Estudio de la dependencia de ácidos grasos esenciales en células de Sarcoma 180 ascítico mediante el análisis de parámetros biológicos y bioquímicos

Objetivos del trabajo: Se ha considerado que para poder establecer la posible dependencia de las células tumorales por los ácidos grasos esenciales es necesario estudiar varios parámetros metabólicos indicativos de esta dependencia. Los parámetros elegidos para el presente estudio fueron: a) Tiempo de duplicación celular: Este parámetro que corresponde al tiempo por medio de duplicación celular es indicativo de la adaptabilidad de la célula al medio en el cual se desarrolla y es una manifestación de la vitalidad de la misma. b) Incorporación selectiva del radiolinoléico en las distintas fracciones lipídicas: Estudios realizados en animales deficientes en AGE han puesto en evidencia que la incorporación in vivo de radiolinoleíco se realiza preferentemente en la fracción de fosfolípídos hepáticos y no en la fracción de lípidos neutros como se observa en los animales controles (117). Este hecho pondría en evidencia la necesidad de la célula deficiente de restablecer la composición en ácidos grasos polietilénicos de sus fosfolípídos estructurales. c) Modificaciones de la composición de ácidos grasos en la deficiencia de AGE: La célula trata de compensar la deficiencia en AGE por el aumento de la síntesis de ácido octadecenoico y eicosatrienoico. Estas modificaciones traen como consecuencia alteraciones de ciertas relaciones entre los ácidos grasos, siendo la más característica la que corresponde al aumento de C20: 3 ω9 / C20: 4 ω6. Este índice es indicativo de deficiencia en AGE cuando su valor es mayor de 0.4 (101). d) Capacidad de síntesis de ácidos no saturados: La actividad de la enzima ∆9 desaturasa se incrementa notablemente en la deficiencia en AGE. Este aumento sería un mecanismo compensador dirigido a aumentar la síntesis de ácidos grasos no saturados y condicionaría al parámetro anterior (118).Tesis digitalizada en SEDICI gracias a la colaboración de la Biblioteca de la Facultad de Ciencias Exactas (UNLP).Facultad de Ciencias Exacta

The p10 FAST protein fusion peptide functions as a cystine noose to induce cholesterol-dependent liposome fusion without liposome tubulation

AbstractThe reovirus p10 fusion-associated small transmembrane (FAST) proteins are the smallest known membrane fusion proteins, and evolved specifically to mediate cell–cell, rather than virus–cell, membrane fusion. The 36–40-residue ectodomains of avian reovirus (ARV) and Nelson Bay reovirus (NBV) p10 contain an essential intramolecular disulfide bond required for both cell–cell fusion and lipid mixing between liposomes. To more clearly define the functional, biochemical and biophysical features of this novel fusion peptide, synthetic peptides representing the p10 ectodomains of ARV and NBV were analyzed by solution-state NMR spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy-based hydrophobicity analysis, and liposome binding and fusion assays. Results indicate that disulfide bond formation promotes exposure of hydrophobic residues, as indicated by bis-ANS binding and time-dependent peptide aggregation under aqueous conditions, implying the disulfide bond creates a small, geometrically constrained, cystine noose. Noose formation is required for peptide partitioning into liposome membranes and liposome lipid mixing, and electron microscopy revealed that liposome–liposome fusion occurs in the absence of liposome tubulation. In addition, p10 fusion peptide activity, but not membrane partitioning, is dependent on membrane cholesterol

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX-and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc

Nonenveloped Avian Reoviruses Released with Small Extracellular Vesicles Are Highly Infectious

Vesicle-encapsulated nonenveloped viruses are a recently recognized alternate form of nonenveloped viruses that can avoid immune detection and potentially increase systemic transmission. Avian orthoreoviruses (ARVs) are the leading cause of various disease conditions among birds and poultry. However, whether ARVs use cellular vesicle trafficking routes for egress and cell-to-cell transmission is still poorly understood. We demonstrated that fusogenic ARV-infected quail cells generated small (~100 nm diameter) extracellular vesicles (EVs) that contained electron-dense material when observed by transmission electron microscope. Cryo-EM tomography indicated that these vesicles did not contain ARV virions or core particles, but the EV fractions of OptiPrep gradients did contain a small percent of the ARV virions released from cells. Western blotting of detergent-treated EVs revealed that soluble virus proteins and the fusogenic p10 FAST protein were contained within the EVs. Notably, virus particles mixed with the EVs were up to 50 times more infectious than virions alone. These results suggest that EVs and perhaps fusogenic FAST-EVs could contribute to ARV virulence

In vitro conversion of saturated to monounsaturated fatty acid by Ehrlich ascites cells

In this paper, evidence is presented on the capacity of Ehrlich ascites cells to synthesize in vitro monounsaturated fatty acids from radioactive palmitate. Localization of the double bond was determined by ozonolysis and subsequent reduction of the ozonides to aldesters followed by gas liquid chromatography. These results proved that Ehrlich ascites cells have a Δ9 desaturase that catalyzes the biosynthesis of palmitoleic acid from palmitic acid and oleic and vaccenic acid via elongation-desaturation and desaturation-elongation, respectively, using palmitic acid as substrate. Furthermore, it is shown that, as in the hepatic cells, Δ9 desaturase enzyme activity of the tumoral cells is associated with the endoplasmic reticulum. The electron transport components involved in the desaturase system, i.e., NADH-cytochrome bs reductase and NADH-cytochrome bs reductase, were also measured. The activities of these enzymes do not appear to be rate-limiting in the desaturase activity of these tumoral cells.Facultad de Ciencias Médica

Differential Cellular Sensing of Fusion from within and Fusion from without during Virus Infection

The physical entry of virus particles into cells triggers an innate immune response that is dependent on both calcium and nucleic acid sensors, with particles containing RNA or DNA genomes detected by RNA or DNA sensors, respectively. While membrane fusion in the absence of viral nucleic acid causes an innate immune response that is dependent on calcium, the involvement of nucleic acid sensors is poorly understood. Here, we used lipoplexes containing purified reovirus p14 fusion protein as a model of exogenous or fusion from without and a cell line expressing inducible p14 protein as a model of endogenous or fusion from within to examine cellular membrane fusion sensing events. We show that the cellular response to membrane fusion in both models is dependent on calcium, IRF3 and IFN. The method of sensing fusion, however, differs between fusion from without and fusion from within. Exogenous p14 lipoplexes are detected by RIG-I-like RNA sensors, whereas fusion by endogenous p14 requires both RIG-I and STING to trigger an IFN response. The source of nucleic acid that is sensed appears to be cellular in origin. Future studies will investigate the source of endogenous nucleic acids recognized following membrane fusion events

Activity of human \u3945 and \u3946 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids

Yeast co-expressing human elongase and desaturase genes were used to investigate whether the same desaturase gene encodes an enzyme able to desaturate n-3 and n-6 fatty acids with the same or different carbon chain length. The results clearly demonstrated that a single human \u3945 desaturase is active on 20:3n-6 and 20:4n-3. Endogenous \u3946 desaturase substrates were generated by providing to the yeast radiolabelled 20:4n-6 or 20:5n-3 which, through two sequential elongations, produced 24:4n-6 and 24:5n-3, respectively. Overall, our data suggest that a single human \u3946 desaturase is active on 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3.Peer reviewed: YesNRC publication: N

Differential Cellular Sensing of Fusion from within and Fusion from without during Virus Infection

The physical entry of virus particles into cells triggers an innate immune response that is dependent on both calcium and nucleic acid sensors, with particles containing RNA or DNA genomes detected by RNA or DNA sensors, respectively. While membrane fusion in the absence of viral nucleic acid causes an innate immune response that is dependent on calcium, the involvement of nucleic acid sensors is poorly understood. Here, we used lipoplexes containing purified reovirus p14 fusion protein as a model of exogenous or fusion from without and a cell line expressing inducible p14 protein as a model of endogenous or fusion from within to examine cellular membrane fusion sensing events. We show that the cellular response to membrane fusion in both models is dependent on calcium, IRF3 and IFN. The method of sensing fusion, however, differs between fusion from without and fusion from within. Exogenous p14 lipoplexes are detected by RIG-I-like RNA sensors, whereas fusion by endogenous p14 requires both RIG-I and STING to trigger an IFN response. The source of nucleic acid that is sensed appears to be cellular in origin. Future studies will investigate the source of endogenous nucleic acids recognized following membrane fusion events

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor.

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation