Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne

Abstract

Ubiquitin is a versatile cellular signaling molecule that can form polymers of eight different linkages, and individual linkage-types have been associated with distinct cellular functions. Though little is currently known about Lys11-linked ubiquitin chains, recent data indicate that they may be as abundant as Lys48-linkages and involved in vital cellular processes. Here we report the generation of Lys11-linked polyubiquitin in vitro, for which the Lys11-specific E2 enzyme UBE2S was fused to a ubiquitin binding domain. Crystallography and NMR analyses of Lys11-linked diubiquitin reveal that Lys11-linked chains adopt compact conformations in which Ile44 is solvent exposed. Furthermore, we identify the OTU family deubiquitinase Cezanne as the first deubiquitinase with Lys11-linkage preference. Our data highlight the intrinsic specificity of the ubiquitin system that extends to Lys11-linked chains, and emphasize that differentially linked polyubiquitin chains must be regarded as independent posttranslational modifications.

A Highly Efficient Synthesis of Polyubiquitin Chains

“…K11‐diUb was found to adopt a compact conformation, which is consistent with previous studies (Figure 4D),31 whereas K63‐diUb took on an open conformation (Figure 4E). The structural alignment of wild‐type Ub with the Ub units in K11‐ and K63‐diUb very strongly suggests that all the Ub units adopted the native conformation (Figure 4F,G).…”

“…In vitro Ub enzyme systems were first developed for the preparation of polyUbs, and their utility has been demonstrated by the synthesis of almost all types of Ub chains 15, 28, 29, 30, 31. However, K27‐polyUbs and heterogeneous polyUbs have yet to be accessed with this technology 32.…”

miR-486 sustains NF-κB activity by disrupting multiple NF-κB-negative feedback loops

“…Thus, our results not only suggest that Cezanne functions as a tumor suppressor in gliomas but also further support the notion that dysregulation of ubiquitin conjugation/deconjugation is involved in human cancer. Although Cezanne was reported to inhibit NF-κB signaling through removing K63-linked polyubiquitin chains from RIP1 [24,25], Bremm et al [40] found that Cezanne preferentially hydrolyzes K11-linked polyubiquitin chains. Interestingly, a recent study reported that K11-linked polyubiquitin chains of RIP1 can also serve as a platform for recruitment of NEMO and participate in TNF-α-stimulated NF-κB activation [41].…”

HectD1 E3 Ligase Modifies Adenomatous Polyposis Coli (APC) with Polyubiquitin to Promote the APC-Axin Interaction

“…2B) (24). Second, full-length, wild type Trabid hydrolyzes Lys-63-linked chains (24,(37)(38)(39). Third, although Lys-48 and Lys-29 chains were the most abundant ubiquitin polymers that co-precipitated with Trabid C443S, Lys-63 chains were also detected, and the majority of these were present in the HMW regions a and b (Fig.…”