Peptide-mediated Interference of TIR Domain Dimerization in MyD88 Inhibits Interleukin-1-dependent Activation of NF-κB

Myeloid differentiation factor 88 (MyD88) plays a crucial role in the signaling pathways triggered by interleukin (IL) -1 and Toll-like receptors in several steps of innate host defense. A crucial event in this signaling pathway is represented by dimerization of MyD88, which allows the recruitment of downstream kinases like IRAK-1 and IRAK-4. Herein, we have investigated the function of the Toll/IL-1 receptor (TIR) domain in MyD88 homodimerization in cell-free and in vitro experimental settings by using epta-peptides that mimic the BB-loop region of the conserved TIR domain of different proteins. By using a pull-down assay with purified glutathione S-transferase-MyD88 TIR or co-immunoprecipitation experiments, we found that epta-peptides derived from the TIR domain of MyD88 and IL-18R are the most effective in inhibiting homodimerization with either the isolated TIR or full-length MyD88. Moreover, we demonstrated that a cell permeable analog of MyD88 epta-peptide inhibits homodimerization of MyD88 TIR domains in an in vitro cell system and significantly reduces IL-1 signaling, as assayed by activation of the downstream transcription factor NF-κB. Our results indicate that the BB-loop in TIR domain of MyD88 is a good target for specific inhibition of MyD88-mediated signaling in vivo. Myeloid differentiation factor 88 (MyD88) plays a crucial role in the signaling pathways triggered by interleukin (IL) -1 and Toll-like receptors in several steps of innate host defense. A crucial event in this signaling pathway is represented by dimerization of MyD88, which allows the recruitment of downstream kinases like IRAK-1 and IRAK-4. Herein, we have investigated the function of the Toll/IL-1 receptor (TIR) domain in MyD88 homodimerization in cell-free and in vitro experimental settings by using epta-peptides that mimic the BB-loop region of the conserved TIR domain of different proteins. By using a pull-down assay with purified glutathione S-transferase-MyD88 TIR or co-immunoprecipitation experiments, we found that epta-peptides derived from the TIR domain of MyD88 and IL-18R are the most effective in inhibiting homodimerization with either the isolated TIR or full-length MyD88. Moreover, we demonstrated that a cell permeable analog of MyD88 epta-peptide inhibits homodimerization of MyD88 TIR domains in an in vitro cell system and significantly reduces IL-1 signaling, as assayed by activation of the downstream transcription factor NF-κB. Our results indicate that the BB-loop in TIR domain of MyD88 is a good target for specific inhibition of MyD88-mediated signaling in vivo. Myeloid differentiation factor 88 (MyD88) 1The abbreviations used are: MyD88, myeloid differentiation factor 88; BSA, bovine serum albumin; DTT, dithio-dl-threitol; GST, glutathione S-transferase; PBS, phosphate-buffered saline; TLR, Toll-like receptor; IL, interleukin; DD, death domain; TIR, Toll/IL-1 receptor; TNF, tumor necrosis factor; MAPK, mitogen-activated protein kinase; LPS, lipopolysaccharide; aa, amino acids; RP, reverse-phase; HPLC, high pressure liquid chromatography; HEK, human embryonic kidney; PLB, passive lysis buffer; RAcP, receptor accessory protein. is a crucial adaptor protein that functions to recruit signaling proteins to receptors of the Toll-like or interleukin-1 receptor (TLR/IL-1R) superfamily (1Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6751) Google Scholar, 2O'Neill L. A. J. Biochem. Soc. Trans. 2003; 31: 643-647Crossref PubMed Scopus (105) Google Scholar). Activation of signaling pathways downstream of this class of receptors is fundamental for several aspects of host defense. The MyD88 protein has a modular structure composed of a death domain (DD) at the N terminus and a Toll/IL-1 receptor (TIR) domain at the C terminus separated by a short linker region, referred to as intermediary domain (3Hardiman G. Rock F. L. Balasubramanian S. Kastelein R. A. Bazan J. F. Oncogene. 1996; 13: 2467-2475PubMed Google Scholar). Upon ligand stimulation, MyD88 is recruited to the membrane by interaction of its TIR domain with the analogous domain in the IL-1R or TLR receptors (4Akira S. J. Biol. Chem. 2003; 278: 38105-38108Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). It has been shown that MyD88 forms homodimers (5Burns K. Martinon F. Esslinger C. Pahl H. Schneider P. Bodmer J. L. Di Marco F. French L. Tschopp J. J. Biol. Chem. 1998; 273: 12203-12209Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar) and promotes the recruitment to the plasma membrane and the activation of two IL-1 receptor-associated kinases: IRAK-4 and IRAK-1. A homophilic interaction between MyD88 DD and the homologous DD found at the N terminus of the kinases is required for such event (6Janssens S. Beyaert R. Mol. Cell. 2003; 11: 293-302Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). A recent model proposes that MyD88 binds to IRAK-4 and promotes phoshorylation of critical IRAK-1 residues by IRAK-4 (7Janssens S. Beyaert R. Trends Biochem. Sci. 2002; 27: 474-482Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). These events stimulate IRAK-1 autophosphorylation and its interaction with TRAF6 (tumor necrosis factor (TNF) receptor-associated factor 6), leading to activation of both the inhibitory κB kinase (IKK) and the mitogen-activated protein kinases (MAPK) JNK and p38. These kinases are pivotal in the ultimate activation of several transcription factors, including NF-κB and activator protein 1 (AP-1), which elicit the production of essential effector molecules for immune and inflammatory responses (8Baud V. Liu Z. G. Bennett B. Suzuki N. Xia Y. Karin M. Genes Dev. 1999; 13: 1297-1308Crossref PubMed Scopus (409) Google Scholar). The generation of MyD88-deficient mice (9Adachi O. Kawai T. Takeda K. Matsumoto M. Tsutsui H. Sakagami M. Nakanishi K. Akira S. Immunity. 1998; 9: 143-150Abstract Full Text Full Text PDF PubMed Scopus (1718) Google Scholar) has shown that this protein is required for the proliferative response of T-cells to IL-1, for the IL-18-mediated production of interferon-γ by Th1 cells, and for the activation of natural killer cells. Thus, MyD88 is an essential mediator for the response of several immune cells to cytokines. Moreover, MyD88-knock-out mice are insensitive to LPS-induced death and fail to secrete cytokines such as IL-6 and TNF-α in vivo (10Kawai T. Adachi O. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 115-122Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar). Most notably, this study also revealed that LPS-induced activation of both NF-κB and MAPK was delayed, rather than abolished, in these mice (11Takeuchi O. Akira S. Curr. Top. Microbiol. Immunol. 2002; 270: 155-167Crossref PubMed Scopus (131) Google Scholar), highlighting the existence of a MyD88-independent pathway of TLR-4 signaling (12O'Neill L. A. J. Science. 2004; 303: 1481-1482Crossref PubMed Scopus (71) Google Scholar). On the other hand, loss of MyD88 expression has an anti-inflammatory effect in early atherosclerosis (13Björkbacka H. Kunjathoor V. V. Moore K. J. Koehn S. Ordija C. M. Lee M. A. Means T. Halmen K. Luster A. D. Golenbock D. T. Freeman M. W. Nat. Med. 2004; 10: 416-421Crossref PubMed Scopus (558) Google Scholar), whereas additional studies have further underlined the key inflammatory role of MyD88 in arthritis. Indeed, MyD88-deficient mice do not develop streptococcal cell wall-induced arthritis (14Joosten L. A. Koenders M. I. Smeets R. L. Heuvelmans-Jacobs M. Helsen M. M. Takeda K. Akira S. Lubberts E. van de Loo F. A. van den Berg W. B. J. Immunol. 2003; 171: 6145-6153Crossref PubMed Scopus (191) Google Scholar) nor a visually detectable synovitis after transfer of arthritogenic sera (15Choe J. Y. Crain B. Wu S. R. Corr M. J. Exp. Med. 2003; 197: 537-542Crossref PubMed Scopus (160) Google Scholar). Taken together, these results suggest that targeting the TLR/IL-1R pathway by interfering with the function of MyD88 may be a novel approach in the therapy of chronic inflammatory disorders. In the present study, we sought to investigate whether it was feasible to attenuate MyD88 signaling by means of peptide-mediated interference of MyD88 TIR domain homodimerization. Computational Methods—The MyD88 sequence was aligned by PSI-Blast to retrieve homologs and the selected sequences were aligned using ClustalW algorithm on the NPS server (npsa-pbil. ibcp. fr). Consensus secondary structure prediction was performed on the Predict-Protein server using different algorithms: Jpred, Psi-pred, PHD, Prof (cubic. bioc. columbia. edu/predictprotein). A structural alignment between the target sequence (GenBank™ accession number: NP₀02459) and the sequences with known crystal structures was performed using FUGUE program and manually adjusted based on secondary structure prediction and conserved regions found (www. cryst. bioc. cam. ac. uk/~fugue/prfsearch. html). The position of gaps and insertions was disfavored within conserved secondary structures. By using a homology modeling technique and Swiss-PDBViewer software (17Guex N. Peitsch M. C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9640) Google Scholar), the three-dimensional model of MyD88 TIR domain (aa 161–295) was built using TLR2 crystal structure (Protein Data Bank code 1fyx) as template, extracted from Protein Data Bank (www. rcsb. org). Molecular dynamics simulation, followed by simulated annealing technique was performed using MacroModel software (AMBER force field, GB/SA solvent model for water, 100 ps each step at different levels of constraint) (18Schrödinger LLC MacroModel 8. 5. Schrödinger LLC, Portland, OR2003Google Scholar). Quality evaluation of the model regarding variable regions and loops was done by the PROCHECK program (19Laskowski R. A. MacArthur M. W. Moss D. S. Thornton J. M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Models of MyD88 homodimers were generated using geometry and hydrophobicity complementary search algorithm GRAMM (20Katchalski-Katzir E. Shariv I. Eisenstein M. Friesem A. A. Aflalo C. Vakser I. A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2195-2199Crossref PubMed Scopus (867) Google Scholar). The docking parameters were set to: step grid at 1. 7 Å, repulsion potential at 30. 0, and intervals of rotation at 10°. The 30 lower energy models were clustered to select the most representative ones and then they were minimized to obtain seven different homodimers. Peptide Synthesis—The peptides were synthesized by the Fmoc (N- (9-fluorenyl) methoxycarbonyl) solid phase method (16Bodanszky M. Principles of Peptide Synthesis. 2nd Ed. Springer-Verlag, Berlin1993Crossref Google Scholar) on Rink-amide (amino methyl) -polystyrene resin using an ABi 431 A (Applied Biosystems) automatic peptide synthesizer and then cleaved from the resin using trifluoroacetic acid/H2O/triisopropylsilane/1, 2-ethanedithiol at 92. 5: 2. 5: 2. 5: 2. 5 (v/v). The peptides were purified by reverse-phase high pressure liquid chromatography (HPLC-RP) on C12 reverse-phase Jupiter-Proteo (Phenomenex) semipreparative columns. The intermediates and product analysis were performed by liquid chromatography/mass spectroscopy by HPLC-RP on C18 reverse-phase Luna (Phenomenex) and mass spectroscopy with Thermofinnigan LCQ-Duo to confirm their molecular weight. The final peptide purity was 90–99%. Plasmids—AU1-tagged MyD88 and AU1-tagged TIR domain of MyD88 expressing plasmids were a kind gift from Dr. Marta Muzio (”Mario Negri“Institute, Milan, Italy). cDNA encoding the TIR domain of MyD88 was amplified by PCR using pCDNA3-AU1-MyD88 as template and oligonucleotides 5′-AGGGATCCCCGACCCCCTGGGGCATATG-3′ (forward) and 5′-AGGAATTCTCAGGGCAGGGACAAGGC-3′ (reverse). The cDNA obtained was subcloned into the BamHI and EcoRI sites of either pCDNA3-N2-Myc or pGEX-3X expression vectors for Myc- or glutathione S-transferase (GST) -tagged TIR domain, respectively. For the NF-κB reporter assays, the NF-κB luciferase and Renilla luciferase constructs were used according to manufacturer's instructions (Promega). Cell Culture and Transfections—The human embryonic kidney (HEK) 293 and HeLa cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen), and grown in a 37 °C humidified atmosphere of 5% CO2. HEK293-TLR3 cells (InvivoGen), stably transfected with human TLR3, were cultured in the same medium supplemented with 10 μg/ml blasticidin S (InvivoGen). The HEK293 cells were cultured in 10-cm-diameter dishes and transfected by the calcium phosphate method with 5–15 μg of the appropriate plasmids. The Antennapedia43–58-fused MyD88196–202 peptides were added to the medium 24 h after transfection. GST-MyD88 TIR Domain Fusion Protein Synthesis and Purification—Escherichia coli cells (BL21) transformed with pGEX-3X-TIR construct were grown at 30 °C in LB medium to an optical density (A600 nm) of 0. 6. GST-TIR fusion protein expression was induced by 3-h induction with 0. 5 mm isopropyl β-thiogalactopyranoside (Sigma-Aldrich). Bacterial pellets were lysed in phosphate-buffered saline (PBS) containing 0. 1% Triton X-100, 1 mm DTT, protease inhibitors, by probe sonication. Bacterial lysates were centrifuged at 12, 000 × g, and supernatant fractions were incubated with glutathione-Sepharose beads (Sigma-Aldrich) for 1 h at 4 °C under constant shaking. After several washes in PBS, GST-fusion proteins were eluted with 50 mm Tris-HCl, pH 8, 100 mm NaCl, containing 10 mm glutathione (Sigma-Aldrich). Purified proteins were stored at –80 °C in the same buffer containing 10% glycerol. Protein purity and integrity was analyzed by SDS-PAGE and Coomassie Blue staining. Immunoprecipitation Assay—HEK293 cells were collected 24 h after transfection, washed in ice-cold PBS, and lysed in buffer containing 50 mm Hepes, pH 7. 4, 150 mm NaCl, 15 mm MgCl2, 15 mm EGTA, 1% Triton X-100, 10% glycerol, 20 mm β-glycerophosphate, 1 mm DTT, 0. 5 μm NaVO4, and protease inhibitors. After 10 min on ice, cell lysates were centrifuged at 10, 000 × g for 10 min at 4 °C and cytosolic fractions collected for immunoprecipitation. Cell extracts (800 μg of total proteins) were incubated with 2 μg of anti-AU1 antibody (Babco) and protein A-Sepharose beads (Sigma-Aldrich) for 2 h at 4 °C under constant shaking. Following incubation, the beads were washed twice with lysis buffer, twice with PBS, and then incubated with 0. 5 μg GST-TIR and 200 μm TIR peptides in PBS for 1 h under constant shaking at 4 °C. Sepharose bead-bound immunocomplexes were washed twice with PBS and eluted in SDS-PAGE sample buffer (62. 5 mm Tris-HCl, pH 6. 8, 10% glycerol, 2% (w/v) SDS, 0. 7 m 2-mercaptoethanol, and 0. 0025% (w/v) bromphenol blue) for Western blot analysis. Co-immunoprecipitation Assay—Cell extracts (700 μg of total proteins) prepared as described above were precleared by incubation for 1 h with a mixture of protein A/G-Sepharose beads (Sigma-Aldrich) under constant shaking at 4 °C. For 1 μg of was for 1 h with a mixture of protein A/G-Sepharose beads (Sigma-Aldrich) in lysis buffer containing bovine serum under constant shaking at 4 °C. After incubation, the beads were washed twice with lysis and then incubated with precleared cell extracts for 2 h at 4 °C under constant shaking. Sepharose bead-bound immunocomplexes were washed in lysis buffer and eluted in SDS-PAGE sample buffer for Western blot analysis. Western extracts or proteins were in sample buffer as described above and for were separated on SDS-PAGE and to using a were with 5% in PBS containing 0. 1% 20 for 1 h at and incubated with the antibody at 4 anti-AU1 from from from to were incubated with the for 1 h at at a in PBS containing 0. 1% were by the method NF-κB cells or HEK293-TLR3 × were cultured in and transfected with 0. 5 μg of an luciferase reporter and Renilla luciferase reporter as an using the according to the manufacturer's after transfection, Antennapedia43–58-fused MyD88196–202 peptides were added to the After 2 additional either or μg/ml were added to the same After additional cells were and lysed in of passive lysis buffer reporter assay for 15 min at Cell lysates were for 30 by at in a and to a to reporter analysis. of cell lysates were with 100 of luciferase assay and the luciferase was using a For the of the Renilla luciferase 100 of were added to the same Data are for by luciferase with that of Renilla Data are as induction from a of of MyD88 to receptors to the TLR/IL-1R superfamily a domain interaction H. S. Immunity. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. L. A. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. H. C. R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). most of the conserved residues in TIR domains within the of the are residues and may to different a referred to as the BB-loop the and in and that a the mice this to A. I. Liu C. E. M. C. M. P. B. B. Science. 1998; PubMed Scopus Google Scholar). Moreover, the also the of TLR2 to with MyD88 in vitro Y. B. T. R. J. L. L. PubMed Scopus Google Scholar). we whether inhibition of MyD88 signaling be by interfering with its TIR homodimerization domain using specific peptides that mimic the the BB-loop be at the we have the TIR domain of MyD88 and generated homodimers using the GRAMM in is the and the two different of interaction of the docking either to (aa or to by an the of this protein for homodimerization we a of peptides peptides the in a a we the of such peptides to with the dimerization of MyD88 TIR domains in AU1-tagged MyD88 was in HEK293 cells and isolated by immunoprecipitation. a purified fusion protein containing the TIR domain of MyD88 (aa was incubated with the beads to in the or of the peptides described in I. GST-TIR was to to full-length MyD88. we that an epta-peptide with the MyD88 sequence this interaction A and whereas a peptide with the same was In the same we found that an epta-peptide based on the receptor sequence (aa was whereas epta-peptides based on the sequence of IL-1 receptor accessory protein or (aa were either effective inhibition for or inhibition for The results of this analysis are in A and and indicate an sequence in the inhibitory potential of based A of referred to as and that the amino be into different an conserved a conserved and a linker in which the most are In a from whereas has a in this to model the the interaction between and the in not the homodimerization. Thus, the experimental results shown in to the model in confirm the of the MyD88 and IL-18R epta-peptides to with dimerization of the TIR we sought an the co-immunoprecipitation The TIR domain of MyD88 (aa was subcloned in expression vectors containing either a or an and were in HEK293 cells they as demonstrated by co-immunoprecipitation in the MyD88 and the IL-18R epta-peptides were of inhibiting this interaction and of were with these peptides were added to the cell extracts 4 and in In experiments, a peptide containing amino to of MyD88196–202 not significantly with TIR domain dimerization we the effect of the MyD88 epta-peptide on cells by it to a short peptide sequence from the of (aa to its into cells L. A. J. Sci. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Peptide was as its to with dimerization of the domains in HEK293 cells. and were in the of either the cell permeable MyD88 epta-peptide or its In with the results from experiments, we found that the cell permeable MyD88 peptide the dimerization of the TIR domains whereas the peptide a effect A and that the epta-peptide MyD88196–202 is of interfering with dimerization of the TIR domains of MyD88 in cells. the MyD88 epta-peptide with dimerization of the TIR domains in cells, we sought to whether it with the activation of the IL-1 signaling The downstream event of with IL-1 is activation of and dimerization of MyD88 is crucial for of this signaling we transfected HeLa cells, which IL-1R and MyD88 not with a reporter construct and then cells with IL-1 in the or of either Antennapedia43–58-fused MyD88196–202 or its Activation of the pathway was by luciferase in cell extracts h after that IL-1 triggered a induction of luciferase in HeLa cells peptide the MyD88196–202 peptide NF-κB induction to whereas the peptide effect that the IL-1 response is by interfering with dimerization of the TIR domain of MyD88. whether the interfering of the MyD88196–202 peptide was we its effect on the NF-κB activation by the which a MyD88-independent pathway M. H. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). HEK293 stably transfected with were with a induction of NF-κB was the MyD88196–202 peptide nor its this that with signaling pathways Our results suggest that the region of MyD88 in the which is conserved in the TIR domains of different is a good for at interfering with MyD88 we by dimerization that the most effective epta-peptides for MyD88 homophilic were from the sequence of the TIR domains of IL-18R and MyD88 A T. M. M. S. J. A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar) that a molecular mimic of the amino in the BB-loop for several MyD88, and between and MyD88. have shown that the in with the is also for dimerization of MyD88 as in both cell-free and in vitro cell Moreover, these are of inhibiting the activation of NF-κB by IL-1, molecules that mimic the structure of MyD88 may for the in vivo role of this protein in signaling leading to the of Nat. Rev. Immunol. 2004; 4: PubMed Scopus Google Scholar).