Research Center for Chronic Inflammatory Diseases

Toll/interleukin-1 (TIR)receptor-containing adapters are critical in orchestrating the different signal transduction pathways following Toll-like receptor (TLR) activation. MyD88 adapter-like (Mal), also termed TIRAP, is involved in bridging MyD88 to the receptor complex for TLR-2 and TLR4 signaling in response to bacterial infection. We have previously reported an interaction between Mal and tumor necrosis factor receptor-associated factor 6 (TRAF6) via a TRAF6-binding motif, the disruption of which inhibited TLR-mediated NF-κB-luciferase reporter activity. Given the recent report of intracellular TRAM localization promoting sequential signaling in TLR4 responses, we further characterized Mal interaction with TRAF6, the cellular localization, and the outcomes of disrupting this association on TLR inflammatory responses. We found that Mal and TRAF6 directly interact in response to TLR2 and TLR4 stimulation, although membrane localization is not necessary to facilitate interaction. Critically, reconstitution of murine Mal-deficient macrophages with MalE190A, containing a mutation within the TRAF6-binding motif, fails to reconstitute the proinflammatory response to TLR2 and TLR4 ligands compared with wild type Mal. Furthermore, Mal interaction with TRAF6 mediates Ser phosphorylation of the p65 subunit of NF-κB and thus controls transcriptional activation but not nuclear translocation of NF-κB. This study characterizes the novel role for Mal in facilitating the direct recruitment of TRAF6 to the plasma membrane, which is necessary for TLR2- and TLR4-induced transactivation of NF-κB and regulation of the subsequent pro-inflammatory response. Toll/interleukin-1 (TIR)receptor-containing adapters are critical in orchestrating the different signal transduction pathways following Toll-like receptor (TLR) activation. MyD88 adapter-like (Mal), also termed TIRAP, is involved in bridging MyD88 to the receptor complex for TLR-2 and TLR4 signaling in response to bacterial infection. We have previously reported an interaction between Mal and tumor necrosis factor receptor-associated factor 6 (TRAF6) via a TRAF6-binding motif, the disruption of which inhibited TLR-mediated NF-κB-luciferase reporter activity. Given the recent report of intracellular TRAM localization promoting sequential signaling in TLR4 responses, we further characterized Mal interaction with TRAF6, the cellular localization, and the outcomes of disrupting this association on TLR inflammatory responses. We found that Mal and TRAF6 directly interact in response to TLR2 and TLR4 stimulation, although membrane localization is not necessary to facilitate interaction. Critically, reconstitution of murine Mal-deficient macrophages with MalE190A, containing a mutation within the TRAF6-binding motif, fails to reconstitute the proinflammatory response to TLR2 and TLR4 ligands compared with wild type Mal. Furthermore, Mal interaction with TRAF6 mediates Ser phosphorylation of the p65 subunit of NF-κB and thus controls transcriptional activation but not nuclear translocation of NF-κB. This study characterizes the novel role for Mal in facilitating the direct recruitment of TRAF6 to the plasma membrane, which is necessary for TLR2- and TLR4-induced transactivation of NF-κB and regulation of the subsequent pro-inflammatory response. Toll-like receptors (TLRs) 2The abbreviations used are: TLRToll-like receptorTIRToll/IL-1 receptorGSTglutathione S-transferaseIFNinterferonILinterleukinIRAKinterleukin-1 receptor-associated kinaseLPSlipopolysaccharideMalMyD88 adapter-likeNF-κBnuclear factor κBPIP2phosphatidylinositol 4,5-biphosphateTNFtumor necrosis factorTRAFTNF receptor-associated factorTRIFTIR domain-containing adapter protein inducing IFNβTRAMTRIF-related adapter moleculeHEKhuman embryonic kidneyGFPgreen fluorescent proteinHAhemagglutininMAPmitogen-activated proteinErkextracellular signal-regulated kinaseJNKc-Jun N-terminal kinase. 2The abbreviations used are: TLRToll-like receptorTIRToll/IL-1 receptorGSTglutathione S-transferaseIFNinterferonILinterleukinIRAKinterleukin-1 receptor-associated kinaseLPSlipopolysaccharideMalMyD88 adapter-likeNF-κBnuclear factor κBPIP2phosphatidylinositol 4,5-biphosphateTNFtumor necrosis factorTRAFTNF receptor-associated factorTRIFTIR domain-containing adapter protein inducing IFNβTRAMTRIF-related adapter moleculeHEKhuman embryonic kidneyGFPgreen fluorescent proteinHAhemagglutininMAPmitogen-activated proteinErkextracellular signal-regulated kinaseJNKc-Jun N-terminal kinase. recognize and respond to both pathogen-associated molecular patterns and endogenous signals associated with danger (1.Kawai T. Akira S. Semin. Immunol. 2007; 19: 24-32Crossref PubMed Scopus (1206) Google Scholar). Upon ligand-induced dimerization the TLRs activate, via their cytosolic Toll/IL-1 receptor (TIR) domains, the homotypic recruitment of one or more proteins of a family of five cytosolic TIR-containing adapter proteins (2.O'Neill L.A. Bowie A.G. Nat. Rev. Immunol. 2007; 7: 353-364Crossref PubMed Scopus (1942) Google Scholar). All TLRs, with the exception of TLR3, recruit MyD88 to their receptor complex, as do members of the IL-1 receptor family. MyD88 recruits interleukin-1 receptor-associated kinase 1 (IRAK1), IRAK4, and then TNF receptor-associated factor 6 (TRAF6), which results in the nuclear translocation of the prototypic inflammatory transcription factor NF-κB, termed the canonical pathway (3.O'Neill L.A. Immunol. Rev. 2008; 226: 10-18Crossref PubMed Scopus (476) Google Scholar, 4.Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8426) Google Scholar), which encodes inflammatory genes such as TNF-α and IL-6. Although TLRs induce common signaling pathways, there is specificity in recruitment of TIR-containing adapter proteins. MyD88 adapter-like (Mal)/TIRAP was the second described adapter capable of mediating NF-κB activation and was responsible for signaling selectively via TLR4 (5.Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Crossref PubMed Scopus (986) Google Scholar, 6.Horng T. Barton G.M. Medzhitov R. Nat. Immunol. 2001; 2: 835-841Crossref PubMed Scopus (821) Google Scholar) and TLR2 signaling (7.Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Crossref PubMed Scopus (677) Google Scholar, 8.Yamamoto M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Crossref PubMed Scopus (804) Google Scholar). TIR domain-containing adapter protein inducing IFNβ (TRIF, also known as TICAM1) was subsequently found to mediate the MyD88-independent pathway leading to TLR4-mediated activation of the transcription factor interferon regulatory factor 3, which regulates Type I IFN production (9.Yamamoto M. Sato S. Hemmi H. Hoshino K. Kaisho T. Sanjo H. Takeuchi O. Sugiyama M. Okabe M. Takeda K. Akira S. Science. 2003; 301: 640-643Crossref PubMed Scopus (2445) Google Scholar). Importantly, TRIF also mediates downstream signaling from TLR3, independent of MyD88. The TRIF-related adapter molecule (TRAM, also known as TICAM2) specifically acts to bridge TLR4 with TRIF, where TRAM-deficient macrophages are ablated in their responses to TLR4 activation but not TLR3 (10.Fitzgerald K.A. Rowe D.C. Barnes B.J. Caffrey D.R. Visintin A. Latz E. Monks B. Pitha P.M. Golenbock D.T. J. Exp. Med. 2003; 198: 1043-1055Crossref PubMed Scopus (901) Google Scholar, 11.Yamamoto M. Sato S. Hemmi H. Uematsu S. Hoshino K. Kaisho T. Takeuchi O. Takeda K. Akira S. Nat. Immunol. 2003; 4: 1144-1150Crossref PubMed Scopus (806) Google Scholar). Toll-like receptor Toll/IL-1 receptor glutathione S-transferase interferon interleukin interleukin-1 receptor-associated kinase lipopolysaccharide MyD88 adapter-like nuclear factor κB phosphatidylinositol 4,5-biphosphate tumor necrosis factor TNF receptor-associated factor TIR domain-containing adapter protein inducing IFNβ TRIF-related adapter molecule human embryonic kidney green fluorescent protein hemagglutinin mitogen-activated protein extracellular signal-regulated kinase c-Jun N-terminal kinase. Toll-like receptor Toll/IL-1 receptor glutathione S-transferase interferon interleukin interleukin-1 receptor-associated kinase lipopolysaccharide MyD88 adapter-like nuclear factor κB phosphatidylinositol 4,5-biphosphate tumor necrosis factor TNF receptor-associated factor TIR domain-containing adapter protein inducing IFNβ TRIF-related adapter molecule human embryonic kidney green fluorescent protein hemagglutinin mitogen-activated protein extracellular signal-regulated kinase c-Jun N-terminal kinase. Mal and TRAM have been described as bridging adapters, responsible for specific recruitment of MyD88 and TRIF proximal to the surface localized TLR2 and TLR4 receptor complexes (10.Fitzgerald K.A. Rowe D.C. Barnes B.J. Caffrey D.R. Visintin A. Latz E. Monks B. Pitha P.M. Golenbock D.T. J. Exp. Med. 2003; 198: 1043-1055Crossref PubMed Scopus (901) Google Scholar, 12.Kagan J.C. Medzhitov R. Cell. 2006; 125: 943-955Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 13.Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1000) Google Scholar). Membrane localization of these bridging adapters has further demonstrated the spatial coordination required for transmission of MyD88 (12.Kagan J.C. Medzhitov R. Cell. 2006; 125: 943-955Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar) and TRAM (14.Kagan J.C. Su T. Horng T. Chow A. Akira S. Medzhitov R. Nat. Immunol. 2008; 9: 361-368Crossref PubMed Scopus (920) Google Scholar) signals, allowing specificity of downstream signaling mediators that induce cytokine production. Mal acts to bridge MyD88 to TLR2 and TLR4 specifically via its TIR domain. The recent description of a Mal functional variant associated with protection against pnuemococcal disease, bacteremia, malaria, and Dunne A. O. A. S. K. A. P. J. G. K. D. K. O'Neill L.A. Nat. 2007; PubMed Scopus Google Scholar) has the of Mal in human Furthermore, the recent of the TIR Mal which is to interact with MyD88 and downstream responses K. J. Monks K.A. Golenbock D.T. J. Google Scholar), has the of in TLR-mediated signaling and the for a of Mal mediates We have previously demonstrated that Mal with TRAF6 A. Brint E. O'Neill L.A. J. Full Text Full Text PDF PubMed Scopus Google Scholar) via a TRAF6-binding H. B. M. Kobayashi T. D. M. M. K. S. H. Nature. 2002; PubMed Scopus Google Scholar) that inhibited TLR2- and TLR4-induced NF-κB activation. the specificity and of this interaction not disruption of Mal to a from disease, we to the specificity of Mal interaction with TRAF6, cellular localization, and the outcomes of disrupting this interaction on TLR-mediated pro-inflammatory responses. this we the direct interaction of Mal with TRAF6 and the localization of this association to the plasma Critically, reconstitution of Mal-deficient macrophages with wild type Mal transactivation of the p65 subunit of NF-κB, which was ablated with disruption of the TRAF6-binding Importantly, NF-κB translocation to the was not in these Mal-deficient macrophages with wild type Mal the pro-inflammatory TNF-α and in response to TLR2 and TLR4 stimulation, with a mutation within the TRAF6-binding a cytokine response to TLR these the novel role for Mal in facilitating the direct recruitment of TRAF6 to the plasma membrane, which is necessary for TLR2- and TLR4-induced transactivation of NF-κB and regulation of the subsequent pro-inflammatory response. and murine Mal-deficient macrophages K. J. Monks K.A. Golenbock D.T. J. Google Scholar) in with and in a in and in a was as described previously M. R. H. J. Immunol. Google Scholar), was from was from was from from and from TRAF6, and from The N-terminal and TIR have been described previously (5.Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Crossref PubMed Scopus (986) Google Scholar). The following Mal described A. Brint E. O'Neill L.A. J. Full Text Full Text PDF PubMed Scopus Google Scholar), Mal and Mal (12.Kagan J.C. Medzhitov R. Cell. 2006; 125: 943-955Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). and the with the or the the from the p65 subunit of NF-κB to was a from The reporter and for of and from and have been described previously A. M. O'Neill L.A. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). was the as for protein was and in bacterial was to was with and and in and 1 of The and and was in and was was on and of to the 6 with the where the of was the in as described A. Smith R. Gray P. J.E. O'Neill L.A. Nat. Immunol. 2006; 7: PubMed Scopus Google Scholar). The or with the for the of of protein for 1 The complexes the of and the the from with used in a for with protein to The complexes in and as in the endogenous in 1 of and as to and with the to to or the and with and with and proteins the for The in in and in The with and with or on with on and was the in a to with was the reporter with the of in with or used to activation of p65 transactivation and The kinase was used to for and was used to and for and for and as with of a containing the NF-κB that previously been with kinase for in the of of as and containing 1 and The to on which subsequently and from used for to of TNF-α or with from to the cellular was the to the of was with and in a of was and for on the from and of the with the endogenous was the the from independent are as the All of the was The of independent was a of The of are as and We have previously demonstrated that Mal with TRAF6 in we to TLR association between the proteins. in the of in TRAF6 Mal in a within of TLR2 of human TLR4 activation also the of although with following Importantly, to induce interaction of with Mal a specific role in TLR2 and TLR4 of was also in following stimulation, that the ligands their results a specific interaction between Mal and TRAF6 following of TLR2 and but not with the described role for Mal in TLR2 and TLR4 signal and Medzhitov (12.Kagan J.C. Medzhitov R. Cell. 2006; 125: 943-955Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar) demonstrated the critical role of Mal localization to the plasma membrane via an N-terminal in TLR2 and TLR4 to the plasma membrane was required to facilitate MyD88 recruitment to the plasma membrane TLR receptor complex leading to of the canonical signaling pathway and subsequent activation and nuclear translocation of NF-κB. of Mal is to to the membrane (12.Kagan J.C. Medzhitov R. Cell. 2006; 125: 943-955Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar) and NF-κB (5.Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Crossref PubMed Scopus (986) Google Scholar, A. Brint E. O'Neill L.A. J. Full Text Full Text PDF PubMed Scopus Google Scholar) TLR stimulation, we to localization of Mal and TRAF6 the plasma membrane or the in We found that Mal localized to the plasma membrane with subunit We that Mal from which of the and Mal membrane localization, and Mal of the of Mal the and both the with (12.Kagan J.C. Medzhitov R. Cell. 2006; 125: 943-955Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar) and of TRAF6 was found in the and not the surface compared with subunit membrane localization was required to facilitate interaction between Mal and TRAF6, we compared wild type Mal association with TRAF6 to Mal this variant an TRAF6-binding but fails to to the membrane and Mal which the and TRAF6-binding of wild type Mal and Mal TRAF6 that Mal proteins are with TRAF6 the plasma membrane type or in the and Mal and is where the of the of are to in between that although Mal and Mal in the as TRAF6 and and Mal is not in the and is not with TRAF6 and of Mal and TRAF6 was of Mal with and or and that the the plasma membrane not from the and a of Mal and TRAF6 association further these we found that both wild type and Mal but not Mal to interact with TRAF6 in that the not the of Mal and TRAF6 to interact and that association independent of cellular that interaction was not on localization, we the on downstream We have previously found that Mal interaction with TRAF6 the activation of the kinase pathway and transactivation of the p65 subunit of NF-κB A. Brint E. O'Neill L.A. J. Full Text Full Text PDF PubMed Scopus Google Scholar). We the of these proteins to NF-κB activation compared with previously reported results Mal. with localization and both wild type Mal and Mal to induce activation of NF-κB Mal to induce a responses. Furthermore, with report A. Brint E. O'Neill L.A. J. Full Text Full Text PDF PubMed Scopus Google Scholar), wild type Mal and Mal to induce NF-κB p65 transactivation and and activation via reporter but not Mal was to induce activation of pathway of its of and TIR these results that Mal and TRAF6 interact the plasma membrane or in the independent of localization to the plasma membrane, that the of Mal to the plasma membrane for spatial signaling or TRAF6 have a but not an association for the Mal. The of an Mal TRAF6-binding for interaction with TRAF6 was study we found that a of in Mal inhibited TLR2 and TLR4 in this mutation was to interact with we to a more specific of the role of the critical in the TRAF6-binding in mediating Mal interaction with with we Mal and TRAF6 association in 3, that both Mal and are both with TRAF6 the plasma membrane, that the are is that Mal and TRAF6 are in the and as such the plasma membrane that as the plasma membrane, TRAF6 in the and is not in the and as such not further this we a mutation of of the TRAF6-binding H. B. M. Kobayashi T. D. M. M. K. S. H. Nature. 2002; PubMed Scopus Google Scholar) from termed which not to with TRAF6, and as such not a of association between the proteins This protein also to with TRAF6 as the results TRAF6 and not that the localization and the of an TRAF6-binding for Mal interaction with TRAF6 of a of Mal and not the of the to the proteins. demonstrated an interaction between Mal and TRAF6 we to further the and the specificity of interaction and the association was of an TRAF6-binding within Mal and independent of bridging proteins. in was to specifically TRAF6 from cellular was to recognize was to Mal from cellular but was to interact with the of an TRAF6-binding to facilitate that the Mal interaction with TRAF6 was we proteins of TRAF6 with in with the TIR-containing proteins. in was to with TRAF6, and TRAF6 a these results that Mal and TRAF6 directly interact the plasma membrane, the interaction on an TRAF6-binding within Mal. Importantly, these also the interaction between Mal and TRAF6 are independent of of Mal or TRAF6 of of a specific complex of proteins. the of Mal and TRAF6 interaction in the response to TLR2 and TLR4 we the of disruption of the Mal TRAF6-binding on TLR inflammatory responses. these we Mal-deficient We that this was to TLR2 and TLR4 but respond to and TLR3 ligands as TNF-α production. also demonstrated the of Mal in these Mal-deficient macrophages then with MalE190A, or to a of proteins of with for protein was used to protein not and of Mal protein in of wild type MalE190A, and we to an TRAF6-binding was required for NF-κB nuclear translocation to the following TLR in macrophages with wild type Mal 1 and and or and NF-κB translocation to the in response to TLR2 TLR4 and stimulation, which that interaction between Mal and TRAF6 is not required for NF-κB translocation via the MyD88 canonical We have previously reported that Mal interaction with TRAF6 is required for transactivation of NF-κB A. Brint E. O'Neill L.A. J. Full Text Full Text PDF PubMed Scopus Google Scholar) and that Mal is for phosphorylation of the p65 subunit of NF-κB A. Smith R. Gray P. J.E. O'Neill L.A. Nat. Immunol. 2006; 7: PubMed Scopus Google Scholar). transactivation of NF-κB is a critical in the transcriptional regulation of the inflammatory response G. S. G. 2002; PubMed Scopus Google Scholar), we Mal interaction with TRAF6 was critical to this response. in with Mal to p65 in response to TLR2 with and compared with 1 and in a Critically, reconstitution of Mal-deficient macrophages with and to phosphorylation of the p65 transactivation of NF-κB. we that with Mal phosphorylation of which is in with Mal has not been previously in this transactivation in response to TLR4 stimulation, p65 or phosphorylation of the MyD88-independent NF-κB transactivation a role in the inflammatory cytokine response to TLR stimulation, we cytokine in Mal-deficient Mal-deficient macrophages with wild type MalE190A, or and with or the J. Immunol. PubMed Scopus Google Scholar) to induce or TNF-α and protein Although reconstitution with Mal was to mediate and and TNF-α and compared with the or both and inhibited TLR2 and TLR4 proinflammatory responses. Importantly, as a response with and was independent of the or of Mal. TLR4 of IFNβ was also not in the or the of Mal with TLR4-induced IFNβ M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Crossref PubMed Scopus (804) Google Scholar). these results the critical role Mal interaction with TRAF6 in the pro-inflammatory response as a of TLR2 and TLR4 activation and that the is via a pathway of NF-κB independent of the canonical NF-κB nuclear this study we have and characterized that the novel interaction of Mal with TRAF6 has a critical role in TLR2- and TLR4-induced inflammatory responses. that the of Mal to the plasma membrane in the spatial of the the direct interaction between Mal and TRAF6, which regulates transactivation of the p65 subunit of NF-κB but not NF-κB nuclear The association of Mal and TRAF6 to the transcriptional of pro-inflammatory of Mal-deficient macrophages with containing a mutation within the TRAF6-binding both TLR2- and TLR4-mediated inflammatory responses compared with wild type Mal. results a specific role for Mal in TLR signaling and a direct interaction between Mal and the signaling TRAF6 that is from that of as a bridging adapter for MyD88 The of Mal in TLR-mediated signal transduction in human was the recent description of the functional variant and its association with protection against disease, malaria, bacteremia, and Dunne A. O. A. S. K. A. P. J. G. K. D. K. O'Neill L.A. Nat. 2007; PubMed Scopus Google Scholar, J. D.C. A. 2008; PubMed Scopus Google Scholar). We found of this variant on the of Mal to recruit TRAF6 not with and and Dunne A. O. A. S. K. A. P. J. G. K. D. K. O'Neill L.A. Nat. 2007; PubMed Scopus Google Scholar) with the of the Mal to interact with MyD88 K. J. Monks K.A. Golenbock D.T. J. Google Scholar) the that to Mal have in the of the plasma membrane proximal signaling Mal has been reported to phosphorylation P. Dunne A. Jefferies C.A. O'Neill L.A. J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, H. K.A. O'Neill L.A. J. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar), E. Dunne A. Jefferies E. K. P. M. Akira S. Golenbock D. K.A. O'Neill L.A. 2007; PubMed Scopus Google Scholar), and the of the of these the of Mal to interact with TRAF6 in TRAF6 not which with the direct interaction of the Mal and TRAF6 proteins that is not required for the interaction to This that there is more to as to the signaling of Mal. Critically, this study the of a within the critical TRAF6-binding of Mal and the response to TLR2 and TLR4 is Mal not as a bridging facilitating MyD88 recruitment to the membrane proximal receptor complex, but directly recruits TRAF6 to the plasma membrane to NF-κB transactivation and transcriptional activation of the inflammatory independent of NF-κB nuclear the TRAF6-binding in Mal is within its TIR the of MyD88 and TRAF6 for recent H. H. A. T. H. M. PubMed Scopus Google Scholar) demonstrated complex that Mal proteins MyD88 on responsible for interaction. that not with between and or the in Mal was to the interaction This is of the of a signaling complex TRAF6, and MyD88 to the signaling has previously been demonstrated that the in the of TRAM is critical for its plasma localization and the to mediate TLR4 signaling D.C. Latz E. Monks M. Akira S. O'Neill L.A. K.A. Golenbock D.T. 2006; PubMed Scopus Google Scholar). (14.Kagan J.C. Su T. Horng T. Chow A. Akira S. Medzhitov R. Nat. Immunol. 2008; 9: 361-368Crossref PubMed Scopus (920) Google Scholar) demonstrated that TRAM also further that recruitment of TRIF and subsequently to the complex to signaling This or to the complex with the to specifically recruit an adapter required for of This description of signaling of TRAM (14.Kagan J.C. Su T. Horng T. Chow A. Akira S. Medzhitov R. Nat. Immunol. 2008; 9: 361-368Crossref PubMed Scopus (920) Google Scholar, Nat. Immunol. 2008; 9: PubMed Scopus Google Scholar) and its to the membrane as a of and the TLR4 adapter have to we have in this study for Mal. recruitment of family members to TIR adapter proteins of localization spatial which signals and coordination of NF-κB, and interferon regulatory factor signaling study found that the Mal variant that was to to the plasma membrane and NF-κB transactivation with wild type that membrane localization was not critical for mediating this of Mal to that with Mal localization to the membrane via activation an for Mal and TRAF6 to which from the complex TIR signaling The described that Mal interaction with TRAF6 not pathway signal transduction leading to NF-κB nuclear the nuclear NF-κB not to transactivation the interaction. that Mal interaction with TRAF6 is in transactivation of the p65 subunit of NF-κB, via the kinase pathway A. Brint E. O'Neill L.A. J. Full Text Full Text PDF PubMed Scopus Google Scholar). the plasma membrane localization of Mal and TRAM to the spatial recruitment of specific signaling as downstream signaling Nat. Immunol. 2008; 9: PubMed Scopus Google Scholar) to that described with TRAM and to IFN in Mal-deficient we to the of interaction between Mal and TRAF6 for a inflammatory response to TLR2 and TLR4 of the TRAF6-binding in Mal inhibited both NF-κB transactivation and G. S. G. 2002; PubMed Scopus Google Scholar) and ablated cytokine and production in response to TLR2 and TLR4 the TRAF6-binding is for of the of critical of the required mutation to TNF-α production. This that pathways a role in TLR4-induced TNF-α This in a of the of TLR4 signaling and MyD88-independent with the of TNF and which the between these and J.E. D. Science. PubMed Scopus Google Scholar) have that TNF-α is the MyD88-independent pathway via interferon regulatory factor 3, which was has been to a role in both TNF-α and protein J. 2003; PubMed Scopus Google Scholar, J. J. A. 2003; PubMed Scopus Google Scholar). We have described previously that Mal interaction with TRAF6 mediates and activation A. Brint E. O'Neill L.A. J. Full Text Full Text PDF PubMed Scopus Google Scholar), but not which the that the is to induce TNF-α we also demonstrated that reconstitution of Mal-deficient with not the of TLR4 to the MyD88-independent pathway a of an on IFNβ that of TNF-α but the MyD88-independent response has for this this study a critical role for Mal in TLR2- and TLR4-mediated inflammatory responses that are novel to its role as a bridging adapter for MyD88. Mal regulation of the transcriptional of NF-κB via TRAF6 interaction and the subsequent of inflammatory is a regulatory in TLR2 and TLR4 inflammatory responses. This interaction activation of both the canonical and transactivation pathways, in the translocation and subsequent transactivation of NF-κB, both of which are critical to production of pro-inflammatory a mutation within the TRAF6-binding of Mal is to the pro-inflammatory response to TLR2 and TLR4 the response for and This study Mal as a of TLR2 and TLR4 signal transduction via a novel interaction with TRAF6, the disruption of which a specific for TLR2- and TLR4-mediated inflammatory on or signaling We Medzhitov for the Mal and Mal and O'Neill of and for the of the with

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at approximately 3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 micro g/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.

Different DNA motifs are required for optimal stimulation of mouse and human immune cells by CpG oligodeoxynucleotides (ODN). These species differences presumably reflect sequence differences in TLR9, the CpG DNA receptor. In this study, we show that this sequence specificity is restricted to phosphorothioate (PS)-modified ODN and is not observed when a natural phosphodiester backbone is used. Thus, human and mouse cells have not evolved to recognize different CpG motifs in natural DNA. Nonoptimal PS-ODN (i.e., mouse CpG motif on human cells and vice versa) gave delayed and less sustained phosphorylation of p38 MAPK than optimal motifs. When the CpG dinucleotide was inverted to GC in each ODN, some residual activity of the PS-ODN was retained in a species-specific, TLR-9-dependent manner. Thus, TLR9 may be responsible for mediating many published CpG-independent responses to PS-ODN.

The Toll-interleukin-1 receptor domain-containing adapter Mal (MyD88 adapter-like protein) is involved in Toll-like receptor (TLR)-2 and TLR4 signal transduction. However, no studies have yet identified a function for Mal distinct from the related adapter MyD88. In this study, we have identified a putative TRAF6 interaction site in Mal but not in MyD88 and we demonstrate that Mal can be co-immunoprecipitated with TRAF6. Overexpression of MalE190A, which contains a mutation within the TRAF6-binding motif, failed to induce the expression of an NF-κB-dependent reporter gene, p65-mediated transactivation of gene expression, or activation of Jun N-terminal kinase or p42/p44 MAP kinase, which are induced with wild type Mal. MalE190A inhibited TLR2- and TLR4-mediated activation of NF-κB. These results identify a specific role for Mal in TLR-mediated signaling in regulating NF-κB-dependent gene transcription via its interaction with TRAF6. The Toll-interleukin-1 receptor domain-containing adapter Mal (MyD88 adapter-like protein) is involved in Toll-like receptor (TLR)-2 and TLR4 signal transduction. However, no studies have yet identified a function for Mal distinct from the related adapter MyD88. In this study, we have identified a putative TRAF6 interaction site in Mal but not in MyD88 and we demonstrate that Mal can be co-immunoprecipitated with TRAF6. Overexpression of MalE190A, which contains a mutation within the TRAF6-binding motif, failed to induce the expression of an NF-κB-dependent reporter gene, p65-mediated transactivation of gene expression, or activation of Jun N-terminal kinase or p42/p44 MAP kinase, which are induced with wild type Mal. MalE190A inhibited TLR2- and TLR4-mediated activation of NF-κB. These results identify a specific role for Mal in TLR-mediated signaling in regulating NF-κB-dependent gene transcription via its interaction with TRAF6. MyD88 adapter-like protein (Mal) 1The abbreviations used are: Mal, MyD88 adapter-like protein; TIR, Toll/IL-1 receptor; TLR, Toll-like receptor; TRIF, TIR-containing adapter inducing interferon β; TRAM, TRIF-related adapter molecule; LPS, lipopolysaccharide; IL, interleukin; HEK, human embryonic kidney; pI:C, poly(I)·poly(C); HA, hemagglutinin; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein. IRAK, IL-1 receptor-associated kinase. (also known as Toll/IL-1 receptor (TIR) domain-containing adapter protein (TIRAP)) (1Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Crossref PubMed Scopus (1005) Google Scholar, 2Horng T. Barton G.M. Medzhitov R. Nat. Immunol. 2001; 2: 835-841Crossref PubMed Scopus (832) Google Scholar) is a member of the family of the TIR domain-containing adapter proteins involved in Toll-like receptor (TLR) signaling (3O'Neill L.A. Fitzgerald K.A. Bowie A.G. Trends Immunol. 2003; 24: 286-290Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 4Yamamoto M. Takeda K. Akira S. Mol. Immunol. 2004; 40: 861-868Crossref PubMed Scopus (304) Google Scholar). MyD88 was the first adapter in the family to be described, and it plays a role in signal transduction by all TLRs, with the exception of TLR3 (5Janssens S. Beyaert R. Trends Biochem. Sci. 2002; 27: 474-482Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). Mal was found as a homologue of MyD88 (1Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Crossref PubMed Scopus (1005) Google Scholar, 2Horng T. Barton G.M. Medzhitov R. Nat. Immunol. 2001; 2: 835-841Crossref PubMed Scopus (832) Google Scholar). In terms of function, it resembles MyD88 in that it is involved in the early activation of NF-κB and MAP kinases, but its use is restricted to signaling by TLR2 and TLR4 (6Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Crossref PubMed Scopus (689) Google Scholar, 7Yamamoto M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Crossref PubMed Scopus (821) Google Scholar). Two further adapters have been found to play a role in TLR signaling. TRIF (TIR-containing adapter inducing interferon β or TICAM-1) (8Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nat. Immunol. 2003; 4: 161-167Crossref PubMed Scopus (1014) Google Scholar, 9Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1025) Google Scholar, 10Hoebe K. Du X. Georgel P. Janssen E. Tabeta K. Kim S.O. Goode J. Lin P. Mann N. Mudd S. Crozat K. Sovath S. Han J. Beutler B. Nature. 2003; 424: 743-748Crossref PubMed Scopus (1037) Google Scholar) is necessary for TLR3- and TLR4-mediated activation of NF-κB and another transcription factor IRF3, while TRAM (TRIF-related adapter molecule, also termed TICAM-2) (11Fitzgerald K.A. Rowe D.C. Barnes B.J. Caffrey D.R. Visintin A. Latz E. Monks B. Pitha P.M. Golenbock D.T. J. Exp. Med. 2003; 198: 1043-1055Crossref PubMed Scopus (939) Google Scholar, 12Yamamoto M. Sato S. Hemmi H. Uematsu S. Hoshino K. Kaisho T. Takeuchi O. Takeda K. Akira S. Nat. Immunol. 2003; 4: 1144-1150Crossref PubMed Scopus (832) Google Scholar, 13Oshiumi H. Sasai M. Shida K. Fujita T. Matsumoto M. Seya T. J. Biol. Chem. 2003; 278: 49751-49762Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar) is essential for TLR4 signals, including IRF3. LPS-stimulated macrophages from MyD88- and Mal-deficient mice both displayed similar absence of cytokine production and delayed NF-κB activation but were normal for IRF3 activation and interferon β production. A double knock-out suggested that neither protein could compensate for the other. While a role for TRIF and TRAM in the IRF3 pathway distinguishes them from MyD88 and Mal, there is still no functional distinction between MyD88 and Mal in terms of function. Recently, two groups reported the identification and functional role TRIF association with TRAF6 via a TRAF6-binding motif (14Jiang Z. Mak T.W. Sen G. Li X. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3533-3538Crossref PubMed Scopus (309) Google Scholar, 15Sato S. Sugiyama M. Yamamoto M. Watanabe Y. Kawai T. Takeda K. Akira S. J. Immunol. 2003; 171: 4304-4310Crossref PubMed Scopus (590) Google Scholar). TRIF was found to mediate TLR3-induced activation of NF-κB via an association with TRAF6, independent of MyD88 and IRAK (15Sato S. Sugiyama M. Yamamoto M. Watanabe Y. Kawai T. Takeda K. Akira S. J. Immunol. 2003; 171: 4304-4310Crossref PubMed Scopus (590) Google Scholar). The TRAF6 interaction motif was based upon work by Pullen et al. (16Pullen S.S. Labadia M.E. Ingraham R.H. McWhirter S.M. Everdeen D.S. Alber T. Crute J.J. Kehry M.R. Biochemistry. 1999; 38: 10168-10177Crossref PubMed Scopus (131) Google Scholar, 17Pullen S.S. Miller H.G. Everdeen D.S. Dang T.T. Crute J.J. Kehry M.R. Biochemistry. 1998; 37: 11836-11845Crossref PubMed Scopus (206) Google Scholar) who first identified a cytoplasmic region of CD40 required to facilitate its binding of the TRAF-C domain of TRAF6. This interaction region was further defined by the elucidation of the crystal structure of the TRAF-C domain of TRAF6 in complex with peptides corresponding to CD40 or TRANCE-R (18Ye H. Arron J.R. Lamothe B. Cirilli M. Kobayashi T. Shevde N.K. Segal D. Dzivenu O.K. Vologodskaia M. Yim M. Du K. Singh S. Pike J.W. Darnay B.G. Choi Y. Wu H. Nature. 2002; 418: 443-447Crossref PubMed Scopus (541) Google Scholar). Structural analysis identified a nominal TRAF6-binding motif consisting of Pro-X-Glu-X-X-aromatic/acidic amino acids. Furthermore, three TRAF6-binding motifs were identified in IRAK, two in IRAK-2, one in IRAK-M, and one in RIP2 (see Table I). Sequential mutation of a critical glutamic acid in one, two, or all three TRAF6-binding motifs in IRAK proportionally attenuated IL-1-induced NF-κB activation.Table IPutative TRAF6-binding motifs in human TIR domain-containing adapters Open table in a new tab In this study we have found that similar to TRIF, Mal has a putative TRAF6-binding motif. The motif is required for Mal to drive signals for NF-κB activation and the interaction between Mal and TRAF6 for downstream signaling events. This provides a distinguishing feature between Mal and MyD88 in TLR2- and TLR4-mediated responses. Cell Lines and Reagents—Human embryonic kidney (HEK) 293, HEK293T, and HEK293 stably transfected cells expressing TLR4 and MD2, were incubated in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mm glutamine and maintained in a 37 °C humidified atmosphere. LPS K235 (Sigma) was repurified as described previously (19Hirschfeld M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (973) Google Scholar), Pam3Cys was obtained from EMC microcollections (Turbingen, Germany), poly(I)·poly(C) (pI:C) was obtained from Amersham Biosciences (Uppsala, Sweden), and anti-FLAG M2-horseradish peroxidase-conjugated antibody and anti-FLAG M2-agarose beads were from Sigma. Plasmids—Full-length pDC304 Mal-HA, pDC304 Mal-HA N-terminal(1–74), and pDC304 Mal-HA TIR domain(74–235) have been described previously (1Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Crossref PubMed Scopus (1005) Google Scholar). MalE190A was generated using the QuikChange site-directed mutagenesis kit with pfu-Turbo (Stratagene, La Jolla, CA) using the pDC304 Mal-HA template. Gal4-p65(1–551) plasmid encoding the full-length p65 subunit of NF-κB fused to Gal4 DNA-binding domain was a kind gift from Lienhard Schmitz (German Cancer Research Centre, Heidelburg, Germany) (20Jefferies C. Bowie A. Brady G. Cooke E.L. Li X. O'Neill L.A. Mol. Cell. Biol. 2001; 21: 4544-4552Crossref PubMed Scopus (71) Google Scholar). The Gal-luciferase reporter gene pFA-Jun and pFA-Elk-1 fusion vectors for analysis of JNK and P42/p44, respectively, were obtained from Stratagene (La Jolla, CA). Transient Transfections and Reporter Gene Assays—HEK293 cells (2 × 104) were seeded in 96-well plates 24 h prior to transfection. Transfections were performed with FuGENE 6 (Roche Diagnostics). Mal vectors (0.5–2 ng) and κB-luciferase were from Stratagene. NF-κB-dependent gene expression was determined using the 5× κB-luciferase reporter construct (Stratagene). Using the PathDetect transient transfection kit (Stratagene), co-transfection of pFR-luciferase in combination with Gal4-p65, pFA-Jun, or pFA-Elk-1 fusions, respectively, were used to analyze Mal/MalE190A activation of MAP kinase and p65 transactivation. The Rous sarcoma virus β-galactosidase construct was used to normalize for transfection efficiency, and pRSV empty vector was used to maintain constant DNA. Cells were left untreated or treated with 100 ng/ml Pam3Cys, 50 ng/ml K235 LPS, or 25 μg/ml pI:C for 4 h where indicated. Transfected cells were lysed using Passive lysis buffer (Promega, Madison, WI) and assayed for luciferase and β-galactosidase activity using luciferase assay reagent (Promega) or β-galactosidase assay reagent. Luminescence readings were corrected for β-Galactosidase activity and expressed as fold increase over non-stimulated control values or percentage stimulation. Statistical Analysis—Significance was evaluated using Student's t test for unpaired data. Immunoprecipitation and Western Blot Analysis—Immunoprecipitation and immunoblotting have been described previously (1Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Crossref PubMed Scopus (1005) Google Scholar). Western blot analysis was performed using anti-HA antibody (Rockland, Gilbertsville, PA) to detect Mal-Ha. Mal Associates with TRAF6 —Structural studies of TRAF6 in a complex with CD40 and TRANCE-R peptides suggested the structural determinant of the target protein contains a Pro-X-Glu-X-X-(aromatic/acidic residue) motif (18Ye H. Arron J.R. Lamothe B. Cirilli M. Kobayashi T. Shevde N.K. Segal D. Dzivenu O.K. Vologodskaia M. Yim M. Du K. Singh S. Pike J.W. Darnay B.G. Choi Y. Wu H. Nature. 2002; 418: 443-447Crossref PubMed Scopus (541) Google Scholar) for TRAF6 interaction. Analysis of the amino acid sequence of Mal indicated a putative TRAF6-binding domain at amino acid position 188–193 consisting of Pro-Pro-Glu-Leu-Arg-Phe similar to that described for IRAK and TRIF (Table I). Further analysis suggested that while Mal, TRIF, and TRAM all contain a putative TRAF6-binding motif, MyD88 does not, since the critical Glu residue (termed the P0 site) that has been found to confer specificity for TRAF6 interaction is changed to Ile in the corresponding MyD88 sequence (Table I). To test the hypothesis that Mal was therefore able to interact with TRAF6, co-precipitation experiments were carried out in HEK293T cells transiently transfected with FLAG-tagged TRAF6 and HA-tagged Mal. As shown in Fig. 1A (lane 1), we were able to detect complexes containing Mal- and TRAF6-tagged proteins by co-immunoprecipitation. To evaluate the functional regions involved in this association, the ability of Mal N-terminal region (amino acids 1–74) and the Mal-TIR region (amino acids 74–235) were also assayed for their ability to co-precipitate with TRAF6. As can be observed in lanes 2 and 3, while the N-terminal region of Mal is unable to associate with TRAF6, the Mal-TIR domain, which harbors the putative TRAF6-binding motif, does co-immunoprecipitate with TRAF6. We next mutated the putative TRAF6-binding motif in Mal by changing the glutamic acid at position 190 to alanine and tested the ability of the mutant to interact with TRAF6. Surprisingly, TRAF6 was found to still co-immunoprecipitate with MalE190A (Fig. 1A, lane 4). The mutated form of Mal did, however, affect the ability of TRAF6 to signal (Figs. 1B, 2, and 3). As can be seen in Fig. 1B, while TRAF6 overexpression induced NF-κB-dependent luciferase expression, MalE190A inhibited this effect in a dose-dependent manner. Furthermore, MalE190A had no effect the activation of NF-κB by overexpressed IKK2 at the maximal dose required to inhibit TRAF6-mediated signaling. This result indicates that the critical Glu at position 190 in Mal is required for TRAF6 to induce downstream signaling events.Fig. 2Mal interaction with TRAF6 mediates NF-κB activation and NF-κB transactivation. HEK293 cells (2 × 104) were transiently co-transfected with κB-luciferase and β-galactosidase reporter plasmids in conjunction with Mal or MalE190A. Readings are normalized for each sample as expressed κB-luciferase over constitutively expressed β-galactosidase and plotted as fold stimulation. Results are ±S.D. for triplicate determinations (n 3). HEK293 cells (2 × 104) were co-transfected with the of the with Mal and MalE190A Results are ±S.D. for triplicate determinations (n 3). HEK293 cells (2 × 104) were co-transfected with the of the pFR-luciferase ng) and (2 ng) and ng) Gal4 fusion respectively, with Mal ng) and MalE190A ng) Results are ±S.D. for triplicate determinations (n TLR2- and TLR4-mediated NF-κB Cells (2 × 104) were co-transfected with κB-luciferase and β-galactosidase reporter in conjunction with a of MalE190A (0.5–2 respectively, for HEK293 TLR2- and cells and respectively, for stably transfected HEK293 Cells were with LPS Pam3Cys or pI:C where indicated for 6 of luciferase activity for triplicate determinations (n The TRAF6-binding in Mal for Mal to type Mal was able to induce an increase in NF-κB-dependent luciferase expression in a dose-dependent manner. MalE190A, however, failed to induce luciferase expression over a corresponding dose (Fig. This result that the TRAF6-binding motif is required to mediate NF-κB activation by Mal. further signals by Mal were by mutation of the TRAF6 interaction motif. As can be seen in Fig. wild type Mal p65-mediated transactivation in a dose-dependent manner. The maximal activation of using this reporter is with the of activation previously reported in transactivation studies (20Jefferies C. Bowie A. Brady G. Cooke E.L. Li X. O'Neill L.A. Mol. Cell. Biol. 2001; 21: 4544-4552Crossref PubMed Scopus (71) Google Scholar). MalE190A failed to induce transactivation control at to wild type Mal. Mal was also unable to JNK or p42/p44 MAP kinase. As shown in Fig. overexpression of an dose of plasmid encoding Mal ng) was to drive and luciferase expression and at with that which we have previously observed using reporter (1Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Crossref PubMed Scopus (1005) Google Scholar, E. M. M. O'Neill J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). MalE190A was unable to induce luciferase expression that of control at These demonstrate that Mal mediates the activation of the MAP JNK and via its interaction with TRAF6. MalE190A TLR-mediated of we the role of interaction in TLR-mediated activation of NF-κB. HEK293 cells were transiently transfected with the luciferase reporter or them to the TLR Pam3Cys, pI:C, and As can be seen in Fig. 3, MalE190A was able to inhibit both TLR2- and TLR4-mediated activation of NF-κB-dependent luciferase expression in a dose-dependent manner. However, MalE190A was unable to inhibit activation of the κB-luciferase with studies no role for Mal in signaling M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Crossref PubMed Scopus (821) Google Scholar, K.A. Rowe D.C. Barnes B.J. Caffrey D.R. Visintin A. Latz E. Monks B. Pitha P.M. Golenbock D.T. J. Exp. Med. 2003; 198: 1043-1055Crossref PubMed Scopus (939) Google Scholar). results that the interaction between Mal and TRAF6 is necessary for the of NF-κB-dependent gene expression, upon and TLR4 stimulation. In this study we have found a feature in Mal that distinguishes it from MyD88. Mal has a TRAF6 interaction motif, which is required for Mal to The motif is for the of Mal, since a mutant form of Mal, MalE190A, which contains a mutation of a critical amino acid within the TRAF6-binding motif failed to p65-mediated transactivation of gene expression, luciferase expression, and activation of JNK and p42/p44 MAP kinase. MyD88 does not contain a putative TRAF6-binding motif and to TRAF6 via IRAK interaction with MyD88 is via domain interaction (5Janssens S. Beyaert R. Trends Biochem. Sci. 2002; 27: 474-482Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). the mutated MalE190A also as a TLR2- and TLR4-mediated activation of a NF-κB reporter results demonstrate that the interaction is required for signal transduction by TLR2 and the we a the role of Mal is to TLR2 and TLR4 with TRAF6, independent of MyD88 and IRAK that induce activation of the MAP kinase pathway and transactivation of the p65 subunit of NF-κB (Fig. 4). studies have shown that a corresponding in the TRAF6-binding motif of TRIF association (14Jiang Z. Mak T.W. Sen G. Li X. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3533-3538Crossref PubMed Scopus (309) Google Scholar), results indicated that MalE190A was still able to associate with TRAF6. however, in the the of the interaction and signaling (14Jiang Z. Mak T.W. Sen G. Li X. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3533-3538Crossref PubMed Scopus (309) Google Scholar, 15Sato S. Sugiyama M. Yamamoto M. Watanabe Y. Kawai T. Takeda K. Akira S. J. Immunol. 2003; 171: 4304-4310Crossref PubMed Scopus (590) Google Scholar). et al. (14Jiang Z. Mak T.W. Sen G. Li X. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3533-3538Crossref PubMed Scopus (309) Google Scholar) found that TRIF could no TRAF6, and overexpression of this mutant failed to drive NF-κB activation as a in NF-κB activation (14Jiang Z. Mak T.W. Sen G. Li X. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3533-3538Crossref PubMed Scopus (309) Google Scholar). Sato et al. (15Sato S. Sugiyama M. Yamamoto M. Watanabe Y. Kawai T. Takeda K. Akira S. J. Immunol. 2003; 171: 4304-4310Crossref PubMed Scopus (590) Google Scholar) a of association between TRIF and TRAF6 by the mutation of all three putative TRAF6-binding motifs in TRIF, with the of the Furthermore, reported that the mutant displayed a of NF-κB activation (15Sato S. Sugiyama M. Yamamoto M. Watanabe Y. Kawai T. Takeda K. Akira S. J. Immunol. 2003; 171: 4304-4310Crossref PubMed Scopus (590) Google Scholar), and the TRIF were required to inhibit NF-κB activation to a similar to that by et al. (14Jiang Z. Mak T.W. Sen G. Li X. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3533-3538Crossref PubMed Scopus (309) Google Scholar). The to the to inhibit association and NF-κB activation suggested that the of an adapter as TRIF affect TRAF6 association and NF-κB of the association of MalE190A and TRAF6 a similar regions of Mal effect TRAF6 association that is not required for signal transduction. for the binding of Mal and TRAF6 in the absence of the P0 Glu is that the two and amino acids of MalE190A are still able to TRAF6 in via as by et al. (18Ye H. Arron J.R. Lamothe B. Cirilli M. Kobayashi T. Shevde N.K. Segal D. Dzivenu O.K. Vologodskaia M. Yim M. Du K. Singh S. Pike J.W. Darnay B.G. Choi Y. Wu H. Nature. 2002; 418: 443-447Crossref PubMed Scopus (541) Google Scholar) for TRANCE-R and TRAF6. However, the absence of the critical P0 Glu with TRAF6 TRAF6 unable to While study that Mal interaction with TRAF6 is there the that Mal interaction with IRAK Dunne and A. also to interaction with TRAF6 as is the with MyD88. The ability of Mal to interact with TRAF6 to TRAF6 to the signaling to that by the This NF-κB inducing a of However, the of cytokine expression in LPS-stimulated Mal-deficient macrophages (6Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Crossref PubMed Scopus (689) Google Scholar, 7Yamamoto M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Crossref PubMed Scopus (821) Google Scholar) that the role of Mal is not to the pathway in TLR4 signaling but to a and necessary with The mutant Mal was unable to drive A signal was p65-mediated transactivation of gene This is the first of Mal this and it is that one for the of NF-κB-dependent gene expression in Mal-deficient NF-κB activation is that the signal for p65-mediated transactivation Mal. While the of regulating NF-κB activity is its in the by the transactivation of NF-κB to control gene expression is of critical to regulating the G. S. G. Biochem. 2002; PubMed Scopus Google Scholar). study that the interaction between Mal and TRAF6 is necessary for this transactivation. In we demonstrate Mal as a TRAF6 protein. The ability to the interaction between Mal and TRAF6 to control signaling and NF-κB-dependent gene expression, while not with the pathway of the TLRs, which are all MyD88. We Golenbock and Fitzgerald for the gift of the cells and for

Acyl-CoA thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoA to free fatty acid and CoA and thereby regulate lipid metabolism and cellular signaling. We present a comprehensive structural and functional characterization of mouse acyl-CoA thioesterase 7 (Acot7). Whereas prokaryotic homologues possess a single thioesterase domain, mammalian Acot7 contains a pair of domains in tandem. We determined the crystal structures of both the N- and C-terminal domains of the mouse enzyme, and inferred the structure of the full-length enzyme using a combination of chemical cross-linking, mass spectrometry, and molecular modeling. The quaternary arrangement in Acot7 features a trimer of hotdog fold dimers. Both domains of Acot7 are required for activity, but only one of two possible active sites in the dimer is functional. Asn-24 and Asp-213 (from N- and C-domains, respectively) were identified as the catalytic residues through site-directed mutagenesis. An enzyme with higher activity than wild-type Acot7 was obtained by mutating the residues in the nonfunctional active site. Recombinant Acot7 was shown to have the highest activity toward arachidonoyl-CoA, suggesting a function in eicosanoid metabolism. In line with the proposal, Acot7 was shown to be highly expressed in macrophages and up-regulated by lipopolysaccharide. Overexpression of Acot7 in a macrophage cell line modified the production of prostaglandins D2 and E2. Together, the results link the molecular and cellular functions of Acot7 and identify the enzyme as a candidate drug target in inflammatory disease.

CSF-1 regulates macrophage differentiation, survival, and function, and is an attractive therapeutic target for chronic inflammation and malignant diseases. Here we describe the effects of a potent and selective inhibitor of CSF-1R-CYC10268-on CSF-1R-dependent signaling. In in vitro kinase assays, CYC10268 was active in the low nanomolar range and showed selectivity over other kinases such as Abl and Kit. CYC10268 blocked survival mediated by CSF-1R in primary murine bone marrow-derived macrophages (BMM) and in the factor-dependent cell line Ba/F3, in which the CSF-1R was ectopically expressed. CYC10268 also inhibited CSF-1 regulated signaling (Akt, ERK-1/2), gene expression (urokinase plasminogen activator, toll-like receptor 9, and apolipoprotein E), and priming of LPS-inducible cytokine production in BMM. In thioglycollate-elicited peritoneal macrophages (TEPM), which survive in the absence of exogenous CSF-1, CYC10268 impaired LPS-induced cytokine production and regulated expression of known CSF-1 target genes. These observations support the conclusion that TEPM are CSF-1 autocrine and that CSF-1 plays a central role in macrophage effector functions during inflammation. CSF-1R inhibitors such as CYC10268 provide a powerful tool to dissect the role of the CSF-1/CSF-1R signaling system in a range of biological systems and have potential for a number of therapeutic applications.

Bacterial DNA activates mouse macrophages, B cells, and dendritic cells in a TLR9-dependent manner. Although short ssCpG-containing phosphodiester oligonucleotides (PO-ODN) can mimic the action of bacterial DNA on macrophages, they are much less immunostimulatory than Escherichia coli DNA. In this study we have assessed the structural differences between E. coli DNA and PO-ODN, which may explain the high activity of bacterial DNA on macrophages. DNA length was found to be the most important variable. Double-strandedness was not responsible for the increased activity of long DNA. DNA adenine methyltransferase (Dam) and DNA cytosine methyltransferase (Dcm) methylation of E. coli DNA did not enhance macrophage NO production. The presence of two CpG motifs on one molecule only marginally improved activity at low concentration, suggesting that ligand-mediated TLR9 cross-linking was not involved. The major contribution was from DNA length. Synthetic ODN >44 nt attained the same levels of activity as bacterial DNA. The response of macrophages to CpG DNA requires endocytic uptake. The length dependence of the CpG ODN response was found to correlate with the presence in macrophages of a length-dependent uptake process for DNA. This transport system was absent from B cells and fibroblasts.

Bacterial CpG-containing (CpG) DNA promotes survival of murine macrophages and triggers production of proinflammatory mediators. The CpG DNA-induced inflammatory response is mediated via TLR9, whereas a recent study reported that activation of the Akt prosurvival pathway occurs via DNA-dependent protein kinase (DNA-PK) and independently of TLR9. We show, in this study, that Akt activation and survival of murine bone marrow-derived macrophages (BMM) triggered by CpG-containing phosphodiester oligodeoxynucleotides or CpG-containing phosphorothioate oligodeoxynucleotides was completely dependent on TLR9. In addition, survival triggered by CpG-containing phosphodiester oligodeoxynucleotides was not compromised in BMM from SCID mice that express a catalytically inactive form of DNA-PK. CpG DNA-induced survival of BMM was inhibited by the PI3K inhibitor, LY294002, but not by the MEK1/2 inhibitor, PD98059. The effect of LY294002 was specific to survival, because treatment of BMM with LY294002 affected CpG DNA-induced TNF-alpha production only modestly. Therefore, CpG DNA activates macrophage survival via TLR9 and the PI3K-Akt pathway and independently of DNA-PK and MEK-ERK.

Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is hampered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under non-denaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an individual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous sample preparation requirements. The efficacy of the screen is demonstrated on small-scale expression samples for 15 proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism.

OBJECTIVE: The optimal agent for thromboprophylaxis following arthroscopic anterior cruciate ligament reconstruction (ACLR) remains unclear, particularly in patients with a low baseline risk for venous thromboembolism (VTE). This retrospective cohort study aims to compare the effectiveness and safety of aspirin versus low molecular weight heparins (LMWHs) in this specific patient population. METHODS: We analyzed data from patients who underwent ACLR between March 2016 and March 2021, focusing on those with a low risk for VTE. High-risk individuals, identified by factors such as cardiac disease, pulmonary disease, diabetes mellitus, previous VTE, inflammatory bowel disease, active cancer, and a BMI > 40, were excluded (n = 33). Our approach included a thorough review of medical charts, surgical reports, and pre-operative assessments, complemented by telephone follow-up conducted over a 3-month period by a single investigator. We assessed the incidence of symptomatic VTE, including deep vein thrombosis and pulmonary thromboembolism, as the primary outcome. The secondary outcomes included to complications related to the surgery and thromboprophylaxis. Statistical analysis included descriptive statistics, univariate logistic regression models, and calculations of incidence rates. RESULT: In our study, 761 patients (761 knees) were included, with 458 (60.18%) receiving aspirin and 303 (39.82%) receiving LMWH. The two groups showed no significant differences in demographic factors except for age. The incidence of VTE was reported at 1.31% (10 individuals). Specifically, five patients in the aspirin group (1.09%) and five patients in the LMWH group (1.65%) developed a symptomatic VTE event (p = 0.53). Additionally, the two groups did not significantly differ in terms of other complications, such as hemarthrosis or surgical site infection (p > 0.05). Logistic regression analysis revealed no statistically significant difference in VTE risk between the two groups. CONCLUSION: This study, focusing on isolated ACLR in patients with a low baseline risk for venous thromboembolism, demonstrated that aspirin is equally effective as low molecular weight heparins for VTE prophylaxis following this surgery. LEVEL OF EVIDENCE: III.

Sessions 4 1 diet L-Threonine is an indispensable amino acid and under normal conditions is synthesised by microbes from oxaloacetate. Threonine degradation occurs

Sessions 4 1 diet L-Threonine is an indispensable amino acid and under normal conditions is synthesised by microbes from oxaloacetate. Threonine degradation occurs