Role of E2-RING Interactions in Governing RNF4-Mediated Substrate Ubiquitination

DiBello, Anthony; Datta, Aritreyee; Zhang, Xiangbin; Wolberger, Cynthia

doi:10.1016/j.jmb.2016.09.018

Cited by 13 publications

(10 citation statements)

References 67 publications

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“…PCR-amplified ORFs corresponding to ZNRF1 full-length, ZNRF1 CTD (residues 139–227), and ZNRF1 RING (residues 166–227), were cloned into pETSUMO2 vector [ 20 ] using the Infusion HD Cloning Kit (Clontech, Inc., U.S.A.) and expressed as fusion proteins containing N-terminal 6xHis–SUMO2 tag. In addition to the N-terminal tag, full-length ZNRF1 also contained a non-cleavable C-terminal strep-tag II for the ease of purification.…”

Section: Methodsmentioning

confidence: 99%

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

Behera

Naskar

Agarwal

et al. 2018

Biochemical Journal

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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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Section: Methodsmentioning

confidence: 99%

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

Behera

Naskar

Agarwal

et al. 2018

Biochemical Journal

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“…Cells were harvested by pelleting at 4000 rpm at 4 °C. Purification of the catalytic mutant OTUB1 C91S ( 9 ), RNF4 ( 51 ), human E1 enzyme ( 52 ), and ubiquitin ( 41 ) were performed as previously described. Cell pellets containing His-UBE2E1 were resuspended in lysis buffer (20 m m HEPES, pH 7.3, 300 m m NaCl, 25 m m imidazole, 2 m m β-mercaptoethanol).…”

Section: Methodsmentioning

confidence: 99%

OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

Pasupala

Morrow

Que

et al. 2018

Journal of Biological Chemistry

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OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

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“…S1). Using genetically encoded linear SUMO2 chains, fused via their C-terminal diglycine and lysine 11 of the distal and proximal moieties (4xSUMO2), a well established model topology [67][68][69][70][71] , we validated eight candidates distributed across a range of gene clusters and M-values ( Fig. 1B; Supplementary Table S1).…”

Section: Biolayer Interferometry Validates Polysumo2 Receptors With Dmentioning

confidence: 99%

“…All XRCC4 recombinant constructs were purified as described previously 78 . The 6xHis-4xSUMO2-Strep expression plasmid was a kind gift from Cynthia Wolberger (Johns Hopkins University, USA) 67 . 6xHis-4xSUMO2-Strep, a linear fusion of a N-terminal full-length SUMO2 fused to truncated SUMO2 (residues 11-92) linked via K11 for the second, third and fourth, was expressed and purified as described previously 67 .…”

Section: Biolayer Interferometry (Bli)mentioning

confidence: 99%

Microarray screening reveals a non-conventional SUMO-binding mode linked to DNA repair by non-homologous end-joining

Cabello-Lobato

Jenner

Loc’h³

et al. 2021

Preprint

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SUMOylation is critical for a plethora of cellular signalling pathways including the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Based on systematic proteome microarray screening combined with widely applicable carbene footprinting and high-resolution structural profiling, we define two non-conventional SUMO2-binding modules on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, interaction of SUMO2 with XRCC4 is incompatible with XRCC4 binding to at least two other NHEJ proteins – XLF and DNA ligase 4 (LIG4). These findings are consistent with SUMO2 interactions of XRCC4 acting as backup pathways at different stages of NHEJ, in the absence of these factors or their dysfunctioning. Such scenarios are not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. This work reveals insights into topology-specific SUMO recognition and its potential for modulating DSB repair by NHEJ. Moreover, it provides a rich resource on binary SUMO receptors that can be exploited for uncovering regulatory layers in a wide array of cellular processes.

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Role of E2-RING Interactions in Governing RNF4-Mediated Substrate Ubiquitination

Cited by 13 publications

References 67 publications

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

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

OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

Microarray screening reveals a non-conventional SUMO-binding mode linked to DNA repair by non-homologous end-joining

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