Inducible Degradation of Target Proteins through a Tractable Affinity-Directed Protein Missile System. Issue 9 (17th September 2020)

• Record Type: Journal Article
• Authors: Simpson, Luke M.; Macartney, Thomas J.; Nardin, Alice; Fulcher, Luke J.; Röth, Sascha; Testa, Andrea; Maniaci, Chiara; Ciulli, Alessio; Ganley, Ian G.; Sapkota, Gopal P.
• Is Part Of: Cell chemical biology. Volume 27:Issue 9(2020)
• Page Start: 1164
• Page End: 1180.e5
• Publication Date: 2020-09-17
• Subjects: AdPROM -- PROTAC -- HaloPROTAC -- nanobody -- monobody -- targeted proteolysis -- ULK1 -- FAM83D -- SGK3 -- RAS; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2020.06.013
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: 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. Graphical Abstract: Highlights: Ligand (L)-inducible AdPROM consists of Halo conjugated to a target protein binder Target protein is degraded with HaloPROTAC in cells expressing L-AdPROM construct HaloPROTAC-mediated target protein degradation using L-AdPROM system is reversible Degradation using HaloPROTAC L-AdPROM impacts target protein biological function Abstract : Simpson et al . combine Halo-tag/VHL-recruiting proteolysis-targeting chimera (HaloPROTAC) technology with high-affinity small polypeptide binders to develop a ligand-inducible degradation system (L-AdPROM) for target proteins of interest (POI). In cells expressing a Halo-POI binder, target protein degradation occurs only in the presence of the HaloPROTAC.