The Lysine 48 and Lysine 63 Ubiquitin Conjugates Are Processed Differently by the 26 S Proteasome

Abstract

The role of Lys-63 ubiquitin chains in targeting proteins for proteasomal degradation is still obscure. We systematically compared proteasomal processing of Lys-63 ubiquitin chains with that of the canonical proteolytic signal, Lys-48 ubiquitin chains. Quantitative mass spectrometric analysis of ubiquitin chains in HeLa cells determines that the levels of Lys-63 ubiquitin chains are insensitive to short-time proteasome inhibition. Also, the Lys-48/Lys-63 ratio in the 26 S proteasome-bound fraction is 1.7-fold more than that in the cell lysates, likely because some cellular Lys-63 ubiquitin conjugates are sequestered by Lys-63 chain-specific binding proteins. In vitro, Lys-48 and Lys-63 ubiquitin chains bind the 26 S proteasome comparably, whereas Lys-63 chains are deubiquitinated 6-fold faster than Lys-48 chains. Also, Lys-63 tetraubiquitin-conjugated UbcH10 is rapidly deubiquitinated into the monoubiquitinated form, whereas Lys-48 tetraubiquitin targets UbcH10 for degradation. Furthermore, we found that both the ubiquitin aldehyde-and 1,10-phenanthroline-sensitive deubiquitinating activities of the 26 S proteasome contribute to Lys-48-and Lys-63-linkage deubiquitination, albeit the inhibitory extents are different. Together, our findings suggest that compared with Lys-48 chains, cellular Lys-63 chains have less proteasomal accessibility, and proteasome-bound Lys-63 chains are more rapidly deubiquitinated, which could cause inefficient degradation of Lys-63 conjugates.

Deubiquitinase Inhibition by Small-Molecule WP1130 Triggers Aggresome Formation and Tumor Cell Apoptosis

“…5C). USP5 and UCH37 are known to deubiquitinate both K48-and K63-linked polyubiquitinated proteins (50,51). The accumulation of K63-linked polyubiquitinated proteins in WP1130-treated HEK293T cells may partially be explained based on these findings, although there are other DUBs, such as CYLD (51), which can break such chains as well.…”

Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21

“…Although Ub is a canonical degradation signal, both Ub-48 Ub and Ub-63 Ub are hydrolyzed by purified proteasomes in vitro (16,17), and some studies have observed Ub-63 Ub specificity in the polyUb amputation activity of Poh1 (18,19). Whether Ub-63 Ub has a role at the proteasome remains unclear; its presence at this location would seem incongruous with a model in which only Ub-48 Ublabeled proteins are genuine substrates (20,21).…”

“…1). Of the three DUBs associated with the proteasome 19S RP, Poh1 uniquely cleaves an isopeptide bond between the proximal Ub in a polyUb adduct and its substrate, thereby releasing a polyUb chain en bloc (16,25,26). To test the notion that Poh1 might deubiquitinate TRIM21, we stably depleted Poh1 in TE671 cells by shRNA expression, assessing efficiency by immunoblot (Fig.…”

Ubiquitin Chain Trimming Recycles the Substrate Binding Sites of the 26 S Proteasome and Promotes Degradation of Lysine 48-linked Polyubiquitin Conjugates

“…It was originally suggested that the chain trimming activity serves as a safeguard to rescue poorly ubiquitinated proteins from the proteasome by editing the conjugates (6). We recently found that Lys-63 polyUb chains are highly susceptible to chain trimming, which renders Lys-63-linked polyUb conjugates to be rapidly deubiquitinated by the 26 S proteasome without efficient degradation (11). Thus, Ub chain trimming could rescue Lys-63-linked substrates from proteasomal degradation.…”