Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

Suryadinata, Randy; Holien, Jessica K.; Yang, George P.; Parker, Michael W.; Papaleo, Elena; Šarčević, Boris

doi:10.4161/cc.24818

Cited by 19 publications

(13 citation statements)

References 46 publications

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“…K6 and K48 of the acceptor ubiquitin are close to the interaction surface of UEV1a (Mms2; Fig 4D) [17,21]. Similarly, structural (UBCH5 and UBE2K) or modeling (CDC34 and UBE2S) analyses indicate that the K6 residue of noncovalently bound ubiquitin is located at the E2 interaction surface ( Supplementary Fig S6A) [5,7,22,23]. Our surface plasmon resonance (SPR) analysis revealed that the affinity of noncovalent interactions between UEV1a and ubiquitin is lower with AcK6 or AcK48 (Fig 4D and Supplementary Fig S6B).…”

Section: Acetylation Of Ubiquitin At K6 or K48 Represses Polyubiquitimentioning

confidence: 96%

Ubiquitin acetylation inhibits polyubiquitin chain elongation

Ohtake

Saeki

Sakamoto³

et al. 2014

EMBO Reports

122

134

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Ubiquitylation is a versatile post-translational modification (PTM). The diversity of ubiquitylation topologies, which encompasses different chain lengths and linkages, underlies its widespread cellular roles. Here, we show that endogenous ubiquitin is acetylated at lysine (K)-6 (AcK6) or K48. Acetylated ubiquitin does not affect substrate monoubiquitylation, but inhibits K11-, K48-, and K63-linked polyubiquitin chain elongation by several E2 enzymes in vitro. In cells, AcK6-mimetic ubiquitin stabilizes the monoubiquitylation of histone H2B-which we identify as an endogenous substrate of acetylated ubiquitin-and of artificial ubiquitin fusion degradation substrates. These results characterize a mechanism whereby ubiquitin, itself a PTM, is subject to another PTM to modulate mono-and polyubiquitylation, thus adding a new regulatory layer to ubiquitin biology.

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Section: Acetylation Of Ubiquitin At K6 or K48 Represses Polyubiquitimentioning

confidence: 96%

Ubiquitin acetylation inhibits polyubiquitin chain elongation

Ohtake

Saeki

Sakamoto³

et al. 2014

EMBO Reports

122

134

View full text Add to dashboard Cite

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“…A ubiquitin chain can be formed via linkage of any of the seven lysine residues in ubiquitin (K6, K11, K27, K29, K33, K48 and K63) [29, 30] or less commonly through the N-terminal methionine of ubiquitin (M1, linear chain) [31, 32]. The complexity of the system is further enhanced by the existence of several different types of chains: (i) single linkage, (ii) mixed linkages with different linkages in one chain, (iii) branched linkages where one ubiquitin molecule in the chain is attached to two ubiquitin molecules and lastly (iiii) chains consisting of a mixture of ubiquitin and other ubiquitin-like molecules e.g.…”

Section: Introductionmentioning

confidence: 99%

Screening for E3-Ubiquitin ligase inhibitors: challenges and opportunities

et al. 2014

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The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, and its deregulation has been implicated in a wide range of human pathologies that include cancer, neurodegenerative and immunological disorders and viral infections. Targeting the UPS by small molecular regulators thus provides an opportunity for the development of therapeutics for the treatment of several diseases. The proteasome inhibitor Bortezomib was approved for treatment of hematologic malignancies by the FDA in 2003, becoming the first drug targeting the ubiquitin proteasome system in the clinic. Development of drugs targeting specific components of the ubiquitin proteasome system, however, has lagged behind, mainly due to the complexity of the ubiquitination reaction and its outcomes. However, significant advances have been made in recent years in understanding the molecular nature of the ubiquitination system and the vast variety of cellular signals that it produces. Additionally, improvement of screening methods, both in vitro and in silico, have led to the discovery of a number of compounds targeting components of the ubiquitin proteasome system, and some of these have now entered clinical trials. Here, we discuss the current state of drug discovery targeting E3 ligases and the opportunities and challenges that it provides.

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“…The Ub E3 ligase works in concert with an E2 to form Ub conjugates on substrates, and it has been reported that the E2s determine the lysine‐linkage specificity of Ub chains and serve as the key mediators of Ub chain assembly (Ye and Rape, ; Sadowski et al ., ; Middleton and Day, ; Hodge et al ., ). In humans, the E2 enzyme Cdc34 or E2‐25K mediates the formation of poly‐Ub chains conjugated via K48, whereas UBC13 with UEV forms K63 Ub chains (Suryadinata et al ., ; Middleton and Day, ; Hodge et al ., ). In plants, an in vitro conjugation assay has indicated that AtUBC35, the homolog of human UBC13, also mediates the generation of Ub chains linked through K63 (Zhao et al ., ).…”

Section: Resultsmentioning

confidence: 98%

“…E2s play key roles in Ub chain assembly and in determination of the lysine specificity of Ub chains (Ye and Rape, ; Sadowski et al ., ; Middleton and Day, ; Hodge et al ., ). Thus, the concerted actions of the E2s and E3s determine the substrate specificities, the types of Ub modification and even the ubiquitination site(s) on the substrate proteins, resulting in different specificities and destinies of the target proteins (VanDemark et al ., ; Petroski and Deshaies, ; Wickliffe et al ., ; Suryadinata et al ., ).…”

Section: Introductionmentioning

confidence: 97%

Reconstitution of the plant ubiquitination cascade in bacteria using a synthetic biology approach

et al. 2017

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Ubiquitination modulates nearly all aspects of plant life. Here, we reconstituted the Arabidopsis thaliana ubiquitination cascade in Escherichia coli using a synthetic biology approach. In this system, plant proteins are expressed and then immediately participate in ubiquitination reactions within E. coli cells. Additionally, the purification of individual ubiquitination components prior to setting up the ubiquitination reactions is omitted. To establish the reconstituted system, we co-expressed Arabidopsis ubiquitin (Ub) and ubiquitination substrates with E1, E2 and E3 enzymes in E. coli using the Duet expression vectors. The functionality of the system was evaluated by examining the auto-ubiquitination of a RING (really interesting new gene)-type E3 ligase AIP2 and the ubiquitination of its substrate ABI3. Our results demonstrated the fidelity and specificity of this system. In addition, we applied this system to assess a subset of Arabidopsis E2s in Ub chain formation using E2 conjugation assays. Affinity-tagged Ub allowed efficient purification of Ub conjugates in milligram quantities. Consistent with previous reports, distinct roles of various E2s in Ub chain assembly were also observed in this bacterial system. Therefore, this reconstituted system has multiple advantages, and it can be used to screen for targets of E3 ligases or to study plant ubiquitination in detail.

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Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

Cited by 19 publications

References 46 publications

Ubiquitin acetylation inhibits polyubiquitin chain elongation

Ubiquitin acetylation inhibits polyubiquitin chain elongation

Screening for E3-Ubiquitin ligase inhibitors: challenges and opportunities

Reconstitution of the plant ubiquitination cascade in bacteria using a synthetic biology approach

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