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1.
Analyst ; 144(9): 3030-3037, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30901016

RESUMO

The design of ion sensors has gained importance for the study of ion dynamics in cells, with fluorescent proton nanosensors attracting particular interest because of their applicability in monitoring pH gradients in biological microcompartments and reconstituted membrane systems. In this work, we describe the improved synthesis, photophysical properties and applications of pH sensors based on amine-reactive pHrodo esters and short-chain lipid derivatives of phosphoethanolamine. The major features of these novel probes include strong fluorescence under acidic conditions, efficient partitioning into membranes, and extractability by back exchange to albumin. These features allow for the selective labeling of the inner liposomal leaflet in reconstituted membrane systems for studying proton pumping activities in a quantitative fashion, as demonstrated by assaying the activity of a plant plasma membrane H+-ATPase. Furthermore, the short-chain lipid-conjugated pH sensors enable the monitoring of pH changes from neutral to acidic conditions in the endocytic pathway of living cells. Collectively, our results demonstrate the applicability of short-chain lipid-conjugated sensors for in vivo and in vitro studies and thus pave the way for the design of lipid-conjugated sensors selective to other biologically relevant ions, e.g. calcium and sodium.


Assuntos
Transporte Biológico/fisiologia , Corantes Fluorescentes/química , Lipossomos/metabolismo , Fosfatidiletanolaminas/química , Rodaminas/química , Animais , Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Células COS , Bovinos , Linhagem Celular Tumoral , Chlorocebus aethiops , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Microscopia Confocal/métodos , Fragmentos de Peptídeos/metabolismo , Fosfatidiletanolaminas/síntese química , Fosfatidiletanolaminas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Rodaminas/síntese química , Rodaminas/metabolismo , Soroalbumina Bovina/química
2.
Int J Mol Sci ; 18(10)2017 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-28973963

RESUMO

Bio-plastics and bio-materials are composed of natural or biomass derived polymers, offering solutions to solve immediate environmental issues. Polysaccharide-based bio-plastics represent important alternatives to conventional plastic because of their intrinsic biodegradable nature. Amylose-only (AO), an engineered barley starch with 99% amylose, was tested to produce cross-linked all-natural bioplastic using normal barley starch as a control. Glycerol was used as plasticizer and citrate cross-linking was used to improve the mechanical properties of cross-linked AO starch extrudates. Extrusion converted the control starch from A-type to Vh- and B-type crystals, showing a complete melting of the starch crystals in the raw starch granules. The cross-linked AO and control starch specimens displayed an additional wide-angle diffraction reflection. Phospholipids complexed with Vh-type single helices constituted an integrated part of the AO starch specimens. Gas permeability tests of selected starch-based prototypes demonstrated properties comparable to that of commercial Mater-Bi© plastic. The cross-linked AO prototypes had composting characteristics not different from the control, indicating that the modified starch behaves the same as normal starch. The data shows the feasibility of producing all-natural bioplastic using designer starch as raw material.


Assuntos
Amilose/química , Plásticos Biodegradáveis/química , Ácido Cítrico/química , Reagentes de Ligações Cruzadas/química , Hordeum/química , Cristalização , Glicerol/química , Permeabilidade , Transição de Fase , Plantas Geneticamente Modificadas/química , Plastificantes/química , Amido/química
3.
Eur Biophys J ; 46(2): 103-119, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27437691

RESUMO

Studying membrane proteins at the molecular level represents a major challenge in biochemistry due to the complexity of the membrane in which they are embedded. As an important step towards a detailed understanding of their action and molecular functioning, current studies focus on membrane proteins reconstituted into artificial lipid environments. Such reconstituted systems allow for a more flexible choice of biochemical, biophysical, and microscopy techniques for characterizing the proteins. This review gives an overview of the methods currently available for reconstituting membrane proteins in a functional state into giant unilamellar vesicles, and discusses some key methods to verify successful reconstitution.


Assuntos
Proteínas de Membrana/química , Lipossomas Unilamelares/química , Fusão de Membrana , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo
4.
Analyst ; 140(18): 6313-20, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26280031

RESUMO

Accurate real-time measurements of the dynamics of proton concentration gradients are crucial for detailed molecular studies of proton translocation by membrane-bound enzymes. To reduce complexity, these measurements are often carried out with purified, reconstituted enzyme systems. Yet the most paramount problem to detect pH changes in reconstituted systems is that soluble pH reporters leak out of the vesicle system during the reconstitution procedure. This requires loading of substantial amounts of pH-sensors into the lumen of unilamellar liposomes during reconstitution. Here, we report the synthesis and detailed characterisation of two lipid-linked pH sensors employing amine-reactive forms of seminaphthorhodafluors (SNARF®-1 dye) and rhodamine probes (pHrodo™ Red dye). Lipid-conjugation of both dyes allowed for efficient detergent-based reconstitution of these pH indicators into liposomes. Vesicle-embedded pHrodo™ displayed excellent photostability and an optimal pH-response between 4 and 7. The suitability of the lipid-linked pHrodo™ probe as a pH reporter was demonstrated by assaying the activity of a plant plasma membrane H(+)-ATPase (proton pump) reconstituted in proteoliposomes.


Assuntos
Corantes Fluorescentes/química , Lipossomos/química , Fosfatidiletanolaminas/química , Fosfolipídeos/química , Amidas/química , Benzopiranos/síntese química , Benzopiranos/química , Etanolamina/química , Corantes Fluorescentes/síntese química , Concentração de Íons de Hidrogênio , Naftóis/síntese química , Naftóis/química , ATPases Translocadoras de Prótons/metabolismo , Prótons , Rodaminas/síntese química , Rodaminas/química
5.
Proc Natl Acad Sci U S A ; 112(29): 9040-5, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26134396

RESUMO

ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor-Rakeb syndrome and Parkinson's disease (PD), providing protection against α-synuclein, Mn(2+), and Zn(2+) toxicity in various model systems. So far, the molecular function and regulation of ATP13A2 remains undetermined. Here, we demonstrate that ATP13A2 contains a unique N-terminal hydrophobic extension that lies on the cytosolic membrane surface of the lysosome, where it interacts with the lysosomal signaling lipids phosphatidic acid (PA) and phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2]. We further demonstrate that ATP13A2 accumulates in an inactive autophosphorylated state and that PA and PI(3,5)P2 stimulate the autophosphorylation of ATP13A2. In a cellular model of PD, only catalytically active ATP13A2 offers cellular protection against rotenone-induced mitochondrial stress, which relies on the availability of PA and PI(3,5)P2. Thus, the N-terminal binding of PA and PI(3,5)P2 emerges as a key to unlock the activity of ATP13A2, which may offer a therapeutic strategy to activate ATP13A2 and thereby reduce α-synuclein toxicity or mitochondrial stress in PD or related disorders.


Assuntos
Lipídeos/química , Doença de Parkinson/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citosol/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Manganês/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Ácidos Fosfatídicos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Homologia Estrutural de Proteína , Zinco/farmacologia
6.
J Biol Chem ; 290(26): 16281-91, 2015 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-25971968

RESUMO

Eukaryotic P-type plasma membrane H(+)-ATPases are primary active transport systems that are regulated at the post-translation level by cis-acting autoinhibitory domains, which can be relieved by protein kinase-mediated phosphorylation or binding of specific lipid species. Here we show that lysophospholipids specifically activate a plant plasma membrane H(+)-ATPase (Arabidopsis thaliana AHA2) by a mechanism that involves both cytoplasmic terminal domains of AHA2, whereas they have no effect on the fungal counterpart (Saccharomyces cerevisiae Pma1p). The activation was dependent on the glycerol backbone of the lysophospholipid and increased with acyl chain length, whereas the headgroup had little effect on activation. Activation of the plant pump by lysophospholipids did not involve the penultimate residue, Thr-947, which is known to be phosphorylated as part of a binding site for activating 14-3-3 protein, but was critically dependent on a single autoinhibitory residue (Leu-919) upstream of the C-terminal cytoplasmic domain in AHA2. A corresponding residue is absent in the fungal counterpart. These data indicate that plant plasma membrane H(+)-ATPases evolved as specific receptors for lysophospholipids and support the hypothesis that lysophospholipids are important plant signaling molecules.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Lisofosfolipídeos/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/química , Membrana Celular/enzimologia , Membrana Celular/genética , Ativação Enzimática , Fosforilação , Estrutura Terciária de Proteína , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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