Response to crowded conditions reveals compact nucleus for amyloid formation of folded protein.

Although the consequences of the crowded cell environments may affect protein folding, function and misfolding reactions, these processes are often studied in dilute solutions in vitro. We here used biophysical experiments to investigate the amyloid fibril formation process of the fish protein apo-ß-parvalbumin in solvent conditions that mimic steric and solvation aspects of the in vivo milieu. Apo-ß-parvalbumin is a folded protein that readily adopts an amyloid state via a nucleation-elongation mechanism. Aggregation experiments in the presence of macromolecular crowding agents (probing excluded volume, entropic effects) as well as small molecule osmolytes (probing solvation, enthalpic effects) revealed that both types of agents accelerate overall amyloid formation, but the elongation step was faster with macromolecular crowding agents but slower in the presence of osmolytes. The observations can be explained by the steric effects of excluded volume favoring assembled states and that amyloid nucleation does not involve monomer unfolding. In contrast, the solvation effects due to osmolyte presence promote nucleation but not elongation. Therefore, the amyloid-competent nuclei must be compact with less osmolytes excluded from the surface than either the folded monomers or amyloid fibers. We conclude that, in contrast to other amyloidogenic folded proteins, amyloid formation of apo-ß-parvalbumin is accelerated by crowded cell-like conditions due to a nucleation process that does not involve large-scale protein unfolding.

Amyloid formation of fish ß-parvalbumin involves primary nucleation triggered by disulfide-bridged protein dimers.

Amyloid formation involves the conversion of soluble protein species to an aggregated state. Amyloid fibrils of ß-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 ß-parvalbumin has not yet been elucidated. We performed biophysical experiments in combination with mathematical modeling of aggregation kinetics and discovered that the aggregation of ß-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 ß-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 ß-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.

Crosstalk Between Alpha-Synuclein and Other Human and Non-Human Amyloidogenic Proteins: Consequences for Amyloid Formation in Parkinson's Disease.

It was recently shown (Sampson et al., Elife9, 2020) that an amyloidogenic protein, CsgA, present in E. coli biofilms in the gut can trigger Parkinson's disease in mice. This study emphasizes the possible role of the gut microbiome in modulation (and even initiation) of human neurodegenerative disorders, such as Parkinson's disease. As the CsgA protein was found to accelerate alpha-synuclein (the key amyloidogenic protein in Parkinson's disease) amyloid formation in vitro, this result suggests that also other amyloidogenic proteins from gut bacteria, and even from the diet (such as stable allergenic proteins), may be able to affect human protein conformations and thereby modulate amyloid-related diseases. In this review, we summarize what has been reported in terms of in vitro cross-reactivity studies between alpha-synuclein and other amyloidogenic human and non-human proteins. It becomes clear from the limited data that exist that there is a fine line between acceleration and inhibition, but that cross-reactivity is widespread, and it is more common for other proteins (among the studied cases) to accelerate alpha-synuclein amyloid formation than to block it. It is of high importance to expand investigations of cross-reactivity between amyloidogenic proteins to both reveal underlying mechanisms and links between human diseases, as well as to develop new treatments that may be based on an altered gut microbiome.

Abundant fish protein inhibits α-synuclein amyloid formation.

The most common allergen in fish, the highly-abundant protein ß-parvalbumin, forms amyloid structures as a way to avoid gastrointestinal degradation and transit to the blood. In humans, the same amyloid structures are mostly associated with neurodegenerative disorders such as Alzheimer's and Parkinson's. We here assessed a putative connection between these amyloids using recombinant Atlantic cod ß-parvalbumin and the key amyloidogenic protein in Parkinson's disease, α-synuclein. Using a set of in vitro biophysical methods, we discovered that ß-parvalbumin readily inhibits amyloid formation of α-synuclein. The underlying mechanism was found to involve α-synuclein binding to the surface of ß-parvalbumin amyloid fibers. In addition to being a new amyloid inhibition mechanism, the data suggest that health benefits of fish may be explained in part by cross-reaction of ß-parvalbumin with human amyloidogenic proteins.

Copper chaperone blocks amyloid formation via ternary complex.

Protein misfolding in cells is avoided by a network of protein chaperones that detect misfolded or partially folded species. When proteins escape these control systems, misfolding may result in protein aggregation and amyloid formation. We here show that aggregation of the amyloidogenic protein α-synuclein (αS), the key player in Parkinson's disease, is controlled by the copper transport protein Atox1 in vitro. Copper ions are not freely available in the cellular environment, but when provided by Atox1, the resulting copper-dependent ternary complex blocks αS aggregation. Because the same inhibition was found for a truncated version of αS, lacking the C-terminal part, it appears that Atox1 interacts with the N-terminal copper site in αS. Metal-dependent chaperoning may be yet another manner in which cells control its proteome.

Therapeutic innovation: Inflammatory-reactive astrocytes as targets of inflammation.

This study aimed to test pharmaceutical compounds targeting astrocytes showing inflammatory dysregulation. The primary rat brain cultures were treated with different batches of serum with or without microglia added to make the cells inflammatory-reactive. Lipopolysaccharide (LPS) and tryptase were used as inflammatory inducers. Expression levels of Toll-like receptor 4 (TLR4), Na+/K+-ATPase, and matrix metalloprotease-13 (MMP-13), as well as actin filament organization, pro-inflammatory cytokines, and intracellular Ca2+ release, were evaluated. LPS combined with tryptase upregulated TLR4 expression, whereas Na+/K+-ATPase expression was downregulated, ATP-evoked Ca2+ transients were increased, actin filaments were reorganized and ring structures instead of stress fibers were observed. Other aims of the study were to prevent astrocytes from becoming inflammatory-reactive and to restore inflammatory dysregulated cellular changes. A combination of the µ-opioid antagonist (-)-naloxone in ultra-low concentrations, the non-addictive µ-opioid agonist (-)-linalool, and the anti-epileptic agent levetiracetam was examined. The results indicated that this drug cocktail prevented the LPS- and tryptase-induced inflammatory dysregulation. The drug cocktail could also restore the LPS- and tryptase-treated cells back to a normal physiological level in terms of the analyzed parameters.