RESUMO
In hormone-responsive breast cancer cells, progesterone (P4) has been shown to act via its nuclear receptor (nPR), a ligand-activated transcription factor. A small fraction of progesterone receptor is palmitoylated and anchored to the cell membrane (mbPR) forming a complex with estrogen receptor alpha (ERα). Upon hormone exposure, either directly or via interaction with ERα, mbPR activates the SRC/RAS/ERK kinase pathway leading to phosphorylation of nPR by ERK. Kinase activation is essential for P4 gene regulation, as the ERK and MSK1 kinases are recruited by the nPR to its genomic binding sites and trigger chromatin remodeling. An interesting open question is whether activation of mbPR can result in gene regulation in the absence of ligand binding to intracellular progesterone receptor (iPR). This matter has been investigated in the past using P4 attached to serum albumin, but the attachment is leaky and albumin can be endocytosed and degraded, liberating P4. Here, we propose a more stringent approach to address this issue by ensuring attachment of P4 to the cell membrane via covalent binding to a stable phospholipid. This strategy identifies the actions of P4 independent from hormone binding to iPR. We found that a membrane-attached progestin can activate mbPR, the ERK signaling pathway leading to iPR phosphorylation, initial gene regulation and entry into the cell cycle, in the absence of detectable intracellular progestin.
Assuntos
Neoplasias , Progesterona , Progesterona/farmacologia , Receptores de Progesterona/genética , Receptor alfa de Estrogênio , Progestinas/farmacologia , Ligantes , Membrana CelularRESUMO
In macroautophagy, the autophagosome (AP) engulfs portions of cytoplasm to allow their lysosomal degradation. AP formation in humans requires the concerted action of the ATG12 and LC3/GABARAP conjugation systems. The ATG12-ATG5-ATG16L1 or E3-like complex (E3 for short) acts as a ubiquitin-like E3 enzyme, promoting LC3/GABARAP proteins anchoring to the AP membrane. Their role in the AP expansion process is still unclear, in part because there are no studies comparing six LC3/GABARAP family member roles under the same conditions, and also because the full human E3 was only recently available. In the present study, the lipidation of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of E3, and the mechanisms by which E3 and LC3/GABARAP proteins participate in vesicle tethering and fusion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggest the existence of a lipidation threshold, lower for the GABARAP subfamily, as a requisite for tethering and inter-vesicular lipid mixing. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However, E3 hampers LC3/GABARAP capacity to induce inter-vesicular lipid mixing or subsequent fusion, presumably through the formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulatory mechanism would allow vesicle incorporation and phagophore growth when E3 is present.
Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos , Humanos , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Autofagossomos/metabolismo , Lipídeos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 12 Relacionada à Autofagia , Proteína 5 Relacionada à Autofagia/genéticaRESUMO
Aß peptides are known to bind neural plasma membranes in a process leading to the deposit of Aß-enriched plaques. These extracellular structures are characteristic of Alzheimer's disease, the major cause of late-age dementia. The mechanisms of Aß plaque formation and deposition are far from being understood. A vast number of studies in the literature describe the efforts to analyze those mechanisms using a variety of tools. The present review focuses on biophysical studies mostly carried out with model membranes or with computational tools. This review starts by describing basic physical aspects of lipid phases and commonly used model membranes (monolayers and bilayers). This is followed by a discussion of the biophysical techniques applied to these systems, mainly but not exclusively Langmuir monolayers, isothermal calorimetry, density-gradient ultracentrifugation, and molecular dynamics. The Methodological Section is followed by the core of the review, which includes a summary of important results obtained with each technique. The last section is devoted to an overall reflection and an effort to understand Aß-bilayer binding. Concepts such as Aß peptide membrane binding, adsorption, and insertion are defined and differentiated. The roles of membrane lipid order, nanodomain formation, and electrostatic forces in Aß-membrane interaction are separately identified and discussed.
Assuntos
Peptídeos beta-Amiloides , Bicamadas Lipídicas , Lipídeos de Membrana , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Humanos , Bicamadas Lipídicas/metabolismo , Bicamadas Lipídicas/química , Lipídeos de Membrana/metabolismo , Lipídeos de Membrana/química , Ligação Proteica , Membrana Celular/metabolismo , Doença de Alzheimer/metabolismo , Animais , Fenômenos Biofísicos , Simulação de Dinâmica MolecularRESUMO
N-maleimide-derivatized phospholipids are often used to facilitate protein anchoring to membranes. In autophagy studies, this is applied to the covalent binding of Atg8, an autophagy protein, to a phosphatidylethanolamine (PE) in the nascent autophagosome. However, the question remains on how closely the N-maleimide PE derivative (PE-mal) mimicks the native PE in the bilayer. In the present paper, spectroscopic and calorimetric techniques have been applied to vesicles containing either PE or PE-mal (together with other phospholipids) to compare the properties of the native and derivatized forms of PE. According to differential scanning calorimetry, and to infrared spectroscopy, the presence of PE-mal did not perturb the fatty acyl chains in the bilayer. Fluorescence spectroscopy and microscopy showed that PE-mal did not alter the bilayer permeability either. However, fluorescence emission polarization of the Laurdan and DPH probes indicated an increased order, or decreased fluidity, in the bilayers containing PE-mal. In addition, the infrared spectral data from the phospholipid phosphate region revealed a PE-mal-induced conformational change in the polar heads, accompanied by increased hydration. Globally considered, the results suggest that PE-mal would be a reasonable substitute for PE in model membranes containing reconstituted proteins.
Assuntos
Bicamadas Lipídicas , Fosfatidiletanolaminas , Bicamadas Lipídicas/química , Fosfatidiletanolaminas/química , Fosfolipídeos/química , Membranas , Maleimidas , Varredura Diferencial de CalorimetriaRESUMO
Cardiolipin (CL) is a key lipid for damaged mitochondrial recognition by the LC3/GABARAP human autophagy proteins. The role of ceramide (Cer) in this process is unclear, but CL and Cer have been proposed to coexist in mitochondria under certain conditions. Varela et al. showed that in model membranes composed of egg sphingomyelin (eSM), dioleoyl phosphatidylethanolamine (DOPE), and CL, the addition of Cer enhanced the binding of LC3/GABARAP proteins to bilayers. Cer gave rise to lateral phase separation of Cer-rich rigid domains but protein binding took place mainly in the fluid continuous phase. In the present study, a biophysical analysis of bilayers composed of eSM, DOPE, CL, and/or Cer was attempted to understand the relevance of this lipid coexistence. Bilayers were studied by differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy. Upon the addition of CL and Cer, one continuous phase and two segregated ones were formed. In bilayers with egg phosphatidylcholine instead of eSM, in which the binding of LC3/GABARAP proteins hardly increased with Cer in the former study, a single segregated phase was formed. Assuming that phase separation at the nanoscale is ruled by the same principles acting at the micrometer scale, it is proposed that Cer-enriched rigid nanodomains, stabilized by eSM:Cer interactions formed within the DOPE- and CL-enriched fluid phase, result in structural defects at the rigid/fluid nanointerfaces, thus hypothetically facilitatingLC3/GABARAP protein interaction.
Assuntos
Cardiolipinas , Ceramidas , Humanos , Ceramidas/química , Bicamadas Lipídicas/química , Macroautofagia , Esfingomielinas/química , MitocôndriasRESUMO
Sphingomyelin has been considered as a merely structural lipid for many years. However, this organelle-specific lipid has many other roles, including increasing membrane molecular order, acting as a source of ceramide in cell signaling and apoptosis, and forming clusters/nanodomains with cholesterol and ceramide. This contribution is dedicated to Professor E. Carafoli, on occasion of his 90th anniversary.
Assuntos
Ceramidas , Esfingomielinas , Esfingomielinas/química , Ceramidas/química , Colesterol/química , Apoptose , Esfingomielina FosfodiesteraseRESUMO
Sphingolipids (SL) are ubiquitous in mammalian cell membranes, yet there is little data on the behavior of cells under SL-restriction conditions. LY-B cells derive from a CHO linein whichserine palmitoyl transferase (SPT), thus de novo SL synthesis, is suppressed, while maintaining the capacity of taking up and metabolizing exogenous sphingoid bases from the culture medium. In this study, LY-B cells were adapted to grow in a fetal bovine serum (FBS)-deficient medium to avoid external uptake of lipids. The lowest FBS concentration that allowed LY-B cell growth, though at a slow rate, under our conditions was 0.04%, that is, 250-fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72 hours. Enriching with sphingomyelin the SL-deficient medium allowed the recovery of growth rates analogous to those of control LY-B cells. Studies including whole cells, plasma membrane preparations, and derived lipid vesicles were carried out. Laurdan fluorescence was recorded to measure membrane molecular order, showing a significant decrease in the rigidity of LY-B cells, not only in plasma membrane but also in whole cell lipid extract, as a result of SL limitation in the growth medium. Plasma membrane preparations and whole cell lipid extracts were also studied using atomic force microscopy in the force spectroscopy mode. Force measurements demonstrated that lower breakthrough forces were required to penetrate samples obtained from SL-poor LY-B cells than those obtained from control cells. Mass-spectroscopic analysis was also a helpful tool to understand the rearrangement undergone by the LY-B cell lipid metabolism. The most abundant SL in LY-B cells, sphingomyelin, decreased by about 85% as a result of SL limitation in the medium, the bioactive lipid ceramide and the ganglioside precursor hexosylceramide decreased similarly, together with cholesterol. Quantitative SL analysis showed that a 250-fold reduction in sphingolipid supply to LY-B cells led only to a sixfold decrease in membrane sphingolipids, underlining the resistance to changes in composition of these cells. Plasma membrane compositions exhibited similar changes, at least qualitatively, as the whole cells with SL restriction. A linear correlation was observed between the sphingomyelin concentration in the membranes, the degree of lipid order as measured by laurdan fluorescence, and membrane breakthrough forces assessed by atomic force microscopy. Smaller, though significant, changes were also detected in glycerophospholipids under SL-restriction conditions.
Assuntos
Membrana Celular/metabolismo , Proliferação de Células , Glicerofosfolipídeos/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Esfingolipídeos/metabolismo , Animais , Células CHO , Cricetinae , CricetulusRESUMO
This work intends to describe the physical properties of red blood cell (RBC) membranes in obese adults. The hypothesis driving this research is that obesity, in addition to increasing the amount of body fat, will also modify the lipid composition of membranes in cells other than adipocytes. Forty-nine control volunteers (16 male, 33 female, BMI 21.8 ± 5.6 and 21.5 ± 4.2 kg/m2, respectively) and 52 obese subjects (16 male and 36 female, BMI 38.2± 11.0 and 40.7 ± 8.7 kg/m2, respectively) were examined. The two physical techniques applied were atomic force microscopy (AFM) in the force spectroscopy mode, which allows the micromechanical measurement of penetration forces, and fluorescence anisotropy of trimethylammonium diphenylhexatriene (TMA-DPH), which provides information on lipid order at the membrane polar-nonpolar interface. These techniques, in combination with lipidomic studies, revealed a decreased rigidity in the interfacial region of the RBC membranes of obese as compared to control patients, related to parallel changes in lipid composition. Lipidomic data show an increase in the cholesterol/phospholipid mole ratio and a decrease in sphingomyelin contents in obese membranes. ω-3 fatty acids (e.g., docosahexaenoic acid) appear to be less prevalent in obese patient RBCs, and this is the case for both the global fatty acid distribution and for the individual major lipids in the membrane phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS). Moreover, some ω-6 fatty acids (e.g., arachidonic acid) are increased in obese patient RBCs. The switch from ω-3 to ω-6 lipids in obese subjects could be a major factor explaining the higher interfacial fluidity in obese patient RBC membranes.
Assuntos
Difenilexatrieno/análogos & derivados , Membrana Eritrocítica/fisiologia , Lipidômica/métodos , Obesidade/diagnóstico por imagem , Adolescente , Adulto , Fenômenos Biomecânicos , Estudos de Casos e Controles , Difenilexatrieno/administração & dosagem , Membrana Eritrocítica/metabolismo , Feminino , Polarização de Fluorescência , Humanos , Masculino , Microscopia de Força Atômica , Pessoa de Meia-Idade , Obesidade/metabolismo , Obesidade/fisiopatologia , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Adulto JovemRESUMO
Lipid model membranes are important tools in the study of biophysical processes such as lipid self-assembly and lipid-lipid interactions in cell membranes. The use of model systems to adequate and modulate complexity helps in the understanding of many events that occur in cellular membranes, that exhibit a wide variety of components, including lipids of different subfamilies (e.g., phospholipids, sphingolipids, sterols ), in addition to proteins and sugars. The capacity of lipids to segregate by themselves into different phases at the nanoscale (nanodomains) is an intriguing feature that is yet to be fully characterized in vivo due to the proposed transient nature of these domains in living systems. Model lipid membranes, instead, have the advantage of (usually) greater phase stability, together with the possibility of fully controlling the system lipid composition. Atomic force microscopy (AFM) is a powerful tool to detect the presence of meso- and nanodomains in a lipid membrane. It also allows the direct quantification of nanomechanical resistance in each phase present. In this review, we explore the main kinds of lipid assemblies used as model membranes and describe AFM experiments on model membranes. In addition, we discuss how these assemblies have extended our knowledge of membrane biophysics over the last two decades, particularly in issues related to the variability of different model membranes and the impact of supports/cytoskeleton on lipid behavior, such as segregated domain size or bilayer leaflet uncoupling.
Assuntos
Bicamadas Lipídicas/química , Lipídeos/química , Microscopia de Força Atômica/métodos , Biofísica , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Fosfolipídeos/química , Esfingolipídeos/químicaRESUMO
This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4-6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30-32 C atoms in their acyl chains but were relatively poor in those containing 34-40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.
Assuntos
Membrana Celular/química , Fenômenos Químicos , Animais , Células CHO , Membrana Celular/ultraestrutura , Cricetulus , Lipídeos de Membrana/química , Lipídeos de Membrana/isolamento & purificação , Microscopia de Força Atômica , Análise Espectral , Coloração e RotulagemRESUMO
The binding of Aß42 peptide monomers to sphingomyelin/cholesterol (1:1 mol ratio) bilayers containing 5 mol% gangliosides (either GM1, or GT1b, or a mixture of brain gangliosides) has been assayed by density gradient ultracentrifugation. This procedure provides a direct method for measuring vesicle-bound peptides after non-bound fraction separation. This centrifugation technique has rarely been used in this context previously. The results show that gangliosides increase by about two-fold the amount of Aß42 bound to sphingomyelin/cholesterol vesicles. Complementary studies of the same systems using thioflavin T fluorescence, Langmuir monolayers or infrared spectroscopy confirm the ganglioside-dependent increased binding. Furthermore these studies reveal that gangliosides facilitate the aggregation of Aß42 giving rise to more extended ß-sheets. Thus, gangliosides have both a quantitative and a qualitative effect on the binding of Aß42 to sphingomyelin/cholesterol bilayers.
Assuntos
Peptídeos beta-Amiloides/química , Colesterol/química , Gangliosídeos/química , Fragmentos de Peptídeos/química , Esfingomielinas/química , Fenômenos Biofísicos , Centrifugação com Gradiente de Concentração , Humanos , Bicamadas Lipídicas/química , Lipossomos/química , Ligação ProteicaRESUMO
Sphingolipids constitute a significant fraction of cellular plasma membrane lipid content. Among sphingolipids, ceramide levels are usually very low. However, in some cell processes like apoptosis, cell membrane ceramide levels increase markedly because of the activation of enzymes like acid sphingomyelinase. This increase can change the physical state of the membrane by promoting molecular order and inducing solid-ordered (So) phase domains. This effect has been observed in a previous 2H NMR study on membranes consisting of palmitoyl sphingomyelin (PSM) and palmitoyl ceramide (PCer). Cholesterol (Chol), too, is present at high concentrations in mammalian plasma membranes and has a favorable interaction with sphingomyelin (SM), together forming domains in the liquid-ordered phase in model membranes. There are reports that Chol is able to displace ceramide (Cer) in SM bilayers and abolish the So phase domains formed by SM:Cer. This ability of Chol appears to be concentration dependent; in membranes with low Chol and high Cer contents, So phase domains rich in Cer coexist with the continuous fluid phase of the membrane. Here, we studied the effect of increasing PCer concentration in PSM:Chol bilayers, using 2H NMR. Chol:PCer mole ratios were 3:1, 3:2, and 3:3, at a fixed 7:3 phospholipid:cholesterol mol ratio. Both PSM and PCer were monitored in separate samples for changes in their physical state by introducing a perdeuterated palmitoyl chain in either molecule. Moreover, the effect of replacing PSM with DPPC was investigated to test the impact on membrane phase behavior of replacing the sphingosine with a palmitoylated glycerol backbone. We found that PCer can increase acyl chain order in both PSM:Chol and DPPC:Chol bilayers. Especially in bilayers with Chol:PCer 1:1 molar ratios, PCer induces highly stable So phase domains in both PSM and DPPC bilayers near 37°C. However, PCer has a more pronounced ordering effect on PSM compared to DPPC bilayers.
Assuntos
Ceramidas/química , Colesterol/química , Deutério/química , Espectroscopia de Ressonância Magnética , Fosfolipídeos/química , TemperaturaRESUMO
Cell membranes have been proposed to be laterally inhomogeneous, particularly in the case of mammalian cells, due to the presence of "domains" enriched in sphingolipids and cholesterol (Chol). Among membrane sphingolipids, sphingomyelin (SM) in the cell plasma membrane is known to be degraded to ceramide (Cer) by acid sphingomyelinases under stress conditions. Since cholesterol (Chol) is abundant in the plasma membrane, the study of ternary mixtures SM:Chol:Cer is interesting from the point of view of membrane biophysics, and it might be physiologically relevant. In previous studies, we have described the homogeneous gel phase formed by phospholipid:Chol:Cer at 54:23:23 mol ratios, where phospholipid was either SM or dipalmitoylphosphatidylcholine (DPPC). We now provide new data, based on trans-parinaric acid and diphenylhexatriene fluorescence, supporting that the gel phase includes all three components in a single bilayer. The main question addressed in this paper is the stability of the ternary gel phase when bilayer composition is changed, specifically when the SM proportion is varied. To this aim, we have prepared bilayers of composition phospholipid:Chol:Cer at X:Y:Y ratios, in which phospholipid increased between 54 and 70 mol %. The N-palmitoyl derivatives of SM (pSM) and Cer (pCer) have been used. We observe that for X = 54 or 60 mol %, a gel phase is clearly predominant. However, when the proportion of phospholipid increases beyond 60 mol %, i.e., in 66:17:17 or 70:15:15 mixtures, a lateral phase separation occurs at the micrometer scale. These data can be interpreted in terms of a pCer:Chol interaction, that would predominate at the lower phospholipid concentrations. The putative pCer:Chol complexes (or nanodomains) would mix well with the phospholipid. At the higher SM concentrations pSM:pCer and pSM:Chol interactions would become more important, giving rise to the coexisting gel and liquid-ordered phases respectively. Heterogeneity, or lateral phase separation, occurs more easily with pSM than with DPPC, indicating a higher affinity of SM over DPPC for Chol or Cer. The observation that heterogeneity, or lateral phase separation, occurs more easily with pSM than with DPPC, indicates a higher affinity of SM over DPPC for Chol or Cer, and can be related to cell regulation through the sphingolipid signaling pathway.
Assuntos
Ceramidas/química , Colesterol/química , Bicamadas Lipídicas/química , 1,2-Dipalmitoilfosfatidilcolina/sangue , Anisotropia , Varredura Diferencial de Calorimetria , Polarização de Fluorescência , Microscopia de Força Atômica , Microscopia Confocal , Fosfolipídeos/química , Esfingomielinas/química , Lipossomas Unilamelares/químicaRESUMO
Sphingosine [(2 S,3 R,4 E)-2-amino-4-octadecene-1,3-diol] is the most common sphingoid base in mammals. Ceramides are N-acyl sphingosines. Numerous small variations on this canonical structure are known, including the 1-deoxy, the 4,5-dihydro, and many others. However, whenever there is a Δ4 double bond, it adopts the trans (or E) configuration. We synthesized a ceramide containing 4 Z-sphingosine and palmitic acid ( cis-pCer) and studied its behavior in the form of monolayers extended on an air-water interface. cis-pCer acted very differently from the trans isomer in that, upon lateral compression of the monolayer, a solid-solid transition was clearly observed at a mean molecular area ≤44 Å2·molecule-1, whose characteristics depended on the rate of compression. The solid-solid transition, as well as states of domain coexistence, could be imaged by atomic force microscopy and by Brewster-angle microscopy. Atomistic molecular dynamics simulations provided results compatible with the experimentally observed differences between the cis and trans isomers. The data can help in the exploration of other solid-solid transitions in lipids, both in vitro and in vivo, that have gone up to now undetected because of their less obvious change in surface properties along the transition, as compared to cis-pCer.
RESUMO
Although detergents are routine tools in biomembrane research, their use remains empirical. We propose that solubilization is the result of a balance between two parameters: (i) the energy associated with bending of phospholipid monolayers into a curved micellar surface, and (ii) the energy associated with filling the void in the center of the resultant mixed micelle. In this review, we show that reliable data on the phase boundaries, and their dependence on various conditions, are consistent with this hypothesis, even if the data might have been interpreted differently. Although most of the experimental data discussed here were obtained with the non-ionic detergent Triton X-100, the conclusions should be applicable to a wide variety of detergents.
Assuntos
Detergentes/química , Bicamadas Lipídicas/química , Micelas , Fosfolipídeos/química , Transferência de Energia , Cinética , Modelos Químicos , Modelos Moleculares , Octoxinol/química , SolubilidadeRESUMO
Ceramide is a sphingolipid involved in several cellular processes, including apoptosis. It has been proposed that ceramide forms large and stable channels in the mitochondrial outer membrane that induce cell death through direct release of cytochrome c. However, this mechanism is still debated because the membrane permeabilizing activity of ceramide remains poorly understood. To determine whether the mechanism of ceramide-induced membrane leakage is consistent with the hypothesis of an apoptotic ceramide channel, we have used here assays of calcein release from liposomes. When assaying liposomes containing sphingomyelin and cholesterol, we observed an overall gradual phenomenon of contents release, together with some all-or-none leakage (at low ceramide concentrations or short times). The presence of channels in the bilayer should cause only an all-or-none leakage. When liposomes poor in sphingomyelin/cholesterol or mimicking the lipid composition of the mitochondrial outer membrane were tested, we did not detect any leakage. In consequence, the hypothesis of formation of large ceramide channels in the membrane is not consistent with our results. Instead we propose that the presence of ceramide in one of the membrane monolayers causes a surface area mismatch between both monolayers, which leads to vesicle collapse. The gradual phenomenon of calcein release would be due to a competition between two ceramide effects; namely, lateral segregation that facilitates permeabilization, and at longer times, trans-bilayer flip-flop that opposes asymmetric lateral segregation and causes a mismatch.
Assuntos
Ceramidas/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colesterol/metabolismo , Relação Dose-Resposta a Droga , Fluoresceínas/metabolismo , Lipossomos/metabolismo , Esfingomielinas/metabolismoRESUMO
The immunity proteins against pore-forming colicins represent a family of integral membrane proteins that reside in the inner membrane of producing cells. Cai, the colicin A immunity protein, was characterized here in detergent micelles by circular dichroism (CD), size exclusion chromatography, chemical cross-linking, nuclear magnetic resonance (NMR) spectroscopy, cysteine accessibility, and colicin A binding in detergent micelles. Bile-salt derivatives induced extensive protein polymerization that precluded further investigation. The physical characterization of detergent-solubilized protein indicates that phosphate-containing detergents are more efficient in extracting, solubilizing and maintaining Cai in a monomeric state. Yet, their capacity to ensure protein activity, reconstitution, helix packing, and high-quality NMR spectra was inferior to that of milder detergents. Solvent ionic strength and composition greatly modified the solubilizing capacity of milder detergents. Most importantly, binding to the colicin A pore-forming domain (pf-ColA) occurred almost exclusively in sugar-derived detergents. The relative performance of the different detergents in each experiment depends on their impact not only on Cai structure, solubility and oligomerization state, but also on other reaction components and technical aspects. Thus, proteoliposomes were best obtained from protein in LDAO micelles, possibly also due to indirect effects on the lipidic bilayer. The compatibility of a detergent with Cai/pf-ColA complex formation is influenced by its effect on the conformational landscape of each protein, where detergent-mediated pf-ColA denaturation could also lead to negative results. The NMR spectra were greatly affected by the solubility, monodispersity, fold and dynamics of the protein-detergent complexes, and none of those tested here provided NMR spectra of sufficient quality to allow for peak assignment. Cai function could be proven in alkyl glycosides and not in those detergents that afforded the best solubility, reconstitution efficiency or spectral quality indicating that these criteria cannot be taken as unambiguous proof of nativeness without the support of direct activity measurements.
Assuntos
Colicinas/química , Colicinas/isolamento & purificação , Detergentes/química , Micelas , Sequência de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Detergentes/farmacologia , Escherichia coli/química , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Análise de Sequência de Proteína , SolubilidadeRESUMO
Human phospholipid scramblase 1 (SCR) is a membrane protein that catalyzes the transmembrane (flip-flop) motion of phospholipids. It can also exist in a non membrane-bound form in the nucleus, where it modulates several aspects of gene expression. Catalysis of phospholipid flip-flop requires the presence of millimolar Ca2+, and occurs in the absence of ATP. Membrane-bound SCR contains a C-terminal α-helical domain embedded in the membrane bilayer. The latter domain can be removed giving rise to a stable truncated mutant SCRΔ that is devoid of scramblase activity. In order to improve our understanding of SCR structure infrared spectra have been recorded of both the native and truncated forms, and the effects of adding Ca2+, or removing detergent, or thermally denaturing the protein have been observed. Under all conditions the main structural component of SCR/SCRΔ is a ß-sheet. Removing the C-terminal 28 aa residues, which anchor SCR to the membrane, leads to a change in tertiary structure and an increased structural flexibility. The main effect of Ca2+ is an increase in the α/ß ratio of secondary structure components, with a concomitant increase in the proportion of non-periodic structures. At least in SCRΔ, detergent (Zwittergent 3-12) decreases the structural flexibility, an effect somewhat opposite to that of increasing temperature. Thermal denaturation is affected by Ca2+, detergent, and by the presence or absence of the C-terminal domain, each of them influencing in different ways the denaturation pattern.
Assuntos
Cálcio/farmacologia , Detergentes/farmacologia , Proteínas de Transferência de Fosfolipídeos/química , Espectrofotometria Infravermelho/métodos , Humanos , Conformação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , TemperaturaRESUMO
The effects of C24:1 sphingolipids have been tested in phospholipid bilayers containing cholesterol. Confocal microscopy, differential scanning calorimetry, and atomic force microscopy imaging and force curves have been used. More precisely, the effects of C24:1 ceramide (nervonoyl ceramide, nCer) were evaluated and compared to those of C16:0 ceramide (palmitoyl ceramide, pCer) in bilayers composed basically of dioleoylphosphatidylcholine, sphingomyelin (either C24:1, nSM or C16:0, pSM) and cholesterol. Combination of equimolecular amounts of C24:1 and C16:0 sphingolipids were also studied under the same conditions. Results show that both pCer and nCer are capable of forming segregated gel domains. Force spectroscopy data point to nCer having a lower stiffening effect than pCer, while the presence of nSM reduces the stiffness. DSC reveals Tm reduction by nSM in every case. Furthermore, pSM seems to better accommodate both ceramides in a single phase of intermediate properties, while nSM partial accommodation of ceramides generates different gel phases with higher stiffnesses caused by interceramide cooperation. If both pSM and nSM are present, a clear preference of both ceramides toward pSM is observed. These findings show the sharp increase in complexity when membranes exhibit different sphingolipids of varying N-acyl chains, which should be a common issue in an actual cell membrane environment.
RESUMO
Toxicity evaluation for the proper use of graphene oxide (GO) in biomedical applications involving intravenous injections is crucial, but the GO circulation time and blood interactions are largely unknown. It is thought that GO may cause physical disruption (hemolysis) of red blood cells. The aim of this work is to characterize the interaction of GO with model and cell membranes and use this knowledge to improve GO hemocompatibility. We have found that GO interacts with both neutral and negatively charged lipid membranes; binding is decreased beyond a certain concentration of negatively charged lipids and favored in high-salt buffers. After this binding occurs, some of the vesicles remain intact, while others are disrupted and spread over the GO surface. Neutral membrane vesicles tend to break down and extend over the GO, while vesicles with negatively charged membranes are mainly bound to the GO without disruption. GO also interacts with red blood cells and causes hemolysis; hemolysis is decreased when GO is previously coated with lipid membranes, particularly with pure phosphatidylcholine vesicles.