Development and utilisation of a ubiquitin directed photoreactive probe to identify endogenous E2-RING E3 interactions

Ubiquitin is a small protein, its addition to other proteins regulates many cellular processes such as protein homeostasis and cell signalling. The addition of ubiquitin to protein substrates is catalysed by an enzymatic cascade. Ubiquitin is transferred from a ubiquitin-activating enzyme (E1) to a ubiquitin conjugating enzyme (E2).

The ubiquitin bound E2 interacts with an E3 ligase which catalyses the transfer of ubiquitin to the substrate. Methods to identify interactions between ubiquitin bound E2s and E3 ligases in cells are limited. To try to capture these interactions I will develop a ubiquitin directed photoreactive probe.

The probe will consist of a photoreactive diazirine group chemically conjugated to recombinant ubiquitin bound to the E2, Ube2W.

The ability of this probe to form covalent attachments with E3 ligases will be validated in vitro using the SUMO-targeted ubiquitin ligase RNF4. Following validation, multiple probes will be made using different E2s.

These will be incubated with cell lysates and irradiated to form photo cross-linked products that will be identified using mass spectrometry.

This approach could potentially identify physiologically relevant E2-E3 pairs and novel E3 ligases which would be beneficial for the development of targeted protein degradation strategies.