title: The TLR signaling adaptor TRAM interacts with TRAF6 to mediate activation of the inflammatory response by TLR4.
abstract: TLRs act as sentinels in professional immune cells to detect and initiate the innate immune response to pathogen challenge. TLR4 is a widely expressed TLR, responsible for initiating potent immune responses to LPS. TRAM acts to bridge TLR4 with TRIF, orchestrating the inflammatory response to pathogen challenge. We have identified a putative TRAF6-binding motif in TRAM that could mediate a novel signaling function for TRAM in TLR4 signaling. TRAM and TRAF6 association was confirmed by immunoprecipitation of endogenous, ectopically expressed and recombinant proteins, which was ablated upon mutation of a key Glu residue in TRAM (TRAM E183A). TRAF6 and TRAM were observed colocalizing using confocal microscopy following ectopic expression in cells and the ability of TRAM and TRAM E183A to activate luciferase-linked reporter assays was determined in HEK293 and TRAF6-deficient cells. Importantly, TRAM-deficient macrophages reconstituted with TRAM E183A display significantly reduced inflammatory TNF-α, IL-6, and RANTES protein production compared with WT TRAM. These results demonstrate a novel role for TRAM in TLR4-mediated signaling in regulating inflammatory responses via its interaction with TRAF6, distinct from its role as a bridging adaptor between TLR4 and TRIF.
authors.affiliation: Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, Melbourne, Victoria, Australia; Department of Biochemistry, University of Cambridge, United Kingdom;
authors.forename: Brett
authors.initials: B
authors.lastname: Verstak
authors.affiliation: Immunology Research Centre, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland;
authors.forename: Julianne
authors.initials: J
authors.lastname: Stack
authors.affiliation: School of Chemistry and Molecular Biosciences, Australian Infectious Diseases Research Centre, and Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia;
authors.forename: Thomas
authors.initials: T
authors.lastname: Ve
authors.affiliation: Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, Melbourne, Victoria, Australia;
authors.forename: Matthew
authors.initials: M
authors.lastname: Mangan
authors.affiliation: Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, Melbourne, Victoria, Australia;
authors.forename: Kathryn
authors.initials: K
authors.lastname: Hjerrild
authors.affiliation: School of Chemistry and Molecular Biosciences, Australian Infectious Diseases Research Centre, and Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia;
authors.forename: Jannah
authors.initials: J
authors.lastname: Jeon
authors.affiliation: Institute of Innate Immunity, University Hospitals, University of Bonn, Germany; and.
authors.forename: Rainer
authors.initials: R
authors.lastname: Stahl
authors.affiliation: Institute of Innate Immunity, University Hospitals, University of Bonn, Germany; and Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Eicke
authors.initials: E
authors.lastname: Latz
authors.affiliation: Department of Biochemistry, University of Cambridge, United Kingdom;
authors.forename: Nick
authors.initials: N
authors.lastname: Gay
authors.affiliation: School of Chemistry and Molecular Biosciences, Australian Infectious Diseases Research Centre, and Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia;
authors.forename: Bostjan
authors.initials: B
authors.lastname: Kobe
authors.affiliation: Immunology Research Centre, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland;
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Bowie
authors.affiliation: Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, Melbourne, Victoria, Australia; ashley.mansell@monash.edu.
authors.forename: Ashley
authors.initials: A
authors.lastname: Mansell
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D033942:Adaptor Proteins, Vesicular Transport / Q000037:antagonists & inhibitors / Q000235:genetics; D020816:Amino Acid Motifs; D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis; D005347:Fibroblasts; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D007249:Inflammation / Q000503:physiopathology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D020125:Mutation, Missense; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D025941:Protein Interaction Mapping; D034741:RNA, Small Interfering / Q000494:pharmacology; D018123:Receptors, Interleukin / Q000172:deficiency; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D048029:TNF Receptor-Associated Factor 6 / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D051197:Toll-Like Receptor 4 / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D016207:Cytokines; D008070:Lipopolysaccharides; D010446:Peptide Fragments; D034741:RNA, Small Interfering; D018123:Receptors, Interleukin; D011993:Recombinant Fusion Proteins; C470409:TICAM1 protein, human; C480228:TICAM2 protein, human; C495345:TLR4 protein, human; D048029:TNF Receptor-Associated Factor 6; C476416:Ticam2 protein, mouse; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4
keywords: Toll-like receptors; inflammation; innate immunity; signal transduction