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

Cell Chemical Biology
Luke M SimpsonGopal P Sapkota

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.

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Citations

Dec 3, 2020·American Journal of Physiology. Cell Physiology·Jan Gettemans, Brian De Dobbelaer
Feb 8, 2021·Current Opinion in Chemical Biology·Ronen Gabizon, Nir London
Dec 20, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Bernat Coll-MartínezBernat Crosas
May 25, 2021·Frontiers in Molecular Biosciences·Matylda Anna IzertMaria Wiktoria Górna
Jul 25, 2021·Journal of Hematology & Oncology·Kun ChenPeng Wang
Aug 9, 2021·Current Opinion in Structural Biology·Sharon L Campbell, Mark R Philips

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Methods Mentioned

BETA
protein knockout
immunoprecipitation
transfection
immunoprecipitations
lipidation
pull down
fluorescence microscopy
flow cytometry
scraping

Software Mentioned

OMERO
GraphPad Prism
ImageJ
Prism®

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