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Proteins in either a native or denatured conformation often aggregate at an isoelectric point (pI), a phenomenon known as pI precipitation. However, only a few studies have addressed the role of pI precipitation in amyloid formation, the crystal-like aggregation of denatured proteins. We found that α-synuclein, an intrinsically disordered protein of 140 amino acid residues associated with Parkinson's disease, formed amyloid fibrils at pI (= 4.7) under the low-sodium phosphate conditions. Although α-synuclein also formed amyloid fibrils at a wide pH range under high concentrations of sodium phosphate, the pI-amyloid formation was characterized by marked amyloid-specific thioflavin T fluorescence and clear fibrillar morphology, indicating highly ordered structures. Analysis by heteronuclear NMR in combination with principal component analysis suggested that amyloid formation under low and high phosphate conditions occurred by distinct mechanisms. The former was likely to be caused by the intermolecular attractive charge-charge interactions, where α-synuclein has +17 and −17 charges even with the zero net charge. On the other hand, the latter was caused by the phosphate-dependent salting-out effects. pI-amyloid formation may play a role in the membrane-dependent amyloid formation of α-synuclein, where the negatively charged membrane surface reduces the local pH to pI and the membrane hydrophobic environment enhances electrostatic interactions. The results extend the supersaturation-limited mechanism of amyloid formation: Amyloid fibrils are formed under a variety of conditions of decreased solubility of denatured proteins triggered by the breakdown of supersaturation.

β2-Microglobulin (β2m) is the causative protein of dialysis-related amyloidosis, and its D76N variant is less stable and more prone to aggregation. Since their crystal structures are indistinguishable from each other, enhanced amyloidogenicity induced by the mutation may be attributed to changes in the structural dynamics of the molecule. We examined pressure and mutation effects on the β2m molecule by NMR and MD simulations, and found that the mutation induced the loosening of the inter-sheet packing of β2m, which is relevant to destabilization and subsequent amyloidogenicity. On the other hand, this loosening was coupled with perturbed dynamics at some peripheral regions. The key result for this conclusion was that both the mutation and pressure induced similar reductions in the mobility of these residues, suggesting that there is a common mechanism underlying the suppression of inherent fluctuations in the β2m molecule. Analyses of data obtained under high pressure conditions suggested that the network of dynamically correlated residues included not only the mutation site, but also distal residues, such as those of the C- and D-strands. Reductions in these local dynamics correlated with the loosening of inter-sheet packing.

β2-Microglobulin (β2m) is the causative protein of dialysisrelated amyloidosis. Its unfolding mainly proceeds along the pathway of NC →UC%UT, whereas refolding follows the UT → IT (→NT) →NC pathway, in which N, I, and U are the native, intermediate, and unfolded states, respectively, with the Pro32 peptidyl-prolyl bond in cis or trans conformation as indicated by the subscript. It is noted that the IT state is a putative amyloidogenic precursor state. Several aggregation-prone variants of β2m have been reported to date. One of these variants is D76N β2m, which is a naturally occurring amyloidogenic mutant. To elucidate the molecular mechanisms contributing to the enhanced amyloidogenicity of the mutant, we investigated the equilibrium and kinetic transitions of pressure-induced folding/unfolding equilibria in the wild type and D76N mutant by monitoring intrinsic tryptophan and 1-anilino-8-naphthalene sulfonate fluorescence. An analysis of kinetic data revealed that the different folding/unfolding behaviors of the wild type and D76N mutant were due to differences in the activation energy between the unfolded and the intermediate states as well as stability of the native state, leading to more rapid accumulation of IT state for D76N in the refolding process. In addition, the IT state was found to assume more hydrophobic nature. These changes induced the enhanced amyloidogenicity of the D76N mutant and the distinct pathogenic symptoms of patients. Our results suggest that the stabilization of the native state will be an effective approach for suppressing amyloid fibril formation of this mutant. [ABSTRACT FROM AUTHOR]

Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of β2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles. [ABSTRACT FROM AUTHOR]

Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. β2-Microglobulin (β2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving long-term hemodialysis. Although amyloid fibrils of β2m in patients are observed at neutral pH, formation of β2m amyloids in vitro has been difficult to discern at neutral pH because of the amyloid-resistant native structure. Here, to further understand the mechanism underlying in vivo amyloid formation, we investigated the relationship between protein folding/unfolding and misfolding leading to amyloid formation. Using thioflavin T assays, CD spectroscopy, and transmission EM analyses, we found that β2m efficiently forms amyloid fibrils even at neutral pH by heating with agitation at high-salt conditions. We constructed temperature- and NaCl concentration-dependent conformational phase diagrams in the presence or absence of agitation, revealing how amyloid formation under neutral pH conditions is related to thermal unfolding and breakdown of supersaturation. Of note, after supersaturation breakdown and following the law of mass action, the β2m monomer equilibrium shifted to the unfolded state, destabilizing the native state and thereby enabling amyloid formation even under physiological conditions with a low amount of unfolded precursor. The amyloid fibrils depolymerized at both lower and higher temperatures, resembling cold- or heat-induced denaturation of globular proteins. Our results suggest an important role for heating in the onset of dialysis-related amyloidosis and related amyloidoses. [ABSTRACT FROM AUTHOR]