E2 enzymes in genome stability: pulling the strings behind the scenes

Osborne, Hugh C.; Irving, Elsa; Forment, Josep V.; Schmidt, Christine K.

doi:10.1016/j.tcb.2021.01.009

Cited by 20 publications

(19 citation statements)

References 111 publications

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“…To explore the electrostatic variability in the E2 HTH , we constructed a deep sequence alignment and consensus sequence for each E2, aligned them using Promals3D, and created a phylogenetic tree. This phylogenetic reconstruction resembled others and indicated that the E2s we tested were sampled broadly across the tree (Fig EV2A; Winn et al, 2004;Osborne et al, 2021). Based on this consensus alignment, we also analyzed the Shannon entropy across E2 families.…”

Section: Helix-turn-helix Motif Of the Ubc Modulates Activity Of Multiple E2ssupporting

confidence: 66%

Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes

et al. 2021

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Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the Nterminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix-turnhelix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2 HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2S HTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2 HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination.

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Section: Helix-turn-helix Motif Of the Ubc Modulates Activity Of Multiple E2ssupporting

confidence: 66%

Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes

et al. 2021

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“…To date, approximately 20 inhibitors of ubiquitin-conjugating E2 enzymes have been discovered. − , Many of the reported inhibitors target the active site Cys, others act by disrupting protein–protein interactions, and some have an allosteric effect on ubiquitin transfer. Because of the conservation and lack of a distinctive active site pocket on E2 enzymes, many of these orthosteric inhibitors have low specificity and relatively high IC 50 values . Display technologies offer powerful ways of isolating protein or peptide-based inhibitors of proteins (including E2 enzymes) that can circumvent these problems.…”

Section: Resultsmentioning

confidence: 99%

“…Together, the machinery covalently links ubiquitin to a substrate lysine or N-terminal methionine residue with an isopeptide bond. Ubiquitin itself can be a substrate as it contains seven Lys residues, and this results in the formation of ubiquitin chains with distinct consequences . For example, chains linked by Lys63 can act as scaffolds for recruiting proteins to signaling cascades, whereas Lys48-linked ubiquitin chains typically result in degradation of the attached substrate by the proteasome .…”

Section: Introductionmentioning

confidence: 99%

“…Ubiquitin itself can be a substrate as it contains seven Lys residues, and this results in the formation of ubiquitin chains with distinct consequences. 8 For example, chains linked by Lys63 can act as scaffolds for recruiting proteins to signaling cascades, whereas Lys48-linked ubiquitin chains typically result in degradation of the attached substrate by the proteasome. 8 In the presence of RING E3 ligases, the nature of the ubiquitin signal is typically dictated by the E2 enzyme, of which there are ∼40 in humans.…”

Section: ■ Introductionmentioning

confidence: 99%

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Identification of Ubiquitin Variants That Inhibit the E2 Ubiquitin Conjugating Enzyme, Ube2k

et al. 2021

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Transfer of ubiquitin to substrate proteins regulates most processes in eukaryotic cells. E2 enzymes are a central component of the ubiquitin machinery, and generally determine the type of ubiquitin signal generated and thus the ultimate fate of substrate proteins. The E2, Ube2k, specifically builds degradative ubiquitin chains on diverse substrates. Here we have identified protein-based reagents, called ubiquitin variants (UbVs), that bind tightly and specifically to Ube2k. Crystal structures reveal that the UbVs bind to the E2 enzyme at a hydrophobic cleft that is distinct from the active site and previously identified ubiquitin binding sites. We demonstrate that the UbVs are potent inhibitors of Ube2k and block both ubiquitin charging of the E2 enzyme and E3-catalyzed ubiquitin transfer. The binding site of the UbVs suggests they directly clash with the ubiquitin activating enzyme, while potentially disrupting interactions with E3 ligases via allosteric effects. Our data reveal the first protein-based inhibitors of Ube2k and unveil a hydrophobic groove that could be an effective target for inhibiting Ube2k and other E2 enzymes.

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“…The homology of the shallow E2 active site across the enzyme superfamily has raised doubts over whether it can be selectively targeted pharmacologically. Prior orthosteric (active site-targeting) E2 pre-clinical inhibitors have mainly consisted of covalent binders, indicative of the UBC domain topology [28]. In 2014, the sesquiterpene lactone IJ-5, a natural product, was identified as a UBE2D1-3 inhibitor, which was further complemented by a later computational study (Figure 1C) [20,29].…”

Section: Bds Activity Is Not Mediated By Cellular Inhibition Of Ube2d Enzymesmentioning

confidence: 97%

Sesquiterpene Lactones Potentiate Olaparib-Induced DNA Damage in p53 Wildtype Cancer Cells

Osborne

Larrosa

Schmidt

2022

IJMS

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Despite notable advances in utilising PARP inhibitor monotherapy, many cancers are not PARP inhibitor-sensitive or develop treatment resistance. In this work, we show that the two structurally-related sesquiterpene lactones, a 2-bromobenzyloxy derivative of dehydrosantonin (BdS) and alantolactone (ATL) sensitise p53 wildtype, homologous recombination-proficient cancer cells to low-dose treatment with the PARP inhibitor, olaparib. Exposure to combination treatments of olaparib with BdS or ATL induces cell-cycle changes, chromosomal instability, as well as considerable increases in nuclear area. Mechanistically, we uncover that mitotic errors likely depend on oxidative stress elicited by the electrophilic lactone warheads and olaparib-mediated PARP-trapping, culminating in replication stress. Combination treatments exhibit moderately synergistic effects on cell survival, probably attenuated by a p53-mediated, protective cell-cycle arrest in the G2 cell-cycle phase. Indeed, using a WEE1 inhibitor, AZD1775, to inhibit the G2/M cell-cycle checkpoint further decreased cell survival. Around half of all cancers diagnosed retain p53 functionality, and this proportion could be expected to increase with improved diagnostic approaches in the clinic. Utilising sublethal oxidative stress to sensitise p53 wildtype, homologous recombination-proficient cancer cells to low-dose PARP-trapping could therefore serve as the basis for future research into the treatment of cancers currently refractory to PARP inhibition.

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E2 enzymes in genome stability: pulling the strings behind the scenes

Cited by 20 publications

References 111 publications

Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes

Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes

Identification of Ubiquitin Variants That Inhibit the E2 Ubiquitin Conjugating Enzyme, Ube2k

Sesquiterpene Lactones Potentiate Olaparib-Induced DNA Damage in p53 Wildtype Cancer Cells

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