Differential Involvement of BB Loops of Toll-IL-1 Resistance (TIR) Domain-Containing Adapter in TLR4- versus TLR2-Mediated Signal This information is current as Transduction of September 28, 2021. Vladimir U. Toshchakov, Subhendu Basu, Matthew J. Fenton and Stefanie N. Vogel J Immunol 2005; 175:494-500; ; doi: 10.4049/jimmunol.175.1.494 Downloaded from http://www.jimmunol.org/content/175/1/494

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Involvement of BB Loops of Toll-IL-1 Resistance (TIR) Domain-Containing Adapter Proteins in TLR4- versus TLR2-Mediated Signal Transduction1

Vladimir U. Toshchakov,* Subhendu Basu,† Matthew J. Fenton,2*† and Stefanie N. Vogel2,3*†

TLRs sense pathogens and transmit intracellular signals via the use of specific adapter proteins. We designed a set of “blocking peptides” (BPs) comprised of the 14 aa that correspond to the sequences of the BB loops of the four known Toll-IL-1 resistance (TIR) domain-containing adapter proteins (i.e., MyD88, TIR domain-containing adapter inducing IFN-␤ (), TRIF-related adapter molecule (TRAM), and TIR-domain containing adapter (TIRAP)) linked to the cell-penetrating segment of the antennapedia homeodomain. LPS (TLR4)-mediated expression, as well as MAPK and transcription factor activation asso- ciated with both MyD88-dependent and -independent signaling pathways, were disrupted by all four BPs (TRAM Ϸ MyD88 > Downloaded from TRIF > TIRAP), but not by a control peptide. In contrast, none of the BPs inhibited TLR2-mediated activation of MAPKs. Only the MyD88 BP significantly blocked Pam3Cys-induced IL-1␤ mRNA; however, the inhibitory effect was much less than observed for LPS. Our data suggest that the interactions required for a fully functional TLR4 signaling “platform” are disrupted by these BPs, and that the adapter BB loops may serve distinct roles in TLR4 and TLR2 signalosome assembly. The Journal of Immu- nology, 2005, 175: 494–500. http://www.jimmunol.org/ oll-like receptors are a family of signaling molecules that domain-containing adapter molecule-1 (7); and TRIF-related act as sensors for recognition of diverse pathogen-asso- adapter molecule (TRAM) (8), also named as TIR-containing pro- T ciated molecular patterns. All TLRs share a similar do- tein (9) or TIR domain-containing adapter molecule-2 (10). main architecture: an extracellular region that contains multiple MyD88 and TRIF have a more complex domain architecture leucine-rich repeat regions (1) that function in recognition of than TIRAP and TRAM. In addition to the TIR domain, MyD88 pathogen-associated molecular patterns and coreceptors (2); a has a mid-region and an N-terminal death domain that enable in- transmembrane domain comprised of one membrane-spanning teraction with IL-1R-associated kinase 4 (IRAK-4) and IRAK-1, ␣-helix; and an intracellular “Toll-IL-1 resistance” (TIR)4 domain respectively, serine/threonine kinases that signal downstream of located within the C terminus (1). IL-1R (11) and TLRs (3), leading to NF-␬B activation (reviewed by guest on September 28, 2021 In addition to TLRs, a group of TIR domain-containing proteins in Ref. 12). TRIF is the largest member of adapter family, with the that lack a transmembrane domain has been identified. MyD88 was TIR domain located in the middle of its sequence. The N terminus the first TIR domain-containing protein that was shown to serve as of TRIF is necessary for activation of NF-␬B and IFN regulatory an adapter for IL-1R, and later, for TLR signaling (3). With rapid factor (IRF)-3 (6), a transcription factor responsible for induction progress in sequencing and gene annotation, this family grew of IFN-␤ and other . I␬B kinase (IKK)-⑀ and TRAF family quickly and now includes five members, four of which are in- member-associated NF-␬B binding kinase (TANK)-binding ki- volved in TLR4 signaling: MyD88; TIR-domain containing nase-1 (TBK-1), act downstream of TRIF to phosphorylate IRF-3 adapter protein (TIRAP) (4), also known as Mal (5); TIR domain- (13). Recruitment of IKK-⑀ and TBK-1 to TRIF does not require containing adapter inducing IFN-␤ (TRIF) (6), also referred as TIR TIR domain of the protein (8), and overexpression of a TRIF mu- tant that lacks the C terminus still activates NF-␬B and IFN-␤ reporter constructs (6, 7). *Department of Microbiology and Immunology and †Department of Medicine, Uni- versity of Maryland, Baltimore, MD 21201 TIRAP and TRAM are the smallest adapters involved in TLR Received for publication January 10, 2005. Accepted for publication April 16, 2005. signaling. The N terminus of TRAM has a myristoylation site, the The costs of publication of this article were defrayed in part by the payment of page mutation of which alters its normal membrane localization (14). charges. This article must therefore be hereby marked advertisement in accordance Both TIRAP and TRAM have been found to associate constitu- with 18 U.S.C. Section 1734 solely to indicate this fact. tively with TLR4 (5, 8). Current models of TLR signaling ascribe 1 This work was supported by National Institutes of Health Grants AI-18797 and different roles to the adapters (10, 14). TIRAP and TRAM have AI-44936 (to S.N.V.) and AI-57490 (to M.J.F.). been suggested to serve as “platform-forming” components re- 2 M.J.F. and S.N.V. contributed equally to this work. sponsible for recruitment of the larger adapters, MyD88 and TRIF, 3 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Depart- respectively, that in turn, recruit downstream effector molecules ment of Microbiology and Immunology, University of Maryland, Baltimore, 655 West Baltimore Street, 13-009, Baltimore, MD 21201. E-mail address: (e.g., IRAK-1 and IRAK-4 to MyD88 and TBK-1, TRAF6, and [email protected] IKK-⑀ to TRIF via non-TIR domains). Distinct combinations of 4 Abbreviations used in this paper: TIR, Toll-IL-1 resistance; BP, blocking peptide; adapters involved in a signaling platform differ among TLRs and CP, control peptide; TIRAP, TIR-domain containing adapter protein; TRIF, TIR do- have been postulated as the basis for the observed specificity of main-containing adapter inducing IFN-␤; TRAM, TRIF-related adapter molecule; TBK-1, TRAF family member-associated NF-␬B binding kinase (TANK)-binding gene subsets induced by different TLR (see Refs. 14 and ⑀ ␬ ⑀ kinase-1; IKK- ,I B kinase ; IRAK-1/4, IL-1R-associated kinase 1/4; Pam3Cys, 15 for review). (S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys4-OH); HPRT, hy- poxanthine phosphoribosyltransferase; IRF, IFN regulatory facto; GI, GenBank ID The structure of TLR TIR domains was first predicted by Rock number. et al. (1). Analysis of sequences of five human TLRs and their

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 495 diverse homologues led the authors to conclude that TIR domains ited TLR2-mediated induction of IL-1␤ mRNA; however, its in- are composed of five ␤-strands alternating with five ␣-helices. hibitory effect was considerably weaker than its effect on TLR4- Resolution of crystal structures of human TLR1 and TLR2 con- mediated IL-1␤ . Collectively, our findings firmed these theoretical findings and suggested that a functionally suggest that these BPs interfere with protein-protein interactions important proline-glycine combination that is highly conserved involved in the assembly and/or stabilization of the TLR4 “plat- among TIR domains is located in the loop connecting the second form” and support the hypothesis that surfaces on MyD88 and ␤-strand with the second helix, the “BB loop,” using the termi- TIRAP apart from the BB loop enable their interaction with TLR2. nology proposed by Xu et al. (16). The importance of BB loops in TLR signaling derives from Materials and Methods observations that the function of TIR domain-containing proteins Animals is highly susceptible to mutations in this region. A naturally oc- curring point mutation (P712H) in murine TLR4, which leads to C3H/OuJ mice were purchased from The Jackson Laboratory. Thioglyco- late-elicited peritoneal macrophages were harvested and cultured in the complete unresponsiveness of the C3H/HeJ mouse to LPS (17, presence of K235 LPS or (S-[2,3-bis(palmitoyloxy)-(2- 681 18), lies in this region. Similarly, replacement of Pro with His RS)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys4-OH) (Pam3Cys) as described in human TLR2 was reported to disrupt in- previously (29). duced by Gram-positive bacteria and essentially abolishes MyD88 Design of peptides recruitment (16); however, this finding was not confirmed by Dunne et al. (19). Mutations of residues in the vicinity of the The peptides were composed of 16-aa carrier sequence from conserved BB loop proline also led to the decreased activity of homeodomain protein (aa 366–381, GenBank ID number (GI): 4389425), Downloaded from the C terminus of which was synthesized in tandem with the N terminus of TLR2 (16) and TLR4 (20). Many TIR-domain containing proteins the 14 aa that surround the conserved P(C)G sequence in BB loops of that bear mutations homologous to the mutation in C3H/HeJ mice murine TIR domain-containing adapter proteins: TRAM (GI: 27734184), exert dominant-negative effects on TLR signaling (e.g., TLR4 MyD88 (GI: 6754772), TRIF (GI: 33859797), and TIRAP (GI: 16905131) (21), TLR2 (22), TIRAP (4), and TRAM (8)). Overexpression of (sequences provided in Table I). A CP was a scrambled amino acid se- the P200H mutation of MyD88, however, fails to prevent induc- quence shown by Basic Local Alignment Search Tool (BLAST) to not be homologous to known proteins. Peptides were synthesized and purified by

␬ http://www.jimmunol.org/ tion of NF- B (4) or its binding to human TLR4 (19). Similarly, HPLC by Biosynthesis. Purity was confirmed by mass spectrometry. Ten- in a yeast two-hybrid system, interaction of TLR4 with TRAM millimolar stocks were dissolved in 25% DMSO and kept frozen at Ϫ80°C. is not disrupted by mutation of the TRAM cysteine 117 in its BB Forty micromolar was the highest concentration used in these experiments loop (19). due to limited solubility of BPs in the cell culture medium. Cell-penetrating cationic peptides are potent and efficient tools Isolation of mRNA and RT-PCR for delivery into the intracellular space of diverse substances that normally would not penetrate plasma membranes (23). Cargoes Total cellular RNA was extracted from macrophage cultures as described that can be carried into cells vary widely in size and chemical elsewhere and reversed transcribed using AMV Reverse Transcriptase (Promega) and poly-T priming as recommended by the manufacturer. The nature and include oligonucleotides and proteins. After the initial resulting cDNA was quantified by real-time PCR using SYBR Green PCR observation that aa 47–57 of HIV-1 TAT protein are crucial for the Master Mix (Applied Biosystems) and ABI Prism 7900HT cycler. Primers by guest on September 28, 2021 ability of this protein to enter cells (24, 25), functionally similar for detection of IL-1␤, IFN-␤, MIP-1␤, RANTES, and hypoxanthine phos- sequences were identified in the homeodomain of the Drosophila phoribosyltransferase (HPRT) mRNAs were designed using the Primer Ex- press 2.0 program (Applied Biosystems). Relative gene expression was transcription factor, antennapedia (26), and the herpes-simplex- calculated using ⌬Ct method with HPRT as a housekeeping gene Ϫ virus-1 DNA-binding protein, VP22 (27). The mechanism by (1.8ˆ(CtHPRT CtGene)). Statistical analysis was performed by one-way which cell-penetrating cationic peptides enter cells is still contro- ANOVA with repeated measures, followed by Dunnett’s multiple compar- versial (28). ison post hoc test with p Յ 0.05 chosen as the level of significance. For this study, we designed a set of “blocking peptides” (BPs) SDS-PAGE and Western analysis comprised of the translocating sequence of the antennapedia ho- meodomain, in tandem with 14-aa sequences corresponding to the SDS-PAGE and Western blot analysis were performed as described pre- BB loops of MyD88, TRAM, TIRAP, and TRIF. This approach viously (30) with minor modifications. Abs for detection of activated MAPKs (pERK, pJNK, and pP38) were purchased from Cell Signaling was first used by Horng et al. (4) in their original studies on the Technology. IRF-1 was detected using a rabbit polyclonal Ab from Santa identification of TIRAP in TLR4 signaling. We tested these four Cruz Biotechnology. Ab for detection of tyrosine 701 phosphorylated constructs and a control peptide (CP) in primary murine macro- STAT1 was purchased from Zymed Laboratories. phages for the ability to interfere with LPS-induced signal trans- Native PAGE for detection of IRF-3 activation was performed with minor modifications (31). A total of 1 ϫ 107 thioglycolate-elicited cells duction. All four BPs blocked TLR4-mediated gene expression, were plated into a 6-cm dish and washed with PBS to remove nonadherent MAPK, and transacting factor activation, but failed to block cells. Primary macrophages were stimulated with 100 ng/ml LPS for 1.5 h TLR2-mediated activation of MAPKs. Only the MyD88 BP inhib- and lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1

Table I. The physical-chemical properties of BPsa

Number of Positively Number of Negatively Peptide Sequence Length Charged Residues Charged Residues AIb Hydropathicity

Control peptide RQIKIWFQNRRMKWKKSLHGRGDPMEAFII 30 8 2 68.33 Ϫ0.877 MyD88-BP RQIKIWFQNRRMKWKKVSDRDVLPGTCVWS 30 8 2 68.00 Ϫ0.850 TRAM-BP RQIKIWFQNRRMKWKKIVFAEMPCGRLHLQ 30 8 1 78.00 Ϫ0.637 TIRAP-BP RQIKIWFQNRRMKWKKLQLRDAAPGGAIVS 30 8 1 71.67 Ϫ0.723 TRIF-BP RQIKIWFQNRRMKWKKFCEEFQVPGRGELH 30 8 3 48.67 Ϫ1.190

a The physical-chemical properties of peptides were calculated using ExPASY-ProtParam Tool (Swiss Institute of Bioinformatics, Geneva, Switzerland). b AI, Peptide aliphatic index; relative volume occupied by aliphatic chains of A, V, I, L. 496 ADAPTER BB LOOP PEPTIDES BLOCK TLR4, NOT TLR2 mM EDTA, 1% Nonidet P-40, 1 mM sodium orthovanadate, and 1ϫ Com- effect of targeted mutations in TRIF or TRAM affects both arms of plete protease inhibitor mixture (Roche). Lysates were mixed with native the TLR4 signaling pathway: macrophages derived from TRIF and lysis buffer (Bio-Rad) supplemented with 1% deoxycholate. Fifteen to 20 TRAM knockouts not only exhibited a complete loss of MyD88- ␮g of protein per lane was electrophoresed on a nonreducing gel and im- munoblotted. IRF-3 dimers and monomers were detected using rabbit anti- independent signaling, but also exhibited a severe loss of signaling IRF-3 Ab (Zymed Laboratories) by Western analysis. associated with the MyD88-dependent pathway (e.g., TNF-␣, IL-6, and IL-12 p40 secretion is essentially eliminated, although there is some retention of early NF-␬B translocation) (35, 36). Results Therefore, the first question we sought to address experimentally is BB loop peptides block LPS-induced gene expression in primary whether interactions of the BB loops of the TIR domains of these macrophages, but do not distinguish between MyD88-dependent adapters affect protein interactions leading to gene expression. To and -independent pathways this end, we designed cell permeable peptides whose sequences Based on the observation that macrophages from MyD88 knockout correspond to the BB loops of each adapter molecule and mea- mice do not synthesize TNF-␣ or IL-6 in response to LPS, while sured the ability of each BP to interfere with the induction of they are still capable of inducing IFN-␤ and IFN-␤-inducible immediate response genes induced via MyD88-dependent and -in- genes (32, 33), two different signaling pathways propagating from dependent pathways. Fig. 1A shows the effect of the BP treatment TLR4 were defined (reviewed in Ref. 15). The “MyD88-indepen- of macrophage cultures on the induction of genes that encode two dent” pathway of TLR4 signaling uses TRAM and TRIF to acti- MyD88-dependent , IL-1␤ and MIP-1␤ (37, 38), and two vate IRF-3 that, in turn, leads to induction of IFN-␤ and IFN-␤- MyD88-independent cytokines, IFN-␤ and RANTES (32, 33, 37), inducible genes, while the “MyD88-dependent” pathway leads to 1 h after LPS stimulation of primary macrophage cultures. All BB Downloaded from induction of genes that encode IL-1␤, TNF-␣, and IL-6 that do not loop-containing peptides exerted an inhibitory effect on LPS-in- depend on IRF-3 for their expression. Mice with targeted muta- duced expression of MyD88-dependent IL-1␤ and MIP-1␤, as well tions in MyD88 (32, 33) or TIRAP (34) retain the capacity to as MyD88-independent IFN-␤ and RANTES mRNA, as measured signal through the MyD88-independent pathway. In contrast, the by real-time PCR (Fig. 1A). In general, the inhibitory effect of the http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Effect of BPs on the induction by LPS. Thioglycolate-elicited primary murine macrophages were preincubated in the presence of 40 ␮M BP for 1 h before stimulation with 10 ng/ml LPS. A, Quantitative real-time PCR analysis of gene expression. B, Secretion of IL-1␤ and IL-6. Concentration of the cytokines was measured after overnight incubation of the cells in the presence of LPS and the peptides. C, Sensitivity of the LPS-stimulated expression of IFN-␤ and IL-1␤ mRNA to varying concentrations of TRAM and MyD88 BP. Total mRNA was isolated 1 h after LPS .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .(stimulation (A and C The Journal of Immunology 497

TRAM BP was strongest, with MyD88 BP being comparable or tion and nuclear translocation of STAT-1, phosphorylation of only slightly less inhibitory; the TIRAP BP was consistently weak- Ser727 modulates its transcriptional activity (reviewed in Ref. 39). est among the four tested. Fig. 1B demonstrates the effect of BB We previously reported that tyrosine phosphorylation of STAT1 loop peptides on IL-1␤ and IL-6 protein expression. The BPs, but by LPS depends on MyD88-independent induction of IFN-␤ and not the CP, also blocked secretion of IL-1␤ and IL-6. The con- the subsequent autocrine activation of type I IFN receptors (33). centration of BP used in Fig. 1 and all other experiments was 40 LPS-induced Tyr701 phosphorylation of STAT-1 was strongly ␮M based on optimal inhibitory activity of the two most active BPs, inhibited by the TRAM and TRIF BPs (Fig. 2B). Consistent with TRAM BP and MyD88 BP, against an MyD88-dependent gene (e.g., its strong ability to block IFN-␤ gene expression (Fig. 1), the IL-1␤) and an MyD88-independent gene (e.g., IFN-␤) (Fig. 1C). Fig. MyD88 BP was also highly effective at inhibiting Tyr701 phos- 1C also shows that BP-mediated inhibition is seen over a very narrow phorylation of STAT-1 (Fig. 2B). However, treatment of macro- concentration range (i.e., between 10 and 40 ␮M). phages with the TIRAP BP did not completely block tyrosine phosphorylation of STAT-1 in response to LPS, although it was BPs inhibit activation of MAPKs by LPS significantly diminished compared with the level seen in the pres- Because LPS-induced gene expression and cytokine secretion are ence of the CP (Fig. 2B). LPS-induced Ser727 phosphorylation of consequences of signaling pathways induced by both the MyD88- STAT-1 was similarly blocked by the BPs (data not shown). The dependent and -independent signaling pathways, the data pre- BPs failed to inhibit IFN-␤-induced Tyr701 phosphorylation of sented in Fig. 1 support the hypothesis that the BPs sequester STAT-1 (data not shown), consistent with the observation that the and/or block target proteins of the adapter BB loops that are es- type I IFN does not depend on any of these adapters to sential for both arms of the TLR signaling pathway. Therefore, we signal (reviewed in Ref. 39). Downloaded from next sought to examine upstream signaling pathways for their sen- sitivity to BPs. Effect of BB loop BPs on activation of IRF-1 and IRF-3 MAPKs regulate various cellular functions including signal IRFs were first identified as important transcriptional mediators of transduction from TLRs and their activation can be detected sig- the antiviral immune response (reviewed in Ref. 40). Later, it was nificantly earlier than gene expression or cytokine secretion. Using recognized that this family of transcription factors plays an essen-

Abs specific for the phosphorylated (activated) forms of JNK, tial role in signal transduction mediated by different TLRs. IRF-1 http://www.jimmunol.org/ ERK, and p38, we next sought to examine the effect of BB loop- is regulated transcriptionally by signaling from different TLRs, as containing inhibitory peptides on LPS-stimulated activation of well as by cytokines induced by TLR engagement (e.g., IL-1, TNF, MAPKs. LPS induces quick and transient activation of MAPKs in and IFN-␤) (41). In contrast, IRF-3 is constitutively expressed in primary macrophages (Fig. 2A) that were not affected by the ad- many cell types (42). Upon stimulation through TLR4 or TLR3, dition of CP. However, preincubation of cells for 1 h with TRAM IRF-3 is phosphorylated at multiple sites in its C terminus, and this or MyD88 BPs strongly blocked activation of ERK and JNK in modification enables it to dimerize and translocate to the nucleus response to LPS (Fig. 2A). Similar results were observed for ac- where it targets IRF-3-responsive genes (32, 43). Using MyD88 tivated p38 (data not shown). The TRIF BP also strongly inhibited knockout mice, Kawai et al. (32) demonstrated that LPS activates phosphorylation of MAPKs, although inhibition of ERK activation IRF-3 via the MyD88-independent pathway, and this, in turn, leads by guest on September 28, 2021 was incomplete. The TIRAP BP was the weakest BP; all MAPKs to the induction of IFN-␤, the signature cytokine gene for the were activated in the presence of it, although to a lesser extent MyD88-independent pathway (33). Pretreatment of cells with when compared with CP. TRAM, TRIF, or MyD88 BPs significantly inhibited LPS-induced dimerization of IRF-3 (Fig. 3). The TIRAP BP also decreased the Effect of BB loop BPs on activation of STAT-1 by LPS amount of detectable IRF-3 dimer; however, inhibition was not as STAT-1 propagates signal transduction emanating from both type complete as with the other BPs. I and type II IFN receptors (reviewed in Ref. 39). Two phosphate acceptor sites are involved in activation of STAT-1. Although phosphorylation of Tyr701 is critical for homo- or heterodimeriza-

FIGURE 3. Effect of BPs on activation of IRF-3 and IRF-1. The upper FIGURE 2. Effect of BPs on the LPS-stimulated activation of MAPKs panel shows native PAGE blot, the two below represent the same lysates and Tyr701 STAT-1. Ten micrograms of total protein was loaded per lane. run on SDS-PAGE. Primary mouse macrophages were stimulated with 100 A representative blot of three separate experiments is shown. Other details ng/ml LPS. Seventeen micrograms of total protein was loaded per lane of are given in Materials and Methods and the legend for Fig. 1. the native gel. Other details are as described in the legend for Fig. 1. 498 ADAPTER BB LOOP PEPTIDES BLOCK TLR4, NOT TLR2

IRF-1 is an immediate response gene, quickly induced by sig- strongly blocked by all BPs, none inhibited Pam3Cys-induced naling from different TLRs, which contributes to the induction of phosphorylation of MAPKs. other important TLR-inducible genes (e.g., inducible NO syn- The effect of BPs on Pam3Cys-induced expression of IL-1␤ thase). Importantly, MyD88 knockout macrophages are capable of mRNA is shown in Fig. 4B. LPS and Pam3Cys induced compa- inducing IRF-1 mRNA (35). Stimulation of macrophages by LPS rable levels of IL-1␤ mRNA, which were not significantly affected for 1.5 h after pretreatment of cells with medium only or CP re- by treatment of cells with CP (data not shown). Similar to their sulted in a strong up-regulation of IRF-1 protein (Fig. 3). As was effect on activation of MAPKs, BPs were much weaker inhibitors observed for IRF-3, TRAM, and MyD88 peptides were the stron- with respect to Pam3Cys- vs LPS-induced expression of IL-1␤ gest inhibitors with respect to both IRF-3 and IRF-1. The TRIF BP mRNA. Only the MyD88 BP exerted a statistically significant in- again showed intermediate efficacy, while the TIRAP peptide was hibitory effect (Fig. 4B) and this effect was considerably less than the weakest of the BPs. that seen in the case of LPS stimulation; in LPS-stimulated cells, MyD88 BP reduced IL-1␤ mRNA expression to Ͻ5% of the level Limited capacity of adapter BB loop inhibitory peptides to block detected in the presence of the CP, while in Pam3Cys-stimulated TLR2-induced signaling cells, IL-1␤ gene expression was reduced to ϳ25% of control lev- Apart from the use of a CP, we sought to analyze further the els. Taken collectively, these data demonstrate that TLR4-induced specificity of the inhibitory effects of BB loop peptides on TLR signaling is much more susceptible to inhibition by the adapter BB signaling. Therefore, we also tested the effect of the peptides on loop BPs than is TLR2-mediated signaling. signal transduction elicited from TLR2. TLR2 was chosen because it shares MyD88-dependent signaling pathway with TLR4, and Discussion Downloaded from TLR2-mediated responses are depreciated in both MyD88 and TI- This study represents an attempt to use penetrating oligopeptides RAP-deficient cells (34, 44); however, TLR2-mediated signaling containing homologous sequences that presumably interfere with does not appear to use MyD88-independent arm for signal trans- the protein-protein interaction surfaces formed between the BB duction (reviewed in Ref. 15). Stimulation of MyD88 knockout loops of four adapter proteins with their target protein(s) to inves- macrophages with the Pam3Cys, a TLR2 , does not activate tigate mechanisms of signal transduction emanating from TLR4

MAPKs (Ref. 44 and our unpublished observation), whereas LPS- and TLR2. The impetus for generating these four adapter BPs http://www.jimmunol.org/ stimulated MyD88 knockout macrophages exhibit MAPKs and stemmed from contradictory results obtained using the TIRAP BP NF-␬B activation, albeit with delayed kinetics (38). Fig. 4A dem- and a TIRAP dominant-negative mutant construct vs macrophages onstrates that the BPs differentially affect phosphorylation of ERK from TIRAP knockout mice. The TIRAP BP was first used by and JNK in response to LPS vs Pam3Cys. Surprisingly, under Horng et al. (4) as a complementary tool to confirm the involve- conditions where LPS-induced MAPK phosphorylation was ment of this adapter in LPS-signaling and was reported to inhibit TLR4-, but not TLR9-mediated signaling (4, 33). However, using this same TIRAP BP, we found that it also inhibited LPS-induced IFN-␤ gene expression and IFN-␤-luciferase reporter activity (33), while later studies using macrophages from TIRAP knockout mice by guest on September 28, 2021 found that induction of IRF-3 and IRF-3/STAT1-dependent genes by LPS was intact (34). Therefore, we sought to carry out a sys- tematic comparison of the effects of BPs based on the homologous sequences of the other known TLR4 adapters to gain insights into the role of adapter BB loops for protein interactions required for TLR4 signaling. Although the mechanism by which cell-penetrat- ing, BB loop peptides inhibit TLR signaling has not been formally demonstrated, it has been postulated that such peptides, once in- side the cell, can occupy the docking site of the cognate adapter on its target and prevent binding of the native adapter. This, in turn, would be predicted to disrupt the formation of a functional signal- ing platform (14, 45). Analysis of the effects of BPs on the expression of early induc- ible genes revealed an interesting and completely unexpected pat- tern; the BPs showed little specificity with respect to genes in- duced by LPS via MyD88-dependent or -independent pathways. For example, the MyD88 peptide did not preferentially block IL-1␤ and MIP-1␤ genes, which, according to data derived from knockout mice, require MyD88 for induction (37, 38). Conversely, TRAM BP was equally effective in inhibiting both MyD88-depen- dent and -independent genes, in contrast to the conclusion drawn from experiments with TRAM knockout mice (35). The fact that all four BPs block TLR4 signaling through either the MyD88- dependent or -independent pathways suggests strongly that the sta- FIGURE 4. Comparative effect of BPs on TLR2- vs TLR4-mediated bility of the TLR4 signaling platform is indeed disrupted or fails to activation of MAPKs (A) and IL-1␤ mRNA expression (B). For Western blot analysis, 4.5 ϫ 106 primary macrophages were preincubated without assemble correctly as a consequence of BP-target protein or with 40 ␮M BP and stimulated for 30 min with LPS (10 ng/ml) or interactions. Pam3Cys (P3C; 150 ng/ml). The results shown are a representative blot of In this regard, the disruption of TLR4 signaling by BPs is more three separate experiments. Experimental details for B are the same as reminiscent of a “receptor-knockout” phenotype, where both reported in the legend for Fig. 1A. branches of the TLR4 signaling pathway are inhibited, rather than The Journal of Immunology 499 an “adapter-knockout” phenotype characterized by expression of IRF-3/7 and NF-␬B involves the Toll adapters TRAM and TRIF. J. Exp. Med. distinct subsets of genes. The observation that BPs are capable of 198: 1043–1055. 9. Bin, L.-X., L. G. Xu, and H. B. Shu. 2003. 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