Author ORCID Identifier

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Jun Yin

Second Advisor

Angela Mabb

Third Advisor

Jenny Yang


SMAD specific E3 ubiquitin protein ligase 2 (Smurf2), a member of the HECT domain family of E3 ubiquitin (UB) ligases, participates in many crucial biological and physiological processes in the cell. Through the E1-E2-E3 ubiquitin transfer cascade, Smurf2 regulates its substrates' stability by ubiquitination-mediated degradation. E6-associated protein (E6AP), another HECT domain E3 ligase, pairs with a papillomavirus oncoprotein E6 as a complex to recruit substrates that result in cervical cancers. Ring finger protein 216 (RNF216) is an RBR type E3 ligase and is involved in neurodegenerative and neurodevelopmental diseases by transferring UB to its substrates. Due to the importance of these E3s in a variety of cellular functions, identifying their substrates to connect the disease-related proteins with E3s is urgent. Our lab has been developing an orthogonal ubiquitin transfer (OUT) cascade, in which a UB variant can be exclusively transferred via an engineered E1-E2-E3 cascade to conjugate with the substrate. By implementation of OUT cascade in mammalian cells, UB-conjugated proteins from cell lysate can be enriched by a tandem purification and then characterized by proteomics to acquire a potential substrate profiling of E3. In this study, yeast surface display was used to engineer the Smurf2 HECT domain, while the point mutation technique was applied to the RBR domain of RNF216, both for incorporation into an OUT cascade to profile their substrate pools in the cell. And based on the existing OUT cascade of E6AP, we further identified the substrates of the E6-E6AP pair and transport proteins to attenuate tumor-suppressive signaling. Moreover, O-GlcNAc transferase (OGT) was identified as a substrate protein of E6AP by OUT cascade and its proteasomal degradation can be accelerated by E6 to suppress O-GlcNAc modification in the cell. Overall, our work demonstrates OUT cascade as a powerful tool for identifying substrates and mapping signaling pathways in cells to provide potential therapeutic insights.


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