Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain
Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain
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DOI：
10.1073/pnas.1313575110
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发表时间：
2013-11-19
影响因子：
11.1
通讯作者：
Toshchakov, Vladimir Y.
中科院分区：
文献类型：
--
作者：
Piao, Wenji;Ru, Lisa W.;Toshchakov, Vladimir Y.
含有Toll/IL-1 R抗性（TIR）结构域的衔接子诱导IFN-β（TRIF）是一种Toll样受体（TLR）衔接子，其通过激活IFN调节因子3和NF κ B介导I型干扰素的MyD 88非依赖性诱导。我们已经检查了源自TRIF TIR结构域的肽抑制TLR 4的能力。除了先前鉴定的BB环肽（TF 4）之外，衍生自TRIF TIR的推定螺旋B的肽（TF 5）强烈抑制野生型细胞中LPS诱导的细胞因子和MAPK活化。TF 5不能抑制LPS诱导的细胞因子和激酶在TRIF缺陷的永生化骨髓源性巨噬细胞中的活化，但在MyD 88敲除细胞中完全抑制。TF 5不阻断由TLR 2、TLR 3、TLR 9或视黄酸诱导基因1/黑素瘤分化相关蛋白5激动剂诱导的巨噬细胞活化。免疫沉淀试验表明，TF 4结合TLR 4，但不TRIF相关衔接分子（TRAM），而TF 5结合TRAM强烈和TLR 4在较小程度上。尽管TF 5阻止TRIF与TRAM和TLR 4的共免疫沉淀，但TRIF B螺旋残基的定点突变选择性地影响TRIF-TRAM共免疫沉淀，因为这些突变不阻断TRIF-TLR 4缔合。这些结果表明，折叠的TRIF TIR结构域通过TRIF B螺旋区与TRAM缔合，但使用不同的区域用于TRIF-TLR 4缔合。然而，B螺旋肽TF 5可以与TRAM或TLR 4缔合。在TLR 4驱动的炎症小鼠模型中，TF 5降低了血浆细胞因子水平，并保护小鼠免受致命的LPS攻击。我们的数据确定了与TLR 4和TRAM相互作用的重要TRIF位点，并证明TF 5是一种有效的TLR 4抑制剂，具有作为人类脓毒症候选治疗药物的显著潜力。
Toll/IL-1R resistance (TIR) domain-containing adapter-inducing IFN-beta (TRIF) is a Toll-like receptor (TLR) adapter that mediates MyD88-independent induction of type I interferons through activation of IFN regulatory factor 3 and NF kappa B. We have examined peptides derived from the TRIF TIR domain for ability to inhibit TLR4. In addition to a previously identified BB loop peptide (TF4), a peptide derived from putative helix B of TRIF TIR (TF5) strongly inhibits LPS-induced cytokine and MAPK activation in wild-type cells. TF5 failed to inhibit LPS-induced cytokine and kinase activation in TRIF-deficient immortalized bone-marrow-derived macrophage, but was fully inhibitory in MyD88 knockout cells. TF5 does not block macrophage activation induced by TLR2, TLR3, TLR9, or retinoic acid-inducible gene 1/melanoma differentiation-associated protein 5 agonists. Immunoprecipitation assays demonstrated that TF4 binds to TLR4 but not TRIF-related adaptor molecule (TRAM), whereas TF5 binds to TRAM strongly and TLR4 to a lesser extent. Although TF5 prevented coimmunoprecipitation of TRIF with both TRAM and TLR4, site-directed mutagenesis of the TRIF B helix residues affected TRIF-TRAM coimmunoprecipitation selectively, as these mutations did not block TRIF-TLR4 association. These results suggest that the folded TRIF TIR domain associates with TRAM through the TRIF B helix region, but uses a different region for TRIF-TLR4 association. The B helix peptide TF5, however, can associate with either TRAM or TLR4. In a mouse model of TLR4-driven inflammation, TF5 decreased plasma cytokine levels and protected mice from a lethal LPS challenge. Our data identify TRIF sites that are important for interaction with TLR4 and TRAM, and demonstrate that TF5 is a potent TLR4 inhibitor with significant potential as a candidate therapeutic for human sepsis.