WEHI PhD Completion Seminar hosted by Dr Bekky Feltham

Hao (Coco) Dong
PhD Student – Feltham Laboratory, Ubiquitin Signalling division, WEHI

Branching Out: Coordinated E3-Driven K29/K48 Ubiquitination Enables Efficient PROTAC-Induced Proteolysis

Davis Auditorium

Join via TEAMS

Including Q&A session

Followed by refreshments in Tapestry Lounge

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.

TRIP12 was found to work in conjunction with PROTAC-recruited CRL2VHL to assemble K29/K48 branched ubiquitin chains on a neo-substrate. This observation contradicts the conventional assumption that only one E3 ubiquitin ligase, or ligase complex is engaged upon PROTAC treatment to generate one ubiquitin linkage type on the neo-substrate, highlighting that complex cooperativity is involved in the underlying mechanism of PROTACs. This observation though was limited to in vitro biochemical classification, and cellular evidence of this phenomenon had not yet been realised.

Through a suite of mass spectrometry-based analyses, I identified PROTAC-induced formation of K29/K48-branched ubiquitin chains on a GFP-tagged model substrate. Further screening revealed that an additional E3 ligase acts in concert with the PROTAC-recruited E3 ligases CRL2VHL or CRL4CRBN complex to assemble these branched chains, expanding the observation of E3 cooperativity in the PROTAC mechanism of action from the singular example of TRIP12 to a broader phenomenon. The resulting K48/K29-branched ubiquitin topology was found to promote complete substrate degradation by the proteasome, thus highlighting the unappreciated mechanistic importance of ubiquitin branching in PROTAC-mediated degradation.

This is the first insight into the ubiquitination events induced on a single in vivo neo-substrate and paves the way for a deeper understanding of PROTAC activity and pathway towards enhancing proteolysis-targeted protein degradation.

All welcome!