Un entorno lipídico desfavorable disminuye la expresión de caveolina-1 en placentas preeclámpticas

La Caveolina-1 (Cav-1) es una proteína crucial en la formación de las caveolas, microdominios rígidos de membrana ricos en colesterol y esfingolípidos. Está involucrada en muchos procesos celulares como la diferenciación celular, donde regularía negativamente del ciclo celular, retardando la mitosis con el fin de detener la proliferación y conducir a la célula a su diferenciación. Contrariamente a lo que ocurre en otros epitelios, la diferenciación del trofoblasto se asocia con una reducción del número de caveolas, acompañado con una disminución de la expresión de Cav-1 y una modificación la composición de fosfolípidos de la membrana plasmática del sincitiotrofoblasto (hST) resultantes de la fusión con las células de citotrofoblasto. Este proceso es esencial para el mantenimiento de un embarazo exitoso y alteraciones se asocian a condiciones patológicas tales como la preeclampsia. En placentas preclámpticas (PE), se encontraron alteraciones en la composición lipídica de la membrana de hST y expresiones anormales de proteínas de transporte que alterarían las funciones de la placenta. Sin embargo, si estos cambios en la composición lipídica de la membrana del hST pueden afectar la expresión de Cav-1 en placentas preeclámpticas se desconocen.Facultad de Ciencias Médica

Un entorno lipídico desfavorable disminuye la expresión de caveolina-1 en placentas preeclámpticas

La Caveolina-1 (Cav-1) es una proteína crucial en la formación de las caveolas, microdominios rígidos de membrana ricos en colesterol y esfingolípidos. Está involucrada en muchos procesos celulares como la diferenciación celular, donde regularía negativamente del ciclo celular, retardando la mitosis con el fin de detener la proliferación y conducir a la célula a su diferenciación. Contrariamente a lo que ocurre en otros epitelios, la diferenciación del trofoblasto se asocia con una reducción del número de caveolas, acompañado con una disminución de la expresión de Cav-1 y una modificación la composición de fosfolípidos de la membrana plasmática del sincitiotrofoblasto (hST) resultantes de la fusión con las células de citotrofoblasto. Este proceso es esencial para el mantenimiento de un embarazo exitoso y alteraciones se asocian a condiciones patológicas tales como la preeclampsia. En placentas preclámpticas (PE), se encontraron alteraciones en la composición lipídica de la membrana de hST y expresiones anormales de proteínas de transporte que alterarían las funciones de la placenta. Sin embargo, si estos cambios en la composición lipídica de la membrana del hST pueden afectar la expresión de Cav-1 en placentas preeclámpticas se desconocen.Facultad de Ciencias Médica

The unfavorable lipid environment reduced caveolin-1 expression in apical membranes from human preeclamptic placentas

Syncytialization process is associated with a reduction in the number of caveolas, and a decreased of caveolin-1 (Cav-1). Differentiation of syncytiotrophoblast affects the membranes phospholipid composition. Thus, disturbances in these processes are related to pathological conditions such as preeclampsia. Objective To analyse the lipid composition of the apical (MVM) and the basal (BM) membranes of syncytiotrophoblast and its relationship with Cav-1 expression in normal and preeclamptic placentas. Molecular expression of Cav-1 was determined in MVM and BM from normal and preeclamptic placentas and in detergent-resistant membranes (DRMs). Phospholipids were analyzed by thin layer chromatography. Cholesterol was also determined by enzymatic assay. Membrane fluidity was evaluated by electron paramagnetic resonance. Sphingomyelin (SM) molecular species were analyzed and quantified by gas-liquid chromatography and mass spectrometry. Cav-1 was significantly reduced in MVM from preeclamptic placentas. Regarding Cav-1 localization, it was barely detectable in syncytiotrophoblast but it was present in the endothelium. Western blots also showed a significantly decrease of Cav-1 in the apical DRMs from preeclamptic placentas. Lipid analysis showed an increase SM in MVM from preeclamptic placentas without changes in cholesterol. Preeclamptic MVM fluidity decreased significantly and we found an increase in C18:1 fatty acids of SM. We concluded that preeclamptic-MVMs are more rigid than normal ones, possible due to an increment on SM. Moreover, the increase of long and unsaturated SM molecular specie found in these vesicles may disrupt the ability of SM to assemble into lipid rafts in the luminal leaflet of the bilayer, creating an unfavorable environment for Cav-1.Instituto de Investigaciones Bioquímicas de La Plat

Un entorno lipídico desfavorable disminuye la expresión de caveolina-1 en placentas preeclámpticas

La Caveolina-1 (Cav-1) es una proteína crucial en la formación de las caveolas, microdominios rígidos de membrana ricos en colesterol y esfingolípidos. Está involucrada en muchos procesos celulares como la diferenciación celular, donde regularía negativamente del ciclo celular, retardando la mitosis con el fin de detener la proliferación y conducir a la célula a su diferenciación. Contrariamente a lo que ocurre en otros epitelios, la diferenciación del trofoblasto se asocia con una reducción del número de caveolas, acompañado con una disminución de la expresión de Cav-1 y una modificación la composición de fosfolípidos de la membrana plasmática del sincitiotrofoblasto (hST) resultantes de la fusión con las células de citotrofoblasto. Este proceso es esencial para el mantenimiento de un embarazo exitoso y alteraciones se asocian a condiciones patológicas tales como la preeclampsia. En placentas preclámpticas (PE), se encontraron alteraciones en la composición lipídica de la membrana de hST y expresiones anormales de proteínas de transporte que alterarían las funciones de la placenta. Sin embargo, si estos cambios en la composición lipídica de la membrana del hST pueden afectar la expresión de Cav-1 en placentas preeclámpticas se desconocen.Facultad de Ciencias Médica

Boundary region between coexisting lipid phases as initial binding sites for Escherichia coli alpha-hemolysin: A real-time study

Abstractα-Hemolysin (HlyA) is a protein toxin, a member of the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenic strands of Escherichia coli. The mechanism of action of this toxin seems to involve three stages that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin within the membrane. Since the influence of phase segregation on HlyA binding and insertion in lipid membranes is not clearly understood, we explored at the meso- and nanoscale—both in situ and in real-time—the interaction of HlyA with lipid monolayers and bilayers. Our results demonstrate that HlyA could insert into monolayers of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/16:0SM/Cho) and DOPC/24:1SM/Cho. The time course for HlyA insertion was similar in both lipidic mixtures. HlyA insertion into DOPC/16:0SM/Cho monolayers, visualized by Brewster-angle microscopy (BAM), suggest an integration of the toxin into both the liquid-ordered and liquid-expanded phases. Atomic-force-microscopy imaging reported that phase boundaries favor the initial binding of the toxin, whereas after a longer time period the HlyA becomes localized into the liquid-disordered (Ld) phases of supported planar bilayers composed of DOPC/16:0SM/Cho. Our AFM images, however, showed that the HlyA interaction does not appear to match the general strategy described for other invasive proteins. We discuss these results in terms of the mechanism of action of HlyA

Efecto de los ácidos grasos W3 sobre la estructura de membranas y actividad del NHE-1 en ratas normotensas y espontáneamente hipertensas (SHR)

Los ácidos grasos de la serie w-3 han sido asociados experimental y epidemiológicamente a diversos estados fisiopatológicos, como inflamación, diabetes, enfermedad cardiovascular y cáncer. Uno de los mecanismos moleculares mediante los cuales estos ácidos grasos ejercen su efecto biológico consiste en su incorporación en membranas celulares, afectando las propiedades biofísicas y bioquímicas de las mismas, impactando específicamente sobre la estructura y organización de lipid rafts, la localización de proteínas de vías de señalización y, en definitiva, sobre numerosas funciones celulares. Los lipid rafts se definen como dominios especializados de membranas biológicas, enriquecidos en esfingolípidos (SLs) y colesterol (Col), que reclutan proteínas específicas. Las caveolas son un tipo especializado de rafts que se caracteriza por su estructura invaginada de 50-100 nm de diámetro, revestida y estabilizada por caveolinas y cavinas, entre otras proteínas. El intercambiador Na+/H+ (NHE-1), una proteína integral de membrana, está involucrado en el mantenimiento del pH intracelular (pH ). Su actividad está regulada por la sensibilidad del sitio alostérico i para el H+, fosforilación y por la unión de ATP, lípidos y factores de crecimiento. Diversos estudios en corazón de ratas SHR han demostrado la hiperactividad de dicho transportador, y que la inhibición del mismo revierte la hipertrofia que éstos presentan. El objetivo es estudiar la composición lipídica y contenidos de PUFAs de la serie w3 en sangre de ratas normotensas (Wistar) y espontáneamente hipertensas (SHR), en relación con la organización de membrana plasmática –lipid rafts/caveolas- y actividad del intercambiador Na+ /H+ miocárdico (NHE-1)

The unfavorable lipid environment reduced caveolin-1 expression in apical membranes from human preeclamptic placentas

Syncytialization process is associated with a reduction in the number of caveolas, and a decreased of caveolin-1 (Cav-1). Differentiation of syncytiotrophoblast affects the membranes phospholipid composition. Thus, disturbances in these processes are related to pathological conditions such as preeclampsia. Objective To analyse the lipid composition of the apical (MVM) and the basal (BM) membranes of syncytiotrophoblast and its relationship with Cav-1 expression in normal and preeclamptic placentas. Molecular expression of Cav-1 was determined in MVM and BM from normal and preeclamptic placentas and in detergent-resistant membranes (DRMs). Phospholipids were analyzed by thin layer chromatography. Cholesterol was also determined by enzymatic assay. Membrane fluidity was evaluated by electron paramagnetic resonance. Sphingomyelin (SM) molecular species were analyzed and quantified by gas-liquid chromatography and mass spectrometry. Cav-1 was significantly reduced in MVM from preeclamptic placentas. Regarding Cav-1 localization, it was barely detectable in syncytiotrophoblast but it was present in the endothelium. Western blots also showed a significantly decrease of Cav-1 in the apical DRMs from preeclamptic placentas. Lipid analysis showed an increase SM in MVM from preeclamptic placentas without changes in cholesterol. Preeclamptic MVM fluidity decreased significantly and we found an increase in C18:1 fatty acids of SM. We concluded that preeclamptic-MVMs are more rigid than normal ones, possible due to an increment on SM. Moreover, the increase of long and unsaturated SM molecular specie found in these vesicles may disrupt the ability of SM to assemble into lipid rafts in the luminal leaflet of the bilayer, creating an unfavorable environment for Cav-1.Instituto de Investigaciones Bioquímicas de La Plat

N-Nervonoylsphingomyelin (C24:1) prevents lateral heterogeneity in cholesterol-containing membranes

This study was conducted to explore how the nature of the acyl chains of sphingomyelin (SM) influence its lateral distribution in the ternary lipid mixture SM/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), focusing on the importance of the hydrophobic part of the SM molecule for domain formation. Atomic force microscopy (AFM) measurements showed that the presence of a double bond in the 24:1 SM molecule in mixtures with cholesterol (CHO) or in pure bilayers led to a decrease in the molecular packing. Confocal microscopy and AFM showed, at the meso- and nanoscales respectively, that unlike 16:0 and 24:0 SM, 24:1 SM does not induce phase segregation in ternary lipid mixtures with DOPC and CHO. This ternary lipid mixture had a nanomechanical stability intermediate between those displayed by liquid-ordered (Lo) and liquid-disordered (Ld) phases, as reported by AFM force spectroscopy measurements, demonstrating that 24:1 SM is able to accommodate both DOPC and CHO, forming a single phase. Confocal experiments on giant unilamellar vesicles made of human, sheep, and rabbit erythrocyte ghosts rich in 24:1 SM and CHO, showed no lateral domain segregation. This study provides insights into how the specific molecular structure of SM affects the lateral behavior and the physical properties of both model and natural membranes. Specifically, the data suggest that unsaturated SM may help to keep membrane lipids in a homogeneous mixture rather than in separate domains.Instituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias Exacta

Histidine 19 Residue Is Essential for Cell Internalization of Antifungal Peptide SmAPα1-21 Derived from the α-Core of the Silybum marianum Defensin DefSm2-D in Fusarium graminearum

The synthetic peptide SmAPα1-21 (KLCEKPSKTWFGNCGNPRHCG) derived from DefSm2-D defensin α-core is active at micromolar concentrations against the phytopathogenic fungus Fusarium graminearum and has a multistep mechanism of action that includes alteration of the fungal cell wall and membrane permeabilization. Here, we continued the study of this peptide’s mode of action and explored the correlation between the biological activity and its primary structure. Transmission electron microscopy was used to study the ultrastructural effects of SmAPα1-21 in conidial cells. New peptides were designed by modifying the parent peptide SmAPα1-21 (SmAPH19R and SmAPH19A, where His19 was replaced by Arg or Ala, respectively) and synthesized by the Fmoc solid phase method. Antifungal activity was determined against F. graminearum. Membrane permeability and subcellular localization in conidia were studied by confocal laser scanning microscopy (CLSM). Reactive oxygen species (ROS) production was assessed by fluorescence spectroscopy and CLSM. SmAPα1-21 induced peroxisome biogenesis and oxidative stress through ROS production in F. graminearum and was internalized into the conidial cells’ cytoplasm. SmAPH19R and SmAPH19A were active against F. graminearum with minimal inhibitory concentrations (MICs) of 38 and 100 µM for SmAPH19R and SmAPH19A, respectively. The replacement of His19 by Ala produced a decrease in the net charge with a significant increase in the MIC, thus evidencing the importance of the positive charge in position 19 of the antifungal peptide. Like SmAPα1-21, SmAP2H19A and SmAP2H19R produced the permeabilization of the conidia membrane and induced oxidative stress through ROS production. However, SmAPH19R and SmAPH19A were localized in the conidia cell wall. The replacement of His19 by Ala turned all the processes slower. The extracellular localization of peptides SmAPH19R and SmAPH19A highlights the role of the His19 residue in the internalization

N-Nervonoylsphingomyelin (C24:1) Prevents Lateral Heterogeneity in Cholesterol-Containing Membranes

This study was conducted to explore how the nature of the acyl chains of sphingomyelin (SM) influence its lateral distribution in the ternary lipid mixture SM/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), focusing on the importance of the hydrophobic part of the SM molecule for domain formation. Atomic force microscopy (AFM) measurements showed that the presence of a double bond in the 24:1 SM molecule in mixtures with cholesterol (CHO) or in pure bilayers led to a decrease in the molecular packing. Confocal microscopy and AFM showed, at the meso- and nanoscales respectively, that unlike 16:0 and 24:0 SM, 24:1 SM does not induce phase segregation in ternary lipid mixtures with DOPC and CHO. This ternary lipid mixture had a nanomechanical stability intermediate between those displayed by liquid-ordered (Lo) and liquid-disordered (Ld) phases, as reported by AFM force spectroscopy measurements, demonstrating that 24:1 SM is able to accommodate both DOPC and CHO, forming a single phase. Confocal experiments on giant unilamellar vesicles made of human, sheep, and rabbit erythrocyte ghosts rich in 24:1 SM and CHO, showed no lateral domain segregation. This study provides insights into how the specific molecular structure of SM affects the lateral behavior and the physical properties of both model and natural membranes. Specifically, the data suggest that unsaturated SM may help to keep membrane lipids in a homogeneous mixture rather than in separate domains.Fil: Maté, Sabina María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Busto, Jon V.. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; EspañaFil: García Arribas, Aritz B.. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Sot, Jesús. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; EspañaFil: Vazquez, Romina Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Herlax, Vanesa Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Wolf, Claude. Universite Pierre et Marie Curie; FranciaFil: Bakas, Laura Susana. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas; ArgentinaFil: Goñi, Felix M. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; Españ