LAUSR

Significance Amyloid fibrils are generally related to neurodegenerative diseases, but they can also be part of normal protein function. Amyloid formation involves numerous steps and intermediate species. In this study, we investigated a fish protein, beta-parvalbumin, which readily forms amyloid on ligand removal. Using biophysical experiments, we provide evidence that the underlying mechanism of amyloid formation includes primary nucleation and elongation processes; we also reveal a key role for a disulfide-bridged dimer in the nucleation step. Little is known about intermolecular disulfides in amyloid formation, but covalent dimers and dimer-induced aggregation may be of clinical relevance, because oxidative stress, which can trigger covalent bond formation, is often a hallmark of human neurodegenerative diseases. Amyloid formation involves the conversion of soluble protein species to an aggregated state. Amyloid fibrils of β-parvalbumin, a protein abundant in fish, act as an allergen but also inhibit the in vitro assembly of the Parkinson protein α-synuclein. However, the intrinsic aggregation mechanism of β-parvalbumin has not yet been elucidated. We performed biophysical experiments in combination with mathematical modeling of aggregation kinetics and discovered that the aggregation of β-parvalbumin is initiated by the formation of dimers stabilized by disulfide bonds and then proceeds via primary nucleation and fibril elongation processes. Dimer formation is accelerated by H2O2 and hindered by reducing agents, resulting in faster and slower aggregation rates, respectively. Purified β-parvalbumin dimers readily assemble into amyloid fibrils with similar morphology as those formed when starting from monomer solutions. Furthermore, addition of preformed dimers accelerates the aggregation reaction of monomers. Aggregation of purified β-parvalbumin dimers follows the same kinetic mechanism as that of monomers, implying that the rate-limiting primary nucleus is larger than a dimer and/or involves structural conversion. Our findings demonstrate a folded protein system in which spontaneously formed intermolecular disulfide bonds initiate amyloid fibril formation by recruitment of monomers. This dimer-induced aggregation mechanism may be of relevance for human amyloid diseases in which oxidative stress is often an associated hallmark.