The affinity-directed protein missile (AdPROM) system utilizes specific polypeptide binders of intracellular proteins of interest (POIs) conjugated to an E3 ubiquitin ligase moiety to enable targeted proteolysis of the POI. However, a chemically tuneable AdPROM system is more desirable. Here, we use Halo-tag/VHL-recruiting proteolysis-targeting chimera (HaloPROTAC) technology to develop a ligand-inducible AdPROM (L-AdPROM) system. When we express an L-AdPROM construct consisting of an anti-GFP nanobody conjugated to the Halo-tag, we achieve robust degradation of GFP-tagged POIs only upon treatment of cells with the HaloPROTAC. For GFP-tagged POIs, ULK1, FAM83D, and SGK3 were knocked in with a GFP-tag using CRISPR/Cas9. By substituting the anti-GFP nanobody for a monobody that binds H- and K-RAS, we achieve robust degradation of unmodified endogenous RAS proteins only in the presence of the HaloPROTAC. Through substitution of the polypeptide binder, the highly versatile L-AdPROM system is useful for the inducible degradation of potentially any intracellular POI.

亲和导向蛋白导弹 (AdPROM) 系统利用与 E3 泛素连接酶部分结合的细胞内感兴趣蛋白 (POI) 的特定多肽结合剂，以实现 POI 的靶向蛋白水解。然而，更需要化学可调的 AdPROM 系统。在这里，我们使用 Halo-tag/VHL-recruiting proteolysis-targeting chimera (HaloPROTAC) 技术开发配体诱导型 AdPROM (L-AdPROM) 系统。当我们表达由与 Halo-tag 缀合的抗 GFP 纳米体组成的 L-AdPROM 构建体时，我们仅在用 HaloPROTAC 处理细胞后才能实现 GFP 标记的 POI 的稳健降解。对于 GFP 标记的 POI，ULK1、FAM83D 和 SGK3 使用 CRISPR/Cas9 用 GFP 标记敲入。通过用抗 GFP 纳米抗体代替结合 H-和 K-RAS 的单体，我们仅在 HaloPROTAC 存在的情况下实现了未修饰的内源性 RAS 蛋白的稳健降解。通过替代多肽结合剂，高度通用的 L-AdPROM 系统可用于潜在地任何细胞内 POI 的可诱导降解。