title: SARM modulates MyD88-mediated TLR activation through BB-loop dependent TIR-TIR interactions.
abstract: Toll-like receptors (TLRs) recognise invading pathogens and initiate an innate immune response by recruiting intracellular adaptor proteins via heterotypic Toll/interleukin-1 receptor (TIR) domain interactions. Of the five TIR domain-containing adaptor proteins identified, Sterile α- and armadillo-motif-containing protein (SARM) is functionally unique; suppressing immune signalling instead of promoting it. Here we demonstrate that the recombinantly expressed and purified SARM TIR domain interacts with both the major human TLR adaptors, MyD88 and TRIF. A single glycine residue located in the BB-loop of the SARM TIR domain, G601, was identified as essential for interaction. A short peptide derived from this motif was also found to interact with MyD88 in vitro. SARM expression in HEK293 cells was found to significantly suppress lipopolysaccharide (LPS)-mediated upregulation of inflammatory cytokines, IL-8 and TNF-α, an effect lost in the G601A mutant. The same result was observed with cytokine activation initiated by MyD88 expression and stimulation of TLR2 with lipoteichoic acid (LTA), suggesting that SARM is capable of suppressing both TRIF- and MyD88- dependent TLR signalling. Our findings indicate that SARM acts on a broader set of target proteins than previously thought, and that the BB-loop motif is functionally important, giving further insight into the endogenous mechanisms used to suppress inflammation in immune cells.
authors.affiliation: Department of Life Sciences, Imperial College London, SW7 2AZ, UK; Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117543, Singapore. Electronic address: e.carlsson11@imperial.ac.uk.
authors.forename: Emil
authors.initials: E
authors.lastname: Carlsson
authors.affiliation: Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117543, Singapore. Electronic address: dbsdjl@nus.edu.sg.
authors.forename: Jeak Ling
authors.initials: JL
authors.lastname: Ding
authors.affiliation: Department of Life Sciences, Imperial College London, SW7 2AZ, UK. Electronic address: b.byrne@imperial.ac.uk.
authors.forename: Bernadette
authors.initials: B
authors.lastname: Byrne
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D051186:Armadillo Domain Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D018613:Microscopy, Confocal; D009154:Mutation; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017472:Receptors, Interleukin-1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013682:Teichoic Acids / Q000494:pharmacology; D051193:Toll-Like Receptors / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D051186:Armadillo Domain Proteins; D003598:Cytoskeletal Proteins; D016209:Interleukin-8; D008070:Lipopolysaccharides; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D017472:Receptors, Interleukin-1; C435689:SARM1 protein, human; C470409:TICAM1 protein, human; D013682:Teichoic Acids; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; C009900:lipoteichoic acid
keywords: Immunomodulation; Innate immunity; Sterile α­ and armadillo­motif­containing protein (SARM); TIR domain; TLR signalling