Date of Award

12-17-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Jun Yin

Second Advisor

Ming Luo

Third Advisor

Binghe Wang

Abstract

Ubiquitin chains of specific linkages are assembled by ubiquitin conjugating enzymes (E2s) and ubiquitin ligases (E3s) to encode unique signals in the cell. Nevertheless, ubiquitin chains are often trimmed by deubiquitinases to reverse encoded signals. Given that there are altogether eight linkage types of polyubiquitin chains, how E2s, E3s and DUBs mediate ubiquitin chains in a linkage-specific manner is still an open question. In this thesis, an interdisciplinary approach including organic synthesis, genetic code expansion, protein engineering has been investigated to stress this intriguing question. To this end, linkage-specific diUB featuring a thiol group embedded near the isopeptide bond site was prepared in large scale and it was readily used for activity-based protein profiling to uncover 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 should provide access for structural biologists to map dynamic, transient, weak protein-protein interactions in UB transfer, therefore to uncover detailed mechanism underlying chain specificities of E2, HECT E3 or DUB with direct evidences and to reconstitute full pictures of E2, E3 mediated ubiquitination on substrates and linkage-specific chain elongation.

DOI

https://doi.org/10.57709/15956625

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