Xie Guojiao, Gao Lin, Lu Renee, Tian Linxia, Zheng Tiantian, Li Xinning, Dang Yongjun, Cole Philip A, Yu Xian, Jiang Hanjie, Chen Zan
Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention, Ministry of Education, College of Pharmacy, Phase I Clinical Trial Center, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02215, USA.
Structure. 2025 Aug 7;33(8):1304-1313.e4. doi: 10.1016/j.str.2025.05.005. Epub 2025 Jun 4.
As a key immune checkpoint ligand, PD-L1 is a critical target in cancer immunotherapy. While multiple E3 ubiquitin ligases including CRL3, ARIH1, and NEDD4 have been implicated in PD-L1 degradation, the precise enzymatic mechanisms remain unclear. In this study, we systematically compared the enzymatic activities of CRL3, ARIH1, and NEDD4 ligases toward the cytoplasmic domain of PD-L1 through in vitro reconstitution with purified components. ARIH1, rather than CRL3, independently ubiquitinates PD-L1. We reveal a mechanism where ARIH1 acts as a substrate receptor and cooperates with CRLs to catalyze PD-L1 ubiquitination. We also biochemically validated the E3 ligase activity of the NEDD4 family E3s toward PD-L1. By using liposomes in the enzymatic assays, we show that phosphorylation enhances PD-L1 ubiquitination through disrupting its membrane association. Our study provides further biochemical insights into PD-L1 ubiquitination, which advances our understanding of the molecular details of PD-L1 regulation.
作为一种关键的免疫检查点配体，程序性死亡配体1（PD-L1）是癌症免疫治疗中的一个关键靶点。虽然包括Cullin-RING连接酶3（CRL3）、含锚蛋白重复序列的E3泛素连接酶1（ARIH1）和神经前体细胞表达发育下调蛋白4（NEDD4）在内的多种E3泛素连接酶都与PD-L1的降解有关，但具体的酶促机制仍不清楚。在本研究中，我们通过用纯化的组分进行体外重组，系统地比较了CRL3、ARIH1和NEDD4连接酶对PD-L1胞质结构域的酶活性。ARIH1而非CRL3能独立地使PD-L1泛素化。我们揭示了一种机制，即ARIH1作为底物受体并与Cullin-RING连接酶（CRLs）协同催化PD-L1的泛素化。我们还通过生物化学方法验证了NEDD4家族E3对PD-L1的E3连接酶活性。通过在酶促测定中使用脂质体，我们表明磷酸化通过破坏PD-L1与膜的结合来增强其泛素化。我们的研究为PD-L1的泛素化提供了进一步的生物化学见解，这加深了我们对PD-L1调控分子细节的理解。
