Identifying E3 Ligase Substrates With Quantitative Degradation Proteomics

Controlled protein degradation by the ubiquitin-proteasome pathway is critical for almost all cellular processes. E3 ubiquitin ligases are responsible for targeting proteins for ubiquitylation and subsequent proteasomal degradation with spatial and temporal precision. While studies have revealed various E3-substrate pairs involved in distinct biological processes, the complete substrate profiles of individual E3 ligases are largely unknown. Here we report a new approach to identify substrates of an E3 ligase for proteasomal degradation using unnatural amino acid incorporation pulse-chase proteomics (degradomics). Applying this approach, we determine the steady-state substrates of the C-terminal to LisH (CTLH) E3 ligase, a multi-component complex with poorly defined substrates. By comparing the proteome degradation profiles of active and inactive CTLH-expressing cells, we successfully identify previously known and new potential substrates of CTLH ligase. Altogether, degradomics can comprehensively identify degradation substrates of an E3 ligase, which can be adapted for other E3 ligases in various cellular contexts.

Automated summary

Controlled protein degradation is crucial for cellular processes, and E3 ubiquitin ligases play a key role in targeting proteins for degradation. However, the substrate profiles of individual E3 ligases are largely unknown. This study presents a new approach, degradomics, which uses unnatural amino acid incorporation pulse-chase proteomics, to identify degradation substrates of an E3 ligase. By comparing the proteome degradation profiles of active and inactive E3 ligase-expressing cells, the researchers successfully identified known and potential substrates of the ligase.