Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) and RING finger protein 38 (RNF38) are members of the RING family of E3 ubiquitin (UB) ligases and regulate key processes in the cell. Through the E1-E2-E3 cascades, Cbl-b and RNF38 can transfer UB to cellular proteins and regulate their stability, subcellular localization, and interaction with other proteins. Identifying the ubiquitination targets of Cbl-b and RNF38 holds the key to decipher their roles in cell regulation. In this study, we used phage display to engineer the Ring domains of the two E3s for their incorporation into an orthogonal UB transfer (OUT) cascade to identify their substrate proteins in the cell. The OUT cascades 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 purifying xUB-conjugated proteins from cells expressing the OUT cascade of an E3 and identifying them by proteomics, we would be able to map the UB transfer pathways from the E3 to its cellular targets. So far, we have successfully constructed the OUT cascade of RNF38 and used it to profile its substrates in HEK293 cells. The newly acquired substrate profile of RNF38 enables us to discover potential roles of the E3 in cell cycle regulation and other cellular processes. We also expect our engineering of the RING domain of Cbl-b will generate an OUT cascade of the E3 for elucidating its functions in the cell. Overall, we have demonstrated that the OUT cascades we generated with RING E3s are powerful tools for profiling E3 substrates and mapping the cellular circuits mediated by the E3 enzymes.

Profiling the substrate proteins of RING-type E3 ubiquitin ligase by the engineering of orthogonal ubiquitin transfer (OUT) cascades

ACS Fall 2021

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