RESUMEN
Ascorbic acid (AsA) is an important antioxidant and enzyme cofactor in many biochemical processes. Most biological activities of AsA are closely related to its redox properties. Recent investigations have demonstrated that AsA is associated with amyloid-related diseases and can inhibit amyloid aggregation of polypeptides. In the present study, we determined the kinetics of AsA degradation and investigated the anti-amyloidogenic activities of AsA and its degradation products by utilizing insulin as a model polypeptide. The results showed that the half-life of AsA varied with the pH of the medium and the incubation temperature. The degradation products of AsA inhibited insulin fibrillation, with an activity positively correlated to the degree of AsA degradation. The degradation species, compared with intact AsA, also showed a stronger disruptive effect on mature amyloid fibrils and significantly decreased fibrillar cytotoxicity. Dehydroascorbic acid and diketogulonic acid, two key intermediates in AsA degradation, had similar anti-amyloidogenic activity toward the degradation species of AsA. The results of this work indicate that degradation of natural antioxidants must be considered when evaluating their anti-amyloidogenic effects. These insights into the action of AsA may also provide a novel route to understand its physiological/pharmacological roles in amyloid-related diseases.
Asunto(s)
Amiloide/química , Ácido Deshidroascórbico/química , Insulina/química , Agregado de Proteínas , Animales , Bovinos , Estabilidad ProteicaRESUMEN
Sodium dodecyl sulfate (SDS), as an anionic surfactant, can induce protein conformational changes. Recent investigations demonstrated different effects of SDS on protein amyloid aggregation. In the present study, the effect of SDS on amyloid aggregation of bovine serum albumin (BSA) was evaluated. BSA transformed to ß-sheet-rich amyloid aggregates upon incubation at pH 7.4 and 65°C, as demonstrated by thioflavin T fluorescence, circular dichroism, and transmission electron microscopy. SDS at submicellar concentrations inhibited BSA amyloid aggregation with IC50 of 47.5 µM. The inhibitory effects of structural analogs of SDS on amyloid aggregation of BSA were determined to explore the structure-activity relationship, with results suggesting that both anionic and alkyl moieties of SDS were critical, and that an alkyl moiety with chain length ≥10 carbon atoms was essential to amyloid inhibition. We attributed the inhibitory effect of SDS on BSA amyloid aggregation to interactions between the detergent molecule and the fatty acid binding sites on BSA. The bound SDS stabilized BSA, thereby inhibiting protein transformation to amyloid aggregates. This study reports for the first time that the inhibitory effect of SDS on albumin fibrillation is closely related to its alkyl structure. Moreover, the specific binding of SDS to albumin is the main driving force in amyloid inhibition. This study not only provides fresh insight into the role of SDS in amyloid aggregation of serum albumin, but also suggests rational design of novel anti-amyloidogenic reagents based on specific-binding ligands.
Asunto(s)
Proteínas Amiloidogénicas/antagonistas & inhibidores , Albúmina Sérica Bovina/antagonistas & inhibidores , Dodecil Sulfato de Sodio/farmacología , Tensoactivos/farmacología , Proteínas Amiloidogénicas/metabolismo , Animales , Bovinos , Relación Dosis-Respuesta a Droga , Micelas , Tamaño de la Partícula , Agregado de Proteínas/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Dodecil Sulfato de Sodio/química , Relación Estructura-Actividad , Propiedades de Superficie , Tensoactivos/químicaRESUMEN
Preformed amyloid fibrils can act as seeds for accelerating protein ï¬brillation. In the present study, we examined the effects of preformed seeds on lysozyme amyloid fibrillation in the presence of two distinct inhibitors - epigallocatechin (EGC) and polyethylene glycol 2000 (PEG). The results demonstrated that the effects of fibrillar seeds on the acceleration of lysozyme fibrillation depended on the aggregation pathway directed by an inhibitor. EGC inhibited lysozyme fibrillation and modified the peptide chains with quinone moieties in a concentration-dependent manner. The resulting aggregates showed amorphous off-pathway morphology. Preformed fibril seeds did not promote lysozyme fibrillation in the presence of EGC. PEG also inhibited lysozyme fibrillation, and the resulting aggregates showed on-pathway protofibrillar morphology. In contrast, the addition of fibril seeds into the mixture of lysozyme and PEG significantly stimulated fibril growth. Assays of cell viability showed that both EGC and PEG inhibited the formation of cytotoxic species. In accordance with thioflavine T data, the seeds failed to alter the cell-damaging potency of the EGC-directed off-pathway aggregates, but increased the cytotoxicity of the PEG-directed on-pathway fibrils. We suggest that the pattern of interaction between lysozyme and an inhibitor determines the pathway of aggregation and therefore the effects of seeding on amyloid formation. EGC covalently modified lysozyme chains with quinones, directing the aggregation to proceed through an off-pathway, whereas PEG affected the protein in a noncovalent manner, and fibril growth could be stimulated under seeding through an on-pathway.
Asunto(s)
Amiloide/química , Catequina/análogos & derivados , Muramidasa/química , Polietilenglicoles/química , Animales , Catequina/química , Células PC12 , RatasRESUMEN
Epigallocatechin gallate (EGCG), the most abundant flavanoid in green tea, is currently being evaluated in the clinic due to its benefits in the treatment of amyloid disorders. Its anti-amyloidogenic effect has been attributed to direct interaction of the intact molecule with misfolded polypeptides. In addition, antioxidant activity is also involved in the anti-amyloidogenic role. The detailed molecular mechanism is still unclear and requires further investigation. In the present study, the kinetics of EGCG oxidation and the anti-amyloidogenic effect of the resultant oxidation substances have been examined. The results indicate that EGCG degrades in a medium at pH 8.0 with a half-life less than 2h. By utilizing lysozyme as an in vitro model, the oxidized EGCG demonstrates a more potent anti-amyloidogenic capacity than the intact molecule, as shown by ThT and ANS fluorescence, TEM determination, and hemolytic assay. The oxidized EGCG also has a stronger disruptive effect on preformed fibrils than the native form. Ascorbic acid eliminates the disruptive role of native EGCG on the fibrils, suggesting that oxidation is a prerequisite in fibril disruption. The results of this work demonstrate that oxidized EGCG plays a more important role than the intact molecule in anti-amyloidogenic activity. These insights into the action of EGCG may provide a novel route to understand the anti-amyloidogenic activity of natural polyphenols.
Asunto(s)
Proteínas Amiloidogénicas/química , Antioxidantes/química , Catequina/análogos & derivados , Muramidasa/química , Proteínas Amiloidogénicas/antagonistas & inhibidores , Animales , Ácido Ascórbico/química , Catequina/química , Pollos , Semivida , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Muramidasa/antagonistas & inhibidores , Oxidación-Reducción , SolucionesRESUMEN
Numerous studies demonstrate that natural polyphenols can inhibit amyloid formation and disrupt preformed amyloid fibrils. In the present study, the fibril-disruptive effects of epigallocatechin-3-gallate (EGCG) were examined using lysozyme as a model protein. The results indicated that EGCG dose dependently inhibited lysozyme fibrillation and modified the peptide chains with quinonoid moieties under acidic conditions, as measured by ThT fluorescence, transmission electron microscopy, and an NBT-staining assay. Moreover, EGCG transformed the preformed lysozyme fibrils to amorphous aggregates through quinopeptide formation. The thiol blocker, N-ethylmaleimide, inhibited the disruptive effect of EGCG on preformed fibrils, suggesting that thiol groups are the binding sites for EGCG. We propose that the formation of quinone intermediates via oxidation and subsequent binding to lysozyme chains are the main processes driving the inhibition of amyloid formation and disruption of preformed fibrils by EGCG. The information presented in this study may provide fresh insight into the link between the antioxidant capacity and anti-amyloid activity of polyphenols.
Asunto(s)
Catequina/análogos & derivados , Muramidasa/química , Péptidos/química , Agregación Patológica de Proteínas , Amiloide/química , Animales , Catequina/química , Catequina/farmacología , Estructura Molecular , Polifenoles/química , Polifenoles/farmacologíaRESUMEN
Numerous phenolic compounds have been reported to have an inhibitory role on amyloid formation of proteins. The present study, utilizing lysozyme as a model system, examined the anti-amyloidogenic effects of phenol and three diphenol epimers. The results indicated that catechol and hydroquinone dose-dependently inhibited lysozyme fibrillation and covalently bound to the peptide chains to form quinoproteins, showing a similar effect to benzoquinone. In contrast, phenol and resorcinol did not modify the peptide with a quinone moiety, showing no effect on lysozyme fibrillation. We suggest that quinone intermediates are the active form for a phenolic compound to inhibit lysozyme fibrillation. The modification of lysozyme with quinone moieties alters the interacting forces between peptide chains and consequently interrupts the process of lysozyme fibrillation.
Asunto(s)
Amiloide/metabolismo , Muramidasa/metabolismo , Fenoles/metabolismo , Amiloide/antagonistas & inhibidores , Amiloide/ultraestructura , Benzoquinonas/química , Benzoquinonas/metabolismo , Benzoquinonas/farmacología , Catecoles/química , Catecoles/metabolismo , Catecoles/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Hidroquinonas/química , Hidroquinonas/metabolismo , Hidroquinonas/farmacología , Cinética , Microscopía Electrónica de Transmisión , Estructura Molecular , Muramidasa/antagonistas & inhibidores , Muramidasa/ultraestructura , Fenoles/química , Fenoles/farmacología , Unión Proteica , Resorcinoles/química , Resorcinoles/metabolismo , Resorcinoles/farmacologíaRESUMEN
Growing interest and research efforts have recently been focused on elucidating the molecular mechanism of amyloid formation and the screening of effective inhibitors to interrupt amyloid structures. In the present study, the anti-amyloidogenic effects of quercetin were investigated in vitro using bovine insulin as a model protein. The results demonstrated that quercetin dose-dependently inhibited amyloid formation of insulin. Moreover, quercetin destabilized the preformed insulin fibrils and transformed the fibrils into amorphous aggregates. Hemolysis was observed when human erythrocytes were co-incubated with insulin fibrils. Quercetin inhibited fibril-induced hemolysis in a dose-dependent manner. SDS-PAGE showed that insulin fibrils induced the aggregation of cytoskeletal proteins of erythrocyte membranes and that quercetin attenuated this fibril-induced cytoskeletal aggregation. The results of the present work suggest that quercetin may serve as a lead structure for the design of novel anti-amyloidogenic drugs.
Asunto(s)
Amiloide/antagonistas & inhibidores , Insulina/química , Quercetina/farmacología , Amiloide/química , Amiloide/ultraestructura , Animales , Bovinos , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Hemólisis/efectos de los fármacos , Humanos , Estabilidad Proteica/efectos de los fármacosRESUMEN
Increasing evidence has demonstrated that EGCG possesses prooxidant potential in biological systems, including modifying proteins, breaking DNA strands and inducing the generation of reactive oxygen species. In the present study, the prooxidant effect of EGCG on erythrocyte membranes was investigated. SDS-PAGE and NBT-staining assay were utilized to detect the catechol-protein adducts that generated upon treating the membranes with EGCG. The results indicated that EGCG was able to bind covalently to sulfhydryl groups of membrane proteins, leading to the formation of protein aggregates with intermolecular cross-linking. We suggested that the catechol-quinone originated from the oxidation of EGCG acted as a cross-linker on which peptide chains were combined through thiol-S-alkylation at the C2- and C6-sites of the gallyl ring. EGC showed similar effects as EGCG on the ghost membranes, whereas ECG and EC did not, suggesting that a structure with a gallyl moiety is a prerequisite for a catechin to induce the aggregation of membrane proteins and to deplete membrane sulfhydryls. EDTA and ascorbic acid inhibited the EGCG-induced aggregation of membrane proteins by blocking the formation of catechol-quinone. The information of the present study may provide a fresh insight into the prooxidant effect and cytotoxicity of tea catechins.
Asunto(s)
Catequina/análogos & derivados , Reactivos de Enlaces Cruzados/farmacología , Flavonoides/farmacología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Fenoles/farmacología , Té/química , Ácido Ascórbico/farmacología , Benzoquinonas/química , Benzoquinonas/farmacología , Catequina/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Ácido Edético/farmacología , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Humanos , Peso Molecular , Polifenoles , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína , Especies Reactivas de Oxígeno/farmacología , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Natural polyphenols are major constituents of plant foods and herbs. Numerous studies have demonstrated that natural polyphenols inhibited amyloid formation and destabilized the preformed amyloid fibrils. However, the molecular mechanism for the antiamyloidogenesis of polyphenols is still unclear and remains to be further explored. In the present study, the preformed lysozyme fibrils were used as an in vitro model to study the disruptive effects of tea catechins on amyloid fibrils. Results showed that tea catechins induced the conversion of lysozyme fibrils to amorphous aggregates and inhibited fibril-induced hemolysis. Hydroquinone also showed disruptive effect on the fibrils, whereas phenol and two typical antioxidants, ascorbic acid and alpha-tocopherol, did not affect the fibrillar structure, suggesting that polyphenolic structure is essential for fibril deposition. Correlation analyses indicate that the fibril-depositing effects were related to both the antioxidative potency and hydrophobicity of tea catechins. These findings provide new evidence for comprehensive understanding of the interaction between natural polyphenols and amyloid fibrils.
Asunto(s)
Amiloide/química , Camellia sinensis/química , Catequina/química , Muramidasa/química , Flavonoides/química , Interacciones Hidrofóbicas e Hidrofílicas , Fenoles/química , Polifenoles , Conformación Proteica , Pliegue de ProteínaRESUMEN
Accumulating evidence has strongly suggested that amyloid fibrils of protein or peptide are cytotoxic. Fibrillar species appear to lead to disruption of cell membrane structures and thereby cause cell death. In this study, human erythrocytes were used as an in vitro model to examine the disruptive effect of lysozyme fibrils on the plasma membrane. Both the protofibrils and mature fibrils induced hemolysis and aggregation of erythrocytes. Treating ghost membranes with the fibrils resulted in aggregation of membrane proteins through intermolecular disulfide cross-linking. LC-ESI-MS/MS and Western blotting analysis showed that lysozyme fragments were incorporated into the aggregates of ghost membrane proteins, which suggested that thio-disulfide exchange among lysozyme and membrane proteins was triggered when the fibrils interacted with erythrocyte membranes. Metal-ion chelators, radical scavengers, and antioxidants had no effect on the amyloid-induced disulfide cross-linking. The exposure of interior hydrophobic residues and the increased level of solvent-accessible disulfides in the lysozyme fibrils are thought to be involved in membrane disruption. These results may unveil a novel pathway for the cytotoxicity of amyloid fibrils.
Asunto(s)
Amiloide/fisiología , Disulfuros/metabolismo , Membrana Eritrocítica/metabolismo , Secuencia de Aminoácidos , Amiloide/química , Amiloide/genética , Amiloide/ultraestructura , Animales , Senescencia Celular/fisiología , Pollos , Reactivos de Enlaces Cruzados/metabolismo , Membrana Eritrocítica/química , Membrana Eritrocítica/genética , Membrana Eritrocítica/ultraestructura , Humanos , Datos de Secuencia Molecular , Muramidasa/química , Muramidasa/genética , Muramidasa/fisiología , Muramidasa/ultraestructuraRESUMEN
Extracts from the leaves of Ginkgo biloba have been used in Chinese medicine for thousands of years. Today, various standardized preparations from G. biloba leaf extract have been developed. G. biloba leaf extract, which contains flavonoids and terpenoids as the major biologically active components, has become one of the most popular and commonly used herbal remedies due to its wide spectrum of beneficial effects on health. In this study, we investigated the effects of G. biloba leaf extract on the properties of human red blood cells in the presence and absence of amyloid peptide (Abeta25-35), peroxide and hypotonic stress. The results suggest that G. biloba leaf extract has a dual action, both protective and disruptive, on red blood cells, depending on whether an exogenous stress is present. G. biloba leaf extract has a protective role on red blood cells against Abeta- and hypotonic pressure-induced haemolysis, peroxide-induced lipoperoxidation, as well as glutathione consumption and methaemoglobin formation. On the other hand, G. biloba leaf extract also exhibited damage to red blood cells by increasing cell fragility, changing cellular morphology and inducing glutathione consumption and methaemoglobin formation, especially when applied at high doses. These anti- and pro-oxidative activities of polyphenolic substances are thought to be involved in the dual function of G. biloba leaf extract. The results of this study suggest that high doses of herbal remedies and dietary supplements can be toxic to cells.