Details

Autor(en) / Beteiligte

• 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.

Titel

Inducible Degradation of Target Proteins through a Tractable Affinity-Directed Protein Missile System

Ist Teil von

• Cell chemical biology, 2020-09, Vol.27 (9), p.1164-1180.e5

Ort / Verlag

United States: Elsevier Ltd

Erscheinungsjahr

2020

Quelle

Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals

Beschreibungen/Notizen

• 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. [Display omitted] •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 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.