title: Orthogonal ubiquitin transfer through engineered E1-E2 cascades for protein ubiquitination.
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. 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.
authors.affiliation: Department of Chemistry, University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhao
authors.forename: Karan
authors.initials: K
authors.lastname: Bhuripanyo
authors.forename: Keya
authors.initials: K
authors.lastname: Zhang
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kiyokawa
authors.forename: Hermann
authors.initials: H
authors.lastname: Schindelin
authors.forename: Jun
authors.initials: J
authors.lastname: Yin
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D016296:Mutagenesis; D019151:Peptide Library; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D019151:Peptide Library; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes