基于MyD88探讨新型TIR诱饵肽上调LAP作用保护脓毒症小鼠的作用机制

LC3-associated phagocytosis (LAP) of monocytes/phagocytes is a newly identified target for treatment of sepsis, but there is no targeted drug up till now. Based on our previous findings of TLR9 TIR domain, we proposed a new hypothesis that TIR decoy peptide D3 was found to promote LAP thereby protect septic mice based on MyD88. In this study, co-localization will be performed to unveil the target process of D3 in the recruitment of MyD88, then pull-down, affinity assays and molecular docking will be applied to further detect D3-target interactions. Subsequently, immunoprecipitation-MS and siRNA knock-down technology will be carried out to seek the downstream adaptors of MyD88 which participate in D3-induced LAP. Next, co-localization and siRNA knock-down experiment will be performed to seek the members involved in PI3K-III complex and NOX complex which participate in D3-induced LAP, such as Rubicon and NOX2. Finally, co-localization will be carried out to confirm the key proteins involved in the MyD88-LAP pathway which contribute to the protective activity of D3 against sepsis. The purpose of this study is to investigate the molecular mechanism of D3.

单核/吞噬细胞的LC3相关吞噬作用（LAP）是治疗细菌脓毒症的新靶点，至今尚无靶向药物。基于前期基金发现，提出“新型TIR诱饵肽D3通过MyD88上调巨噬细胞LAP作用，从而保护脓毒症小鼠”的假说。为验证该假说，首先，采用共定位实验确定D3促进MyD88招募的作用环节，进而通过pull-down、亲和力实验和分子对接模拟探讨D3与靶点TIR的相互作用及互作细节；其次，采用免疫沉淀—蛋白质谱结合siRNA干扰实验，确认参与D3作用的MyD88下游接头分子成员；再次，采用共定位结合siRNA干扰实验，确认参与D3作用的PI3K-III和NOX复合物成员如Rubicon、NOX2等；最后，利用共定位实验在大肠杆菌攻击脓毒症小鼠（野生型和MyD88敲除）中，确认参与D3作用的MyD88–LAP通路重要分子。本项目旨在阐明D3通过MyD88上调LAP从而保护脓毒症小鼠的分子机制。

单核/吞噬细胞的LC3相关吞噬作用（LAP）是治疗细菌脓毒症的新靶点，至今尚无靶向药物。基于前期发现，提出“新型TIR诱饵肽D3通过MyD88上调巨噬细胞LAP作用，从而保护脓毒症小鼠”的假说。为验证该假说，本项目开展了以下研究：首先，采用共定位实验确定D3促进MyD88招募的作用环节，进而通过免疫沉淀、亲和力实验和分子对接模拟探讨D3与靶点TIR的相互作用及互作细节；其次，采用免疫沉淀结合siRNA干扰实验，确认参与D3作用的MyD88下游接头蛋白；再次，采用免疫印迹、共定位结合siRNA干扰实验，确认参与D3作用的PI3K-III复合物和Nox2复合物成员；最后，利用体内荧光示踪及共定位实验，确认参与D3作用的MyD88–LAP通路重要分子。结果表明，D3的直接作用靶点为MyD88，其通过特异性结合MyD88（KD = 29.4 μM），促进细菌招募衔接蛋白MyD88，继而依次招募下游接头分子IRAK4和TRAF6，及PI3K-III复合物（关键分子为PI3K-III、Beclin1）、Nox2复合物（关键分子为Nox2）及两个复合物的共同成员Rubicon，从而上调巨噬细胞LAP通路，促进细菌的吞噬和清除，最终发挥对大肠杆菌攻击脓毒症小鼠的保护作用。本项目在脓毒症小鼠中，阐明了大肠杆菌LAP通路的重要环节及关键分子，并发现D3通过MyD88上调LAP从而保护脓毒症小鼠的分子机制，为脓毒症防治提供了候选药物。