Date of Award

12-14-2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Dr. Jun Yin

Second Advisor

Dr. Binghe Wang

Third Advisor

Dr. Angela Mabb

Abstract

RING finger protein 38 (RNF38) is a member of the RING family of E3 ubiquitin (UB) ligases while HHARI is one of RBR family members: both regulate key processes in the cell. Through the E1-E2-E3 cascades, RNF38 and HHARI transfer UB to cellular proteins and regulate their stability, subcellular localization, and interaction with other proteins. Identifying the ubiquitination targets of RNF38 and HHARI holds the key to deciphering their roles in cell regulation. In this study, we used phage display to engineer the RING domains of the two E3 ligases for their incorporation into an orthogonal UB transfer (OUT) cascade to identify their substrate proteins in the cell. The OUT cascade consisting of engineered E1, E2 and E3 enzymes enable the exclusive transfer of an engineered UB (xUB) through a designated E3 to its substrate proteins. By affinity purification of xUB-conjugated proteins from cells expressing the OUT cascade of an E3 ligase and subsequent identification by proteomics, we hope to map the UB transfer pathways from the E3 ligase to its cellular targets. So far, we have successfully constructed the OUT cascade of RNF38 and HHARI and we used them to profile their substrates in HEK293T cells. The newly acquired substrate profiles of RNF38 and HHARI enables us to discover potential roles of the E3s in the cell. Additionally, we screened out a cyclic peptidomimetics P6 targeting E6AP, an E3 ubiquitinating p53 driven by the human papillomavirus and regulating pathways involved in neurodevelopmental diseases. The ubiquitin ligase activity of E6AP ubiquitinating its substrate proteins UbxD8, HHR23A, and β-catenin was substantially increased by P6 both in vitro and in vivo. Such results suggest that synthetic ligands may be used to enhance E3 activity in cells. Overall, we have demonstrated that the OUT cascades we generated with RING E3 and RBR E3 are powerful tools for profiling E3 substrates and mapping the cellular circuits mediated by the E3 enzymes. Additionally, the identified substrates can be further used as indicators to test the effects on the activity of E3 ligases targeted by specific peptides.

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