Major yolk protein and HSC70 are essential for the activation of the TLR pathway via interacting with MyD88 in Apostichopus japonicus
Major yolk protein and HSC70 are essential for the activation of the TLR pathway via interacting with MyD88 in Apostichopus japonicus
复制标题
主要卵黄蛋白和 HSC70 通过与刺参中的 MyD88 相互作用，对于 TLR 通路的激活至关重要
DOI：
10.1016/j.abb.2019.02.019
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发表时间：
2019-04-15
影响因子：
3.9
通讯作者：
Zhao，Xuelin
中科院分区：
文献类型：
--
作者：
Lv，Zhimeng;Li，Chenghua;Zhao，Xuelin
Toll 级联在动物针对传染性病原体的先天免疫中发挥着重要作用。 Toll Cascade 作为一种古老的免疫防御系统，在不同物种之间是保守的。不同物种之间TLR通路的激活通常涉及不同的相互作用蛋白。该途径的核心成员已在多种生物体中得到证实，包括海洋无脊椎动物海参。然而，这些蛋白质并不作为单个孤立的实体发挥作用，而是与细胞生物分子环境中的其他蛋白质参与动态物理网络。为了填补这方面的知识空白，通过 GST 下拉和质谱分析，刺参中主要卵黄蛋白 (MYP) 和热休克同源蛋白 70 (HSC70) 的两个新成员被鉴定为骨髓分化因子 88 (MyD88) 相互作用蛋白。它们的相互作用通过免疫共沉淀分析得到进一步证实。共聚焦显微镜分析显示这三种蛋白质共定位于细胞质中。功能实验表明，通过 siRNA 干扰测定，每种蛋白质单独可以抑制培养的体腔细胞核中 NF-κB 的易位，表明这三种蛋白质作为复合物发挥作用。为了更好地解决这些相互作用，我们使用 ZDOCK 对接平台来模拟 MyD88-HSC70-MYP 复合体的结构。 MyD88 的死亡结构域在不同的空间位置与 HSC70 和 MYP 结合。 MyD88与HSC70的相互作用程度为HSC70中的K574、D591、E592和E619以及MyD88中的E75、R76、K197和R203。在MYP-MyD88模型中，MYP的K260、K452、K467和E839以及MyD88的D29、R40和E62被认为是必需位点。这些位点的位点特异性诱变表明，通过生物层干涉测定法，大多数残基是它们相互作用的关键位点，其结合常数相对于其天然对应物明显降低，其中只有MyD88突变体的K197和R203对这些相互作用没有影响。我们的结果首次证明 HSC70 和 MYP 通过与 MyD88 相互作用并激活刺参中的 TLR 通路在免疫调节中发挥作用。
The Toll cascade plays important functions in innate immunity against infectious pathogens in animals. Toll cascade as an ancient immune defender were conserved among different species. The activation of the TLR pathway between different species often involves different interacting proteins. The core members of this pathway have been well established in a wide range of organisms, including the marine invertebrate sea cucumber. However, these proteins do not function as single isolated entities but are engaged in a dynamic physical network with other proteins in the biomolecular context of a cell. To fill the knowledge gap in this context, two novel members of major yolk protein (MYP) and heat shock cognate protein 70 (HSC70) were identified as myeloid differentiation factor 88 (MyD88) interacting proteins by GST pull-down and mass spectrometry assays inApostichopus japonicus. Their interactions were further confirmed by a co-immunoprecipitation analysis. Confocal microscopy analysis revealed that these three proteins were co-localized in the cytoplasm. A functional experiment indicated that each protein alone could suppress NF-κB translocated in the nucleus in cultured coelomocytes via a siRNA interference assay, suggesting that the three proteins functioned as a complex. To better address these interactions, we used the ZDOCK docking platform to mock the structure of the MyD88-HSC70-MYP complex. The death domain of MyD88 bound to HSC70 and MYP in separate spatial positions. The extent of interaction between MyD88 and HSC70 were K574, D591, E592and E619in HSC70 and E75, R76, K197and R203in MyD88. In the MYP-MyD88 model, K260, K452, K467and E839of MYP and D29, R40and E62of MyD88 were considered essential sites. Site-specific mutagenesis of these sites showed that most residues were key sites for their interaction with distinctly reduced binding constants relative to those of their native counterparts by biolayer interferometry assays, in which only K197and R203of MyD88 mutants displayed no effect on these interactions. Our results provide the first evidence of the roles of HSC70 and MYP in immune regulation via interacting with MyD88 and activating the TLR pathway inApostichopus japonicus.