Author ORCID Identifier

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Jun Yin

Second Advisor

Kathryn Grant

Third Advisor

Lei Li


Ubiquitination is a multifaceted post translational modification (PTM) that plays a substantial role in the regulation of protein homeostasis for numerous biological processes, including proteasomal degradation, DNA repair, and signal transduction. The 76-amino acid protein ubiquitin (UB) covalently modifies a substrate through attachment of its C-terminal end to any lysine (Lys) residue on the substrate, forming an isopeptide bond. Furthermore, UB contains seven Lys residues: K6, K11, K27, K29, K33, K48, and K63, in addition to the M1 residue, which all serve as acceptor sites for conjugation to another UB monomer. Consequently, a substrate can be modified by an indefinite number of intricate UB patterns that exposes it to a multitude of cellular functions. Substrate ubiquitination is controlled by a network of governing enzymes that act coherently to write, edit, and code UB signals. How these enzymes function together to modify a ubiquitinated substrate is still poorly understood. Among them, are a group of deubiquitinating enzymes (DUBs) as editors that catalyze the cleavage of isopeptide bond linkages between UB monomers or UB and substrate to modify a protein’s cellular function. However, DUB enzymes have different catalytic activities and binding modes toward UB, which makes it rather difficult to understand their involvement in UB regulation. DUB dysregulation often leads to cancer or neurodegenerative diseases, such as Parkinson’s disease. As a result, numerous tools have been developed to elucidate complicated UB editing mechanisms exploited by DUBs, which rely profoundly on structural and analytical studies. One method, site-specific mutagenesis through unnatural amino acid (UAA) incorporation, has provided proteins with unique properties that enable them to be utilized as chemical probes for observing protein interactions. This thesis covers the design of activity-based diUB probes that were efficiently constructed though synthetic applications of UAA incorporation to profile the activity of linkage-specific DUBs. Here, a lysine derivative Nε-L-Thiaprolyl-L-lysine (ThzK), is site-specifically incorporated into the K48 and K63 UB sites using an orthogonal pyrrolysl-tRNA synthetase and tRNAPyl pair. The incorporated protein is deprotected to generate a reactive N-terminal cysteine (Cys) for further chemical ligation with a C-terminal thioester, forming a polyUB chain linked through a native peptide bond with thiol functionality. The thiol group is converted into dehydroalanine (Dha) by an elimination reaction, which serves as an electrophilic trap towards catalytic DUBs that could potentially recognize specificity of the probe both in vitro and in vivo. Proteomic methods can subsequently be employed to identify DUB binding components toward linkage-specific diUB probes in hopes of revealing their differential catalytic activities and homeostatic involvement in the UB transfer pathway, which can potentially help in therapeutic development against diseases.


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