基于编码非天然氨基酸研究PD-L1蛋白翻译后调控机制

The underling mechanism of drug resistance to PD-1/PD-L1 immunotherapy is still unclear. Researches have indicated that post-translational regulation of PD-L1 protein plays key roles in cancer immunotherapy. We will employ the genetic code expansion technology to reveal the mechanism of PD-L1 regulation on protein level. In our preliminary study, we incorporated unnature amino acids (UAAs) with reactive function group into PD-L1 precise position to covalent crosslinking transient and weak interaction proteins in vivo, and identified multiple PD-L1 interaction proteins (PdlDBP) that involved in protein regulation. Based on these findings, we propose to further investigate how these PdlDBP impact PD-L1 protein by studying PD-L1 post-translational modification, stability, localization and cell cycle-dependent expression in PdlDBP defective and over-expressed mutants. Using RNA interference, photocaged UAAs and post-translational modification UAAs, we are aiming to reveal the function and mechanism of PdlDBP in vivo. Studies in this proposal are expected to elucidate novel mechanism of cancer resistance to PD-1/PD-L1 immunotherapy, and potentially provide novel targets for cancer therapy and combination therapies strategy for PD-1/PD-L1 blockade.

PD-1/PD-L1免疫治疗的耐药性机制仍不十分清楚。研究显示PD-L1蛋白翻译后调控在免疫治疗中发挥关键作用。基于此,课题将利用遗传编码非天然氨基酸技术研究PD-L1蛋白层面的调控机制。我们前期通过PD-L1蛋白精确位点引入反应性非天然氨基酸（UAAs），利用共价交联方式富集瞬时及弱蛋白相互作用，鉴定了活体肿瘤细胞内参与翻译后修饰的PD-L1相互作用蛋白（PdlDBP）多个。本项目拟进一步利用shRNA干扰、遗传编码翻译后修饰UAAs及光致降解UAAs等手段，研究PdlDBP缺失和过表达对PD-L1翻译后修饰、稳定性、空间定位及周期性表达等的影响，阐明PdlDBP的生物学功能和作用机制，并探索PdlDBP在PD-L1免疫治疗中的作用。预期我们的研究将揭示PD-1/PD-L1药物耐药性的新机制，并有望发现新的肿瘤治疗靶点，为联合治疗提供新思路。

肿瘤免疫治疗是当前肿瘤治疗领域中最具前景的方法之一。PD-1/PD-L1相关耐药性机制仍不十分清楚。项目在建立高效引入非经典氨基酸的哺乳动物细胞系基础上，在活体肿瘤细胞内发现并鉴定了多个新的PD-L1的相互作用蛋白(PdlDBPs)，深入揭示了PD-L1二聚化、PGRMC1、PdlDBP1调控PD-L1蛋白的作用及机制，丰富了PD-L1的翻译后水平调控机制，为肿瘤的联合疗法提供新思路。