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 hom multiple ubiquitins linked to each other via the C terminus of one Ub and the E-amine of Lys(48) Of another Uh. Among Ub-conjugating enzymes, E2-25K is unique in its ability to synthesize in vitro unanchored Lys(48)-linked poly-Uh chains from mono- or poly-Uh, 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 Lys48 Side chain available on the distal end Ub. E2-25K similarly can catalyze the cyclization of longer poly-Uh 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-Uh forms in vivo, it will not accumulate as a deadend product.