Uncovering the regulation of transthiolating and RING E3 ligase activity by metabolites

The ubiquitin system, a central regulator of cellular processes, relies on the activity of approximately 650 E3 ligases to mediate the ubiquitination of cellular entities. This activity is tightly regulated to avoid aberrant ubiquitination, a hallmark of numerous diseases. While several regulatory layers of ligase activity have been documented, a novel factor – activity control by metabolites – remains vastly underexplored. A key bottleneck for uncovering metabolite-driven regulation has been the lack of appropriate tools for activity read-out of cellular ligases. However, in the past few years, innovative activity-based probes have been added to our toolbox. In this proposal, I will exploit complementary E2~Ub-mimicking probes to monitor the proteome-wide activity of both transthiolating and single subunit RING E3 ligases after manipulation of intracellular metabolite pools. By coupling mass-spectrometry-aided ligase-agnostic profiling with biophysical characterisation of the interaction, I will chart the metabolite E3 landscape, deepen our mechanistic understanding of ubiquitin regulation, and uncover new therapeutic entry points for diseases such as cancer and neurodegeneration.