Ubiquitin is a small regulatory protein known to target proteins for degradation. On one hand, ubiquitin chains are assembled by the ubiquitination cascade pathway which involves three different enzymes: ubiquitin activating enzymes (E1s), ubiquitin conjugating enzymes (E2s), and ubiquitin ligases (E3s). On the other hand, deubiquitinases (DUBs) oppose the role of ubiquitination by removing ubiquitin from substrate proteins. There are altogether eight linkage types of ubiquitin chains. What is more interesting is that E2s, E3s, or DUBs show linkage specificity. However, how these enzymes mediate ubiquitin chains in a linkage-specific manner is still an open question. Here, we developed an interdisciplinary approach including organic synthesis, genetic code expansion, and protein engineering to synthesize a linkage-specific diUB to help us answer this question. To the end, a linkage-specific diUB featuring a thiol group embedded near the isopeptide bond was prepared and it was readily used for activity-based protein profiling to discover more linkage-specific ubiquitin chain regulators by converting it into a probe with an electrophilic trap, or for structure determination by conjugating it to corresponding chain-specific E2 or E3s. Taken together, these linkage-specific diUB probes help us to uncover detailed mechanisms underlying chain specificities of E2, E3, or DUB with direct evidence and to draw a big picture of E2, E3 mediated ubiquitination on substrates and linkage-specific chain elongation.

Tools for probing the catalytic mechanism of protein ubiquitination enabled by unnatural amino acid incorporation

ACS Fall 2021

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