotting, interactions were detected using anti-FLAG antibody (Sigma) andanti-E antibody (GE Healthcare).

Caspase-cleavage assaysWe transfected 4�105 HEK293T cells with different combinations of pTRE-tight-Mal or pTRE-tight-MalD198E, pCAGGSE-p10, pCAGGSE-p20 together with thepTET-ON advanced vector. 24 hours after transfection, cells were stimulated with1 g/ml doxycyclin, resulting in the expression of Mal or MalD198E. 18 hourslater, cells were incubated with 20 M of the proteasomal inhibitor MG132(Calbiochem) for 6 hours before cell lysis. Cleared lysates (modified RIPA lysisbuffer) were analysed after SDS-PAGE and western blotting, with an monoclonalanti-E antibody.

The pET30HIS-Mal and pET30HIS-MalD198E plasmids were electroporated intoE. coli BL21(DE3) cells. This allows the expression of His-tagged human Mal in thecytoplasm of E. coli BL21(DE3) cells. The cytoplasmic fraction was prepared byFrench pressing using a lysis buffer containing 20 mM Tris-HCl, pH 8, 2 mM MgCl2and EDTA-free complete protease inhibitor (Roche Applied Science). TheMal(mutant) protein was purified from the cytoplasmic fraction by affinitychromatography with a HiTrap FF column (GE Healthcare) chelated with Ni2+. Afterloading of the cytoplasmic fraction, the column was washed with IMAC buffer (20mM Tris-HCl, pH 8, 300 mM NaCl, 20 mM imidazole and 5 mM DTT). Protein waseluted from the column using a gradient of imidazole. A buffer exchange wasperformed on the eluate using a HiTrap Desalting Column (GE Healthcare), resultingin a protein eluate dissolved in 20 mM HEPES, pH 7.2, 50 mM NaCl, 10 mM EDTAand 5 mM DTT. Approximately 3 g protein was incubated for 2 hours at 37°C withor without 1 unit of active recombinant human caspase-1 (Biovision) according tothe manufacturer’s instructions. Samples were analysed after SDS-PAGE and westernblotting, with a monoclonal anti-HIS antibody (Clontech) and a polyclonal anti-Malantibody (FL-235; Santa Cruz Biotechnology).

Transduction of immortalized Mal knockout macrophagesThe immortalized Mal-knockout macrophage cell line (a gift from Jonathan Kagan,Harvard Medical School, Boston, MA) was transduced with retrovirus, produced usingthe pMX-Mal(mutant)-IRES-GFP vectors. GFP-positive cells were sorted using aDakoCytomation Mo-Flo fluorescence-activated cell sorter. Expression of Mal or itsmutants was assayed using an anti-Mal antibody (FL-235; Santa Cruz Biotechnology).

TNF- ELISAReconstituted macrophages were seeded at a density of 4�104 cells per well andstimulated for 6 hours with various TLR ligands. Supernatants were collected andassayed for TNF- using the DuoSET ELISA kit from R&D Systems, according tothe manufacturer’s instructions.

Sequence analysis and homology modeling of Mal and TLR4 The crystal structures of the TIR domains of TLR1, TLR2, TLR10 and IL-1RAPLand an NMR structure of MyD88 were structurally superposed via the molecularoperating environment (moe) program (chemical computing group). In moe, thisstructural superposition was aligned with the TIR domains of orthologs of Mal, MyD88and TLR4 collected from the nr and Ensembl databases and with the TIR domainsequences of all human TLRs. This alignment was used to build homology modelsfor the TIR domain of human Mal and TLR4 in moe. The structure of the TIR domainof TLR1 (pdb code 1FYV) (Xu et al., 2000) was used as template for the TLR4model. The model of Mal is based on the NMR structure of MyD88 (Protein DataBank code 2Z5V) (Ohnishi et al., 2009).

Using the alignment of the orthologs and the render by conservation function ofthe Multialign Viewer of the UCSF Chimera package (Pettersen et al., 2004), wevisualized the amino acid conservation of Mal or TLR4 orthologs onto thecorresponding model structures. The conservation is colored from red (highlyconserved) to blue (low conservation) on a linear scale. The program WHISCY (deVries et al., 2006) was used to predict possible protein-protein interaction sites onthe surface of our Mal homology model. The alignment of the TIR domains of theMal orthologs and the Mal TIR model structure were submitted to the WHISCYserver (http://nmr.chem.uu.nl/Software/whiscy/index.html). The server returns a pdbfile where the B-factor is replaced by the WHISCY prediction scores. The WHISCYprediction scores were visualized in UCSF Chimera by coloring residues accordingto their B-factor using the ‘render by attribute’ function. The residues are colored ona linear scale according to their WHISCY sore (red, high WHISCY score; blue, lowWHISCY score).

We thank Jonathan Kagan (Harvard Medical School, Boston) for thegenerous gift of the immortalized Mal-knockout macrophage cell line.This work was supported by grants of the Fund of Scientific ResearchFlanders (G.0031.06), Ghent University (GOA 12051401 and BOF01Z03306) and IUAP-6 (P6:28).

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/123/2/256/DC1

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