To elucidate the role of β-hairpin motifs in amyloid formation, we studied the effects of hairpin modifications in amyloidogenic proteins. For specific modifications, we found a strong inhibitory effect on fibril elongation. This effect was highly dependent on the precise arrangement of the hairpin, suggesting that specific hairpin conformers act as inhibitors in amyloid fibril elongation. In a proteome-wide by bioinformatics, we found a high number of protein sequence regions with the potential to form amyloidogenic β-hairpins. One of these β-hairpin could promote formation of functional amyloid, indicating that β-hairpins can also play a role in cross-interaction of different amyloidogenic proteins.
We investigated how engineered binding-proteins called β-wrapins, which specifically bind to β-hairpin motifs, are able to prevent aggregation of the protein α-synuclein, the core component of protein deposits found in the Parkinson’s disease-affected brains. First, we found out that β-wrapins prevent α-synuclein monomers from elongating the amyloid fibrils, by capturing the monomers and forming chemical complexes with them. Another mechanism, however, is responsible for the particular effectivity of β-wrapins at low concentrations: They prevent seed fibrils from forming in the first place. Very small amounts of the β-wrapins are sufficient for this to happen, explaining the sub-stoichiometric effect that makes the process especially effective. We found that it is the aforementioned complexes comprising binding proteins and monomers that are responsible for inhibiting seed formation. The inhibition of α-synuclein aggregation could be reproduced in a cell culture model, in a Drosophila melanogaster model, and in a mouse model of Parkinson’s disease.
A different mode of inhibition was found for hairpin-enriched variants of amyloidogenic proteins: The inhibitor binds to growing amyloid fibril ends. Intriguingly, it cooperates with the wild-type amyloidogenic monomers in ‘contaminating’ the amyloid fibril end, which blocks further fibril elongation. The identified substrate-inhibitor cooperativity allowed to design fusion constructs with increased potency, which are effective at low nanomolar concentrations.
In order to get a better picture of the structures that our inhibitors are acting on, we applied cryo-electron microscopy to determine atomic resolution images of amyloid fibrils. These structures, for example of the Alzheimer disease-associated amyloid-β peptide (Aβ) and the type 2 diabetes-related islet amyloid polypeptide (IAPP), display patterns of grooves and ridges at the lateral fibril surface and at the fibril ends. The cryo-electron microscopy structures thereby reveal the binding sites for new monomers of the aggregating protein that are added during fibril growth, but also for inhibitors of amyloid fibril growth.
Smaller clusters of amyloid-forming proteins termed oligomers are particularly toxic. β-Hairpins might be the building blocks of such oligomers. We have developed oligomer models of the amyloid-beta peptide that can be produced with high yield and homogeneity. This enabled the analysis of the mechanism of oligomer formation. In addition, we found that the oligomers interact with amyloid fibrils. This interplay of amyloid oligomers and fibrils affects their formation, stability, and toxic activities.
