The ability to selectively and rapidly degrade a protein of interest (POI) inside cells is desirable for studying its function and in therapeutics. Previously, the Sapkota Lab had developed the Affinity-directed PROtein Missile (AdPROM) system for constitutive degradation of POIs (Fulcher et al., 2016 & 2017).
Now, research led by Luke Simpson (Sapkota Lab; MRC-PPU, University of Dundee) has developed a ligand-inducible AdPROM (L-AdPROM) system for tractable degradation of POIs inside cells. This work was supported by excellent collaboration from members of the Ciulli (BCDD, University of Dundee) and Ganley Labs (MRC-PPU, University of Dundee). The AdPROM system utilises small, high-affinity polypeptide binders of intracellular POIs (as “protein missiles”) that are physically linked to an E3 ubiquitin ligase moiety (as the “destroyer”) to enable targeted destruction of the POI. The team combined Halo-tag/VHL-recruiting proteolysis-targeting chimera (HaloPROTAC-E) technology with the AdPROM system to develop an inducible POI degradation system.
Using the L-AdPROM system, the team were able to target four distinct proteins, namely FAM83D (involved in mitosis), ULK1 (involved in autophagy), SGK3 (involved in cell growth), and RAS GTPases (oncogenes), for ligand-inducible and reversible degradation. The inducible degradation of these proteins was able to impact known biological and signalling roles of some of these proteins. Moreover, the level of SGK3 degradation by L-AdPROM mirrored that caused by a SGK3-directed PROTAC.
Gopal Sapkota said, “The L-AdPROM technology is versatile and adaptable and could potentially target any protein inside cells for inducible degradation, as long as there is a small polypeptide binder of the POI.” He added, “Alternatively, we can tag the POIs with green fluorescent protein (GFP) using CRISPR/Cas genome editing and use a GFP-binding polypeptide nanobody to exploit L-AdPROM, as we have done here”.
Luke Simpson said, “This technology offers an excellent opportunity for any researcher wishing to dissect the function of potentially any intracellular POI. In addition, the L-AdPROM system can be exploited to rapidly inform the utility of protein degradation as a therapeutic approach, prior to developing more resource- and time-intensive small molecule degraders, such as PROTACs”.
Luke added, “It is incredibly rewarding when all the work you put in at the bench is published in an internationally reputable peer-reviewed journal. Despite the COVID-19 lockdown, we were able to address all reviewers’ concerns timely and I am thrilled that the paper is now published”.
This team effort included contributions from Thomas Macartney (MRC-PPU Reagents & Services), Luke Fulcher and Sascha Röth (Sapkota Lab; MRC-PPU, University of Dundee), Alice Nardin (Ganley Lab; MRC-PPU, University of Dundee), and Andrea Testa and Chiara Maniaci (Ciulli Lab; BCDD, University of Dundee).
The findings are published in the current issue of Cell Chemical Biology (https://www.cell.com/cell-chemical-biology/fulltext/S2451-9456(20)30236-1)
Related publications from the Sapkota lab: