• Protein kinase C delta is phosphorylated on five novel Ser/Thr sites following inducible overexpression in human colorectal cancer cells

      Welman, Arkadiusz; Griffiths, John R; Whetton, Anthony D; Dive, Caroline; Cancer Research UK, Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom. awelman@picr.man.ac.uk (2007-12)
      Phosphorylation plays an important role in regulation of protein kinase C delta (PKCdelta). To date, three Ser/Thr residues (Thr 505, Ser 643, and Ser 662) and nine tyrosine residues (Tyr 52, Tyr 64, Tyr 155, Tyr 187, Tyr 311, Tyr 332, Tyr 512, Tyr 523, and Tyr 565) have been defined as regulatory phosphorylation sites for this protein (rat PKCdelta numbering). We combined doxycycline-regulated inducible gene expression technology with a hypothesis-driven mass spectrometry approach to study PKCdelta phosphorylation pattern in colorectal cancer cells. We report identification of five novel Ser/Thr phosphorylation sites: Thr 50, Thr 141, Ser 304, Thr 451, and Ser 506 (human PKCdelta numbering) following overexpression of PKCdelta in HCT116 human colon carcinoma cells grown in standard tissue culture conditions. Identification of potential novel phosphorylation sites will affect further functional studies of this protein, and may introduce additional complexity to PKCdelta signaling.
    • Protein kinase cδ deficiency causes mendelian systemic lupus erythematosus with B cell-defective apoptosis and hyperproliferation.

      Belot, A; Kasher, P; Trotter, Eleanor W; Foray, A-P; Debaud, A-L; Rice, G; Szynkiewicz, M; Zabot, M-T; Rouvet, I; Bhaskar, S; et al. (2013-08)
      Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that is assumed to occur via a complex interplay of environmental and genetic factors. Rare causes of monogenic SLE have been described, providing unique insights into fundamental mechanisms of immune tolerance. The aim of this study was to identify the cause of an autosomal-recessive form of SLE.
    • Protein kinase signaling networks in cancer.

      Brognard, J; Hunter, Tony; Signalling Networks in Cancer Group, Cancer Research UK, Paterson Institute for Cancer Research, The University of Manchester, Manchester, UK. (2010-11-29)
      Protein kinases orchestrate the activation of signaling cascades in response to extracellular and intracellular stimuli to control cell growth, proliferation, and survival. The complexity of numerous intracellular signaling pathways is highlighted by the number of kinases encoded by the human genome (539) and the plethora of phosphorylation sites identified in phosphoproteomic studies. Perturbation of these signaling networks by mutations or abnormal protein expression underlies the cause of many diseases including cancer. Recent RNAi screens and cancer genomic sequencing studies have revealed that many more kinases than anticipated contribute to tumorigenesis and are potential targets for inhibitor drug development intervention. This review will highlight recent insights into known pathways essential for tumorigenesis and discuss exciting new pathways for therapeutic intervention.
    • Protein Z: a putative novel biomarker for early detection of ovarian cancer

      Russell, M; Walker, M; Williamson, A; Gentry-Maharaj, A; Ryan, A; Kalsi, J; Skates, S; D'Amato, A; Dive, Caroline; Pernemalm, M; et al. (2016-01-27)
      Ovarian cancer (OC) has the highest mortality of all gynaecological cancers. Early diagnosis offers an approach to achieving better outcomes. We conducted a blinded-evaluation of prospectively collected preclinical serum from participants in the multimodal group of the United Kingdom Collaborative Trial of Ovarian Cancer Screening. Using isobaric tags (iTRAQ) we identified 90 proteins differentially expressed between OC cases and controls. A second targeted mass spectrometry analysis of twenty of these candidates identified Protein Z as a potential early detection biomarker for OC. This was further validated by ELISA analysis in 482 serial serum samples, from 80 individuals, 49 OC cases and 31 controls, spanning up to 7 years prior to diagnosis. Protein Z was significantly down-regulated up to 2 years pre-diagnosis (p = 0.000000411) in 8 of 19 Type I patients whilst in 5 Type II individuals, it was significantly up-regulated up to 4 years before diagnosis (p=0.01). ROC curve analysis for CA-125 and CA-125 combined with Protein Z showed a statistically significant (p= 0.00033) increase in the AUC from 77% to 81% for Type I and a statistically significant (p= 0.00003) increase in the AUC from 76% to 82% for Type II. Protein Z is a novel independent early detection biomarker for Type I and Type II ovarian cancer; which can discriminate between both types. Protein Z also adds to CA-125 and potentially the Risk of Ovarian Cancer algorithm in the detection of both subtypes. This article is protected by copyright. All rights reserved. © 2014 Wiley Periodicals, Inc.
    • Proteoglycan-specific molecular switch for RPTPσ clustering and neuronal extension.

      Coles, Charlotte H; Shen, Yingjie; Tenney, Alan P; Siebold, Christian; Sutton, Geoffrey C; Lu, Weixian; Gallagher, John T; Jones, E Yvonne; Flanagan, John G; Aricescu, A Radu; et al. (2011-04-22)
      Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) regulate numerous cell surface signaling events, with typically opposite effects on cell function. CSPGs inhibit nerve regeneration through receptor protein tyrosine phosphatase sigma (RPTPσ). Here we report that RPTPσ acts bimodally in sensory neuron extension, mediating CSPG inhibition and HSPG growth promotion. Crystallographic analyses of a shared HSPG-CSPG binding site reveal a conformational plasticity that can accommodate diverse glycosaminoglycans with comparable affinities. Heparan sulfate and analogs induced RPTPσ ectodomain oligomerization in solution, which was inhibited by chondroitin sulfate. RPTPσ and HSPGs colocalize in puncta on sensory neurons in culture, whereas CSPGs occupy the extracellular matrix. These results lead to a model where proteoglycans can exert opposing effects on neuronal extension by competing to control the oligomerization of a common receptor.
    • Proteoglycans in cellular differentiation and neoplasia.

      Gallagher, John T; Hampson, Ian N (1984-06)
    • Proteoglycans: a regulatory role in the proliferation of intestinal epithelia.

      Flint, Neil; Evans, Gareth S; Cove, Frances L; CRC Department of Epithelial Biology, Paterson Institute, Christie Hospital, Manchester. (1992-05)
    • Proteoglycans: pericellular and cell surface multireceptors that integrate external stimuli in the mammary gland.

      Delehedde, Maryse; Lyon, Malcolm; Sergeant, Nicolas; Rahmoune, Hassan; Fernig, David G; School of Biological Sciences, University of Liverpool, United Kingdom. (2001-07)
      Proteoglycans consist of a core protein and an associated glycosaminoglycan (GAG) chain of heparan sulfate, chondroitin sulfate, dermatan sulfate or keratan sulfate, which are attached to a serine residue. The core proteins of cell surface proteoglycans may be transmembrane, e.g., syndecan, or GPI-anchored, e.g., glypican. Many different cell surface and matrix proteoglycan core proteins are expressed in the mammary gland and in mammary cells in culture. The level of expression of these core proteins, the structure of their GAG chains, and their degradation are regulated by many of the effectors that control the development and function of the mammary gland. Regulatory proteins of the mammary gland that bind GAG include many growth factors and morphogens (fibroblast growth factors, hepatocyte growth factor/scatter factor, members of the midkine family, wnts), matrix proteins (collagen, fibronectin, and laminin), enzymes (lipoprotein lipase) and microbial surface proteins. Structural diversity within GAG chains ensures that each protein-GAG interaction is as specific as necessary and a number of sequences of saccharides that recognize individual proteins have been elucidated. The GAG-protein interactions serve to regulate the signal output of growth factor receptor tyrosine kinase and hence cell fate as well as the storage and diffusion of extracellular protein effectors. In addition, GAGs clearly coordinate stromal and epithelial development, and they are active participants in mediating cell-cell and cell-matrix interactions. Since a single proteoglycan, even if it carries a single GAG chain, can bind multiple proteins, proteoglycans are also likely to act as multireceptors which promote the integration of cellular signals.
    • Proteome biology of stem cells: a new joint HUPO and ISSCR initiative.

      Krijgsveld, Jeroen; Whetton, Anthony D; Lee, Bong Hee; Lemischka, Ihor; Oh, Steve; Pera, Martin; Mummery, Christine; Heck, Albert J R; Department of Biomolecular Mass Spectrometry, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. j.krijgsveld@uu.nl (2008-01)
    • Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors.

      Marei, Hadir; Carpy, A; Macek, B; Malliri, Angeliki; Cell Signaling Group, Cancer Research UK Manchester Institute, The University of Manchester , Manchester , UK (2016-05-06)
      The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects.
    • Proteomic analysis reveals a novel mechanism induced by the leukemic oncogene Tel/PDGFRβ in stem cells: activation of the interferon response pathways.

      Dobbin, E; Graham, C; Freeburn, R; Unwin, Richard D; Griffiths, John R; Pierce, Andrew; Whetton, Anthony D; Wheadon, H; Paul O'Gorman Leukaemia Research Centre, University of Glasgow, Glasgow, G12 0YN, UK. (2010-11)
      Objective proteomic analysis offers opportunities for hypothesis generation on molecular events associated with pathogenesis in stem cells. Relative quantification mass spectrometry was employed to identify pathways affected by Tel/PDGFRβ, an oncogene associated with myeloproliferative neoplasia (MPN). Its effects on over 1800 proteins were quantified with high confidence. Of those up-regulated by Tel/PDGFRβ several were involved in the interferon gamma (IFNγ) response. To validate these observations we employed embryonic and myeloid stem cells models which revealed Tel/PDGFRβ-induced STAT1 up-regulation and activation was responsible for modulating the interferon response. A STAT1 target highly up-regulated was ICSBP, a transcriptional regulator of myeloid and eosinophilic differentiation. ICSBP interacts with CBP/p300 and Ets transcription factors, to promote transcription of additional genes, including the Egr family, key regulators of myelopoiesis. These interferon responses were recapitulated using IFNγ stimulation of stem cells. Thus Tel/PDGFRβ induces aberrant IFN signaling and downstream targets, which may ultimately impact the hematopoietic transcriptional factor network to bias myelomonocytic differentiation in this MPN.
    • Proteomic identification of prognostic tumour biomarkers, using chemotherapy-induced cancer-associated fibroblasts.

      Peiris-Pagès, Maria; Smith, Duncan L; Győrffy, B; Sotgia, Federica; Lisanti, Michael P; The Breast Cancer Now Research Unit, Institute of Cancer Sciences, University of Manchester (2015-10)
      Cancer cells grow in highly complex stromal microenvironments, which through metabolic remodelling, catabolism, autophagy and inflammation nurture them and are able to facilitate metastasis and resistance to therapy. However, these changes in the metabolic profile of stromal cancer-associated fibroblasts and their impact on cancer initiation, progression and metastasis are not well-known. This is the first study to provide a comprehensive proteomic portrait of the azathioprine and taxol-induced catabolic state on human stromal fibroblasts, which comprises changes in the expression of metabolic enzymes, myofibroblastic differentiation markers, antioxidants, proteins involved in autophagy, senescence, vesicle trafficking and protein degradation, and inducers of inflammation. Interestingly, many of these features are major contributors to the aging process. A catabolic stroma signature, generated with proteins found differentially up-regulated in taxol-treated fibroblasts, strikingly correlates with recurrence, metastasis and poor patient survival in several solid malignancies. We therefore suggest the inhibition of the catabolic state in healthy cells as a novel approach to improve current chemotherapy efficacies and possibly avoid future carcinogenic processes.
    • Proteomic plasma membrane profiling reveals an essential role for gp96 in the cell surface expression of LDLR family members, including the LDL receptor and LRP6.

      Weekes, M; Antrobus, R; Talbot, S; Hör, S; Simecek, N; Smith, Duncan L; Bloor, S; Randow, F; Lehner, P; Cambridge Institute for Medical Research, University of Cambridge , Hills Road, Cambridge, CB2 0XY, United Kingdom. (2012-03-02)
      The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.
    • Proteomic response of Schizosaccharomyces pombe to static and oscillating extremely low-frequency electromagnetic fields.

      Sinclair, John; Weeks, Mark E; Butt, Amna; Worthington, Jessica L; Akpan, Akunna; Jones, Nic; Waterfield, Michael D; Allan, Donald; Timms, John F; Ludwig Institute for Cancer Research, Department of Biochemistry and Molecular Biology University College London, UK. (2006-09)
      There is considerable public concern regarding the health effects of exposure to low-frequency electromagnetic fields. In addition, the association between exposure and disease incidence or the possible biological effects of exposure are unclear. Using 2D-DIGE and MS in a blind study, we have investigated the effects of static and oscillating extremely low-frequency electromagnetic fields (ELF EMFs) on the proteomes of wild type Schizosaccharomyces pombe and a Sty1p deletion mutant which displays increased sensitivity to a variety of cellular stresses. Whilst this study identifies a number of protein isoforms that display significant differential expression across experimental conditions, there was no correlation between their patterns of expression and the ELF EMF exposure regimen. We conclude that there are no significant effects of either static or oscillating EMF on the yeast proteome at the sensitivity afforded by 2D-DIGE. We hypothesise that the proteins identified must be sensitive to subtle changes in culture and/or handling conditions, and that the identification of these proteins in other proteomic studies should be treated with some caution when the results of such studies are interpreted in a biological context.
    • Proteomics profiling of interactome dynamics by colocalisation analysis (COLA).

      Mardakheh, F; Sailem, H; Kümper, S; Tape, C; McCully, R; Paul, A; Anjomani-Virmouni, S; Jørgensen, Claus; Poulogiannis, G; Marshall, C; et al. (2016-11-08)
      Localisation and protein function are intimately linked in eukaryotes, as proteins are localised to specific compartments where they come into proximity of other functionally relevant proteins. Significant co-localisation of two proteins can therefore be indicative of their functional association. We here present COLA, a proteomics based strategy coupled with a bioinformatics framework to detect protein-protein co-localisations on a global scale. COLA reveals functional interactions by matching proteins with significant similarity in their subcellular localisation signatures. The rapid nature of COLA allows mapping of interactome dynamics across different conditions or treatments with high precision.
    • Proteomics techniques and their application to hematology.

      Cristea, Ileana M; Gaskell, Simon J; Whetton, Anthony D; Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Manchester, United Kingdom. (2004-05-15)
      The recent sequencing of a number of genomes has raised the level of opportunities for studies on proteins. This area of research has been described with the all-embracing term, proteomics. In proteomics, the use of mass spectrometric techniques enables genomic databases to be used to establish the identity of proteins with relatively little data, compared to the era before genome sequencing. The use of related analytical techniques also offers the opportunity to gain information on regulation, via posttranslational modification, and potential new diagnostic and prognostic indicators. Relative quantification of proteins and peptides in cellular and extracellular material remains a challenge for proteomics and mass spectrometry. This review presents an analysis of the present and future impact of these proteomic technologies with emphasis on relative quantification for hematologic research giving an appraisal of their potential benefits.
    • A protocol for isolating Xenopus oocyte nuclear envelope for visualization and characterization by scanning electron microscopy (SEM) or transmission electron microscopy (TEM).

      Allen, Terence D; Rutherford, Sandra A; Murray, Stephen M; Sanderson, Helen S; Gardiner, Fiona; Kiseleva, Elena; Goldberg, Martin W; Drummond, Sheona P; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Withington, Manchester M20 4BX, UK. tallen@picr.man.ac.uk (2007)
      This protocol details methods for the isolation of oocyte nuclear envelopes (NEs) from the African clawed toad Xenopus laevis, immunogold labeling of component proteins and subsequent visualization by field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). This procedure involves the initial removal of the ovaries from mature female X. laevis, the dissection of individual oocytes, then the manual isolation of the giant nucleus and subsequent preparation for high-resolution visualization. Unlike light microscopy, and its derivative technologies, electron microscopy enables 3-5 nm resolution of nuclear structures, thereby giving unrivalled opportunities for investigation and immunological characterization in situ of nuclear structures and their structural associations. There are a number of stages where samples can be stored, although we recommend that this protocol take no longer than 2 d. Samples processed for FESEM can be stored for weeks under vacuum, allowing considerable time for image acquisition.
    • A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM).

      Kiseleva, Elena; Allen, Terence D; Rutherford, Sandra A; Murray, Stephen M; Morozova, Ksenia N; Gardiner, Fiona; Goldberg, Martin W; Drummond, Sheona P; Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, Russia. elka@bionet.nsu.ru (2007)
      This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.
    • A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy.

      Murray, Stephen M; Kiseleva, Elena; TEM Service Facility, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom. (2008)
      This article describes a protocol that details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immunogold labelling of proteins, and visualization by Field Emission Scanning Electron Microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high resolution microscopy. The nuclear isolation step is performed by enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of nuclei by centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs), and associated cytoskeletal structures. Samples, once processed for FESEM, can be stored under vacuum for weeks, allowing considerable time for image acquisition.
    • Proton nuclear magnetic resonance imaging as a predictor of the outcome of photodynamic therapy of tumours.

      Moore, James V; Dodd, Nicholas J F; Wood, B; Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester. (1989-09)