The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.

LisH C 端 (CTLH) 复合物是一种泛素连接酶复合物，可通过其底物受体葡萄糖诱导降解 4 (GID4) 识别带有 Pro/N-降解决定子的底物，但其在人类中的功能和底物仍不清楚。在此，我们报道了 PFI-7，这是一种有效的、选择性的、具有细胞活性的化学探针，可拮抗 Pro/N-degron 与人 GID4 的结合。在邻近依赖性生物素化和定量蛋白质组学中使用 PFI-7 能够鉴定 GID4 相互作用因子和 GID4 调节蛋白。 GID4 相互作用子富含核仁蛋白，包括含有 Pro/N-degron 的 RNA 解旋酶 DDX21 和 DDX50。我们还鉴定了一个独特的蛋白质子集，其细胞水平受 GID4 调节，包括 HMGCS1（一种含有 Pro/N-degron 的代谢酶）。这些数据揭示了人类 GID4 Pro/N-degron 靶标通过降解和非降解功能的组合进行调节。展望未来，PFI-7 将成为研究 CTLH 复杂生物学并促进开发劫持 CTLH E3 连接酶活性的靶向蛋白质降解策略的宝贵研究工具。