RESUMEN
Molecular chaperones are key components of the cellular proteostasis network whose role includes the suppression of the formation and proliferation of pathogenic aggregates associated with neurodegenerative diseases. The molecular principles that allow chaperones to recognize misfolded and aggregated proteins remain, however, incompletely understood. To address this challenge, here we probe the thermodynamics and kinetics of the interactions between chaperones and protein aggregates under native solution conditions using a microfluidic platform. We focus on the binding between amyloid fibrils of α-synuclein, associated with Parkinson's disease, to the small heat-shock protein αB-crystallin, a chaperone widely involved in the cellular stress response. We find that αB-crystallin binds to α-synuclein fibrils with high nanomolar affinity and that the binding is driven by entropy rather than enthalpy. Measurements of the change in heat capacity indicate significant entropic gain originates from the disassembly of the oligomeric chaperones that function as an entropic buffer system. These results shed light on the functional roles of chaperone oligomerization and show that chaperones are stored as inactive complexes which are capable of releasing active subunits to target aberrant misfolded species.
Asunto(s)
Amiloide/metabolismo , Proteínas de Choque Térmico Pequeñas/metabolismo , Cadena B de alfa-Cristalina/metabolismo , alfa-Sinucleína/metabolismo , Entropía , Humanos , Enfermedad de Parkinson/metabolismo , Agregado de Proteínas/fisiología , Proteostasis/fisiologíaRESUMEN
The formation of reactive oxygen species (ROS) is linked to the pathogenesis of neurodegenerative diseases. Here we have investigated the effect of soluble and aggregated amyloid-ß (Aß) and α-synuclein (αS), associated with Alzheimer's and Parkinson's diseases, respectively, on the Cu(2+)-catalyzed formation of ROS in vitro in the presence of a biological reductant. We find that the levels of ROS, and the rate by which ROS is generated, are significantly reduced when Cu(2+) is bound to Aß or αS, particularly when they are in their oligomeric or fibrillar forms. This effect is attributed to a combination of radical scavenging and redox silencing mechanisms. Our findings suggest that the increase in ROS associated with the accumulation of aggregated Aß or αS does not result from a particularly ROS-active form of these peptides, but rather from either a local increase of Cu(2+) and other ROS-active metal ions in the aggregates or as a downstream consequence of the formation of the pathological amyloid structures.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Cobre/química , Especies Reactivas de Oxígeno , alfa-Sinucleína/metabolismo , Catálisis , Depuradores de Radicales Libres/metabolismo , Humanos , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
High-throughput studies have been widely used to identify protein-protein interactions; however, few of these candidate interactions have been confirmed in vitro. We have used a combination of isothermal titration calorimetry and fluorescence anisotropy to screen candidate interactions within the pantothenate biosynthetic pathway. In particular, we observed no interaction between the next enzyme in the pathway, pantothenate synthetase (PS), and aspartate decarboxylase, but did observe an interaction between PS and the putative Nudix hydrolase, YfcD. Confirmation of the interaction by fluorescence anisotropy was dependent upon labelling an adventitiously formed glycine on the protein N-terminal affinity purification tag by using Sortase. Subsequent formation of the protein-protein complex led to apparent restriction of the dynamics of this tag, thus suggesting that this approach could be generally applied to a subset of other protein-protein interaction complexes.
Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Ácido Pantoténico/biosíntesis , Aminoaciltransferasas/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Vías Biosintéticas , Cisteína Endopeptidasas/aislamiento & purificación , Polarización de Fluorescencia , Estructura Molecular , Ácido Pantoténico/química , Péptido Sintasas/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of ß-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their ß-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the ß-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.