The End of The End: Terminal Degrons Discovery using a Synthetic Human Peptidome

Degrons are the minimal elements within proteins that are recognized by E3 ligases to promote proteolysis. Despite the central role of degrons in proteostasis, thus far only a limited number of degron motifs have been characterized and matched to their cognate E3 ligase.

To characterize degron motifs in human proteins in a high-throughput manner, we developed a genome-wide approach called GPS-peptidome. This technology is a hybrid of the Global Protein Stability (GPS) system combined with a synthetic human peptidome library. GPS is based on a lentiviral construct encoding two fluorescent proteins: DsRed, which serves as an internal reference, and a GFP-fusion peptide that is translated from an IRES. As both DsRed and the GFP-fusion peptide are expressed from the same transcript, the GFP/DsRed ratio can be used to readout the effect of the fusion peptide on the stability of GFP.

This powerful approach has allowed us to identify a large number of terminal degrons lying at both the N- and C-termini of protein substrates. A series of CRISPR screens identified the dedicated E3 ligases that recognize these degrons. The C-terminome screen uncovered the first examples of C-degrons, while the N-terminome screen revealed a novel N-degron pathway centered on N-terminal glycine.

Terminal degron motifs are depleted from the human proteome, suggesting evolutionary pressure to avoid degradation by E3 ligases targeting terminal degrons. In addition, important roles of terminal degrons may be to degrade proteins that fail to localize properly to membranes and to destroy proteolytic fragments generated by caspases during apoptosis.