RESUMEN
BACKGROUND: The extracellular matrix (ECM) is a complex tridimensional scaffold that actively participates in physiological and pathological events. The objective of this study was to test whether structural proteins of the ECM and glycosaminoglycans (GAGs) may favor the retention of human apolipoprotein A-I (apoA-I) variants associated with amyloidosis and atherosclerosis. METHODS: Biopolymeric matrices containing collagen type I (Col, a main macromolecular component of the ECM) with or without heparin (Hep, a model of GAGs) were constructed and characterized, and used to compare the binding of apoA-I having the native sequence (Wt) or Arg173Pro, a natural variant inducing cardiac amyloidosis. Protein binding was observed by fluorescence microscopy and unbound proteins quantified by a colorimetric assay. RESULTS: Both, Wt and Arg173Pro bound to the scaffolds containing Col, but the presence of Hep diminished the binding efficiency. Col-Hep matrices retained Arg173Pro more than the Wt. The retained protein was only partially removed from the matrices with saline solutions, indicating that electrostatic interactions may occur but are not the main driving force. Using in addition thermodynamic molecular simulations and size exclusion chromatography approaches, we suggest that the binding of apoA-I variants to the biopolymeric matrices is driven by many low affinity interactions. CONCLUSIONS: Under this scenario Col-Hep scaffolds contribute to the binding of Arg173Pro, as a cooperative platform which could modify the native protein conformation affecting protein folding. GENERAL SIGNIFICANCE: We show that the composition of the ECM is key to the protein retention, and well characterized biosynthetic matrices offer an invaluable in vitro model to mimic the hallmark of pathologies with interstitial infiltration such as cardiac amyloidosis.
Asunto(s)
Amiloidosis , Heparina , Humanos , Amiloidosis/metabolismo , Apolipoproteína A-I/genética , Apolipoproteína A-I/química , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Heparina/metabolismoRESUMEN
Apolipoprotein A-I (apoA-I) has a key function in the reverse cholesterol transport. However, aggregation of apoA-I single point mutants can lead to hereditary amyloid pathology. Although several studies have tackled the biophysical and structural consequences introduced by these mutations, there is little information addressing the relationship between the evolutionary and structural features that contribute to the amyloid behavior of apoA-I. We combined evolutionary studies, in silico mutagenesis and molecular dynamics (MD) simulations to provide a comprehensive analysis of the conservation and pathogenic role of the aggregation-prone regions (APRs) present in apoA-I. Sequence analysis demonstrated that among the four amyloidogenic regions described for human apoA-I, only two (APR1 and APR4) are evolutionary conserved across different species of Sarcopterygii. Moreover, stability analysis carried out with the FoldX engine showed that APR1 contributes to the marginal stability of apoA-I. Structural properties of full-length apoA-I models suggest that aggregation is avoided by placing APRs into highly packed and rigid portions of its native fold. Compared to silent variants extracted from the gnomAD database, the thermodynamic and pathogenic impact of amyloid mutations showed evidence of a higher destabilizing effect. MD simulations of the amyloid variant G26R evidenced the partial unfolding of the alpha-helix bundle with the concomitant exposure of APR1 to the solvent, suggesting an insight into the early steps involved in its aggregation. Our findings highlight APR1 as a relevant component for apoA-I structural integrity and emphasize a destabilizing effect of amyloid variants that leads to the exposure of this region.
Asunto(s)
Apolipoproteína A-I , Secuencia de Aminoácidos , Amiloide/química , Amiloide/metabolismo , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Secuencia Conservada , Evolución Molecular , Humanos , Simulación de Dinámica Molecular , Mutación/genética , Estabilidad Proteica , TermodinámicaRESUMEN
BACKGROUND: The identification of dysfunctional human apolipoprotein A-I (apoA-I) in atherosclerotic plaques suggests that protein structure and function may be hampered under a chronic pro inflammatory scenario. Moreover, the fact that natural mutants of this protein elicit severe cardiovascular diseases (CVD) strongly indicates that the native folding could shift due to the mutation, yielding a structure more prone to misfold or misfunction. To understand the events that determine the failure of apoA-I structural flexibility to fulfill its protective role, we took advantage of the study of a natural variant with a deletion of the residue lysine 107 (K107del) associated with atherosclerosis. METHODS: Biophysical approaches, such as electrophoresis, fluorescence and spectroscopy were used to characterize proteins structure and function, either in native conformation or under oxidation or intramolecular crosslinking. RESULTS: K107del structure was more flexible than the protein with the native sequence (Wt) but interactions with artificial membranes were preserved. Instead, structural restrictions by intramolecular crosslinking impaired the Wt and K107del lipid solubilization function. In addition, controlled oxidation decreased the yield of the native dimer conformation for both variants. CONCLUSIONS: We conclude that even though mutations may alter protein structure and spatial arrangement, the highly flexible conformation compensates the mild shift from the native folding. Instead, post translational apoA-I modifications (probably chronic and progressive) are required to raise a protein conformation with significant loss of function and increased aggregation tendency. GENERAL SIGNIFICANCE: The results learnt from this variant strength a close association between amyloidosis and atherosclerosis.
Asunto(s)
Apolipoproteína A-I/metabolismo , Aterosclerosis/metabolismo , Procesamiento Proteico-Postraduccional , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Aterosclerosis/genética , Humanos , Lípidos de la Membrana/metabolismo , Mutación , Oxidación-Reducción , Conformación ProteicaRESUMEN
The reason that determines the pathological deposition of human apolipoprotein A-I variants inducing organ failure has been under research since the early description of natural mutations in patients. To shed light into the events associated with protein aggregation, we studied the structural perturbations that may occur in the natural variant that shows a substitution of a Leucine by an Arginine in position 60 (L60R). Circular dichroism, intrinsic fluorescence measurements, and proteolysis analysis indicated that L60R was more unstable, more sensitive to cleavage and the N-terminus was more disorganized than the protein with the native sequence (Wt). A higher tendency to aggregate was also detected when L60R was incubated at physiological pH. In addition, the small structural rearrangement observed for the freshly folded variant led to the release of tumor necrosis factor-α and interleukin-1ß from a model of macrophages. However, the mutant preserved both its dimeric conformation and its lipid-binding capacity. Our results strongly suggest that the chronic disease may be a consequence of the native conformation loss which elicits the release of protein conformations that could be either cytotoxic or precursors of amyloid conformations.
Asunto(s)
Proteínas Amiloidogénicas/metabolismo , Apolipoproteína A-I/metabolismo , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/genética , Amiloidosis/etiología , Amiloidosis/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Humanos , Mutación Puntual , Multimerización de Proteína , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: Different protein conformations may be involved in the development of clinical manifestations associated with human amyloidosis. Although a fibrillar conformation is usually the signature of damage in the tissues of patients, it is not clear whether this species is per se the cause or the consequence of the disease. Hereditary amyloidosis due to variants of apolipoprotein A-I (apoA-I) with a substitution of a single amino acid is characterized by the presence of fibrillar protein within the lesions. Thus mutations result in increased protein aggregation. Here we set up to characterize the folding of a natural variant with a mutation leading to a deletion at position 107 (apoA-I Lys107-0). Patients carrying this variant show amyloidosis and severe atherosclerosis. METHODS: We oxidized this variant under controlled concentrations of hydrogen peroxide and analyzed the structure obtained after 30-day incubation by fluorescence, circular dichroism and microscopy approaches. Neutrophils activation was characterized by confocal microscopy. RESULTS: We obtained a high yield of well-defined stable fibrillar structures of apoA-I Lys107-0. In an in vitro neutrophils system, we were able to detect the induction of Neutrophils Extracellular Traps (NETs) when we incubated with oxidized apoA-I variants. This effect was exacerbated by the fibrillar structure of oxidized Lys 107-0. CONCLUSIONS: We conclude that a pro-inflammatory microenvironment could result in the formation of aggregation-prone species, which, in addition may induce a positive feed-back in the activation of an inflammatory response. GENERAL SIGNIFICANCE: These events may explain a close association between amyloidosis due to apoA-I Lys107-0 and atherosclerosis.
Asunto(s)
Amiloidosis Familiar/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Aterosclerosis/genética , Mutación , Amiloidosis Familiar/metabolismo , Apolipoproteína A-I/metabolismo , Aterosclerosis/metabolismo , Humanos , Conformación ProteicaRESUMEN
Among other components of the extracellular matrix (ECM), glycoproteins and glycosaminoglycans (GAGs) have been strongly associated to the retention or misfolding of different proteins inducing the formation of deposits in amyloid diseases. The composition of these molecules is highly diverse and a key issue seems to be the equilibrium between physiological and pathological events. In order to have a model in which the composition of the matrix could be finely controlled, we designed and synthesized crosslinked hydrophilic polymers, the so-called hydrogels varying the amounts of negative charges and hydroxyl groups that are prevalent in GAGs. We checked and compared by fluorescence techniques the binding of human apolipoprotein A-I and a natural mutant involved in amyloidosis to the hydrogel scaffolds. Our results indicate that both proteins are highly retained as long as the negative charge increases, and in addition it was shown that the mutant is more retained than the Wt, indicating that the retention of specific proteins in the ECM could be part of the pathogenicity. These results show the importance of the use of these polymers as a model to get deep insight into the studies of proteins within macromolecules.
Asunto(s)
Proteínas Amiloidogénicas/química , Apolipoproteína A-I/química , Materiales Biomiméticos/química , Hidrogeles/química , Metacrilatos/química , Polímeros/química , Ácidos Sulfónicos/química , Proteínas Amiloidogénicas/genética , Apolipoproteína A-I/genética , Sitios de Unión , Matriz Extracelular/química , Glicosaminoglicanos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Mutación , Unión Proteica , Electricidad EstáticaRESUMEN
The catabolism and structure of high-density lipoproteins (HDL) may be the determining factor of their atheroprotective properties. To better understand the role of the kidney in HDL catabolism, here we characterized HDL subclasses and the catabolic rates of apo A-I in a rabbit model of proteinuria. Proteinuria was induced by intravenous administration of doxorubicin in New Zealand white rabbits (n = 10). HDL size and HDL subclass lipids were assessed by electrophoresis of the isolated lipoproteins. The catabolic rate of HDL-apo A-I was evaluated by exogenous radiolabelling with iodine-131. Doxorubicin induced significant proteinuria after 4 weeks (4.47 ± 0.55 vs. 0.30 ± 0.02 g/L of protein in urine, P < 0.001) associated with increased uremia, creatininemia, and cardiotoxicity. Large HDL2b augmented significantly during proteinuria, whereas small HDL3b and HDL3c decreased compared to basal conditions. HDL2b, HDL2a, and HDL3a subclasses were enriched with triacylglycerols in proteinuric animals as determined by the triacylglycerol-to-phospholipid ratio; the cholesterol content in HDL subclasses remained unchanged. The fractional catabolic rate (FCR) of [(131)I]-apo A-I in the proteinuric rabbits was faster (FCR = 0.036 h(-1)) compared to control rabbits group (FCR = 0.026 h(-1), P < 0.05). Apo E increased and apo A-I decreased in HDL, whereas PON-1 activity increased in proteinuric rabbits. Proteinuria was associated with an increased number of large HDL2b particles and a decreased number of small HDL3b and 3c. Proteinuria was also connected to an alteration in HDL subclass lipids, apolipoprotein content of HDL, high paraoxonase-1 activity, and a rise in the fractional catabolic rate of the [(131)I]-apo A-I.
Asunto(s)
Apolipoproteína A-I/metabolismo , Doxorrubicina/efectos adversos , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Proteinuria/inducido químicamente , Proteinuria/metabolismo , Administración Intravenosa , Animales , Apolipoproteína A-I/química , Doxorrubicina/administración & dosificación , Masculino , Tamaño de la Partícula , ConejosRESUMEN
BACKGROUND: Low plasma levels of high-density lipoprotein-cholesterol (HDL-C) are typical of acute myocardial infarction (MI) and predict risk of recurrent cardiovascular events. The potential relationships between modifications in the molecular composition and the functionality of HDL subpopulations in acute MI however remain indeterminate. METHODS AND RESULTS: ST segment elevation MI (STEMI) patients were recruited within 24h after diagnosis (n=16) and featured low HDL-C (-31%, p<0.05) and acute-phase inflammation (determined as marked elevations in C-reactive protein, serum amyloid A (SAA) and interleukin-6) as compared to age- and sex-matched controls (n=10). STEMI plasma HDL and its subpopulations (HDL2b, 2a, 3a, 3b, 3c) displayed attenuated cholesterol efflux capacity from THP-1 cells (up to -32%, p<0.01, on a unit phospholipid mass basis) vs. CONTROLS: Plasma HDL and small, dense HDL3b and 3c subpopulations from STEMI patients exhibited reduced anti-oxidative activity (up to -68%, p<0.05, on a unit HDL mass basis). HDL subpopulations in STEMI were enriched in two proinflammatory bioactive lipids, lysophosphatidylcholine (up to 3.0-fold, p<0.05) and phosphatidic acid (up to 8.4-fold, p<0.05), depleted in apolipoprotein A-I (up to -23%, p<0.05) and enriched in SAA (up to +10.2-fold, p<0.05); such changes were most marked in the HDL3b subfraction. In vitro HDL enrichment in both lysophosphatidylcholine and phosphatidic acid exerted deleterious effects on HDL functionality. CONCLUSIONS: In the early phase of STEMI, HDL particle subpopulations display marked, concomitant alterations in both lipidome and proteome which are implicated in impaired HDL functionality. Such modifications may act synergistically to confer novel deleterious biological activities to STEMI HDL. SIGNIFICANCE: Our present data highlight complex changes in the molecular composition and functionality of HDL particle subpopulations in the acute phase of STEMI, and for the first time, reveal that concomitant modifications in both the lipidome and proteome contribute to functional deficiencies in cholesterol efflux and antioxidative activities of HDL particles. These findings may provide new biomarkers and new insights in therapeutic strategy to reduce cardiovascular risk in this clinical setting where such net deficiency in HDL function, multiplied by low circulating HDL concentrations, can be expected to contribute to accelerated atherogenesis.
Asunto(s)
Lipoproteínas HDL3/sangre , Lisofosfatidilcolinas/sangre , Infarto del Miocardio/sangre , Ácidos Fosfatidicos/sangre , Proteína Amiloide A Sérica/metabolismo , Adulto , Anciano , Apolipoproteína A-I/química , Apolipoproteína A-I/deficiencia , Apolipoproteína A-I/metabolismo , Proteína C-Reactiva/metabolismo , Estudios de Casos y Controles , Línea Celular , Femenino , Humanos , Interleucina-6/sangre , Lipoproteínas HDL3/química , Lisofosfatidilcolinas/química , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Infarto del Miocardio/patología , Ácidos Fosfatidicos/química , Proteoma/química , Proteoma/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Amiloidogénicas/química , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Heparina/metabolismo , Mutación Puntual , Proteínas Amiloidogénicas/genética , Proteínas Amiloidogénicas/metabolismo , Apolipoproteína A-I/genética , Arginina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Fosfolípidos/metabolismo , Prolina/metabolismo , Agregado de Proteínas , Unión Proteica , Estabilidad Proteica , Dodecil Sulfato de Sodio/metabolismoRESUMEN
Discoidal high-density lipoproteins (D-HDL) are critical intermediates in reverse cholesterol transport. Most of the present knowledge of D-HDL is based on studies with reconstituted lipoprotein complexes of apolipoprotein A-I (apoA-I) obtained by cholate dialysis (CD). D-HDL can also be generated by the direct microsolubilization (DM) of phospholipid vesicles at the gel/fluid phase transition temperature, a process mechanistically similar to the "in vivo" apoAI lipidation via ABCA1. We compared the apoA-I configuration in D-HDL reconstituted with dimyristoylphosphatidylcholine by both procedures using fluorescence resonance energy transfer measurements with apoA-I tryptophan mutants and fluorescently labeled cysteine mutants. Results indicate that apoA-I configuration in D-HDL depends on the reconstitution process and are consistent with a "double belt" molecular arrangement with different helix registry. As reported by others, a configuration with juxtaposition of helices 5 of each apoAI monomer (5/5 registry) predominates in D-HDL obtained by CD. However, a configuration with helix 5 of one monomer juxtaposed with helix 2 of the other (5/2 registry) would predominate in D-HDL generated by DM. Moreover, we also show that the kinetics of cholesterol efflux from macrophage cultures depends on the reconstitution process, suggesting that apoAI configuration is important for this HDL function.
Asunto(s)
Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Lipoproteínas/metabolismo , Sustitución de Aminoácidos , Animales , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Línea Celular , Colesterol/química , Colesterol/genética , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Humanos , Lipoproteínas/química , Lipoproteínas/genética , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Mutación Missense , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins. This protein has key functions in lipoprotein metabolism and its plasma concentration is inversely correlated with the incidence of atherosclerosis and cardiovascular diseases. There is an increasing need to develop methods for efficient production of recombinant apoA-I for using it in basic research or pharmacological therapy. An apoA-I variant lacking two amino acid residues at the N-terminus can be easily produced by bacterial expression. We report here the characterization of this variant comparing its properties with those of the protein isolated from human serum. The results validate the use of this variant in future assays and investigations.
Asunto(s)
Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Apolipoproteína A-I/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Guanidina , Humanos , Desnaturalización Proteica , Estabilidad Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Reproducibilidad de los Resultados , TermodinámicaRESUMEN
Human apolipoprotein A-I (apoA-I)-derived amyloidosis can present with either wild-type (Wt) protein deposits in atherosclerotic plaques or as a hereditary form in which apoA-I variants deposit causing multiple organ failure. More than 15 single amino acid replacement amyloidogenic apoA-I variants have been described, but the molecular mechanisms involved in amyloid-associated pathology remain largely unknown. Here, we have investigated by fluorescence and biochemical approaches the stabilities and propensities to aggregate of two disease-associated apoA-I variants, apoA-IGly26Arg, associated with polyneuropathy and kidney dysfunction, and apoA-ILys107-0, implicated in amyloidosis in severe atherosclerosis. Results showed that both variants share common structural properties including decreased stability compared to Wt apoA-I and a more flexible structure that gives rise to formation of partially folded states. Interestingly, however, distinct features appear to determine their pathogenic mechanisms. ApoA-ILys107-0 has an increased propensity to aggregate at physiological pH and in a pro-inflammatory microenvironment than Wt apoA-I, whereas apoA-IGly26Arg elicited macrophage activation, thus stimulating local chronic inflammation. Our results strongly suggest that some natural mutations in apoA-I variants elicit protein tendency to aggregate, but in addition the specific interaction of different variants with macrophages may contribute to cellular stress and toxicity in hereditary amyloidosis.
Asunto(s)
Sustitución de Aminoácidos , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Animales , Línea Celular , Humanos , Macrófagos/metabolismo , Ratones , Pliegue de Proteína , Multimerización de Proteína , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
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.
Asunto(s)
Amiloide/metabolismo , Apolipoproteína A-I/metabolismo , Aterosclerosis/metabolismo , Naftalenosulfonatos de Anilina/metabolismo , Animales , Apolipoproteína A-I/química , Apolipoproteína A-I/ultraestructura , Benzotiazoles , Células CHO , Colesterol/metabolismo , Cricetinae , Cricetulus , Heparina/metabolismo , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Ácido Hipocloroso/farmacología , Metaloproteinasa 12 de la Matriz/metabolismo , Activación Neutrófila/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteolisis/efectos de los fármacos , Solventes , Acetato de Tetradecanoilforbol/farmacología , Tiazoles/metabolismo , Triptófano/metabolismoRESUMEN
Apolipoprotein A-I (apoAI) contains several amphipathic α-helices. To carry out its function, it exchanges between lipid-free and different lipidated states as bound to membranes or to lipoprotein complexes of different morphology, size, and composition. When bound to membranes or to spherical lipoprotein surfaces, it is thought that most α-helices arrange with their long axis parallel to the membrane surface. However, we previously found that a central region spanning residues 87-112 is exclusively labeled by photoactivable reagents deeply located into the membrane (Córsico et al. (2001) J. Biol. Chem. 276, 16978-16985). A pair of amphipathic α-helical repeats with a particular charge distribution is predicted in this region. In order to study their insertion topology, three single tryptophan mutants, each one containing the tryptophan residue at a selected position in the hydrophobic face of the central Y-helices (W@93, W@104, and W@108), were used. From the accessibility to quenchers located at different membrane depths, distances from the bilayer center of 13.4, 10.5, and 15.7 Å were estimated for positions 93, 104, and 108, respectively. Reported data also indicate that distances between homologous positions (in particular for W@93 and W@104) are very short in dimers in aqueous solution, but they are larger in membrane-bound dimers. Data indicate that an intermolecular central Y-helix bundle would penetrate the membrane perpendicularly to the membrane surface. Intermolecular helix-helix interactions would occur through the hydrophilic helix faces in the membrane-bound bundle but through the hydrophobic faces in the case of dimers in solution.
Asunto(s)
Apolipoproteína A-I/química , Membrana Dobles de Lípidos/química , Triptófano/genética , Apolipoproteína A-I/metabolismo , Dimiristoilfosfatidilcolina/química , Polarización de Fluorescencia , Humanos , Membrana Dobles de Lípidos/metabolismo , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Fenilalanina/genética , Fosfatidilcolinas/química , Valor Predictivo de las Pruebas , Unión Proteica/genética , Multimerización de Proteína/genética , Estructura Secundaria de Proteína , Transporte de Proteínas/genética , Secuencias Repetitivas de Aminoácido/genética , Espectrometría de FluorescenciaRESUMEN
We studied the role of a central domain of human apolipoprotein AI (apoAI) in cholesterol mobilization and removal from cells. In order to check different protein conformations, we tested different sized and cholesterol-content reconstituted apoAI particles (rHDL). Meanwhile cholesterol-free discs were active to induce mobilization, only small cholesterol-containing rHDL were active. To test the influence of a central domain in such events, we used two apoAI variants: one, with its central Y helix pair replaced by the C-terminal domain, and the other having a lysine deleted in central region. The helix-swapping variant decrease the cholesterol pool available to acyl-CoA cholesterol acyl transferase and increase mobilization of newly synthesized cholesterol. Instead, the deletion mutant had no effect on both events. We conclude that the central domain of apoAI is involved in cholesterol cell traffic and solubilization, and that a Y-type charge distribution in polar face may be required, as well as a correct helices-polar face orientation.
Asunto(s)
Apolipoproteína A-I/fisiología , Colesterol/metabolismo , Líquido Intracelular/metabolismo , Movilización Lipídica/fisiología , Péptidos/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Células CHO , Colesterol/química , Colesterol/deficiencia , HDL-Colesterol/química , HDL-Colesterol/fisiología , Cricetinae , Cricetulus , Humanos , Líquido Intracelular/química , Movilización Lipídica/genética , Lisina/genética , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína , Solubilidad , Electricidad EstáticaRESUMEN
We studied the effect of bacterial lipopolysaccharide (LPS)-apolipoprotein A-I (apo A-I) interaction on the structure and function of this protein. The micellization process of dimirystoil phosphatidylcholine liposomes (MLV-DMPC) by apo A-I in the presence of LPS was characterized. Apo A-I may interact with MLV-DMPC at the lipid transition temperature, forming micellar complexes. The kinetics of MLV-DMPC micellization was studied by turbidimetry. In the absence of LPS, a monoexponential decrease in turbidity is observed. Preincubation of apo A-I with LPS impairs the micellization reaction, resulting in biphasic kinetics. The amplitude of the fast phase decreases with increasing concentrations of LPS. In the absence or in the presence of low amounts of LPS (1:0.1 protein:LPS weight ratio), two major micellization products-containing two and three apo A-I molecules per particle-were observed. However, in the presence of higher amounts of LPS (1:1 protein:LPS weight ratio), particles mainly contained two apo A-I molecules. In contrast, a decrease in intrinsic fluorescence intensity of the protein was observed in the presence of an increasing LPS concentration. Finally, we studied the effect of LPS on the transition temperature (Tt) of MLV-DMPC without detecting changes in Tt. In conclusion, the changes found in the micellization process are likely to be mainly caused by changes in the apo A-I conformation by LPS interaction in solution.
Asunto(s)
Apolipoproteína A-I/química , Lipopolisacáridos/química , Lipoproteínas HDL/química , Liposomas/química , Micelas , Pared Celular/química , Dimiristoilfosfatidilcolina/química , Endotoxinas/química , Escherichia coli/química , Fluorescencia , Cinética , Lipopolisacáridos/aislamiento & purificación , Nefelometría y Turbidimetría , Unión Proteica , Solubilidad , Soluciones/química , Espectrometría de FluorescenciaRESUMEN
Pioglitazone is an agonist of the peroxisome proliferator-activated receptor gamma (PPARgamma) that raises HDL-cholesterol plasma in humans. Whether pioglitazone-mediated modifications in HDL-apolipoprotein AI (apo AI) turnover in vivo contribute to this effect has not been completely elucidated. Therefore, we performed kinetic studies of HDL-apo AI radiolabeled with 125I in male New Zealand White rabbits after 6 weeks of 0.6 (n = 8), 1.75 (n = 8), and 2.6 mg/kg/day (n = 7) pioglitazone and vehicle (n = 12) treatment. Fractional catabolic rate (FCR) of HDL-apo AI was significantly higher in 1.75 and 2.6 mg/kg pioglitazone-treated animals, as compared with control rabbits (0.057+/-0.014 and 0.049+/-0.01 versus 0.025+/-0.005 pools/h, respectively); these changes were associated to a similar increase in apo AI production rates (PR) (1.24+/-0.62 and 1.14+/-0.40 versus 0.53+/-0.17 mg/kg/h, p < 0.01). Consequently, apo AI plasma levels in pioglitazone-treated animals were similar to those of controls. The apo AI-FRC and -PR correlated with the relative proportion of the HDL3c subclass, as determined by polyacrylamide gradient electrophoresis. Our data demonstrate that pioglitazone markedly modifies apo AI kinetics and enhances the proportion of small HDL3c particles, despite the unchanged apo AI concentration. Whether or not the pioglitazone-induced structural changes of HDL contribute to the anti-atherosclerotic effects of the drug remains to be determined.
Asunto(s)
Apolipoproteína A-I/farmacocinética , Aterosclerosis/tratamiento farmacológico , Hipoglucemiantes/farmacología , Lipoproteínas HDL/farmacocinética , Tiazolidinedionas/farmacología , Animales , Apolipoproteína A-I/sangre , Apolipoproteína A-I/química , Aterosclerosis/metabolismo , Radioisótopos de Yodo , Lipoproteínas HDL/sangre , Lipoproteínas HDL/química , Hígado/metabolismo , Masculino , Tamaño de la Partícula , Pioglitazona , ConejosRESUMEN
Previous results [J. Biol. Chem. 276 (2001) 16978] indicated that an apolipoprotein A-I (apoAI) central region swings away from lipid contact in discoidal high density lipoproteins (HDL), but it is able to penetrate into the bilayer of lipid vesicles. In this work, we have studied the interaction with lipid membranes of a synthetic peptide with the sequence of apoAI region between residues 77 and 120 (AI 77-120). Like apoAI, AI 77-120 binds to phospholipid vesicles and shows selectivity for cholesterol-containing membranes. Moreover, AI 77-120 promotes cholesterol desorption from membranes in a similar fashion as apoAI and can stimulate cholesterol efflux from Chinese hamster ovary cells. AI 77-120 has a considerable alpha-helical content in water solution, and its secondary structure is not largely modified after binding to membranes. Both apoA-I and AI 77-120 are oligomeric in the lipid-bound state, suggesting that dimerization of the central domain could be required for the membrane binding activity of apoA-I in HDL.
Asunto(s)
Apolipoproteína A-I/química , Péptidos/química , Animales , Células CHO , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Dicroismo Circular , Cricetinae , Relación Dosis-Respuesta a Droga , Humanos , Luz , Metabolismo de los Lípidos , Lípidos/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Factores de TiempoRESUMEN
Previous evidence indicated that discoidal reconstituted high density lipoproteins (rHDL) of apolipoprotein A-I (apoA-I) can interact with lipid membranes (Tricerri, M. A., Córsico, B., Toledo, J. D., Garda, H. A., and Brenner, R. R. (1998) Biochim. Biophys. Acta 1391, 67-78). With the aim of studying this interaction, photoactivable reagents and protein cleavage with CNBr and hydroxylamine were used. The generic hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine gave information on the apoA-I regions in contact with the lipid phase in the rHDL discs. Two protein regions loosely bound to lipids were detected: a C-terminal domain and a central one located between residues 87 and 112. They consist of class Y amphipathic alpha-helices that have a different distribution of the charged residues in their polar faces by comparison with class A helices, which predominate in the rest of the apoA-I molecule. The phospholipid analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoro-methyl-3-H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, which does not undergo significant exchange between membranes and lipoproteins, was used to identify the apoA-I domain directly involved in the interaction of rHDL discs with membranes. By incubating either rHDL or lipid-free apoA-I with lipid vesicles containing 125I-TID-PC, only the 87-112 apoA-I segment becomes labeled after photoactivation. These results indicate that the central domain formed by two type Y helices swings away from lipid contact in the discoidal lipoproteins and is able to insert into membrane bilayers, a process that may be of great importance for the mechanism of cholesterol exchange between high density lipoproteins and cell membranes.
Asunto(s)
Apolipoproteína A-I/sangre , Apolipoproteína A-I/química , Membrana Dobles de Lípidos/química , Secuencia de Aminoácidos , Apolipoproteína A-I/aislamiento & purificación , Azirinas/farmacocinética , Sitios de Unión , Bromuro de Cianógeno , Humanos , Hidroxilamina , Radioisótopos de Yodo , Modelos Biológicos , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fosfatidilcolinas/farmacocinética , Etiquetas de Fotoafinidad/farmacocinética , Estructura Secundaria de ProteínaRESUMEN
Two monoclonal antibodies (MAbs) against apolipoprotein A-I (apo A-I), 6B9 and FF9B10, and one MAb against apolipoprotein A-II (apo A-II), 3F5, were characterized. To establish the epitope of apo A-I recognized by these antibodies, different experimental approaches were performed. First, competition between MAbs and the related epitopes on the same antigen was performed using double-determinant tests with previously characterized MAbs. Second, competition of different synthetic peptides of apo A-I in solution with apo A-I immobilized to solid phase was carried out. The MAbs against apo A-I (6B9 and FF9B10) appear to recognize discontinuous epitopes located in the amino-terminal region of the apo A-I. In competition experiments MAb 3F5 did not recognize central- or carboxy-terminal peptides of apo A-II. Furthermore, apo A-II was stronger recognized when it was included in HDL or LpA-I:A-II than in its purified form. So the epitope for 3F5 is better expressed in the lipoprotein structure. Finally, to establish the epitopes expression in different antigens in solution, competition of purified apo A-I, apo A-II, LpA-I, and LpA-I:A-II particles or HDL3, with apo A-I or HDL immobilized to solid phase, was carried out. The results showed that both MAbs against apo A-I reacted with poor affinity against free apo A-I, with high and similar affinities against Lp A-I and Lp A-I:A-II lipoparticles and with the highest affinity against HDL3. The MAb 3F5 against apo A-II recognized only LpA-I:A-II and not LpA-I lipoparticles.