Axel Abelein

Biophysical Studies of the Amyloid β-Peptide: Interactions with Metal Ions and Small Molecules

Alzheimers disease is the most common of the protein misfolding (“amyloid”) diseases. The deposits in the brains of afflicted patients contain as a major fraction an aggregated insoluble form of the so‐called amyloid β‐peptides (Aβ peptides): fragments of the amyloid precursor protein of 39–43 residues in length. This review focuses on biophysical studies of the Aβ peptides: that is, of the aggregation pathways and intermediates observed during aggregation, of the molecular structures observed along these pathways, and of the interactions of Aβ with Cu and Zn ions and with small molecules that modify the aggregation pathways. Particular emphasis is placed on studies based on high‐resolution and solid‐state NMR methods. Theoretical studies relating to the interactions are also included. An emerging picture is that of Aβ peptides in aqueous solution undergoing hydrophobic collapse together with identical partners. There then follows a relatively slow process leading to more ordered secondary and tertiary (quaternary) structures in the growing aggregates. These aggregates eventually assemble into elongated fibrils visible by electron microscopy. Small molecules or metal ions that interfere with the aggregation processes give rise to a variety of aggregation products that may be studied in vitro and considered in relation to observations in cell cultures or in vivo. Although the heterogeneous nature of the processes makes detailed structural studies difficult, knowledge and understanding of the underlying physical chemistry might provide a basis for future therapeutic strategies against the disease. A final part of the review deals with the interactions that may occur between the Aβ peptides and the prion protein, where the latter is involved in other protein misfolding diseases.

Zinc as chaperone-mimicking agent for retardation of amyloid β peptide fibril formation

Significance One histologic hallmark of Alzheimer’s disease is the self-assembly of amyloid β peptide (Aβ) into insoluble amyloid aggregates. This aggregation process is strongly dependent on environmental conditions and metal ions, such as zinc, have been shown to modulate Aβ aggregation. To understand the underlying molecular mechanism of how zinc affects fibril formation we analyzed the aggregation kinetics and could conclude that zinc causes a significant reduction in elongation rate (i.e., monomer addition to the fibril ends). We used NMR methods to elucidate the details of zinc binding and we found that the N terminus of Aβ transiently folds around the zinc ion, forming a metastable dynamic complex. Metal ions have emerged to play a key role in the aggregation process of amyloid β (Aβ) peptide that is closely related to the pathogenesis of Alzheimer’s disease. A detailed understanding of the underlying mechanistic process of peptide–metal interactions, however, has been challenging to obtain. By applying a combination of NMR relaxation dispersion and fluorescence kinetics methods we have investigated quantitatively the thermodynamic Aβ–Zn2+ binding features as well as how Zn2+ modulates the nucleation mechanism of the aggregation process. Our results show that, under near-physiological conditions, substoichiometric amounts of Zn2+ effectively retard the generation of amyloid fibrils. A global kinetic profile analysis reveals that in the absence of zinc Aβ40 aggregation is driven by a monomer-dependent secondary nucleation process in addition to fibril-end elongation. In the presence of Zn2+, the elongation rate is reduced, resulting in reduction of the aggregation rate, but not a complete inhibition of amyloid formation. We show that Zn2+ transiently binds to residues in the N terminus of the monomeric peptide. A thermodynamic analysis supports a model where the N terminus is folded around the Zn2+ ion, forming a marginally stable, short-lived folded Aβ40 species. This conformation is highly dynamic and only a few percent of the peptide molecules adopt this structure at any given time point. Our findings suggest that the folded Aβ40–Zn2+ complex modulates the fibril ends, where elongation takes place, which efficiently retards fibril formation. In this conceptual framework we propose that zinc adopts the role of a minimal antiaggregation chaperone for Aβ40.

The hairpin conformation of the amyloid β peptide is an important structural motif along the aggregation pathway

The amyloid β (Aβ) peptides are 39–42 residue-long peptides found in the senile plaques in the brains of Alzheimer’s disease (AD) patients. These peptides self-aggregate in aqueous solution, going from soluble and mainly unstructured monomers to insoluble ordered fibrils. The aggregation process(es) are strongly influenced by environmental conditions. Several lines of evidence indicate that the neurotoxic species are the intermediate oligomeric states appearing along the aggregation pathways. This minireview summarizes recent findings, mainly based on solution and solid-state NMR experiments and electron microscopy, which investigate the molecular structures and characteristics of the Aβ peptides at different stages along the aggregation pathways. We conclude that a hairpin-like conformation constitutes a common motif for the Aβ peptides in most of the described structures. There are certain variations in different hairpin conformations, for example regarding H-bonding partners, which could be one reason for the molecular heterogeneity observed in the aggregated systems. Interacting hairpins are the building blocks of the insoluble fibrils, again with variations in how hairpins are organized in the cross-section of the fibril, perpendicular to the fibril axis. The secondary structure propensities can be seen already in peptide monomers in solution. Unfortunately, detailed structural information about the intermediate oligomeric states is presently not available. In the review, special attention is given to metal ion interactions, particularly the binding constants and ligand structures of Aβ complexes with Cu(II) and Zn(II), since these ions affect the aggregation process(es) and are considered to be involved in the molecular mechanisms underlying AD pathology.

Hydrophobicity and Conformational Change as Mechanistic Determinants for Nonspecific Modulators of Amyloid β Self-Assembly

The link between many neurodegenerative disorders, including Alzheimers and Parkinsons diseases, and the aberrant folding and aggregation of proteins has prompted a comprehensive search for small organic molecules that have the potential to inhibit such processes. Although many compounds have been reported to affect the formation of amyloid fibrils and/or other types of protein aggregates, the mechanisms by which they act are not well understood. A large number of compounds appear to act in a nonspecific way affecting several different amyloidogenic proteins. We describe here a detailed study of the mechanism of action of one representative compound, lacmoid, in the context of the inhibition of the aggregation of the amyloid β-peptide (Aβ) associated with Alzheimers disease. We show that lacmoid binds Aβ(1-40) in a surfactant-like manner and counteracts the formation of all types of Aβ(1-40) and Aβ(1-42) aggregates. On the basis of these and previous findings, we are able to rationalize the molecular mechanisms of action of nonspecific modulators of protein self-assembly in terms of hydrophobic attraction and the conformational preferences of the polypeptide.

Formation of dynamic soluble surfactant-induced amyloid β peptide aggregation intermediates

Background: β-Structured oligomers of the amyloid β peptide are considered neurotoxic and on-pathway to amyloid fibril formation. Results: Surfactant-induced co-aggregated oligomers show dynamic rapid exchange with free peptide during a slow fibril formation process. Conclusion: β-Structure inducing small molecules kinetically promote peptide assembly into co-aggregates. Significance: Knowledge about molecular mechanisms of peptide aggregation modulators is potentially helpful for therapeutic purposes. Intermediate amyloidogenic states along the amyloid β peptide (Aβ) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aβ aggregation, we here investigate surfactant-induced Aβ aggregation. This process leads to co-aggregates featuring a β-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aβ in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via β-structure to the fully formed α-helical state at high surfactant concentration. The β-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aβ co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (kex ∼1100 s−1) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with β-structure promoting substances, such as surfactants, Aβ is kinetically driven toward an aggregation-prone state.

Transient small molecule interactions kinetically modulate amyloid β peptide self-assembly

Small organic molecules, like Congo red and lacmoid, have been shown to modulate the self‐assembly of the amyloid β peptide (Aβ). Here, we show that Aβ forms NMR invisible non‐toxic co‐aggregates together with lacmoid as well as Congo red. We find that the interaction involves two distinct kinetic processes and at every given time point only a small fraction of Aβ is in the co‐aggregate. These weak transient interactions kinetically redirect the aggregation prone Aβ from self‐assembling into amyloid fibrils. These findings suggest that even such weak binders might be effective as therapeutics against pathogenic protein aggregation.

Ionic Strength Modulation of the Free Energy Landscape of Aβ40 Peptide Fibril Formation

Protein misfolding and formation of cross-β structured amyloid fibrils are linked to many neurodegenerative disorders. Although recently developed quantitative approaches have started to reveal the molecular nature of self-assembly and fibril formation of proteins and peptides, it is yet unclear how these self-organization events are precisely modulated by microenvironmental factors, which are known to strongly affect the macroscopic aggregation properties. Here, we characterize the explicit effect of ionic strength on the microscopic aggregation rates of amyloid β peptide (Aβ40) self-association, implicated in Alzheimers disease. We found that physiological ionic strength accelerates Aβ40 aggregation kinetics by promoting surface-catalyzed secondary nucleation reactions. This promoted catalytic effect can be assigned to shielding of electrostatic repulsion between monomers on the fibril surface or between the fibril surface itself and monomeric peptides. Furthermore, we observe the formation of two different β-structured states with similar but distinct spectroscopic features, which can be assigned to an off-pathway immature state (Fβ*) and a mature stable state (Fβ), where salt favors formation of the Fβ fibril morphology. Addition of salt to preformed Fβ* accelerates transition to Fβ, underlining the dynamic nature of Aβ40 fibrils in solution. On the basis of these results we suggest a model where salt decreases the free-energy barrier for Aβ40 folding to the Fβ state, favoring the buildup of the mature fibril morphology while omitting competing, energetically less favorable structural states.

Formation of Dynamic Soluble Surfactant-Induced Amyloid Beta Peptide Aggregation Intermediates

The 40-42 residue Amyloid β (Aβ) peptide forms β-structured oligomers on-pathway to amyloid fibril formation and are linked to neuronal damage characteristic for Alzheimers disease. Surfactants such as SDS induce relatively stable β-structured Aβ co-aggregates and may be considered as a model system for lipids. We have characterized different intermediate aggregation states appearing during the Aβ aggregation process, as well as the kinetics of their formation and dynamic exchange between free and bound peptide. A broad range of biophysical techniques were used, including small angle X-ray scattering (SAXS) and NMR spectroscopy, particularly 15N-CPMG relaxation dispersion experiments.Aβ shows a three-state secondary structure transition depending on surfactant concentration, from random coil-like, via β-structure to α-helix at high surfactant concentration. Structural information on the β-structured co-aggregates was obtained by SAXS experiments that at the beginning showed a large fraction of globular co-aggregates (diameter ∼ 75 A). This fraction gradually vanished on a min to hr timescale and elongated co-aggregated fibrils were formed (diameter ∼ 60 A and length > 350 A), in line with transmission electron microscopy images. A fast dynamic exchange process (kex ∼ 1100 s-1) between free and co-aggregate bound peptide takes place, as monitored by NMR relaxation dispersion experiments.This study of the surfactant-induced Aβ aggregation may serve as a model for aggregation of Aβ alone or in the presence of lipids.Reference:1. Abelein, A. et al., J. Biol. Chem. (2013), 288, 23518-23528.