E3 UB ligases regulate diverse processes in the cell, including protein quality control and degradation, gene activation and repair, and cell cycle and apoptosis, etc. The multifaceted roles of E3s are underpinned by their diverse substrate specificities, yet it has been a challenge to profile E3 substrates due to the transient interactions of the E3-substrate pairs. To overcome such a challenge, we developed a method to enable the direct tracking of UB transfer from an E3 to its substrate proteins through an engineered orthogonal UB transfer cascade (OUT). The OUT cascade is constituted by an engineered set of xE1, xE2 and xE3 that would enable the exclusive transfer of an engineered xUB to the substrates of a specific E3. xUB shares no cross-reactivities with the native UB transferring enzymes, so its labeling of the E3 substrates through the OUT cascade would enable the unambiguous identification of the ubiquitination targets of an E3. So far, we have used phage and yeast cell surface display to engineer OUT cascades with various classes of E3s and used them to profile substrate specificities of HECT E3s E6AP and Rsp5, RBR E3s Parkin and HHARI, and U-box E3s E4B and CHIP. Based on the newly generated substrate profiles of various E3s, we have discovered new pathways of cell regulation through E3 catalyzed ubiquitination of kinases, phosphatases, transcription factors, and chaperon proteins that suggested essential roles of the E3s in regulating cell senescence, metastasis, mitophagy, and stress response. Overall, our work has established OUT as an empowering proteomics platform for deconvoluting E3-substrate relationships and deciphering the role of E3s in cell biology.

Engineering ubiquitin transfer in the cell – the orthogonal way

ACS Fall 2021

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