Aβ Aggregation Process Research Articles

The amyloid‐β peptide in Alzheimer's disease: molecular interactions and structure conversions

The amyloid β (Aβ) peptide consists of 39–43 residues and is the major component of the neuritic plaques in the brains of Alzheimer's disease (AD) patients. It is possible to study the peptide self‐aggregation process (“amyloid formation”, structures and kinetics) using biophysical methods such as NMR, fluorescence or CD spectroscopy. The interactions of Aβ(1–40) with small molecules, or metal ions such as Zn(II) or Cu(II), that modulate the aggregation process can be studied in semi‐stationary states by these methods. Kinetic effects on the amyloid formation process can be followed by fluorescence, after labeling the material rich in β‐structure by the amyloid‐specific dye Thioflavin T. The β‐hairpin structure of the peptide with a mainly unstructured N‐terminus appears to be a basic unit for formation of the larger amyloid structures associated with insoluble fibrils. Smaller oligomeric peptide aggregates, possibly the major cell toxic agents, can be studied by Soft Ionization Mass Spectrometry.Fluorescence Correlation Spectroscopy (FCS) shows the heterogeneity of the aggregation process of ThT‐labeled Aβ, in terms of time dependent amyloid aggregate sizes, on a single particle level. Modulation of the amyloid formation by Zn(II) or Cu(II) ions binding to specific residues in the N‐terminus of the Aβ peptide can be followed by the biophysical methods, showing changes in structures as well as kinetics for the aggregation process.Understanding the basic properties and reactions of the major participants in the chemical processes leading to AD gives a firm basis for the future development of agents that may be used in therapies against AD.Support or Funding InformationThe Swedish Research Council, The Alzheimer Foundation, The Magn. Bergvall FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Trace amounts of pyroglutaminated Aβ-(3–42) enhance aggregation of Aβ-(1–42) on neuronal membranes at physiological concentrations: FCS analysis of cell surface

Minor species of amyloid β-peptide (Aβ), such as Aβ-(1–43) and pyroglutaminated Aβ-(3–42) (Aβ-(3pE–42)), have been suggested to be involved in the initiation of the Aβ aggregation process, which is closely associated with the etiology of Alzheimer's disease. They can play important roles in aggregation not only in the aqueous phase but also on neuroral membranes; however, the latter behaviors remain mostly unexplored. Here, initial aggregation processes of Aβ on living cells were monitored at physiological nanomolar concentrations by fluorescence correlation spectroscopy. Membrane-bound Aβ-(1–42) and Aβ-(1–40) formed oligomers composed of ~4 Aβ molecules during 48-h incubation, whereas the peptides remained monomeric in the culture medium, indicating that the membranes facilitated Aβ aggregation. The presence of 5 mol% Aβ-(3pE–42), but not Aβ-(1–43), significantly enhanced the aggregation of Aβ-(1–42) up to ~10-mers. On the other hand, neither trace amounts of Aβ-(1–42) nor Aβ-(3pE–42) enhanced the aggregation of Aβ-(1–40). The observed small Aβ oligomers are expected to act as pathogenic seeds for amyloid fibrils responsible for neurotoxicity. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.

Production and Use of Recombinant Aβ for Aggregation Studies.

The amyloid β-protein (Aβ) is believed to play a central role in Alzheimer's disease (AD) pathogenesis and there is great interest in understanding the process of Aβ aggregation, its underlying mechanism and the species generated during aggregation and their biological activity. Although Aβ has been studied for more than 30years, analysis of its aggregation has been hampered by structural and chemical impurities. Here we provide a detailed protocol for the expression and purification of chemically and structurally homogeneous Aβ monomer. We also describe a method to produce covalent Aβ dimers linked by phenolic coupling of tyrosine residues.

Alzheimer\u2019s disease and cigarette smoke components: effects of nicotine, PAHs, and Cd(II), Cr(III), Pb(II), Pb(IV) ions on amyloid-\u03b2 peptide aggregation

Cigarette smoking is a significant risk factor for Alzheimer’s disease (AD), which is associated with extracellular brain deposits of amyloid plaques containing aggregated amyloid-β (Aβ) peptides. Aβ aggregation occurs via multiple pathways that can be influenced by various compounds. Here, we used AFM imaging and NMR, fluorescence, and mass spectrometry to monitor in vitro how Aβ aggregation is affected by the cigarette-related compounds nicotine, polycyclic aromatic hydrocarbons (PAHs) with one to five aromatic rings, and the metal ions Cd(II), Cr(III), Pb(II), and Pb(IV). All PAHs and metal ions modulated the Aβ aggregation process. Cd(II), Cr(III), and Pb(II) ions displayed general electrostatic interactions with Aβ, whereas Pb(IV) ions showed specific transient binding coordination to the N-terminal Aβ segment. Thus, Pb(IV) ions are especially prone to interact with Aβ and affect its aggregation. While Pb(IV) ions affected mainly Aβ dimer and trimer formation, hydrophobic toluene mainly affected formation of larger aggregates such as tetramers. The uncharged and hydrophilic nicotine molecule showed no direct interactions with Aβ, nor did it affect Aβ aggregation. Our Aβ interaction results suggest a molecular rationale for the higher AD prevalence among smokers, and indicate that certain forms of lead in particular may constitute an environmental risk factor for AD.

SEN1500, a novel oral amyloid-β aggregation inhibitor, attenuates brain pathology in a mouse model of Alzheimer's disease.

Amyloid-β (Aβ) aggregation is thought to be a major pathogenic event underlying the neuropathology of Alzheimer's disease (AD). The development of new drugs inhibiting the Aβ aggregation process is, therefore, important. SEN1500, an orally bioavailable and CNS-penetrant Aβ aggregation inhibitor, has previously been shown to reduce spatial learning and memory deficits in an APP transgenic mouse model. To verify that the pharmacological properties of SEN1500 are not unique to this model, we investigated brain Aβ pathology, neuroinflammation, as well as memory in a different mouse model of AD expressing the human amyloid precursor protein with Swedish and London mutations (APPSL). APPSL transgenic mice and non-transgenic littermates were treated with SEN1500 via food pellets from three months of age for four months. At the end of the treatment, animals were tested for memory deficits using the contextual fear conditioning test and brain tissue was analyzed for soluble and insoluble amyloid-β1-38, -40, -42, β-amyloid plaques, β-sheet plaque cores, as well as for astrocytosis and activated microglia. SEN1500 treatment lowered insoluble Aβ levels and β-amyloid plaque load in the brain compared with control-treated APPSL mice. Activated microglia were significantly reduced in the cortex but not the hippocampus of SEN1500-treated APPSL mice. Memory deficits of APPSL mice could not be rescued by SEN1500. SEN1500 is not only able to reduce Aβ pathology and activated microglia but also to improve learning and memory as previously shown, making SEN1500 a potential candidate for human AD treatment. This Aβ aggregation inhibitor could be a promising therapeutic agent for the disease-modifying treatment of AD.

Molybdenum Disulfide Nanoparticles as Multifunctional Inhibitors against Alzheimer's Disease.

The complex pathogenic mechanisms of Alzheimer's disease (AD) include the aggregation of β-amyloid peptides (Aβ) into oligomers or fibrils as well as Aβ-mediated oxidative stress, which require comprehensive treatment. Therefore, the inhibition of Aβ aggregation and free-radical scavenging are essential for the treatment of AD. Nanoparticles (NPs) have been found to influence Aβ aggregation process in vitro. Herein, we report the inhibition effects of molybdenum disulfide (MoS2) NPs on Aβ aggregation. Polyvinylpyrrolidone-functionalized MoS2 NPs were fabricated by a pulsed laser ablation method. We find that MoS2 NPs exhibit multifunctional effects on Aβ peptides: inhibiting Aβ aggregation, destabilizing Aβ fibrils, alleviating Aβ-induced oxidative stress, as well as Aβ-mediated cell toxicity. Moreover, we show that MoS2 NPs can block the formation of the Ca2+ channel induced by Aβ fibrils in the cell membrane for the first time. Thus, these observations suggest that MoS2 NPs have great potential for a multifunctional therapeutic agent against amyloid-related diseases.

Possible Molecular Interactions of Bexarotene - A Retinoid Drug and Alzheimer's Aβ Peptide: A Docking Study.

Alzheimer's disease (AD) pathogenesis is primarily hallmarked by the production and accumulation of amyloid beta (Aβ) peptide. Along with the understanding of the neurodegenerative disease progression and its pathophysiological mechanisms, development of anti-Aβ targeted effective therapeutics is essential for AD management. Numerous therapeutic approaches targeting the production, toxicity and removal of Aβ are being attempted worldwide. Prime need is to design inhibitors which can slow down the Aβ aggregation process in a physiological environment. Bexarotene (targretin) is the first of the U.S. Food and Drug Administration approved oral retinoid X receptors (RXR)-selective retinoids, or rexinoids. It is effectively used for the treatment of advanced, refractory cutaneous T cell lymphoma, and also reportedly reduces Aβ levels in AD mouse models. Administration of bexarotene facilitates intracellular Aβ clearance via RXR regulated apolipoprotein E (ApoE) production. To the best of our knowledge, this is the first structural attempt to find binding interactions of the drug bexarotene with monomeric Aβ peptide. We checked binding possibilities of bexarotene by using structural bioinformatics method. We found in our study the basic amino acids His13 and Lys 16 of Aβ peptide to be crucial for the interaction with bexarotene. We speculate that direct binding of bexarotene to free Aβ peptide may lessen the concentration of free Aβ peptides in the brain and hamper the propensity of the peptide's clumping and aggregating behavior. Further experimental validation of the results of this study would be required for its therapeutic success.

Biophysical Studies of the Amyloid Beta Peptide Involved in Alzheimer's Disease

The amyloid β (Aβ) peptide consists of 39-43 residues and is the major component of the neuritic plaques in the brains of Alzheimer's disease (AD) patients. It is possible to study the peptide self-aggregation process (“amyloid formation”, structures as well as kinetics) using biophysical methods such as NMR, fluorescence or CD spectroscopy. The interactions of Aβ with small molecules or metal ions that modulate the aggregation process can be studied in semi-stationary states. Kinetic effects on the aggregation process can be followed by fluorescence, after labeling the amyloid material rich in β-structure by the amyloid-specific dye Thioflavin T (ThT). The β-hairpin structure of the peptide with a mainly unstructured N-terminus appears to be a basic unit for formation of the higher order amyloid structures associated with insoluble fibrils. Fluorescence Correlation Spectroscopy (FCS) shows the heterogeneity of the aggregation process of ThT-labeled Aβ, in terms of time dependent amyloid aggregate sizes on a single particle level. Modulation of the amyloid formation by Zn(II) or Cu(II) ions binding to specific residues in the N-terminus of the Aβ peptide can be followed by FCS, showing changes in structures as well as kinetics for the aggregation process. Understanding the basic properties and reactions of the major participants in the chemical processes leading to AD gives a firm basis for the future development of agents that may be used in therapies against AD.

Biofunctionalized Silica Nanoparticles: Standards in Amyloid-β Oligomer-Based Diagnosis of Alzheimer's Disease.

Amyloid-β (Aβ) oligomers represent a promising biomarker for the early diagnosis of Alzheimer's disease (AD). However, state-of-the-art methods for immunodetection of Aβ oligomers in body fluids show a large variability and lack a reliable and stable standard that enables the reproducible quantitation of Aβ oligomers. At present, the only available standard applied in these assays is based on a random aggregation process of synthetic Aβ and has neither a defined size nor a known number of epitopes. In this report, we generated a highly stable standard in the size range of native Aβ oligomers that exposes a defined number of epitopes. The standard consists of a silica nanoparticle (SiNaP), which is functionalized with Aβ peptides on its surface (Aβ-SiNaP). The different steps of Aβ-SiNaP synthesis were followed by microscopic, spectroscopic and biochemical analyses. To investigate the performance of Aβ-SiNaPs as an appropriate standard in Aβ oligomer immunodetection, Aβ-SiNaPs were diluted in cerebrospinal fluid and quantified down to a concentration of 10 fM in the sFIDA (surface-based fluorescence intensity distribution analysis) assay. This detection limit corresponds to an Aβ concentration of 1.9 ng l-1 and lies in the sensitivity range of currently applied diagnostic tools based on Aβ oligomer quantitation. Thus, we developed a highly stable and well-characterized standard for the application in Aβ oligomer immunodetection assays that finally allows the reproducible quantitation of Aβ oligomers down to single molecule level and provides a fundamental improvement for the worldwide standardization process of diagnostic methods in AD research.

Metal Ions and Metal Complexes in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia that seriously affects daily life. Even if AD pathogenesis is still subject of debate, it is generally accepted that cerebral cortex plaques formed by aggregated amyloid-β (Aβ) peptides can be considered a characteristic pathological hallmark. It is well known that metal ions play an important role in the aggregation process of Aβ. This review focuses on the anti-Aβ aggregation activity of chelating ligands as well as on the use of metal complexes as diagnostic probes and as potential drugs. While chelating agents, such as curcumin or flavonoid derivatives, are currently used to capture metal ions responsible for Aβ aggregation, the potential application of platinum, ruthenium and cobalt complexes, among others, of several heterocyclic ligands, represents a promising new strategy to fight AD.

Ac-LPFFD-Th: A Trehalose-Conjugated Peptidomimetic as a Strong Suppressor of Amyloid-β Oligomer Formation and Cytotoxicity.

The inhibition of amyloid formation is a promising therapeutic approach for the treatment of neurodegenerative diseases. Peptide-based inhibitors, which have been widely investigated, are generally derived from original amyloid sequences. Most interestingly, trehalose, a nonreducing disaccharide of α-glucose, is effective in preventing the aggregation of numerous proteins. We have determined that the development of hybrid compounds could provide new molecules with improved properties that might synergically increase the potency of their single moieties. In this work, the ability of Ac-LPFFD-Th, a C-terminally trehalose-conjugated derivative, to slow down the Aβ aggregation process was investigated by means of different biophysical techniques, including thioflavin T fluorescence, dynamic light scattering, ESI-MS, and NMR spectroscopy. Moreover, we demonstrate that Ac-LPFFD-Th modifies the aggregation features of Aβ and protects neurons from Aβ oligomers' toxic insult.

The Structure-Activity Relationship of Glycosaminoglycans and Their Analogues with β-Amyloid Peptide.

Alzheimer's disease (AD) is a serious neurodegenerative disorder. β-amyloid peptide (Aβ) aggregation is believed to be the major cause of the disease. The process of Aβ aggregation can be enhanced by sulfated glycosaminoglycans. However, cell experiments have shown that sulfated glycosaminoglycan oligosaccharides or analogues may have significant neuroprotective properties and could inhibit the aggregation by competitive inhibition. The length and species of oligosaccharides or analogues can inhibit the toxicity of Aβ by inducing conformational changes of proteins in different manners. This review presents the conformational changes of Aβ in the presence of glycosaminoglycan, glycosaminoglycan oligosaccharides and analogues. The review might be helpful to comprehend the mechanism of β-amyloid fibrillations and the aggregation process.

A new structural model of Alzheimer's Aβ42 fibrils based on electron paramagnetic resonance data and Rosetta modeling.

Brain deposition of Aβ in the form of amyloid plaques is a pathological hallmark of Alzheimer's disease. There are two major species of Aβ in the brain: Aβ42 and Aβ40. Although Aβ40 is several-fold more abundant than Aβ42 in soluble form, Aβ42 is the major component of amyloid plaques. Structural knowledge of Aβ42 fibrils is important both for understanding the process of Aβ aggregation and for designing fibril-targeting drugs. Here we report site-specific structural information of Aβ42 fibrils at 22 residue positions based on electron paramagnetic resonance data. In combination with structure prediction program Rosetta, we modeled Aβ42 fibril structure at atomic resolution. Our Aβ42 fibril model consists of four parallel in-register β-sheets: βN (residues ∼7-13), β1 (residues ∼17-20), β2 (residues ∼32-36), and βC (residues 39-41). The region of β1-loop-β2 in Aβ42 fibrils adopts similar structure as that in Aβ40 fibrils. This is consistent with our cross seeding data that Aβ42 fibril seeds shortened the lag phase of Aβ40 fibrillization. On the other hand, Aβ42 fibrils contain a C-terminal β-arc-β motif with a special turn, termed "arc", at residues 37-38, which is absent in Aβ40 fibrils. Our results can explain both the higher aggregation propensity of Aβ42 and the importance of Aβ42 to Aβ40 ratio in the pathogenesis of Alzheimer's disease.

Selective amyloid β oligomer assay based on abasic site-containing molecular beacon and enzyme-free amplification

Amyloid-beta (Aβ) oligomers are highly toxic species in the process of Aβ aggregation and are regarded as potent therapeutic targets and diagnostic markers for Alzheimer's disease (AD). Herein, a label-free molecular beacon (MB) system integrated with enzyme-free amplification strategy was developed for simple and highly selective assay of Aβ oligomers. The MB system was constructed with abasic site (AP site)-containing stem-loop DNA and a fluorescent ligand 2-amino-5,6,7-trimethyl-1,8-naphyridine (ATMND), of which the fluorescence was quenched upon binding to the AP site in DNA stem. Enzyme-free amplification was realized by target-triggered continuous opening of two delicately designed MBs (MB1 and MB2). Target DNA hybridization with MB1 and then MB2 resulted in the release of two ATMND molecules in one binding event. Subsequent target recycling could greatly amplify the detection sensitivity due to the greatly enhanced turn-on emission of ATMND fluorescence. Combining with Aβ oligomers aptamers, the strategy was applied to analyze Aβ oligomers and the results showed that it could quantify Aβ oligomers with high selectivity and monitor the Aβ aggregation process. This novel method may be conducive to improve the diagnosis and pathogenic study of Alzheimer's disease.

(R)‐α‐trifluoromethylalanine containing short peptide in the inhibition of amyloid peptide fibrillation

The extracellular deposition of insoluble amyloid fibrils resulting from the aggregation of the amyloid-β (Aβ) is a pathological feature of neuronal loss in Alzheimer's disease (AD). Numerous small molecules have been reported to interfere with the process of Aβ aggregation. Compounds containing aromatic structures, hydrophobic amino acids and/or the α-aminoisobutyric acid (Aib) as β-sheet breaker elements have been reported to be effective inhibitors of Aβ aggregation. We synthesized two peptides, one containing the Aib amino acid and the other including its trifluoromethylated analog (R)-α-Trifluoromethylalanine ((R)-Tfm-Alanine) and we evaluated the impact of these peptides on Aβ amyloid formation. The compounds were tested by standard methods such as thioflavin-T fluorescence spectroscopy and transmission electron microscopy but also by circular dichroism, liquid state nuclear magnetic resonance (NMR) and NMR saturation transfer difference (STD) experiments to further characterize the effect of the two molecules on Aβ structure and on the kinetics of depletion of monomeric, soluble Aβ. Our results demonstrate that the peptide containing Aib reduces the quantity of aggregates containing β-sheet structure but slightly inhibits Aβ fibril formation, while the molecule including the trifluoromethyl (Tfm) group slows down the kinetics of Aβ fibril formation, delays the random coil to β-sheet structure transition and induces a change in the oligomerization pathway. These results suggest that the hydrophobic Tfm group has a better affinity with Aβ than the methyl groups of the Aib and that this Tfm group is effective and important in preventing the Aβ aggregation.

Polymorphic Associations and Structures of the Cross-Seeding of Aβ1-42 and hIAPP1-37 Polypeptides.

Emerging evidence have shown that the patients with Alzheimer's disease (AD) often have a higher risk of later developing type II diabetes (T2D), and vice versa, suggesting a potential pathological link between AD and T2D. Amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) are the principle causative components responsible for the pathologies of AD and T2D, respectively. The cross-sequence interactions between Aβ and hIAPP may provide a molecular basis for better understanding the potential link between AD and T2D. Herein, we systematically modeled and simulated the cross-sequence aggregation process, molecular interactions, and polymorphic structures of full-length Aβ and hIAPP peptides using a combination of coarse-grained (CG) replica-exchange molecular dynamics (REMD) and all-atom molecular dynamics (MD) simulations, with particular focus on the effect of association models between Aβ and hIAPP on the structural stability and polymorphic populations of hybrid Aβ-hIAPP aggregates. Four distinct association models (double-layer, elongation, tail-tail, and block models) between Aβ and hIAPP oligomers were identified, and the associated polymorphic Aβ-hIAPP structures were determined as well. Among them, different association models led to different Aβ-hIAPP aggregates, with large differences in structural morphologies and populations, interacting interfaces, and underlying association forces. The computational models support the cross-sequence interactions between Aβ and hIAPP pentamers, which would lead to the complex hybrid Aβ-hIAPP assemblies. This computational work may also provide a different point of view to a better understanding of a potential link between AD and T2D.

Neuroprotective effects of the monoamine oxidase inhibitor tranylcypromine and its amide derivatives against Aβ(1–42)-induced toxicity

Monoamine oxidase (MAO) enzymes play a central role in the pathogenesis of Alzheimer's disease (AD) and MAO inhibitors (MAOIs) are antidepressant drugs currently studied for their neuroprotective properties in neurodegenerative disorders. In the present work MAOIs such as tranylcypromine [trans-(+)-2-phenylcyclopropanamine, TCP] and its amide derivatives, TCP butyramide (TCP-But) and TCP acetamide (TCP-Ac), were tested for their ability to protect cortical neurons challenged with synthetic amyloid-β (Aβ)-(1–42) oligomers (100nM) for 48h. TCP significantly prevented Aβ-induced neuronal death in a concentration-dependent fashion and was maximally protective only at 10µM. TCP-But was maximally protective in mixed neuronal cultures at 1µM, a lower concentration compared to TCP, whereas the new derivative, TCP-Ac, was more efficacious than TCP and TCP-But and significantly protected cortical neurons against Aβ toxicity at nanomolar concentrations (100nM).Experiments carried out with the Thioflavin-T (Th-T) fluorescence assay for fibril formation showed that TCP and its amide derivatives influenced the early events of the Aβ aggregation process in a concentration-dependent manner. TCP-Ac was more effective than TCP-But and TCP in slowing down the Aβ(1–42) aggregates formation through a lengthening at the lag phase. In our experimental model co-incubation of Aβ(1–42) oligomers with TCP-Ac was able to almost completely prevent Aβ-induced neurodegeneration. These results suggest that inhibition of Aβ oligomer-mediated aggregation significantly contributes to the overall neuroprotective activity of TCP-Ac and also raise the possibility that TCP, and in particular the new compound TCP-Ac, might represent new pharmacological tools to yield neuroprotection in AD.

Thioflavin T templates amyloid β(1–40) conformation and aggregation pathway

Aβ(1–40) peptide supramolecular assembly and fibril formation processes are widely recognized to have direct implications in the progression of Alzheimer's disease. The molecular basis of this biological process is still unknown and there is a strong need of developing effective strategies to control the occurring events. To this purpose the exploitation of small molecules interacting with Aβ aggregation represents one of the possible routes. Moreover, the use specific labeling has represented so far one of the most common and effective methods to investigate such a process. This possibility in turn rests on the reliability of the probe/labels involved.Here we present evidences of the effect of Thioflavin T (ThT), a worldwide used fluorescent dye to monitor amyloid growth, on the Aβ(1–40) conformation, stability and aggregation. By combining experimental information and Molecular Dynamics simulation results, we show that the presence of ThT in solution affects peptide conformation inducing peculiar supramolecular association. In particular ThT interactions with specific Aβ(1–40) residues promote a rigid partially-folded conformation which shifts the balance between different species in solution toward a more aggregation-prone ensemble of peptides, leading to aggregation. Our findings suggest ways for developing strategies to reverse and block aggregation or to stimulate supramolecular assembly and consequently reduce the presence of transient oligomers. This investigation underlines the need of developing label-free techniques for unbiased quantitative studies of Aβ(1–40) aggregation processes.

Oxidation of Methionine-35 in Alzheimer’s amyloid-beta peptide and the aggregation of the oxidized peptide

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive loss of brain tissue and accumulation of amyloid-β(Aβ) and tau. Aggregation of Aβ peptides into amyloid plaques is considered to be a causative factor in AD, however the precise mechanism behind the AD onset has remained elusive. Oxidative stress (OS) is also characteristic to AD, but it is not known whether OS is a risk factor or a consequence of AD. It is assumed that Aβ aggregates generate free radicals in the presence of copper ions by participation of the Met35 residue, which can increase the OS levels. The aim of our study was to establish the role of Met35 residue in the oxidation of Aβ and peptide aggregation processes. Oxidation of Aβ was studied in the presence of two redox-active compounds: H2O2 and copper ions. In the absence of copper ions the Met35 residue was readily oxidized by H2O2 in a two electron process. The fibrillization of Aβ with Met35 oxidized to sulfoxide was threefold slower compared to that of the native peptide. TEM analysis showed that the fibrils of native and oxidized peptides are similar. The relatively small inhibitory effect of Met35 oxidation on the fibrillization suggests that the possible variation in the Met oxidation state should not affect the in vivo plaque formation. In the presence of copper ions (one-electron process) the oxidation was more complex: addition of the first oxygen was still the fastest process, however, it was accompanied by multiple unspecific modifications of several amino acid residues. Addition of copper ions to the already oxidized Aβ Met35 by H2O2, resulted in a similar pattern of nonspecific modifications, suggesting that the one-electron oxidation processes in Aβ do not depend on the oxidation state of Met35. Thus, it can be concluded that Met35 residue is not a part of the radical generating mechanism of Aβ-Cu(II) complex.

Transient Binding of Zn(II) Redirects Amyloid Beta Peptide from Fibril Formation

Metal ions have emerged to play a key role in the aggregation process of amyloid β (Aβ) peptide which is closely related to the pathogenesis of Alzheimer's disease. A detailed understanding of the underlying mechanistic process of peptide-metal interactions has however been difficult to obtain. Applying a combination of NMR relaxation dispersion and fluorescence based aggregation kinetics methods enabled us to investigate quantitatively thermodynamic Aβ-Zn(II) binding features as well as the nucleation mechanism of the aggregation process. Our results reveal that Zn(II) binds to the N-terminus of the peptide which transiently forms a marginally stable fold that encloses the metal ion. Aggregation kinetics studies show that Zn(II) inhibits generation of amyloid fibrils probed by the Thioflavin T fluorescence assay. Kinetic analyses of aggregation half times in the absence and presence of Zn(II) reveal mechanistic details of the Aβ(1-40) aggregation processes. The inhibition impact by Zn(II) on the aggregation process shows an exponential dependence on the Zn(II) ion concentration, and exerts its main effect on the secondary nucleation. Taken together, these findings suggest that transient binding of Zn(II) to the N-terminus of the peptide prevents Aβ(1-40) monomers to participate in secondary nucleation reactions and thereby decelerates Aβ(1-40) self-assembly.