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

34 Pages Posted: 24 Mar 2020 Sneak Peek Status: Review Complete

See all articles by Luke Simpson

Luke Simpson

University of Dundee - MRC Protein Phosphorylation and Ubiquitylation Unit

Thomas Macartney

University of Dundee - MRC Protein Phosphorylation and Ubiquitylation Unit

Alice Nardin

University of Dundee - MRC Protein Phosphorylation and Ubiquitylation Unit

Luke Fulcher

University of Dundee - MRC Protein Phosphorylation and Ubiquitylation Unit

Andrea Testa

University of Dundee

Chiara Maniaci

University of Dundee

Alessio Ciulli

University of Dundee

Ian Ganley

University of Dundee - MRC Protein Phosphorylation and Ubiquitylation Unit

Gopal Sapkota

University of Dundee - MRC Protein Phosphorylation and Ubiquitylation Unit

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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 ULK1FAM83D 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.

Keywords: L-AdPROM, AdPROM, PROTAC, Halo, VHL, nanobody, degradation, ULK1, FAM83D, SGK3

Suggested Citation

Simpson, Luke and Macartney, Thomas and Nardin, Alice and Fulcher, Luke and Testa, Andrea and Maniaci, Chiara and Ciulli, Alessio and Ganley, Ian and Sapkota, Gopal, Inducible Degradation of Target Proteins Through a Tractable Affinity-Directed PROtein Missile (AdPROM) System. CELL-CHEMICAL-BIOLOGY-D-20-00073. Available at SSRN: https://ssrn.com/abstract=3552150 or http://dx.doi.org/10.2139/ssrn.3552150
This is a paper under consideration at Cell Press and has not been peer-reviewed.

Luke Simpson

University of Dundee - MRC Protein Phosphorylation and Ubiquitylation Unit

United Kingdom

Thomas Macartney

University of Dundee - MRC Protein Phosphorylation and Ubiquitylation Unit

United Kingdom

Alice Nardin

University of Dundee - MRC Protein Phosphorylation and Ubiquitylation Unit

United Kingdom

Luke Fulcher

University of Dundee - MRC Protein Phosphorylation and Ubiquitylation Unit

United Kingdom

Andrea Testa

University of Dundee

Dundee, Scotland DD1 4HN
United Kingdom

Chiara Maniaci

University of Dundee

Dundee, Scotland DD1 4HN
United Kingdom

Alessio Ciulli

University of Dundee

Dundee, Scotland DD1 4HN
United Kingdom

Ian Ganley

University of Dundee - MRC Protein Phosphorylation and Ubiquitylation Unit

United Kingdom

Gopal Sapkota (Contact Author)

University of Dundee - MRC Protein Phosphorylation and Ubiquitylation Unit ( email )

United Kingdom

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