TY - JOUR
T1 - Orthogonal ubiquitin transfer through engineered E1-E2 cascades for protein ubiquitination
AU - Zhao, Bo
AU - Bhuripanyo, Karan
AU - Zhang, Keya
AU - Kiyokawa, Hiroaki
AU - Schindelin, Hermann
AU - Yin, Jun
N1 - Funding Information:
This work was supported by a lab startup grant from the University of Chicago and a National Science Foundation CAREER award (1057092). This work was also funded in part by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust and the Deutsche Forschungsgemeinschaft (FZ82 and Schi 425/5-1). We thank Professors Carlos F. Barbas (The Scripps Research Institute), Bernard Roizman (University of Chicago), Suzanne D. Conzen (University of Chicago), Carl G. Maki (Rush University Medical Center), Linda Hicke (Northwestern University), Jon M. Huibregtse (University of Texas, Austin), and Cam Patterson (University of North Carolina) for generously providing materials for this research. We thank Jeffrey Schneider and David Boon (University of Chicago) for proofreading the manuscript.
PY - 2012/10/26
Y1 - 2012/10/26
N2 - 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. One way to solve this problem is to engineer an orthogonal UB transfer (OUT) cascade in which the engineered UB (xUB) is relayed by engineered E1, E2 and E3 enzymes (xE1, xE2, xE3) to modify the substrate proteins of a specific E3. Here, we use phage display and mutagenesis to construct xUB-xE1 and xE1-xE2 pairs that are orthogonal to the native E1 and E2 enzymes. Our work on engineering the UB transfer cascades will enable us to use OUT to map the signal transduction networks mediated by protein ubiquitination.
AB - 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. One way to solve this problem is to engineer an orthogonal UB transfer (OUT) cascade in which the engineered UB (xUB) is relayed by engineered E1, E2 and E3 enzymes (xE1, xE2, xE3) to modify the substrate proteins of a specific E3. Here, we use phage display and mutagenesis to construct xUB-xE1 and xE1-xE2 pairs that are orthogonal to the native E1 and E2 enzymes. Our work on engineering the UB transfer cascades will enable us to use OUT to map the signal transduction networks mediated by protein ubiquitination.
UR - http://www.scopus.com/inward/record.url?scp=84868015450&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84868015450&partnerID=8YFLogxK
U2 - 10.1016/j.chembiol.2012.07.023
DO - 10.1016/j.chembiol.2012.07.023
M3 - Article
C2 - 23102221
AN - SCOPUS:84868015450
VL - 19
SP - 1265
EP - 1277
JO - Cell Chemical Biology
JF - Cell Chemical Biology
SN - 2451-9448
IS - 10
ER -