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The formation of amyloid fibrils by the intrinsically disordered protein α-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that α-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of α-synuclein aggregation and aggregate spreading in the context of Parkinson disease.

The aggregation of proteins into oligomers and amyloid fibrils is characteristic of several neurodegenerative diseases, including Parkinson disease (PD). In PD, the process of aggregation of α-synuclein (α-syn) from monomers, via oligomeric intermediates, into amyloid fibrils is considered the disease-causative toxic mechanism. We developed α-syn mutants that promote oligomer or fibril formation and tested the toxicity of these mutants by using a rat lentivirus system to investigate loss of dopaminergic neurons in the substantia nigra. The most severe dopaminergic loss in the substantia nigra is observed in animals with the α-syn variants that form oligomers (i.e., E57K and E35K), whereas the α-syn variants that form fibrils very quickly are less toxic. We show that α-syn oligomers are toxic in vivo and that α-syn oligomers might interact with and potentially disrupt membranes.

The generation of toxic oligomers during the aggregation of the amyloid-β (Aβ) peptide Aβ42 into amyloid fibrils and plaques has emerged as a central feature of the onset and progression of Alzheimer’s disease, but the molecular pathways that control pathological aggregation have proved challenging to identify. Here, we use a combination of kinetic studies, selective radiolabeling experiments, and cell viability assays to detect directly the rates of formation of both fibrils and oligomers and the resulting cytotoxic effects. Our results show that once a small but critical concentration of amyloid fibrils has accumulated, the toxic oligomeric species are predominantly formed from monomeric peptide molecules through a fibril-catalyzed secondary nucleation reaction, rather than through a classical mechanism of homogeneous primary nucleation. This catalytic mechanism couples together the growth of insoluble amyloid fibrils and the generation of diffusible oligomeric aggregates that are implicated as neurotoxic agents in Alzheimer’s disease. These results reveal that the aggregation of Aβ42 is promoted by a positive feedback loop that originates from the interactions between the monomeric and fibrillar forms of this peptide. Our findings bring together the main molecular species implicated in the Aβ aggregation cascade and suggest that perturbation of the secondary nucleation pathway identified in this study could be an effective strategy to control the proliferation of neurotoxic Aβ42 oligomers.

The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale—including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy—we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.

The transthyretin amyloidoses (ATTR) are invariably fatal diseases characterized by progressive neuropathy and/or cardiomyopathy. ATTR are caused by aggregation of transthyretin (TTR), a natively tetrameric protein involved in the transport of thyroxine and the vitamin A–retinol-binding protein complex. Mutations within TTR that cause autosomal dominant forms of disease facilitate tetramer dissociation, monomer misfolding, and aggregation, although wild-type TTR can also form amyloid fibrils in elderly patients. Because tetramer dissociation is the rate-limiting step in TTR amyloidogenesis, targeted therapies have focused on small molecules that kinetically stabilize the tetramer, inhibiting TTR amyloid fibril formation. One such compound, tafamidis meglumine (Fx-1006A), has recently completed Phase II/III trials for the treatment of Transthyretin Type Familial Amyloid Polyneuropathy (TTR-FAP) and demonstrated a slowing of disease progression in patients heterozygous for the V30M TTR mutation. Herein we describe the molecular and structural basis of TTR tetramer stabilization by tafamidis. Tafamidis binds selectively and with negative cooperativity (K ds ∼2 nM and ∼200 nM) to the two normally unoccupied thyroxine-binding sites of the tetramer, and kinetically stabilizes TTR. Patient-derived amyloidogenic variants of TTR, including kinetically and thermodynamically less stable mutants, are also stabilized by tafamidis binding. The crystal structure of tafamidis-bound TTR suggests that binding stabilizes the weaker dimer-dimer interface against dissociation, the rate-limiting step of amyloidogenesis.

Amyloid β-protein (Aβ) is linked to neuronal injury and death in Alzheimer's disease (AD). Of particular relevance for elucidating the role of Aβ in AD is new evidence that oligomeric forms of Aβ are potent neurotoxins that play a major role in neurodegeneration and the strong association of the 42-residue form of Aβ, Aβ42, with the disease. Detailed knowledge of the structure and assembly dynamics of Aβ thus is important for the development of properly targeted AD therapeutics. Recently, we have shown that Aβ oligomers can be cross-linked efficiently, and their relative abundances quantified, by using the technique of photo-induced cross-linking of unmodified proteins (PICUP). Here, PICUP, size-exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy, and electron microscopy have been combined to elucidate fundamental features of the early assembly of Aβ40 and Aβ42. Carefully prepared aggregate-free Aβ40 existed as monomers, dimers, trimers, and tetramers, in rapid equilibrium. In contrast, Aβ42 preferentially formed pentamer/hexamer units (paranuclei) that assembled further to form beaded superstructures similar to early protofibrils. Addition of Ile-41 to Aβ40 was sufficient to induce formation of paranuclei, but the presence of Ala-42 was required for their further association. These data demonstrate that Aβ42 assembly involves formation of several distinct transient structures that gradually rearrange into protofibrils. The strong etiologic association of Aβ42 with AD may thus be a result of assemblies formed at the earliest stages of peptide oligomerization.

Greater understanding of the mechanisms contributing to chemical and enzymatic solubilization of plant cell walls is critical for enabling cost-effective industrial conversion of cellulosic biomass to biofuels. Here, we report the use of correlative imaging in real time to assess the impact of pretreatment as well as the resulting nanometer-scale changes in cell wall structure, upon subsequent digestion by two commercially relevant cellulase systems. We demonstrate that the small, noncomplexed fungal cellulases deconstruct cell walls using mechanisms that differ considerably from those of the larger, multienzyme complexes (cellulosomes). Furthermore, high-resolution measurement of the microfibrillar architecture of cell walls suggests that digestion is primarily facilitated by enabling enzyme access to the hydrophobic cellulose face. The data support the conclusion that ideal pretreatments should maximize lignin removal and minimize polysaccharide modification, thereby retaining the essentially native microfibrillar structure.

Wild-type, full-length (40- and 42-residue) amyloid β-peptide (Aβ) fibrils have been shown by a variety of magnetic resonance techniques to contain cross-β structures in which the β-sheets have an in-register parallel supramolecular organization. In contrast, recent studies of fibrils formed in vitro by the Asp23-to-Asn mutant of 40-residue Aβ (D23N-Aβ1–40), which is associated with early onset neurodegeneration, indicate that D23N-Aβ1–40 fibrils can contain either parallel or antiparallel β-sheets. We report a protocol for producing structurally pure antiparallel D23N-Aβ1–40 fibril samples and a series of solid state nuclear magnetic resonance and electron microscopy measurements that lead to a specific model for the antiparallel D23N-Aβ1–40 fibril structure. This model reveals how both parallel and antiparallel cross-β structures can be constructed from similar peptide monomer conformations and stabilized by similar sets of interactions, primarily hydrophobic in nature. We find that antiparallel D23N-Aβ1–40 fibrils are thermodynamically metastable with respect to conversion to parallel structures, propagate less efficiently than parallel fibrils in seeded fibril growth, and therefore must nucleate more efficiently than parallel fibrils in order to be observable. Experiments in neuronal cell cultures indicate that both antiparallel and parallel D23N-Aβ1–40 fibrils are cytotoxic. Thus, our antiparallel D23N-Aβ1–40 fibril model represents a specific \"toxic intermediate\" in the aggregation process of a disease-associated Aβ mutant.

Self-assembly of misfolded proteins into ordered fibrillar aggregates known as amyloid results in numerous human diseases. Despite an increasing number of proteins and peptide fragments being recognised as amyloidogenic, how these amyloid aggregates assemble remains unclear. In particular, the identity of the nucleating species, an ephemeral entity that defines the rate of fibril formation, remains a key outstanding question. Here, we propose a new strategy for analyzing the self-assembly of amyloid fibrils involving global analysis of a large number of reaction progress curves and the subsequent systematic testing and ranking of a large number of possible assembly mechanisms. Using this approach, we have characterized the mechanism of the nucleation-dependent formation of β₂-microglobulin (β₂m) amyloid fibrils. We show, by defining nucleation in the context of both structural and thermodynamic aspects, that a model involving a structural nucleus size approximately the size of a hexamer is consistent with the relatively small concentration dependence of the rate of fibril formation, contrary to expectations based on simpler theories of nucleated assembly. We also demonstrate that fibril fragmentation is the dominant secondary process that produces higher apparent cooperatively in fibril formation than predicted by nucleated assembly theories alone. The model developed is able to explain and predict the behavior of β₂m fibril formation and provides a rationale for explaining generic properties observed in other amyloid systems, such as fibril growth acceleration and pathway shifts under agitation.

Significance The assembly of normally soluble proteins into large fibrils, known as amyloid aggregation, is associated with a range of pathologies. Prefibrillar protein oligomers but not the grown fibers are believed to be the main toxic agents. It is unresolved if these oligomers are necessary for fibril assembly or just a dangerous byproduct. We show using computer simulations that, at physiological concentrations, amyloid formation must proceed through a two-step process including prefibrillar oligomers. We find that there is an optimal oligomeric size for amyloid nucleation and that classical nucleation theory cannot be applied to this process. Formation of oligomers and hence, fibrils, is controlled by the strength of nonspecific attractions, whose weakening may be crucial in preventing amyloid aggregation. Protein oligomers have been implicated as toxic agents in a wide range of amyloid-related diseases. However, it has remained unsolved whether the oligomers are a necessary step in the formation of amyloid fibrils or just a dangerous byproduct. Analogously, it has not been resolved if the amyloid nucleation process is a classical one-step nucleation process or a two-step process involving prenucleation clusters. We use coarse-grained computer simulations to study the effect of nonspecific attractions between peptides on the primary nucleation process underlying amyloid fibrillization. We find that, for peptides that do not attract, the classical one-step nucleation mechanism is possible but only at nonphysiologically high peptide concentrations. At low peptide concentrations, which mimic the physiologically relevant regime, attractive interpeptide interactions are essential for fibril formation. Nucleation then inevitably takes place through a two-step mechanism involving prefibrillar oligomers. We show that oligomers not only help peptides meet each other but also, create an environment that facilitates the conversion of monomers into the β -sheet–rich form characteristic of fibrils. Nucleation typically does not proceed through the most prevalent oligomers but through an oligomer size that is only observed in rare fluctuations, which is why such aggregates might be hard to capture experimentally. Finally, we find that the nucleation of amyloid fibrils cannot be described by classical nucleation theory: in the two-step mechanism, the critical nucleus size increases with increases in both concentration and interpeptide interactions, which is in direct contrast with predictions from classical nucleation theory.