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Abstract
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.
Original language | English |
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Pages (from-to) | 1164-1180.e5 |
Number of pages | 17 |
Journal | Cell Chemical Biology |
Volume | 27 |
Issue number | 9 |
Early online date | 14 Jul 2020 |
DOIs | |
Publication status | Published - 17 Sept 2020 |
Keywords
- AdPROM
- PROTAC
- HaloPROTAC
- nanobody
- monobody
- targeted proteolysis
- ULK1
- FAM83D
- SGK3
- RAS
ASJC Scopus subject areas
- Biochemistry
- Molecular Medicine
- Molecular Biology
- Pharmacology
- Drug Discovery
- Clinical Biochemistry
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- 1 Finished
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DrugE3CRL's: Probing Druggability of Multisubunit Complexes: E3 Cullin RING Ligases (ERC Starting Grant)
Ciulli, A. (Investigator)
COMMISSION OF THE EUROPEAN COMMUNITIES
1/05/13 → 30/04/18
Project: Research