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Alzheimer's disease (AD) is a neurological disorder associated with amyloid ß-protein (Aß) assembly into toxic oligomers. In addition to the two predominant alloforms, Aß1-40 and Aß1-42, other C-terminally truncated Aß peptides, including Aß1-38 and Aß1-43, are produced in the brain. Here, we use discrete molecular dynamics (DMD) and a four-bead protein model with amino acid-specific hydropathic interactions, DMD4B-HYDRA, to examine oligomer formation of Aß1-38, Aß1-40, Aß1-42, and Aß1-43. Self-assembly of 32 unstructured monomer peptides into oligomers is examined using 32 replica DMD trajectories for each of the four peptides. In a quasi-steady state, Aß1-38 and Aß1-40 adopt similar unimodal oligomer size distributions with a maximum at trimers, whereas Aß1-42 and Aß1-43 oligomer size distributions are multimodal with the dominant maximum at trimers or tetramers, and additional maxima at hexamers and unidecamers (for Aß1-42) or octamers and pentadecamers (for Aß1-43). The free energy landscapes reveal isoform- and oligomer-order specific structural and morphological features of oligomer ensembles. Our results show that oligomers of each of the four isoforms have unique features, with Aß1-42 alone resulting in oligomers with disordered and solvent-exposed N-termini. Our findings help unravel the structure-function paradigm governing oligomers formed by various Aß isoforms.

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Molecular dynamics (MD) is a great tool for elucidating conformational dynamics of proteins and peptides in water at the atomistic level that often surpasses the level of detail available experimentally. Structure predictions, however, are limited by the accuracy of the underlying MD force field. This limitation is particularly stark in the case of intrinsically disordered peptides and proteins, which are characterized by solvent-accessible and disordered peptide regions and domains. Recent studies show that most additive MD force fields, including CHARMM36m, do not reproduce the intrinsic conformational distributions of guest amino acid residues x in cationic GxG peptides in water in line with experimental data. Positing that a lack of polarizability in additive MD force fields may be the culprit for the reported discrepancies, we here examine the conformational dynamics of guest glycine and alanine residues in cationic GxG peptides in water using two polarizable MD force fields, CHARMM Drude and AMOEBA. Our results indicate that while AMOEBA captures the experimental data better than CHARMM Drude, neither of the two polarizable force fields offers an improvement of the Ramachandran distributions of glycine and alanine residues in cationic GGG and GAG peptides, respectively, over CHARMM36m.

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Disordered protein segments called short linear motifs (SLiM) serve as recognition sites for a variety of biological processes and act as targeting signals, modification, and ligand binding sites. While SLiMs do not adopt one of the known regular secondary structures, the conformational distribution might still reflect the structural propensities of their amino acid residues and possible interactions between them. In the past, conformational analyses of short peptides provided compelling evidence for the notion that individual residues are less conformationally flexible than locally expected for a random coil. Here, we combined various spectroscopies (NMR, IR, vibrational, and UV circular dichroism) to determine the Ramachandran plots of two SLiM motifs, i.e., GRRDSG and GRRTSG. They are two representatives of RxxS motifs that are capable of being phosphorylated by protein kinase A, an enzyme that plays a fundamental role in a variety of biological processes. Our results reveal that the nearest and non-nearest interactions between residues cause redistributions between polyproline II and ß-strand basins while concomitantly stabilizing extended relative to turn-forming and helical structures. They also cause shifts in basin positions. With increasing temperature, ß-strand populations become more populated at the expense of polyproline II. While molecular dynamics simulations with Amber ff14SB and CHARMM 36m force fields indicate residue-residue interactions, they do not account for the observed structural changes.

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It is well established that amyloid ß-protein (Aß) self-assembly is involved in triggering of Alzheimer's disease. On the other hand, evidence of physiological function of Aß interacting with lipids has only begun to emerge. Details of Aß-lipid interactions, which may underlie physiological and pathological activities of Aß, are not well understood. Here, the effects of salt and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids on conformational dynamics of Aß42 monomer in water are examined by all-atom molecular dynamics (MD). We acquired six sets of 250 ns long MD trajectories for each of the three lipid concentrations (0, 27, and 109 mM) in the absence and presence of 150 mM salt. Ten replica trajectories per set are used to enhance sampling of Aß42 conformational space. We show that salt facilitates long-range tertiary contacts in Aß42, resulting in more compact Aß42 conformations. By contrast, addition of lipids results in lipid-concentration dependent Aß42 unfolding concomitant with enhanced stability of the turn in the A21-A30 region. At the high lipid concentration, salt enables the N-terminal region of Aß42 to form long-range tertiary contacts and interact with lipids, which results in formation of a parallel ß-strand. Aß42 forms stable lipid-protein complexes whereby the protein is adhered to the lipid cluster rather than embedded into it. We propose that the inability of Aß42 monomer to get embedded into the lipid cluster may be important for facilitating repair of leaks in the blood-brain barrier without penetrating and damaging cellular membranes.

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Molecular dynamics (MD) is a powerful tool for studying intrinsically disordered proteins, however, its reliability depends on the accuracy of the force field. We assess Amber ff19SB, Amber ff14SB, OPLS-AA/M, and CHARMM36m with respect to their capacity to capture intrinsic conformational dynamics of 14 guest residues x (=G, A, L, V, I, F, Y, DP, EP, R, C, N, S, T) in GxG peptides in water. The MD-derived Ramachandran distribution of each guest residue is used to calculate 5 J-coupling constants and amide I' band profiles to facilitate a comparison to spectroscopic data through reduced χ2 functions. We show that the Gaussian model, optimized to best fit the experimental data, outperforms all MD force fields by an order of magnitude. The weaknesses of the MD force fields are: (i) insufficient variability of the polyproline II (pPII) population among the guest residues; (ii) oversampling of antiparallel at the expense of transitional ß-strand region; (iii) inadequate sampling of turn-forming conformations for ionizable and polar residues; and (iv) insufficient guest residue-specificity of the Ramachandran distributions. Whereas Amber ff19SB performs worse than the other three force fields with respect to χ2 values, it accounts for residue-specific pPII content better than the other three force fields. Additional testing of residue-specific RSFF1 and Amber ff14SB combined with TIP4P/2005 on six guest residues x (=A, I, F, DP, R, S) reveals that residue specificity derived from protein coil libraries or an improved water model alone do not result in significantly lower χ2 values.

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Protein self-assembly plays an important role in cellular processes. Whereas molecular dynamics (MD) represents a powerful tool in studying assembly mechanisms, its predictions depend on the accuracy of underlying force fields, which are known to overly promote protein assembly. We here examine villin headpiece domain, HP36, which remains soluble at concentrations amenable to MD studies. The experimental characterization of soluble HP36 at concentrations of 0.05 to 1 mM reveals concentration-independent 90% monomeric and 10% dimeric populations. Extensive all-atom MD simulations at two protein concentrations, 0.9 and 8.5 mM, probe the HP36 dimer population, stability, and kinetics of dimer formation within two MD force fields, Amber ff14SB and CHARMM36m. MD results demonstrate that whereas CHARMM36m captures experimental HP36 monomer populations at the lower concentration, both force fields overly promote HP36 association at the higher concentration. Moreover, contacts stabilizing HP36 dimers are force-field-dependent. CHARMM36m produces consistently higher HP36 monomer populations, lower association rates, and weaker dependence of these quantities on the protein concentration than Amber ff14SB. Nonetheless, the highest monomer populations and dissociation constants are observed when the TIP3P water model in Amber ff14SB is replaced by TIP4P/2005, showcasing the critical role of the water model in addressing the protein solubility problem in MD.

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Intrinsically disordered proteins and intrinsically disordered regions are frequently enriched in charged amino acids. Intrinsically disordered regions are regularly involved in important biological processes in which one or more charged residues is the driving force behind a protein-biomolecule interaction. Several lines of experimental and computational evidence suggest that polypeptides and proteins that carry high net charges have a high preference for extended conformations with average end-to-end distances exceeding expectations for self-avoiding random coils. Here, we show that charged arginine residues even in short glycine-capped model peptides (GRRG and GRRRG) significantly affect the conformational propensities of each other when compared with the intrinsic propensities of a mostly unperturbed arginine in the tripeptide GRG. A conformational analysis based on experimentally determined J-coupling constants from heteronuclear NMR spectroscopy and amide I' band profiles from vibrational spectroscopy reveals that nearest-neighbor interactions stabilize extended ß-strand conformations at the expense of polyproline II and turn conformations. The results from molecular dynamics simulations with a CHARMM36m force field and TIP3P water reproduce our results only to a limited extent. The use of the Ramachandran distribution of the central residue of GRRRG in a calculation of end-to-end distances of polyarginines of different length yielded the expected power law behavior. The scaling coefficient of 0.66 suggests that such peptides would be more extended than predicted by a self-avoiding random walk. Our findings thus support in principle theoretical predictions.

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Amyloid ß-protein (Aß) oligomers are broadly viewed as the proximate mediators of toxicity in Alzheimer's disease (AD). Recent studies, however, provide substantial evidence that Aß is involved in protection and repair of the central nervous system whereby Aß oligomer and subsequent fibril formation are integral to its normal antimicrobial and antiviral function. These developments raise a question of what exactly makes Aß oligomers toxic in the context of AD. This Perspective describes a paradigm shift in the search for toxic Aß oligomer species that involves oxidative-stress-induced stabilization of Aß oligomers via cross-linking and reviews most recent research elucidating structural aspects of cross-linked Aß oligomers and potential inhibition of their toxicity.

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In vitro, cationic glycylalanylglycine (GAG) forms a hydrogel in binary mixtures of water and ethanol. In water, alanine residue is known for its high polyproline II (pPII) content. Spectroscopic data, including three J-coupling constants and amide I' profiles, indicate that addition of 42% ethanol to water significantly reduces the pPII content of alanine residue in GAG. Here, experiment-based Gaussian Ramachandran distributions of alanine in GAG at different ethanol fractions are examined and three MD force fields are evaluated with respect to their ability to capture these ethanol-induced conformational changes. MD simulations on monomeric GAG in eight different water/ethanol mixtures within Amber ff14SB, OPLS-AA/M, and CHARMM36m reveal that only Amber ff14SB partially captures the ethanol-induced conformational changes of alanine residue in monomeric GAG when 42% ethanol is added to water. MD simulations of 200 mM GAG ensembles in pure water and in the aqueous solution with 42% ethanol showcase the ability of CHARMM36m to capture the effect of ethanol on the average pPII content of alanine in GAG and provide a plausible explanation for this effect, which may stem from an increased propensity of GAG to form oligomers in the presence of ethanol.

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Conformational preferences of amino acid residues in water are determined by the backbone and side-chain properties. Alanine is known for its high polyproline II (pPII) propensity. The question of relative contributions of the backbone and side chain to the conformational preferences of alanine and other amino acid residues in water is not fully resolved. Because glycine lacks a heavy-atom side chain, glycine-based peptides can be used to examine to which extent the backbone properties affect the conformational space. Here, we use published spectroscopic data for the central glycine residue of cationic triglycine in water to demonstrate that its conformational space is dominated by the pPII state. We assess three commonly used molecular dynamics (MD) force fields with respect to their ability to capture the conformational preferences of the central glycine residue in triglycine. We show that pPII is the mesostate that enables the functional backbone groups of the central residue to form the most hydrogen bonds with water. Our results indicate that the pPII propensity of the central glycine in GGG is comparable to that of alanine in GAG, implying that the water-backbone hydrogen bonding is responsible for the high pPII content of these residues.

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We examine the ability of six molecular dynamics (MD) force fields (Amber ff14SB, Amber ff99SBnmr1, Amber ff03ws, OPLS-AA/L, OPLS-AA/M, and CHARMM36) to reproduce conformational ensembles of the central alanine in GAG and AAA in a way that is consistent with five (GAG) or six (AAA) J coupling constants and amide I' profiles. MD-derived Ramachandran plots for all six force fields under study differ from those obtained by the Gaussian fit to experimental data in three major ways: (i) the polyproline II (pPII) basin in the Ramachandran plot is too concentrated, (ii) the antiparallel ß (aß) basin is overpopulated, and (iii) the transitional ß (ßt) basin is underpopulated. Amber ff14SB outperforms the other five MD force fields and yields the highest pPII populations of the central alanine residue in GAG (55%) and AAA (63%), in good agreement with the predictions of the Gaussian model (59 and 76%). The analysis of the hydration layer around the central alanine residue reveals considerable reorientation of water molecules and reduction in both the average number of water molecules and the average number of water-water hydrogen bonds when glycines (in GAG) are replaced by alanines (in AAA), elucidating water-mediated nearest neighbor effects on alanine's conformational dynamics.

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Alzheimer's disease (AD) is associated with self-assembly of amyloid ß-protein (Aß) into soluble oligomers. Of the two predominant Aß alloforms, Aß40 and Aß42, the latter is particularly strongly linked to AD. Longitudinal studies revealed a correlation between AD and type 2 diabetes (T2D), characterized by abnormal insulin levels and insulin resistance. Although administration of intranasal insulin is explored as a therapy against AD, the extent to which insulin affects Aß dynamics and activity is unclear. We here investigate the effect of insulin on Aß42 self-assembly and characterize the capacity of insulin, Aß42, and Aß42 co-incubated with insulin to disrupt the integrity of biomimetic lipid vesicles. We demonstrate that quiescently incubated insulin, which does not form amyloid fibrils, over time develops membrane-disrupting capacity, which we propose to originate in misfolded insulin monomers. These hypothetically toxic misfolded monomers might contribute to the development of insulin resistance in early stages of T2D that are associated with abnormally high insulin levels. We show that in contrast to quiescent incubation, insulin incubated under agitated conditions readily forms amyloid fibrils, which protect against membrane permeation. Insulin quiescently incubated with Aß42 attenuates both Aß42 fibril formation and the ability of Aß42 to disrupt membranes in a concentration-dependent manner. Our findings offer insights into interactions between insulin and Aß42 that are relevant to understanding the molecular basis of intranasal insulin as a therapy against Aß-induced AD pathology, thereby elucidating a plausible mechanism underlying the observed correlations between AD and T2D.

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Amyloid ß-protein (Aß) oligomers play a seminal role in Alzheimer's disease (AD). Cross-linking (X-linking), which can be used to determine Aß oligomer size distributions experimentally, was reported to stabilize Aß oligomers. Aß oligomers X-linked in the presence of copper and hydrogen peroxide may represent the proximate neurotoxic species in AD. Our previous computational study demonstrated that X-linking of Aß40 and Aß42 oligomers via tyrosines alone cannot explain experimental findings. Here, we explore three plausible X-linking mechanisms, which involve, in addition to tyrosine, also lysine (mechanism 1), histidine (mechanism 2), and hydroxylated phenylalanine (mechanism 3). By examining the effect of X-linking on oligomer size distributions, we show that only mechanism 3 is consistent with experimental data. Our findings provide important insights into the two-step X-linking via mechanism 3, which consists of a simple covalent bonding via tyrosines in the presence of hydroxylated phenylalanines, followed by covalent bonding among tyrosines and hydroxylated phenylalanines. Structural analysis of X-linked Aß oligomers revealed increased solvent exposure at the N-terminal region, which was previously associated with increased oligomer toxicity. Our results elucidate a potentially important role of phenylalanine hydroxylation and increased toxicity of Aß oligomers induced by X-linking.

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The CTCF protein has emerged as a key architectural protein involved in genome organization. Although hypothesized to initiate DNA looping, direct evidence of CTCF-induced DNA loop formation is still missing. Several studies have shown that the 11 zinc finger (11 ZF) domain of CTCF is actively involved in DNA binding. We here use atomic force microscopy to examine the effect of the 11 ZF domain comprising residues 266-579 (11 ZF CTCF) and the 3 ZF domain comprising residues 402-494 (6-8 ZF CTCF) of human CTCF on the DNA morphology. Our results show that both domains alter the DNA architecture from the relaxed morphology observed in control DNA samples to compact circular complexes, meshes, and networks, offering important insights into the multivalent character of the 11 ZF CTCF domain. Atomic force microscopy images reveal quasi-circular DNA/CTCF complexes, which are destabilized upon replacing the 11 ZF CTCF by the 6-8 ZF CTCF domain, highlighting the role of the 11 ZF motif in loop formation. Intriguingly, the formation of circular DNA/CTCF complexes is dominated by non-specific binding, whereby contour length and height profiles suggest a single DNA molecule twice wrapped around the protein.

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Deficiency in insulin secretion and function that characterize type 2 diabetes often requires administration of extraneous insulin, leading to injection-site amyloidosis. Insulin aggregation at neutral pH is not well understood. Although oligomer formation is believed to play an important role, insulin oligomers have not been fully characterized yet. Here, we elucidate similarities and differences between in vitro insulin aggregation at acidic and neutral pH for a range of insulin concentrations (2.5-100 µM) by using kinetic thioflavin T fluorescence, circular dichroism, atomic force and electron microscopy imaging. Importantly, we characterize the size distribution of insulin oligomers at different assembly stages by the application of covalent cross-linking and gel electrophoresis. Our results show that at the earliest assembly stage, oligomers comprise up to 40% and 70% of soluble insulin at acidic and neutral pH, respectively. While the highest oligomer order increases with insulin concentration at acidic pH, the opposite tendency is observed at neutral pH, where oligomers up to heptamers are formed in 10 µM insulin. These findings suggest that oligomers may be on- and off-pathway assemblies for insulin at acidic and neutral pH, respectively. Agitation, which is required to induce insulin aggregation at neutral pH, is shown to increase fibril formation rate and fibrillar mass both by an order of magnitude. Insulin incubated under agitated conditions at neutral pH rapidly aggregates into large micrometer-sized aggregates, which may be of physiological relevance and provides insight into injection-site amyloidosis and toxic pulmonary aggregates induced by administration of extraneous insulin.

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Oligomers formed by amyloid ß-protein (Aß) are central to Alzheimer's disease (AD) pathology, yet their structure remains elusive. Of the two predominant Aß alloforms, Aß40 and Aß42, the latter is more strongly associated with AD. Here, we structurally characterized Aß40 and Aß42 monomers through pentamers which were converted from previously derived coarse-grained (DMD4B-HYDRA) simulations into all-atom conformations and subjected to explicit-solvent MD. Free energy landscapes revealed that structural differences between Aß40 and Aß42 conformations increase with oligomer order up to trimers. All conformations display high statistical coil and turn content (40-50%) with minor ß-strand and α-helical content (<10%). Aß40 tetramers and pentamers exhibit significantly more elongated morphologies than the respective Aß42 conformations. Unlike the initial DMD4B-HYDRA conformations, fully atomistic Aß40 and Aß42 trimers, tetramers, and pentamers form water-permeable pores, whereby the tendency for pore formation sharply increased with oligomer order and is the highest for Aß42 pentamers. Previous studies reported that Aß oligomers form ion channels when embedded into a cellular membrane, which causes an abnormal ion flux and eventually leads to cell death. Our findings reveal an extraordinary ability of Aß oligomers to form pores in pure water prior to their insertion into a membrane and thus provide support to the ion channel hypothesis of AD.

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Alzheimer's disease (AD) pathology is hypothesized to be triggered by amyloid ß-protein (Aß) assembly into oligomers. Oligomer size distributions of both predominant Aß alloforms, Aß40 and Aß42, can be determined in vitro using cross-linking followed by gel electrophoresis. Cross-linking, which can occur in vivo in the presence of copper and hydrogen peroxide, was recently shown to stabilize Aß oligomers by inhibiting their conversion into fibrils. Whereas several studies showed that cross-linking is facilitated by dityrosine bond formation, the molecular-level mechanism of cross-linking remains unclear. Here, we use efficient discrete molecular dynamics with DMD4B-HYDRA force field to examine the effect of cross-linking via tyrosines on Aß oligomer formation. Our results show that cross-linking via tyrosines promotes Aß self-assembly, in particular that of Aß40, but does not account for cross-linked oligomers larger than Aß40 trimers and Aß42 tetramers. Cross-linking via tyrosines profoundly alters Aß40 and Aß42 oligomer conformations by increasing the solvent exposure of hydrophobic residues, resulting in elongated oligomeric morphologies that differ from globular structures of noncross-linked oligomers. When compared to available experimental data, our findings imply that amino acids other than tyrosines are involved in Aß cross-linking, a proposition that is currently under investigation.

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Amyloid ß-protein (A ß) assembles into oligomers that play a seminal role in Alzheimer's disease (AD), a leading cause of dementia among the elderly. Despite undisputed importance of A ß oligomers, their structure and the basis of their toxicity remain elusive. Previous experimental studies revealed that the [K16A] substitution strongly inhibits toxicity of the two predominant A ß alloforms in the brain, A ß 40 and A ß 42, whereas the [K28A] substitution exerts only a moderate effect. Here, folding and oligomerization of [A16]A ß 40, [A28]A ß 40, [A16]A ß 42, and [A28]A ß 42 are examined by discrete molecular dynamics (DMD) combined with a four-bead implicit solvent force field, DMD4B-HYDRA, and compared to A ß 40 and A ß 42 oligomer formation. Our results show that both substitutions promote A ß 40 and A ß 42 oligomerization and that structural changes to oligomers are substitution- and alloform-specific. The [K28A] substitution increases solvent-accessible surface area of hydrophobic residues and the intrapeptide N-to-C terminal distance within oligomers more than the [K16A] substitution. The [K16A] substitution decreases the overall ß-strand content, whereas the [K28A] substitution exerts only a modest change. Substitution-specific tertiary and quaternary structure changes indicate that the [K16A] substitution induces formation of more compact oligomers than the [K28A] substitution. If the structure-function paradigm applies to A ß oligomers, then the observed substitution-specific structural changes in A ß 40 and A ß 42 oligomers are critical for understanding the structural basis of A ß oligomer toxicity and correct identification of therapeutic targets against AD.

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Oligomeric assemblies are postulated to be proximate neurotoxic species in human diseases associated with aberrant protein aggregation. Their heterogeneous and transient nature makes their structural characterization difficult. Size distributions of oligomers of several amyloidogenic proteins, including amyloid ß-protein (Aß) relevant to Alzheimer's disease (AD), have been previously characterized in vitro by photo-induced cross-linking of unmodified proteins (PICUP) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Due to non-physiological conditions associated with the PICUP chemistry, Aß oligomers cross-linked by PICUP may not be representative of in vivo conditions. Here, we examine an alternative Copper and Hydrogen peroxide Induced Cross-linking of Unmodified Proteins (CHICUP), which utilizes naturally occurring divalent copper ions and hydrogen peroxide and does not require photo activation. Our results demonstrate that CHICUP and PICUP applied to the two predominant Aß alloforms, Aß40 and Aß42, result in similar oligomer size distributions. Thioflavin T fluorescence data and atomic force microscopy images demonstrate that both CHICUP and PICUP stabilize Aß oligomers and attenuate fibril formation. Relative to noncross-linked peptides, CHICUP-treated Aß40 and Aß42 cause prolonged disruption to biomimetic lipid vesicles. CHICUP-stabilized Aß oligomers link the amyloid cascade, metal, and oxidative stress hypotheses of AD into a more comprehensive understanding of the molecular basis of AD pathology. Because copper and hydrogen peroxide are elevated in the AD brain, CHICUP-stabilized Aß oligomers are biologically relevant and should be further explored as a new therapeutic target.

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