J B Findlay

University of Leeds, Leeds, ENG, United Kingdom

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Publications (207)745.91 Total impact

  • Kathryn L Chapman, John B C Findlay
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    ABSTRACT: This study involves the structural and functional properties of the recombinant melanocortin 4 receptor (MC(4)R) expressed in the HEK-293 cell line. Using co-immuno-purification approaches, the receptor appears to be an oligomer, which can be crosslinked through disulphide bonds involving a native cysteine residue (84) to give a dimeric species. This position is located near the cytosolic region of transmembrane segment 2 and it is suggested that this is an interacting interface between MC(4)R monomers. Using co-expression of the native protein and a C84A mutant, it appears that the receptor also forms higher order oligomers via alternative interfaces. Interestingly, disulphide crosslink formation does not occur if the receptor is uncoupled from its G-protein, even though the oligomeric state is preserved. This suggests that the conformational changes, which occur on activation, affect the TM2 interface. The pharmacology of the agonist, NDP-MSH, indicates that the MC(4)R retains high affinity for the ligand in the absence of the G-protein but occupancy for the ligand is increased. The data can be interpreted to suggest that the G-protein exerts a negative allosteric effect on the receptor. Co-expression of one receptor lacking the ability to signal with another, which cannot bind the agonist, restored ligand-dependent activation of the G-protein to situations in which neither receptor on its own could activate the G-protein. Such transactivation suggests meaningful cross talk between the receptor subunits in the oligomeric complex. These studies demonstrate further unique features of the MC(4)R.
    Biochimica et Biophysica Acta 10/2012; · 4.66 Impact Factor
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    ABSTRACT: Mutated versions of membrane proteins often fail to express at the plasma membrane, but instead are trapped in the secretory pathway, resulting in disease. The retention of these mutant proteins is thought to result from local misfolding, which prevents export from the ER (endoplasmic reticulum), targeting the receptor for degradation via the ER-associated quality control system. The rhodopsin-like G-protein-coupled MC4R (melanocortin 4 receptor) is an example of such a membrane protein. Over 100 natural MC4R mutations are linked with an obese phenotype and to date represent the most common monogenic cause of severe early-onset obesity. More than 80% of these mutations result in a substantial proportion of MC4R being retained intracellularly. If these receptors were expressed at the plasma membrane, many could be functional, as mutations often occur in regions distinct from those associated with ligand or G-protein binding. Our aim is to show proof of concept that selective compounds can rescue the function of MC4R mutants by increasing their cell-surface expression, and further to this, examine whether the rescue profile differs between mutants. Whole-cell ELISA and 96-well fluorescence-based assays with N-terminally HA (haemagglutinin)-tagged and C-terminally mCherry-tagged mutant MC4Rs were used to screen a number of novel MC4R-selective compounds. A total of four related compounds increased the cell-surface expression of wild-type and three intracellularly retained mutant MC4Rs, thus acting as pharmacological chaperones. There appears to be a unique rescue efficacy profile for each compound that does not correlate with potency, suggesting distinct receptor conformations induced by the different mutations. A degree of functionality of V50M and S58C was also rescued following relocation to the cell surface.
    Biochemical Society Transactions 08/2012; 40(4):717-20. · 2.59 Impact Factor
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    ABSTRACT: The mycotoxin deoxynivalenol (DON) commonly contaminates cereal grains. It is ubiquitous in the Western European diet, although chronic, low-dose effects in humans are not well described, but immunotoxicity has been reported. In this study, two-dimensional gel electrophoresis was used to identify phosphoproteomic changes in human B (RPMI1788) and T (Jurkat E6.1) lymphocyte cell lines after exposure to modest concentrations of DON (up to 500ng/mL) for 24h. Proteins identified as having altered phosphorylation state post-treatment (C-1-tetrahydrofolate synthase, eukaryotic elongation factor 2, nucleoside diphosphate kinase A, heat shock cognate 71kDa protein, eukaryotic translation initiation factor 3 subunit I and growth factor receptor-bound protein 2) are involved in regulation of metabolic pathways, protein biosynthesis and signaling transduction. All exhibited a greater than 1.4-fold change, reproducible in three separate experiments consisting of 36 gels in total. Flow cytometry validated the observations for eukaryotic elongation factor 2 and growth factor receptor-bound protein 2. These findings provide further insights as to how low dose exposure to DON may affect human immune function and may have potential as mechanism-based phosphoprotein biomarkers for DON exposure.
    Biochimica et Biophysica Acta 07/2011; 1814(7):850-7. · 4.66 Impact Factor
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    ABSTRACT: Serum retinol binding protein (sRBP) is released from the liver as a complex with transthyretin (TTR), a process under the control of dietary retinol. Elevated levels of sRBP may be involved in inhibiting cellular responses to insulin and in generating first insulin resistance and then type 2 diabetes, offering a new target for therapeutic attack for these conditions. A series of retinoid analogues were synthesized and examined for their binding to sRBP and their ability to disrupt the sRBP-TTR and sRBP-sRBP receptor interactions. A number inhibit the sRBP-TTR and sRBP-sRBP receptor interactions as well as or better than Fenretinide (FEN), presenting a potential novel dual mechanism of action and perhaps offering a new therapeutic intervention against type 2 diabetes and its development. Shortening the chain length of the FEN derivative substantially abolished binding to sRBP, indicating that the strength of the interaction lies in the polyene chain region. Differences in potency against the sRBP-TTR and sRBP-sRBP receptor interactions suggest variant effects of the compounds on the two loops of sRBP guarding the entrance of the binding pocket that are responsible for these two protein-protein interactions.
    Journal of Medicinal Chemistry 06/2011; 54(13):4378-87. · 5.61 Impact Factor
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    ABSTRACT: The mycotoxin deoxynivalenol (DON) contaminates cereals worldwide and is a common contaminant in the Western European diet. At high doses, DON induces acute gastrointestinal toxicity; chronic, low-dose effects in humans are not well described, but immunotoxicity has been reported. In this study, 2-DE was used to identify proteomic changes in human B (RPMI1788) and T (JurkatE6.1) lymphocyte cell lines after exposure to minimally toxic concentrations (up to 500 ng/mL) for 24 h. Proteins which changed their abundance post treatment, by a greater than 1.4-fold change reproducible in three separate experiments consisting of 36 gels in total, are ubiquitin carboxyl-terminal hydrolase isozyme L3, proteasome subunit β type-4 and α type-6, inosine-5'-monophosphate dehydrogenase 2, GMP synthase, microtubule-associated protein RP/EB family member 1 (EB1), RNA polymerases I, II, III subunit ABC1, triosephosphate isomerase and transketolase. Flow cytometry was used to validate changes to protein expression, except for EB1. These findings provide insights as to how low-dose exposure to DON may affect human immune function and may provide mechanism-based biomarkers for DON exposure.
    Proteomics 03/2011; 11(10):1903-14. · 4.43 Impact Factor
  • Ref. No: PG13583IE00, Year: 01/2011
  • Source
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    ABSTRACT: Melanocortin-4 receptor (MC4R) has an important regulatory role in energy homeostasis and food intake. Peptide agonists of the MC4R are characterized by the conserved sequence His(6)-Phe(7)-Arg(8)-Trp(9), which is crucial for their interaction with the receptor. This investigation utilized the covalent attachment approach to identify receptor residues in close proximity to the bound ligand [Nle(4),D-Phe(7)]melanocyte-stimulating hormone (NDP-MSH), thereby differentiating between residues directly involved in ligand binding and those mutations that compromise ligand binding by inducing conformational changes in the receptor. Also, recent X-ray structures of G-protein-coupled receptors were utilized to refine a model of human MC4R in the active state (R(*)), which was used to generate a better understanding of the binding mode of the ligand NDP-MSH at the atomic level. The mutation of residues in the human MC4R--such as Leu106 of extracellular loop 1, and Asp122, Ile125, and Asp126 of transmembrane (TM) helix 3, His264 (TM6), and Met292 (TM7)--to Cys residues produced definitive indications of proximity to the side chains of residues in the core region of the peptide ligand. Of particular interest was the contact between D-Phe(7) on the ligand and Ile125 of TM3 on the MC4R. Additionally, Met292 (TM7) equivalent to Lys(7.45) (Ballesteros numbering scheme) involved in covalently attaching retinal in rhodopsin is shown to be in close proximity to Trp(9). For the first time, the interactions between the terminal regions of NDP-MSH and the receptor are described. The amino-terminus appears to be adjacent to a series of hydrophilic residues with novel interactions at Cys196 (TM5) and Asp189 (extracellular loop 2). These interactions are reminiscent of sequential ligand binding exhibited by the beta(2)-adrenergic receptor, with the former interaction being equivalent to the known interaction involving Ser204 of the beta(2)-adrenergic receptor.
    Journal of Molecular Biology 08/2010; 401(3):433-50. · 3.91 Impact Factor
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    ABSTRACT: To operate as a rotary motor, the ATP-hydrolyzing domain of the vacuolar H(+)-ATPase must be connected to a fixed structure in its membrane-bound proton pump domain by a mechanical stator. Although low-resolution structural data and spectroscopic analysis indicate that a filament-like subunit E/subunit G heterodimer performs this role, more detailed information about the relative arrangement of these subunits is limited. We have used a site-directed cross-linking approach to show that, in both bacterial and yeast V-type ATPases, the N-terminal alpha-helical segments of the G and E subunits are closely aligned over a distance of up to 40 A. Furthermore, cross-linking coupled to mass spectrometry shows that the C-terminal end of G is anchored at the C-terminal globular domain of subunit E. These data are consistent with a stator model comprising two approximately 150 A long parallel alpha-helices linked to each other at both ends, stabilized by a coiled-coil arrangement and capped by the globular C-terminal domain of E that connects the cytoplasmic end of the helical structure to the V-ATPase catalytic domain.
    Molecular Membrane Biology 05/2010; 27(4-6):147-59. · 3.13 Impact Factor
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    ABSTRACT: Plasma retinol-binding protein (RBP4) is the principal carrier of vitamin A in blood. Recent studies have suggested that RBP4 may have also a role in insulin resistance. To date the recombinant protein is usually produced by refolding inclusion bodies in Escherichia coli. Here we report the expression and characterization of recombinant human plasma RBP4 using the Pichia pastoris expression system. Simple and rapid purification allowed us to obtain 5mg/L of purified protein from the fermentation supernatant with no need to perform denaturing and refolding steps. The identity of the protein was verified by ion-trap MS and Western blotting. The functionality of recombinant RBP4, i.e., the binding to its physiologic ligand, retinol, and the interaction with transthyretin (TTR), was tested by fluorimetric and pull-down assays, respectively. The apparent dissociation constant for retinol to the recombinant protein of 2 x 10(-7)M was consistent with published data for native human protein. The recombinant protein interacted specifically with TTR. These results suggest that expression of recombinant human RBP4 in P. pastoris provides an efficient source of fully functional protein in soluble form for biochemical and biophysical studies.
    Protein Expression and Purification 05/2010; 71(1):28-32. · 1.43 Impact Factor
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    ABSTRACT: The synthesis and reactivity of fully protected thioamide analogues of asparagine and glutamine are described. A key feature of the synthetic strategies employed was the ability to perform selective thiations on multiple carbonyl-containing substrates. Also described are the preparations of thioamide derivatives of phenylalanine. The utility of these amino acid derivatives for solid-phase peptide synthesis is discussed.
    ChemInform 01/2010; 32(7).
  • Toxicology Letters - TOXICOL LETT. 01/2010; 196.
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 01/2010; 28(28).
  • I. D. KERSEY, C. W. G. FISHWICK, J. B. C. FINDLAY, P. WARD
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 01/2010; 26(43).
  • [Show abstract] [Hide abstract]
    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 01/2010; 32(27).
  • M. A. Rodgers, J. B. C. Findlay, P. A. Millner
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    ABSTRACT: Lipocalins, which are compact binding proteins for odorants, have potential as bioreceptors for small hydrophobic analytes. Studies on several isoforms of the lipocalin major mouse urinary protein (MUP) showed that self-assembled monolayers (SAMs) containing affinity lipid chelating groups were inadequate for building responsive sensors. Non-specific surface immobilisation generated a more responsive but less reproducible sensor. A new SAM was designed that contained a thiolated Ni2+-nitrilotriacetic acid (NTA) moiety to ensure chelating groups were present over the entire surface, allowing specific immobilisation over the whole SAM. This new SAM was found to give improved reproducibility without sacrificing the responsiveness of the sensor. Further development of this SAM type should result in highly sensitive sensors based on lipocalins.
    Sensors and Actuators B-chemical - SENSOR ACTUATOR B-CHEM. 01/2010; 150(1):12-18.
  • C. W. G. FISHWICK, J. M. SANDERSON, J. B. C. FINDLAY
    ChemInform 01/2010; 25(46).
  • J. B. C. FINDLAY, C. W. G. FISHWICK, I. D. KERSEY, P. WARD
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 01/2010; 26(30).
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    ABSTRACT: Peripherin/RDS is a member of the tetraspanin family of integral membrane proteins and plays a major role in the morphology of photoreceptor outer segments. Peripherin/RDS has a long extracellular loop (hereafter referred to as the LEL domain), which is vital for its function. Point mutations in the LEL domain often lead to impaired photoreceptor formation and function, making peripherin/RDS an important drug target. Being a eukaryotic membrane protein, acquiring sufficient peripherin/RDS for biophysical characterisation represents a significant challenge. Here, we describe the expression and characterisation of peripherin/RDS in Drosophila melangolaster Schneider (S2) insect cells and in the methylotrophic yeast Pichia pastoris. The wild-type peripherin/RDS and the retinitis pigmentosa causing P216L mutant from S2 cells are characterised using circular dichroism (CD) spectroscopy. The structure of peripherin/RDS and of a pathogenic mutant is assessed spectroscopically for the first time. These findings are evaluated in relation to a three-dimensional model of the functionally important LEL domain obtained by protein threading.
    Biophysics of Structure and Mechanism 11/2009; 39(4):679-88. · 2.44 Impact Factor
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    ABSTRACT: A simple and rapid protein chemical approach for determining the transmembrane structure of membrane proteins is described. The method involves single substitutions of consecutive amino acid residues, within putative transmembrane segments, to cysteine. This is followed by the analysis of their susceptibility to modification by maleimides with different physico-chemical properties. Fluorescein-5-maleimide (FM), being hydrophilic, modified only residues located in the aqueous environment, while the hydrophobic reagent, benzophenone-4-maleimide (BM) modified residues exposed to the lipid phase. These probes are large enough to cause an increase in the molecular weight of relatively small membrane proteins or polypeptide fragments, which is detectable by SDS-PAGE. Modification by much smaller probes, such as N-ethylmaleimide (NEM), could also be monitored indirectly by the ability to prevent SDS-solubilized protein from being modified with fluorescein-5-maleimide. The approach is demonstrated with the proteolipid complex of the vacuolar H(+)-ATPase expressed in yeast and with the putative Isk K(+)-channel expressed and radiolabelled in E. coli. The advantages of this approach are: (1)it is rapid, easy and inexpensive, (2) detection of the modification of engineered cysteines is simple, (3) it requires only minute quantities of the protein, (4) the protein does not require purification, (5) a broad range of maleimides with different physico-chemical properties can be used, (6) the structure can be investigated under native conditions and does not require protein reconstitution into artificial bilayers.
    Molecular Membrane Biology 07/2009; 13(1):53-60. · 3.13 Impact Factor
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    ABSTRACT: The LAD2 cell line is a relatively recent addition to the range of mast cell analogues and is of particular importance as it is the only human analogue which can be stimulated to degranulate in an IgE-dependent manner. Mast cells are tissue-based effector cells which have historically been shown to play an important role in the adaptive immune response, though there is now gathering evidence of their significance as a component of the innate immune system. These functions can be attributed to the ability of mast cells to regulate secretion of a wide variety of potent biologically active mediators through immediate and delayed responses. This well-orchestrated secretory mechanism of the mast cell makes it an ideal model in which to study this event. In this investigation, two-dimensional electrophoresis was employed as part of the proteomic characterization of the LAD2 human mast cell line, focusing in particular on a global analysis of membrane protein relocation after an IgE-mediated stimulatory event. This investigation has identified six membrane-associated protein spots which became phosphorylated upon IgE-mediated activation, 31 protein spots which displayed consistent recruitment to the membrane fraction, and three which were consistently lost from the soluble fraction. The scenario which emerges reveals a series of substantial changes which affect every compartment of the cell, providing evidence for a coordinated response to a secretory stimulus.
    Journal of Proteome Research 07/2009; 8(8):4116-25. · 5.06 Impact Factor

Publication Stats

3k Citations
745.91 Total Impact Points

Institutions

  • 1982–2012
    • University of Leeds
      • • Faculty of Biological Sciences
      • • School of Biomedical Sciences
      • • School of Biochemistry and Microbiology
      Leeds, ENG, United Kingdom
  • 2009–2011
    • National University of Ireland, Maynooth
      • Department of Biology
      Maynooth, L, Ireland
  • 1995–2008
    • Max Planck Institute for Biophysical Chemistry
      Göttingen, Lower Saxony, Germany
    • University of East Anglia
      • School of Chemistry
      Norwich, ENG, United Kingdom
  • 2005
    • AstraZeneca
      Tukholma, Stockholm, Sweden
  • 1989–2005
    • The University of Sheffield
      • • Department of Biomedical Science
      • • Department of Molecular Biology and Biotechnology
      Sheffield, ENG, United Kingdom
  • 2004
    • The American Society for Biochemistry and Molecular Biology
      Johnson Lane, Nevada, United States
  • 1999
    • University of Oxford
      Oxford, England, United Kingdom
    • EMBL-EBI
      Cambridge, England, United Kingdom
    • University of Copenhagen
      København, Capital Region, Denmark
  • 1996
    • University of Groningen
      • Groningen Biomolecular Sciences and Biotechnology Institute (GBB)
      Groningen, Groningen, Netherlands
  • 1991–1992
    • Beatson Institute for Cancer Research
      Glasgow, Scotland, United Kingdom
  • 1988
    • University of Birmingham
      Birmingham, England, United Kingdom
  • 1986
    • John Innes Centre
      Norwich, England, United Kingdom