Major Recent Grants

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Major Recent Grants

  1. 1. Department of Physiology NEWSLETTER, Sept, 2003 Head of Department However the recent award, together with My term of office as Head of Department other awards earlier in the session, mean ends on 30th Sept. I would like to take this that members of the Department have opportunity to thank all the colleagues in collectively been awarded nearly £4M from the Department for their support and help Research Council sources in the year in making my job a stimulating and 2002-3. This is the clearest possible enjoyable one over the last five years. The statement of the continued Department was in excellent shape when I competitiveness of the research efforts in took over from Ole Petersen in October the Department. 1998, and since then I believe our trajectory has been maintained. We have In addition, other members of the had major successes in Teaching Quality Department have had success in research Assessment (1999 – rated Excellent) and grant applications. Ian Prior has been in the Research Assessment Exercise awarded a prestigious Royal Society (2001 – rated 5**); many major grants University Research Fellowship. Andrea have been renewed (see also below) and Varro, Mark Pritchard (Medicine) and important new ones awarded; moreover, myself have attracted a NWCRF project the Department has expanded its estate grant (£116,101), Bob Burgoyne was (the Robert’s Wing) and has grown in size awarded a Wellcome Trust Prize (new academic appointments and Fellowship (£41,431) to support Dermott sustained expansions of our post- and O’Callaghan, and Judy Coulson has undergraduate programmes) while received a NWCRF project grant maintaining quality. All of this reflects huge (£36,273). effort at every level of the Department. It has been an honour as well as a personal Rod Dimaline joins Wellcome Trust pleasure to see at first hand the Panel commitment of many members of the I am pleased to report that Rod Dimaline Department, and to be able to report them has accepted an invitation from the in Departmental Newsletters. Bob Wellcome Trust to join the Physiology & Burgoyne will be taking over as Head of Pharmacology Panel. This is an important Department from 1st October 2003. I am recognition of the esteem in which Rod is certain that Bob will be an excellent leader held and comes after he has served a full and I am delighted to offer him my term as a member of the MRC Advisory congratulations on his appointment, and Board. my continued support as a member of the Department. Sue Wray joins Academy of Medical Sciences Sectional Committee 2 Major New Grants The Academy of Medical Sciences has There have been a number of important invited Sue Wray to serve on its Sectional items of good news since the last Committee 2 which considers candidates Newsletter. Bob Burgoyne was telephoned for election to the Academy in the by the Wellcome Trust last week to Physiology, Pharmacology, Neuroscience confirm that our 4-year PhD programme area. I am delighted to congratulate Sue has been renewed for another five years. on this appointment. Bob prepared our submission for the renewal of this programme and it is a great Papers in high impact journals pleasure to be able to acknowledge his Members of the Department have immense contribution in seeing the continued to publish in high impact factor renewal process through to a highly peer-reviewed journals since the last satisfactory conclusion. Newsletter. Many recent successes are I am also pleased to report the renewal by set out on the following pages. The MRC of the Programme Grant held by Rod Dimaline, Andrea Varro and myself. The abstract and further details can be Graham Dockray, Sept 2003 found on the attached pages. It is, of course, well known that competition for Research Council funding has been especially intense in the last two sessions.
  2. 2. Major Recent Publications Mol. Cell (2003) 11, 1685-1692 (Impact Factor 16.47) Suppression of homologous recombination by the Saccharomyces cerevisiae linker histone. Downs JA, Kosmidou E, Morgan A. and Jackson SP. The basic unit of chromatin in eukaryotes is the nucleosome, comprising 146 bp of DNA wound around two copies of each of four core histones. Chromatin is further condensed by association with linker histones. Saccharomyces cerevisiae Hho1p has sequence homology to other known linker histones and interacts with nucleosomes in vitro. However, disruption of HHO1 results in no significant changes in the phenotypes examined thus far. Here, we show that Hho1p is inhibitory to DNA repair by homologous recombination (HR). We find Hho1p is abundant and associated with the genome, consistent with a global role in DNA repair. Furthermore, we establish that Hho1p is required for a full life span and propose that this is mechanistically linked to its role in HR. Finally, we show that Hho1p is inhibitory to the recombination-dependent mechanism of telomere maintenance. The role of linker histones in genome stability, aging, and tumorigenesis is discussed. J.Clin.Invest., in press (2003) (Impact Factor: 14.05) Severe small intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency. Jackson RS, Creemers JWM, Farooqi IS, Raffin-Sanson ML, Varro A, Dockray GJ, Holst JJ, Brubaker PL, Corvol P, Polonsky KS, Ostrega D, Becker KL, Bertagna X, Hutton JC, White A, Dattani M.T, Hussain K, Middleton SJ, Nicholl TM, Lindley KJ, O’Rahilly S. We have previously described the only reported case of human proprotein convertase 1 deficiency in a female (Subject A) who presented with obesity, hypogonadism, hypoadrenalism and reactive hypoglycemia. We now report the second case of human PC1 deficiency (Subject B), also due to compound heterozygosity for novel missense and nonsense mutations. While both subjects shared the phenotypes of obesity, hypoadrenalism, reactive hypoglycemia and massively elevated circulating levels of certain prohormones, the clinical presentation of Subject B was dominated by severe refractory neonatal diarrhea, malabsorptive in type. Subsequent investigation of Subject A revealed marked small intestinal absorptive dysfunction, which was not previously clinically suspected. Thus, PC1, presumably in the enteroendocrine cells, is essential for the normal absorptive function of the human small intestine. The differences in the nature and severity of presentation between the two cases cannot readily be explained on the basis of allelic heterogeneity as the nonsense and missense mutations from both subjects had comparably severe effects on the catalytic activity of PC1. Despite Subject A’s negligible PC1 activity some mature adrenocorticotropin and GLP-17-36amide were detectable in her plasma suggesting that the production of these hormones, at least in humans, does not have an absolute dependence on PC1. The presence of severe obesity and the absence of growth retardation in both subjects contrast markedly with mice lacking PC1 and suggest that the precise physiological repertoire of this enzyme may vary between mammalian species. Gastroenterology (2003) Vol 125: 510-521 (Impat Factor 13.44) Transcriptional Regulation of the Human Trefoil Factor, TFF1, by Gastrin. Khan ZE, Wang TC, Cui G, Chi AL and Dimaline R. Background & Aims. This study aimed to identify gastrin-sensitive genes that may mediate the effects of this hormone on gastric epithelial architecture. Methods: Gastrin-sensitive genes were identified by mRNA differential display of gastric fundus from gastrin-deficient (GAS-KO) or wild type mice. Gastrin-stimulated expression of the trefoil peptide TFF1 in mouse fundus and in the gastric cancer cell line AGS-GR was determined by Northern blot and real time PCR. Transcriptional regulation of TFF1 in AGS- GR cells was studied using promoter-reporter assays and EMSA. Expression of TFF1 and CCKB receptor in response to gastric mucosal injury was determined by immunohistochemistry. Results: mRNA differential display identified TFF1 as a gastrin- regulated gene. TFF1 mRNA was reversibly reduced in GAS-KO mice and increased in a hypergastrinemic transgenic strain (INS-GAS) versus respective background strains. TFF1
  3. 3. mRNA expression was rapidly and potently induced by gastrin in a gastric cancer cell line that expresses the gastrin/CCKB receptor. Gastrin responsiveness of the human TFF1 promoter mapped to a G-C rich region 300bp upstream of the transcriptional start site. This region bound the transcription factors SP3 and MAZ. Gastrin activated transcription through a Raf-, Mek- and Erk-dependent but Ras-independent pathway. TFF1 expression was induced both directly and by transactivation between neighboring cells. Neither direct nor indirect gastrin- induced TFF1 expression required activation of the EGF receptor. Conclusions: Gastrin exerts tonic control of TFF1 expression, but also has the potential for rapid up-regulation of this trefoil factor. TFF1 is a potential candidate to counterbalance the proliferative effects of gastrin. J. Cell Biol. (2003) 160: 165-70. (Impact Factor: 12.52) Direct visualization of Ras proteins in spatially distinct cell surface microdomains. Prior IA, Muncke C, Parton RG and Hancock JF. Localization of signaling complexes to specific microdomains co-ordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44nm that occupy 35% of the cell surface. Cross- linking an outer leaflet raft protein results in the redistribution of inner leaflet rafts but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in non-overlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these non-raft microdomains, whereas K-ras clustering is supported by farnesylation but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins. Human Molecular Genetics (2003) 12, 1415-1435 (Impact factor 9.31) IL1 receptor accessory protein like, a protein involved in X-linked mental retardation, interacts with Neuronal Calcium Sensor-1 and regulates exocytosis. Bahi N, Friocourt G, Carrie A, Graham ME, Weiss JL, Chafet P, Fauchereau F, Burgoyne RD and Chelley J. Previously, human genetics-based approaches allowed us to show that mutations in the IL-1 receptor accessory protein-like gene (IL1RAPL) are responsible for a non-specific form of X- linked mental retardation. This gene encodes a predicted protein of 696 amino acids that belongs to a novel class of the IL-1/Toll receptor family. In addition to the extracellular portion consisting of three Ig-like domains and the intracellular TIR domain characteristic of the IL-1/Toll receptor family, IL1RAPL contains a specific 150 amino acid carboxy terminus that has no significant homology with any protein of known function. In order to begin to elucidate the function of this IL-1/Toll receptor-like protein, we have assessed the effect of recombinant IL1RAPL on the binding affinity of type I IL-1R for its ligands IL-1a and b and searched for proteins interacting with the specific carboxy terminus domain of IL1RAPL. Our results show that IL1RAPL is not a protein receptor for IL-1. In addition we present here the identification of Neuronal Calcium Sensor-1 (NCS-1) as an IL1RAPL interactor. Remarkably, although NCS-1 and its non-mammalian homologue, frequenin, are members of a highly conserved EF-hand Ca2+ binding protein family, our data show that IL1RAPL interacts only with NCS-1 through its specific C-terminal domain. The functional relevance of IL1RAPL activity was further supported by the inhibitory effect on exocytosis in PC12 cells overexpressing IL1RAPL. Taken together, our data suggest that IL1RAPL may regulate calcium-dependent exocytosis and provide insight into the understanding of physiopathological mechanisms underlying cognitive impairment resulting from IL1RAPL dysfunction.
  4. 4. Journal of Cell Science (2003) 116, 4833-4845 (Impact factor 6.95) Residues within the myristoylation motif determine intracellular targeting of the neuronal Ca2+ sensor protein KChIP1 to post-ER transport vesicles and traffic of Kv4 K+ channels. O’Callaghan DW*, Hasdemir B*, Leighton M and Burgoyne RD (* equal first authors) KChIPs (K+ channel interacting proteins) regulate the function of A-type Kv4 potassium channels by modifying channel properties and by increasing their cell surface expression. We have explored factors affecting the localization of Kv4.2 and the targeting of KChIP1 and other NCS proteins by using GFP-variant fusion proteins expressed in HeLa cells. Kv4.2- ECFP expressed alone was not retained in the ER but reached the Golgi complex. In cells co- expressing ECFP-Kv4.2 and KChIP1-EYFP, the two proteins were co-localized and were mainly present on the plasma membrane. When KChIP1-EYFP was expressed alone it was instead targeted to punctate structures. This was distinct from the localization of the NCS proteins NCS-1 and hippocalcin, which were targeted, to the trans-Golgi network (TGN) and plasma membrane. The membrane localization of each NCS protein required myristoylation and minimal myristoylation motifs of hippocalcin or KChIP1 were sufficient to target fusion proteins to either TGN/plasma membrane or to punctate structures. The existence of targeting information within the N-terminal motifs was confirmed by mutagenesis of residues corresponding to three conserved basic amino acids in hippocalcin and NCS-1 at positions 3, 7 and 9. Residues at these positions determined intracellular targeting to the different organelles. Myristoylation and correct targeting of KChIP1 was required for the efficient traffic of Kv4.2-ECFP to the plasma membrane. Expression of KChIP1(1-11)-EYFP resulted in the formation of enlarged structures which were positive for ERGIC-53 and r -COP. ECFP-Kv4.2 was also accumulated in these structures suggesting that KChIP1(1-11)-EYFP inhibited traffic out of the ERGIC. We suggest that KChIP1 is targeted by its myristoylation motif to post-ER transport vesicles where it could interact with and regulate the traffic of Kv4 channels to the plasma membrane under the influence of localized Ca2+ signals. J.Cell Sci 116, 4169-4179 (2003) (Impact factor 6.95) The UIM domain of Hrs couples receptor sorting to vesicle formation. Urbé S, Sachse M, Row PE, Preisinger C, Barr FA, Strous G, Klumperman J, Clague MJ. Hepatocyte growth factor regulated tyrosine kinase substrate (Hrs), a main component of the "bilayered" clathrin coat on sorting endosomes, was originally identified as a substrate of activated tyrosine kinase receptors. We have analysed Hrs phosphorylation in response to Epidermal Growth Factor (EGF) stimulation and show that the evolutionary conserved tyrosines Y329 and Y334 provide the principal phosphorylation sites. Hrs is proposed to concentrate ubiquitinated receptors within clathrin-coated regions via direct interaction with its UIM (Ubiquitin Interaction Motif) domain. We show that the same UIM domain is necessary for EGF-stimulated tyrosine phosphorylation of Hrs. Over-expression of wild type Hrs or a double mutant, Y329/334F, defective in EGF-dependent phosphorylation, both substantially retard EGF-receptor (EGFR) degradation by inhibiting internal vesicle formation and thereby preventing EGFR incorporation into lumenal vesicles of the multivesicular bodies. In contrast, mutation or deletion of the Hrs-UIM domain strongly suppresses this effect. In addition the UIM-deletion and point mutants are also observed on internal membranes, indicating a failure to dissociate from the endosomal membrane prior to incorporation of the receptor complex into lumenal vesicles. Our data suggest a role for the UIM-domain of Hrs in actively retaining EGFR at the limiting membrane of endosomes as a prelude to lumenal vesicle formation. J. Biol. Chem. (2003) April 13 issue (Impact Factor: 6.69) Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane. Wyse BD, Prior IA, Qian, H, Morrow, IC, Nixon S, Muncke, C, Kurzchalia TV, Thomas WG, Parton RG and Hancock JF. The mechanisms involved in angiotensin II (AngII) type 1 receptor (AT1-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT1-R is not concentrated in caveolae, but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT1-R in caveolae, AT1-R/ caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain (CSD) because mutant caveolins
  5. 5. that lack a functional CSD do not interact with AT1-R. Expression of an N-terminally truncated caveolin-3, CavDGV that localizes to lipid bodies, or a point mutant Cav3-P104L that accumulates in the Golgi, mislocalize AT1-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT1-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin binding site abrogates AT1-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT1-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT1-R levels compared to controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT1-R through the exocytic pathway, but this does not result in AT1-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT1-R. Gut, in press, (2003) (Impact Factor 6.32) Keeping neuroendocrine cells in check: roles for TGFβ, Smads and menin? Dockray GJ The endocrine cells of the gastrointestinal epithelium sense the luminal contents and through secretions at their basolateral side signal both to other epithelial cells and to sub-epithelial cells including smooth muscle, neurones and inflammatory cells (1). Some of the features of these cells are clearly neurone-like and for a time it was thought that during development they might be derived, like enteric neurones, from the neural crest. This now seems unlikely, and instead it is thought that normally they arise from the pluripotent stem cells that also give rise to the other epithelial cell lineages (2). However, in some circumstances at least, these cells appear to have the capacity for proliferation, and in extreme cases this gives rise to tumours that are called “neuroendocrine” since they exhibit some of the features of neurones and endocrine cells. There are many similarities between neuroendocrine tumours of the gastrointestinal tract and pancreas. In general these tumours grow slowly and the reasons for this are unknown. Wimmel et al. now present evidence that transforming growth factor (TGF)β is produced by neuroendocrine tumours and through autocrine and paracrine mechanisms restrains tumour cell proliferation (3). Sciences STKE (2003) pl9. Observing cell surface signaling domains using electron microscopy Prior IA, Parton RG, Hancock JF. The plasma membrane is made up of a complex mosaic of different functional microdomains but the tools required to accurately visualize them have been limited. We present a protocol that allows both inner and outer leaflet domains to be visualized on a nanometer scale. By using a combined electron microscopic-statistical analysis approach it is possible to screen proteins associating with, and co-localizing within, lipid rafts and other morphologically featureless microdomains. The approach has enormous potential to determine plasma membrane organization, and the spatial dynamics of regulated signaling and membrane trafficking events associated with the cell surface. Major Recent Grants MRC Programme Grant, £1,121, 924 (2003-2008). The endocrinology of the upper gastrointestinal tract Dockray GJ, Dimaline R, Varro A Diseases of the upper gastrointestinal tract including gastric cancer and acid-peptic disorders are a major burden on health and a major cost to health services. The hormone gastrin acutely regulates acid secretion, and determines the capacity for acid secretion by regulating parietal cell maturation. Recent work suggests an unexpectedly diverse family of active products generated from the gastrin gene. Moreover, a number of new target genes have been identified that are regulated by gastrin and that may be involved in mucosal organisation. We propose experimental studies (a) to define the physiological role in the stomach of peptides generated from the gastrin precursor, (b) to determine the mechanisms by which gastrin induces gene expression in primary cells, and (c) to characterise endocrine and paracrine regulated events in the gastric mucosa that determine mucosal organisation.
  6. 6. Royal Society Fellowship, £186,370 + £11,000pa consumables (5 years) Nanoscale characterisation of cell surface signalling domains Prior I The fellowship will allow me to continue my work investigating the mechanisms whereby cell surface and intracellular microdomains regulate signalling. I am developing new electron microscopy techniques to study protein-protein interactions and protein S-nitrosylation and applying them to studies of RTK-Ras-Raf-MAPK signalling and also regulation of endothelial nitric oxide synthase signalling and trafficking. North West Cancer Research Fund, project grant, £116,101 (2003-2006) The role of MMP-7 in the pathogenesis of gastric cancer Varro A, Dockray GJ, Pritchard DM (Medicine). Gastric cancer is the second commonest cause of death due to malignancy worldwide. There is a clear association between infection with the gastric bacterium H.pylori and gastric adenocarcinoma. Our recent work indicates that H.pylori infection increases expression of matrix metalloproteinase (MMP)-7, or matrilysin, in the gastric epithelium and that this leads to increased cell migration. Pilot studies also indicate that the hormone gastrin, which is elevated with H.pylori infection may stimulate MMP-7 expression. We propose to test the specific hypothesis that H.pylori and gastrin interact to regulate MMP-7 expression and that MMP-7 in turn liberates paracrine factors that influence the proliferation, migration and apoptosis of both epithelial cells and gastric myofibroblasts leading to the development of premalignant changes in the gastric mucosa. Wellcome Trust Prize Fellowship, £44,431 (1 year) Mechanisms underlying the intracellular targeting of neuronal calcium sensor proteins Dermott O.Callaghan Changes in the concentration of cytosolic free Ca 2+ regulates many different physiological process. The effect of Ca2+ are transduced through its binding to Ca2+ sensor proteins of which the most studied is calmodulin. In neurons Ca2+ is the signal for neurotransmitter release and also for the regulation of many aspects of neuronal function linked to neuronal plasticity ranging from rapid changes in ion channel activity to long-term changes in gene expression. Amongst the Ca2+-binding proteins that may mediate these effects are the neuronal Ca2+ sensor (NCS) proteins which have recently become a focus for study. They include NCS-1 (frequenin), the VILIPs (neurocalcins), KChIPs, recoverin and the GCAPs that have a diverse array of functions. NCS-1 is involved in the regulation of neurotransmitter release, learning, channel function and activation of phosphotidylinositol-4-kinase. The KChIPs were identified as regulators of A-type K channels. This project derives from the finding made by Dermott O’Callaghan during his Ph.D. project that different members of the neuronal calcium sensor (NCS) protein family localise to distinct organelles when expressed in HeLa cells as GFP-fusion proteins. NCS-1, hippocalcin and neurocalcinl are associated with or translocate to the trans-Golgi network. In contrast KChIP1 is associated with distinct organelles identified as post-ER transport vesicles. These different localizations could be functionally important in determining the spatial nature of the calcium signals recognised by these proteins. Membrane association of all of the proteins requires N-terminal myristoylation and the myristoylation motif is sufficient for localization. Additional characterisation of these motifs led to the discovery that the exact amino acid sequence within the 11-14 amino acid motif required for myristoylation determines the intracellular targeting of these proteins. The aim of the project is to determine the molecular basis for this differential localisation, in particular to find out whether this is determined by distinct interactions with proteins or with different lipids NWCRF, £36,273 (one year pilot project) Expression and function of neural-specific stathmins in lung cancer Coulson JM There are four stathmin family members, which destabilise microtubules playing a key role in cell division and neuronal axon growth. Stathmin-1 is ubiquitous, but has been associated with certain tumours including breast cancer. Stathmins 2-4 are neural-specific and we have shown for the first time that stathmin-2 is highly expressed in neuroendocrine small cell lung cancer (SCLC), but not non-SCLC cell lines. We propose to study expression and function of
  7. 7. the neural-specific stathmins in lung cancer. Expression will be determined at the mRNA and protein levels in cell lines and compared to other tumour types, findings will be validated in clinical material. Interference RNA vectors will be used to knock down expression of specific stathmins and the effects on microtubule dynamics, cell morphology, proliferation and signalling will be studied. The relevance of findings to chemotherapy drugs that target microtubules and are used for lung cancer (paclitaxel and vinblastine) will be investigated in vitro.

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