RESUMO
Lipid hydroperoxides are the primary reaction products of lipid oxidation, a natural outcome of life under oxygen. While playing a major role in cell metabolism, the microscopic origins of the effects of lipid hydroperoxidation on biomembranes remain elusive. Here we probe the polar structure of partially to fully hydroperoxidized bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) by a combination of environment-sensitive fluorescent probes and coarse-grained Martini numerical simulations. We find that the inserted organic hydroperoxide group -OOH migrates preferentially to the surface for bilayers with small fractions of hydroperoxidized lipids, but populates also significantly the bilayer interior for larger fractions. Our findings suggest that by modifying the intimate polarity of biomembranes, lipid peroxidation will have a significant impact on the activity of transmembrane proteins and on the bio-medical efficiency of membrane active molecules such as cell-penetrating and antimicrobial peptides.
Assuntos
Peróxido de Hidrogênio/química , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Espectrometria de FluorescênciaRESUMO
Ureases are metalloenzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. Jack bean (Canavalia ensiformis) produces three isoforms of urease (Canatoxin, JBU and JBURE-II). Canatoxin and JBU display several biological properties independent of their ureolytic activity, such as neurotoxicity, exocytosis-inducing and pro-inflammatory effects, blood platelets activation, insecticidal and antifungal activities. The Canatoxin entomotoxic activity is mostly due to an internal peptide, named pepcanatox, released upon the hydrolysis of the protein by insect cathepsin-like digestive enzymes. Based on pepcanatox sequence, Jaburetox-2Ec was produced in Escherichia coli. JBU and its peptides were shown to permeabilize membranes through an ion channel-based mechanism. Here we studied the JBU and Jaburetox-2Ec interaction with platelet-like multilamellar liposomes (PML) using Dynamic Light Scattering and Small Angle X-ray Scattering techniques. We also analyzed the interaction of JBU with giant unilamellar vesicles (GUVs) using Fluorescence Microscopy. The interaction of vesicles with JBU led to a slight reduction of hydrodynamic radius, and caused an increase in the lamellar repeat distance of PML, suggesting a membrane disordering effect. In contrast, Jaburetox-2Ec decreased the lamellar repeat distance of PML membranes, while also diminishing their hydrodynamic radius. Fluorescence microscopy showed that the interaction of GUVs with JBU caused membrane perturbation with formation of tethers. In conclusion, JBU can interact with PML, probably by inserting its Jaburetox "domain" into the PML external membrane. Additionally, the interaction of Jaburetox-2Ec affects the vesicle's internal bilayers and hence causes more drastic changes in the PML membrane organization in comparison with JBU.
Assuntos
Canavalia/enzimologia , Lipossomos/metabolismo , Peptídeos/metabolismo , Urease/metabolismo , Difusão Dinâmica da Luz , Microscopia de Fluorescência , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Using giant unilamellar vesicles (GUVs) made from POPC, DPPC, cholesterol and a small amount of a porphyrin-based photosensitizer that we name PE-porph, we investigated the response of the lipid bilayer under visible light, focusing in the formation of domains during the lipid oxidation induced by singlet oxygen. This reactive species is generated by light excitation of PE-porf in the vicinity of the membrane, and thus promotes formation of hydroperoxides when unsaturated lipids and cholesterol are present. Using optical microscopy we determined the lipid compositions under which GUVs initially in the homogeneous phase displayed Lo-Ld phase separation following irradiation. Such an effect is attributed to the in situ formation of both hydroperoxized POPC and cholesterol. The boundary line separating homogeneous Lo phase and phase coexistence regions in the phase diagram is displaced vertically towards the higher cholesterol content in respect to ternary diagram of POPC:DPPC:cholesterol mixtures in the absence of oxidized species. Phase separated domains emerge from sub-micrometer initial sizes to evolve over hours into large Lo-Ld domains completely separated in the lipid membrane. This study provides not only a new tool to explore the kinetics of domain formation in mixtures of lipid membranes, but may also have implications in biological signaling of redox misbalance.
Assuntos
Luz , Peroxidação de Lipídeos/efeitos da radiação , Lipídeos de Membrana/química , Membranas Artificiais , Transição de Fase/efeitos da radiação , Processos Fotoquímicos/efeitos da radiaçãoRESUMO
We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid headgroup. We studied by optical microscopy the response to light irradiation of giant unilamellar vesicles of mixtures of unsaturated phosphatidylcholine lipids and PE-porph. In this configuration, singlet oxygen is produced at the bilayer surface by the anchored porphyrin. Under irradiation, the PE-porph decorated giant unilamellar vesicles exhibit a rapid increase in surface area with concomitant morphological changes. We quantify the surface area increase of the bilayers as a function of time and photosensitizer molar fraction. We attribute this expansion to hydroperoxide formation by the reaction of the singlet oxygen with the unsaturated bonds. Considering data from numeric simulations of relative area increase per phospholipid oxidized (15%), we measure the efficiency of the oxidative reactions. We conclude that for every 270 singlet oxygen molecules produced by the layer of anchored porphyrins, one eventually reacts to generate a hydroperoxide species. Remarkably, the integrity of the membrane is preserved in the full experimental range explored here, up to a hydroperoxide content of 60%, inducing an 8% relative area expansion.
Assuntos
Luz , Bicamadas Lipídicas/química , Estresse Oxidativo , Fosfatidiletanolaminas/química , Fármacos Fotossensibilizantes/química , Porfirinas/química , Lipossomas Unilamelares/química , Simulação por Computador , Fluorescência , Bicamadas Lipídicas/efeitos da radiação , Microscopia de Fluorescência , Modelos Químicos , Oxigênio/química , Fosfatidilcolinas/química , Fosfatidilcolinas/efeitos da radiação , Fosfatidiletanolaminas/efeitos da radiação , Fármacos Fotossensibilizantes/efeitos da radiação , Porfirinas/efeitos da radiação , Fatores de Tempo , Lipossomas Unilamelares/efeitos da radiaçãoRESUMO
Gliadin is a fraction of wheat gluten, a protein supramolecular complex known for its remarkable and biotechnologically relevant viscoelastic properties. Very high molecular mass characterise these systems, thus hindering high-resolution structural investigations. It is known, however, that these proteins comprise rather extended, extensively interassociated structures, which respond for their peculiar mechanical behaviour. Besides these properties, some of gluten's fractions, such as gliadin, are also known to be involved in a nutritionally relevant pathology of auto-immune character, the celiac disease, supposedly related to some unusual structural features of the protein. Despite its medical relevance, however, the role played by gliadin in the etiology of the celiac disease is not sufficiently understood to date. In this work, we investigated the role of gliadin on mechanical properties of a membrane model of dioleoylphosphatidylcholine (DOPC) giant unilamellar vesicles. The technique of micropipette aspiration, coupled to videomicroscopy, was applied. The microvesicles, produced by electric field pulsing over metal-covered plates, were suctioned into the micropipettes under varying applied pressures. A significant increase in the values of the bilayer curvature constant, k(c), was observed, with a saturation effect being verified at around 0.02-0.03 gliadin/DOPC mass ratio, indicating that the membrane becomes less elastic in the presence of the protein. Possible correlations between the observed membrane fluctuation properties and the celiac disease etiology are suggested and discussed.