对膜蛋白的相互作用网络进行分析对于理解其调控机制和功能特性至关重要，但这仍然是一项具有挑战性的任务。在本研究中，我们通过遗传修饰交联剂、串联变性纯化和蛋白质组学的方法，为抑制T细胞活性的癌细胞表面蛋白PD-L1添加了交互伴侣。特定位点修饰的交联剂在生理条件下介导了相互作用的共价捕获，并使得PD-L1复合物能够耐受膜蛋白的严苛提取条件。随后的实验鉴定了潜在的PD-L1相互作用候选者，并验证了膜相关孕激素受体组分1（PGRMC1）作为哺乳动物细胞中PD-L1的新型相互作用伴侣。重要的是，我们证明了PGRMC1通过调节GSK3β介导的PD-L1降解在癌细胞中正向调控PD-L1表达。此外，PGRMC1的敲除显著增强了T细胞对癌细胞的细胞毒作用。总之，我们的研究阐明了PD-L1的相互作用组，并发现了PD-L1调控机制中的一个新参与者。版权所有 © 2023 Elsevier Ltd. 保留所有权利。

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.Copyright © 2023 Elsevier Ltd. All rights reserved.