Browsing by Author "Valenzuela, SM"

Now showing 1 - 6 of 6
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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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    The impact of pH on packing in tethered lipid bilayers
    (Australian Institute of Nuclear Science and Engineering, 2016-11-29) Cranfield, CG; Berry, T; Holt, SA; Le Brun, AP; Valenzuela, SM; Coster, HGL; Cornell, BA
    We report that increasing the H3O+ concentration when lowering the pH reduces the intrinsic ionic conduction through phospholipid bilayers (Fig 1A), which is counter to what might be expected from increasing the H3O+ concentration. We attribute the conduction decrease to a reduction of the molecular area per lipid (ao)[1]. These effects are seen at H3O+ concentrations in the range nM to µM despite these being very low concentrations compared to that of a typical bathing electrolyte solution of 135mM ionic concentration. We present a model, in which the pH dependent reduction in ao favours an increase in lipid packing. To support this model, we provide evidence of the effects of the hydronium ion on lipid geometry using neutron reflectometry (Fig 1C). Further examples will be given of the impact of the H3O ion concentration on the hydrogen bonding within the polar groups of lipid.
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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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    Stereoselective synthesis of perdeuterated phytanic acid, its phospholipid derivatives and their formation into lipid model membranes for neutron reflectivity studies
    (Elsevier, 2014-10-01) Yepuri, NR; Holt, SA; Moraes, G; Holden, PJ; Hossain, KR; Valenzuela, SM; Darwish, TA; James, M
    We describe a straightforward method, for synthesis of large scale (gram quantities) of highly deuterated phytanic acid from commercially available phytol while preserving the stereochemistry around the chiral centres. The subsequent synthesis of tail-deuterated analogues of the archeabacterial membrane lipids 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPEPC) and 1,2-di(3RS,7R,11R-phytanoyl)-sn-glycero-3-phosphocholine (DPhyPC) from perdeuterated phytanic acid is also described. Both lipids were employed in construction of two different model membranes, namely Langmuir monolayers and a tethered bilayer membrane (TBM) on a solid substrate, characterised by pressure area isotherm and neutron reflectometry techniques. At 10 mN/m pressure the head-group thickness of both monolayers was similar while the thickness of the tail region was significantly larger for tail-deuterated DPhyPC, which was evident from a smaller area per molecule. At 20 mN/m the thickness of the head and tail regions in both lipids was comparable, yet the area per molecule of tail-deuterated DPhyPC was 10% smaller than tail-deuterated DPEPC. In the TBM bilayer model membrane, the thickness of the lipid tails in both inner and outer leaflets was 8.2 Å, giving a total of 16.4 Å. Deuteration enabled unambiguous determination of the relative proportion of the hydrogenous tether, phospholipid and subphase. © 2014, Elsevier Ireland Ltd.
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    X-ray and neutron reflectivity study of the membrane-bound CLIC1 protein at the air-water interface
    (Australian Institute of Nuclear Science and Engineering, 2016-11-29) Hossain, KR; Holt, SA; Khamici, HA; Valenzuela, SM
    The CLIC proteins are a ubiquitous family of Chloride Intracellular Ion Channel proteins that are evolutionarily conserved across species and exist as a soluble form and an integral membrane-bound form 1. The X-ray structure of the soluble form of a number of CLIC proteins has been solved and their putative transmembrane domain identified 2-5. However, the factors which facilitate the membrane insertion, the structural transition between these two forms and the structural features of the membrane-bound form for this class of proteins remain largely unknown. In an attempt to answer these questions, we have employed biophysical techniques to study the interaction of wild-type and mutant versions of the protein CLIC1 with monolayers prepared using various mixtures of different phospholipids and sterol molecules. Our findings have demonstrated that cholesterol plays a crucial role for the penetration of CLIC1 into the hydrophobic tails of the lipid monolayer with the protein occupying an area per molecule between 5-7 nm2. We have also demonstrated for the first time that CLIC1 interaction with cholesterol is dependent on the intact 3β-OH group in the sterol ring and that the GXXXG motif in CLIC1 acts as the cholesterol-binding site used by the protein for its initial recognition and binding to membrane cholesterol.
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    X-ray and neutron reflectivity study shows that CLIC1 undergoes cholesterol-dependent structural reorganization in lipid monolayers
    (ACS Publications, 2017-10-17) Hossain, KR; Holt, SA; Le Brun, AP; Al Khamici, H; Valenzuela, SM
    CLIC1 belongs to the ubiquitous family of chloride intracellular ion channel proteins that are evolutionarily conserved across species. The CLICs are unusual in that they exist mainly as soluble proteins but possess the intriguing property of spontaneous conversion from the soluble to an integral membrane-bound form. This conversion is regulated by the membrane lipid composition, especially by cholesterol, together with external factors such as oxidation and pH. However, the precise physiological mechanism regulating CLIC1 membrane insertion is currently unknown. In this study, X-ray and neutron reflectivity experiments were performed to study the interaction of CLIC1 with different phospholipid monolayers prepared using POPC, POPE, or POPS with and without cholesterol in order to better understand the regulatory role of cholesterol in CLIC1 membrane insertion. Our findings demonstrate for the first time two different structural orientations of CLIC1 within phospholipid monolayers, dependent upon the absence or presence of cholesterol. In phospholipid monolayers devoid of cholesterol, CLIC1 was unable to insert into the lipid acyl chain region. However, in the presence of cholesterol, CLIC1 showed significant insertion within the phospholipid acyl chains occupying an area per protein molecule of 6−7 nm2 with a total CLIC1 thickness ranging from ∼50 to 56 Å across the entire monolayer. Our data strongly suggests that cholesterol not only facilitates the initial docking or binding of CLIC1 to the membrane but also promotes deeper penetration of CLIC1 into the hydrophobic tails of the lipid monolayer. © 2017 American Chemical Society