Cyclization of polyubiquitin by the E2-25K ubiquitin conjugating enzyme.
For most substrates of ubiquitin (Ub)-dependent degradation, recognition by the proteasome is mediated by a covalently attached signal assembled from multiple ubiquitins linked to each other via the C terminus of one Ub and the epsilon-amine of Lys(48) of another Ub. Among Ub-conjugating enzymes, E2-25K is unique in its ability to synthesize in vitro unanchored Lys(48)-linked poly-Ub chains from mono- or poly-Ub, E1, and ATP; thus, E2-25K has distinct binding sites for donor and acceptor (poly)Ub. During studies of chain assembly by E2-25K, we observed that Lys(48)-linked tri-Ub was efficiently converted to a new species that upon SDS-polyacrylamide gel electrophoresis migrated between linear di-Ub and tri-Ub. Analysis of this product by mass spectrometry and tryptic digestion showed that it was a cyclic form of tri-Ub. Cyclization of tri-Ub requires E1, E2-25K, ATP, and that the linear substrate has a free Gly(76) C terminus on the proximal end Ub and a Lys(48) side chain available on the distal end Ub. E2-25K similarly can catalyze the cyclization of longer poly-Ub chains, including tetra- and penta-Ub. Although cyclic tri-Ub resists hydrolysis by the PA700 or isopeptidase T deubiquitinating enzymes, it can be disassembled to Ub monomers by isopeptidase(s) in a red blood cell extract. Thus, if cyclic poly-Ub forms in vivo, it will not accumulate as a dead-end product.
Yao
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T
Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, USA.
Cohen
R E
RE
D000818:Animals; D001704:Biopolymers / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D025821:Polyubiquitin; D011487:Protein Conformation; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*
D001704:Biopolymers; D014452:Ubiquitins; D025821:Polyubiquitin; C100879:UBE2K protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D008239:Lysine