Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
Robarts Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.
Nat Chem Biol. 2024 Sep;20(9):1164-1175. doi: 10.1038/s41589-024-01618-0. Epub 2024 May 21.
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.
C 端到 LisH（CTLH）复合物是一种泛素连接酶复合物，通过其底物受体葡萄糖诱导降解 4（GID4）识别具有 Pro/N-降解结构域的底物，但它在人类中的功能和底物仍不清楚。在这里，我们报告了 PFI-7，一种有效的、选择性的和细胞活性的化学探针，可拮抗 Pro/N-降解结构域与人类 GID4 的结合。使用 PFI-7 进行邻近依赖性生物素化和定量蛋白质组学，可鉴定 GID4 的相互作用蛋白和 GID4 调节的蛋白。GID4 的相互作用蛋白富含核仁蛋白，包括含有 Pro/N-降解结构域的 RNA 解旋酶 DDX21 和 DDX50。我们还鉴定了一组不同的蛋白质，其细胞水平受 GID4 调节，包括 HMGCS1，一种含有 Pro/N-降解结构域的代谢酶。这些数据揭示了人类 GID4 通过结合降解和非降解功能来调节 Pro/N-降解结构域靶标。展望未来，PFI-7 将成为研究 CTLH 复合物生物学和促进靶向蛋白质降解策略发展的有价值的研究工具，这些策略可以利用 CTLH E3 连接酶活性。
