RESUMEN
This study examined the influence of nanomaterials (NMs) on the organization of membrane lipids and the resulting morphological changes. The cell plasma membrane is heterogeneous, featuring specialized lipid domains in the liquid-ordered (Lo) phase surrounded by regions in the liquid-disordered (Ld) phase. We utilized model membranes composed of various lipids and lipid mixtures in different phase states to investigate the interactions between the NMs and membrane lipids. Specifically, we explored the interactions of pure chitosan (CS) and CS-modified nanocomposites (NCs) with ZnO, CuO, and SiO2 with four lipid mixtures: egg-phosphatidylcholine (EggPC), egg-sphingomyelin/cholesterol (EggSM/Chol), EggPC/Chol, and EggPC/EggSM/Chol, which represent the coexistence of Ld, Lo, and Ld/Lo, respectively. The data show that CS NMs increase the membrane lipid order at glycerol level probed by Laurdan spectroscopy. Additionally, the interaction of CS-based NMs with membranes leads to an increase in bending elasticity modulus, zeta potential, and vesicle size. The lipid order changes are most significant in the highly fluid Ld phase, followed by the Lo/Ld coexistence phase, and are less pronounced in the tightly packed Lo phase. CS NMs induced egg PC vesicle adhesion, fusion, and shrinking. In heterogeneous Lo/Ld membranes, inward invaginations and vesicle shrinking via the Ld phase were observed. These findings highlight mechanisms involved in CS NM-lipid interactions in membranes that mimic plasma membrane heterogeneity.
Asunto(s)
Quitosano , Quitosano/química , Nanoestructuras/química , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Nanocompuestos/química , Membranas Artificiales , Fosfatidilcolinas/química , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacosRESUMEN
The phytoalexin resveratrol has received increasing attention for its potential to prevent oxidative damages in human organism. To shed further light on molecular mechanisms of its interaction with lipid membranes we study resveratrol influence on the organisation and mechanical properties of biomimetic lipid systems composed of synthetic phosphatidylcholines with mixed aliphatic chains and different degree of unsaturation at sn-2 position (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC, and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, PDPC). High-sensitivity isothermal titration calorimetric measurements reveal stronger spontaneous resveratrol association to polyunsaturated phosphatidylcholine bilayers compared to the monounsaturated ones resulting from hydrophobic interactions, conformational changes of the interacting species and desolvation of molecular surfaces. The latter is supported by the results from Laurdan spectroscopy of large unilamellar vesicles providing data on hydration at the glycerol backbones of glycerophospholipides. Higher degree of lipid order is reported for POPC membranes compared to PDPC. While resveratrol mostly enhances the hydration of PDPC membranes, increasing POPC dehydration is reported upon treatment with the polyphenol. Dehydration of the polyunsaturated lipid bilayers is measured only at the highest phytoalexin content studied (resveratrol/lipid 0.5â¯mol/mol) and is less pronounced than the effect reported for POPC membranes. The polyphenol effect on membrane mechanics is probed by thermal shape fluctuation analysis of quasispherical giant unilamellar vesicles. Markedly different trend of the bending elasticity with increasing resveratrol concentration is reported for the two types of phospholipid bilayers studied. POPC membranes become more rigid in the presence of resveratrol, whereas PDPC-containing bilayers exhibit softening at lower concentrations of the polyphenol followed by a slight growth without bilayer stiffening even at the highest resveratrol content explored. The new data on the structural organization and membrane properties of resveratrol-treated phosphatidylcholine membranes may underpin the development of future liposomal applications of the polyphenol in medicinal chemistry.
Asunto(s)
Membrana Dobles de Lípidos , Resveratrol , Resveratrol/química , Membrana Dobles de Lípidos/química , Fosfatidilcolinas/química , Glicerofosfolípidos/química , Glicerofosfolípidos/metabolismo , Estilbenos/química , Materiales Biomiméticos/química , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismoRESUMEN
Resveratrol (Resv) is considered to exert a beneficial impact due to its radical scavenger, anti-microbial and anti-inflammatory properties through several mechanisms that could include its interaction with the cell plasma membrane. To address this issue, we investigated the influence of Resv on membrane lipid order and organization in large unilamellar vesicles composed of different lipids and ratios. The studied lipid membrane models were composed of phosphatidylcholine (PC) species (either palmitoyl-docosahexaenoyl phosphatidylcholine (PDPC) or palmitoyl-oleoyl phosphatidylcholine (POPC)), sphingomyelin (SM) and cholesterol (Chol). This study found that the addition of Resv resulted in complex membrane reorganization depending on the degree of fatty acid unsaturation at the sn-2 position, and the Lipid/Resv and SM/Chol ratios. Resv rigidified POPC-containing membranes and increased liquid-ordered (Lo) domain formation in 40/40/20 POPC/SM/Chol mixtures as this increase was lower at a 33/33/34 ratio. In contrast, Resv interacted with PDPC/SM/Chol mixtures in a bimodal manner by fluidizing/rigidifying the membranes in a dose-dependent way. Lo domain formation upon Resv addition occurred via the following bimodal mode of action: Lo domain size increased at low Resv concentrations; then, Lo domain size decreased at higher ones. To account for the variable effect of Resv, we suggest that it may act as a "spacer" at low doses, with a transition to a more "filler" position in the lipid bulk. We hypothesize that one of the roles of Resv is to tune the lipid order and organization of cell plasma membranes, which is closely linked to important cell functions such as membrane sorting and trafficking.
RESUMEN
The interplay between inflammatory and redox processes is a ubiquitous and critical phenomenon in cell biology that involves numerous biological factors. Among them, secretory phospholipases A2 (sPLA2) that catalyze the hydrolysis of the sn-2 ester bond of phospholipids are key players. They can interact or be modulated by the presence of truncated oxidized phosphatidylcholines (OxPCs) produced under oxidative stress from phosphatidylcholine (PC) species. The present study examined this important, but rarely considered, sPLA2 modulation induced by the changes in biophysical properties of PC vesicles comprising various OxPC ratios in mono- or poly-unsaturated PCs. Being the most physiologically active OxPCs, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) have been selected for our study. Using fluorescence spectroscopy methods, we compared the effect of OxPCs on the lipid order as well as sPLA2 activity in large unilamellar vesicles (LUVs) made of the heteroacid PC, either monounsaturated [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)], or polyunsaturated [1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC)] at a physiological temperature. The effect of OxPCs on vesicle size was also assessed in both the mono- and polyunsaturated PC matrices. Results: OxPCs decrease the membrane lipid order of POPC and PDPC mixtures with PGPC inducing a much larger decrease in comparison with POVPC, indicative that the difference takes place at the glycerol level. Compared with POPC, PDPC was able to inhibit sPLA2 activity showing a protective effect of PDPC against enzyme hydrolysis. Furthermore, sPLA2 activity on its PC substrates was modulated by the OxPC membrane content. POVPC down-regulated sPLA2 activity, suggesting anti-inflammatory properties of this truncated oxidized lipid. Interestingly, PGPC had a dual and opposite effect, either inhibitory or enhancing on sPLA2 activity, depending on the protocol of lipid mixing. This difference may result from the chemical properties of the shortened sn-2-acyl chain residues (aldehyde group for POVPC, and carboxyl for PGPC), being, respectively, zwitterionic or anionic under hydration at physiological conditions.
Asunto(s)
Biomimética , Fosfolipasas A2 Secretoras , Fosforilcolina , Fosfatidilcolinas/química , Fosfolípidos/metabolismo , LecitinasRESUMEN
Endogenous hemorphins are being intensively investigated as therapeutic agents in neuropharmacology, and also as biomarkers in mood regulation, inflammation and oncology. The datasets collected herein report physicochemical parameters of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes in the presence of VV-hemorphin-5 (Val-Val-Tyr-Pro-Trp-Thr-Gln) and analogues, modified at position 1 and 7 by the natural amino acid isoleucine or the non-proteinogenic 2-aminoisobutyric, 2,3-diaminopropanoic or 2,4-diaminobutanoic amino acids. These peptides have been previously screened for nociceptive activity and were chosen accordingly. The present article contains fluorescence spectroscopy data of Laurdan- and di-8-ANEPPS- labelled large unilamellar vesicles (LUV) providing the degree of hydration and dipole potential of lipid bilayers in the presence of VV-hemorphin-5 analogues. Lipid packing is accessible from Laurdan intensity profiles and generalized polarization datasets reported herein. The data presented on fluorescence intensity ratios of di-8-ANEPPS dye provide dipole potential values of phosphatidylcholine-valorphin membranes. Vesicle size and electrophoretic mobility datasets included refer to the effect of valorphins on the size distribution and ζ -potential of POPC LUVs. Investigation of physicochemical properties of peptides such as diffusion coefficients and heterogeneous rate constant relates to elucidation of transport mechanisms in living cells. Voltammetric data of valorphins are presented together with square-wave voltammograms of investigated peptides for calculation of their heterogeneous electron transfer rate constants. Datasets from the thermal shape fluctuation analysis of quasispherical 'giant' unilamellar vesicles (GUV) are provided to quantify the influence of hemorphin incorporation on the membrane bending elasticity. Isothermal titration calorimetric data on the thermodynamics of peptide-lipid interactions and the binding affinity of valorphin analogues to phosphatidylcholine membranes are reported. Data of frequency-dependent deformation of GUVs in alternating electric field are included together with the values of the specific electrical capacitance of POPC-valorphin membranes. The datasets reported in this article can underlie the formulation and implementation of peptide-based strategies in pharmacology and biomedicine.
RESUMEN
New analogues of the endogenous heptapeptide VV-hemorphin-5 (valorphin) synthesised by amino acid replacement allow for tailoring the peptide activity in vivo. Investigation of hemorphin-induced alterations of lipid bilayers' physicochemical parameters unravels membrane-mediated mechanisms of interaction with cells and subcellular structures. We studied the effect of modified valorphins with nociceptive activity on the structure, mechanical and electrical properties of lipid membrane models. Lower bending rigidity and higher specific capacitance of phosphatidylcholine bilayers were found in the presence of VV-hemorphin-5 analogues. Peptide partition constants for the transfer from the aqueous solution into the membrane were determined by isothermal titration calorimetry. It was found that the inclusion of non-proteinogenic acids with different number of methylene groups lead to alterations of hemorphin-membrane binding. The highest membrane affinity was obtained for a hemorphin derivative with dose-dependent variable effects on visceral nociception in mice. The valorphin analogue with the most pronounced anti-nociceptive effect in vivo induced the highest dipole and zeta potential change without significantly affecting the lipid packing at glycerol level in phosphatidylcholine bilayers.
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Hemoglobinas , Fosfatidilcolinas , Animales , Ratones , Hemoglobinas/química , Membrana Celular/metabolismo , Membrana Dobles de LípidosRESUMEN
Resveratrol is a naturally occurring polyphenol which has various beneficial effects, such as anti-inflammatory, anti-tumor, anti-aging, antioxidant, and neuroprotective effects, among others. The anti-cancer activity of resveratrol has been related to alterations in sphingolipid metabolism. We analyzed the effect of resveratrol on the enzymes responsible for accumulation of the two sphingolipids with highest functional activity-apoptosis promoting ceramide (CER) and proliferation-stimulating sphingosine-1-phosphate (S1P)-in human lung adenocarcinoma A549 cells. Resveratrol treatment induced an increase in CER and sphingosine (SPH) and a decrease in sphingomyelin (SM) and S1P. Our results showed that the most common mode of CER accumulation, through sphingomyelinase-induced hydrolysis of SM, was not responsible for a CER increase despite the reduction in SM in A549 plasma membranes. However, both the activity and the expression of CER synthase 6 were upregulated in resveratrol-treated cells, implying that CER was accumulated as a result of stimulated de novo synthesis. Furthermore, the enzyme responsible for CER hydrolysis, alkaline ceramidase, was not altered, suggesting that it was not related to changes in the CER level. The enzyme maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was downregulated, and its expression was reduced, resulting in a decrease in S1P levels in resveratrol-treated lung adenocarcinoma cells. In addition, incubation of resveratrol-treated A549 cells with the SK1 inhibitors DMS and fingolimod additionally downregulated SK1 without affecting its expression. The present studies provide information concerning the biochemical processes underlying the influence of resveratrol on sphingolipid metabolism in A549 lung cancer cells and reveal possibilities for combined use of polyphenols with specific anti-proliferative agents that could serve as the basis for the development of complex therapeutic strategies.
Asunto(s)
Adenocarcinoma del Pulmón , Fenómenos Bioquímicos , Fármacos Neuroprotectores , Células A549 , Adenocarcinoma del Pulmón/tratamiento farmacológico , Ceramidasa Alcalina/metabolismo , Antioxidantes , Ceramidas/metabolismo , Clorhidrato de Fingolimod , Humanos , Lisofosfolípidos/metabolismo , Polifenoles , Resveratrol/farmacología , Esfingolípidos/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
Studies have been carried out on the effects of the phenyl glycoside myconoside, extracted from the relict, Balkan endemic resurrection plant Haberlea rhodopensis on the plasma membrane structural organization and the actin cytoskeleton. Because the plasma membrane is the first target of exogenous bioactive compounds, we focused our attention on the influence of myconoside on the membrane lipid order and actin cytoskeleton in human lung adenocarcinoma A549 cells, using fluorescent spectroscopy and microscopy techniques. We found that low myconoside concentration (5 µg/ml) did not change cell viability but was able to increase plasma membrane lipid order of the treated cells. Higher myconoside concentration (20 µg/ml) inhibited cell viability by decreasing plasma membrane lipid order and impairing actin cytoskeleton. We hypothesize that the observed changes in the plasma membrane structural organization and the actin cytoskeleton are functionally connected to cell viability. Biomimetic membranes were used to demonstrate that myconoside is able to reorganize the membrane lipids by changing the fraction of sphingomyelin-cholesterol enriched domains. Thus, we propose a putative mechanism of action of myconoside on A549 cells plasma membrane lipids as well as on actin filaments in order to explain its cytotoxic effect at high myconoside concentration.
Asunto(s)
Actinas , Adenocarcinoma del Pulmón , Células A549 , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Adenocarcinoma del Pulmón/metabolismo , Membrana Celular/metabolismo , HumanosRESUMEN
The phase separation of aminophospholipids in glycerophospholipid matrix and the effect of cholesterol were studied by means of fluorescence microscopy of giant unilamellar vesicles (GUV). GUVs were composed of binary mixtures, egg yolk phosphatidylcholine (eggPC)/egg yolk phosphatidylethanolamine (eggPE) and egg yolk phosphatidylcholine (eggPC)/brain phosphatidylserine (brainPS), and ternary ones with both aminophospholipids (eggPC/eggPE/brainPS). Gel/liquid-disordered phase coexistence was detected in these mixtures, where aminophospholipids segregate in gel leaf-like domains. When cholesterol (CHOL) was added, the phase separation was shifted at lower temperatures. CHOL increases miscibility of aminophospholipids in PC matrix. Addition of PE and PS to the ternary mixtures (eggPC/eggSM/CHOL) induced liquid-ordered domain formation at higher temperatures. Based on these results, one can conclude that aminophospholipids promote the formation of Lo domains.
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Colesterol/química , Membrana Dobles de Lípidos/química , Microdominios de Membrana/química , Membranas Artificiales , Microscopía Fluorescente/métodos , Fosfolípidos/química , Liposomas Unilamelares/químicaRESUMEN
The effect of integrin receptors on the level and transmembrane localization of cholesterol molecules was investigated in beta1 integrin-expressing (beta1) and beta1 integrin-deficient (beta1 null) cells. We found that the content of specific raft components-cholesterol, sphingomyelin, and caveolin-was increased in integrin-expressing cells. Integrin presence affected as well the transmembrane distribution of cholesterol-a higher percent was found in the plasma membrane outer monolayer of beta1 compared to beta1 null cells. Sphingomyelin depletion reduced the presence of cholesterol in the outer membrane monolayer of both cell lines, but the differences in cholesterol asymmetry, observed between beta1 and beta1 null cells before sphingomyelinase treatment were preserved. These findings implied that integrin receptors affected the non-random transmembrane distribution of cholesterol. Finally, a higher percent of detergent-resistant membranes was obtained from beta1 integrin-expressing cells, suggesting that the presence of these receptors in the membranes influenced the formation and/or stabilization of lipid raft domains.