The integrity of the α-helical domain of intestinal fatty acid binding protein is essential for the collision-mediated transfer of fatty acids to phospholipid membranes

Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the αI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the 'body' (ligand binding domain) of IFABP and the αI-helix of LFABP (α(I)LβIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from α(I)LβIFABP compared to IFABP. The results indicate that the αI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the αII-helix in the formation of a protein-membrane "collisional complex". Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further support the importance of the αI helix of IFABP in its physical interaction with membranes.Fil: Franchini, Gisela Raquel. 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"; ArgentinaFil: Storch, J.. Rutgers University; Estados UnidosFil: 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"; Argentin

Técnicas bioquímicas para el estudio de proteínas

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 Exacta

Fatty acid transfer from intestinal fatty acid binding protein to membranes: Electrostatic and hydrophobic interactions

Intestinal fatty acid binding protein (IFABP) is thought to participate in the intracellular transport of fatty acids (FAs). Fatty acid transfer from IFABP to phospholipid membranes is proposed to occur during protein-membrane collisional interactions. In this study, we analyzed the participation of electrostatic and hydrophobic interactions in the collisional mechanism of FA transfer from IFABP to membranes. Using a fluorescence resonance energy transfer assay, we examined the rate and mechanism of transfer of anthroyloxy-fatty acid analogs a) from IFABP to phospholipid membranes of different composition; b) from chemically modified IFABPs, in which the acetylation of surface lysine residues eliminated positive surface charges; and c) as a function of ionic strength. The results show clearly that negative charges on the membrane surface and positive charges on the protein surface are important for establishing the "collisional complex," during which fatty acid transfer occurs. In addition, changes in the hydrophobicity of the protein surface, as well as the hydrophobic volume of the acceptor vesicles, also influenced the rate of fatty acid transfer. Thus, ionic interactions between IFABP and membranes appear to play a primary role in the process of fatty acid transfer to membranes, and hydrophobic interactions can also modulate the rates of ligand transfer.Instituto de Investigaciones Bioquímicas de La Plat

Fatty acid transfer from intestinal fatty acid binding protein to membranes: Electrostatic and hydrophobic interactions

Intestinal fatty acid binding protein (IFABP) is thought to participate in the intracellular transport of fatty acids (FAs). Fatty acid transfer from IFABP to phospholipid membranes is proposed to occur during protein-membrane collisional interactions. In this study, we analyzed the participation of electrostatic and hydrophobic interactions in the collisional mechanism of FA transfer from IFABP to membranes. Using a fluorescence resonance energy transfer assay, we examined the rate and mechanism of transfer of anthroyloxy-fatty acid analogs a) from IFABP to phospholipid membranes of different composition; b) from chemically modified IFABPs, in which the acetylation of surface lysine residues eliminated positive surface charges; and c) as a function of ionic strength. The results show clearly that negative charges on the membrane surface and positive charges on the protein surface are important for establishing the "collisional complex," during which fatty acid transfer occurs. In addition, changes in the hydrophobicity of the protein surface, as well as the hydrophobic volume of the acceptor vesicles, also influenced the rate of fatty acid transfer. Thus, ionic interactions between IFABP and membranes appear to play a primary role in the process of fatty acid transfer to membranes, and hydrophobic interactions can also modulate the rates of ligand transfer.Instituto de Investigaciones Bioquímicas de La Plat

Fatty acid and retinol-binding protein: Unusual protein conformational and cavity changes dictated by ligand fluctuations

Lipid-binding proteins (LBPs) are soluble proteins responsible for the uptake, transport, and storage of a large variety of hydrophobic lipophilic molecules including fatty acids, steroids, and other lipids in the cellular environment. Among the LBPs, fatty acid binding proteins (FABPs) present preferential binding affinities for long-chain fatty acids. While most of FABPs in vertebrates and invertebrates present similar β-barrel structures with ligands accommodated in their central cavity, parasitic nematode worms exhibit additional unusual α-helix rich fatty acid- and retinol-binding proteins (FAR). Herein, we report the comparison of extended molecular dynamics (MD) simulations performed on the ligand-free and palmitic acid-bond states of the Necator americanus FAR-1 (Na-FAR-1) with respect to other classical β-barrel FABPs. Principal component analysis (PCA) has been used to identify the different conformations adopted by each system during MD simulations. The α-helix fold encompasses a complex internal ligand-binding cavity with a remarkable conformational plasticity that allows reversible switching between distinct states in the holo-Na-FAR-1. The cavity can change up to one-third of its size affected by conformational changes of the protein-ligand complex. Besides, the ligand inside the cavity is not fixed but experiences large conformational changes between bent and stretched conformations. These changes in the ligand conformation follow changes in the cavity size dictated by the transient protein conformation. On the contrary, protein-ligand complex in β-barrel FABPs fluctuates around a unique conformation. The significantly more flexible holo-Na-FAR-1 ligand-cavity explains its larger ligand multiplicity respect to β-barrel FABPs.Fil: Barletta Roldan, Patricio German. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Franchini, Gisela Raquel. 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"; ArgentinaFil: 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"; ArgentinaFil: Fernández Alberti, Sebastián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Fatty acid transfer from intestinal fatty acid binding protein to membranes: Electrostatic and hydrophobic interactions

Intestinal fatty acid binding protein (IFABP) is thought to participate in the intracellular transport of fatty acids (FAs). Fatty acid transfer from IFABP to phospholipid membranes is proposed to occur during protein-membrane collisional interactions. In this study, we analyzed the participation of electrostatic and hydrophobic interactions in the collisional mechanism of FA transfer from IFABP to membranes. Using a fluorescence resonance energy transfer assay, we examined the rate and mechanism of transfer of anthroyloxy-fatty acid analogs a) from IFABP to phospholipid membranes of different composition; b) from chemically modified IFABPs, in which the acetylation of surface lysine residues eliminated positive surface charges; and c) as a function of ionic strength. The results show clearly that negative charges on the membrane surface and positive charges on the protein surface are important for establishing the "collisional complex," during which fatty acid transfer occurs. In addition, changes in the hydrophobicity of the protein surface, as well as the hydrophobic volume of the acceptor vesicles, also influenced the rate of fatty acid transfer. Thus, ionic interactions between IFABP and membranes appear to play a primary role in the process of fatty acid transfer to membranes, and hydrophobic interactions can also modulate the rates of ligand transfer.Instituto de Investigaciones Bioquímicas de La Plat

Análise estrutural e funcional de proteínas solúveis que ligam lipídios de parasitas helmintos

Los parásitos helmintos producen y secretan una gran variedad de proteínas que unen lípidos (LBPs, del inglés lipid binding proteins) que podrían participar en la obtención de nutrientes tales como ácidos grasos y colesterol desde el hospedador. Asimismo, se postula que las LBPs podrían intervenir en la regulación de la respuesta inmune del hospedador. Conocer más acerca de las estructuras de estas proteínas, así como de sus interacciones con ligandos y membranas, es claramente pertinente para comprender las interacciones parásito-hospedador que ellas pudieran mediar. Por otra parte, dichos estudios permitirán profundizar en el conocimiento de los mecanismos de infección helmíntica y en el papel que estas proteínas juegan en la biología de los helmintos en general. Asimismo, esta información podría contribuir al establecimiento de medidas terapéuticas y de prevención de las enfermedades causadas por estos parásitos.Helminth parasites produce and secrete a great variety of lipid binding proteins (LBPs) that may participate in the acquisition of nutrients such as fatty acids and cholesterol from their host. It is also postulated that LBPs might interfere in the regulation of the host's immune response. Knowing more about the structure of these proteins as well as their interactions with ligands and membranes is important in order to understand the host-parasite interaction that they could mediate. On the other hand, these studies will contribute to obtain further knowledge about the mechanisms of helminth infection and the role that these proteins play in helminth biology. Moreover, this information would be useful to set new therapeutic and prevention measures for the diseases caused by these parasites.Os parasitas helmintos produzem e secretam uma grande variedade de proteínas que ligam lipídios, LBPs (Lipid Binding Proteins, por sua sigla em inglês), que poderiam estar envolvidas na obtenção de nutrientes tais como ácidos graxos e colesterol a partir do hospedeiro. Do mesmo modo, é postulado que as LBPs poderiam intervir na regulação da resposta imune do hospedeiro. Saber mais sobre as estruturas dessas proteínas, bem como sobre as suas interações com ligantes e membranas é claramente pertinente para compreender as interações parasita-hospedeiro que elas pudessem mediar. Além disso, estes estudos irão permitir um melhor entendimento dos mecanismos de infecção helmíntica e o papel que estas proteínas desempenham na biologia de helmintos em geral. Também, essa informação poderia ajudar a estabelecer medidas terapêuticas e de prevenção das doenças provocadas por esses parasitas.Fil: Franchini, Gisela Raquel. 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 ; ArgentinaFil: Corsico, 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 ; ArgentinaFil: Pórfido, Jorge Luis. 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 ; ArgentinaFil: Silva, Valeria.Fil: Ibañez Shimabukuro, Marina.Fil: Rey Burusco, Florencia

Analysis of triglyceride synthesis unveils a green algal soluble diacylglycerol acyltransferase and provides clues to potential enzymatic components of the chloroplast pathway

Background: Microalgal triglyceride (TAG) synthesis has attracted considerable attention. Particular emphasis has been put towards characterizing the algal homologs of the canonical rate-limiting enzymes, diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). Less work has been done to analyze homologs from a phylogenetic perspective. In this work, we used HMMER iterative profiling and phylogenetic and functional analyses to determine the number and sequence characteristics of algal DGAT and PDAT, as well as related sequences that constitute their corresponding superfamilies. We included most algae with available genomes, as well as representative eukaryotic and prokaryotic species. Results: Amongst our main findings, we identified a novel clade of DGAT1-like proteins exclusive to red algae and glaucophyta and a previously uncharacterized subclade of DGAT2 proteins with an unusual number of transmembrane segments. Our analysis also revealed the existence of a novel DGAT exclusive to green algae with moderate similarity to plant soluble DGAT3. The DGAT3 clade shares a most recent ancestor with a group of uncharacterized proteins from cyanobacteria. Subcellular targeting prediction suggests that most green algal DGAT3 proteins are imported to the chloroplast, evidencing that the green algal chloroplast might have a soluble pathway for the de novo synthesis of TAGs. Heterologous expression of C. reinhardtii DGAT3 produces an increase in the accumulation of TAG, as evidenced by thin layer chromatography. Conclusions: Our analysis contributes to advance in the knowledge of complex superfamilies involved in lipid metabolism and provides clues to possible enzymatic players of chloroplast TAG synthesis.Instituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias Médica

Análisis estructural y funcional de macromoléculas

El objetivo de este libro es incentivar a alumnos y docentes del área de bioquímica y afines a acercarse a un grupo de metodologías modernas que se aplican a la comprensión de los principios moleculares responsables de los procesos biológicos, en particular aquellos referidos a las proteínas. Desde los años ´90, el advenimiento de la genómica y proteómica ha permitido identificar un gran número de proteínas para las cuáles sólo se cuenta con su estructura primaria. Para profundizar la caracterización estructural y funcional de dichas proteínas, ha sido necesario contar con diversas herramientas bioquímicas y biofísicas. Hoy en día se han desdibujado los límites entre las distintas disciplinas de las ciencias naturales, siendo necesaria una visión integral, provista por la biofisicoquímica, que abarque las técnicas complementarias disponibles actualmente. (Párrafo extraído del texto a modo de resumen)Facultad de Ciencias Exacta

Lipid-Free Antigen B Subunits from <i>Echinococcus granulosus</i>: Oligomerization, Ligand Binding, and Membrane Interaction Properties

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 PlataFacultad de Ciencias Médica