Degron technology for conditional protein depletion

Abstract

Degron systems such as the degradation tag (dTAG) and auxin-inducible degron (AID) allow conditional, rapid, and reversible protein depletion in response to a small molecule stimulus. However, current tags show variable efficiency across proteins and require optimisation to expand their utility across more challenging target proteins and tissue environments. Using Green Fluorescent Protein (GFP) as a fluorescent reporter protein, this study evaluated how the placement of dTAG, one of the best characterised degron tags, on a target protein influences degradation efficiency. It also addressed whether the efficiency of dTAG-induced protein depletion depended upon the specific E3 ligase complex that is recruited for ubiquitination. Finally, I began to investigate the potential for modifying the linker peptide through which degrons connect to their target protein in order to improve degron function. Experiments testing the extent of GFP degradation via flow cytometry have highlighted differences in depletion kinetics dependent on tag position, and the specific E3 ligase that is recruited for degradation. Cereblon (CRBN)-mediated degradation using the dTAG-13 ligand was evidently more efficient when the degron tag was positioned at the C-terminus of GFP compared to the N-terminus. Opposingly, VHL-mediated depletion using the dTAGv1 ligand was more effective with the degron attached at the N-terminus of GFP. The addition of extra protein domains to recombinant protein constructs for which GFP was N-terminally tagged with dTAG significantly improved CRBN-mediated GFP depletion. Yet, the same protein modifications did not significantly affect VHL-mediated GFP depletion.

Collectively, these results show that many factors can influence the speed and extent of protein degradation using degron-tagging systems. Positional context of the tag can be a key factor in designing proteins that are competent for rapid and complete degradation. In the future, a better understanding of the structural basis for ternary complex formation will be important for designing optimal degron tagging strategies, which could be particularly important when applying degron tagging in mouse models of human disease.