基于小分子化合物SND67发现的TRAF6分子开关样作用驱动巨噬细胞表型转换

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.

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。

作为天然免疫系统的重要组成成分，，巨噬细胞的功能表型变化在众多炎症性疾病中起到至关重要的作用。因此，探究巨噬细胞极化的调控机制，寻找选择性的干预手段，将为炎症的诊断和治疗打下基础。.本项目筛选了一系列化合物，首次发现青藤碱衍生物SND67，可诱导巨噬细胞表型从M1向M2转化。机制研究证实，在SND67作用下，TRAF6与IRF5的结合被抑制，导致IRF5信号通路阻断、巨噬细胞的M1极化减弱。同时，SND67促进TRAF6与IRF4的结合，并且增强IRF4的泛素化水平。进一步通过胞内热迁移、Pulldown、SPR等多种方法证实，SND67能够与TRAF6蛋白直接结合，即SND67发挥调节巨噬细胞极化表型的作用是通过直接靶向TRAF6实现的。另一方面，通过对SND67结构类似物的筛选，我们发现SND67 的A环和C环均对巨噬细胞的表型调控作用至关重要，A环添加长链取代基或者C环取代基可能对SND67 与TRAF6的相互作用起到阻碍作用。借助于系列TRAF6截短体与突变体蛋白，揭示了SND67通过靶向调控TRAF6改变巨噬细胞极化表型的机制。由此得出结论，在SND67 作用下，TRAF6 构象发生了一定变化，从而使得与IRF4的亲和性增强，改变了下游信号转导过程，最终导致致炎的M1型巨噬细胞转为抗炎的M2型巨噬细胞。体内实验也证实了SND67具有良好的抗炎功效，可以显著缓解胶原诱导的小鼠类风湿性关节炎。给予SND67 的小鼠临床评分以及发病率下降。病理分析显示，给药组小鼠关节的骨破坏被抑制，炎症细胞浸润减少。血清学检测则表明，小鼠血液中Ⅱ型胶原抗体和促炎因子均被显著抑制。.因此，本项目基于对小分子化合物SND67作用机制的研究，发现TRAF6 介导的“分子开关”样作用对巨噬细胞极化的新型调控方式。SND67的独特作用最终表现为通过合理干预某些关键步骤扭转已偏移的极化失衡，这种分子开关转换的思想为今后相关药物的开发提供了新的方向。