Proteolysis-Targeting Chimera (PROTAC) technology holds the promise of enabling the rapid and reversible depletion of therapeutically-relevant protein targets through induced ubiquitination. However, PROTAC development is hampered by a limited understanding of the sequential steps of ternary complex formation (target-PROTAC-E3 ligase), ubiquitination of the target protein, and proteasomal degradation of the target protein. As such, PROTAC candidate design relies heavily on costly trial and error processes.
A recent publication by Kaiho-Soma et al. has identified an additional E3 ligase, TRIP12, which is also recruited upon PROTAC treatment and works in conjunction with CRL2VHL to assemble K29/K48 branched ubiquitin chains on the substrate in vitro, thereby enhancing the PROTAC efficiency. This observation contradicts the conventional assumption that only the recruited E3 Ubiquitin ligase (typically either CRL2VHL or CRL4CRBN) is recruited upon PROTAC treatment to generate one ubiquitin linkage type on the substrate, highlighting the cooperativity in the underlying mechanism of PROTAC 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 PROTAC 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 PROTAC activity and pathway towards enhancing proteolysis-targeted protein degradation.