Targeted Protein Degradation (TPD) technology holds the promise of enabling the rapid and reversible depletion of therapeutically-relevant protein targets through induced ubiquitination. However, TPD degrader development is hampered by a limited understanding of the sequential steps of ternary complex formation (target-degrader-E3 ligase), ubiquitination of the target protein, and proteasomal degradation of the target protein. As such, degrader candidate design relies heavily on costly trial and error processes.
The 2021 publication by Kaiho-Soma et al. has identified an additional E3 ligase, TRIP12, which is also recruited upon degrader treatment and works in conjunction with CRL2VHL to assemble K29/K48 branched ubiquitin chains on the substrate in vitro, thereby enhancing the degrader efficiency. This observation contradicts the conventional assumption that only the intended E3 Ubiquitin ligase (typically either CRL2VHL or CRL4CRBN) is recruited upon degrader treatment to generate one ubiquitin linkage type on the substrate, highlighting the cooperativity in the underlying mechanism of degrader function.
Using a range of mass spectrometry methods and ubiquitin clipping I have defined the distinct architecture and composition of polyubiquitin chains formed on a model substrate as well as specific ubiquitination sites on the model substrate upon degrader treatment. This is the first insight into the ubiquitination events induced on a single in vivo target and paves the way for a deeper understanding of degrader activity and pathway towards enhancing proteolysis-targeted protein degradation.