小分子配体与hnRNP A1的相互作用及其调控活化T细胞的研究

Based on our previous findings obtained in the former key project of NSFC, in which we found the target protein hnRNP A1 of Astilbin,a flavone, and elucidated the role of hnRNP A1 in the transformation of T cells from activation to apoptosis, we want to optimize the small molecule ligands of hnRNP A1 in the aspects of compounds' structure and their binding to A1 from the non-chromone derivatives of Astilbin. Then we will examine the effects of dificiency of hnRNP A1 on Akt signaling and p38 signaling in the activation of T cells and on the transformation of activated T cells to apoptosis by using the hnRNP A1-conditional knocked out mice and its T cells; invstigatate the binding domain to hnRNP A1 of the optimized compound using various mutants such as S192A, S199A, S192D, S199D, L102A, and K179A of hnRNP A1; and establish new assays for evaluating compound-hnRNP A1 interaction. Furthermore, we will examine the mechanisms underlying the apoptosis induction of the selected compound via promoting hnRNP A1-mediated apoptosis of activated T cells and the therapeutic efficacy of the compound on experimental autoimmune encephalomyelitis and collagen-induced arthritis. By this project, we will provide a reasonable explanation for the transformation of activated T cells into apoptosis and confirm the role of the event involved in the transformation as the target of novel immunosuppressive agents. This study will also be beneficial for finding a new lead compound or drug canididate.

基于前一自然基金重点项目发现小分子Astilbin的靶蛋白核不均一核糖核白A1(hnRNP A1，以下称A1）及其介导的T细胞从活化向凋亡转向事件，本项目拟选择Astilbin的非色酮衍生物，从结构及与A1结合的角度优化A1的小分子配体；在A1条件性敲除小鼠及其T细胞上，观察A1缺失对T细胞活化中Akt、p38通路的影响及细胞从活化向凋亡转换的影响；借助A1的非磷酸化突变S192A、S199A，拟磷酸化突变S192D、S199D和小分子结合位点突变L102A、K179A等，探讨小分子与A1结合的分子基础，建立小分子与A1蛋白功能域相互作用的评价方法；探讨小分子通过A1诱导活化T细胞凋亡的分子机制及其对实验性脑脊髓炎、胶原性关节炎等的治疗作用。为活化T细胞的凋亡转向提供新的理论诠释，明确A1介导的活化T细胞向凋亡转向的事件作为免疫抑制剂新靶标的地位，为新型先导化合物或候选药物的发现打下基础。

我们的前期研究发现了小分子化合物Astilbin的靶蛋白——核不均一核糖核蛋白A1(hnRNP A1)，并证实其可介导T细胞从活化转向凋亡。在此基础上，本项目合成了多种Astilbin衍生物，优化了hnRNP A1 的小分子配体；借助hnRNP A1 的截短变体(J1-J4)和位点突变体(L102A、K179A)等，探讨了小分子与hnRNP A1 结合的分子基础，结果提示hnRNP A1的RGG-M9-F domain应该是这些小分子的主要结合域；进一步阐明了Astilbin诱导活化T细胞转向凋亡的机理，即hnRNP A1在Astilbin的作用下向线粒体转位，并与线粒体膜孔道蛋白VDAC结合，从而开启线粒体凋亡通路；在T细胞上敲除hnRNP A1能够显著抑制Astilbin的选择性诱导活化T 细胞凋亡的作用，说明Astilbin的选择性作用系通过靶向hnRNP A1而实现；在T 细胞条件性敲除 hnRNP A1小鼠上，实验性脑脊髓炎的发病和炎症浸润均明显降低。此外，我们还研究了巨噬细胞中hnRNP A1的功能，发现hnRNP A1能够调控巨噬细胞M1极化，进而影响脂肪组织炎症状态和整体的胰岛素抵抗水平。通过本项目的研究，明确了 hnRNP A1 的小分子可靶向性，其介导的活化 T 细胞向凋亡转向作用可作为免疫抑制剂的新型靶标，本项目针对其小分子配体的研究为先导化合物或候选药物的发现打下了基础。已发表SCI论文9篇，其中影响因子大于5的有4篇，待发表数篇。