Inducible Degradation of Target Proteins Through a Tractable Affinity-Directed PROtein Missile (AdPROM) System

Abstract

Previously we described an Affinity-directed PROtein Missile (AdPROM) system that utilises 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 combined Halo/VHL-recruiting proteolysis-targeting chimeras (HaloPROTACs) with AdPROM for ligand-inducible (L-AdPROM) degradation of POIs. For POIs, cells were generated in which ULK1, FAM83D or SGK3 were knocked in with a GFP-tag using CRISPR/Cas9. When we express the L-AdPROM construct consisting of an anti-GFP nanobody conjugated to a Halo-tag, we achieve degradation of GFP-ULK1, FAM83D-GFP or SGK3-GFP only upon treatment of cells with the HaloPROTAC. The degradation of GFP-ULK1 and FAM83D-GFP interrupts with their cellular roles, namely starvation-induced autophagy and recruitment of CK1α to the mitotic spindle, respectively. The highly versatile and tractable L-AdPROM system is useful for the inducible degradation of potentially any intracellular POI.