RESUMEN
Arctic (E22G) mutation in amyloid-ß (Aß enhances Aß40 fibril accumulation in Alzheimer's disease (AD). Unlike sporadic AD, familial AD (FAD) patients with the mutation exhibit more Aß40 in the plaque core. However, structural details of E22G Aß40 fibrils remain elusive, hindering therapeutic progress. Here, we determine a distinctive W-shaped parallel ß-sheet structure through co-analysis by cryo-electron microscopy (cryoEM) and solid-state nuclear magnetic resonance (SSNMR) of in-vitro-prepared E22G Aß40 fibrils. The E22G Aß40 fibrils displays typical amyloid features in cotton-wool plaques in the FAD, such as low thioflavin-T fluorescence and a less compact unbundled morphology. Furthermore, kinetic and MD studies reveal previously unidentified in-vitro evidence that E22G Aß40, rather than Aß42, may trigger Aß misfolding in the FAD, and prompt subsequent misfolding of wild-type (WT) Aß40/Aß42 via cross-seeding. The results provide insight into how the Arctic mutation promotes AD via Aß40 accumulation and cross-propagation.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Microscopía por Crioelectrón , Mutación , Fragmentos de Péptidos , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Humanos , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/ultraestructura , Cinética , Pliegue de Proteína , Amiloide/metabolismo , Amiloide/química , Simulación de Dinámica MolecularRESUMEN
Filament assembly of amyloid-ß peptides ending at residue 42 (Aß42) is a central event in Alzheimer's disease. Here, we report the cryoelectron microscopy (cryo-EM) structures of Aß42 filaments from human brains. Two structurally related S-shaped protofilament folds give rise to two types of filaments. Type I filaments were found mostly in the brains of individuals with sporadic Alzheimer's disease, and type II filaments were found in individuals with familial Alzheimer's disease and other conditions. The structures of Aß42 filaments from the brain differ from those of filaments assembled in vitro. By contrast, in AppNL-F knock-in mice, Aß42 deposits were made of type II filaments. Knowledge of Aß42 filament structures from human brains may lead to the development of inhibitors of assembly and improved imaging agents.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/ultraestructura , Química Encefálica , Fragmentos de Péptidos/química , Fragmentos de Péptidos/ultraestructura , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Péptidos beta-Amiloides/genética , Animales , Microscopía por Crioelectrón , Femenino , Técnicas de Sustitución del Gen , Humanos , Masculino , Ratones , Persona de Mediana Edad , Modelos Animales , Modelos Moleculares , Fragmentos de Péptidos/genética , Conformación Proteica , Conformación Proteica en Lámina beta , Dominios Proteicos , Pliegue de ProteínaRESUMEN
Gangliosides induced a smelting process in nanostructured amyloid fibril-like films throughout the surface properties contributed by glycosphingolipids when mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/Aß(1-40) amyloid peptide. We observed a dynamical smelting process when pre-formed amyloid/phospholipid mixture is laterally mixed with gangliosides. This particular environment, gangliosides/phospholipid/Aß(1-40) peptide mixed interfaces, showed complex miscibility behavior depending on gangliosides content. At 0% of ganglioside covered surface respect to POPC, Aß(1-40) peptide forms fibril-like structure. In between 5 and 15% of gangliosides, the fibrils dissolve into irregular domains and they disappear when the proportion of gangliosides reach the 20%. The amyloid interfacial dissolving effect of gangliosides is taken place at lateral pressure equivalent to the organization of biological membranes. Domains formed at the interface are clearly evidenced by Brewster Angle Microscopy and Atomic Force Microscopy when the films are transferred onto a mica support. The domains are thioflavin T (ThT) positive when observed by fluorescence microscopy. We postulated that the smelting process of amyloids fibrils-like structure at the membrane surface provoked by gangliosides is a direct result of a new interfacial environment imposed by the complex glycosphingolipids. We add experimental evidence, for the first time, how a change in the lipid environment (increase in ganglioside proportion) induces a rapid loss of the asymmetric structure of amyloid fibrils by a simple modification of the membrane condition (a more physiological situation).
Asunto(s)
Péptidos beta-Amiloides/química , Gangliósidos/química , Glicoesfingolípidos/química , Lípidos de la Membrana/química , Nanoestructuras/química , Fragmentos de Péptidos/química , Amiloide/química , Péptidos beta-Amiloides/ultraestructura , Microscopía de Fuerza Atómica , Nanoestructuras/ultraestructura , Fragmentos de Péptidos/ultraestructura , Fosfatidilcolinas/química , Propiedades de SuperficieRESUMEN
Amyloid ß (Aß) peptide aggregation plays a central role in Alzheimer's disease (AD) etiology. AD drug candidates have included small molecules or peptides directed towards inhibition of Aß fibrillogenesis. Although some Aß-derived peptide fragments suppress Aß fibril growth, comprehensive analysis of inhibitory potencies of peptide fragments along the whole Aß sequence has not been reported. The aim of this work is (a) to identify the region(s) of Aß with highest propensities for aggregation and (b) to use those fragments to inhibit Aß fibrillogenesis. Structural and aggregation properties of the parent Aß1-42 peptide and seven overlapping peptide fragments have been studied, i.e. Aß1-10 (P1), Aß6-15 (P2), Aß11-20 (P3), Aß16-25 (P4), Aß21-30 (P5), Aß26-36 (P6), and Aß31-42 (P7). Structural transitions of the peptides in aqueous buffer have been monitored by circular dichroism and Fourier transform infrared spectroscopy. Aggregation and fibrillogenesis were analyzed by light scattering and thioflavin-T fluorescence. The mode of peptide-peptide interactions was characterized by fluorescence resonance energy transfer. Three peptide fragments, P3, P6, and P7, exhibited exceptionally high propensity for ß-sheet formation and aggregation. Remarkably, only P3 and P6 exerted strong inhibitory effect on the aggregation of Aß1-42, whereas P7 and P2 displayed moderate inhibitory potency. It is proposed that P3 and P6 intercalate between Aß1-42 molecules and thereby inhibit Aß1-42 aggregation. These findings may facilitate therapeutic strategies of inhibition of Aß fibrillogenesis by Aß-derived peptides.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Agregación Patológica de Proteínas/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Dicroismo Circular , Humanos , Fragmentos de Péptidos/uso terapéutico , Fragmentos de Péptidos/ultraestructura , Agregación Patológica de Proteínas/patologíaRESUMEN
Self-assembly of disordered amyloid-beta (Aß) peptides results in highly ordered amyloid fibrils. The structural information of the early-stage events and also in the presence of inhibitors is of great significance. It is challenging to acquire due to the nature of the amyloids and experimental constraints. Here, we demonstrate the cascade of aggregation (early to late) of the Aß25-35 peptide in the absence and presence of carvedilol, a nonselective ß-adrenergic receptor blocker. The aggregation process of Aß25-35 peptide is monitored using Thioflavin T (ThT) fluorescence, dynamic light scattering (DLS), circular dichroism (CD), Raman spectroscopic techniques, and imaging experiments. We find that the Aß25-35 peptide undergoes an early-stage (3-6 h) helical intermediate formation across the fibrillation pathway using CD and Raman measurements. Carvedilol obstructs the helical intermediate formation of Aß25-35 peptide resulting in inhibition. CD spectra and deconvolution of the Raman bands suggest the ß-sheet formation (24-100 h) in the absence of carvedilol. Spectroscopic results indicate a disordered structure for the peptide in the presence of carvedilol (24-100 h). Electron microscopy (EM) shows the formation of polymorphic fibrils for the peptide alone and non-amyloidal aggregates in the presence of carvedilol. Molecular docking study suggests that the plausible mode of interaction with carvedilol involves the C-terminal residues of the peptide.
Asunto(s)
Péptidos beta-Amiloides/química , Amiloide/química , Carvedilol/química , Fragmentos de Péptidos/química , Amiloide/antagonistas & inhibidores , Amiloide/ultraestructura , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/ultraestructura , Carvedilol/farmacología , Dicroismo Circular , Fluorescencia , Humanos , Microscopía Electrónica , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/ultraestructuraRESUMEN
It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimer's amyloid-ß (Aß). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on Aß degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of Aß(1-40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of Aß(1-40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of Aß fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length Aß(1-40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to Aß(1-40) inactivates cf-E111Q-IDE's catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on Aß degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDE's function.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Insulisina/metabolismo , Proteínas Mutantes/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Biocatálisis , Cromatografía Líquida de Alta Presión , Humanos , Insulisina/química , Insulisina/genética , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Simulación de Dinámica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación Missense , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/ultraestructura , Unión Proteica , Proteolisis , Especificidad por Sustrato , Zinc/química , Zinc/metabolismoRESUMEN
Amyloid-ß (Aß) fibrils exhibit self-propagating, molecular-level polymorphisms that may contribute to variations in clinical and pathological characteristics of Alzheimer's disease (AD). We report the molecular structure of a specific fibril polymorph, formed by 40-residue Aß peptides (Aß40), that is derived from cortical tissue of an AD patient by seeded fibril growth. The structure is determined from cryogenic electron microscopy (cryoEM) images, supplemented by mass-per-length (MPL) measurements and solid-state NMR (ssNMR) data. Previous ssNMR studies with multiple AD patients had identified this polymorph as the most prevalent brain-derived Aß40 fibril polymorph from typical AD patients. The structure, which has 2.8-Å resolution according to standard criteria, differs qualitatively from all previously described Aß fibril structures, both in its molecular conformations and its organization of cross-ß subunits. Unique features include twofold screw symmetry about the fibril growth axis, despite an MPL value that indicates three Aß40 molecules per 4.8-Å ß-sheet spacing, a four-layered architecture, and fully extended conformations for molecules in the central two cross-ß layers. The cryoEM density, ssNMR data, and MPL data are consistent with ß-hairpin conformations for molecules in the outer cross-ß layers. Knowledge of this brain-derived fibril structure may contribute to the development of structure-specific amyloid imaging agents and aggregation inhibitors with greater diagnostic and therapeutic utility.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/ultraestructura , Amiloide/ultraestructura , Corteza Cerebral/química , Fragmentos de Péptidos/ultraestructura , Enfermedad de Alzheimer/patología , Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Sitios de Unión , Corteza Cerebral/patología , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , TermodinámicaRESUMEN
Peptide sequence modulates amyloid fibril formation and triggers Alzheimer's disease. The N-terminal region of amyloid peptide is disordered and lack any specific secondary structure. An ionic interaction of Aß1-11 with factor XII is critical for the activation of the contact system in Alzheimer's disease. In this study, we report the self-assembly of fluctuating N-terminal Aß1-11 into nanotubes using atomic force micrography, transmission electron microscopy, circular dichroism studies and molecular modeling studies. The effect of four polyphenols: baicalein, rutin, vanillin and cyanidin-3-O-glucoside (C3G) was also explored on the amyloid fibril inhibitor perspective using amyloid specific dye Thioflavin T (ThT). AFM micrographs suggested the self-assembly of Aß1-11 into nanotubes after three weeks of incubation. Microwave treatment results in the conformational variation of disordered structure to ß-sheet rich amyloid fibrils. The presence of salts (sodium and potassium chloride) induces the structural transformation of Aß1-11 to super-helix. Fluorescence spectroscopy studies using ThT suggested differential inhibition of amyloid fibrils formation in the presence of polyphenols. Molecular modeling studies suggested that binding of polyphenols to Aß1-11 through hydrophobic interaction (Phe4 and Tyr 10) and hydrogen bonding (Glu3 and Arg5) play a substantial role in stabilizing Aß1-11-polyphenols complex. In the presence of polyphenols, Aß1-11 transforms to hybrid nanostructures thus hindering amyloid fibril formation. These results provide structural insights and importance of the N-terminal residues in the Aß1-42 self-assembly mechanism.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/ultraestructura , Humanos , Modelos Moleculares , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/ultraestructura , Polifenoles/farmacología , Estructura Secundaria de Proteína/efectos de los fármacosRESUMEN
Formation of Aß oligomers and fibrils plays a central role in the pathogenesis of Alzheimer's disease. There are two major forms of Aß in the brain: Aß42 and Aß40. Aß42 is the major component of the amyloid plaques, but the overall abundance of Aß40 is several times that of Aß42. In vitro experiments show that Aß42 and Aß40 affect each other's aggregation. In mouse models of Alzheimer's disease, overexpression of Aß40 has been shown to reduce the plaque pathology, suggesting that Aß42 and Aß40 also interact in vivo. Here we address the question of whether Aß42 and Aß40 interact with each other in the formation of oligomers using electron paramagnetic resonance (EPR) spectroscopy. When the Aß42 oligomers were formed using only spin-labeled Aß42, the dipolar interaction between spin labels that are within 20 Å range broadened the EPR spectrum and reduced its amplitude. Oligomers formed with a mixture of spin-labeled Aß42 and wild-type Aß42 gave an EPR spectrum with higher amplitude due to weakened spin-spin interactions, suggesting molecular mixing of labeled and wild-type Aß42. When spin-labeled Aß42 and wild-type Aß40 were mixed to form oligomers, the resulting EPR spectrum also showed reduced amplitude, suggesting that wild-type Aß40 can also form oligomers with spin-labeled Aß42. Therefore, our results suggest that Aß42 and Aß40 form mixed oligomers with direct molecular interactions. Our results point to the importance of investigating Aß42-Aß40 interactions in the brain for a complete understanding of Alzheimer's pathogenesis and therapeutic interventions.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/ultraestructura , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Técnicas In Vitro , Ratones , Microscopía Electrónica de Transmisión , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/ultraestructura , Placa Amiloide/metabolismo , Placa Amiloide/patología , Placa Amiloide/ultraestructura , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestructura , Marcadores de SpinRESUMEN
Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.
Asunto(s)
Actinas/química , Proteínas de Microfilamentos/química , Actinas/metabolismo , Actinas/ultraestructura , Animales , Toxinas Bacterianas/química , Sitios de Unión , Unión Competitiva , Cofilina 1/química , Cofilina 1/ultraestructura , Microscopía por Crioelectrón , Colorantes Fluorescentes/química , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/ultraestructura , Microscopía Confocal , Modelos Moleculares , Miosinas/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/ultraestructura , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
Natural products have played a dominant role in the discovery of lead compounds for the development of drugs aimed at the treatment of human diseases. This electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)-based study demonstrates that dietary antioxidants, isolated components from the stigmas of saffron (Crocus sativus L.) may be effective in inhibiting Aß fibrillogenesis, a neuropathological hallmark of Alzheimer's Disease (AD). This study reveals a substantial alteration in the monomer/oligomer distribution of Aß1-40, concomitant with re-direction of fibril formation, induced by the natural product interaction. These alterations on the Aß1-40 aggregation pathway are most prominent for trans-crocin-4 (TC4). Use of ESI-IMS-MS, electron microscopy alongside Thioflavin-T kinetics, and the interpretation of 3-dimensional Driftscope plots indicate a correlation of these monomer/oligomer distribution changes with alterations to Aß1-40 amyloid formation. The latter could prove instrumental in the development of novel aggregation inhibitors for the prevention, or treatment of AD.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Antioxidantes/farmacología , Crocus/química , Extractos Vegetales/farmacología , Agregación Patológica de Proteínas/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Antioxidantes/uso terapéutico , Carotenoides/farmacología , Humanos , Microscopía Electrónica de Transmisión , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/ultraestructura , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Agregación Patológica de Proteínas/patología , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Revealing the aggregation and fibrillation process of variant amyloid proteins is critical for understanding the molecular mechanism of related amyloidosis diseases. Here we characterized the fibrillation morphology and kinetics of type 2 diabetes (T2D) related human islet amyloid polypeptide (hIAPP1-37) fibril formation process using negative staining transmission electron microscopy (NS-TEM), cryo-electron microscopy (cryo-EM) analysis, and 3D cryo-electron tomography (cryo-ET) reconstruction, together with circular dichroism (CD) and Thioflavin-T (ThT) assays. Our results showed that various amyloid fibrils can be observed at different time points of hIAPP1-37 fibrillization process, while the winding of protofibrils presents in different growth stages, which suggests a synchronous process of hIAPP1-37 amyloid fibrillization. This work provides insights into the understanding of hIAPP1-37 amyloid aggregation process and the pathogenesis of Type 2 diabetes disease.
Asunto(s)
Amiloide/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Amiloide/química , Amiloide/ultraestructura , Microscopía por Crioelectrón , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/ultraestructura , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/ultraestructura , Agregación Patológica de Proteínas/metabolismoRESUMEN
Self-assembly of proteins into amyloid fibrils is a hallmark of various diseases, including Alzheimer's disease (AD) and Type-2 diabetes Mellitus (T2DM). Aggregation of specific peptides, like Aß42 in AD and hIAPP in T2DM, causes cellular dysfunction resulting in the respective pathology. While these amyloidogenic proteins lack sequence homology, they all contain aromatic amino acids in their hydrophobic core that play a major role in their self-assembly. Targeting these aromatic residues by small molecules may be an attractive approach for inhibiting amyloid aggregation. Here, various biochemical and biophysical techniques revealed that a panel of tryptophan-galactosylamine conjugates significantly inhibit fibril formation of Aß42 and hIAPP, and disassemble their pre-formed fibrils in a dose-dependent manner. They are also not toxic to mammalian cells and can reduce the cytotoxicity induced by Aß42 and hIAPP aggregates. These tryptophan-galactosylamine conjugates can therefore serve as a scaffold for the development of therapeutics towards AD and T2DM.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Amiloide/metabolismo , Galactosamina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/toxicidad , Fragmentos de Péptidos/toxicidad , Agregado de Proteínas , Triptófano/metabolismo , Secuencia de Aminoácidos , Amiloide/ultraestructura , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/ultraestructura , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Concentración 50 Inhibidora , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/ultraestructura , Fragmentos de Péptidos/química , Fragmentos de Péptidos/ultraestructura , Agregado de Proteínas/efectos de los fármacosRESUMEN
Aß deposition is a pathological hallmark of Alzheimer's disease (AD). Besides the full-length amyloid forming peptides (Aß1-40 and Aß1-42), biochemical analyses of brain deposits have identified a variety of N- and C-terminally truncated Aß variants in sporadic and familial AD patients. However, their relevance for AD pathogenesis remains largely understudied. We demonstrate that Aß4-42 exhibits a high tendency to form ß-sheet structures leading to fast self-aggregation and formation of oligomeric assemblies. Atomic force microscopy and electrophysiological studies reveal that Aß4-42 forms highly stable ion channels in lipid membranes. These channels that are blocked by monoclonal antibodies specifically recognizing the N-terminus of Aß4-42. An Aß variant with a double truncation at phenylalanine-4 and leucine 34, (Aß4-34), exhibits unstable channel formation capability. Taken together the results presented herein highlight the potential benefit of C-terminal proteolytic cleavage and further support an important pathogenic role for N-truncated Aß species in AD pathophysiology.
Asunto(s)
Péptidos beta-Amiloides/ultraestructura , Encéfalo/ultraestructura , Canales Iónicos/metabolismo , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/metabolismo , Anticuerpos Monoclonales/farmacología , Encéfalo/metabolismo , Humanos , Canales Iónicos/genética , Microscopía de Fuerza Atómica , Fragmentos de Péptidos/ultraestructura , Conformación Proteica en Lámina betaRESUMEN
Atypical low-oxidation-state iron phases in Alzheimer's disease (AD) pathology are implicated in disease pathogenesis, as they may promote elevated redox activity and convey toxicity. However, the origin of low-oxidation-state iron and the pathways responsible for its formation and evolution remain unresolved. Here we investigate the interaction of the AD peptide ß-amyloid (Aß) with the iron storage protein ferritin, to establish whether interactions between these two species are a potential source of low-oxidation-state iron in AD. Using X-ray spectromicroscopy and electron microscopy we found that the co-aggregation of Aß and ferritin resulted in the conversion of ferritin's inert ferric core into more reactive low-oxidation-states. Such findings strongly implicate Aß in the altered iron handling and increased oxidative stress observed in AD pathogenesis. These amyloid-associated iron phases have biomarker potential to assist with disease diagnosis and staging, and may act as targets for therapies designed to lower oxidative stress in AD tissue.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Ferritinas/metabolismo , Hierro/metabolismo , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/ultraestructura , Biomarcadores/química , Biomarcadores/metabolismo , Ferritinas/química , Ferritinas/ultraestructura , Humanos , Hierro/química , Microscopía Electrónica de Transmisión de Rastreo , Oxidación-Reducción , Estrés Oxidativo , Fragmentos de Péptidos/ultraestructura , Agregado de Proteínas , Espectrometría por Rayos XRESUMEN
Human pancreatic islet amyloid polypeptide (hIAPP) and beta amyloid (Aß) can accumulate in Type 2 diabetes (T2D) and Alzheimer's disease (AD) brains and evidence suggests that interaction between the two amyloidogenic proteins can lead to the formation of heterocomplex aggregates. However, the structure and consequences of the formation of these complexes remains to be determined. The main objective of this study was to characterise the different types and morphology of Aß-hIAPP heterocomplexes and determine if formation of such complexes exacerbate neurotoxicity. We demonstrate that hIAPP promotes Aß oligomerization and formation of small oligomer and large aggregate heterocomplexes. Co-oligomerized Aß42-hIAPP mixtures displayed distinct amorphous structures and a 3-fold increase in neuronal cell death as compared to Aß and hIAPP alone. However, in contrast to hIAPP, non-amyloidogenic rat amylin (rIAPP) reduced oligomer Aß-mediated neuronal cell death. rIAPP exhibited reductions in Aß induced neuronal cell death that was independent of its ability to interact with Aß and form heterocomplexes; suggesting mediation by other pathways. Our findings reveal distinct effects of IAPP peptides in modulating Aß aggregation and toxicity and provide new insight into the potential pathogenic effects of Aß-IAPP hetero-oligomerization and development of IAPP based therapies for AD and T2D.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Diabetes Mellitus Tipo 2/patología , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Neuronas/patología , Fragmentos de Péptidos/metabolismo , Agregación Patológica de Proteínas/patología , Enfermedad de Alzheimer/etiología , Péptidos beta-Amiloides/toxicidad , Péptidos beta-Amiloides/ultraestructura , Animales , Encéfalo/patología , Línea Celular Tumoral , Diabetes Mellitus Tipo 2/complicaciones , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/toxicidad , Polipéptido Amiloide de los Islotes Pancreáticos/ultraestructura , Microscopía Electrónica de Transmisión , Resonancia Magnética Nuclear Biomolecular , Páncreas/metabolismo , Fragmentos de Péptidos/toxicidad , Fragmentos de Péptidos/ultraestructura , Agregado de Proteínas , Multimerización de Proteína , RatasRESUMEN
The nucleation of Alzheimer-associated Aß peptide monomers can be catalyzed by preexisting Aß fibrils. This leads to autocatalytic amplification of aggregate mass and underlies self-replication and generation of toxic oligomers associated with several neurodegenerative diseases. However, the nature of the interactions between the monomeric species and the fibrils during this key process, and indeed the ultrastructural localization of the interaction sites have remained elusive. Here we used NMR and optical spectroscopy to identify conditions that enable the capture of transient species during the aggregation and secondary nucleation of the Aß42 peptide. Cryo-electron microscopy (cryo-EM) images show that new aggregates protrude from the entire length of the progenitor fibril. These protrusions are morphologically distinct from the well-ordered fibrils dominating at the end of the aggregation process. The data provide direct evidence that self-replication through secondary nucleation occurs along the sides of fibrils, which become heavily decorated under the current solution conditions (14 µM Aß42, 20 mM sodium phosphate, 200 µM EDTA, pH 6.8).
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/ultraestructura , Enfermedad de Alzheimer/patología , Amiloide/metabolismo , Amiloide/ultraestructura , Péptidos beta-Amiloides/química , Benzotiazoles/química , Benzotiazoles/metabolismo , Microscopía por Crioelectrón , Humanos , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Fragmentos de Péptidos/química , Imagen de Lapso de TiempoRESUMEN
Amyloids are protein aggregates observed in several diseases, for example in Alzheimer's and Parkinson's diseases. An aggregate has a very regular beta structure with a tightly packed core, which spontaneously assumes a steric zipper form. Experimental methods enable studying such peptides, however they are tedious and costly, therefore inappropriate for genomewide studies. Several bioinformatic methods have been proposed to evaluate protein propensity to form an amyloid. However, the knowledge of aggregate structures is usually not taken into account. We propose PATH (Prediction of Amyloidogenicity by THreading) - a novel structure-based method for predicting amyloidogenicity and show that involving available structures of amyloidogenic fragments enhances classification performance. Experimental aggregate structures were used in templatebased modeling to recognize the most stable representative structural class of a query peptide. Several machine learning methods were then applied on the structural models, using their energy terms. Finally, we identified the most important terms in classification of amyloidogenic peptides. The proposed method outperforms most of the currently available methods for predicting amyloidogenicity, with its area under ROC curve equal to 0.876. Furthermore, the method gave insight into significance of selected structural features and the potentially most stable structural class of a peptide fragment if subjected to crystallization.
Asunto(s)
Amiloide/ultraestructura , Fragmentos de Péptidos/ultraestructura , Conformación Proteica en Lámina beta/genética , Programas Informáticos , Algoritmos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Amiloide/química , Biología Computacional/métodos , Humanos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Fragmentos de Péptidos/química , Agregado de Proteínas/genética , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patologíaRESUMEN
Celiac disease (CeD) is a highly prevalent chronic immune-mediated enteropathy developed in genetically predisposed individuals after ingestion of a group of wheat proteins (called gliadins and glutenins). The 13mer α-gliadin peptide, p31-43, induces proinflammatory responses, observed by in vitro assays and animal models, that may contribute to innate immune mechanisms of CeD pathogenesis. Since a cellular receptor for p31-43 has not been identified, this raises the question of whether this peptide could mediate different biological effects. In this work, we aimed to characterize the p31-43 secondary structure by different biophysical and in silico techniques. By dynamic light scattering and using an oligomer/fibril-sensitive fluorescent probe, we showed the presence of oligomers of this peptide in solution. Furthermore, atomic force microscopy analysis showed p31-43 oligomers with different height distribution. Also, peptide concentration had a very strong influence on peptide self-organization process. Oligomers gradually increased their size at lower concentration. Whereas, at higher ones, oligomers increased their complexity, forming branched structures. By CD, we observed that p31-43 self-organized in a polyproline II conformation in equilibrium with ß-sheets-like structures, whose pH remained stable in the range of 3-8. In addition, these findings were supported by molecular dynamics simulation. The formation of p31-43 nanostructures with increased ß-sheet structure may help to explain the molecular etiopathogenesis in the induction of proinflammatory effects and subsequent damage at the intestinal mucosa in CeD.
Asunto(s)
Enfermedad Celíaca/tratamiento farmacológico , Gliadina/farmacología , Inmunidad Innata/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Células CACO-2 , Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Gliadina/genética , Gliadina/inmunología , Gliadina/ultraestructura , Humanos , Inmunidad Innata/inmunología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Microscopía de Fuerza Atómica , Conformación Molecular/efectos de los fármacos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/ultraestructura , Péptidos/química , Péptidos/inmunología , Péptidos/farmacología , Conformación Proteica en Lámina beta , Soluciones/química , Agua/químicaRESUMEN
Alzheimer's disease is linked to amyloid ß (Aß) peptide aggregation in the brain, and a detailed understanding of the molecular mechanism of Aß aggregation may lead to improved diagnostics and therapeutics. While previous studies have been performed in pure buffer, we approach the mechanism in vivo using cerebrospinal fluid (CSF). We investigated the aggregation mechanism of Aß42 in human CSF through kinetic experiments at several Aß42 monomer concentrations (0.8-10 µM). The data were subjected to global kinetic analysis and found consistent with an aggregation mechanism involving secondary nucleation of monomers on the fibril surface. A mechanism only including primary nucleation was ruled out. We find that the aggregation process is composed of the same microscopic steps in CSF as in pure buffer, but the rate constant of secondary nucleation is decreased. Most importantly, the autocatalytic amplification of aggregate number through catalysis on the fibril surface is prevalent also in CSF.