Division of Respiratory and Critical Care Medicine, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu 610041, China.
Division of Respiratory and Critical Care Medicine, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu 610041, China; Collaborative Innovation Center of Biotherapy, West China Hospital of Sichuan University, Chengdu 610041, China.
Cell Chem Biol. 2023 Nov 16;30(11):1488-1497.e5. doi: 10.1016/j.chembiol.2023.07.004. Epub 2023 Aug 3.
Profiling membrane proteins' interacting networks is crucial for understanding their regulatory mechanisms and functional characteristics, but it remains a challenging task. Here, by combining genetic incorporation of crosslinkers, tandem denatured purification, and proteomics, we added interaction partners for PD-L1, a cancer cell surface protein that inhibits T cell activity. The site-specifically incorporated crosslinker mediates the covalent capture of interactions under physiological conditions and enabled the PD-L1 complexes to withstand the harsh extraction conditions of membrane proteins. Subsequent experiments led to the identification of potential PD-L1 interaction candidates and verified membrane-associated progesterone receptor component 1 as a novel PD-L1 interaction partner in mammalian cells. Importantly, we demonstrated that PGRMC1 positively regulates PD-L1 expression by regulating GSK3β-mediated PD-L1 degradation in cancer cells. Furthermore, PGRMC1 knockdown results in dramatically enhanced T cell-mediated cytotoxicity in cancer cells. In conclusion, our study elucidated the interactome of PD-L1 and uncovered a new player in the PD-L1 regulation mechanism.
解析膜蛋白相互作用网络对于理解其调控机制和功能特征至关重要，但这仍然是一项具有挑战性的任务。在这里，我们结合了交联剂的遗传掺入、串联变性纯化和蛋白质组学，为 PD-L1 添加了相互作用伙伴，PD-L1 是一种癌细胞表面蛋白，可抑制 T 细胞活性。特异性掺入的交联剂介导了生理条件下相互作用的共价捕获，并使 PD-L1 复合物能够耐受膜蛋白的苛刻提取条件。随后的实验鉴定出了潜在的 PD-L1 相互作用候选物，并验证了膜相关孕激素受体成分 1 是哺乳动物细胞中 PD-L1 的一个新的相互作用伙伴。重要的是，我们证明了 PGRMC1 通过调节 GSK3β 介导的 PD-L1 降解来正向调节 PD-L1 的表达。此外，PGRMC1 的敲低导致癌细胞中 T 细胞介导的细胞毒性显著增强。总之，我们的研究阐明了 PD-L1 的相互作用组，并揭示了 PD-L1 调节机制中的一个新角色。
