A luminescence-based biosensor to measure endogenous UBE3A activity.

ABSTRACT: Loss- and gain-of-function (LOF and GOF) mutations in the E3 ubiquitin ligase UBE3A cause distinct neurodevelopmental disorders. The AZUL domain of UBE3A binds with low nanomolar affinity to a 45 amino acid (aa) region of PSMD4. We fused this 45-aa sequence to Firefly luciferase to generate a luminescence-based biosensor, Firefly-45aa, that acts as an artificial UBE3A substrate and exhibits exceptional sensitivity in measuring UBE3A activity. Testing UBE3A variants using Firefly-45aa in HEK293T cells revealed distinct biosensor activity profiles, enabling the classification of LOF and GOF mutations based on maximum activity, inhibitory concentration-50, and activity per unit of protein. Some strong LOF mutations had dominant negative activity. In addition, Firefly-45aa can be used to quant

SUBMITTER: Xing L 

PROVIDER: S-EPMC12554127 | biostudies-literature | 2025 Nov

REPOSITORIES: biostudies-literature