Activation of E6AP/UBE3A-Mediated Protein Ubiquitination and Degradation Pathways by a Cyclic γ‑AA Peptide

Manipulating the activities of E3
ubiquitin ligases with chemical
ligands holds promise for correcting E3 malfunctions and repurposing
the E3s for induced protein degradation in the cell. Herein, we report
an alternative strategy to proteolysis-targeting chimeras (PROTACs)
and molecular glues to induce protein degradation by constructing
and screening a γ-AA peptide library for cyclic peptidomimetics
binding to the HECT domain of E6AP, an E3 ubiquitinating p53 coerced
by the human papillomavirus and regulating pathways implicated in
neurodevelopmental disorders such as Angelman syndrome. We found that
a γ-AA peptide P6, discovered from the affinity-based
screening with the E6AP HECT domain, can significantly stimulate the
ubiquitin ligase activity of E6AP to ubiquitinate its substrate proteins
UbxD8, HHR23A, and β-catenin in reconstituted reactions and
HEK293T cells. Furthermore, P6 can accelerate the degradation
of E6AP substrates in the cell by enhancing the catalytic activities
of E6AP. Our work demonstrates the feasibility of using synthetic
ligands to stimulate E3 activities in the cell. The E3 stimulators
could be developed alongside E3 inhibitors and substrate recruiters
such as PROTACs and molecular glues to leverage the full potential
of protein ubiquitination pathways for drug development.