Engineering Ubiquitin Transfer – the Orthogonal Way

Abstract

Protein modification by ubiquitin (UB) controls diverse cellular processes. UB is conjugated to cellular proteins by sequential transfer through an E1-E2-E3 enzymatic cascade. The cross-activities of 2 E1s, 50 E2s and thousands of E3s encoded by the human genome make it difficult to identify the substrate proteins of a specific E3 enzyme in the cell. To solve this problem, we used phage display to engineer orthogonal pairs of UB-E1 and E1-E2 so that the engineered UB (xUB) can only be activated by an engineered E1 (xE1) and transferred to an engineered E2 (xE2). We have constructed stable cell lines to express the xUB-xE1-xE2 cascade for orthogonal UB transfer (OUT). We are now carrying out proteomic profiling to identify the downstream targets of individual E1 and E2 enzymes. We are also engineering orthogonal xE2-xE3 pairs by phage and yeast cell surface display to bridge the transfer of xUB to the direct substrates of a specific E3 enzyme. Besides engineering the OUT cascade, we used yeast cell surface display to enable the β-propeller domain of the E3 enzyme to recognize new targets. We also used phage display to identified short peptides that mimic the actions of UB, Nedd8 and SUMO for the modification of cellular proteins. These peptides provide a new way to inhibit protein modification by UB and UB-like proteins (UBLs).