Nucleation of α-Synuclein Amyloid Fibrils Induced by Cross-Interaction with ß-Hairpin Peptides Derived from Immunoglobulin Light Chains.

Heterologous interactions between different amyloid-forming proteins, also called cross-interactions, may have a critical impact on disease-related amyloid formation. ß-hairpin conformers of amyloid-forming proteins have been shown to affect homologous interactions in the amyloid self-assembly process. Here, we applied two ß-hairpin-forming peptides derived from immunoglobulin light chains as models to test how heterologous ß-hairpins modulate the fibril formation of Parkinson's disease-associated protein α-synuclein (αSyn). The peptides SMAhp and LENhp comprise ß-strands C and C' of the κ4 antibodies SMA and LEN, which are associated with light chain amyloidosis and multiple myeloma, respectively. SMAhp and LENhp bind with high affinity to the ß-hairpin-binding protein ß-wrapin AS10 according to isothermal titration calorimetry and NMR spectroscopy. The addition of SMAhp and LENhp affects the kinetics of αSyn aggregation monitored by Thioflavin T (ThT) fluorescence, with the effect depending on assay conditions, salt concentration, and the applied ß-hairpin peptide. In the absence of agitation, substoichiometric concentrations of the hairpin peptides strongly reduce the lag time of αSyn aggregation, suggesting that they support the nucleation of αSyn amyloid fibrils. The effect is also observed for the aggregation of αSyn fragments lacking the N-terminus or the C-terminus, indicating that the promotion of nucleation involves the interaction of hairpin peptides with the hydrophobic non-amyloid-ß component (NAC) region.

The hydrophobic effect characterises the thermodynamic signature of amyloid fibril growth.

Many proteins have the potential to aggregate into amyloid fibrils, protein polymers associated with a wide range of human disorders such as Alzheimer's and Parkinson's disease. The thermodynamic stability of amyloid fibrils, in contrast to that of folded proteins, is not well understood: the balance between entropic and enthalpic terms, including the chain entropy and the hydrophobic effect, are poorly characterised. Using a combination of theory, in vitro experiments, simulations of a coarse-grained protein model and meta-data analysis, we delineate the enthalpic and entropic contributions that dominate amyloid fibril elongation. Our prediction of a characteristic temperature-dependent enthalpic signature is confirmed by the performed calorimetric experiments and a meta-analysis over published data. From these results we are able to define the necessary conditions to observe cold denaturation of amyloid fibrils. Overall, we show that amyloid fibril elongation is associated with a negative heat capacity, the magnitude of which correlates closely with the hydrophobic surface area that is buried upon fibril formation, highlighting the importance of hydrophobicity for fibril stability.

ß-Turn exchanges in the α-synuclein segment 44-TKEG-47 reveal high sequence fidelity requirements of amyloid fibril elongation.

The folding of turns and ß-hairpins has been implicated in amyloid formation, with diverse potential consequences such as promotion or inhibition of fibril nucleation, fibril elongation, or off-pathway oligomer formation. In the Parkinson's disease-associated protein α-synuclein (αS), a ß-hairpin comprised of residues 36-56 was detected in complex with an engineered binding protein, with a turn formed by the αS sequence segment 44-TKEG-47. Molecular dynamics simulations revealed extensive populations of transient ß-hairpin conformations in this region in free, monomeric αS. Here, we investigated potential effects of turn formation on αS fibril formation by studying the aggregation kinetics of an extensive set of αS variants with between two and four amino acid exchanges in the 44-TKEG-47 segment. The exchanges were chosen to specifically promote formation of ß1-, ß1'-, or ß2'-turns. All variants assembled into amyloid fibrils, with increased ß1'- or ß2'-turn propensity associated with faster aggregation and increased ß1-turn propensity with slower aggregation compared to wild-type (WT) αS. Atomic force microscopy demonstrated that ß-turn exchanges altered fibril morphology. In cross-elongation experiments, the turn variants showed a low ability to elongate WT fibril seeds, and, vice versa, WT monomer did not efficiently elongate turn variant fibril seeds. This demonstrates that sequence identity in the turn region is crucial for efficient αS fibril elongation. Elongation experiments of WT fibril seeds in the presence of both WT and turn variant monomers suggest that the turn variants can bind and block WT fibril ends to different degrees, but cannot efficiently convert into the WT fibril structure. Our results indicate that modifications in the 44-TKEG-47 segment strongly affect amyloid assembly by driving αS into alternative fibril morphologies, whose elongation requires high sequence fidelity.

Endo-lysosomal Aß concentration and pH trigger formation of Aß oligomers that potently induce Tau missorting.

Amyloid-ß peptide (Aß) forms metastable oligomers >50 kDa, termed AßOs, that are more effective than Aß amyloid fibrils at triggering Alzheimer's disease-related processes such as synaptic dysfunction and Tau pathology, including Tau mislocalization. In neurons, Aß accumulates in endo-lysosomal vesicles at low pH. Here, we show that the rate of AßO assembly is accelerated 8,000-fold upon pH reduction from extracellular to endo-lysosomal pH, at the expense of amyloid fibril formation. The pH-induced promotion of AßO formation and the high endo-lysosomal Aß concentration together enable extensive AßO formation of Aß42 under physiological conditions. Exploiting the enhanced AßO formation of the dimeric Aß variant dimAß we furthermore demonstrate targeting of AßOs to dendritic spines, potent induction of Tau missorting, a key factor in tauopathies, and impaired neuronal activity. The results suggest that the endosomal/lysosomal system is a major site for the assembly of pathomechanistically relevant AßOs.