Identifying the Substrate Proteins of E3 Ubiquitin Ligase by Orthogonal Ubiquitin Transfer (OUT)

Abstract

Ubiquitin (UB) is transferred through an E1‐E2‐E3 cascade to cellular proteins and encodes diverse signals such as proteasome degradation, subcellular trafficking, interacting with other proteins, and enzyme catalysis. The 2 E1s, 50 E2s, and more than 600 E3s constitute a complex network of UB transfer and make it a significant challenge to identify the substrates of a specific E3. Here, we developed a method that we called “orthogonal UB transfer (OUT)” to identify the direct ubiquitination targets of an E3. In this approach, an engineered UB (xUB) is exclusively transferred through an engineered cascade of xE1‐xE2‐xE3 to the substrates of a specific E3 (“x” designates engineered enzymes that are free of cross‐reactivities with their native partners). We used phage and yeast cell surface display to construct the OUT cascades of HECT E3 E6AP, U‐box E3s E4B and CHIP, and Ring‐between‐Ring (RBR) E3s HHARI and Parkin to profile their substrates. We found key signaling proteins such as kinases, phosphatases, and methyltransferases are ubiquitinated by these E3s. Based on the substrate profiles from the OUT screen, we discovered new pathways to regulate protein ubiquitination and degradation in the cell. Overall, we validated OUT as a powerful proteomic platform to identify the substrates of an E3 and map its position in the cell signaling networks.Support or Funding InformationThis work is supported by the National Institutes of Health (GM104498) and the National Science Foundation (1420193 and 1710460).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.