Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (PROTAC)

The p97 protein is a member of the AAA + family of ATPases. It is a mechanoenzyme that uses energy from ATP hydrolysis to promote protein unfolding and segregation actively. The unfolded products of p97 are presented to the 26S Proteasome for degradation. p97 substrate recognition is mediated by adaptors, which interact with substrates directly or indirectly through ubiquitin modifications, resulting in substrate funnelling into the central pore of the p97 hexamer and unfolding. We have engineered synthetic adaptors to target specific substrates to p97, using the extraordinary intracellular binding capabilities of camelid nanobodies fused to the UBX domain of the p97 Adapter FAF1. In such a way, we created a p97-directed proteolysis-targeting chimera (PROTAC), representing a unique E3 ubiquitin ligase-independent strategy to promote specific proteolysis.