Journal
CELL CHEMICAL BIOLOGY
Volume 27, Issue 9, Pages 1164-+Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2020.06.013
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Funding
- UK Medical Research Council (MRC) [MC_UU_00018/6, MC_UU_12016/3]
- Division of Signal Transduction Therapy (Boehringer-Ingelheim)
- Division of Signal Transduction Therapy (GlaxoSmithKline)
- Division of Signal Transduction Therapy (Merck-Serono)
- UK MRC Prize PhD studentship
- European Research Council (ERC) under the European Union's Seventh Framework Program [ERC-2012-StG-311460]
- Boehringer Ingelheim
- Eisai Inc.
- Nurix Inc.
- Ono Pharma
- Amphista Therapeutics
- MRC [MC_UU_12016/3, MC_UU_00018/6] Funding Source: UKRI
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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-tagNHL-recruiting proteolysis-targeting chimera (HaloPROTAC) technology to develop a ligand-inducible AdPROM (L-Ad-PROM) 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 POls only upon treatment of cells with the HaloPROTAC. For GFP-tagged POls, 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 HaIoPROTAC. Through substitution of the polypeptide binder, the highly versatile L-AdPROM system is useful for the inducible degradation of potentially any intracellular POI.
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