The poly-ubiquitin (UB) chains attached to the cellular proteins carry diverse signals that regulate virtually all aspect of cell biology including protein stability, enzyme catalysis, and gene activation. The poly UB chain is built by UB transfer through a E1-E2-E3 enzymatic cascade to the cellular proteins. E1 activates UB and loads it on E2 as a thioester conjugate. The E2~UB conjugate is then bound to E3 that recruit cellular proteins for the UB transfer reaction. There are more than 45 E2s and 1,000 E3s in the cell. Each E2 can pair with multiple E3s. Each E3 can pair with different E2s to transfer UB to multiple target proteins in the cell. Due to the complex cross reactivity among the E2 and E3 enzymes, it has been a significant challenge to identify the substrate proteins of a specific E3. I has also not been possible to compare the substrate pool of the same E3 pairing with different E2s in order to reveal the effect of various E2 on the reactivity of E3. The lack of efficient approaches to profile the substrate specificity of an E3 enzyme or an E2-E3 pair prevents the elucidation of the function of E2 and E3 enzymes in the regulatory circuits of the cell. As a resul the ubiquitination targets of many E3 enzymes such as Mmd2 and Smurf2 are poorly characterized despite their strong connection with cancer or neurodegenerative diseases. In this application, we plan to develop a method to profile the substrate specificities of a E3 or a E2-E3 pair in the protein ubiquitination reaction. We have proven by preliminary results that we can use protein engineering methods based on phage display, structure-based design and site-directed mutagenesis to create pair wise interactions between UB and E1, and E1 and E2. These engineered pairs shares no cross reactivity with native E1 and E2 enzymes in the cell and we have demonstrated that the engineered E1 (xE1) can transfer engineered UB (xUB) to a specific E2 (xE2) that is engineered to match with xE1. We plan to engineer specific xE2-xE3 interactions so that a UB transfer pathway through the xE1-xE2-xE3 cascade can be installed in the cell to transfer xUB to the substrate proteins of an engineered E3 (xE3). xUB is fused to an affinity tag to allow the identification of the ubiquitination targets of xE3 by affinity purificaton. The orthogonality of the xE1-xE2-xE3 cascade with their native counterparts ensures xUB can only be utilized by the xE2-xE3 pair to be attached to the substrate proteins of xE3. We thus call such a method to profile E3 substrate specificity Orthogonal UB Transfer or OUT. By engineering various E2s to create specific xE2-xE3 pairs with the same xE3, we will be able to use OUT to compare the difference of the UB transfer targets of various xE2-xE3 pairs in order to reveal the regulatory role of E2 on E3. We plan to use OUT to profile the ubiquitination targets of Mdm2 and Smurf2 in combination with various E2s. Overall our work will generate a platform to map the UB transfer networks associated with key E3 enzymes for the discovery of new signal transduction pathways in the cell.