Supramolecular non-amyloid intermediates in the early stages of ?-synuclein aggregation.
ABSTRACT: The aggregation of ?-synuclein is associated with progression of Parkinson's disease. We have identified submicrometer supramolecular structures that mediate the early stages of the overall mechanism. The sequence of structural transformations between metastable intermediates were captured and characterized by atomic force microscopy guided by a fluorescent probe sensitive to preamyloid species. A novel ~0.3-0.6 ?m molecular assembly, denoted the acuna, nucleates, expands, and liberates fibers with distinctive segmentation and a filamentous fuzzy fringe. These fuzzy fibers serve as precursors of mature amyloid fibrils. Cryo-electron tomography resolved the acuna inner structure as a scaffold of highly condensed colloidal masses interlinked by thin beaded threads, which were perceived as fuzziness by atomic force microscopy. On the basis of the combined data, we propose a sequential mechanism comprising molecular, colloidal, and fibrillar stages linked by reactions with disparate temperature dependencies and distinct supramolecular forms. We anticipate novel diagnostic and therapeutic approaches to Parkinson's and related neurodegenerative diseases based on these new insights into the aggregation mechanism of ?-synuclein and intermediates, some of which may act to cause and/or reinforce neurotoxicity.
Project description:Amyloid fibrils are a hallmark of a range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. A detailed understanding of the physico-chemical properties of the different aggregated forms of proteins, and of their interactions with other compounds of diagnostic or therapeutic interest, is crucial for devising effective strategies against such diseases. Protein aggregates are situated at the boundary between soluble and insoluble structures, and are challenging to study because classical biophysical techniques, such as scattering, spectroscopic and calorimetric methods, are not well adapted for their study. Here we present a detailed characterization of the thermophoretic behavior of different forms of the protein ?-synuclein, whose aggregation is associated with Parkinson's disease. Thermophoresis is the directed net diffusional flux of molecules and colloidal particles in a temperature gradient. Because of their low volume requirements and rapidity, analytical methods based on this effect have considerable potential for high throughput screening for drug discovery. In this paper we rationalize and describe in quantitative terms the thermophoretic behavior of monomeric, oligomeric and fibrillar forms of ?-synuclein. Furthermore, we demonstrate that microscale thermophoresis (MST) is a valuable method for screening for ligands and binding partners of even such highly challenging samples as supramolecular protein aggregates.
Project description:The morphological features of ?-synuclein (AS) amyloid aggregation in vitro and in cells were elucidated at the nanoscale by far-field subdiffraction fluorescence localization microscopy. Labeling AS with rhodamine spiroamide probes allowed us to image AS fibrillar structures by fluorescence stochastic nanoscopy with an enhanced resolution at least 10-fold higher than that achieved with conventional, diffraction-limited techniques. The implementation of dual-color detection, combined with atomic force microscopy, revealed the propagation of individual fibrils in vitro. In cells, labeled protein appeared as amyloid aggregates of spheroidal morphology and subdiffraction sizes compatible with in vitro supramolecular intermediates perceived independently by atomic force microscopy and cryo-electron tomography. We estimated the number of monomeric protein units present in these minute structures. This approach is ideally suited for the investigation of the molecular mechanisms of amyloid formation both in vitro and in the cellular milieu.
Project description:The deposition of ?-synuclein fibrils is one hallmark of Parkinson's disease. Here, we investigate how ganglioside lipids, present in high amounts in neurons and exosomes, influence the aggregation kinetics of ?-synuclein. Gangliosides, as well as, other anionic lipid species with small or large headgroups were found to induce conformational changes of ?-synuclein monomers and catalyse their aggregation at mildly acidic conditions. Although the extent of this catalytic effect was slightly higher for gangliosides, the results imply that charge interactions are more important than headgroup chemistry in triggering aggregation. In support of this idea, uncharged lipids with large headgroups were not found to induce any conformational change and only weakly catalyse aggregation. Intriguingly, aggregation was also triggered by free ganglioside headgroups, while these caused no conformational change of ?-synuclein monomers. Our data reveal that partially folded ?-synuclein helical intermediates are not required species in triggering of ?-synuclein aggregation.
Project description:DJ-1 is a deglycase enzyme which exhibits a redox-sensitive chaperone-like activity. The partially oxidized state of DJ-1 is active in inhibiting the aggregation of ?-synuclein, a key protein associated with Parkinson's disease. The underlying molecular mechanism behind ?-synuclein aggregation inhibition remains unknown. Here we report that the partially oxidized DJ-1 possesses an adhesive surface which sequesters ?-synuclein monomers and blocks the early stages of ?-synuclein aggregation and also restricts the elongation of ?-synuclein fibrils. DJ-1 remodels mature ?-synuclein fibrils into heterogeneous toxic oligomeric species. The remodeled fibers show loose surface topology due to a decrease in elastic modulus and disrupt membrane architecture, internalize easily and induce aberrant nitric oxide release. Our results provide a mechanism by which partially oxidized DJ-1 counteracts ?-synuclein aggregation at initial stages of aggregation and provide evidence of a deleterious effect of remodeled ?-synuclein species generated by partially oxidized DJ-1.
Project description:Intrinsically Disordered Proteins (IDPs) are characterized by the lack of well-defined 3-D structure and show high conformational plasticity. For this reason, they are a strong challenge for the traditional characterization of structure, supramolecular assembly and biorecognition phenomena. We show here how the fine tuning of protein orientation on a surface turns useful in the reliable testing of biorecognition interactions of IDPs, in particular ?-Synuclein. We exploited atomic force microscopy (AFM) for the selective, nanoscale confinement of ?-Synuclein on gold to study the early stages of ?-Synuclein aggregation and the effect of small molecules, like dopamine, on the aggregation process. Capitalizing on the high sensitivity of AFM topographic height measurements we determined, for the first time in the literature, the dissociation constant of dopamine-?-Synuclein adducts.
Project description:Neurodegeneration in patients with Parkinson's disease is correlated with the occurrence of Lewy bodies-intracellular inclusions that contain aggregates of the intrinsically disordered protein ?-synuclein1. The aggregation propensity of ?-synuclein in cells is modulated by specific factors that include post-translational modifications2,3, Abelson-kinase-mediated phosphorylation4,5 and interactions with intracellular machineries such as molecular chaperones, although the underlying mechanisms are unclear6-8. Here we systematically characterize the interaction of molecular chaperones with ?-synuclein in vitro as well as in cells at the atomic level. We find that six highly divergent molecular chaperones commonly recognize a canonical motif in ?-synuclein, consisting of the N terminus and a segment around Tyr39, and hinder the aggregation of ?-synuclein. NMR experiments9 in cells show that the same transient interaction pattern is preserved inside living mammalian cells. Specific inhibition of the interactions between ?-synuclein and the chaperone HSC70 and members of the HSP90 family, including HSP90?, results in transient membrane binding and triggers a remarkable re-localization of ?-synuclein to the mitochondria and concomitant formation of aggregates. Phosphorylation of ?-synuclein at Tyr39 directly impairs the interaction of ?-synuclein with chaperones, thus providing a functional explanation for the role of Abelson kinase in Parkinson's disease. Our results establish a master regulatory mechanism of ?-synuclein function and aggregation in mammalian cells, extending the functional repertoire of molecular chaperones and highlighting new perspectives for therapeutic interventions for Parkinson's disease.
Project description:Hairpin peptides bearing cross-strand Trp-Trp and Tyr-Tyr pairs at non-H-bonded strand sites modulate the aggregation of two unrelated amyloidogenic systems, human pancreatic amylin (hAM) and ?-synuclein (?-syn), associated with type II diabetes and Parkinson's disease, respectively. In the case of hAM, we have previously reported that inhibition of amyloidogenesis is observed as an increase in the lag time to amyloid formation and a diminished thioflavin (ThT) fluorescence response. In this study, a reduced level of hAM fibril formation is confirmed by transmission electron microscopy imaging. Several of the hairpins tested were significantly more effective inhibitors than rat amylin. Moreover, a marked inhibitory effect on hAM-associated cytotoxicity by the more potent hairpin peptide is demonstrated. In the case of ?-syn, the dominant effect of active hairpins was, besides a weakened ThT fluorescence response, the earlier appearance of insoluble aggregates that do not display amyloid characteristics with the few fibrils observed having abnormal morphology. We attribute the alteration of the ?-synuclein aggregation pathway observed to the capture of a preamyloid state and diversion to nonamyloidogenic aggregates. These ?-hairpins represent a new class of amyloid inhibitors that bear no sequence similarity to the amyloid-producing polypeptides that are inhibited. A mechanistic rationale for these effects is proposed.
Project description:Conventional methods to determine the aggregation number, that is, the number of monomers per oligomer, struggle to yield reliable results for large protein aggregates, such as amyloid oligomers. We have previously demonstrated the use of a combination of single-molecule photobleaching and substoichiometric fluorescent labeling to determine the aggregation number of oligomers of human ?-synuclein, implicated in Parkinson's disease. We show here that this approach is capable of accurately resolving mixtures of multiple distinct molecular species present in the same sample of dopamine-induced ?-synuclein oligomers, and that we can determine the respective aggregation numbers of each species from a single histogram of bleaching steps. We found two distinct species with aggregation numbers of 15-19 monomers and 34-38 monomers. These results show that this single-molecule approach allows for the systematic study of the aggregation numbers of complex supramolecular assemblies formed under different aggregation conditions.
Project description:Parkinson's disease neurodegenerative brain tissue exhibits two biophysically distinct ?-synuclein fiber isoforms-single stranded fibers that appear to be steric-zippers and double-stranded fibers with an undetermined structure. Herein, we describe a ?-helical homology model of ?-synuclein that exhibits stability in probabilistic and Monte Carlo simulations as a candidate for stable prional dimer conformers in equilibrium with double-stranded fibers and cytotoxic pore assemblies. Molecular models of ?-helical pore assemblies are consistent with ?-synucleinA53T transfected rat immunofluorescence epitope maps. Atomic force microscopy reveals that ?-synuclein peptides aggregate into anisotropic fibrils lacking the density or circumference of a steric-zipper. Moreover, fibrillation was blocked by mutations designed to hinder ?-helical but not steric-zipper conformations. ?-helical species provide a structural basis for previously described biophysical properties that are incompatible with a steric-zipper, provide pathogenic mechanisms for familial human ?-synuclein mutations, and offer a direct cytotoxic target for therapeutic development.
Project description:A compelling link is emerging between the posttranslational modification O-GlcNAc and protein aggregation. A prime example is ?-synuclein, which forms toxic aggregates that are associated with neurodegeneration in Parkinson's and related diseases. ?-Synuclein has been shown to be O-GlcNAcylated at nine different positions in in vivo proteomics experiments from mouse and human tissues. This raises the possibility that O-GlcNAc may alter the aggregation of this protein and could be both an important biological mediator of neurodegeneration and also a therapeutic target. Here, we expand upon our previous research in this area through the chemical synthesis of six site-specifically O-GlcNAcylated variants of ?-synuclein. We then use a variety of biochemical experiments to show that O-GlcNAc in general inhibits the aggregation of ?-synuclein but can also alter the structure of ?-synuclein aggregates in site-specific ways. Additionally, an ?-synuclein protein bearing three O-GlcNAc modifications can inhibit the aggregation of unmodified protein. Primary cell culture experiments also show that several of the O-GlcNAc sites inhibit the toxicity of extracellular ?-synuclein fibers that are likely culprits in the spread of Parkinson's disease. We also demonstrate that O-GlcNAcylation can inhibit the aggregation of an aggressive mutant of ?-synuclein, indicating that therapies currently in development that increase this modification might be applied in animal models that rely on this mutant. Finally, we also show that the pan-selective antibody for O-GlcNAc does not generally recognize this modification on ?-synuclein, potentially explaining why it remains understudied. These results support further development of O-GlcNAcylation tools and therapeutic strategies in neurodegenerative diseases.