Cyclization of Polyubiquitin by the E2-25K Ubiquitin Conjugating Enzyme

Abstract

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 ⑀-amine of Lys 48 of another Ub. Among Ubconjugating 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 tetraand 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 deadend product.

Conformational Dynamics of Wild-type Lys-48-linked Diubiquitin in Solution

“…Preparation of Wild-type and Cyclic Lys-48-linked diUbs-E2-25K catalyzes the formation of cyclic polyUb chains as well as the non-cyclic variety in vitro (36). To prepare wild-type Lys-48-linked diUb by suppressing the formation of the cyclic forms, we used Ub-His.…”

E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1)

“…RING finger E3 ligases have been reported to catalyze autoubiquitinylation (15) and substrate ubiquitinylation (34), as well as the synthesis of unanchored polyubiquitin chains in vitro (23,24). Although previously unrecognized substrate(s) for many proteins containing RING finger domain(s) continue to be identified, for the majority, including ARD1, the physiological substrates remain unknown.…”

“…In the in vitro ubiquitinylation assays containing only ATP and free ubiquitin plus three other purified proteins (E1, E2͞UbcH6, and E3͞ARD1), products should be only ubiquitinylated E1, E2, E3, and͞or heterogeneous ''free,'' unanchored polyubiquitin chains (23)(24)(25), and͞or contaminants in the protein preparations. Immunoblotting with antibodies specific for GST, ARD1, E1, or UbcH6 was used to try to detect ubiquitinylated forms of these proteins.…”

Distinct Functional Surface Regions on Ubiquitin

“…with ubiquitin leads to the accumulation of circular polyubiquitin chains that are inefficiently recognized (38). Circular chains were absent from the preparations used here, based on the finding that Ͼ85% of the Ub 4 and Ub 5 in each mixture was disassembled to Ub 1 upon incubation with purified isopeptidase T. Proteasome assays employed 100 nM Ub 5 DHFR and 10 nM proteasomes (10), plus 0.5 mg/ml monoubiquitin to prevent absorptive loss of Ub 5 DHFR.…”