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The Babraham Institute Publications database contains details of all publications resulting from our research groups and scientific facilities. Pre-prints by Institute authors can be viewed on the Institute's bioRxiv channel. We believe that free and open access to the outputs of publicly‐funded research offers significant social and economic benefits, as well as aiding the development of new research. We are working to provide Open Access to as many publications as possible and these can be identified below by the padlock icon. Where this hasn't been possible, subscriptions may be required to view the full text.
 

Alvarez-Jarreta J, Rodrigues PRS, Fahy E, O'Connor A, Price A, Gaud C, Andrews S, Benton P, Siuzdak G, Hawksworth JI, Valdivia-Garcia M, Allen SM, O'Donnell VB Bioinformatics

We present LipidFinder 2.0, incorporating four new modules that apply artefact filters, remove lipid and contaminant stacks, in-source fragments and salt clusters, and a new isotope deletion method which is significantly more sensitive than available open-access alternatives. We also incorporate a novel false discovery rate (FDR) method, utilizing a target-decoy strategy, which allows users to assess data quality. A renewed lipid profiling method is introduced which searches three different databases from LIPID MAPS and returns bulk lipid structures only, and a lipid category scatter plot with color blind friendly pallet. An API interface with XCMS Online is made available on LipidFinder's online version. We show using real data that LipidFinder 2.0 provides a significant improvement over non-lipid metabolite filtering and lipid profiling, compared to available tools.

+view abstract Bioinformatics , PMID: 33027502

Parry A, Rulands S, Reik W Epigenetics

DNA methylation is a key layer of epigenetic regulation. The deposition of methylation marks relies on the catalytic activity of DNA methyltransferases (DNMTs), and their active removal relies on the activity of ten-eleven translocation (TET) enzymes. Paradoxically, in important biological contexts these antagonistic factors are co-expressed and target overlapping genomic regions. The ensuing cyclic biochemistry of cytosine modifications gives rise to a continuous, out-of-thermal equilibrium transition through different methylation states. But what is the purpose of this intriguing turnover of DNA methylation? Recent evidence demonstrates that methylation turnover is enriched at gene distal regulatory elements, including enhancers, and can give rise to large-scale oscillatory dynamics. We discuss this phenomenon and propose that DNA methylation turnover might facilitate key lineage decisions.

+view abstract Nature reviews. Genetics , PMID: 33024290

Gerri C, Menchero S, Mahadevaiah SK, Turner JMA, Niakan KK Epigenetics

Understanding human embryology has historically relied on comparative approaches using mammalian model organisms. With the advent of low-input methods to investigate genetic and epigenetic mechanisms and efficient techniques to assess gene function, we can now study the human embryo directly. These advances have transformed the investigation of early embryogenesis in nonrodent species, thereby providing a broader understanding of conserved and divergent mechanisms. Here, we present an overview of the major events in human preimplantation development and place them in the context of mammalian evolution by comparing these events in other eutherian and metatherian species. We describe the advances of studies on postimplantation development and discuss stem cell models that mimic postimplantation embryos. A comparative perspective highlights the importance of analyzing different organisms with molecular characterization and functional studies to reveal the principles of early development. This growing field has a fundamental impact in regenerative medicine and raises important ethical considerations.

+view abstract Annual review of cell and developmental biology , PMID: 33021826

Streeter MD, Rowan S, Ray J, McDonald DM, Volkin J, Clark J, Taylor A, Spiegel DA Biological Chemistry

Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to age-related morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch's membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.

+view abstract ACS chemical biology , PMID: 32975399

Ili C, Buchegger K, Demond H, Castillo-Fernandez J, Kelsey G, Zanella L, Abanto M, Riquelme I, López J, Viscarra T, García P, Bellolio E, Saavedra D, Brebi P Epigenetics

Colorectal cancer is a heterogeneous disease caused by both genetic and epigenetics factors. Analysing DNA methylation changes occurring during colorectal cancer progression and metastasis formation is crucial for the identification of novel epigenetic markers of patient prognosis. Genome-wide methylation sequencing of paired samples of colon (normal adjacent, primary tumour and lymph node metastasis) showed global hypomethylation and CpG island (CGI) hypermethylation of primary tumours compared to normal. In metastasis we observed high global and non-CGI regions methylation, but lower CGI methylation, compared to primary tumours. Gene ontology analysis showed shared biological processes between hypermethylated CGIs in metastasis and primary tumours. After complementary analysis with The Cancer Genome Atlas (TCGA) cohort, , , , and genes were found associated with poor survival. We mapped the methylation landscape of colon normal tissues, primary tumours and lymph node metastasis, being capable of identified methylation changes throughout the genome. Furthermore, we found five genes with potential for methylation biomarkers of poor prognosis in colorectal cancer patients.

+view abstract Cancers , PMID: 32971738

Gerri C, McCarthy A, Alanis-Lobato G, Demtschenko A, Bruneau A, Loubersac S, Fogarty NME, Hampshire D, Elder K, Snell P, Christie L, David L, Van de Velde H, Fouladi-Nashta AA, Niakan KK Epigenetics

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human and cow. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.

+view abstract Nature , PMID: 32968278

Varner EL, Trefely S, Bartee D, von Krusenstiern E, Izzo L, Bekeova C, O'Connor RS, Seifert EL, Wellen KE, Meier JL, Snyder NW Epigenetics

Lysine lactoylation is a recently described protein post-translational modification (PTM). However, the biochemical pathways responsible for this acylation remain unclear. Two metabolite-dependent mechanisms have been proposed: enzymatic histone lysine lactoylation derived from lactoyl-coenzyme A (lactoyl-CoA, also termed lactyl-CoA), and non-enzymatic lysine lactoylation resulting from acyl-transfer via lactoyl-glutathione. While the former has precedent in the form of enzyme-catalysed lysine acylation, the lactoyl-CoA metabolite has not been previously quantified in mammalian systems. Here, we use liquid chromatography-high-resolution mass spectrometry (LC-HRMS) together with a synthetic standard to detect and validate the presence of lactoyl-CoA in cell and tissue samples. Conducting a retrospective analysis of data from previously analysed samples revealed the presence of lactoyl-CoA in diverse cell and tissue contexts. In addition, we describe a biosynthetic route to generate CN-isotopically labelled lactoyl-CoA, providing a co-eluting internal standard for analysis of this metabolite. We estimate lactoyl-CoA concentrations of 1.14 × 10 pmol per cell in cell culture and 0.0172 pmol mg tissue wet weight in mouse heart. These levels are similar to crotonyl-CoA, but between 20 and 350 times lower than predominant acyl-CoAs such as acetyl-, propionyl- and succinyl-CoA. Overall our studies provide the first quantitative measurements of lactoyl-CoA in metazoans, and provide a methodological foundation for the interrogation of this novel metabolite in biology and disease.

+view abstract Open biology , PMID: 32961073

Rönkkö J, Molchanova S, Revah-Politi A, Pereira EM, Auranen M, Toppila J, Kvist J, Ludwig A, Neumann J, Bultynck G, Humblet-Baron S, Liston A, Paetau A, Rivera C, Harms MB, Tyynismaa H, Ylikallio E Immunology

ITPR3, encoding inositol 1,4,5-trisphosphate receptor type 3, was previously reported as a potential candidate disease gene for Charcot-Marie-Tooth neuropathy. Here, we present genetic and functional evidence that ITPR3 is a Charcot-Marie-Tooth disease gene.

+view abstract Annals of clinical and translational neurology , PMID: 32949214

Galvão A, Kelsey G Epigenetics

Single-cell bisulfite sequencing (scBS-seq) enables profiling of DNA methylation at single-nucleotide resolution and across all genomic features. It can explore methylation differences between cells in mixed cell populations and profile methylation in very rare cell types, such as mammalian oocytes and cells from early embryos. Here, we outline the scBS-seq protocol in a 96-well plate format applicable to studies of moderate throughput.

+view abstract Methods in molecular biology , PMID: 32944913

Rayon T, Stamataki D, Perez-Carrasco R, Garcia-Perez L, Barrington C, Melchionda M, Exelby K, Lazaro J, Tybulewicz VLJ, Fisher EMC, Briscoe J Epigenetics

Although many molecular mechanisms controlling developmental processes are evolutionarily conserved, the speed at which the embryo develops can vary substantially between species. For example, the same genetic program, comprising sequential changes in transcriptional states, governs the differentiation of motor neurons in mouse and human, but the tempo at which it operates differs between species. Using in vitro directed differentiation of embryonic stem cells to motor neurons, we show that the program runs more than twice as fast in mouse as in human. This is not due to differences in signaling, nor the genomic sequence of genes or their regulatory elements. Instead, there is an approximately two-fold increase in protein stability and cell cycle duration in human cells compared with mouse cells. This can account for the slower pace of human development and suggests that differences in protein turnover play a role in interspecies differences in developmental tempo.

+view abstract Science , PMID: 32943498

Staels F, Betrains A, Willemsen M, Corvelyn A, Tousseyn T, Dierickx D, Humblet-Baron S, Liston A, Vanderschueren S, Schrijvers R Immunology

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+view abstract Rheumatology , PMID: 32940674

Ross SH, Cantrell DA Immunology

Personalized medicines require understanding the molecular causes of disease. In this issue of Immunity, Gruber et al. reveal that a gain-of-function JAK1 genetic variant results in a mutant protein with mosaic expression that drives multi-organ immune dysregulation via kinase dependent and independent mechanisms. The work highlights how biochemistry can inform therapies to resolve complex immune disorders.

+view abstract Immunity , PMID: 32937149

Cook SJ, Tucker JA, Lochhead PA Signalling

ERK5 is a protein kinase that also contains a nuclear localisation signal and a transcriptional transactivation domain. Inhibition of ERK5 has therapeutic potential in cancer and inflammation and this has prompted the development of ERK5 kinase inhibitors (ERK5i). However, few ERK5i programmes have taken account of the ERK5 transactivation domain. We have recently shown that the binding of small molecule ERK5i to the ERK5 kinase domain stimulates nuclear localisation and paradoxical activation of its transactivation domain. Other kinase inhibitors paradoxically activate their intended kinase target, in some cases leading to severe physiological consequences highlighting the importance of mitigating these effects. Here, we review the assays used to monitor ERK5 activities (kinase and transcriptional) in cells, the challenges faced in development of small molecule inhibitors to the ERK5 pathway, and classify the molecular mechanisms of paradoxical activation of protein kinases by kinase inhibitors.

+view abstract Biochemical Society transactions , PMID: 32915196

Rajewsky N, Almouzni G, Gorski SA, Aerts S, Amit I, Bertero MG, Bock C, Bredenoord AL, Cavalli G, Chiocca S, Clevers H, De Strooper B, Eggert A, Ellenberg J, Fernández XM, Figlerowicz M, Gasser SM, Hubner N, Kjems J, Knoblich JA, Krabbe G, Lichter P, Linnarsson S, Marine JC, Marioni J, Marti-Renom MA, Netea MG, Nickel D, Nollmann M, Novak HR, Parkinson H, Piccolo S, Pinheiro I, Pombo A, Popp C, Reik W, Roman-Roman S, Rosenstiel P, Schultze JL, Stegle O, Tanay A, Testa G, Thanos D, Theis FJ, Torres-Padilla ME, Valencia A, Vallot C, van Oudenaarden A, Vidal M, Voet T, Epigenetics

LifeTime aims to track, understand and target human cells during the onset and progression of complex diseases and their response to therapy at single-cell resolution. This mission will be implemented through the development and integration of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during progression from health to disease. Analysis of such large molecular and clinical datasets will discover molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. Timely detection and interception of disease embedded in an ethical and patient-centered vision will be achieved through interactions across academia, hospitals, patient-associations, health data management systems and industry. Applying this strategy to key medical challenges in cancer, neurological, infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.

+view abstract Nature , PMID: 32894860

Yu JJS, Maugarny-Calès A, Pelletier S, Alexandre C, Bellaiche Y, Vincent JP, McGough IJ Signalling

Planar cell polarity (PCP) organizes the orientation of cellular protrusions and migratory activity within the tissue plane. PCP establishment involves the subcellular polarization of core PCP components. It has been suggested that Wnt gradients could provide a global cue that coordinates local PCP with tissue axes. Here, we dissect the role of Wnt ligands in the orientation of hairs of Drosophila wings, an established system for the study of PCP. We found that PCP was normal in quintuple mutant wings that rely solely on the membrane-tethered Wingless for Wnt signaling, suggesting that a Wnt gradient is not required. We then used a nanobody-based approach to trap Wntless in the endoplasmic reticulum, and hence prevent all Wnt secretion, specifically during the period of PCP establishment. PCP was still established. We conclude that, even though Wnt ligands could contribute to PCP, they are not essential, and another global cue must exist for tissue-wide polarization.

+view abstract Developmental cell , PMID: 32888416

Guarnieri G, Sarchielli E, Comeglio P, Herrera-Puerta E, Piaceri I, Nacmias B, Benelli M, Kelsey G, Maggi M, Gallina P, Vannelli GB, Morelli A Epigenetics

TNFα is the main proinflammatory cytokine implicated in the pathogenesis of neurodegenerative disorders, but it also modulates physiological functions in both the developing and adult brain. In this study, we investigated a potential direct role of TNFα in determining phenotypic changes of a recently established cellular model of human basal forebrain cholinergic neuroblasts isolated from the nucleus basalis of Meynert (hfNBMs). Exposing hfNBMs to TNFα reduced the expression of immature markers, such as nestin and β-tubulin III, and inhibited primary cilium formation. On the contrary, TNFα increased the expression of TNFα receptor TNFR2 and the mature neuron marker MAP2, also promoting neurite elongation. Moreover, TNFα affected nerve growth factor receptor expression. We also found that TNFα induced the expression of DNA-methylation enzymes and, accordingly, downregulated genes involved in neuronal development through epigenetic mechanisms, as demonstrated by methylome analysis. In summary, TNFα showed a dual role on hfNBMs phenotypic plasticity, exerting a negative influence on neurogenesis despite a positive effect on differentiation, through mechanisms that remain to be elucidated. Our results help to clarify the complexity of TNFα effects in human neurons and suggest that manipulation of TNFα signaling could provide a potential therapeutic approach against neurodegenerative disorders.

+view abstract International journal of molecular sciences , PMID: 32854421

Keating SM, Waltemath D, König M, Zhang F, Dräger A, Chaouiya C, Bergmann FT, Finney A, Gillespie CS, Helikar T, Hoops S, Malik-Sheriff RS, Moodie SL, Moraru II, Myers CJ, Naldi A, Olivier BG, Sahle S, Schaff JC, Smith LP, Swat MJ, Thieffry D, Watanabe L, Wilkinson DJ, Blinov ML, Begley K, Faeder JR, Gómez HF, Hamm TM, Inagaki Y, Liebermeister W, Lister AL, Lucio D, Mjolsness E, Proctor CJ, Raman K, Rodriguez N, Shaffer CA, Shapiro BE, Stelling J, Swainston N, Tanimura N, Wagner J, Meier-Schellersheim M, Sauro HM, Palsson B, Bolouri H, Kitano H, Funahashi A, Hermjakob H, Doyle JC, Hucka M,

Systems biology has experienced dramatic growth in the number, size, and complexity of computational models. To reproduce simulation results and reuse models, researchers must exchange unambiguous model descriptions. We review the latest edition of the Systems Biology Markup Language (SBML), a format designed for this purpose. A community of modelers and software authors developed SBML Level 3 over the past decade. Its modular form consists of a core suited to representing reaction-based models and packages that extend the core with features suited to other model types including constraint-based models, reaction-diffusion models, logical network models, and rule-based models. The format leverages two decades of SBML and a rich software ecosystem that transformed how systems biologists build and interact with models. More recently, the rise of multiscale models of whole cells and organs, and new data sources such as single-cell measurements and live imaging, has precipitated new ways of integrating data with models. We provide our perspectives on the challenges presented by these developments and how SBML Level 3 provides the foundation needed to support this evolution.

+view abstract Molecular systems biology , PMID: 32845085

Kelsey G Epigenetics

No abstract available

+view abstract Nature reviews. Molecular cell biology , PMID: 32839539

Schacker M, Cheng YH, Eckersley-Maslin M, Snaith RM, Colledge WH Epigenetics

Mouse embryonic stem cells (ESCs) have played a crucial role in biomedical research where they can be used to elucidate gene function through the generation of genetically modified mice. A critical requirement for the success of this technology is the ability of ESCs to contribute to viable chimaeras with germ-line transmission of the genetically modified allele. We have identified several ESC clones that cause embryonic death of chimaeras at mid to late gestation stages. These clones had a normal karyotype, were pathogen free and their in vitro differentiation potential was not compromised. Chimaeric embryos developed normally up to E13.5 but showed a significant decrease in embryo survival by E17.5 with frequent haemorrhaging. We investigated the relationship between the ESCs transcriptional and epigenomic state and their ability to contribute to viable chimaeras. RNA sequencing identified four genes (Gtl2, Rian, Mirg and Rtl1as) located in the Dlk1-Dio3 imprinted locus that were expressed at lower levels in the compromised ESC clones and this was confirmed by qRT-PCR. Bisulphite sequencing analysis showed significant hypermethylation at the Dlk1-Dio3 imprinted locus with no consistent differences in methylation patterns at other imprinted loci. Treatment of the compromised ESCs with 5-azacytidine reactivated stable expression of Gtl2 and rescued the lethal phenotype but only gave low level chimaeras.

+view abstract Stem cell research , PMID: 32822966

Linterman MA Immunology

T cell regulation of antibody-mediated immunity is critical for health. In this issue of JEM, Li et al. (https://doi.org/10.1084/jem.20191537) identify the Cbl family of E3 ubiquitin ligases as B cell-intrinsic gatekeepers of T cell-dependent humoral immunity.

+view abstract The Journal of experimental medicine , PMID: 32813871

Ortmann D, Brown S, Czechanski A, Aydin S, Muraro D, Huang Y, Tomaz RA, Osnato A, Canu G, Wesley BT, Skelly DA, Stegle O, Choi T, Churchill GA, Baker CL, Rugg-Gunn PJ, Munger SC, Reinholdt LG, Vallier L Epigenetics

Variability among pluripotent stem cell (PSC) lines is a prevailing issue that hampers not only experimental reproducibility but also large-scale applications and personalized cell-based therapy. This variability could result from epigenetic and genetic factors that influence stem cell behavior. Naive culture conditions minimize epigenetic fluctuation, potentially overcoming differences in PSC line differentiation potential. Here we derived PSCs from distinct mouse strains under naive conditions and show that lines from distinct genetic backgrounds have divergent differentiation capacity, confirming a major role for genetics in PSC phenotypic variability. This is explained in part through inconsistent activity of extra-cellular signaling, including the Wnt pathway, which is modulated by specific genetic variants. Overall, this study shows that genetic background plays a dominant role in driving phenotypic variability of PSCs.

+view abstract Cell stem cell , PMID: 32795399

Lau D, Garçon F, Chandra A, Lechermann LM, Aloj L, Chilvers ER, Corrie PG, Okkenhaug K, Gallagher FA

Efficient T-cell targeting, infiltration and activation within tumors is crucial for successful adoptive T-cell therapy. Intravital microscopy is a powerful tool for the visualization of T-cell behavior within tumors, as well as spatial and temporal heterogeneity in response to immunotherapy. Here we describe an experimental approach for intravital imaging of adoptive T-cell morphology, mobility and trafficking in a skin-flap tumor model, following immune modulation with immune checkpoint inhibitors (ICIs) targeting PD-L1 and CTLA-4. A syngeneic model of ovalbumin and mCherry-expressing amelanotic mouse melanoma was used in conjunction with adoptively transferred OT-1 cytotoxic T-cells expressing GFP to image antigen-specific live T-cell behavior within the tumor microenvironment. Dynamic image analysis of T-cell motility showed distinct CD8 T-cell migration patterns and morpho-dynamics within different tumor compartments in response to ICIs: this approach was used to cluster T-cell behavior into four groups based on velocity and meandering index. The results showed that most T-cells within the tumor periphery demonstrated Lévy-like trajectories, consistent with tumor cell searching strategies. T-cells adjacent to tumor cells had reduced velocity and appeared to probe the local environment, consistent with cell-cell interactions. An increased number of T-cells were detected following treatment, traveling at lower mean velocities than controls, and demonstrating reduced displacement consistent with target engagement. Histogram-based analysis of immunofluorescent images from harvested tumors showed that in the ICI-treated mice there was a higher density of CD31 vessels compared to untreated controls and a greater infiltration of T-cells towards the tumor core, consistent with increased cellular trafficking post-treatment.

+view abstract Frontiers in immunology , PMID: 32793206

Morello GM, Hultgren J, Capas-Peneda S, Wiltshire M, Thomas A, Wardle-Jones H, Brajon S, Gilbert C, Olsson IAS

High and variable pre-weaning mortality is a persistent problem in laboratory mouse breeding. Assuming a modest 15% mortality rate across mouse strains, means that approximately 1 million more pups are produced yearly in the EU to compensate for those which die. This paper presents the first large study under practical husbandry conditions to determine the risk factors associated with mouse pre-weaning mortality. We analysed historical records from 219,975 pups from two breeding facilities, collected as part of their management routine and including information on number of pups born and weaned per litter, parents' age and identification, and dates of birth and death of all animals. Pups were counted once in their first week of life and at weaning, and once every one or two weeks, depending on the need for cage cleaning. Dead pups were recorded as soon as these were found during the daily cage screening (without opening the cage). It was hypothesized that litter overlap (i.e. the presence of older siblings in the cage when new pups are born), a recurrent social configuration in trio-housed mice, is associated with increased newborn mortality, along with advanced dam age, large litter size, and a high number and age of older siblings in the cage. The estimated probability of pup death was two to seven percentage points higher in cages with litter overlap compared to those without. Litter overlap was associated with an increase in death of the entire litter of five and six percentage points, which represent an increase of 19% and 103% compared to non-overlapped litters in the two breeding facilities, respectively. Increased number and age of older siblings, advanced dam age, small litter size (less than four pups born) and large litter size (over 11 pups born) were associated with increased probability of pup death.

+view abstract PloS one , PMID: 32785214

Mabbitt PD, Loreto A, Déry MA, Fletcher AJ, Stanley M, Pao KC, Wood NT, Coleman MP, Virdee S

MYCBP2 is a ubiquitin (Ub) E3 ligase (E3) that is essential for neurodevelopment and regulates axon maintenance. MYCBP2 transfers Ub to nonlysine substrates via a newly discovered RING-Cys-Relay (RCR) mechanism, where Ub is relayed from an upstream cysteine to a downstream substrate esterification site. The molecular bases for E2-E3 Ub transfer and Ub relay are unknown. Whether these activities are linked to the neural phenotypes is also unclear. We describe the crystal structure of a covalently trapped E2~Ub:MYCBP2 transfer intermediate revealing key structural rearrangements upon E2-E3 Ub transfer and Ub relay. Our data suggest that transfer to the dynamic upstream cysteine, whilst mitigating lysine activity, requires a closed-like E2~Ub conjugate with tempered reactivity, and Ub relay is facilitated by a helix-coil transition. Furthermore, neurodevelopmental defects and delayed injury-induced degeneration in RCR-defective knock-in mice suggest its requirement, and that of substrate esterification activity, for normal neural development and programmed axon degeneration.

+view abstract Nature chemical biology , PMID: 32747811