Center of Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, Essen, Germany.
Center of Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, Essen, Germany.
J Biol Chem. 2022 Jun;298(6):101976. doi: 10.1016/j.jbc.2022.101976. Epub 2022 Apr 22.
The protease SPRTN degrades DNA-protein crosslinks (DPCs) that threaten genome stability. SPRTN has been connected to the ubiquitin-directed protein unfoldase p97 (also called VCP or Cdc48), but a functional cooperation has not been demonstrated directly. Here, we biochemically reconstituted p97-assisted proteolysis with purified proteins and showed that p97 targets ubiquitin-modified DPCs and unfolds them to prepare them for proteolysis by SPRTN. We demonstrate that purified SPRTN alone was unable to degrade a tightly-folded Eos fluorescent reporter protein even when Eos was crosslinked to DNA (Eos-DPC). However, when present, p97 unfolded poly-ubiquitinated Eos-DPC in a manner requiring its ubiquitin adapter, Ufd1-Npl4. Notably, we show that, in cooperation with p97 and Ufd1-Npl4, SPRTN proteolyzed unfolded Eos-DPC, which relied on recognition of the DNA-crosslink by SPRTN. In a simplified unfolding assay, we further demonstrate that p97, while unfolding a protein substrate, can surmount the obstacle of a DNA crosslink site in the substrate. Thus, our data demonstrate that p97, in conjunction with Ufd1-Npl4, assists SPRTN-mediated proteolysis of tightly-folded proteins crosslinked to DNA, even threading bulky protein-DNA adducts. These findings will be relevant for understanding how cells handle DPCs to ensure genome stability and for designing strategies that target p97 in combination cancer therapy.
蛋白酶 SPRTN 可降解威胁基因组稳定性的 DNA-蛋白质交联物 (DPC)。SPRTN 与泛素导向的蛋白质 unfoldase p97（也称为 VCP 或 Cdc48）有关联，但尚未直接证明其具有功能上的合作关系。在这里，我们使用纯化的蛋白质重新构建了 p97 辅助的蛋白水解，并表明 p97 靶向泛素修饰的 DPC 并将其展开，以准备 SPRTN 进行蛋白水解。我们证明，单独的纯化 SPRTN 甚至不能降解紧密折叠的 Eos 荧光报告蛋白，即使 Eos 与 DNA 交联（Eos-DPC）也是如此。然而，当存在时，p97 以需要其泛素接头 Ufd1-Npl4 的方式展开多泛素化的 Eos-DPC。值得注意的是，我们表明，在与 p97 和 Ufd1-Npl4 合作的情况下，SPRTN 可蛋白水解解折叠的 Eos-DPC，这依赖于 SPRTN 对 DNA 交联的识别。在简化的展开测定中，我们进一步表明，p97 虽然可以展开蛋白质底物，但可以克服底物中 DNA 交联位点的障碍。因此，我们的数据表明，p97 与 Ufd1-Npl4 一起，协助 SPRTN 介导的紧密折叠的蛋白质与 DNA 交联的蛋白水解，即使是穿入体积庞大的蛋白质-DNA 加合物。这些发现对于理解细胞如何处理 DPC 以确保基因组稳定性以及设计靶向 p97 的癌症联合治疗策略将具有重要意义。
