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

Cell Chem Biol. 2020 Sep 17;27(9):1164-1180.e5. doi: 10.1016/j.chembiol.2020.06.013. Epub 2020 Jul 14.

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.

Keywords: AdPROM; FAM83D; HaloPROTAC; PROTAC; RAS; SGK3; ULK1; monobody; nanobody; targeted proteolysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels
  • Autophagy-Related Protein-1 Homolog / genetics
  • Autophagy-Related Protein-1 Homolog / metabolism
  • CRISPR-Cas Systems / genetics
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Line
  • Gene Knock-In Techniques
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / immunology
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Peptides / chemistry
  • Peptides / metabolism
  • Proteolysis*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Single-Chain Antibodies / chemistry
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / metabolism*
  • Ubiquitination
  • ras Proteins / metabolism

Substances

  • Affinity Labels
  • Cell Cycle Proteins
  • FAM83D protein, human
  • Microtubule-Associated Proteins
  • Peptides
  • Recombinant Fusion Proteins
  • Single-Chain Antibodies
  • Green Fluorescent Proteins
  • Autophagy-Related Protein-1 Homolog
  • ras Proteins