The covalent attachment of ubiquitin to proteins is a vital posttranslational modification that controls regulated proteolysis and non-degradative signaling essential to all aspects of cellular function. When misregulated, perturbations within the ubiquitin-proteasome system (UPS) can lead to many human pathologies including cancer. Ubiquitination relies on the precise transfer of a small protein, called ubiquitin, between three enzyme families (E1, E2, and E3) before becoming linked to its final protein target. The development of next-generation inhibitors that can selectively target specific steps within the vast UPS is an important frontier for cancer therapy. However, current therapies focus primarily on E3-substrate interactions, and current methods cannot unravel specific E1-E2-E3 sequences relevant for a particular protein target. Given that the UPS requires the coordination of hundreds of enzymes and thousands of specific interactions, it is extraordinarily difficult to study. As a result, there is a striking need for tools that can follow ubiquitin through the enzymatic E1-E2-E3 cascade to its target protein in live cells.
Our work in this area focuses on the development and implementation of such a method, called targeted Charging of Ubiquitin to E2 (tCUbE). tCUbE channels a tagged ubiquitin (Ub*) to a specific E2 within a living cell, and can therefore identify distinct interactions relevant for ubiquitination of specific protein targets. This strategy enables us to track the fate of ubiquitin from a single member of the E2 family to a specific subset of E3 enzymes and/or target proteins with which the E2 interacts. As a result, this strategy is unmatched in its ability to illuminate the subset of Ub*-products that arise from the activity of a single E2, thus identifying new targets for disrupting the UPS for the treatment of cancer.