Sequence-based identification of amyloidogenic ß-hairpins reveals a prostatic acid phosphatase fragment promoting semen amyloid formation.

ß-Structure-rich amyloid fibrils are hallmarks of several diseases, including Alzheimer's (AD), Parkinson's (PD), and type 2 diabetes (T2D). While amyloid fibrils typically consist of parallel ß-sheets, the anti-parallel ß-hairpin is a structural motif accessible to amyloidogenic proteins in their monomeric and oligomeric states. Here, to investigate implications of ß-hairpins in amyloid formation, potential ß-hairpin-forming amyloidogenic segments in the human proteome were predicted based on sequence similarity with ß-hairpins previously observed in Aß, α-synuclein, and islet amyloid polypeptide, amyloidogenic proteins associated with AD, PD, and T2D, respectively. These three ß-hairpins, established upon binding to the engineered binding protein ß-wrapin AS10, are characterized by proximity of two sequence segments rich in hydrophobic and aromatic amino acids, with high ß-aggregation scores according to the TANGO algorithm. Using these criteria, 2505 potential ß-hairpin-forming amyloidogenic segments in 2098 human proteins were identified. Characterization of a test set of eight protein segments showed that seven assembled into Thioflavin T-positive aggregates and four formed ß-hairpins in complex with AS10 according to NMR. One of those is a segment of prostatic acid phosphatase (PAP) comprising amino acids 185-208. PAP is naturally cleaved into fragments, including PAP(248-286) which forms functional amyloid in semen. We find that PAP(185-208) strongly decreases the protein concentrations required for fibril formation of PAP(248-286) and of another semen amyloid peptide, SEM1(86-107), indicating that it promotes nucleation of semen amyloids. In conclusion, ß-hairpin-forming amyloidogenic protein segments could be identified in the human proteome with potential roles in functional or disease-related amyloid formation.

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.

Atomic Resolution Insights into pH Shift Induced Deprotonation Events in LS-Shaped Aß(1-42) Amyloid Fibrils.

Alzheimer's disease is a neurodegenerative disorder associated with the deposition of misfolded aggregates of the amyloid-ß protein (Aß). Aß(1-42) is one of the most aggregation-prone components in senile plaques of AD patients. We demonstrated that relatively homogeneous Aß(1-42) fibrils with one predominant fold visible in solid-state NMR spectra can be obtained at acidic pH. The structure of these fibrils differs remarkably from some other polymorphs obtained at neutral pH. In particular, the entire N-terminal region is part of the rigid fibril core. Here, we investigate the effects of a pH shift on the stability and the fold of these fibrils at higher pH values. Fibril bundling at neutral pH values renders cryo-EM studies impractical, but solid-state NMR spectroscopy, molecular dynamics simulations, and biophysical methods provide residue-specific structural information under these conditions. The LS-fold of the Aß(1-42) fibrils does not change over the complete pH range from pH 2 to pH 7; in particular, the N-terminus remains part of the fibril core. We observe changes in the protonation state of charged residues starting from pH 5 on a residue-specific level. The deprotonation of the C-terminal carboxyl group of A42 in the intermolecular salt bridge with D1 and K28 is slow on the NMR time scale, with a local pKa of 5.4, and local conformations of the involved residues are affected by deprotonation of A42. Thus, we demonstrate that this fibril form is stable at physiological pH values.

Structural basis for the inhibition of IAPP fibril formation by the co-chaperonin prefoldin.

Chaperones, as modulators of protein conformational states, are key cellular actors to prevent the accumulation of fibrillar aggregates. Here, we integrated kinetic investigations with structural studies to elucidate how the ubiquitous co-chaperonin prefoldin inhibits diabetes associated islet amyloid polypeptide (IAPP) fibril formation. We demonstrated that both human and archaeal prefoldin interfere similarly with the IAPP fibril elongation and secondary nucleation pathways. Using archaeal prefoldin model, we combined nuclear magnetic resonance spectroscopy with electron microscopy to establish that the inhibition of fibril formation is mediated by the binding of prefoldin's coiled-coil helices to the flexible IAPP N-terminal segment accessible on the fibril surface and fibril ends. Atomic force microscopy demonstrates that binding of prefoldin to IAPP leads to the formation of lower amounts of aggregates, composed of shorter fibrils, clustered together. Linking structural models with observed fibrillation inhibition processes opens perspectives for understanding the interference between natural chaperones and formation of disease-associated amyloids.

Alpha-Synuclein-Specific Naturally Occurring Antibodies Inhibit Aggregation In Vitro and In Vivo.

Parkinson's disease (PD) is associated with motor and non-motor symptoms and characterized by aggregates of alpha-synuclein (αSyn). Naturally occurring antibodies (nAbs) are part of the innate immune system, produced without prior contact to their specific antigen, and polyreactive. The abundance of nAbs against αSyn is altered in patients with PD. In this work, we biophysically characterized nAbs against αSyn (nAbs-αSyn) and determined their biological effects. nAbs-αSyn were isolated from commercial intravenous immunoglobulins using column affinity purification. Biophysical properties were characterized using a battery of established in vitro assays. Biological effects were characterized in HEK293T cells transiently transfected with fluorescently tagged αSyn. Specific binding of nAbs-αSyn to monomeric αSyn was demonstrated by Dot blot, ELISA, and Surface Plasmon Resonance. nAbs-αSyn did not affect viability of HEK293T cells as reported by Cell Titer Blue and LDH Assays. nAbs-αSyn inhibited fibrillation of αSyn reported by the Thioflavin T aggregation assay. Altered fibril formation was confirmed with atomic force microscopy. In cells transfected with EGFP-tagged αSyn we observed reduced formation of aggresomes, perinuclear accumulations of αSyn aggregates. The results demonstrate that serum of healthy individuals contains nAbs that specifically bind αSyn and inhibit aggregation of αSyn in vitro. The addition of nAbs-αSyn to cultured cells affects intracellular αSyn aggregates. These findings help understanding the role of the innate immune systems for the pathogenesis of PD and suggest that systemic αSyn binding agents could potentially affect neuronal αSyn pathology.

The role of heat shock proteins in preventing amyloid toxicity.

The oligomerization of monomeric proteins into large, elongated, ß-sheet-rich fibril structures (amyloid), which results in toxicity to impacted cells, is highly correlated to increased age. The concomitant decrease of the quality control system, composed of chaperones, ubiquitin-proteasome system and autophagy-lysosomal pathway, has been shown to play an important role in disease development. In the last years an increasing number of studies has been published which focus on chaperones, modulators of protein conformational states, and their effects on preventing amyloid toxicity. Here, we give a comprehensive overview of the current understanding of chaperones and amyloidogenic proteins and summarize the advances made in elucidating the impact of these two classes of proteins on each other, whilst also highlighting challenges and remaining open questions. The focus of this review is on structural and mechanistic studies and its aim is to bring novices of this field "up to speed" by providing insight into all the relevant processes and presenting seminal structural and functional investigations.

Clustering of human prion protein and α-synuclein oligomers requires the prion protein N-terminus.

The interaction of prion protein (PrP) and α-synuclein (αSyn) oligomers causes synaptic impairment that might trigger Parkinson's disease and other synucleinopathies. Here, we report that αSyn oligomers (αSynO) cluster with human PrP (huPrP) into micron-sized condensates. Multivalency of αSyn within oligomers is required for condensation, since clustering with huPrP is not observed for monomeric αSyn. The stoichiometry of the heteroassemblies is well defined with an αSyn:huPrP molar ratio of about 1:1. The αSynO-huPrP interaction is of high affinity, signified by slow dissociation. The huPrP region responsible for condensation of αSynO, residues 95-111 in the intrinsically disordered N-terminus, corresponds to the region required for αSynO-mediated cognitive impairment. HuPrP, moreover, achieves co-clustering of αSynO and Alzheimer's disease-associated amyloid-ß oligomers, providing a case of a cross-interaction of two amyloidogenic proteins through an interlinking intrinsically disordered protein region. The results suggest that αSynO-mediated condensation of huPrP is involved in the pathogenesis of synucleinopathies.

Cryo-EM structure of islet amyloid polypeptide fibrils reveals similarities with amyloid-ß fibrils.

Amyloid deposits consisting of fibrillar islet amyloid polypeptide (IAPP) in pancreatic islets are associated with beta-cell loss and have been implicated in type 2 diabetes (T2D). Here, we applied cryo-EM to reconstruct densities of three dominant IAPP fibril polymorphs, formed in vitro from synthetic human IAPP. An atomic model of the main polymorph, built from a density map of 4.2-Å resolution, reveals two S-shaped, intertwined protofilaments. The segment 21-NNFGAIL-27, essential for IAPP amyloidogenicity, forms the protofilament interface together with Tyr37 and the amidated C terminus. The S-fold resembles polymorphs of Alzheimer's disease (AD)-associated amyloid-ß (Aß) fibrils, which might account for the epidemiological link between T2D and AD and reports on IAPP-Aß cross-seeding in vivo. The results structurally link the early-onset T2D IAPP genetic polymorphism (encoding Ser20Gly) with the AD Arctic mutation (Glu22Gly) of Aß and support the design of inhibitors and imaging probes for IAPP fibrils.