title: Mutational analysis identifies residues crucial for homodimerization of myeloid differentiation factor 88 (MyD88) and for its function in immune cells.
abstract: Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu(183), Ser(244), and Arg(288) impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88151-189 and GFP-MyD88168-189), comprising the Glu(183) residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.
authors.affiliation: From the Department of Biomedicine and Prevention, University of Rome "Tor Vergata", 00133 Rome, Italy,; the Laboratory of Neuroembryology and.
authors.forename: Maria
authors.initials: M
authors.lastname: Loiarro
authors.affiliation: Laboratory of Neuroimmunology, Santa Lucia Foundation, 00143 Rome, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Volpe
authors.affiliation: the Research & Development Sigma-Tau S.p.A., 00040 Pomezia (Rome), Italy, and.
authors.forename: Vito
authors.initials: V
authors.lastname: Ruggiero
authors.affiliation: the Research & Development Sigma-Tau S.p.A., 00040 Pomezia (Rome), Italy, and.
authors.forename: Grazia
authors.initials: G
authors.lastname: Gallo
authors.affiliation: the Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Furlan
authors.affiliation: the Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Maiorino
authors.affiliation: Laboratory of Neuroimmunology, Santa Lucia Foundation, 00143 Rome, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Battistini
authors.affiliation: From the Department of Biomedicine and Prevention, University of Rome "Tor Vergata", 00133 Rome, Italy,; the Laboratory of Neuroembryology and. Electronic address: claudio.sette@uniroma2.it.
authors.forename: Claudio
authors.initials: C
authors.lastname: Sette
mesh_terms: D019943:Amino Acid Substitution; D045744:Cell Line, Tumor; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D009000:Monocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology*; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D053592:Interleukin-1 Receptor-Associated Kinases
keywords: Cellular Immune Response; Mutagenesis; MyD88; Signal Transduction; Toll IL-1 Receptor (TIR) Domain; Toll-like Receptor (TLR)
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