Search

Las proteínas que unen ácidos grasos (FABPs) son proteínas pequeñas citoplasmáticas que se expresan en casi todos los tejidos de mamíferos. Estudios previos han ayudado a elucidar los mecanismos de transferencia de ácidos grasos (FA) por distintas FABPs, así como sus estructuras y distribución tisular. Sus funciones, sin embargo, aún están en discusión. Se ha propuesto que estas proteínas podrían amortiguar el contenido de FA libres así comodireccionarlos hacia distintas rutas metabólicas. Las FABPs podrían transportar FA a diferentes compartimientos celulares dirigiéndolos hacia su oxidación, utilización en síntesis de lípidos complejos y/o regulación de factores transcripcionales. Así mismo, una de las funciones propuestas de las FABPs es la regulación de la expresión génica por medio del direccionamiento de ligandos hacia receptores nucleares (NR). La regulación de los NR por FABPs es compleja y, en muchos casos, no clara., Universidad Nacional de La Plata

Background:\ud \ud The giant kidney worm, Dioctophyme renale, is a debilitating and potentially lethal parasite that inhabits and destroys, typically host’s right kidney, and may also be found in ectopic sites. It is circumglobally distributed, mainly in dogs, and is increasingly regarded as a threat to other domestic animals and humans. There is little information on the parasite’s true incidence, or immune responses to it, and none on its biochemistry and molecular biology.\ud Results:\ud \ud We characterised the soluble proteins of body wall, intestine, gonads and pseudocelomic fluid (PCF) of adult parasites. Two proteins, P17 and P44, dominate the PCF of both male and females. P17 is of 16,622 Da by mass spectrometry, and accounts for the intense red colour of the adult parasites. It may function to carry or scavenge oxygen and be related to the ‘nemoglobins’ found in other nematode clades. P44 is of 44,460 Da and was found to associate with fatty acids by thin layer chromatography. Using environment-sensitive fluorescent lipid probes, P44 proved to be a hydrophobic ligand-binding protein with a binding site that is highly apolar, and competitive displacement experiments showed that P44 binds fatty acids. It may therefore have a role in distributing lipids within the parasites and, if also secreted, might influence local inflammatory and tissue responses. N-terminal and internal peptide amino-acid sequences of P44 indicate a relationship with a cysteine- and histidine-rich protein of unknown function from Trichinella spiralis.\ud Conclusions:\ud \ud The dominant proteins of D. renale PCF are, like those of large ascaridids, likely to be involved in lipid and oxygen handling, although there is evidence of strong divergence between the two groups.

Background: The giant kidney worm, Dioctophyme renale, is a debilitating and potentially lethal parasite that inhabits and destroys, typically host’s right kidney, and may also be found in ectopic sites. It is circumglobally distributed, mainly in dogs, and is increasingly regarded as a threat to other domestic animals and humans. There is little information on the parasite’s true incidence, or immune responses to it, and none on its biochemistry and molecular biology. Results: We characterised the soluble proteins of body wall, intestine, gonads and pseudocelomic fluid (PCF) of adult parasites. Two proteins, P17 and P44, dominate the PCF of both male and females. P17 is of 16,622 Da by mass spectrometry, and accounts for the intense red colour of the adult parasites. It may function to carry or scavenge oxygen and be related to the ‘nemoglobins’ found in other nematode clades. P44 is of 44,460 Da and was found to associate with fatty acids by thin layer chromatography. Using environment-sensitive fluorescent lipid probes, P44 proved to be a hydrophobic ligand-binding protein with a binding site that is highly apolar, and competitive displacement experiments showed that P44 binds fatty acids. It may therefore have a role in distributing lipids within the parasites and, if also secreted, might influence local inflammatory and tissue responses. N-terminal and internal peptide amino-acid sequences of P44 indicate a relationship with a cysteine- and histidinerich protein of unknown function from Trichinella spiralis. Conclusions: The dominant proteins of D. renale PCF are, like those of large ascaridids, likely to be involved in lipid and oxygen handling, although there is evidence of strong divergence between the two groups., Facultad de Ciencias Veterinarias

El cáncer es la principal causa de muerte a nivel mundial. El estudio de los mecanismos que intervienen en el desarrollo del cáncer es importante para la detección de nuevos blancos terapéuticos. Las células cancerosas presentan una reprogramación metabólica direccionada hacia la obtención de biomoléculas necesarias para la duplicación celular. Si bien se demostró la importancia de la síntesis de membranas para la proliferación celular, se desconocen diversos aspectos del metabolismo lipídico en células tumorales. Las proteínas que unen ácidos grasos (FABPs) son proteínas de bajo peso molecular 14- 15kDa, citosólicas que unen ácidos grasos de cadena larga con alta afinidad. Se ha estudiado su estructura, distribución tisular y su mecanismo de transferencia de ácidos grasos, sin embargo, su función aún está en discusión. La existencia de nueve FABPs y la presencia simultánea de más de una isoforma en un tejido sugiere que las mismas difieren en su función. Se propone que podrían transportar ácidos grasos a diferentes compartimentos celulares dirigiéndolos así hacia su oxidación, utilización en síntesis de lípidos complejos y regulación de factores transcripcionales. FABP5, epidermal o de queratinocito, presenta una expresión ubicua. A diferencia de otras isoformas FABP5 ha sido asociada positivamente con la progresión y el desarrollo de ciertos cánceres, en especial próstata y mama. Su función en el metabolismo de los mismos es, sin embargo, desconocida., Facultad de Ciencias Médicas

Fatty acid and retinol binding proteins (FARs) comprise a family of unusual α-helix rich lipid binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein (Ce-FAR-7) is from a subfamily of FARs that does not appear to be important at the host-parasite interface. We have therefore examined Na-FAR-1 from the blood-feeding intestinal parasite of humans, Necator americanus . The three dimensional structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, respectively, reveals an a-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand binding cavity and an additional C-terminal a-helix. Titration of apo -Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein:ligand complexes can be formed. Na-FAR-1, and possibly other FARs, may have a wider repertoire for hydrophobic ligand binding, as confirmed here by our finding that a range of neutral and polar lipids co-purify with the bacterial recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male.

Background:\ud \ud The hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied.\ud \ud Methodology/Principal Findings:\ud \ud Lipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles.\ud \ud Conclusions/Significance:\ud \ud We show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy.

The hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied. Lipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles. We show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy., Instituto de Investigaciones Bioquímicas de La Plata, Facultad de Ciencias Médicas

Las enfermedades parasitarias y particularmente las enteroparasitosis, afectan a todos los grupos etarios sin distinción de sexo, siendo los niños los más expuestos con perjuicios severos en su crecimiento y desarrollo, tanto físico como psíquico que compromete su salud hasta inclusive producirles la muerte. Se estima que el 12% de las enfermedades de la niñez son debidas a parásitos intestinales. En los últimos diez años años como consecuencia de la crisis económica, las familias de bajos recursos se ven obligadas a migrar en busca de trabajo y un mejor bienestar, produciendo cambios socioambientales que determinan la emergencia de nuevas parasitosis y un aumento de la incidencia de las ya instaladas. Las parasitosis intestinales son endémicas en los países en desarrollo siendo un indicador de las condiciones sanitarias y ecológicas. En la ciudad de La Plata se ha observado un notorio crecimiento de los asentamientos en la perifería, encontrándose familias numerosas que conviven en viviendas precarias con pisos de tierra, sin instalaciones sanitarias con eliminación de excretas a los arroyos, eliminación incorrecta de la basura, sin agua potable y mascotas sin desparasitar. En este marco de situación, la combinación de pobreza, ignorancia y desnutrición acompañadas de un saneamiento ambiental deficiente, favorecen la instalación de las infecciones parasitarias intestinales, convirtiéndose en un verdadero problema de salud pública., Facultad de Ciencias Exactas

Luego de la ingesta, el epitelio del intestino delgado está encargado de asimilar grandes cantidades de nutrientes, como aminoácidos, glúcidos y ácidos grasos. Las proteínas solubles que unen lípidos cumplirían un rol determinante en este proceso, sobre todo protegiendo la integridad del tejido contra el efecto simil-detergente de los ácidos grasos provenientes de la dieta. En enterocitos se expresan dos proteínas que unen ácidos grasos de cadena larga, IFABP y LFABP, para las cuales no se conocen bien aún sus funciones específicas, o el porqué de la necesidad de dos proteínas aparentemente equivalentes. Este laboratorio se ha enfocado en el estudio comparativo de estas dos proteínas empleando distintas variantes estructurales y métodos bioquímicos, biofísicos, y de biología molecular y celular. Así, se han podido definir los determinantes moleculares de cada proteína responsables de la interacción con membranas, los mecanismos de transferencia de ligandos y los factores que modulan estas propiedades. Más recientemente, se han extendido estos ensayos a cultivos celulares donde se ha correlacionado la expresión de estas proteínas con la secreción de citoquinas, la proliferación y la diferenciación celular. El estudio de estas proteínas es de gran importancia por su potencial como blancos terapéuticos y su utilidad en el diagnóstico de injurias tisulares., After ingestion, the epithelium of the small intestine is responsible for assimilating large amounts of nutrients such as amino acids, sugars and fatty acids. Soluble lipid binding proteins fulfill a determining role in this process, especially protecting the tissue integrity against the detergent-like effect of fatty acids from the diet. Two proteins that bind long-chain fatty acids are expressed in enterocytes, IFABP and LFABP, whose specific functions are still poorly understood, or the reason for the need of two apparently equivalent proteins. Our laboratory has focused on the comparative study of these two proteins using structural variants and biochemical, biophysical, and molecular and cellular biology approaches. Thus, the molecular determinants responsible for the interaction with membranes were defined for each protein, their ligand transfer mechanism and the factors that modulate these properties. More recently, these assays have been extended to cell culture studies which correlate the expression of these proteins with cytokine secretion, cell proliferation and differentiation. The study of these proteins is of great importance due to their potential as therapeutic targets and their usefulness in the diagnosis of tissue injury., Após a ingestão, o epitélio do intestino delgado é responsável pela assimilação de uma grande quantidade de nutrientes, tais como aminoácidos, glicídios e ácidos graxos. As proteínas solúveis que ligam lipídeos desempenhariam um papel determinante neste processo, principalmente protegendo a integridade do tecido contra o efeito detergente dos ácidos graxos da dieta. Nos enterócitos se expressam duas proteínas que ligam ácidos graxos de cadeia longa, IFABP e LFABP; cujas funções específicas ainda não são muito conhecidas, ou não se conhece o motivo pelo qual são necessárias duas proteínas aparentemente equivalentes. Nosso laboratório tem se focado no estudo comparativo destas duas proteínas utilizando variantes estruturais e métodos bioquímicos, biofísicos, e de biologia molecular e celular. Assim, foi possível definir os determinantes moleculares de cada proteína responsáveis pela interação com membranas, os mecanismos da transferência de ligantes e os fatores que modulam essas propriedades. Mais recentemente, estendemos estes ensaios para culturas celulares, correlacionando a expressão destas proteínas com a secreção de citocinas, a proliferação e a diferenciação celular. O estudo destas proteínas é de grande importância por seu potencial como alvos terapêuticos e sua utilidade no diagnóstico de lesões teciduais., Instituto de Investigaciones Bioquímicas de La Plata

Con la finalidad de realizar estudios estructurales y funcionales de una proteína, uno de los pasos fundamentales es obtenerla en forma pura. Luego del proceso de purificación deben llevarse a cabo una serie de procedimientos que permitan controlar el grado de pureza y también realizar una caracterización inicial de dichas proteínas. Entre las técnicas bioquímicas más comúnmente utilizadas y que aportan información complementaria podemos mencionar las siguientes: electroforesis, cromatografía de exclusión molecular, centrifugación analítica y análisis de entrecruzamiento químico, entre otras. Este capítulo está destinado a que el alumno tenga una primera aproximación a una serie de técnicas que permiten caracterizar y controlar el grado de pureza de una proteína. Si bien esto no pretende ser un manual técnico, se aportarán los fundamentos, los procedimientos básicos y el análisis de la información obtenida a partir de las técnicas que se describen., Facultad de Ciencias Exactas

The role of fatty acid binding proteins as intracellular fatty acid transporters may require their direct interaction with membranes. In this way different mechanisms have been previously characterized through experimental studies suggesting different models for FABPs-membrane association, although the process in which the molecule adsorbs to the membrane remains to be elucidated. To estimate the importance of the electrostatic energy in the FABP-membrane interaction, we computationally modeled the interaction of different FABPs with both anionic and neutral membranes. Free Electrostatic Energy of Binding (dE), was computed using Finite Difference Poisson Boltzmann Equation (FDPB) method as implemented in APBS (Adaptive Poisson Boltzmann Solver). Based on the computational analysis, it is found that recruitment to membranes is facilitated by non-specific electrostatic interactions. Also energetic analysis can quantitatively differentiate among the mechanisms of membrane association proposed and determinate the most energetically favorable configuration for the membrane-associated states of different FABPs. This type of calculations could provide a starting point for further computational or experimental analysis. Fil: Zamarreño, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Sur. Departamento de Física; Argentina Fil: Herrera, Fernando Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Córsico, Betina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Costabel, Marcelo Daniel. Universidad Nacional del Sur. Departamento de Física; Argentina

We used a combination of in vitro and in cell studies to assess the differential functionality of abundantly coexpressed FABP in the enterocyte, liver and intestinal fatty acid binding proteins., Facultad de Ciencias Médicas

Los parásitos helmintos expresan ciertas proteínas de unión a lípidos (LBPs) estructuralmente diferentes a las de sus hospedadores. Estas proteínas son capaces de unir un amplio espectro de ligandos, tales como ácidos grasos, retinoides, eicosanoides y fosfolípidos. Su función en la biología de los parásitos aún es desconocida, aunque se asume que podrían participar de la relación parásito-hospedador., Facultad de Ciencias Médicas

The objective of this work was to analyze LFABP´s capacity to transfer acyl CoAs to artificial membranes and compare it to LCFA transfer employing natural ligands, in order to better understand the specific physiological role of LFABP in the cell., Facultad de Ciencias Médicas

We expect to identify the interaction partners of intestinal FABPs in order to describe the molecular crosstalk relevant for its biological functions., Facultad de Ciencias Médicas

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the α-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the α-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the α-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the α2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the α2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the α2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP., Instituto de Investigaciones Bioquímicas de La Plata