Prion Protein Octarepeat Domain Forms Transient ß-Sheet Structures upon Residue-Specific Binding to Cu(II) and Zn(II) Ions.

Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in PrPC misfolding. PrPC is a combined Cu(II) and Zn(II) metal-binding protein, where the main metal-binding site is located in the octarepeat (OR) region. Thus, the biological function of PrPC may involve the transport of divalent metal ions across membranes or buffering concentrations of divalent metal ions in the synaptic cleft. Recent studies have shown that an excess of Cu(II) ions can result in PrPC instability, oligomerization, and/or neuroinflammation. Here, we have used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region of PrPC. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Binding of the first metal ion results in a structural transition from the polyproline II helix to the ß-turn structure, while the binding of additional metal ions induces the formation of ß-sheet structures. Fluorescence spectroscopy data indicate that the OR region can bind both Cu(II) and Zn(II) ions at neutral pH, but under acidic conditions, it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of either metal ion to the OR region results in the formation of ß-hairpin structures. As the formation of ß-sheet structures can be a first step toward amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSE diseases.

Metal ion coordination delays amyloid-ß peptide self-assembly by forming an aggregation-inert complex.

A detailed understanding of the molecular pathways for amyloid-ß (Aß) peptide aggregation from monomers into amyloid fibrils, a hallmark of Alzheimer's disease, is crucial for the development of diagnostic and therapeutic strategies. We investigate the molecular details of peptide fibrillization in vitro by perturbing this process through addition of differently charged metal ions. Here, we used a monovalent probe, the silver ion, that, similarly to divalent metal ions, binds to monomeric Aß peptide and efficiently modulates Aß fibrillization. On the basis of our findings, combined with our previous results on divalent zinc ions, we propose a model that links the microscopic metal-ion binding to Aß monomers to its macroscopic impact on the peptide self-assembly observed in bulk experiments. We found that substoichiometric concentrations of the investigated metal ions bind specifically to the N-terminal region of Aß, forming a dynamic, partially compact complex. The metal-ion bound state appears to be incapable of aggregation, effectively reducing the available monomeric Aß pool for incorporation into fibrils. This is especially reflected in a decreased fibril-end elongation rate. However, because the bound state is significantly less stable than the amyloid state, Aß peptides are only transiently redirected from fibril formation, and eventually almost all Aß monomers are integrated into fibrils. Taken together, these findings unravel the mechanistic consequences of delaying Aß aggregation via weak metal-ion binding, quantitatively linking the contributions of specific interactions of metal ions with monomeric Aß to their effects on bulk aggregation.

Effects of in vivo conditions on amyloid aggregation.

One of the grand challenges of biophysical chemistry is to understand the principles that govern protein misfolding and aggregation, which is a highly complex process that is sensitive to initial conditions, operates on a huge range of length- and timescales, and has products that range from protein dimers to macroscopic amyloid fibrils. Aberrant aggregation is associated with more than 25 diseases, which include Alzheimer's, Parkinson's, Huntington's, and type II diabetes. Amyloid aggregation has been extensively studied in the test tube, therefore under conditions that are far from physiological relevance. Hence, there is dire need to extend these investigations to in vivo conditions where amyloid formation is affected by a myriad of biochemical interactions. As a hallmark of neurodegenerative diseases, these interactions need to be understood in detail to develop novel therapeutic interventions, as millions of people globally suffer from neurodegenerative disorders and type II diabetes. The aim of this review is to document the progress in the research on amyloid formation from a physicochemical perspective with a special focus on the physiological factors influencing the aggregation of the amyloid-ß peptide, the islet amyloid polypeptide, α-synuclein, and the hungingtin protein.

Copper ions induce dityrosine-linked dimers in human but not in murine islet amyloid polypeptide (IAPP/amylin).

Dysregulation and aggregation of the peptide hormone IAPP (islet amyloid polypeptide, a.k.a. amylin) into soluble oligomers that appear to be cell-toxic is a known aspect of diabetes mellitus (DM) Type 2 pathology. IAPP aggregation is influenced by several factors including interactions with metal ions such as Cu(II). Because Cu(II) ions are redox-active they may contribute to metal-catalyzed formation of oxidative tyrosyl radicals, which can generate dityrosine cross-links. Here, we show that such a process, which involves Cu(II) ions bound to the IAPP peptide together with H2O2, can induce formation of large amounts of IAPP dimers connected by covalent dityrosine cross-links. This cross-linking is less pronounced at low pH and for murine IAPP, likely due to less efficient Cu(II) binding. Whether IAPP can carry out its hormonal function as a cross-linked dimer is unknown. As dityrosine concentrations are higher in blood plasma of DM Type 2 patients - arguably due to disease-related oxidative stress - and as dimer formation is the first step in protein aggregation, generation of dityrosine-linked dimers may be an important factor in IAPP aggregation and thus relevant for DM Type 2 progression.

Metal binding to the amyloid-ß peptides in the presence of biomembranes: potential mechanisms of cell toxicity.

The amyloid-ß (Aß) peptides are key molecules in Alzheimer's disease (AD) pathology. They interact with cellular membranes, and can bind metal ions outside the membrane. Certain oligomeric Aß aggregates are known to induce membrane perturbations and the structure of these oligomers-and their membrane-perturbing effects-can be modulated by metal ion binding. If the bound metal ions are redox active, as e.g., Cu and Fe ions are, they will generate harmful reactive oxygen species (ROS) just outside the membrane surface. Thus, the membrane damage incurred by toxic Aß oligomers is likely aggravated when redox-active metal ions are present. The combined interactions between Aß oligomers, metal ions, and biomembranes may be responsible for at least some of the neuronal death in AD patients.

The Neuronal Tau Protein Blocks in Vitro Fibrillation of the Amyloid-ß (Aß) Peptide at the Oligomeric Stage.

In Alzheimer's disease, amyloid-ß (Aß) plaques and tau neurofibrillary tangles are the two pathological hallmarks. The co-occurrence and combined reciprocal pathological effects of Aß and tau protein aggregation have been observed in animal models of the disease. However, the molecular mechanism of their interaction remain unknown. Using a variety of biophysical measurements, we here show that the native full-length tau protein solubilizes the Aß40 peptide and prevents its fibrillation. The tau protein delays the amyloid fibrillation of the Aß40 peptide at substoichiometric ratios, showing different binding affinities toward the different stages of the aggregated Aß40 peptides. The Aß monomer structure remains random coil in the presence of tau, as observed by nuclear magnetic resonance (NMR), circular dichroism (CD) spectroscopy and photoinduced cross-linking methods. We propose a potential interaction mechanism for the influence of tau on Aß fibrillation.

Shortfall of B3LYP in Reproducing NMR JCH Couplings in Some Isomeric Epoxy Structures with Strong Stereoelectronic Effects: A Benchmark Study on DFT Functionals.

Unprecedented scatter plots of calculated versus measured NMR 2,3 JCH coupling constants in six densely oxygen functionalized epoxides are found with some B3LYP protocols, an effect attributed to stereoelectronic effects. Hence, 26 other exchange-correlation density functionals (xc DFs) are benchmarked in this work. Very good results are achieved with mPW1PW91 and PBE0 in conjunction with the pcJ-1 basis set (BS) of moderate size. A thorough statistical analysis of 53 relationships between the predicted and observed 2,3 JCH datasets is presented. The effects of some xc DFs, including their x and c parts, and BSs on the calculation results are discussed, also in the context of DFT modeling of electron-density distributions. Moreover, related 1 JCH datasets predicted with 11 different DF methods are considered and compared with the experimental data. Finally, some proposals for further improvement of existing DFs based on the available n JCH (n=1-3) values are briefly outlined, in line with recent results on the DFT electron densities.

Ionic Strength Modulation of the Free Energy Landscape of Aß40 Peptide Fibril Formation.

Protein misfolding and formation of cross-ß 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 ß peptide (Aß40) self-association, implicated in Alzheimer's disease. We found that physiological ionic strength accelerates Aß40 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 ß-structured states with similar but distinct spectroscopic features, which can be assigned to an off-pathway immature state (Fß*) and a mature stable state (Fß), where salt favors formation of the Fß fibril morphology. Addition of salt to preformed Fß* accelerates transition to Fß, underlining the dynamic nature of Aß40 fibrils in solution. On the basis of these results we suggest a model where salt decreases the free-energy barrier for Aß40 folding to the Fß state, favoring the buildup of the mature fibril morphology while omitting competing, energetically less favorable structural states.

Heterogeneity and Turnover of Intermediates during Amyloid-ß (Aß) Peptide Aggregation Studied by Fluorescence Correlation Spectroscopy.

Self-assembly of amyloid ß (Aß) peptide molecules into large aggregates is a naturally occurring process driven in aqueous solution by a dynamic interplay between hydrophobic interactions among Aß molecules, which promote aggregation, and steric and overall electrostatic hindrance, which stifles it. Aß self-association is entropically unfavorable, as it implies order increase in the system, but under favorable kinetic conditions, the process proceeds at appreciable rates, yielding Aß aggregates of different sizes and structures. Despite the great relevance and extensive research efforts, detailed kinetic mechanisms underlying Aß aggregation remain only partially understood. In this study, fluorescence correlation spectroscopy (FCS) and Thioflavin T (ThT) were used to monitor the time dependent growth of structured aggregates and characterize multiple components during the aggregation of Aß peptides in a heterogeneous aqueous solution. To this aim, we collected data during a relatively large number of observation periods, 30 consecutive measurements lasting 10 s each, at what we consider to be a constant time point in the slow aggregation process. This approach enabled monitoring the formation of nanomolar concentrations of structured amyloid aggregates and demonstrated the changing distribution of amyloid aggregate sizes throughout the aggregation process. We identified aggregates of different sizes with molecular weight from 260 to more than 1 × 10(6) kDa and revealed the hitherto unobserved kinetic turnover of intermediates during Aß aggregation. The effect of different Aß concentrations, Aß:ThT ratios, differences between the 40 (Aß40) and 42 (Aß42) residue long variants of Aß, and the effect of stirring were also examined.

The role of pro-inflammatory S100A9 in Alzheimer's disease amyloid-neuroinflammatory cascade.

Pro-inflammatory S100A9 protein is increasingly recognized as an important contributor to inflammation-related neurodegeneration. Here, we provide insights into S100A9 specific mechanisms of action in Alzheimer's disease (AD). Due to its inherent amyloidogenicity S100A9 contributes to amyloid plaque formation together with Aß. In traumatic brain injury (TBI) S100A9 itself rapidly forms amyloid plaques, which were reactive with oligomer-specific antibodies, but not with Aß and amyloid fibrillar antibodies. They may serve as precursor-plaques for AD, implicating TBI as an AD risk factor. S100A9 was observed in some hippocampal and cortical neurons in TBI, AD and non-demented aging. In vitro S100A9 forms neurotoxic linear and annular amyloids resembling Aß protofilaments. S100A9 amyloid cytotoxicity and native S100A9 pro-inflammatory signaling can be mitigated by its co-aggregation with Aß, which results in a variety of micron-scale amyloid complexes. NMR and molecular docking demonstrated transient interactions between native S100A9 and Aß. Thus, abundantly present in AD brain pro-inflammatory S100A9, possessing also intrinsic amyloidogenic properties and ability to modulate Aß aggregation, can serve as a link between the AD amyloid and neuroinflammatory cascades and as a prospective therapeutic target.

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

The amyloid ß (Aß) 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 Aß peptides at different stages along the aggregation pathways. We conclude that a hairpin-like conformation constitutes a common motif for the Aß 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 Aß 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.

An NMR and MD modeling insight into nucleation of 1,2-alkanediols: selective crystallization of lipase-catalytically resolved enantiomers from the reaction mixtures.

The work on developing a scalable lipase-catalytic method for the kinetic resolution of long-chain 1,2-alkanediols, complemented by crystallization of the pure enantiomers from the reaction mixtures, offered the possibility of a more detailed study of the aggregation of such diols. MD modeling, mass spectrometry, (1)H NMR, and DOSY studies provided a novel insight into the nucleation process. An efficient protocol for stereo- and chemoselective crystallization of (S)-1,2-dodecanediol and related compounds from the crude bioconversion mixtures was developed.

13C- and 15N-labeling of amyloid-ß and inhibitory peptides to study their interaction via nanoscale infrared spectroscopy.

Interactions between molecules are fundamental in biology. They occur also between amyloidogenic peptides or proteins that are associated with different amyloid diseases, which makes it important to study the mutual influence of two polypeptides on each other's properties in mixed samples. However, addressing this research question with imaging techniques faces the challenge to distinguish different polypeptides without adding artificial probes for detection. Here, we show that nanoscale infrared spectroscopy in combination with 13C, 15N-labeling solves this problem. We studied aggregated amyloid-ß peptide (Aß) and its interaction with an inhibitory peptide (NCAM1-PrP) using scattering-type scanning near-field optical microscopy. Although having similar secondary structure, labeled and unlabeled peptides could be distinguished by comparing optical phase images taken at wavenumbers characteristic for either the labeled or the unlabeled peptide. NCAM1-PrP seems to be able to associate with or to dissolve existing Aß fibrils because pure Aß fibrils were not detected after mixing.

Erratum: The amyloid-inhibiting NCAM-PrP peptide targets Aß peptide aggregation in membrane-mimetic environments.

[This corrects the article DOI: 10.1016/j.isci.2021.102852.].

Characterization of Uranyl (UO22+) Ion Binding to Amyloid Beta (Aß) Peptides: Effects on Aß Structure and Aggregation.

Uranium (U) is naturally present in ambient air, water, and soil, and depleted uranium (DU) is released into the environment via industrial and military activities. While the radiological damage from U is rather well understood, less is known about the chemical damage mechanisms, which dominate in DU. Heavy metal exposure is associated with numerous health conditions, including Alzheimer's disease (AD), the most prevalent age-related cause of dementia. The pathological hallmark of AD is the deposition of amyloid plaques, consisting mainly of amyloid-ß (Aß) peptides aggregated into amyloid fibrils in the brain. However, the toxic species in AD are likely oligomeric Aß aggregates. Exposure to heavy metals such as Cd, Hg, Mn, and Pb is known to increase Aß production, and these metals bind to Aß peptides and modulate their aggregation. The possible effects of U in AD pathology have been sparsely studied. Here, we use biophysical techniques to study in vitro interactions between Aß peptides and uranyl ions, UO22+, of DU. We show for the first time that uranyl ions bind to Aß peptides with affinities in the micromolar range, induce structural changes in Aß monomers and oligomers, and inhibit Aß fibrillization. This suggests a possible link between AD and U exposure, which could be further explored by cell, animal, and epidemiological studies. General toxic mechanisms of uranyl ions could be modulation of protein folding, misfolding, and aggregation.

Residue-specific binding of Ni(II) ions influences the structure and aggregation of amyloid beta (Aß) peptides.

Alzheimer's disease (AD) is the most common cause of dementia worldwide. AD brains display deposits of insoluble amyloid plaques consisting mainly of aggregated amyloid-ß (Aß) peptides, and Aß oligomers are likely a toxic species in AD pathology. AD patients display altered metal homeostasis, and AD plaques show elevated concentrations of metals such as Cu, Fe, and Zn. Yet, the metal chemistry in AD pathology remains unclear. Ni(II) ions are known to interact with Aß peptides, but the nature and effects of such interactions are unknown. Here, we use numerous biophysical methods-mainly spectroscopy and imaging techniques-to characterize Aß/Ni(II) interactions in vitro, for different Aß variants: Aß(1-40), Aß(1-40)(H6A, H13A, H14A), Aß(4-40), and Aß(1-42). We show for the first time that Ni(II) ions display specific binding to the N-terminal segment of full-length Aß monomers. Equimolar amounts of Ni(II) ions retard Aß aggregation and direct it towards non-structured aggregates. The His6, His13, and His14 residues are implicated as binding ligands, and the Ni(II)·Aß binding affinity is in the low µM range. The redox-active Ni(II) ions induce formation of dityrosine cross-links via redox chemistry, thereby creating covalent Aß dimers. In aqueous buffer Ni(II) ions promote formation of beta sheet structure in Aß monomers, while in a membrane-mimicking environment (SDS micelles) coil-coil helix interactions appear to be induced. For SDS-stabilized Aß oligomers, Ni(II) ions direct the oligomers towards larger sizes and more diverse (heterogeneous) populations. All of these structural rearrangements may be relevant for the Aß aggregation processes that are involved in AD brain pathology.

Choosing an Optimal Solvent Is Crucial for Obtaining Cell-Penetrating Peptide Nanoparticles with Desired Properties and High Activity in Nucleic Acid Delivery.

Cell-penetrating peptides (CPPs) are highly promising transfection agents that can deliver various compounds into living cells, including nucleic acids (NAs). Positively charged CPPs can form non-covalent complexes with negatively charged NAs, enabling simple and time-efficient nanoparticle preparation. However, as CPPs have substantially different chemical and physical properties, their complexation with the cargo and characteristics of the resulting nanoparticles largely depends on the properties of the surrounding environment, i.e., solution. Here, we show that the solvent used for the initial dissolving of a CPP determines the properties of the resulting CPP particles formed in an aqueous solution, including the activity and toxicity of the CPP-NA complexes. Using different biophysical methods such as dynamic light scattering (DLS), atomic force microscopy (AFM), transmission and scanning electron microscopy (TEM and SEM), we show that PepFect14 (PF14), a cationic amphipathic CPP, forms spherical particles of uniform size when dissolved in organic solvents, such as ethanol and DMSO. Water-dissolved PF14, however, tends to form micelles and non-uniform aggregates. When dissolved in organic solvents, PF14 retains its α-helical conformation and biological activity in cell culture conditions without any increase in cytotoxicity. Altogether, our results indicate that by using a solvent that matches the chemical nature of the CPP, the properties of the peptide-cargo particles can be tuned in the desired way. This can be of critical importance for in vivo applications, where CPP particles that are too large, non-uniform, or prone to aggregation may induce severe consequences.

Specific binding of a ß-cyclodextrin dimer to the amyloid ß peptide modulates the peptide aggregation process.

Alzheimer's disease involves progressive neuronal loss. Linked to the disease is the amyloid ß (Aß) peptide, a 38-43-amino acid peptide found in extracellular amyloid plaques in the brain. Cyclodextrins are nontoxic, cone-shaped oligosaccharides with a hydrophilic exterior and a hydrophobic cavity making them suitable hosts for aromatic guest molecules in water. ß-Cyclodextrin consists of seven α-d-glucopyranoside units and has been shown to reduce the level of fibrillation and neurotoxicity of Aß. We have studied the interaction between Aß and a ß-cyclodextrin dimer, consisting of two ß-cyclodextrin monomers connected by a flexible linker. The ß-cyclodextrin monomer has been found to interact with Aß(1-40) at sites Y10, F19, and/or F20 with a dissociation constant (K(D)) of 3.9 ± 2.0 mM. Here (1)H-(15)N and (1)H-(13)C heteronuclear single-quantum correlation nuclear magnetic resonance (NMR) spectra show that in addition, the ß-cyclodextrin monomer and dimer bind to the histidines. NMR translational diffusion experiments reveal the increased affinity of the ß-cyclodextrin dimer (apparent K(D) of 1.1 ± 0.5 mM) for Aß(1-40) compared to that of the ß-cyclodextrin monomer. Kinetic aggregation experiments based on thioflavin T fluorescence indicate that the dimer at 0.05-5 mM decreases the lag time of Aß aggregation, while a concentration of 10 mM increases the lag time. The ß-cyclodextrin monomer at a high concentration decreases the lag time of the aggregation. We conclude that cyclodextrin monomers and dimers have specific, modulating effects on the Aß(1-40) aggregation process. Transmission electron microscopy shows that the regular fibrillar aggregates formed by Aß(1-40) alone are replaced by a major fraction of amorphous aggregates in the presence of the ß-cyclodextrin dimer.

Mercury Ion Binding to Apolipoprotein E Variants ApoE2, ApoE3, and ApoE4: Similar Binding Affinities but Different Structure Induction Effects.

Mercury intoxication typically produces more severe outcomes in people with the APOE-ε4 gene, which codes for the ApoE4 variant of apolipoprotein E, compared to individuals with the APOE-ε2 and APOE-ε3 genes. Why the APOE-ε4 allele is a risk factor in mercury exposure remains unknown. One proposed possibility is that the ApoE protein could be involved in clearing of heavy metals, where the ApoE4 protein might perform this task worse than the ApoE2 and ApoE3 variants. Here, we used fluorescence and circular dichroism spectroscopies to characterize the in vitro interactions of the three different ApoE variants with Hg(I) and Hg(II) ions. Hg(I) ions displayed weak binding to all ApoE variants and induced virtually no structural changes. Thus, Hg(I) ions appear to have no biologically relevant interactions with the ApoE protein. Hg(II) ions displayed stronger and very similar binding affinities for all three ApoE isoforms, with K D values of 4.6 µM for ApoE2, 4.9 µM for ApoE3, and 4.3 µM for ApoE4. Binding of Hg(II) ions also induced changes in ApoE superhelicity, that is, altered coil-coil interactions, which might modify the protein function. As these structural changes were most pronounced in the ApoE4 protein, they could be related to the APOE-ε4 gene being a risk factor in mercury toxicity.

The amyloid-inhibiting NCAM-PrP peptide targets Aß peptide aggregation in membrane-mimetic environments.

Substantial research efforts have gone into elucidating the role of protein misfolding and self-assembly in the onset and progression of Alzheimer's disease (AD). Aggregation of the Amyloid-ß (Aß) peptide into insoluble fibrils is closely associated with AD. Here, we use biophysical techniques to study a peptide-based approach to target Aß amyloid aggregation. A peptide construct, NCAM-PrP, consists of a largely hydrophobic signal sequence linked to a positively charged hexapeptide. The NCAM-PrP peptide inhibits Aß amyloid formation by forming aggregates which are unavailable for further amyloid aggregation. In a membrane-mimetic environment, Aß and NCAM-PrP form specific heterooligomeric complexes, which are of lower aggregation states compared to Aß homooligomers. The Aß:NCAM-PrP interaction appears to take place on different aggregation states depending on the absence or presence of a membrane-mimicking environment. These insights can be useful for the development of potential future therapeutic strategies targeting Aß at several aggregation states.