Roles of intermolecular interactions in amyloid fibril formation mechanisms

Abstract: Amyloid fibrils, a major pathological feature of several neurodegenerative disorders, are highly stable, insoluble aggregates of misfolded proteins. The formation of such aggregates involves a complex equilibrium between protein monomers, different on- and off-pathway transient oligomeric species, and amyloid fibrils. Amyloid fibril formation in vivo may be induced by a myriad of factors, including oxidative stress and alteration of metal ion homeostasis. The work in this thesis involves biophysical studies of the amyloid fibril formation mechanisms of the natively folded Ca2+-binding fish protein, β-parvalbumin (β-PV); and how it is modulated by cell conditions including macromolecular crowding and stabilizing osmolytes, both of which tend to stabilize compact folded protein conformations through an excluded volume- or osmophobic effect, respectively. It was found that when β-PV aggregation is triggered, as occurs upon Ca2+ removal from the protein, spontaneous cystine formation between β-PV monomers initiates the process, whereafter the dimers template monomers into the amyloid conformation, resulting in polymerization. Furthermore, it was discovered that both excluded volume and the osmophobic effect promote the overall aggregation of β-PV, likely by facilitating protein dimerization. Together, these results highlight the potentially detrimental effects of ligand loss and oxidative stress on proteins, whose destabilization might induce amyloid fibril formation that is further exacerbated by otherwise protective in-cell conditions. Amyloid fibril formation by fish β-PV at acidic pH is thought to confer protection against proteolytic degradation in the human gut. In addition, since recent evidence suggests that many incidences of the neurodegenerative disorder Parkinson’s disease (PD) might originate from the gut, a putative interaction between β-PV and α-synuclein (αS), which forms amyloid fibrils in PD, was tested in vitro. Amyloid fibrils of β-PV block αS aggregation, likely by sequestering αS monomers onto the surface, thus potentially implying a protective effect of a diet rich in fish against PD. Lastly, in light of the fact that copper is reduced in affected brain regions of PD patients, as well as the presence of copper binding sites on αS, aggregation of αS in the presence of the endogenous cytoplasmic copper chaperone, Atox1, was studied. It was found that copper-Atox1 interacts with αS and can prevent its aggregation, while apo-Atox1 is ineffective, indicating a copper dependent interaction. The reduced copper levels associated with PD might thus play a role in PD progression by abolishing this protective interaction.