Publications

Title / Authors / Details Open Access Download

Cell-Surface Proteomics Identifies Differences in Signaling and Adhesion Protein Expression between Naive and Primed Human Pluripotent Stem Cells.
Wojdyla K, Collier AJ, Fabian C, Nisi PS, Biggins L, Oxley D, Rugg-Gunn PJ

Naive and primed human pluripotent stem cells (hPSC) provide valuable models to study cellular and molecular developmental processes. The lack of detailed information about cell-surface protein expression in these two pluripotent cell types prevents an understanding of how the cells communicate and interact with their microenvironments. Here, we used plasma membrane profiling to directly measure cell-surface protein expression in naive and primed hPSC. This unbiased approach quantified over 1,700 plasma membrane proteins, including those involved in cell adhesion, signaling, and cell interactions. Notably, multiple cytokine receptors upstream of JAK-STAT signaling were more abundant in naive hPSC. In addition, functional experiments showed that FOLR1 and SUSD2 proteins are highly expressed at the cell surface in naive hPSC but are not required to establish human naive pluripotency. This study provides a comprehensive stem cell proteomic resource that uncovers differences in signaling pathway activity and has identified new markers to define human pluripotent states.

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Stem cell reports, 1, 1, , 10 Apr 2020

PMID:32302559
DOI: 10.1016/j.stemcr.2020.03.017

Open Access

Paradoxical activation of the protein kinase-transcription factor ERK5 by ERK5 kinase inhibitors.
Lochhead PA, Tucker JA, Tatum NJ, Wang J, Oxley D, Kidger AM, Johnson VP, Cassidy MA, Gray NS, Noble MEM, Cook SJ

The dual protein kinase-transcription factor, ERK5, is an emerging drug target in cancer and inflammation, and small-molecule ERK5 kinase inhibitors have been developed. However, selective ERK5 kinase inhibitors fail to recapitulate ERK5 genetic ablation phenotypes, suggesting kinase-independent functions for ERK5. Here we show that ERK5 kinase inhibitors cause paradoxical activation of ERK5 transcriptional activity mediated through its unique C-terminal transcriptional activation domain (TAD). Using the ERK5 kinase inhibitor, Compound 26 (ERK5-IN-1), as a paradigm, we have developed kinase-active, drug-resistant mutants of ERK5. With these mutants, we show that induction of ERK5 transcriptional activity requires direct binding of the inhibitor to the kinase domain. This in turn promotes conformational changes in the kinase domain that result in nuclear translocation of ERK5 and stimulation of gene transcription. This shows that both the ERK5 kinase and TAD must be considered when assessing the role of ERK5 and the effectiveness of anti-ERK5 therapeutics.

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Nature communications, 11, 1, , 13 Mar 2020

PMID:32170057
DOI: 10.1038/s41467-020-15031-3

An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis.
Odle RI, Walker SA, Oxley D, Kidger AM, Balmanno K, Gilley R, Okkenhaug H, Florey O, Ktistakis NT, Cook SJ

Since nuclear envelope breakdown occurs during mitosis in metazoan cells, it has been proposed that macroautophagy must be inhibited to maintain genome integrity. However, repression of macroautophagy during mitosis remains controversial and mechanistic detail limited to the suggestion that CDK1 phosphorylates VPS34. Here, we show that initiation of macroautophagy, measured by the translocation of the ULK complex to autophagic puncta, is repressed during mitosis, even when mTORC1 is inhibited. Indeed, mTORC1 is inactive during mitosis, reflecting its failure to localize to lysosomes due to CDK1-dependent RAPTOR phosphorylation. While mTORC1 normally represses autophagy via phosphorylation of ULK1, ATG13, ATG14, and TFEB, we show that the mitotic phosphorylation of these autophagy regulators, including at known repressive sites, is dependent on CDK1 but independent of mTOR. Thus, CDK1 substitutes for inhibited mTORC1 as the master regulator of macroautophagy during mitosis, uncoupling autophagy regulation from nutrient status to ensure repression of macroautophagy during mitosis.

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Molecular cell, S1097-2765, 19, , 06 Nov 2019

PMID:31733992
DOI: 10.1016/j.molcel.2019.10.016

Open Access

MEK1/2 inhibitor withdrawal reverses acquired resistance driven by BRAF amplification whereas KRAS amplification promotes EMT-chemoresistance.
Sale MJ, Balmanno K, Saxena J, Ozono E, Wojdyla K, McIntyre RE, Gilley R, Woroniuk A, Howarth KD, Hughes G, Dry JR, Arends MJ, Caro P, Oxley D, Ashton S, Adams DJ, Saez-Rodriguez J, Smith PD, Cook SJ

Acquired resistance to MEK1/2 inhibitors (MEKi) arises through amplification of BRAF or KRAS to reinstate ERK1/2 signalling. Here we show that BRAF amplification and MEKi resistance are reversible following drug withdrawal. Cells with BRAF amplification are addicted to MEKi to maintain a precise level of ERK1/2 signalling that is optimal for cell proliferation and survival, and tumour growth in vivo. Robust ERK1/2 activation following MEKi withdrawal drives a p57-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, selecting against those cells with amplified BRAF. p57 expression is required for loss of BRAF amplification and reversal of MEKi resistance. Thus, BRAF amplification confers a selective disadvantage during drug withdrawal, validating intermittent dosing to forestall resistance. In contrast, resistance driven by KRAS amplification is not reversible; rather ERK1/2 hyperactivation drives ZEB1-dependent epithelial-to-mesenchymal transition and chemoresistance, arguing strongly against the use of drug holidays in cases of KRAS amplification.

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Nature communications, 10, 2041-1723, , 2019

PMID:31048689

Correction to: Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.
Olova N, Krueger F, Andrews S, Oxley D, Berrens RV, Branco MR, Reik W

Following publication of the original article [1], it was reported that the incorrect "Additional file 3" was published. The correct additional file is given below.

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Genome biology, 20, 1474-760X, , 2019

PMID:30795792

Open Access

Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors.
Tsolakos N, Durrant TN, Chessa T, Suire SM, Oxley D, Kulkarni S, Downward J, Perisic O, Williams RL, Stephens L, Hawkins PT

Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and β bound equivalently to p110α or p110β but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85β, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5-6× more efficiently than p110β-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kβ. Nevertheless, PI3Kβ contributes substantially to acute PDGF-stimulation of PIP and PKB in MEFs because it is synergistically, and possibly sequentially, activated by receptor-recruitment and small GTPases (Rac/CDC42) via its RBD, whereas parallel activation of PI3Kα is independent of its RBD. These results begin to provide molecular clarity to the rules of engagement between class IA PI3K subunits in vivo and past work describing "excess p85," p85α as a tumor suppressor, and differential receptor activation of PI3Kα and PI3Kβ.

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Proceedings of the National Academy of Sciences of the United States of America, 115, 1091-6490, , 2018

PMID:30442661

Open Access

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.
Olova N, Krueger F, Andrews S, Oxley D, Berrens RV, Branco MR, Reik W

Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing.

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Genome biology, 19, 1474-760X, , 2018

PMID:29544553

Open Access

Contractile responses to endothelin-1 are regulated by PKC phosphorylation of cardiac myosin binding protein-C in rat ventricular myocytes.
Smyrnias I, Goodwin N, Wachten D, Skogestad J, Aronsen JM, Robinson EL, Demydenko K, Segonds-Pichon A, Oxley D, Sadayappan S, Sipido K, Bootman MD, Roderick HL

The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by electrically-mediated increases in [Ca]. This process of excitation-contraction coupling (ECC) is subject to modulation by neurohormonal mediators that tune the output of the heart to meet the needs of the organism. Endothelin-1 (ET-1) is a potent modulator of cardiac function with effects on contraction amplitude, chronotropy and automaticity. The actions of ET-1 are evident during normal adaptive physiological responses and increased under pathophysiological conditions, such as following myocardial infarction and during heart failure, where ET-1 levels are elevated. In myocytes, ET-1 acts through ET- or ET-G protein-coupled receptors (GPCRs). Although well studied in atrial myocytes, the influence and mechanisms of action of ET-1 upon ECC in ventricular myocytes are not fully resolved. We show in rat ventricular myocytes that ET-1 elicits a biphasic effect on fractional shortening (initial transient negative and sustained positive inotropy) and increases the peak amplitude of systolic Ca transients in adult rat ventricular myocytes. The negative inotropic phase was ET receptor-dependent, whereas the positive inotropic response and increase in peak amplitude of systolic Ca transients required ET receptor engagement. Both effects of ET-1 required phospholipase C (PLC)-activity, although distinct signalling pathways downstream of PLC elicited the effects of each ET receptor. The negative inotropic response involved inositol 1,4,5-trisphosphate (InsP) signalling and protein kinase C epsilon (PKCε). The positive inotropic action and the enhancement in Ca transient amplitude induced by ET-1 were independent of InsP signalling, but suppressed by PKCε. Serine 302 in cardiac myosin binding protein-C was identified as a PKCε substrate that when phosphorylated contributed to the suppression of contraction and Ca transients by PKCε following ET-1 stimulation. Thus, our data provide a new role and mechanism of action for InsP and PKCε in mediating the negative inotropic response and in restraining the positive inotropy and enhancement in Ca transients following ET-1 stimulation.

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Journal of molecular and cellular cardiology, 117, 1, , 04 2018

PMID:29470978
DOI: 10.1016/j.yjmcc.2018.02.012

Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human.
Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F, Oxley D, Santos F, Clarke J, Mansfield W, Reik W, Bertone P, Smith A

Cell, 162, 1097-4172, , 2015

PMID:28843285

Open Access

The Ageing Brain: Effects on DNA Repair and DNA Methylation in Mice.
Langie SA, Cameron KM, Ficz G, Oxley D, Tomaszewski B, Gorniak JP, Maas LM, Godschalk RW, van Schooten FJ, Reik W, von Zglinicki T, Mathers JC

Base excision repair (BER) may become less effective with ageing resulting in accumulation of DNA lesions, genome instability and altered gene expression that contribute to age-related degenerative diseases. The brain is particularly vulnerable to the accumulation of DNA lesions; hence, proper functioning of DNA repair mechanisms is important for neuronal survival. Although the mechanism of age-related decline in DNA repair capacity is unknown, growing evidence suggests that epigenetic events (e.g., DNA methylation) contribute to the ageing process and may be functionally important through the regulation of the expression of DNA repair genes. We hypothesize that epigenetic mechanisms are involved in mediating the age-related decline in BER in the brain. Brains from male mice were isolated at 3-32 months of age. Pyrosequencing analyses revealed significantly increased Ogg1 methylation with ageing, which correlated inversely with Ogg1 expression. The reduced Ogg1 expression correlated with enhanced expression of methyl-CpG binding protein 2 and ten-eleven translocation enzyme 2. A significant inverse correlation between Neil1 methylation at CpG-site2 and expression was also observed. BER activity was significantly reduced and associated with increased 8-oxo-7,8-dihydro-2'-deoxyguanosine levels. These data indicate that Ogg1 and Neil1 expression can be epigenetically regulated, which may mediate the effects of ageing on DNA repair in the brain.

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Genes, 8, , , 2017

PMID:28218666

Open Access

Retinol and ascorbate drive erasure of epigenetic memory and enhance reprogramming to naïve pluripotency by complementary mechanisms.
Hore TA, von Meyenn F, Ravichandran M, Bachman M, Ficz G, Oxley D, Santos F, Balasubramanian S, Jurkowski TP, Reik W

Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe(2+) recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome.

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Proceedings of the National Academy of Sciences of the United States of America, , 1091-6490, , 2016

PMID:27729528

Open Access

Exosomes bind autotaxin and act as a physiological delivery mechanism to stimulate LPA receptor signalling in cells.
Jethwa SA, Leah EJ, Zhang Q, Bright NA, Oxley D, Bootman MD, Rudge SA, Wakelam MJ

Autotaxin (ATX) the lysophospholipase responsible for generating the lipid receptor agonist lysophosphatidic acid (LPA) is a secreted enzyme. Here we show that once secreted it can bind to the surface of cell secreted exosomes. Exosome-bound ATX is catalytically active and carries generated LPA. Once bound to a cell, through specific integrin interaction, ATX releases the LPA to activate cell surface G-protein coupled LPA receptors; inhibition of signaling by the receptor antagonist Ki1642 suggests these are either LPAR1 or LPAR3. The binding stimulates downstream signaling including AKT and MAPK phosphorylation, the release of intracellular stored calcium and cell migration. We propose that exosomal binding of LPA-loaded ATX provides a means of efficiently delivering the lipid agonist to cell surface receptors to promote signalling. We further propose that this is a means whereby autotaxin-LPA signaling operates physiologically.

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Journal of cell science, , 1477-9137, , 2016

PMID:27557622

Open Access

Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1.
Pan D, Barber MA, Hornigold K, Baker MJ, Toth JM, Oxley D, Welch HC

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates the small G protein (GTPase) Rac1 to control Rac1-dependent cytoskeletal dynamics, and thus cell morphology. Three mechanisms of P-Rex1 regulation are currently known: (i) binding of the phosphoinositide second messenger PIP3, (ii) binding of the Gβγ subunits of heterotrimeric G proteins, and (iii) phosphorylation of various serine residues. Using recombinant P-Rex1 protein to search for new binding partners, we isolated the G-protein coupled receptor (GPCR)-adaptor protein Norbin (Neurochondrin, NCDN) from mouse brain fractions. Coimmunoprecipitation confirmed the interaction between overexpressed P-Rex1 and Norbin in COS-7 cells, as well as between endogenous P-Rex1 and Norbin in HEK-293 cells. Binding assays with purified recombinant proteins showed that their interaction is direct, and mutational analysis revealed that the PH domain of P-Rex1 is required. Rac-GEF activity assays with purified recombinant proteins showed that direct interaction with Norbin increases the basal, PIP3- and Gβγ-stimulated Rac-GEF activity of P-Rex1. Pak-CRIB pull-down assays demonstrated that Norbin promotes the P-Rex1 mediated activation of endogenous Rac1 upon stimulation of HEK-293 cells with lysophosphatidic acid. Finally, immunofluorescence microscopy and subcellular fractionation showed that coexpression of P-Rex1 and Norbin induces a robust translocation of both proteins from the cytosol to the plasma membrane, as well as promoting cell spreading, lamellipodia formation and membrane ruffling, cell morphologies generated by active Rac1. In summary, we have identified a novel mechanism of P-Rex1 regulation through the GPCR-adaptor protein Norbin, a direct P-Rex1 interacting protein that promotes the Rac-GEF activity and membrane localization of P-Rex1.

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The Journal of biological chemistry, , 1083-351X, , 2016

PMID:26792863

Open Access

Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks.
Latos PA, Sienerth AR, Murray A, Senner CE, Muto M, Ikawa M, Oxley D, Burge S, Cox BJ, Hemberger M

Elf5 is a transcription factor with pivotal roles in the trophoblast compartment, where it reinforces a trophoblast stem cell (TSC)-specific transcriptional circuit. However, Elf5 is also present in differentiating trophoblast cells that have ceased to express other TSC genes such as Cdx2 and Eomes. In the present study, we aimed to elucidate the context-dependent role of Elf5 at the interface between TSC self-renewal and the onset of differentiation. We demonstrate that precise levels of Elf5 are critical for normal expansion of the TSC compartment and embryonic survival, as Elf5 overexpression triggers precocious trophoblast differentiation. Through integration of protein interactome, transcriptome, and genome-wide chromatin immunoprecipitation data, we reveal that this abundance-dependent function is mediated through a shift in preferred Elf5-binding partners; in TSCs, Elf5 interaction with Eomes recruits Tfap2c to triply occupied sites at TSC-specific genes, driving their expression. In contrast, the Elf5 and Tfap2c interaction becomes predominant as their protein levels increase. This triggers binding to double- and single-occupancy sites that harbor the cognate Tfap2c motif, causing activation of the associated differentiation-promoting genes. These data place Elf5 at the center of a stoichiometry-sensitive transcriptional network, where it acts as a molecular switch governing the balance between TSC proliferation and differentiation.

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Genes & development, , 1549-5477, , 2015

PMID:26584622

Open Access

Fgf and Esrrb integrate epigenetic and transcriptional networks that regulate self-renewal of trophoblast stem cells.
Latos PA, Goncalves A, Oxley D, Mohammed H, Turro E, Hemberger M

Esrrb (oestrogen-related receptor beta) is a transcription factor implicated in embryonic stem (ES) cell self-renewal, yet its knockout causes intrauterine lethality due to defects in trophoblast development. Here we show that in trophoblast stem (TS) cells, Esrrb is a downstream target of fibroblast growth factor (Fgf) signalling and is critical to drive TS cell self-renewal. In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes. To elucidate the mechanisms whereby Esrrb controls the expression of its targets, we characterized its TS cell-specific interactome using mass spectrometry. Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex. Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.

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Nature communications, 6, 2041-1723, , 2015

PMID:26206133

Open Access

DYRK1A-mediated Cyclin D1 Degradation in Neural Stem Cells Contributes to the Neurogenic Cortical Defects in Down Syndrome.
Najas S, Arranz J, Lochhead PA, Ashford AL, Oxley D, Delabar JM, Cook SJ, Barallobre MJ, Arbonés ML

Alterations in cerebral cortex connectivity lead to intellectual disability and in Down syndrome, this is associated with a deficit in cortical neurons that arises during prenatal development. However, the pathogenic mechanisms that cause this deficit have not yet been defined. Here we show that the human DYRK1A kinase on chromosome 21 tightly regulates the nuclear levels of Cyclin D1 in embryonic cortical stem (radial glia) cells, and that a modest increase in DYRK1A protein in transgenic embryos lengthens the G1 phase in these progenitors. These alterations promote asymmetric proliferative divisions at the expense of neurogenic divisions, producing a deficit in cortical projection neurons that persists in postnatal stages. Moreover, radial glial progenitors in the Ts65Dn mouse model of Down syndrome have less Cyclin D1, and Dyrk1a is the triplicated gene that causes both early cortical neurogenic defects and decreased nuclear Cyclin D1 levels in this model. These data provide insights into the mechanisms that couple cell cycle regulation and neuron production in cortical neural stem cells, emphasizing that the deleterious effect of DYRK1A triplication in the formation of the cerebral cortex begins at the onset of neurogenesis, which is relevant to the search for early therapeutic interventions in Down syndrome.

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EBioMedicine, 2, 2352-3964, , 2015

PMID:26137553

Open Access

Resetting transcription factor control circuitry toward ground-state pluripotency in human.
Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F, Oxley D, Santos F, Clarke J, Mansfield W, Reik W, Bertone P, Smith A

Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.

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Cell, 158, 1097-4172, , 2014

PMID:25215486

Open Access

The nuclear exosome is active and important during budding yeast meiosis.
Frenk S, Oxley D, Houseley J

Nuclear RNA degradation pathways are highly conserved across eukaryotes and play important roles in RNA quality control. Key substrates for exosomal degradation include aberrant functional RNAs and cryptic unstable transcripts (CUTs). It has recently been reported that the nuclear exosome is inactivated during meiosis in budding yeast through degradation of the subunit Rrp6, leading to the stabilisation of a subset of meiotic unannotated transcripts (MUTs) of unknown function. We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis that are undetectable in wild-type cells, showing that the nuclear exosome remains functional for CUT degradation, and we further report that the meiotic exosome complex contains Rrp6. Indeed Rrp6 over-expression is insufficient to suppress MUT transcripts, showing that the reduced amount of Rrp6 in meiotic cells does not directly cause MUT accumulation. Lack of TRAMP activity stabilises ∼ 1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC). CBC mutants display defects in the formation of meiotic double strand breaks (DSBs), and we see similar defects in TRAMP mutants, suggesting that a key function of the nuclear exosome is to prevent saturation of the CBC complex by CUTs. Together, our results show that the nuclear exosome remains active in meiosis and has an important role in facilitating meiotic recombination.

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PloS one, 9, 1932-6203, , 2014

PMID:25210768

Open Access

Dictyostelium uses ether-linked inositol phospholipids for intracellular signalling.
Clark J,Kay RR,Kielkowska A,Niewczas I,Fets L,Oxley D,Stephens LR,Hawkins PT

Inositol phospholipids are critical regulators of membrane biology throughout eukaryotes. The general principle by which they perform these roles is conserved across species and involves binding of differentially phosphorylated inositol head groups to specific protein domains. This interaction serves to both recruit and regulate the activity of several different classes of protein which act on membrane surfaces. In mammalian cells, these phosphorylated inositol head groups are predominantly borne by a C38:4 diacylglycerol backbone. We show here that the inositol phospholipids of Dictyostelium are different, being highly enriched in an unusual C34:1e lipid backbone, 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), in which the sn-1 position contains an ether-linked C16:0 chain; they are thus plasmanylinositols. These plasmanylinositols respond acutely to stimulation of cells with chemoattractants, and their levels are regulated by PIPKs, PI3Ks and PTEN. In mammals and now in Dictyostelium, the hydrocarbon chains of inositol phospholipids are a highly selected subset of those available to other phospholipids, suggesting that different molecular selectors are at play in these organisms but serve a common, evolutionarily conserved purpose.

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The EMBO journal, , 1460-2075, , 2014

PMID:25180230

Open Access

The immune system GTPase GIMAP6 interacts with the Atg8 homologue GABARAPL2 and is recruited to autophagosomes.
Pascall JC, Rotondo S, Mukadam AS, Oxley D, Webster J, Walker SA, Piron J, Carter C, Ktistakis NT, Butcher GW

The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux.

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PloS one, 8, 1932-6203, , 2013

PMID:24204963

Open Access

A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation.
Iurlaro M, Ficz G, Oxley D, Raiber EA, Bachman M, Booth MJ, Andrews S, Balasubramanian S, Reik W

DNA methylation (5mC) plays important roles in epigenetic regulation of genome function. Recently, TET hydroxylases have been found to oxidise 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC) and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins which showed preferential binding to 5-methylcytosine (5mC) and its oxidised forms, where readers for 5mC and 5hmC showed little overlap, and proteins bound to further oxidation forms were enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine using DNA from mouse embryonic stem cell extracts.

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Genome biology, 14, 1465-6914, , 2013

PMID:24156278

Open Access

A novel DYRK1B inhibitor AZ191 demonstrates that DYRK1B acts independently of GSK3β to phosphorylate cyclin D1 at Thr(286), not Thr(288).
AL Ashford, D Oxley, J Kettle, K Hudson, S Guichard, SJ Cook, PA Lochhead

DYRK1B (dual-specificity tyrosine phosphorylation-regulated kinase 1B) is amplified in certain cancers and may be an oncogene; however, our knowledge of DYRK1B has been limited by the lack of selective inhibitors. In the present study we describe AZ191, a potent small molecule inhibitor that selectively inhibits DYRK1B in vitro and in cells. CCND1 (cyclin D1), a key regulator of the mammalian G1-S-phase transition, is phosphorylated on Thr(286) by GSK3β (glycogen synthase kinase 3β) to promote its degradation. DYRK1B has also been proposed to promote CCND1 turnover, but was reported to phosphorylate Thr(288) rather than Thr(286). Using in vitro kinase assays, phospho-specific immunoblot analysis and MS in conjunction with AZ191 we now show that DYRK1B phosphorylates CCND1 at Thr(286), not Thr(288), in vitro and in cells. In HEK (human embryonic kidney)-293 and PANC-1 cells (which exhibit DYRK1B amplification) DYRK1B drives Thr(286) phosphorylation and proteasome-dependent turnover of CCND1 and this is abolished by AZ191 or DYRK1B RNAi, but not by GSK3β inhibitors or GSK3β RNAi. DYRK1B expression causes a G1-phase cell-cycle arrest, but overexpression of CCND1 (wild-type or T286A) fails to overcome this; indeed, DYRK1B also promotes the expression of p21CIP1 (21 kDa CDK-interacting protein 1) and p27KIP1 (CDK-inhibitory protein 1). The results of the present study demonstrate for the first time that DYRK1B is a novel Thr(286)-CCND1 kinase that acts independently of GSK3β to promote CCND1 degradation. Furthermore, we anticipate that AZ191 may prove useful in defining further substrates and biological functions of DYRK1B.

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The Biochemical journal, 457, 1, , 2014

PMID:24134204
DOI: 10.1042/BJ20130461

Differential isolation and identification of PI(3)P and PI(3,5)P2 binding proteins from Arabidopsis thaliana using an agarose-phosphatidylinositol-phosphate affinity chromatography.
D Oxley, N Ktistakis, T Farmaki

A phosphatidylinositol-phosphate affinity chromatographic approach combined with mass spectrometry was used in order to identify novel PI(3)P and PI(3,5)P2 binding proteins from Arabidopsis thaliana suspension cell extracts. Most of the phosphatidylinositol-phosphate interacting candidates identified from this differential screening are characterized by lysine/arginine rich patches. Direct phosphoinositide binding was identified for important membrane trafficking regulators as well as protein quality control proteins such as the ATG18p orthologue involved in autophagosome formation and the lipid Sec14p like transfer protein. A pentatricopeptide repeat (PPR) containing protein was shown to directly bind to PI(3,5)P2 but not to PI(3)P. PIP chromatography performed using extracts obtained from high salt (0.4M and 1M NaCl) pretreated suspensions showed that the association of an S5-1 40S ribosomal protein with both PI(3)P and PI(3,5)P2 was abolished under salt stress whereas salinity stress induced an increase in the phosphoinositide association of the DUF538 domain containing protein SVB, associated with trichome size. Additional interacting candidates were co-purified with the phosphoinositide bound proteins. Binding of the COP9 signalosome, the heat shock proteins, and the identified 26S proteasomal subunits, is suggested as an indirect effect of their interaction with other proteins directly bound to the PI(3)P and the PI(3,5)P2 phosphoinositides.

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Journal of proteomics, , , , 2013

PMID:24007659
DOI: 10.1016/j.jprot.2013.08.020

FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency.
G Ficz, TA Hore, F Santos, HJ Lee, W Dean, J Arand, F Krueger, D Oxley, YL Paul, J Walter, SJ Cook, S Andrews, MR Branco, W Reik

Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency.

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Cell stem cell, 13, 3, , 2013

PMID:23850245
DOI: 10.1016/j.stem.2013.06.004

Open Access

Regulation of lineage specific DNA hypomethylation in mouse trophectoderm.
Oda M, Oxley D, Dean W, Reik W

DNA methylation is reprogrammed during early embryogenesis by active and passive mechanisms in advance of the first differentiation event producing the embryonic and extraembryonic lineage cells which contribute to the future embryo proper and to the placenta respectively. Embryonic lineage cells re-acquire a highly methylated genome dependent on the DNA methyltransferases (DNMTs) Dnmt3a and Dnmt3b that are required for de novo methylation. By contrast, extraembryonic lineage cells remain globally hypomethylated but the mechanisms that underlie this hypomethylation remain unknown.

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PloS one, 8, 1932-6203, , 2013

PMID:23825703

Open Access

Maternal folate depletion and high-fat feeding from weaning affects DNA methylation and DNA repair in brain of adult offspring.
Langie SA, Achterfeldt S, Gorniak JP, Halley-Hogg KJ, Oxley D, van Schooten FJ, Godschalk RW, McKay JA, Mathers JC

The mechanisms through which environmental and dietary factors modulate DNA repair are still unclear but may include dysregulation of gene expression due to altered epigenetic markings. In a mouse model, we investigated the effect of maternal folate depletion during pregnancy and lactation, and high-fat feeding from weaning, on base excision repair (BER) and DNA methylation and expression of selected BER-related genes in the brain of adult offspring. While folate depletion did not affect BER activity of the mothers, BER increased in the offspring at weaning (P=0.052). In the long term, as observed in 6-mo-old offspring, the double insult, i.e., maternal low-folate supply and high-fat feeding from weaning, decreased BER activity significantly in the cortex, cerebellum, hippocampus, and subcortical regions (P≤0.017). This fall in BER activity was associated with small changes in methylation or expression of BER-related genes. Maternal folate depletion led to slightly increased oxidative DNA damage levels in subcortical regions of adult offspring, which may increase sensitivity to oxidative stress and predispose to neurological disorders. In summary, our data suggest that low-folate supply during early life may leave an epigenetic mark that can predispose the offspring to further dietary insults, causing adverse effects during adult life.

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FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 27, 1530-6860, , 2013

PMID:23603834

p38α MAPK regulates adult muscle stem cell fate by restricting progenitor proliferation during postnatal growth and repair.
Brien P, Pugazhendhi D, Woodhouse S, Oxley D, Pell JM

Stem cell function is essential for the maintenance of adult tissue homeostasis. Controlling the balance between self-renewal and differentiation is crucial to maintain a receptive satellite cell pool capable of responding to growth and regeneration cues. The mitogen-activated protein kinase p38α has been implicated in the regulation of these processes but its influence in adult muscle remains unknown. Using conditional satellite cell p38α knockout mice we have demonstrated that p38α restricts excess proliferation in the postnatal growth phase while promoting timely myoblast differentiation. Differentiation was still able to occur in the p38α-null satellite cells, however, but was delayed. An absence of p38α resulted in a postnatal growth defect along with the persistence of an increased reservoir of satellite cells into adulthood. This population was still capable of responding to cardiotoxin-induced injury, resulting in complete, albeit delayed, regeneration, with further enhancement of the satellite cell population. Increased p38γ phosphorylation accompanied the absence of p38α, and inhibition of p38γ ex vivo substantially decreased the myogenic defect. We have used genome-wide transcriptome analysis to characterize the changes in expression that occur between resting and regenerating muscle, and the influence p38α has on these expression profiles. This study provides novel evidence for the fundamental role of p38α in adult muscle homeostasis in vivo.

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Stem cells (Dayton, Ohio), 31, 1549-4918, , 2013

PMID:23592450

The Arabidopsis thaliana immunophilin ROF1 directly interacts with PI(3)P and PI(3,5)P2 and affects germination under osmotic stress.
D Karali, D Oxley, J Runions, N Ktistakis, T Farmaki

A direct interaction of the Arabidopsis thaliana immunophilin ROF1 with phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate was identified using a phosphatidylinositol-phosphate affinity chromatography of cell suspension extracts, combined with a mass spectrometry (nano LC ESI-MS/MS) analysis. The first FK506 binding domain was shown sufficient to bind to both phosphatidylinositol-phosphate stereoisomers. GFP-tagged ROF1 under the control of a 35S promoter was localised in the cytoplasm and the cell periphery of Nicotiana tabacum leaf explants. Immunofluorescence microscopy of Arabidopsis thaliana root tips verified its cytoplasmic localization and membrane association and showed ROF1 localization in the elongation zone which was expanded to the meristematic zone in plants grown on high salt media. Endogenous ROF1 was shown to accumulate in response to high salt treatment in Arabidopsis thaliana young leaves as well as in seedlings germinated on high salt media (0.15 and 0.2 M NaCl) at both an mRNA and protein level. Plants over-expressing ROF1, (WSROF1OE), exhibited enhanced germination under salinity stress which was significantly reduced in the rof1(-) knock out mutants and abolished in the double mutants of ROF1 and of its interacting homologue ROF2 (WSrof1(-)/2(-)). Our results show that ROF1 plays an important role in the osmotic/salt stress responses of germinating Arabidopsis thaliana seedlings and suggest its involvement in salinity stress responses through a phosphatidylinositol-phosphate related protein quality control pathway.

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PloS one, 7, 11, , 2012

PMID:23133621
DOI: 10.1371/journal.pone.0048241

Open Access

DNA methylation profiles define stem cell identity and reveal a tight embryonic-extraembryonic lineage boundary.
CE Senner, F Krueger, D Oxley, S Andrews, M Hemberger

Embryonic (ES) and epiblast (EpiSC) stem cells are pluripotent but committed to an embryonic lineage fate. Conversely, trophoblast (TS) and extraembryonic endoderm (XEN) stem cells contribute predominantly to tissues of the placenta and yolk sac, respectively. Here we show that each of these four stem cell types is defined by a unique DNA methylation profile. Despite their distinct developmental origin, TS and XEN cells share key epigenomic hallmarks, chiefly characterized by robust DNA methylation of embryo-specific developmental regulators, as well as a subordinate role of 5-hydroxymethylation. We also observe a substantial methylation reinforcement of pre-existing epigenetic repressive marks that specifically occurs in extraembryonic stem cells compared to in vivo tissue, presumably due to continued high Dnmt3b expression levels. These differences establish a major epigenetic barrier between the embryonic and extraembryonic stem cell types. In addition, epigenetic lineage boundaries also separate the two extraembryonic stem cell types by mutual repression of key lineage-specific transcription factors. Thus, global DNA methylation patterns are a defining feature of each stem cell type that underpin lineage commitment and differentiative potency of early embryo-derived stem cells. Our detailed methylation profiles identify a cohort of developmentally regulated sequence elements, such as orphan CpG islands, that will be most valuable to uncover novel transcriptional regulators and pivotal "gatekeeper" genes in pluripotency and lineage differentiation.

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Stem cells (Dayton, Ohio), 30, 12, , 2012

PMID:23034951
DOI: 10.1002/stem.1249

Open Access

Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase.
EA Raiber, D Beraldi, G Ficz, HE Burgess, MR Branco, P Murat, D Oxley, MJ Booth, W Reik, S Balasubramanian

ABSTRACT: BACKGROUND: Methylation of cytosine in DNA (5mC) is an important epigenetic mark that is involved in the regulation of genome function. During early embryonic development in mammals, the methylation landscape is dynamically reprogrammed in part through active demethylation. Recent advances have identified key players involved in active demethylation pathways, including oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) by the TET enzymes, and excision of 5fC by the base excision repair enzyme thymine DNA glycosylase (TDG). Here, we provide the first genome-wide map of 5fC in mouse embryonic stem (ES) cells and evaluate potential roles for 5fC in differentiation. RESULTS: Our method exploits the unique reactivity of 5fC for pulldown and high-throughput sequencing. Genome-wide mapping revealed 5fC enrichment in CpG islands (CGIs) of promoters and exons. CGI promoters in which 5fC was relatively more enriched than 5mC or 5hmC corresponded to transcriptionally active genes. Accordingly, 5fC-rich promoters had elevated H3K4me3 levels, associated with active transcription, and were frequently bound by RNA polymerase II. TDG down-regulation led to 5fC accumulation in CGIs in ES cells, which correlates with increased methylation in these genomic regions during differentiation of ES cells in wild-type and TDG knockout contexts. CONCLUSIONS: Collectively, our data suggest that 5fC plays a role in epigenetic reprogramming within specific genomic regions, which is controlled in part by TDG-mediated excision. Notably, 5fC excision in ES cells is necessary for the correct establishment of CGI methylation patterns during differentiation and hence for appropriate patterns of gene expression during development.

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Genome biology, 13, 8, , 2012

PMID:22902005
DOI: 10.1186/gb-2012-13-8-r69

Open Access

The H19 lincRNA is a developmental reservoir of miR-675 that suppresses growth and Igf1r.
A Keniry, D Oxley, P Monnier, M Kyba, L Dandolo, G Smits, W Reik

The H19 large intergenic non-coding RNA (lincRNA) is one of the most highly abundant and conserved transcripts in mammalian development, being expressed in both embryonic and extra-embryonic cell lineages, yet its physiological function is unknown. Here we show that miR-675, a microRNA (miRNA) embedded in H19's first exon, is expressed exclusively in the placenta from the gestational time point when placental growth normally ceases, and placentas that lack H19 continue to grow. Overexpression of miR-675 in a range of embryonic and extra-embryonic cell lines results in their reduced proliferation; targets of the miRNA are upregulated in the H19 null placenta, including the growth-promoting insulin-like growth factor 1 receptor (Igf1r) gene. Moreover, the excision of miR-675 from H19 is dynamically regulated by the stress-response RNA-binding protein HuR. These results suggest that H19's main physiological role is in limiting growth of the placenta before birth, by regulated processing of miR-675. The controlled release of miR-675 from H19 may also allow rapid inhibition of cell proliferation in response to cellular stress or oncogenic signals.

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Nature cell biology, 14, 7, , 2012

PMID:22684254
DOI: 10.1038/ncb2521

Open Access

Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution.
MJ Booth, MR Branco, G Ficz, D Oxley, F Krueger, W Reik, S Balasubramanian

5-Methylcytosine can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. We introduce oxidative bisulfite sequencing (oxBS-Seq), the first method for quantitative mapping of 5hmC in genomic DNA at single-nucleotide resolution. Selective chemical oxidation of 5hmC to 5-formylcytosine (5fC) enables bisulfite conversion of 5fC to uracil. We demonstrate the utility of oxBS-Seq to map and quantify 5hmC at CpG islands (CGIs) in mouse embryonic stem (ES) cells and identify 800 5hmC-containing CGIs that have on average 3.3% hydroxymethylation. High levels of 5hmC were found in CGIs associated with transcriptional regulators and in long interspersed nuclear elements, suggesting that these regions might undergo epigenetic reprogramming in ES cells. Our results open new questions on 5hmC dynamics and sequence-specific targeting by TETs.

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Science (New York, N.Y.), 336, 6083, , 2012

PMID:22539555
DOI: 10.1126/science.1220671

Open Access

RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.
L Tavares, E Dimitrova, D Oxley, J Webster, R Poot, J Demmers, K Bezstarosti, S Taylor, H Ura, H Koide, A Wutz, M Vidal, S Elderkin, N Brockdorff

Polycomb-repressive complex 1 (PRC1) has a central role in the regulation of heritable gene silencing during differentiation and development. PRC1 recruitment is generally attributed to interaction of the chromodomain of the core protein Polycomb with trimethyl histone H3K27 (H3K27me3), catalyzed by a second complex, PRC2. Unexpectedly we find that RING1B, the catalytic subunit of PRC1, and associated monoubiquitylation of histone H2A are targeted to closely overlapping sites in wild-type and PRC2-deficient mouse embryonic stem cells (mESCs), demonstrating an H3K27me3-independent pathway for recruitment of PRC1 activity. We show that this pathway is mediated by RYBP-PRC1, a complex comprising catalytic subunits of PRC1 and the protein RYBP. RYBP-PRC1 is recruited to target loci in mESCs and is also involved in Xist RNA-mediated silencing, the latter suggesting a wider role in Polycomb silencing. We discuss the implications of these findings for understanding recruitment and function of Polycomb repressors.

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Cell, 148, 4, , 2012

PMID:22325148
DOI: 10.1016/j.cell.2011.12.029

Open Access

The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α.
MA Barber, A Hendrickx, M Beullens, H Ceulemans, D Oxley, S Thelen, M Thelen, M Bollen, HC Welch

P-Rex1 is a GEF (guanine-nucleotide-exchange factor) for the small G-protein Rac that is activated by PIP3 (phosphatidylinositol 3,4,5-trisphosphate) and Gβγ subunits and inhibited by PKA (protein kinase A). In the present study we show that PP1α (protein phosphatase 1α) binds P-Rex1 through an RVxF-type docking motif. PP1α activates P-Rex1 directly in vitro, both independently of and additively to PIP3 and Gβγ. PP1α also substantially activates P-Rex1 in vivo, both in basal and PDGF (platelet-derived growth factor)- or LPA (lysophosphatidic acid)-stimulated cells. The phosphatase activity of PP1α is required for P-Rex1 activation. PP1β, a close homologue of PP1α, is also able to activate P-Rex1, but less effectively. PP1α stimulates P-Rex1-mediated Rac-dependent changes in endothelial cell morphology. MS analysis of wild-type P-Rex1 and a PP1α-binding-deficient mutant revealed that endogenous PP1α dephosphorylates P-Rex1 on at least three residues, Ser834, Ser1001 and Ser1165. Site-directed mutagenesis of Ser1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1α, confirming Ser1165 as a dephosphorylation site important in regulating P-Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P-Rex1 through PP1α-dependent dephosphorylation.

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The Biochemical journal, 443, 1, , 2012

PMID:22242915
DOI: 10.1042/BJ20112078

Open Access

Protein identification by MALDI-TOF mass spectrometry.
Webster J, Oxley D

MALDI-TOF mass spectrometers are now commonplace and their relative ease of use means that most non-specialist labs can readily access the technology for the rapid and sensitive analysis of biomolecules. One of the main uses of MALDI-TOF-MS is in the identification of proteins, by peptide mass fingerprinting (PMF). Here we describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by 1D or 2D gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and destaining, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF-MS of the tryptic peptides and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method.

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Methods in molecular biology (Clifton, N.J.), 800, 1940-6029, , 2012

PMID:21964792

CDK1, not ERK1/2 or ERK5, is required for mitotic phosphorylation of BIMEL.
R Gilley, PA Lochhead, K Balmanno, D Oxley, J Clark, SJ Cook

The pro-apoptotic BH3 only protein BIM(EL) is phosphorylated by ERK1/2 and this targets it for proteasome-dependent degradation. A recent study has shown that ERK5, an ERK1/2-related MAPK, is activated during mitosis and phosphorylates BIM(EL) to promote cell survival. Here we show that treatment of cells with nocodazole or paclitaxel does cause phosphorylation of BIM(EL), which is independent of ERK1/2. However, this was not due to ERK5-catalysed phosphorylation, since it was not reversed by the MEK5 inhibitor BIX02189 and proceeded normally in ERK5-/- fibroblasts. Indeed, although ERK5 is phosphorylated at multiple sites in the C-terminal transactivation region during mitosis, these do not include the activation-loop and ERK5 kinase activity does not increase. Mitotic phosphorylation of BIM(EL) occurred at proline-directed phospho-acceptor sites and was abolished by selective inhibition of CDK1. Furthermore, cyclin B1 was able to interact with BIM and cyclin B1/CDK1 complexes could phosphorylate BIM in vitro. Finally, we show that CDK1-dependent phosphorylation of BIM(EL) drives its polyubiquitylation and proteasome-dependent degradation to protect cells during mitotic arrest. These results provide new insights into the regulation of BIM(EL) and may be relevant to the therapeutic use of agents such as paclitaxel.

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Cellular signalling, 24, 1, , 2012

PMID:21924351
DOI: 10.1016/j.cellsig.2011.08.018

Maintenance of silent chromatin through replication requires SWI/SNF-like chromatin remodeler SMARCAD1.
SP Rowbotham, L Barki, A Neves-Costa, F Santos, W Dean, N Hawkes, P Choudhary, WR Will, J Webster, D Oxley, CM Green, P Varga-Weisz, JE Mermoud

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.

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Molecular cell, 42, 3, , 2011

PMID:21549307
DOI: 10.1016/j.molcel.2011.02.036

Open Access

PI3Kβ plays a critical role in neutrophil activation by immune complexes.
S Kulkarni, C Sitaru, Z Jakus, KE Anderson, G Damoulakis, K Davidson, M Hirose, J Juss, D Oxley, TA Chessa, F Ramadani, H Guillou, A Segonds-Pichon, A Fritsch, GE Jarvis, K Okkenhaug, R Ludwig, D Zillikens, A Mocsai, B Vanhaesebroeck, LR Stephens, PT Hawkins

Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the β isoform of phosphoinositide 3-kinase (PI3Kβ) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kβ alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kβ and PI3Kδ, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3Kβ by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B₄. Coincident activation by a tyrosine kinase-coupled receptor (FcγR) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the β isoform of PI3K. PI3Kβ-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kβ and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kβ as a potential therapeutic target in inflammatory disease.

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Science signaling, 4, 168, , 2011

PMID:21487106
DOI: 10.1126/scisignal.2001617

Open Access

A novel foregut mucin characterized by a murine monoclonal autoantibody.
Binos S, Royce SG, Oxley D, Bacic A, Bhathal PS, Underwood JR

Autoantibodies to gastric cellular antigens and glycoproteins including mucins and Lewis X and Y antigens have been implicated in the induction of autoimmune gastritis. Monoclonal antibody D10 (D10 MAb) recognizes a highly conserved mucin expressed in the foregut of mammals and other vertebrates. The objective of this study was to biochemically characterize the autoantigen identified by D10 MAb and examine its autoimmunogenicity in the mouse. Characterization of the mucin autoantigen was undertaken following purification, by amino acid and carbohydrate analyses, deglycosylation, SDS-PAGE, and immunoblotting using D10 MAb. Autoimmune reactivity and specificity of D10 MAb were validated by immunohistochemistry and ELISA using mouse tissue. Induction of autoimmune gastritis was investigated following immunization of mice with D10 MAb-reactive heterologous mucin. D10 MAb was shown to be a murine anti-mucin autoantibody with a unique pattern of immunohistochemical staining of Brunner's glands of the duodenum and the cardiac glands, mucous neck cells, and pyloric glands of the stomach from inbred Balb/c mice in patterns identical to that previously reported in human tissue. Amino acid and carbohydrate analysis of purified D10 mucin reflected a compositional profile of a typical mucin molecule. Confirmation that D10 MAb recognizes a mucin was also provided by demonstration that the carbohydrate epitope resides on a high molecular weight (>1x10(6)Da), high-density (>1.40 g/mL) molecule comprised of greater than 60% carbohydrate. Mice immunized with D10 MAb-reactive, purified, heterologous mucin produced autoantibodies of identical specificity to the original D10 MAb. These data demonstrate the autoimmunogenic properties of a novel foregut mucin and raise the potential of anti-mucin autoantibodies in the induction of autoimmune gastritis.

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Hybridoma (2005), 29, 1557-8348, , 2010

PMID:20455280

Fission yeast Iec1-ino80-mediated nucleosome eviction regulates nucleotide and phosphate metabolism.
CJ Hogan, S Aligianni, M Durand-Dubief, J Persson, WR Will, J Webster, L Wheeler, CK Mathews, S Elderkin, D Oxley, K Ekwall, PD Varga-Weisz

Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenine-responsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.

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Molecular and cellular biology, 30, 3, , 2010

PMID:19933844
DOI: 10.1128/MCB.01117-09

Open Access

Immunoglobulin aggregation leading to Russell body formation is prevented by the antibody light chain.
D Corcos, MJ Osborn, LS Matheson, F Santos, X Zou, JA Smith, G Morgan, A Hutchings, M Hamon, D Oxley, M Brüggemann

Russell bodies (RBs) are intracellular inclusions filled with protein aggregates. In diverse lymphoid disorders these occur as immunoglobulin (Ig) deposits, accumulating in abnormal plasma or Mott cells. In heavy-chain deposition disease truncated antibody heavy-chains (HCs) are found, which bear a resemblance to diverse polypeptides produced in Ig light-chain (LC)-deficient (L(-/-)) mice. In L(-/-) animals, the known functions of LC, providing part of the antigen-binding site of an antibody and securing progression of B-cell development, may not be required. Here, we show a novel function of LC in preventing antibody aggregation. L(-/-) mice produce truncated HC naturally, constant region (C)gamma and Calpha lack C(H)1, and Cmicro is without C(H)1 or C(H)1 and C(H)2. Most plasma cells found in these mice are CD138(+) Mott cells, filled with RBs, formed by aggregation of HCs of different isotypes. The importance of LC in preventing HC aggregation is evident in knock-in mice, expressing Cmicro without C(H)1 and C(H)2, which only develop an abundance of RBs when LC is absent. These results reveal that preventing antibody aggregation is a major function of LC, important for understanding the physiology of heavy-chain deposition disease, and in general recognizing the mechanisms, which initiate protein conformational diseases.

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Blood, 115, 2, , 2010

PMID:19822901
DOI: 10.1182/blood-2009-07-234864

Light chain-deficient mice produce novel multimeric heavy-chain-only IgA by faulty class switching.
LS Matheson, MJ Osborn, JA Smith, D Corcos, M Hamon, R Chaouaf, J Coadwell, G Morgan, D Oxley, M Brüggemann

Recently, we identified that diverse heavy chain (H-chain)-only IgG is spontaneously produced in light chain (L-chain)-deficient mice (L(-/-) with silenced kappa and lambda loci) despite a block in B cell development. In murine H-chain IgG, the first Cgamma exon, C(H)1, is removed after DNA rearrangement and secreted polypeptides are comparable with camelid-type H-chain IgG. Here we show that L(-/-) mice generate a novel class of H-chain Ig with covalently linked alpha chains, not identified in any other healthy mammal. Surprisingly, diverse H-chain-only IgA can be released from B cells at levels similar to conventional IgA and is found in serum and sometimes in milk and saliva. Surface IgA without L-chain is expressed in B220(+) spleen cells, which exhibited a novel B cell receptor, suggesting that associated conventional differentiation events occur. To facilitate the cellular transport and release of H-chain-only IgA, chaperoning via BiP association seems to be prevented as only alpha chains lacking C(H)1 are released from the cell. This appears to be accomplished by imprecise class-switch recombination (CSR) from Smu into the alpha constant region, which removes all or part of the Calpha1 exon at the genomic level.

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International immunology, 21, 8, , 2009

PMID:19561045
DOI: 10.1093/intimm/dxp062

Effect of enzymatic deimination on the conformation of recombinant prion protein.
DS Young, F Meersman, D Oxley, J Webster, AC Gill, I Bronstein, CR Lowe, DV Dear

Deimination is the post-translational conversion of arginine residues to citrulline. It has been implicated as a causative factor in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis and more recently, as a marker of neurodegeneration. We have investigated the effect of the post-translational modification of arginine residues on the structure of recombinant ovine prion protein. Deiminated prion protein exhibited biophysical properties characteristic of the scrapie-associated conformer of prion protein viz. an increased beta-sheet secondary structure, congophilic structures indicative of amyloid and proteinase K resistance which could be templated onto normal unmodified prion protein. In the light of these findings, a potential role of post-translational modifications to prion protein in disease initiation or propagation is discussed.

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Biochimica et biophysica acta, 1794, 8, , 2009

PMID:19341825
DOI: 10.1016/j.bbapap.2009.03.013

The equatorial subsegment in mammalian spermatozoa is enriched in tyrosine phosphorylated proteins.
R Jones, PS James, D Oxley, J Coadwell, F Suzuki-Toyota, EA Howes

The equatorial subsegment (EqSS) was originally identified by atomic force microscopy as a discrete region within the equatorial segment of Artiodactyl spermatozoa. In this investigation, we show that the EqSS is enriched in tyrosine phosphorylated proteins and present preliminary evidence for its presence in mouse and rat spermatozoa. The anti-phosphotyrosine monoclonal antibody (McAb) 4G10 bound strongly and discretely to the EqSS of permeabilized boar, ram, and bull spermatozoa. It also bound to a small patch on the posterior acrosomal region of permeabilized mouse and rat spermatozoa, suggesting that the EqSS is not restricted to the order Artiodactyla. An anti-HSPA1A (formerly Hsp70) antibody recognized the EqSS in boar spermatozoa. Immunogold labeling with McAb 4G10 localized the tyrosine phosphorylated proteins to the outer acrosomal membrane. This was verified by freeze-fracture electron microscopy, which identified the EqSS in three overlying membranes, the plasma membrane, outer acrosomal membrane, and inner acrosomal membrane. In all five species, tyrosine phosphorylated proteins became restricted to the EqSS during sperm maturation in the epididymis. The major tyrosine phosphorylated proteins in the EqSS of boar and ram spermatozoa were identified by mass spectrometry as orthologs of human SPACA1 (formerly SAMP32). Immunofluorescence with a specific polyclonal antibody localized SPACA1 to the equatorial segment in boar spermatozoa. We speculate that the EqSS is an organizing center for assembly of multimolecular complexes that initiate fusion competence in this area of the plasma membrane following the acrosome reaction.

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Biology of reproduction, 79, 3, , 2008

PMID:18448843
DOI: 10.1095/biolreprod.107.067314

Heavy chain-only antibodies are spontaneously produced in light chain-deficient mice.
X Zou, MJ Osborn, DJ Bolland, JA Smith, D Corcos, M Hamon, D Oxley, A Hutchings, G Morgan, F Santos, PJ Kilshaw, MJ Taussig, AE Corcoran, M Brüggemann

In healthy mammals, maturation of B cells expressing heavy (H) chain immunoglobulin (Ig) without light (L) chain is prevented by chaperone association of the H chain in the endoplasmic reticulum. Camelids are an exception, expressing homodimeric IgGs, an antibody type that to date has not been found in mice or humans. In camelids, immunization with viral epitopes generates high affinity H chain-only antibodies, which, because of their smaller size, recognize clefts and protrusions not readily distinguished by typical antibodies. Developmental processes leading to H chain antibody expression are unknown. We show that L(-/-) (kappa(-/-)lambda(-/-)-deficient) mice, in which conventional B cell development is blocked at the immature B cell stage, produce diverse H chain-only antibodies in serum. The generation of H chain-only IgG is caused by the loss of constant (C) gamma exon 1, which is accomplished by genomic alterations in C(H)1-circumventing chaperone association. These mutations can be attributed to errors in class switch recombination, which facilitate the generation of H chain-only Ig-secreting plasma cells. Surprisingly, transcripts with a similar deletion can be found in normal mice. Thus, naturally occurring H chain transcripts without C(H)1 (V(H)DJ(H)-hinge-C(H)2-C(H)3) are selected for and lead to the formation of fully functional and diverse H chain-only antibodies in L(-/-) animals.

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The Journal of experimental medicine, 204, 13, , 2007

PMID:18086860
DOI: 10.1084/jem.20071155

Open Access

Characterisation of secreted polysaccharides and (glyco)proteins from suspension cultures of Pyrus communis.
Webster JM, Oxley D, Pettolino FA, Bacic A

High molecular weight material recovered from the culture filtrate of cell suspension cultured Pyrus communis was composed of 81% carbohydrate, 13% protein and 5% inorganic material. This material was separated into three fractions (one neutral (Fraction A) and two acidic (Fractions B and C)), by anion-exchange chromatography on DEAE-Sepharose CL-6B using a gradient of imidazole-HCl at pH 7.0. The monosaccharide and linkage composition of each fraction was determined after carboxyl reduction of uronic acid residues. From the combined results of the carbohydrate analyses, we conclude that the high molecular weight extracellular material consists of three major and two minor polysaccharides: a (fucogalacto)xyloglucan (36%) in the unbound neutral Fraction A; a type II arabinogalactan (as an arabinogalactan-protein, 29%) and an acidic (glucurono)arabinoxylan (2%) in Fraction B; and a galacturonan (33%) and a trace of heteromannan in Fraction C. The main amino acids in the proteins were Glx, Thr, Ser, Hyp/Pro and Gly. Further separation of Fraction B by solvent partition, SDS-PAGE and analysis by LC-MS/MS identified the major proteins as two chitanases, two thaumatin-like proteins, a beta-1,3-glucanase, an extracellular dermal glycoprotein and a pathogenesis-related protein.

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Phytochemistry, 69, 0031-9422, , 2008

PMID:18037144

Independent protein-profiling studies show a decrease in apolipoprotein A1 levels in schizophrenia CSF, brain and peripheral tissues.
JT Huang, L Wang, S Prabakaran, M Wengenroth, HE Lockstone, D Koethe, CW Gerth, S Gross, D Schreiber, K Lilley, M Wayland, D Oxley, FM Leweke, S Bahn

Although some insights into the etiology of schizophrenia have been gained, an understanding of the illness at the molecular level remains elusive. Recent advances in proteomic profiling offer great promise for the discovery of markers underlying pathophysiology of diseases. In the present study, we employed two high-throughput proteomic techniques together with traditional methods to investigate cerebrospinal fluid (CSF), brain and peripheral tissues (liver, red blood cells and serum) of schizophrenia patients in an attempt to identify peripheral/surrogate disease markers. The cohorts used to investigate each tissue were largely independent, although some CSF and serum samples were collected from the same patient. To address the major confounding factor of antipsychotic drug treatment, we also included a large cohort of first-onset drug-naive patients. Apolipoprotein A1 (apoA1) showed a significant decrease in expression in schizophrenia patients compared to controls in all five tissues examined. Specifically, using SELDI-TOF mass spectrometry, apoA1 was found decreased in CSF from schizophrenia patients (-35%, P=0.00001) and, using 2D-DIGE, apoA1 was also found downregulated in liver (-30%, P=0.02) and RBCs (-60%, P=0.003). Furthermore, we found a significant reduction of apoA1 in sera of first-onset drug-naive schizophrenia patients using enzyme-linked immunosorbent assay (-18%, P=0.00008) and in two investigations of post-mortem brain tissue using western blot analysis (-35%, P=0.05; -51%, P=0.05). These results show that apoA1 is consistently downregulated in the central nervous system as well as peripheral tissues of schizophrenia patients and may be linked to the underlying disease mechanism.

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Molecular psychiatry, 13, 12, , 2008

PMID:17938634
DOI: 10.1038/sj.mp.4002108

Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro.
DV Dear, DS Young, J Kazlauskaite, F Meersman, D Oxley, J Webster, TJ Pinheiro, AC Gill, I Bronstein, CR Lowe

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrP(Sc), an aberrant conformer of a normal cellular protein PrP(C). It is thought PrP(Sc) is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrP(Sc), or a sub-population thereof, have been reported. In this study we have investigated whether chemical modification of amino acids within PrP might cause this protein to exhibit aberrant properties and whether these properties can be propagated onto unmodified prion protein. Of particular interest were post-translational modifications resulting from physiological conditions shown to be associated with TSE disease. Here we report that in vitro exposure of recombinant PrP to conditions that imitate the end effects of oxidative/nitrative stress in TSE-infected mouse brains cause the protein to adopt many of the physical characteristics of PrP(Sc). Most interestingly, these properties could be propagated onto unmodified PrP protein when the modified protein was used as a template. These data suggest that post-translational modifications of PrP might contribute to the initiation and/or propagation of prion protein-associated plaques in vivo during prion disease, thereby high-lighting novel biochemical pathways as possible therapeutic targets for these conditions.

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Biochimica et biophysica acta, 1774, 7, , 2007

PMID:17572162
DOI: 10.1016/j.bbapap.2007.05.005

Conversion of platelets from a proaggregatory to a proinflammatory adhesive phenotype: role of PAF in spatially regulating neutrophil adhesion and spreading.
S Kulkarni, KJ Woollard, S Thomas, D Oxley, SP Jackson

The ability of platelets to provide a highly reactive surface for the recruitment of other platelets and leukocytes to sites of vascular injury is critical for hemostasis, atherothrombosis, and a variety of inflammatory diseases. The mechanisms coordinating platelet-platelet and platelet-leukocyte interactions have been well defined and, in general, it is assumed that increased platelet activation correlates with enhanced reactivity toward other platelets and neutrophils. In the current study, we demonstrate a differential role for platelets in supporting platelet and neutrophil adhesive interactions under flow. We demonstrate that the conversion of spread platelets to microvesiculated procoagulant (annexin A5-positive [annexin A5+ve]) forms reduces platelet-platelet adhesion and leads to a paradoxical increase in neutrophil-platelet interaction. This enhancement in neutrophil adhesion and spreading is partially mediated by the proinflammatory lipid, platelet-activating factor (PAF). PAF production, unlike other neutrophil chemokines (IL-8, GRO-alpha, NAP-2, IL-1beta) is specifically and markedly up-regulated in annexin A5+ve cells. Physiologically, this spatially controlled production of PAF plays an important role in localizing neutrophils on the surface of thrombi. These studies define for the first time a specific proinflammatory function for annexin A5+ve platelets. Moreover, they demonstrate an important role for platelet-derived PAF in spatially regulating neutrophil adhesion under flow.

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Blood, 110, 6, , 2007

PMID:17548580
DOI: 10.1182/blood-2006-08-040980

Urinary pheromones promote ERK/Akt phosphorylation, regeneration and survival of vomeronasal (V2R) neurons.
Xia J, Sellers LA, Oxley D, Smith T, Emson P, Keverne EB

The G protein-coupled pheromone receptor neurons (V1R and V2R) of the vomeronasal organ (VNO) are continually replaced throughout the lifetime of the mouse. Moreover, active signalling of V2Rs via the transient receptor potential 2(TRPC2) channel is necessary for regeneration of receptors, as the TRPC2 null mutant mouse showed a 75% reduction of V2Rs by the age of two months. Here we describe V2R mediated signalling in a neuronal line established from vomeronasal stem cells taken from postnatal female mice. Cells were immunoreactive for Galpha(o) and V2R, whereas V1R and Galpha(i) immunoreactivity could not be detected. Biological ligands (dilute urine and its protein fractions) were found to increase proliferation and survival of these neurons. Dilute mouse urine but not artificial urine also induced ERK, Akt and CREB signalling in a dose dependent way. The volatile fraction of male mouse urine alone was without effect while the fraction containing peptides (> 5 kDa) also stimulated ERK and Akt phosphorylation. The ERK, Akt and CREB phosphorylation response was sensitive to pertussis toxin, confirming the involvement of V2R linked Galpha(o). Dilute mouse urine or its high molecular weight protein fraction increased survival and proliferation of these neurons. Hence, urinary pheromones, which signal important social information via mature neurons, also promote survival and proliferation of their regenerating precursors. These data show that regenerating V2Rs respond to urine and the urinary peptides by activation of the Ras-ERK and PI3-Akt pathways, which appear to be important for vomeronasal neural survival and proliferation.

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The European journal of neuroscience, 24, 0953-816X, , 2006

PMID:17229082

DNA damage-induced Bcl-xL deamidation is mediated by NHE-1 antiport regulated intracellular pH.
R Zhao, D Oxley, TS Smith, GA Follows, AR Green, DR Alexander

The pro-survival protein Bcl-xL is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage-induced Bcl-xL deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-xL deamidation remains unknown and its functional consequences unclear. We show here that rBcl-xL deamidation generates an iso-Asp(52)/iso-Asp(66) species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-xL deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage-induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-xL deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-xL deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.

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PLoS biology, 5, 1, , 2007

PMID:17177603
DOI: 10.1371/journal.pbio.0050001

Disease biomarkers in cerebrospinal fluid of patients with first-onset psychosis.
Huang JT, Leweke FM, Oxley D, Wang L, Harris N, Koethe D, Gerth CW, Nolden BM, Gr.oss S, Schreiber D, Reed B, Bahn S

Psychosis is a severe mental condition that is characterized by a loss of contact with reality and is typically associated with hallucinations and delusional beliefs. There are numerous psychiatric conditions that present with psychotic symptoms, most importantly schizophrenia, bipolar affective disorder, and some forms of severe depression referred to as psychotic depression. The pathological mechanisms resulting in psychotic symptoms are not understood, nor is it understood whether the various psychotic illnesses are the result of similar biochemical disturbances. The identification of biological markers (so-called biomarkers) of psychosis is a fundamental step towards a better understanding of the pathogenesis of psychosis and holds the potential for more objective testing methods.

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PLoS medicine, 3, 1549-1676, , 2006

PMID:17090210

Eukaryotic localization, activation and ubiquitinylation of a bacterial type III secreted toxin.
Stirling FR, Cuzick A, Kelly SM, Oxley D, Evans TJ

Type III secretion is a widespread method whereby Gram-negative bacteria introduce toxins into eukaryotic cells. These toxins mimic or subvert a normal cellular process by interacting with a specific target, although how toxins reach their site of action is unclear. We set out to investigate the intracellular localization of a type III toxin of Pseudomonas aeruginosa called ExoU, which has phospholipase activity and requires a eukaryotic factor for activity. We found that ExoU is localized to the plasma membrane and undergoes modification within the cell by addition of two ubiquitin molecules at lysine-178. A region of five amino acids at position 679-683 near the C-terminus of the ExoU protein controls both membrane localization and ubiquitinylation. Site-directed mutagenesis identified a tryptophan at position 681 as crucial for these effects. We found that the same region at position 679-683 was also required for cell toxicity produced by ExoU as well as in vitro phospholipase activity. Localization of the phospholipase ExoU to the plasma membrane is thus required for activation and allows efficient utilization of adjacent substrate phospholipids.

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Cellular microbiology, 8, 1462-5814, , 2006

PMID:16882033

Peptide mass fingerprinting: protein identification using MALDI-TOF mass spectrometry.
Webster J, Oxley D

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-staining, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method.

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Methods in molecular biology (Clifton, N.J.), 310, 1064-3745, , 2005

PMID:16350956

Primary iron overload with inappropriate hepcidin expression in V162del ferroportin disease.
Zoller H, McFarlane I, Theurl I, Stadlmann S, Nemeth E, Oxley D, Ganz T, Halsall DJ, Cox TM, Vogel W

Ferroportin disease (hemochromatosis type 4) is a recently recognized disorder of human iron metabolism, characterized by iron deposition in macrophages, including Kupffer cells. Mutations in the gene encoding ferroportin 1, a cellular iron exporter, are responsible for this iron storage disease, inherited as an autosomal dominant trait. We present clinical, histopathological, and radiological findings in a family with the most common ferroportin mutation, V162del. In the index case, the disorder is characterized by abundant deposition of hemosiderin in all tissues investigated (mesenteric lymph node, liver, gastric and duodenal mucosa, and also in squamous cell carcinoma of the lung). The radiological findings indicated the presence of excess iron in bone marrow and spleen. Despite a significant burden of iron, no features of chronic liver disease were found in affected members of the family, including individuals aged up to 80 years. Hyperferritinemia greater than 1,000 microg/L was a penetrant biochemical finding before the second decade in life and was associated with significantly increased serum concentrations of pro-hepcidin that correlated positively with urinary hepcidin concentrations. In conclusion, the systemic iron burden in ferroportin disease is not a sufficient cause for chronic liver disease. In patients with most, but not all, ferroportin mutations, retention of iron in macrophages of the liver and other organs may protect against damage to parenchymal cells. Finally, macrophage iron storage in ferroportin disease is associated with elevated serum pro-hepcidin levels.

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Hepatology (Baltimore, Md.), 42, 0270-9139, , 2005

PMID:15986403

Beta-elimination: an unexpected artefact in proteome analysis.
Herbert B, Hopwood F, Oxley D, McCarthy J, Laver M, Grinyer J, Goodall A, Williams K, Castagna A, Righetti PG

Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namely carbamylation and deamidation, have been recently dispelled (Herbert et al., J. Proteome Res. 2002, in press). We report here, for the first time, a noxious and unexpected artefact in proteome analysis: beta-elimination (or desulfuration), which results on the loss of an H(2)S group (34 Da) from cysteine (Cys) residues for protein focusing in the alkaline pH region. With such an elimination event, a dehydro alanine residue is generated at the Cys site. In turn, the presence of a double bond in this position elicits lysis of the peptide bond, generating a number of peptides of fairly large size from an intact protein. The first process seems to be favored by the electric field, probably due to the continuous harvesting of the SH(-) anion produced. The only remedy found to this noxious degradation pathway is the reduction and alkylation of all Cys residues prior to their exposure to the electric field. Alkylation appears to substantially reduce both beta-elimination and the subsequent amido bond lysis.

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Proteomics, 3, 1615-9853, , 2003

PMID:12833505

Carbamylation of proteins in 2-D electrophoresis--myth or reality?
McCarthy J, Hopwood F, Oxley D, Laver M, Castagna A, Righetti PG, Williams K, Herbert B

Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.

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Journal of proteome research, 2, 1535-3893, , 0

PMID:12814262

Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears.
Schulz BL, Oxley D, Packer NH, Karlsson NG

Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270 kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200 kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Le(a). After the release of oligosaccharides by reductive beta-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex(0-->2)NeuAc(1-->)(x)Hex(x)HexNAc(x)(-ol), x=1-6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Le(a) epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.

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The Biochemical journal, 366, 0264-6021, , 2002

PMID:12015815

NaAGP4 is an arabinogalactan protein whose expression is suppressed by wounding and fungal infection in Nicotiana alata.
Gilson P, Gaspar YM, Oxley D, Youl JJ, Bacic A

Arabinogalactan proteins (AGPs) are proteoglycans secreted by plant cells that have been implicated in plant growth and development. Most AGPs cloned to date possess highly labile glycosylphosphatidylinositol (GPI) lipid anchors. These anchors transiently attach AGPs to the plasma membrane before they are released into the cell wall following GPI anchor hydrolysis. We have isolated and partially sequenced the protein core of an AGP purified from styles of Nicotiana alata. The protein sequence data were utilised to clone the AGP's gene, NaAGP4. This AGP shares about 78% sequence identity with the tomato AGP LeAGP-1. RNA gel blot analyses of different plant organs indicate that NaAGP4 is expressed in the same tissues and at similar levels as LeAGP-1. Furthermore, NaAGP4 like LeAGP-1 is rapidly suppressed by tissue wounding and by pathogen infection. We believe NaAGP4 and LeAGP-1 are the first described examples of orthologous AGPs from different plant species. In contrast, another AGP from N. alata, NaAGP1, is comparatively unaffected by wounding and pathogen infection, although this AGP is expressed in similar tissues and at similar levels as NaAGP4.

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Protoplasma, 215, 0033-183X, , 2001

PMID:11732052

Structure of the glycosylphosphatidylinositol anchor of an arabinogalactan protein from Pyrus communis suspension-cultured cells.
Oxley D, Bacic A

Arabinogalactan proteins (AGPs) are proteoglycans of higher plants, which are implicated in growth and development. We recently have shown that two AGPs, NaAGP1 (from Nicotiana alata styles) and PcAGP1 (from Pyrus communis cell suspension culture), are modified by the addition of a glycosylphosphatidylinositol (GPI) anchor. However, paradoxically, both AGPs were buffer soluble rather than membrane associated. We now show that pear suspension cultured cells also contain membrane-bound GPI-anchored AGPs. This GPI anchor has the minimal core oligosaccharide structure, D-Manalpha(1-2)-D-Manalpha(1-6)-D-Manalpha(1-4)-D-GlcN -inositol, which is consistent with those found in animals, protozoa, and yeast, but with a partial beta(1-4)-galactosyl substitution of the 6-linked Man residue, and has a phosphoceramide lipid composed primarily of phytosphingosine and tetracosanoic acid. The secreted form of PcAGP1 contains a truncated GPI lacking the phosphoceramide moiety, suggesting that it is released from the membrane by the action of a phospholipase D. The implications of these findings are discussed in relation to the potential mechanisms by which GPI-anchored AGPs may be involved in signal transduction pathways.

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Proceedings of the National Academy of Sciences of the United States of America, 96, 0027-8424, , 1999

PMID:10588691

Arabinogalactan-proteins from Nicotiana alata and Pyrus communis contain glycosylphosphatidylinositol membrane anchors.
Youl JJ, Bacic A, Oxley D

Arabinogalactan-proteins (AGPs) are a class of proteoglycans found in cell secretions and plasma membranes of plants. Attention is currently focused on their structure and their potential role in growth and development. We present evidence that two members of a major class of AGPs, the classical AGPs, AGPNa1 from styles of Nicotiana alata and AGPPc1 from cell suspension cultures of Pyrus communis, undergo C-terminal processing involving glycosylphosphatidylinositol membrane anchors. The evidence is that (i) the transmembrane helix at the C terminus predicted from the cDNA encoding these proteins is not present-the C-terminal amino acid is Asn87 and Ser97 for AGPNa1 and AGPPc1, respectively; (ii) both AGP protein backbones are substituted with ethanolamine at the C-terminal amino acid; and (iii) inositol, glucosamine, and mannose are present in the native AGPs. An examination of the deduced amino acid sequences of other classical AGP protein backbones shows that glycosylphosphatidylinositol-anchors may be a common feature of this class of AGPs.

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Proceedings of the National Academy of Sciences of the United States of America, 95, 0027-8424, , 1998

PMID:9653116

Structure and distribution of N-glycans on the S7-allele stylar self-incompatibility ribonuclease of Nicotiana alata.
Oxley D, Munro SL, Craik DJ, Bacic A

S-RNases are the stylar products of the self-incompatibility (S)-locus in solanaceous plants (including Nicotiana alata), and as such, are involved in the prevention of self-pollination. All cDNA sequences of S-RNase products of functional S-alleles contain potential N-glycosylation sites, with one site being conserved in all cases, suggesting that N-glycosylation is important in self-incompatibility. In this study, we report on the structure and localization of the N-glycans on the S7-allele RNase of N. alata. A total of nine N-glycans, belonging to the high-mannose- and xylosylated hybrid-classes, were identified and characterized by a combination of electrospray-ionization mass-spectrometry (ESI-MS), 1H-NMR spectroscopy, and methylation analyses. The glycosylation pattern of individual glycosylation sites was determined by ESI-MS of the glycans released from isolated chymotryptic glycopeptides. All three N-glycosylation sites showed microheterogeneity and each had a unique complement of N-glycans. The N-glycosylation pattern of the S7-RNase is significantly different to those of the S1- and S2-RNases.

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Journal of biochemistry, 123, 0021-924X, , 1998

PMID:9562634

Structural analysis and molecular model of a self-incompatibility RNase from wild tomato.
Parry S, Newbigin E, Craik D, Nakamura KT, Bacic A, Oxley D

Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S3-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S3-RNase has a single N-glycosylation site (Asn-28) to which one of three N-glycans is attached. S3-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S3-RNase shows that four of the most variable regions of the S-RNases are clustered on one surface of the molecule. This is discussed in the context of recent experiments that set out to determine the regions of the S-RNase important for recognition during the self-incompatibility response.

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Plant physiology, 116, 0032-0889, , 1998

PMID:9489006

Identification of active-site histidine residues of a self-incompatibility ribonuclease from a wild tomato.
Parry S, Newbigin E, Currie G, Bacic A, Oxley D

The style component of the self-incompatibility (S) locus of the wild tomato Lycopersicon peruvianum (L.) Mill. is an allelic series of glycoproteins with ribonuclease activity (S-RNases). Treatment of the S3-RNase from L. peruvianum with iodoacetate at pH 6.1 led to a loss of RNase activity. In the presence of a competitive inhibitor, guanosine 3'-monophosphate (3'-GMP), the rate of RNase inactivation by iodoacetate was reduced significantly. Analysis of the tryptic digestion products of the iodoacetate-modified S-RNase by reversed-phase high-performance liquid chromatography and electrospray-ionization mass spectrometry showed that histidine-32 was preferentially modified in the absence of 3'-GMP. Histidine-88 was also modified, but this occurred both in the presence and absence of 3'-GMP, suggesting that this residue is accessible when 3'-GMP is in the active site. Cysteine-150 was modified by iodoacetate in the absence of 3'-GMP and, to a lesser extent, in its presence. The results are discussed with respect to the related fungal RNase T2 family and the mechanism of S-RNase action.

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Plant physiology, 115, 0032-0889, , 1997

PMID:9414554

Disulphide bonding in a stylar self-incompatibility ribonuclease of Nicotiana alata.
Oxley D, Bacic A

Many flowering plants have developed a self-incompatibility mechanism, which is controlled by a single polyallelic locus (the S-locus), to prevent inbreeding. The products of the S-locus in the styles of solanaceous plants are an allelic series of glycoproteins with RNase activity [McClure, B. A., Haring, V., Ebert, P. R., Anderson, M. A., Simpson, R. J., Sakiyama, F. & Clarke, A. E. (1989) Nature 342, 955-957]. These S-RNases show some amino-acid-sequence similarity with two fungal RNases (T2 and Rh), including the presence of two active-site His residues, which suggests a common three-dimensional structure. Disulphide bonding is important in the maintenance of the three-dimensional structure of the fungal RNases [Kurihara, H., Mitsui, Y., Ohgi, K., Irie, M., Mizuno, H. & Nakamura, T. (1992) FEBS Lett. 306, 189-192] and the S-RNases [Tsai, D. S., Lee, H.-S., Post, L. C., Kreiling, K. M. & Kao, T.-H. (1992) Sex. Plant Reprod. 5, 256-263]. We have used the S2-allele RNase of Nicotiana alata, which has nine Cys residues, to establish the pattern of disulphide bonding. The disulphide bonds Cys16-Cys21, Cys45-Cys94, Cys153-Cys182 and Cys165-Cys176 are consistent with the S2-RNase having a similar three-dimensional structure to RNase Rh. A free Cys residue (Cys95) adjacent to Cys45-Cys94 promotes a rapid specific disulphide migration when the protein is exposed to denaturing conditions.

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European journal of biochemistry / FEBS, 242, 0014-2956, , 1996

PMID:8954155

Structure of N-glycans on the S3- and S6-allele stylar self-incompatibility ribonucleases of Nicotiana alata.
Oxley D, Munro SL, Craik DJ, Bacic A

Self-incompatibility is a mechanism developed by many plants to prevent inbreeding. The products of the self-incompatibility (S)-locus in the styles of solanaceous plants are a series of glycoproteins with ribonuclease activity. In this study, we report on the N-glycans from the stylar self-incompatibility S3- and S6-ribonucleases of Nicotiana alata, which were enzymically released and fractionated by high-pH anion-exchange HPLC. A total of 14 N-glycans were identified and characterized by a combination of electrospray-ionization mass-spectrometry, 1H-NMR spectros-copy, chemical degradation, and methylation analyses. This patterns of N-glycosylation is much more complex than that previously found on the N.alata S1- and S2-RNases, each of which contained only four N-glycans.

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Glycobiology, 6, 0959-6658, , 1996

PMID:8922956

Microheterogeneity of N-glycosylation on a stylar self-incompatibility glycoprotein of Nicotiana alata.
Oxley D, Bacic A

Gametophytic self-incompatibility, a mechanism that prevents inbreeding in some families of flowering plants, is mediated by the products of a single genetic locus, the S-locus. The products of the S-gene in the female sexual tissues of Nicotiana alata are an allelic series of glycoproteins with RNase activity. In this study, we report on the microheterogeneity of N-linked glycosylation at the four potential N-glycosylation sites of the S2-glycoprotein. The S-glycoproteins from N.alata contain from one to five potential N-glycosylation sites based on the consensus sequence Asn-Xaa-Ser/Thr. The S2-glycoprotein contains four potential N-glycosylation sites at Asn27, Asn37, Asn38 and Asn 150, designated sites I, II, IV and V, respectively. Site III is absent from the S2-glycoprotein. Analysis of glycopeptides generated from the S2-glycoprotein by trypsin and chymotrypsin digestions revealed the types of glycans and the degree of microheterogeneity present at each site. Sites I (Asn27) and IV (Asn138) display microheterogeneity, site II (Asn37) contains only a single type of N-glycan, and site V (Asn150) is not glycosylated. The microheterogeneity observed at site I on the S2-glycoprotein is the same as that observed at the only site, site I, on the S1-glycoprotein (Woodward et al., Glycobiology, 2, 241-250, 1992). Since the N-glycosylation consensus sequence at site I is conserved in all S-glycoproteins from other species of self-incompatible solanaceous plants, glycosylation at this site may be important to their function. No other post-translational modifications (e.g. O-glycosylation, phosphorylation) were detected on the S2-glycoprotein.

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Glycobiology, 5, 0959-6658, , 1995

PMID:8563138

Structural and serological characterisation of an O-specific polysaccharide from Serratia plymuthica.
Aucken HM, Oxley D, Wilkinson SG

The surface polysaccharides of a strain of Serratia plymuthica were characterised and shown to consist of a linear, acidic galactoglucomannan as well as a major and a minor neutral galactan. Immunoblotting results demonstrated cross-reactions between this strain and others with similar galactans (S. marcescens O16 and O20, Klebsiella O1, and Pasteurella haemolytica T4 and T10).

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FEMS microbiology letters, 111, 0378-1097, , 1993

PMID:7691682

Structure of the N-linked oligosaccharides from tridacnin, a lectin found in the haemolymph of the giant clam Hippopus hippopus.
Puanglarp N, Oxley D, Currie GJ, Bacic A, Craik DJ, Yellowlees D

Tridacnin, a glycoprotein lectin, was isolated from the symbiotic marine clam Hippopus hippopus and the structure of its major N-glycan chains determined. Tridacnin contains only N-linked glycans which were quantitatively cleaved by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Following purification by anion-exchange HPLC, the structures of the oligosaccharides were established using a combination of electrospray ionisation mass spectrometry, 1H-NMR spectroscopy and linkage analysis. The N-glycans are primarily of the oligomannose type but, in addition, some contain a novel 6-O-Me group on the terminal mannose residue of the chain. The N-glycan chains had the following structures. [formula: see text]

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European journal of biochemistry / FEBS, 232, 0014-2956, , 1995

PMID:7588729

Structural studies of the putative O-specific polysaccharide of Serratia marcescens O9.
Oxley D, Wilkinson SG

A polymeric fraction containing the putative O-antigen has been isolated from the lipopolysaccharide of the reference strain (CDC 4534-60) for serogroup O9 of Serratia marcescens. The major component of the fraction was a polymer with a disaccharide repeating-unit of L-rhamnose (Rha) and 2-acetamido-2-deoxy-D-galactose (GalNAc) with the following structure:----3)D-GalpNAc(beta 1----3)L-Rhap(alpha 1----. Evidence for the presence in the fraction of a similar, minor polymer containing 4-substituted rhamnose residues was provided by the NMR spectra, methylation analysis, and Smith degradation.

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European journal of biochemistry / FEBS, 166, 0014-2956, , 1987

PMID:3301342

Structural studies of glucorhamnans isolated from the lipopolysaccharides of reference strains for Serratia marcescens serogroups O4 and O7, and of an O14 strain.
Oxley D, Wilkinson SG

Partially acetylated glucorhamnans have been isolated from the lipopolysaccharides of three strains of Serratia marcescens. The polymer from the reference strain (C.D.C. 864-57) for serogroup O4 has the disaccharide repeating-unit shown below, in which acetylation at position 2 of the rhamnosyl residue is approximately 90% complete. Similar glucorhamnans from the reference strain (C.D.C. 843-57) for serogroup O7 and from a pigmented strain (NM) of serogroup O14 differ only in the configuration of the L-rhamnopyranosyl residue (beta) and the extent of O-acetylation (O7, almost stoichiometric; NM, 80-90%). Glucorhamnans of the second type have been isolated previously from the lipopolysaccharides of other strains of S. marcescens, including the reference strain for serogroup O6 and another pigmented O14 strain (N.C.T.C. 1377). In all cases, the lipopolysaccharide extracts also contained acidic glycans, but the glucorhamnans are believed to constitute the integral side-chains. (Formula: see text).

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Carbohydrate research, 175, 0008-6215, , 1988

PMID:3288341

Structure of a neutral polymer isolated from the lipopolysaccharide of Serratia marcescens O5 (C.D.C. 867-57).
Oxley D,Wilkinson SG

Carbohydrate research, 172, 0008-6215, , 1988

PMID:3286000

Studies of lipopolysaccharides from two strains (C.D.C. 3607-60 and IP 421) of Serratia marcescens O13: structure of the putative O13 antigen.
Oxley D,Wilkinson SG

Structural studies have been carried out on the putative O-specific polysaccharide of the reference strain (C.D.C. 3607-60) for Serratia marcescens O13. Circumstantial evidence that the O13 antigen is a microcapsular, acidic polymer, rather than an integral part of the lipopolysaccharide, has been obtained. Degradative and spectroscopic studies established that the polymer is based on the repeating unit shown, in which the glucuronic acid residue of the linear pentasaccharide carries the lateral 2-acetamido-2-deoxy-beta-D-glucopyranosyl substituent in only about half of the units. The same polymer, again with non-stoichiometric substitution, is also produced by strain IP 421 (O13:H7). The latter strain also produces a neutral polymer which appears to constitute the side chain of the lipopolysaccharide. This polymer, which has a disaccharide repeating-unit of 2-substituted beta-D-ribofuranosyl and 4-substituted 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residues, has been isolated previously from the lipopolysaccharides of the reference strains for S. marcescens serogroups O12 and O14, and appears to be the antigen known to be shared by these strains. (Formula: see text).

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Carbohydrate research, 172, 0008-6215, , 1988

PMID:3285999

Structural studies of an acidic galactomannan from the reference strain for Serratia marcescens serogroup O4.
Oxley D,Wilkinson SG

An acidic, partially acetylated galactomannan has been isolated from the lipopolysaccharide of the reference strain (C.D.C. 864-57) for Serratia marcescens serogroup O4. From the results of methylation analysis, Smith degradations, and n.m.r. spectroscopic studies of the O-deacetylated polymer, it was concluded that the repeating unit has the structure shown, in which the acetal-linked pyruvic acid has the R configuration. The polymer is believed to confer O specificity on the organism, but not to constitute the side chain of the lipopolysaccharide. (formula; see text).

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Carbohydrate research, 179, 0008-6215, , 1988

PMID:3061646

Structure of a neutral polymer isolated from the lipopolysaccharide of the reference strain (C.D.C. 4523-60) for Serratia marcescens serogroup O15.
Oxley D,Wilkinson SG

Carbohydrate research, 177, 0008-6215, , 1988

PMID:3048660

Structure of the O-specific galactan from the lipopolysaccharide of the reference strain for Serratia marcescens serogroup O24.
Oxley D,Wilkinson SG

The putative O-specific polysaccharide for Serratia marcescens serogroup O24 is a galactan with a branched, trisaccharide repeating-unit of the structure shown. The structure of the backbone is identical to that of the linear galactans isolated from the reference strains for S. marcescens serogroups O16 and O20, presumably accounting for the serological cross-reactions observed. (Formula: see text)

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Carbohydrate research, 195, 0008-6215, , 1989

PMID:2699831

Structure of a neutral polymer isolated from the lipopolysaccharide of the reference strain for Serratia marcescens serogroup O18.
Oxley D,Wilkinson SG

A neutral polymer (the putative O antigen) has been isolated from the lipopolysaccharide of the reference strain for Serratia marcescens serogroup 018. From the results of spectroscopic and degradative studies, the repeating unit of the polymer was identified as a linear tetrasaccharide having the structure shown. ----2)-alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----6)-alpha-D- GlcpNAc-(1----

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Carbohydrate research, 195, 0008-6215, , 1989

PMID:2699830

Structures of neutral glycans isolated from the lipopolysaccharides of reference strains for Serratia marcescens serogroups O16 and O20.
Oxley D,Wilkinson SG

Neutral polymers have been isolated from the lipopolysaccharides of the reference strains for Serratia marcescens O16 and O20, serogroups which exhibit significant cross-reactivity. Both organisms produce a galactan with the disaccharide repeating-unit shown, and which apparently accounts for the serological observations. The same galactan has also been reported as the O4-specific polysaccharide of Pasteurella haemolytica. In S. marcescens O16, the galactan is apparently accompanied by a polymer of 2-substituted beta-D-ribofuranosyl residues.

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Carbohydrate research, 193, 0008-6215, , 1989

PMID:2692814

Structural studies of an acidic galactoglucomannan from the O15 reference strain (C.D.C. 4523-60) of Serratia marcescens.
Oxley D,Wilkinson SG

Both neutral and acidic polymers have been isolated from the lipopolysaccharide extract of the reference strain (C.D.C. 4523-60) for Serratia marcescens serogroup O15. By means of n.m.r. spectroscopy, methylation analysis, and studies of degradation products, the acidic polysaccharide was shown to have a branched pentasaccharide repeating-unit with the following structure. (Formula: see text)

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Carbohydrate research, 186, 0008-6215, , 1989

PMID:2660988

Structural studies of a neutral polymer (the putative O10 antigen) isolated from the lipopolysaccharide of Serratia marcescens strain C.D.C. 1287-54 (O10:H8).
Oxley D,Wilkinson SG

A neutral glucorhamnan has been isolated from the lipopolysaccharide of the O10 reference strain (C.D.C. 1287-54) of Serratia marcescens. By means of n.m.r. spectroscopy, methylation analysis, and degradative studies, the polymer (the putative O-specific antigen) was found to have the branched, pentasaccharide repeating-unit shown. (formula; see text).

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Carbohydrate research, 187, 0008-6215, , 1989

PMID:2472885

Structural studies of an acidic galactoglucomannan from the O3 reference strain (C.D.C. 863-57) of Serratia marcescens.
Oxley D,Wilkinson SG

A partially acetylated acidic galactoglucomannan has been isolated from the lipopolysaccharide of the O3 reference strain (C.D.C. 863-57) of Serratia marcescens. By means of n.m.r. spectroscopy, methylation analysis, and degradative studies, the polymer was found to have the branched pentasaccharide repeating-unit shown. The position(s) of partial acetylation were not determined. Although the polymer is believed to confer O specificity on the parent organism, it is probably not an integral component of the lipopolysaccharide. (Formula: see text).

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Carbohydrate research, 187, 0008-6215, , 1989

PMID:2472884

Structure of the O-specific polysaccharide from the lipopolysaccharide of Serratia marcescens O8.
Oxley D,Wilkinson SG

Structural studies have been carried out on the O-specific polysaccharide from the lipopolysaccharide of the reference strain (CDC 1604-55) for serogroup O8 of Serratia marcescens. The polymer has a branched, tetrasaccharide repeating unit of D-galactose(Gal),D-glucose(Glc), and 2-acetamido-2-deoxy-D-glucose(GlcNAc) with the following structure: (Formula: see text). The anomeric configuration assigned to the glucose residue differs from that (beta) previously proposed [Tarcsay, L., Wang, C. S., Li, S.-C. and Alaupovic, P. (1973) Biochemistry 12, 1948-1955]. The structure of the O8 polymer is identical with that of one of two polymers present in the cell envelope of a strain (CDC 1783-57) of S. marcescens O14.

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European journal of biochemistry / FEBS, 156, 0014-2956, , 1986

PMID:2422032

Structure of a neutral glycan isolated from the lipopolysaccharide of the reference strain for Serratia marcescens serogroup O22.
Oxley D,Wilkinson SG

Both a neutral and an acidic polymer have been isolated from a lipopolysaccharide extract of the reference strain for Serratia marcescens serogroup O22. The neutral polymer has a linear structure with the repeating unit shown. The same tetrasaccharide unit also forms the backbone of the branched neutral polymer isolated from the reference strain for serogroup O10, which cross-reacts strongly with O22. ----2)-alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----3)-alpha-L-+ ++Rhap-(1----3)-alpha- D-GlcpNAc-(1----

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Carbohydrate research, 203, 0008-6215, , 1990

PMID:2276124

A common structure for neutral polymers isolated from the lipopolysaccharides of reference strains for Serratia marcescens serogroups O17 and O19.
Oxley D,Wilkinson SG

Carbohydrate research, 198, 0008-6215, , 1990

PMID:2191776

Structural studies of acidic polymers produced by the O23 reference strain of Serratia marcescens: presence of amide-linked glutamic acid.
Oxley D,Wilkinson SG

The major fraction of an acidic galactoglucomannan present in lipopolysaccharide extracts from cell walls of the O23 reference strain of Serratia marcescens has the tetrasaccharide repeating-unit shown. In a minor fraction, L-glutamic acid was amide-linked to about half of the D-glucuronic acid residues. The possible contributions of the acidic polymers and a neutral polymer produced by the organism to cross-reactions with other serogroups are discussed.

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Carbohydrate research, 204, 0008-6215, , 1990

PMID:1980630

Structure of a mannan isolated from the lipopolysaccharide of the reference strain (S3255) for a new serogroup of Serratia marcescens.
Oxley D,Wilkinson SG

Both a neutral and an acidic polymer have been isolated from a lipopolysaccharide extract of Serratia marcescens strain S3255. The neutral polymer is a linear mannan with the repeating unit shown. The same repeating unit has been described for the O-specific polymers from Escherichia coli O8 and Klebsiella O5.

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Carbohydrate research, 212, 0008-6215, , 1991

PMID:1959117

Structure of an acidic glycan present in the lipopolysaccharide extract from the reference strain for Serratia marcescens serogroup O18.
Oxley D,Wilkinson SG

The lipopolysaccharide extract from the cell wall of the reference strain for Serratia marcescens serogroup O18 contained, in addition to a neutral glycan characterised previously, an acidic glycan. Acidity was contributed both by D-glucuronic acid and by 4-O-[(R)-1-carboxyethyl]-D-glucose (4-O-Lac-D-Glc). By using n.m.r. spectroscopy, methylation analysis, and chemical degradations, the repeating unit of the acidic glycan was identified as a branched hexasaccharide having the structure shown; an O-acetyl group also present was not located. The glycan is believed to define the O18 serogroup, but is probably not an integral component of the lipopolysaccharide. [formula: see text].

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Carbohydrate research, 215, 0008-6215, , 1991

PMID:1794127

Structure of the 021 antigen from Serratia marcescens.
Oxley D,Wilkinson SG

Lipopolysaccharide was isolated from both phases of an aqueous-phenol extraction of defatted cell walls from the reference strain for Serratia marcescens serogroup 021. The product from the aqueous phase was of the R type, lacking a polymeric side-chain. The polymeric fraction of the lipopolysaccharide from the phenolic phase (the 021 antigen) had a disaccharide repeating-unit with the following structure: ----4)-alpha-D-Glcp-(1----4)-beta-D-ManpNAc-(1----.

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Carbohydrate research, 212, 0008-6215, , 1991

PMID:1720344

Structure of a glucorhamnan from the lipopolysaccharide of Serratia marcescens strain S1254.
Oxley D,Wilkinson SG

Carbohydrate research, 209, 0008-6215, , 1991

PMID:1709820

Structure of the putative O23 antigen of Serratia marcescens.
Oxley D,Wilkinson SG

A neutral glycan containing L-rhamnose and 2-acetamido-2-deoxy-D-galactose is one of two polymers present in the lipopolysaccharide extract from the reference strain for Serratia marcescens serogroup O23. The glycan, which has the disaccharide repeating-unit shown, shares structural features with polymers from several other O serogroups. ----4)-alpha-L-Rhap-(1----4)-beta-D-GalpNAc-(1----.

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Carbohydrate research, 196, 0008-6215, , 1990

PMID:1693310

Structure of an acidic glycan from the reference strain for Serratia marcescens serogroup O22.
Oxley D,Wilkinson SG

In addition to a neutral glycan, lipopolysaccharide extracts from the reference strain for Serratia marcescens serogroup O22 contain an acidic polymer which probably defines the serogroup and is of microcapsular origin. The polymer is doubly branched with a heptasaccharide repeating unit and a galactan backbone. By means of spectroscopic and degradative studies, the structure of the repeating unit was established as that shown. [formula: see text]

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Carbohydrate research, 231, 0008-6215, , 1992

PMID:1394317

Structure of a neutral glycan from the lipopolysaccharides of reference strains for Serratia marcescens serogroups O2 and O3.
Oxley D,Wilkinson SG

Serogroups O2 and O3 of Serratia marcescens are differentiated by acidic glycans present in the aqueous phase when lipopolysaccharides are extracted from the reference strains by the aqueous-phenol method. The phenolic phases of these extracts from both strains also contain lipopolysaccharides, from which the same neutral glycan is released on milk acid hydrolysis. The neutral glycan has the disaccharide repeating-unit shown, and accounts for the cross-reactions between the two serogroups and also with serogroup O21: --> 4)-alpha-D-GlcpNAc-(1-->4)-beta-D-ManpNAc-(1--.

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FEMS microbiology letters, 78, 0378-1097, , 1992

PMID:1283379