Amyloid Oligomers and Protofibrils, but Not Filaments, Self-Replicate from Native Lysozyme

Abstract

Self-assembly of amyloid fibrils is the molecular mechanism best known for its connection with debilitating human disorders such as Alzheimer’s disease but is also associated with various functional cellular responses. There is increasing evidence that amyloid formation proceeds along two distinct assembly pathways involving either globular oligomers and protofibrils or rigid monomeric filaments. Oligomers, in particular, have been implicated as the dominant molecular species responsible for pathogenesis. Yet the molecular mechanisms regulating their self-assembly have remained elusive. Here we show that oligomers/protofibrils and monomeric filaments, formed along distinct assembly pathways, display critical differences in their ability to template amyloid growth at physiological vs denaturing temperatures. At physiological temperatures, amyloid filaments remained stable but could not seed growth of native monomers. In contrast, oligomers and protofibrils not only remained intact but were capable of self-replication using native monomers as the substrate. Kinetic data further suggested that this prion-like growth mode of oligomers/protofibrils involved two distinct activities operating orthogonal from each other: autocatalytic self-replication of oligomers from native monomers and nucleated polymerization of oligomers into protofibrils. The environmental changes to stability and templating competence of these different amyloid species in different environments are likely to be important for understanding the molecular mechanisms underlying both pathogenic and functional amyloid self-assembly.

Kinetic barriers to α-synuclein protofilament formation and conversion into mature fibrils

“…20 Protofibrils, which are morphologically distinguished from mature amyloid fibrils, have been shown to have distinct kinetic properties, including a reduced tendency to act as templates for further growth. 21,22 The kinetics of conversion from protofibrillar to fibrillar species of a-synuclein have not yet been well characterised and, as in other systems, 22 this may be a critical step in the self-replication and proliferation of aggregates.…”

What Can the Kinetics of Amyloid Fibril Formation Tell about Off-pathway Aggregation?

“…By expressing the driving force for protein aggregation as the thermodynamic supersaturation ϭ (C Ϫ C a * )/C a * (an approximation to the variation in chemical potential), the steady-state monomer concentration is expected to correspond to the amyloid solubility C ∞ ϭ C a * . At the same time that the CLM was being proposed, Yoshimura et al (33) urged the need to recognize amyloidogenicity as a property determined by the monomer concentration relative to solubility; since then, a wealth of new evidence has unanimously confirmed supersaturation as a major driving force for protein aggregation (6,11,34,35,36,37,38). Although this parallel with crystallization had been hinted at before (21,39), the common practice in literature models is to assume the monomer concentration alone as the driving force for phase transition (18,28,29,30,31), meaning that, for example, amyloid fibrils would continue to grow until the solution became completely depleted from soluble protein.…”

Monoubiquitination Inhibits the Actin Bundling Activity of Fascin

“…Static Light Scattering Assay-Static light scattering (SLS) assays were performed as previously described (40). A Zetasizer Nano S (Malvern Instruments, Worchestershire, UK) dynamic light scattering unit with a 4-milliwatt HeNe laser and an avalanche photodiode detector was used to determine SLS intensities.…”