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

TY - JOUR

T1 - Inducible Degradation of Target Proteins through a Tractable Affinity-Directed Protein Missile System

AU - Simpson, Luke M.

AU - Macartney, Thomas J.

AU - Nardin, Alice

AU - Fulcher, Luke J.

AU - Röth, Sascha

AU - Testa, Andrea

AU - Maniaci, Chiara

AU - Ciulli, Alessio

AU - Ganley, Ian G.

AU - Sapkota, Gopal P.

N1 - This study was funded by the UK Medical Research Council (MRC), awarded to GPS (MC_UU_00018/6 and MC_UU_12016/3). European Union’s Seventh Framework Programme as a Starting Grant (ERC-2012-StG- 311460 DrugE3CRLs)

PY - 2020/9/17

Y1 - 2020/9/17

N2 - 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.

AB - 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.

KW - AdPROM

KW - PROTAC

KW - HaloPROTAC

KW - nanobody

KW - monobody

KW - targeted proteolysis

KW - ULK1

KW - FAM83D

KW - SGK3

KW - RAS

UR - https://www.scopus.com/pages/publications/85089292851

U2 - 10.1016/j.chembiol.2020.06.013

DO - 10.1016/j.chembiol.2020.06.013

M3 - Article

C2 - 32668203

AN - SCOPUS:85089292851

SN - 2451-9456

VL - 27

SP - 1164-1180.e5

JO - Cell Chemical Biology

JF - Cell Chemical Biology

IS - 9

ER -