Aterosclerosis y amiloidosis: ¿dos patologías crónicas interrelacionadas?

A fin de poder llevar a cabo las distintas funciones biológicas, es esencial que las proteínas conserven su conformación nativa. Algunas proteínas son estructuralmente inestables, y entonces pequeños cambios en el microambiente en el que se encuentran pueden ser clave para alterar el equilibrio hacia una conformación patológica. Las amiloidosis se caracterizan por la presencia de depósitos extracelulares de proteínas que adoptan estructura fibrilar. La apolipoproteína A-I humana no está normalmente asociada a esta patología, aunque fueron detectados agregados de la misma con la secuencia nativa en placas ateroscleróticas seniles. A pesar de ser frecuente, se conoce relativamente poco de la patogénesis y significancia de la agregación patológica de la apoA-I

Amyloidogenic propensity of a natural variant of human apolipoprotein A-I: Stability and interaction with ligands

A number of naturally occurring mutations of human apolipoprotein A-I (apoA-I) have been associated with hereditary amyloidoses. The molecular mechanisms involved in amyloid-associated pathology remain largely unknown. Here we examined the effects of the Arg173Pro point mutation in apoA-I on the structure, stability, and aggregation propensity, as well as on the ability to bind to putative ligands. Our results indicate that the mutation induces a drastic loss of stability, and a lower efficiency to bind to phospholipid vesicles at physiological pH, which could determine the observed higher tendency to aggregate as pro-amyloidogenic complexes. Incubation under acidic conditions does not seem to induce significant desestabilization or aggregation tendency, neither does it contribute to the binding of the mutant to sodium dodecyl sulfate. While the binding to this detergent is higher for the mutant as compared to wt apoA-I, the interaction of the Arg173Pro variant with heparin depends on pH, being lower at pH 5.0 and higher than wt under physiological pH conditions. We suggest that binding to ligands as heparin or other glycosaminoglycans could be key events tuning the fine details of the interaction of apoA-I variants with the micro-environment, and probably eliciting the toxicity of these variants in hereditary amyloidoses.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La PlataInstituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

Role of plasma membrane lipid composition on cellular homeostasis: learning from cell line models expressing fatty acid desaturases

Experimental evidence has suggested that plasma membrane (PM)-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids (PUFAs) effect on PM properties and analyze its influence on cholesterol (Chol) homeostasis. We have previously shown that by using a cell line over-expressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and cell viability by different mechanisms. Now, we expanded our studies to a cell model over-expressing both Δ5 and Δ6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein A-I-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the cell functionality is preserved by regulating PM organization and Chol exportation and homeostasis.Instituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias Médica

Propiedades de la membrana y viabilidad celular: importancia de la fluidez

Numerosos estudios sugieren que las vías de señalización y por ende la funcionalidad celular dependen de la organización de dominios en la membrana, que a su vez está determinada por la composición lipídica de la misma. El colesterol (Col) interviene en la regulación de la fluidez al particionar de manera selectiva en dominios específicos de la membrana, y se ha demostrado que su homeostasis es crucial para la viabilidad celular. Además, se sabe que el exceso de Col puede resultar citotóxico. Este lípido no puede ser degradado o utilizado como combustible, por lo que su exceso debe ser removido por aceptores o almacenado en compartimientos intracelulares. Las lipoproteínas de alta densidad (HDL) y en particular su apolipoproteína mayoritaria, la apoA-I, cumplen un rol fundamental en el transporte reverso del Col, que consiste en transportar el excedente desde los tejidos periféricos hacia el hígado para su eliminación en forma de sales biliares, o para ser redirigido desde los hepatocitos hacia los tejidos esteroidogénicos.Universidad Nacional de La Plat

Amyloidogenic propensity of a natural variant of human apolipoprotein A-I: Stability and interaction with ligands

A number of naturally occurring mutations of human apolipoprotein A-I (apoA-I) have been associated with hereditary amyloidoses. The molecular mechanisms involved in amyloid-associated pathology remain largely unknown. Here we examined the effects of the Arg173Pro point mutation in apoA-I on the structure, stability, and aggregation propensity, as well as on the ability to bind to putative ligands. Our results indicate that the mutation induces a drastic loss of stability, and a lower efficiency to bind to phospholipid vesicles at physiological pH, which could determine the observed higher tendency to aggregate as pro-amyloidogenic complexes. Incubation under acidic conditions does not seem to induce significant desestabilization or aggregation tendency, neither does it contribute to the binding of the mutant to sodium dodecyl sulfate. While the binding to this detergent is higher for the mutant as compared to wt apoA-I, the interaction of the Arg173Pro variant with heparin depends on pH, being lower at pH 5.0 and higher than wt under physiological pH conditions. We suggest that binding to ligands as heparin or other glycosaminoglycans could be key events tuning the fine details of the interaction of apoA-I variants with the micro-environment, and probably eliciting the toxicity of these variants in hereditary amyloidoses.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La PlataInstituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

Role of plasma membrane lipid composition on cellular homeostasis: learning from cell line models expressing fatty acid desaturases

Experimental evidence has suggested that plasma membrane (PM)-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids (PUFAs) effect on PM properties and analyze its influence on cholesterol (Chol) homeostasis. We have previously shown that by using a cell line over-expressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and cell viability by different mechanisms. Now, we expanded our studies to a cell model over-expressing both Δ5 and Δ6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein A-I-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the cell functionality is preserved by regulating PM organization and Chol exportation and homeostasis.Instituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias Médica

Aterosclerosis y amiloidosis: ¿dos patologías crónicas interrelacionadas?

A fin de poder llevar a cabo las distintas funciones biológicas, es esencial que las proteínas conserven su conformación nativa. Algunas proteínas son estructuralmente inestables, y entonces pequeños cambios en el microambiente en el que se encuentran pueden ser clave para alterar el equilibrio hacia una conformación patológica. Las amiloidosis se caracterizan por la presencia de depósitos extracelulares de proteínas que adoptan estructura fibrilar. La apolipoproteína A-I humana no está normalmente asociada a esta patología, aunque fueron detectados agregados de la misma con la secuencia nativa en placas ateroscleróticas seniles. A pesar de ser frecuente, se conoce relativamente poco de la patogénesis y significancia de la agregación patológica de la apoA-I.Facultad de Ciencias Médica

Human apolipoprotein A-I-derived amyloid: its association with atherosclerosis

Amyloidoses constitute a group of diseases in which soluble proteins aggregate and deposit extracellularly in tissues. Nonhereditary apolipoprotein A-I (apoA-I) amyloid is characterized by deposits of nonvariant protein in atherosclerotic arteries. Despite being common, little is known about the pathogenesis and significance of apoA-I deposition. In this work we investigated by fluorescence and biochemical approaches the impact of a cellular microenvironment associated with chronic inflammation on the folding and pro-amyloidogenic processing of apoA-I. Results showed that mildly acidic pH promotes misfolding, aggregation, and increased binding of apoA-I to extracellular matrix elements, thus favoring protein deposition as amyloid like-complexes. In addition, activated neutrophils and oxidative/proteolytic cleavage of the protein give rise to pro amyloidogenic products. We conclude that, even though apoA-I is not inherently amyloidogenic, it may produce non hereditary amyloidosis as a consequence of the pro-inflammatory microenvironment associated to atherogenesis.Facultad de Ciencias Médica

Human Apolipoprotein A-I-Derived Amyloid: Its Association with Atherosclerosis

Amyloidoses constitute a group of diseases in which soluble proteins aggregate and deposit extracellularly in tissues. Nonhereditary apolipoprotein A-I (apoA-I) amyloid is characterized by deposits of nonvariant protein in atherosclerotic arteries. Despite being common, little is known about the pathogenesis and significance of apoA-I deposition. In this work we investigated by fluorescence and biochemical approaches the impact of a cellular microenvironment associated with chronic inflammation on the folding and pro-amyloidogenic processing of apoA-I. Results showed that mildly acidic pH promotes misfolding, aggregation, and increased binding of apoA-I to extracellular matrix elements, thus favoring protein deposition as amyloid like-complexes. In addition, activated neutrophils and oxidative/proteolytic cleavage of the protein give rise to pro amyloidogenic products. We conclude that, even though apoA-I is not inherently amyloidogenic, it may produce non hereditary amyloidosis as a consequence of the pro-inflammatory microenvironment associated to atherogenesis

Lipid packing determines protein-membrane interactions: challenges for apolipoprotein A-I and high density lipoproteins.

Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction, and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. Fluorescence Correlation Spectroscopy (FCS) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan Generalized Polarization (GP) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis and offer a methodological design suited to different biological systems