From the Department of Biomedicine and Prevention, University of Rome "Tor Vergata", 00133 Rome, Italy,; the Laboratory of Neuroembryology and.
Laboratory of Neuroimmunology, Santa Lucia Foundation, 00143 Rome, Italy.
J Biol Chem. 2013 Oct 18;288(42):30210-30222. doi: 10.1074/jbc.M113.490946. Epub 2013 Sep 9.
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.
髓样分化因子 88（MyD88）是一种衔接蛋白，可转导 Toll 样受体（TLR）和白介素-1 受体（IL-1R）引发的细胞内信号通路。MyD88 由一个 N 端死亡结构域（DD）和一个 C 端 Toll/IL-1 受体（TIR）结构域组成，两者之间被一个短区域隔开。配体结合后，TLR/IL-1R 异源或同源二聚体通过各自的 TIR 结构域招募 MyD88。然后，MyD88 通过其 DD 和 TIR 结构域寡聚化，并与白介素-1 受体相关激酶（IRAKs）相互作用形成 Myddosome 复合物。我们对位于 MyD88-TIR 结构域暴露区域的保守残基进行了定点突变，并分析了突变对 MyD88 信号转导的影响。研究表明，突变 Glu（183）、Ser（244）和 Arg（288）会损害 MyD88-TIR 结构域的同源二聚化、IRAKs 的募集以及 NF-κB 的激活。此外，过表达包含 Glu（183）残基的两个绿色荧光蛋白（GFP）标记的 MyD88 小蛋白（GFP-MyD88151-189 和 GFP-MyD88168-189）也能再现这些效应。重要的是，这些显性负性 MyD88 小蛋白的表达与内源性 MyD88 的功能竞争，并干扰 TLR2/4 介导的人单核细胞系（THP-1）和人原代单核细胞衍生的树突状细胞中的反应。因此，我们的研究确定了 TIR 结构域中对免疫细胞中 MyD88 同源二聚化和 TLR 信号转导至关重要的新残基。
