A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.

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Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~Ub conformation

10.1101/848788 ◽

2019 ◽

Author(s):

Emma Branigan ◽

J. Carlos Penedo ◽

Ronald T. Hay

Keyword(s):

Single Molecule ◽

Active Form ◽

Resonance Energy Transfer ◽

E3 Ligase ◽

Resonance Energy ◽

Post Translational Modification ◽

E3 Ligases ◽

Closed Conformation ◽

Ring E3 Ligase ◽

Ring E3

AbstractUbiquitination is a eukaryotic post-translational modification that modulates a host of cellular processes1. Modification is mediated by an E1 activating enzyme (E1), an E2 conjugating enzyme (E2) and an E3 ligase (E3). The E1 catalyses formation of a highly reactive thioester linked conjugate between ubiquitin and E2 (E2~Ub)2. The largest class of ubiquitin E3 ligases, which is represented by RING E3s, bind both substrate and E2~Ub and facilitate transfer of ubiquitin from the E2 to substrate. Based on extensive structural analysis3–5 it has been proposed that RING E3s prime the E2~Ub conjugate for catalysis by locking it into a “closed” conformation where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by a substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer is a model that has yet to be experimentally tested. Here we use single molecule Förster Resonance Energy Transfer (smFRET) to test this hypothesis and demonstrate that ubiquitin is transferred from the closed conformation during an E3 catalysed reaction. Using Ubc13 as an E2, we designed a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. Firstly, we defined the high FRET state as the closed conformation using a stable isopeptide linked E2~Ub conjugate, while the low FRET state represents more open conformations. Secondly, we developed a real-time smFRET assay to monitor RING E3 catalysed ubiquitination with a thioester linked E2~Ub conjugate and determined the catalytically active conformation. Our results demonstrate that the reaction proceeds from the high FRET or closed conformation and confirm the hypothesis that the closed conformation is the active form of the conjugate. These findings are not only relevant to RING E3 catalysed ubiquitination but are also broadly applicable to E3 mediated ligation of other ubiquitin-like proteins (Ubls) to substrates.

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Regulation of ubiquitin transfer by XIAP, a dimeric RING E3 ligase

Biochemical Journal ◽

10.1042/bj20121702 ◽

2013 ◽

Vol 450 (3) ◽

pp. 629-638 ◽

Cited By ~ 34

Author(s):

Yoshio Nakatani ◽

Torsten Kleffmann ◽

Katrin Linke ◽

Stephen M. Condon ◽

Mark G. Hinds ◽

...

Keyword(s):

Biochemical Analysis ◽

E3 Ligase ◽

Ring Domain ◽

Aromatic Residue ◽

E3 Ligases ◽

Dimer Interface ◽

Ring E3 Ligase ◽

Thioester Bond ◽

Ring E3 ◽

Apoptosis Proteins

RING domains of E3 ligases promote transfer of Ub (ubiquitin) from the E2~Ub conjugate to target proteins. In many cases interaction of the E2~Ub conjugate with the RING domain requires its prior dimerization. Using cross-linking experiments we show that E2 conjugated ubiquitin contacts the RING homodimer interface of the IAP (inhibitor of apoptosis) proteins, XIAP (X-linked IAP) and cIAP (cellular IAP) 2. Structural and biochemical analysis of the XIAP RING dimer shows that an aromatic residue at the dimer interface is required for E2~Ub binding and Ub transfer. Mutation of the aromatic residue abolishes Ub transfer, but not interaction with Ub. This indicates that nuleophilic attack on the thioester bond depends on precise contacts between Ub and the RING domain. RING dimerization is a critical activating step for the cIAP proteins; however, our analysis shows that the RING domain of XIAP forms a stable dimer and its E3 ligase activity does not require an activation step.

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The Not4 RING E3 Ligase: A Relevant Player in Cotranslational Quality Control

ISRN Molecular Biology ◽

10.1155/2013/548359 ◽

2013 ◽

Vol 2013 ◽

pp. 1-19 ◽

Cited By ~ 9

Author(s):

Martine A. Collart

Keyword(s):

Quality Control ◽

Ubiquitin Ligase ◽

E3 Ligase ◽

Rna Degradation ◽

The Other ◽

Ring E3 Ligase ◽

Ideal Position ◽

Evolutionarily Conserved ◽

A Subunit ◽

Ring E3

The Not4 RING E3 ligase is a subunit of the evolutionarily conserved Ccr4-Not complex. Originally identified in yeast by mutations that increase transcription, it was subsequently defined as an ubiquitin ligase. Substrates for this ligase were characterized in yeast and in metazoans. Interestingly, some substrates for this ligase are targeted for polyubiquitination and degradation, while others instead are stable monoubiquitinated proteins. The former are mostly involved in transcription, while the latter are a ribosomal protein and a ribosome-associated chaperone. Consistently, Not4 and all other subunits of the Ccr4-Not complex are present in translating ribosomes. An important function for Not4 in cotranslational quality control has emerged. In the absence of Not4, the total level of polysomes is reduced. In addition, translationally arrested polypeptides, aggregated proteins, and polyubiquitinated proteins accumulate. Its role in quality control is likely to be related on one hand to its importance for the functional assembly of the proteasome and on the other hand to its association with the RNA degradation machines. Not4 is in an ideal position to signal to degradation mRNAs whose translation has been aborted, and this defines Not4 as a key player in the quality control of newly synthesized proteins.

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The deubiquitylase USP9X controls ribosomal stalling

10.1101/2020.04.15.042291 ◽

2020 ◽

Author(s):

Anne Clancy ◽

Claire Heride ◽

Adán Pinto-Fernández ◽

Andreas Kallinos ◽

Katherine J. Kayser-Bricker ◽

...

Keyword(s):

Protein Stability ◽

E3 Ligases ◽

Molecule Inhibitor ◽

Steady State Levels ◽

Chemical Inhibition ◽

The Stability ◽

Ring E3 Ligases ◽

Ring E3 ◽

Inhibitor Treatment ◽

Hct116 Cells

AbstractWhen a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases respectively, through ubiquitylation of the eS10 and uS10 sub-units of the ribosome. It is common for the stability of RING E3 ligases to be regulated by an interacting deubiquitylase (DUB), which often opposes auto-ubiquitylation of the E3. Here, we show that the DUB USP9X directly interacts with ZNF598 and regulates its abundance through the control of protein stability in human cells. We have developed a highly specific small molecule inhibitor of USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome associated protein stability and reveals loss of ZNF598 and Makorin 2. In the absence of USP9X or following chemical inhibition of its catalytic activity, steady state levels of Makorins and ZNF598 are diminished and the ribosomal quality control pathway is impaired.

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Structural insights into the nanomolar affinity of RING E3 ligase ZNRF1 for Ube2N and its functional implications

Biochemical Journal ◽

10.1042/bcj20170909 ◽

2018 ◽

Vol 475 (9) ◽

pp. 1569-1582 ◽

Cited By ~ 5

Author(s):

Adaitya Prasad Behera ◽

Pritam Naskar ◽

Shubhangi Agarwal ◽

Prerana Agarwal Banka ◽

Asim Poddar ◽

...

Keyword(s):

Dissociation Constants ◽

E3 Ligase ◽

E3 Ligases ◽

Ring E3 Ligase ◽

New Gene ◽

Functional Analyses ◽

Affinity Interaction ◽

Ring E3 Ligases ◽

Ring E3 ◽

Structural Insights

RING (Really Interesting New Gene) domains in ubiquitin RING E3 ligases exclusively engage ubiquitin (Ub)-loaded E2s to facilitate ubiquitination of their substrates. Despite such specificity, all RINGs characterized till date bind unloaded E2s with dissociation constants (Kds) in the micromolar to the sub-millimolar range. Here, we show that the RING domain of E3 ligase ZNRF1, an essential E3 ligase implicated in diverse cellular pathways, binds Ube2N with a Kd of ∼50 nM. This high-affinity interaction is exclusive for Ube2N as ZNRF1 interacts with Ube2D2 with a Kd of ∼1 µM, alike few other E3s. The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N coupled with mutational analyses reveals the molecular basis of this unusual affinity. We further demonstrate that the ubiquitination efficiency of ZNRF1 : E2 pairs correlates with their affinity. Intriguingly, as a consequence of its high E2 affinity, an excess of ZNRF1 inhibits Ube2N-mediated ubiquitination at concentrations ≥500 nM instead of showing enhanced ubiquitination. This suggests a novel mode of activity regulation of E3 ligases and emphasizes the importance of E3-E2 balance for the optimum activity. Based on our results, we propose that overexpression-based functional analyses on E3 ligases such as ZNRF1 must be approached with caution as enhanced cellular levels might result in aberrant modification activity.

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