Allosteric Targeting of the Fanconi Anemia Ubiquitin-Conjugating Enzyme Ube2T by Fragment Screening

Morreale, Francesca; Bortoluzzi, Alessio; Chaugule, Viduth K.; Arkinson, Connor; Walden, Helen; Ciulli, Alessio

doi:10.1021/acs.jmedchem.7b00147

Cited by 31 publications

(41 citation statements)

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“…Biochemical, structural and computational studies have revealed how RING/U-box E3s stimulate internal dynamics in different E2s that are linked to their ubiquitination activity (Benirschke et al, 2010; Chakrabarti et al, 2017; Das et al, 2013; Ozkan et al, 2005). Furthermore, recent efforts in fragment-based inhibitor discovery have revealed promising lead compounds that bind Ube2T (Morreale et al, 2017) as well as the unrelated Ube2I (Hewitt et al, 2016) at regions distal from their active/E3 binding sites, nevertheless can allosterically regulate the respective E2 activities. These observations collectively suggest that allosteric networks could operate across the E2 family and that future investigations into such networks could prove instrumental for basic and translational research in ubiquitin biology.…”

Section: Discussionmentioning

confidence: 99%

Allosteric network in Ube2T drives specificity for RING E3 catalysed ubiquitin signals

Chaugule

Arkinson

Toth

et al. 2018

Preprint

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10In eukaryotes, DNA damage repair is implemented by a host of proteins that are coordinated 11 by defined molecular signals. One such signal that transpires during the Fanconi Anemia 12 (FA) -interstrand crosslink (ICL) repair pathway is the site-specific monoubiquitination of 13 FANCD2 and FANCI proteins by a large, multi-protein FA core complex. The mechanics for 14 this exquisitely specific monoubiquitin signal has been elusive. Here we show FANCL, the 15 RING E3 module of the FA core complex, allosterically activates its cognate E2 Ube2T for 16 monoubiquitination by a mechanism distinct from the typical RING-based catalysis. FANCL 17 triggers intricate re-wiring of Ube2T's intra-residue network thus activating the E2 for 18 precision targeting. This network is intrinsically regulated by conserved gates and loops 19 which can be engineered to yield Ube2T variants that enhance FANCD2 ubiquitination by 20 ~30-fold without compromising on target specificity. Finally, we also uncover allosteric 21 networks in other ubiquitin E2s that can be leveraged by RING E3 ligases to drive specific 22 ubiquitination. 23 24 25 Keywords: DNA repair / E2 / Enzyme allostery / RING E3 / Ubiquitination 26 109 110 7 Results 111The E2 -E3 pair Ube2T -FANCL ubiquitinates its substrates via an atypical 112 mechanism 113 Previous in vitro studies using recombinant chicken (Alpi et al., 2008; Sato et al., 2012), frog 114 (Hodson et al., 2014) and human (Longerich et al., 2014) proteins report the isolated FANCL 115 enzyme with Ube2T directs FANCD2 monoubiquitination at its physiological target site. 116However, the underlying mechanism for the site-specific and strict mono-modification is 117 unclear. In order to understand how the Ube2T and FANCL enzyme pair catalyse this 118 specific signal we reconstituted a minimal E2 -E3 module with human proteins. Based on 119 our earlier work we designed and purified a FANCL URD-RING fragment (FANCL UR , 120 residues 109-375) that is stable, monomeric ( Supplementary Fig 1A) and comprises both the 121 substrate (UBC-RWD domain) and the E2 (RING domain) binding regions (Hodson et al., 122 2011; Hodson et al., 2014). We then tested the activity of the FANCL UR fragment in in vitro 123 FANCD2 ubiquitination assays using fluorescently labelled ubiquitin (Ub IR800 ). Previous 124 studies have shown the additional requirements of FANCI and DNA in complex with 125 FANCD2 for efficient monoubiquitination of this substrate. Consistent with this we observe 126Ube2T -FANCL UR mediated FANCD2 modification when present as a FANCD2-FANCI-127 dsDNA complex ( Fig 1A). Further, an arginine mutant of the physiological FANCD2 target 128 site (FANCD2 K561R) prevents ubiquitination thus confirming the minimal E2 -E3 module 129 is both active and site-specific. We wondered if the FANCL UR fragment could also 130 specifically ubiquitinate FANCI present in the FANCD2-FANCI-dsDNA complex. To test 131 this we titrated increasing amounts of the E2 -E3 module in reactions containing single 132 target site complexes ...

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Section: Discussionmentioning

confidence: 99%

Allosteric network in Ube2T drives specificity for RING E3 catalysed ubiquitin signals

Chaugule

Arkinson

Toth

et al. 2018

Preprint

Self Cite

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“…UBE2T is the E2 enzyme of the Fanconi anemia pathway, which is indispensable to the repair of DNA interchain cross-links 59 . Prior research support the argument that UBE2T is associated with adverse outcomes in breast and lung cancers 60 .…”

Section: Analysis Of Core Genes By the Kaplan Meier Plotter And Gepiamentioning

confidence: 99%

Identification of grade-specific diagnostic and prognostic biomarkers of key candidate genes and pathways in breast cancer by integrated bioinformatics analysis

Zhang

Zhou

Zhang

et al. 2020

Preprint

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“…However, the biological role and action mechanism of these enzymes in ovarian cancer have not been fully elucidated. UBE2T, a member of the UBE2 family, also known as FANCT or HSPC150, combines with FANCL (the E3 ligase) to catalyze the monoubiquitination of the heterodimeric FANCI/FANCD2 complex, contributes to DNA repair in the Fanconi anemia pathway, and plays a critical role in cell proliferation and the maintenance of chromosome stability [8]. Furthermore, several studies have con rmed that UBE2T plays a carcinogenic role in various types of cancer, including hepatocellular carcinoma, as well as lung, breast, stomach, bladder, and prostate cancer, but its expression level and prognostic value in ovarian cancer are still unclear [9][10][11][12][13][14].…”

Section: Introductionmentioning

confidence: 99%

Increased expression of UBE2T predicting poor survival of ovarian cancer: based on bioinformatics analysis of UBE2s, clinical samples and the GEO database

Zou

et al. 2020

Preprint

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Background：Ubiquitin-conjugating enzymes E2 (UBE2) have been reported in the microenvironment of various malignant tumors, but their correlation with ovarian cancer remains elusive. Methods: The Oncomine, GEPIA, Kaplan-Meier Plotter, cBioPortal, and STRING databases were used to systematically analyze the expression pattern, prognostic value, genetic variation, and biological function of 12 members of the UBE2 gene family in ovarian cancer. UBE2T exhibited a strong correlation with ovarian cancer and was thus further examined. Gene set enrichment analysis (GSEA), as well as analyses of function and pathway enrichment, somatic mutations, copy number variation, and methylation were performed, and the correlation with immune cell infiltration was examined to explore the mechanism underlying aberrant UBE2T expression. Finally, the expression and prognostic value of UBE2T in ovarian cancer were verified by immunohistochemical evaluation of 131 clinical ovarian samples and the Gene Expression Omnibus (GEO) database (GSE51088, GSE73614, and GSE63885 datasets) analysis.Results: The mRNA levels of UBE2C, UBE2N, UBE2S, and UBE2T were significantly upregulated in ovarian cancer compared with those in normal ovarian tissue. In patients with ovarian serous carcinoma, UBE2A, UBE2B, UBE2C, UBE2G, UBE2R2 and UBE2T upregulation were associated with poor overall survival. Moreover, UBE2A, UBE2N, UBE2R2 and UBE2T upregulation and UBE2G downregulation were associated with poor progression-free survival. Immunohistochemistry revealed that UBE2T was significantly upregulated in ovarian malignant tumors compared with that in borderline tumors, benign tumors, and normal ovarian tissues, and its high expression was associated with poor prognosis. The Cox model showed that UBE2T upregulation was an independent risk factor affecting the prognosis of ovarian cancer (hazard ratio: 4.095, P=0.029). The above results were verified in the GEO database. In addition, UBE2T was associated with specific immune cells and mainly involved in cell cycle-related events. Genomic analysis showed that TP53 and TTN mutations were associated with UBE2T expression. Gene copy number amplification and hypomethylation may be responsible for UBE2T upregulation in ovarian cancer. Conclusions: UBE2 family members may play a role in the development of ovarian cancer. Specifically, UBE2T could serve as a new prognostic marker and therapeutic target for this disease.

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Allosteric Targeting of the Fanconi Anemia Ubiquitin-Conjugating Enzyme Ube2T by Fragment Screening

Cited by 31 publications

References 40 publications

Allosteric network in Ube2T drives specificity for RING E3 catalysed ubiquitin signals

Allosteric network in Ube2T drives specificity for RING E3 catalysed ubiquitin signals

Identification of grade-specific diagnostic and prognostic biomarkers of key candidate genes and pathways in breast cancer by integrated bioinformatics analysis

Increased expression of UBE2T predicting poor survival of ovarian cancer: based on bioinformatics analysis of UBE2s, clinical samples and the GEO database

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