Browsing by Author "Curmi, PMG"

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    CLIC proteins, ezrin, radixin, moesin and the coupling of membranes to the actin cytoskeleton: A smoking gun?
    (Elsevier, 2014-02-01) Jiang, L; Phang, JM; Yu, J; Harrop, SJ; Sokolova, AV; Duff, AP; Wilik, KE; Alkhamici, H; Breit, SN; Valenzuela, SM; Brown, LJ; Curmi, PMG
    The CLIC proteins are a highly conserved family of metazoan proteins with the unusual ability to adopt both soluble and integral membrane forms. The physiological functions of CLIC proteins may include enzymatic activity in the soluble form and anion channel activity in the integral membrane form. CLIC proteins are associated with the ERM proteins: ezrin, radixin and moesin. ERM proteins act as cross-linkers between membranes and the cortical actin cytoskeleton. Both CLIC and ERM proteins are controlled by Rho family small GTPases. CLIC proteins, ERM and Rho GTPases act in a concerted manner to control active membrane processes including the maintenance of microvillar structures, phagocytosis and vesicle trafficking. All of these processes involve the interaction of membranes with the underlying cortical actin cytoskeleton. The relationships between Rho GTPases, CLIC proteins, ERM proteins and the membrane:actin cytoskeleton interface are reviewed. Speculative models are proposed involving the formation of localised multi-protein complexes on the membrane surface that assemble via multiple weak interactions. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. © 2013, Elsevier B.V.
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    Novel approach for enhancing the catalytic efficiency of a protease at low temperature: reduction in substrate inhibition by chemical modification.
    (Wiley-Blackwell, 2009-07-01) Siddiqui, KS; Parkin, DM; Curmi, PMG; De Francisci, D; Poljak, A; Barrow, KD; Noble, MH; Trewhella, J; Cavicchioli, R
    The alkaline protease, savinase was chemically modified to enhance the productivity of the enzyme at low temperatures on a complex polymeric protein (azocasein) substrate. At 5 and 15 degrees C, savinase modified with ficol or dextran hydrolyzed fivefold more azocasein than the unmodified savinase. Kinetic studies showed that the catalytic improvements are associated with changes in uncompetitive substrate inhibition with K-i values of modified savinases sixfold higher than the unmodified savinase. Modeling of small-angle scattering data indicates that two substrate molecules bind on opposing sides of the enzyme. The combined kinetic and structural data indicate that the polysaccharide modifier sterically blocks the allosteric site and reduces substrate inhibition. In contrast to the properties of cold-active enzymes that generally manifest as low activation enthalpy and high flexibility, this study shows that increased activity and productivity at low temperature can be achieved by reducing uncompetitive substrate inhibition, and that this can be achieved using chemical modification with an enzyme in a commercial enzyme-formulation. Biotechnol. Bioeng. 2009;103: 676-686. © 2009, Wiley-Blackwell.
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    Regulation of the membrane insertion and conductance activity of the metamorphic chloride intracellular channel protein CLIC1 by cholesterol
    (Public Library of Science, 2013-02-14) Valenzuela, SM; Alkhamici, H; Brown, LJ; Almond, OC; Goodchild, SC; Carne, S; Curmi, PMG; Holt, SA; Cornell, BA
    The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer. © 2013, Valenzuela et al.
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    Selective inhibition of human group IIA-secreted phospholipase A(2) (hGIIA) signaling reveals arachidonic acid metabolism Is associated with colocalization of hGIIA to vimentin in rheumatoid synoviocytes
    (Americal Society Biochemistry Molecular Biology Inc., 2013-05-24) Lee, LK; Bryant, KJ; Bouveret, R; Lei, PW; Duff, AP; Harrop, SJ; Huang, EP; Harvey, RP; Gelb, MH; Gray, PP; Curmi, PMG; Cunningham, AM; Church, WB; Scott, KF
    Human group IIA secreted phospholipase A(2) (hGIIA) promotes tumor growth and inflammation and can act independently of its well described catalytic lipase activity via an alternative poorly understood signaling pathway. With six chemically diverse inhibitors we show that it is possible to selectively inhibit hGIIA signaling over catalysis, and x-ray crystal structures illustrate that signaling involves a pharmacologically distinct surface to the catalytic site. We demonstrate in rheumatoid fibroblast-like synoviocytes that non-catalytic signaling is associated with rapid internalization of the enzyme and colocalization with vimentin. Trafficking of exogenous hGIIA was monitored with immunofluorescence studies, which revealed that vimentin localization is disrupted by inhibitors of signaling that belong to a rare class of small molecule inhibitors that modulate protein-protein interactions. This study provides structural and pharmacological evidence for an association between vimentin, hGIIA, and arachidonic acid metabolism in synovial inflammation, avenues for selective interrogation of hGIIA signaling, and new strategies for therapeutic hGIIA inhibitor design. © 2013, American Society for Biochemistry and Molecular Biology