Song Wei, Zhao Yichen, Ruggiano Annamaria, Redfield Christina, Newman Joseph A, Zhu Xiaosheng, García-Flores Marta, Cruz-Migoni Abimael, Roddan Rebecca, Pérez-Ràfols Anna, McHugh Peter J, Elliott Paul R, Ramadan Kristijan
Department of Oncology, MRC Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DS, UK.
Center for Biological Research Margarita Salas (CIB-CSIC), Spanish National Research Council, Madrid, 28040, Spain.
Nucleic Acids Res. 2025 Jul 8;53(13). doi: 10.1093/nar/gkaf638.
DNA-protein crosslinks (DPCs) are endogenous and chemotherapy-induced genotoxic DNA lesions and, if not repaired, lead to embryonic lethality, neurodegeneration, premature ageing, and cancer. DPCs are heavily polyubiquitinated, and the SPRTN protease and 26S proteasome emerged as two central enzymes for DPC proteolysis. The proteasome recognizes its substrates by their ubiquitination status. How SPRTN protease, an essential enzyme for DPC proteolysis, achieves specificity for DPCs is still not entirely clear. We found that the N-terminal SPRTN catalytic region (SprT) possesses a ubiquitin-binding domain that we named the Ubiquitin Interface of SprT Domain (USD). Using multiple biochemical, biophysical, and structural approaches, we reveal that USD binds ubiquitin chains in an avidity manner. SPRTN binding to ubiquitin chains via USD leads to ∼67-fold higher activation of SPRTN proteolysis towards polyubiquitinated DPCs than the unmodified DPCs. In contrast, the constitutive components of the replisome during unperturbed or translesional DNA synthesis, namely proliferating cell nuclear antigen (PCNA) or monoUb-PCNA, respectively, were poorly degraded, if at all, by SPRTN. This study reveals that the poly-ubiquitination of DPCs serves as the key signal for SPRTN's rapid proteolysis and determines its substrate specificity towards DPCs, rather than the replisome.
DNA-蛋白质交联（DPCs）是内源性的以及化疗诱导产生的具有基因毒性的DNA损伤，若不进行修复，会导致胚胎致死、神经退行性变、早衰和癌症。DPCs被大量多聚泛素化，SPRTN蛋白酶和26S蛋白酶体成为DPC蛋白水解的两种核心酶。蛋白酶体通过底物的泛素化状态识别它们。作为DPC蛋白水解必需酶的SPRTN蛋白酶如何实现对DPCs的特异性识别仍不完全清楚。我们发现，SPRTN的N端催化区域（SprT）拥有一个泛素结合结构域，我们将其命名为SprT结构域的泛素界面（USD）。使用多种生化、生物物理和结构方法，我们揭示USD以亲合力方式结合泛素链。通过USD与泛素链结合的SPRTN对多聚泛素化DPCs的蛋白水解激活作用比未修饰的DPCs高约67倍。相比之下，在正常或跨损伤DNA合成过程中复制体的组成成分，即增殖细胞核抗原（PCNA）或单泛素化PCNA，即使有降解，也很少被SPRTN降解。这项研究表明，DPCs的多聚泛素化作为SPRTN快速蛋白水解的关键信号，并决定其对DPCs而非复制体的底物特异性。
