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

Luke M. Simpson, Thomas J. Macartney, Alice Nardin, Luke J. Fulcher, Sascha Röth, Andrea Testa, Chiara Maniaci, Alessio Ciulli, Ian G. Ganley, Gopal P. Sapkota (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

16 Downloads (Pure)

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 languageEnglish
Pages (from-to)1164-1180.e5
Number of pages17
JournalCell Chemical Biology
Volume27
Issue number9
Early online date14 Jul 2020
DOIs
Publication statusPublished - 17 Sep 2020

Keywords

  • AdPROM
  • PROTAC
  • HaloPROTAC
  • nanobody
  • monobody
  • targeted proteolysis
  • ULK1
  • FAM83D
  • SGK3
  • RAS

Fingerprint Dive into the research topics of 'Inducible Degradation of Target Proteins through a Tractable Affinity-Directed Protein Missile System'. Together they form a unique fingerprint.

Cite this