Biology subjects

Hay, R. T.

Publications and source records attributed to Hay, R. T..

12 recordsLinked to original sources

bioRxiv2026

Photocrosslinking Activity-Based Probes to Capture the Dynamics of Ubiquitin RING E3 Ligase Interactions

Almost all cellular processes are influenced by ubiquitination. A large family of enzymes known as E3 ligases provide the specificity for ubiquitination, with the largest class among them, the RING E3s, comprising over 600 members in humans. RING E3s facilitate transfer of ubiquitin to substrates by constraining the highly dynamic E2-Ub thioester linkage to be primed for attack from the substrate nucleophile. We have established a workflow that uses a modified ubiquitin carrying a photoactivatable crosslinker that once stably linked to the active site of an E2, creates an activity based probe (ABP) to monitor interactions with E3 ligases. Using this, regions of interaction between ubiquitin and a selection of different RING E3 were determined, which not only confirmed existing structures of E2-Ub-RING-E3 complexes but was also used to assess new Ub-E2-E3 models generated in absence of existing structures.

molecular biology↗

bioRxiv2025

E2A SUMOylation promotes HAdV mediated inhibition of the tumor suppressor p53 during infection

HAdV express early viral genes to modulate the activity of the cellular tumor suppressor p53 and ensure efficient viral replication, through various processes, whilst some of them are not completely understood. HAdV oncoprotein E1B-55K interactions with SUMO, PML-IV/V and Sp100A are tightly connected to the transforming potential of the viral factor in non-lytic infections. SUMO modified HAdV DNA binding protein (DBP) E2A interacts with PML and Sp100A acting as a molecular bridge between viral replication centers (RCs) and PML tracks. Here, we provide a novel concept showing that HAdV E2A is involved in the inhibition of p53 activity and apoptosis; thus we identified a novel route of exhibiting virus induced oncogenic potential. We revealed an E1B-55K independent localization of p53 within HAdV replication centers marked by E2A. In the absence of any functional E1B-55K protein expression, p53 interacts with E2A. E2A SUMOylation promotes HAdV- mediated inhibition of p53/DNA binding and p53-dependent transactivation by supporting p53 degradation, E1B-55K interaction and colocalization with PML-V that leads to E1B-55K induced upregulation of p53 SUMOylation. In sum, we provide evidence for a novel mechanism by which the HAdV DBP E2A protein inhibits p53- mediated transcriptional activation and apoptotic host cell response. Intriguingly, these findings change the longstanding dogma of host DNA damage response (DDR) inactivation by HAdV infection and offer crucial insights to improve future p53 selective oncolytic virus therapies and adenoviral vectors.

cell biology↗

bioRxiv2024

Mechanism of degrader-targeted protein ubiquitinability

Small molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and the potential to tackle previously undrugged targets. Thermodynamically stable and kinetically long-lived degrader-mediated ternary complexes can drive faster, more profound and durable target degradation, however the mechanistic features by which they impact on target ubiquitination remain elusive. Here, we solve cryo-EM structures of the VHL Cullin 2 RING E3 ligase complexed with degrader MZ1, target protein Brd4BD2 and primed for catalysis with its cognate E2-ubiquitin bound. We find that Brd4BD2 adopts a favourable orientation towards the E2 active site. In vitro ubiquitination coupled with mass spectrometry illuminates a patch of ubiquitinable lysines on one face of Brd4BD2, with Lys456 showing optimal distance and geometry for nucleophilic attack. Our results demonstrate the proficiency of MZ1 in directing the substrate towards catalysis, explains the favourability of Brd4BD2 for ubiquitination, and reveals the flexibility of the enzyme in capturing sub-optimal lysines. We propose a model for ubiquitinability of degrader-recruited targets that provides a mechanistic blueprint for further rational drug design and optimization. One-Sentence SummaryStructural assembly a PROTAC-mediated complex of whole Cullin RING E3 ligase with bound target and E2-ubiquitin reveals structural and mechanistic insights of specificity for target protein ubiquitination.

biochemistry↗

bioRxiv2023

Concerted SUMO-targeted ubiquitin ligase activities of TOPORS and RNF4 are essential for stress management and cell proliferation

Protein SUMOylation provides a principal driving force for cellular stress responses including DNA-protein crosslink (DPC) repair and arsenic-induced PML body degradation. In genome-scale screens, we identified the human E3 ligase TOPORS as a key effector of SUMO-dependent DPC resolution. We demonstrate that TOPORS promotes DPC repair by functioning as a SUMO-targeted ubiquitin ligase (STUbL) for DPCs, combining ubiquitin ligase activity through its RING domain with poly-SUMO chain binding via a cluster of SUMO-interacting motifs, analogous to the STUbL RNF4. Surprisingly, the STUbL activities of TOPORS and RNF4 are both required for SUMO-dependent DPC repair, PML degradation and other stress responses, making overlapping and distinct contributions to ubiquitin chain formation on SUMOylated targets to enable p97/VCP unfoldase recruitment. Combined loss of TOPORS and RNF4 is synthetic lethal even in unstressed cells, leading to defective clearance of SUMOylated proteins from chromatin accompanied by cell cycle arrest and apoptosis. Together, our findings establish TOPORS as a novel STUbL whose concerted action with RNF4 defines a general mechanistic principle in crucial cellular processes governed by direct SUMO-ubiquitin crosstalk. HighlightsO_LIThe RING E3 ligase TOPORS is required for SUMO-dependent DPC repair C_LIO_LITOPORS is a novel SUMO-targeted ubiquitin ligase (STUbL) C_LIO_LITOPORS promotes multiple STUbL-driven processes in conjunction with RNF4 C_LIO_LICombined TOPORS and RNF4 loss is synthetic lethal in human cells C_LI

molecular biology↗

bioRxiv2023

PML mutants resistant to arsenic induced degradation fail to generate the appropriate SUMO and ubiquitin signals required for RNF4 and p97 recruitment

Arsenic is an effective treatment for Acute Promyelocytic Leukaemia as it induces degradation of the Promyelocytic Leukaemia (PML) - retinoic acid receptor alpha (RARA) oncogenic fusion protein. Some patients relapse with arsenic resistant disease because of missense mutations in PML. To determine the mechanistic basis for arsenic resistance we reconstituted PML-/- cells with YFP fusions of wild type (WT) and two mutant forms of PMLV found in patients refractory to arsenic, A216T and L217F. Both mutants formed PML bodies that were larger, but less numerous than WT and neither responded to arsenic by degradation. Analysis of immunoprecipitated PML bodies indicated that while WT PML experiences increased SUMO1, SUMO2/3 and ubiquitin conjugation, A216T PML is almost completely unresponsive and therefore does not recruit the SUMO targeted ubiquitin E3 ligase RNF4. Compared to WT PML, L217F PML was modified to a similar extent by SUMO2 but not SUMO1 and although it recruited RNF4, it failed to develop the appropriate poly-ubiquitin conjugates required to recruit the segregase p97, which is essential for PML degradation.

cell biology↗

bioRxiv2021

Negative modulation of macroautophagy by HERPUD1 is counteracted by an increased ER-lysosomal network with impact in drug-induced stress cell survival

Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation but independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER protein of short-half life and a well-known substrate of the proteasome. We found that increased HERPUD1 stability by deletion of its ubiquitin-like domain (UBL) plays a negative role on basal and induced macroautophagy. Moreover, we found it triggers ER expansion by reordering the ER in crystalloid structures, but in the absence of unfolded protein response activation. Surprisingly, we found ER expansion led to an increase in the number and function of lysosomes establishing a tight network with the presence of membrane-contact sites. Importantly, a phosphomimetic S59D mutation within the UBL mimics UBL deletion on its stability and the ER-lysosomal network expansion revealing an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/447273v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1136b35org.highwire.dtl.DTLVardef@1e39b7eorg.highwire.dtl.DTLVardef@1f59211org.highwire.dtl.DTLVardef@148b2b0_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

SUMO maintains the chromatin environment of human induced pluripotent stem cells.

To determine the role of SUMO modification in the maintenance of pluripotent stem cells we used ML792, a potent and selective inhibitor of SUMO Activating Enzyme. Treatment of human induced pluripotent stem cells with ML792 initiated changes associated with loss of pluripotency such as reduced expression of key pluripotency markers. To identify putative effector proteins and establish sites of SUMO modification, cells were engineered to stably express either SUMO1 or SUMO2 with TGG to KGG mutations that facilitate GlyGly-K peptide immunoprecipitation and identification. A total of 976 SUMO sites were identified in 427 proteins. STRING enrichment created 3 networks of proteins with functions in regulation of gene expression, ribosome biogenesis and RNA splicing, although the latter two categories represented only 5% of the total GGK peptide intensity. The remainder have roles in transcription and chromatin structure regulation. Many of the most heavily SUMOylated proteins form a network of zinc-finger transcription factors centred on TRIM28 and associated with silencing of retroviral elements. At the level of whole proteins there was only limited evidence for SUMO paralogue-specific modification, although at the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. We show that SUMO is involved in the maintenance of the pluripotent state in hiPSCs and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells.

cell biology↗

bioRxiv2019

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

Ubiquitination 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 Forster 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.

biochemistry↗

bioRxiv2019

SUMOylation targets shugoshin to stabilize sister kinetochore biorientation

The accurate segregation of chromosomes during mitosis relies on the attachment of sister chromatids to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore-microtubule remains unclear. Here we identify SUMOylation as a mechanism that promotes anaphase onset upon biorientation. SUMO ligases modify the tension-sensing pericentromere-localized chromatin protein, shugoshin, to stabilize bioriented sister kinetochore-microtubule attachments. In the absence of SUMOylation, Aurora B kinase removal from kinetochores is delayed. Shugoshin SUMOylation prevents its binding to protein phosphatase 2A (PP2A) and release of this interaction is important for stabilizing sister kinetochore biorientation. We propose that SUMOylation modulates the kinase-phosphatase balance within pericentromeres to inactivate the error correction machinery, thereby allowing anaphase entry in response to biorientation.

cell biology↗

bioRxiv2019

Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer

Post-translational modification of proteins with ubiquitin represents a widely used mechanism for cellular regulation. Ubiquitin is activated by an E1 enzyme, transferred to an E2 conjugating enzyme and covalently linked to substrates by one of an estimated 600 E3 ligases (1). RING E3 ligases play a pivotal role in selecting substrates and priming the ubiquitin loaded E2 (E2~Ub) for catalysis (2,3). RING E3 RNF4 is a SUMO targeted ubiquitin ligase (4) with important roles in arsenic therapy for cancer (4,5) and in DNA damage responses (6,7). RNF4 has a RING domain and a substrate recognition domain containing multiple SUMO Interaction Motifs (SIMO_SCPLOWSC_SCPLOW) embedded in a region thought to be intrinsically disordered (8). While molecular details of SUMO recognition by the SIMs (8-10) and RING engagement of ubiquitin loaded E2 (3,11-15) have been determined, the mechanism by which SUMO substrate is delivered to the RING to facilitate ubiquitin transfer is an important question to be answered. Here, we show that the intrinsically disordered substrate-recognition domain of RNF4 maintains the SIMs in a compact global architecture that facilitates SUMO binding, while a highly-basic region positions substrate for nucleophilic attack on RING-bound ubiquitin loaded E2. Contrary to our expectation that the substrate recognition domain of RNF4 was completely disordered, distance measurements using single molecule Fluorescence Resonance Energy Transfer (smFRET) and NMR paramagnetic relaxation enhancement (PRE) revealed that it adopts a defined conformation primed for SUMO interaction. Mutational and biochemical analysis indicated that electrostatic interactions involving the highly basic region linking the substrate recognition and RING domains juxtaposed those regions and mediated substrate ubiquitination. Our results offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.

biophysics↗

bioRxiv2019

Sumoylation regulates central spindle protein dynamics during chromosome segregation in oocyte

Meiotic spindles in most species lack centrosomes and the mechanisms that underlie faithful chromosome segregation in acentrosomal meiotic spindles are not well understood. In C. elegans oocytes, spindle microtubules exert a poleward force on chromosomes dependent on the microtubule-stabilising protein CLS-2CLASP. The kinase BUB-1Bub1 and CLS-2CLASP localise in the central-spindle and display a dynamic localisation pattern throughout anaphase but the signals regulating their anaphase-specific localisation remains unknown. We have shown that SUMO regulates BUB-1 localisation during metaphase I. Here, we found that SUMO modification of BUB-1Bub1 is regulated by the SUMO E3 ligase GEI-17 and the SUMO protease ULP-1. SUMO is required for BUB-1 localisation in between segregating chromosomes during early anaphase I, and SUMO depletion partially phenocopies BUB-1Bub1 depletion. We also show that CLS-2CLASP is subject to SUMO-mediated regulation. Over-all, we provide evidence for a novel, SUMO-mediated control of protein dynamics during early anaphase I in oocytes.

cell biology↗

bioRxiv2017

Characterisation Of The Biflavonoid Hinokiflavone As A Pre-mRNA Splicing Modulator That Inhibits SENP

Here, we identify the plant biflavanoid hinokiflavone as an inhibitor of splicing in vitro and modulater of alternative splicing in multiple human cell lines. Hinokiflavone inhibits splicing in vitro by blocking one or more early steps of spliceosome assembly, leading to accumulation of the A complex. Multiple human cell lines treated with hinokiflavone show changes in the alternative splicing of different pre-mRNA substrates, but little or no change in transcription. They also show altered subnuclear organization, specifically of splicing factors required for A complex formation, which relocalized together with SUMO1 and SUMO2 into enlarged nuclear speckles. While most cell lines treated with hinokiflavone showed cell cycle arrest and eventual cell death, dependent on time and concentration, the promyelocytic NB4 cell line, which expresses the SUMO target PML-RARalpha fusion protein, was exquisitely sensitive to apoptosis following hinokiflavone treatment. Hinokiflavone treatment increased protein SUMOylation levels, both in in vitro splicing reactions and in cells, with little or no effect on levels of ubiquitinylated proteins. Hinokiflavone also inhibited the catalytic activity of purified E. coli expressed SUMO protease, SENP1 in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased following hinokiflavone treatment, with the major targets including 6 proteins that are associated with U2 snRNP and required for A complex formation. These data identify hinokiflavone as a SUMO protease inhibitor and indicate SUMOylation of splicing factors may be important for modulating splice site selection.

biochemistry↗