High-throughput luminescence assays for ubiquitin ligases

Ubiquitin ligases (E3s) play a pivotal role in regulating protein stability and cellular signaling by catalyzing the transfer of ubiquitin to substrate proteins, marking them for degradation or functional modulation. Given their involvement in numerous diseases and targeted protein degradation modalities, E3 ligases have emerged as attractive therapeutic targets. Developing biochemical assays to measure E3 activity is essential for drug discovery and mechanistic studies. One accessible and robust approach involves luminescence-based assays that monitor ATP consumption. These assays not only provide a high-throughput means to measure E3 function, but also enable selectivity profiling across the broader family of ATP-dependent enzymes, including helicases and kinases, ensuring target specificity.