Nanoparticles present enormous surface areas and are found to enhance the rate of protein fibrillation by decreasing the lag time for nucleation. Protein fibrillation is involved in many human diseases, including Alzheimer's, Creutzfeld-Jacob disease, and dialysis-related amyloidosis. Fibril formation occurs by nucleationdependent kinetics, wherein formation of a critical nucleus is the key rate-determining step, after which fibrillation proceeds rapidly. We show that nanoparticles (copolymer particles, cerium oxide particles, quantum dots, and carbon nanotubes) enhance the probability of appearance of a critical nucleus for nucleation of protein fibrils from human 2-microglobulin. The observed shorter lag (nucleation) phase depends on the amount and nature of particle surface. There is an exchange of protein between solution and nanoparticle surface, and 2-microglobulin forms multiple layers on the particle surface, providing a locally increased protein concentration promoting oligomer formation. This and the shortened lag phase suggest a mechanism involving surface-assisted nucleation that may increase the risk for toxic cluster and amyloid formation. It also opens the door to new routes for the controlled self-assembly of proteins and peptides into novel nanomaterials.amyloid ͉ nanotoxicology ͉ surface-assisted nucleation
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 nucleationdependent formation of 2-microglobulin (2m) 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 2m 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.AIC model comparison analysis ͉ amyloid fibril formation ͉ fibril brittleness ͉ global analysis S elf-assembly of misfolded forms of normally soluble and functional proteins or peptides into amyloid fibrils results in numerous human diseases (1). Understanding how amyloid self-assembly occurs, therefore, is of paramount importance for a molecular interpretation of amyloidosis and for the development of therapies against amyloid disease. Over the past decade, advances have been made toward a more complete description of amyoid fibril formation, including the determination of increasingly refined models of fibril structures (reviewed in ref. 1) and the identification of amyloid precursors and oligomeric states reviewed in ref. 2, one or more of which could be the culprits of cytotoxicity associated with several amyloid diseases (e.g., ref.3). However, the molecular events occurring during the self-assembly process itself remain obscure because of the heterogeneity and the complexity of the early association events.Amyloid fibril self-assembly reactions are generally accepted as a form of nucleated polymerization (4). These reactions are characterized by an initial lag phase where little or no change in fibril concentration can be detected. This is followed by an elongation phase where a large mass percentage of the starting protein material is converted into fibrils. A shared feature among amyloid formation and other nuclea...
Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity.
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