我们前期研究发现一种生物碱类化合物SND67具有调控巨噬细胞表型转换的作用，这一转换作用的机制是SDN67开启了TRAF6介导的分子开关，即SND67通过靶向TRAF6分子，引发蛋白构象改变，关闭与IRF5的结合，打开与IRF4的结合，实现巨噬细胞M1向M2转换，达到抗炎目的。本项目拟用SPR、ITC、FRET等方法验证SND67调控从TRAF6-IRF5向TRAF6-IRF4的转换；用蛋白截短、位点突变、计算机模拟等方法阐明TRAF6分子开关的结构基础，并探讨其胞内的调控机制；利用亲和柱层析和二维核磁共振明确SND67的靶蛋白为TRAF6，寻找开启TRAF6分子开关的化学基础；比较野生鼠和巨噬细胞条件性敲除TRAF6的小鼠在免疫炎症模型中的差异，探讨TRAF6分子开关在巨噬细胞极化中的重要作用，并在临床病人组织样本中加以验证。本课题为开辟免疫炎症治疗的新途径打下基础。

Our previous study found that SND67, an alkaloid compound, facilitated the phenotype shift from M1 macrophages to M2 macrophages. The underlying mechanism was that SND67 targeted to TRAF6-mediated molecular switcher, which is triggering the change of TRAF6 3D conformation, straitening TRAF6-IRF5 binding, promoting TRAF6-IRF4 binding, and finally achieving the conversion of macrophage phenotype from M1 to M2. In this project, we will elucidate the shit from TRAF6-IRF5 to TRAF6-IRF4 by various methods including SPR, ITC and FRET. We also want to clarify the molecular basis for the TRAF6 switcher by protein truncation, amino acid mutation and molecular docking to find the interaction interface of TRAF6-IRF5 and TRAF6-IRF4. Then the upstream and downstream signals in the regulation of TRAF6 molecular switcher will be investigated to provide the new insight of macrophage polarization. Affinity chromatography and two-dimensional NMR are used to clarify that TRAF6 is the main target of SND67 in macrophage phenotype shift and to elucidate binding sites of SND67 in TRAF6 sequence. On the basis of these efforts, we will design and synthesize a series of analogues of SND67 targeting TRAF6 molecular switcher and evaluate the effect of the chosen compounds on macrophage polarization. We have established the TRAF6 macrophage conditional knockout mice. We will examine the differences between wild type and Lyz2-Cre:TRAF6 flox/flox mice in collagen induced arthritis, LPS/CLP induced peritonitis or TNBS/DSS induced colitis to manifest the important role of TRAF6 molecular switcher in immuno-inflammation, and confirm it by using joint tissues and cells isolated from patients with arthritis. This study will be useful for the discovery of a novel approach to the cure of immuno-inflammation through specific regulation of macrophage polarization.