Monomeric Beta Research Articles

Effect of diffusivity of amyloid beta monomers on the formation of senile plaques.

Alzheimer's disease (AD) presents a perplexing question: why does its development span decades, even though individual amyloid beta (Aβ) deposits (senile plaques) can form rapidly in as little as 24 hours, as recent publications suggest? This study investigated whether the formation of senile plaques can be limited by factors other than polymerization kinetics alone. Instead, their formation may be limited by the diffusion-driven supply of Aβ monomers, along with the rate at which the monomers are produced from amyloid precursor protein (APP) and the rate at which Aβ monomers undergo degradation. A mathematical model incorporating the nucleation and autocatalytic process (via the Finke-Watzky model), as well as Aβ monomer diffusion, was proposed. The obtained system of partial differential equations was solved numerically, and a simplified version was investigated analytically. The computational results predicted that it takes approximately 7 years for Aβ aggregates to reach a neurotoxic concentration of 50 μM. Additionally, a sensitivity analysis was performed to examine how the diffusivity of Aβ monomers and their production rate impact the concentration of Aβ aggregates.

The growth rate of senile plaques is determined by the competition between the rate of deposition of free Aβ aggregates into plaques and the autocatalytic production of free Aβ aggregates

The formation of amyloid beta (Aβ) deposits (senile plaques) is one of the hallmarks of Alzheimer’s disease (AD). This study investigates what processes are primarily responsible for their formation. A model is developed to simulate the diffusion of amyloid beta (Aβ) monomers, the production of free Aβ aggregates through nucleation and autocatalytic processes, and the deposition of these aggregates into senile plaques. The model suggests that efficient degradation of Aβ monomers alone may suffice to prevent the growth of senile plaques, even without degrading Aβ aggregates and existing plaques. This is because the degradation of Aβ monomers interrupts the supply of reactants needed for plaque formation. The impact of Aβ monomer diffusivity is demonstrated to be small, enabling the application of the lumped capacitance approximation and the derivation of approximate analytical solutions for limiting cases with both small and large rates of Aβ aggregate deposition into plaques. It is found that the rate of plaque growth is governed by two competing processes. One is the deposition rate of free Aβ aggregates into senile plaques. If this rate is small, the plaque grows slowly. However, if the rate of deposition of Aβ aggregates into senile plaques is very large, the free Aβ aggregates are removed from the intracellular fluid by deposition into the plaques, leaving insufficient free Aβ aggregates to catalyze the production of new aggregates. This suggests that under certain conditions, Aβ plaques may offer neuroprotection and impede their own growth. Additionally, it indicates that there exists an optimal rate of deposition of free Aβ aggregates into the plaques, at which the plaques attain their maximum size.

Numerical modeling of senile plaque development under conditions of limited diffusivity of amyloid-β monomers

This paper introduces a new model to simulate the progression of senile plaques, focusing on scenarios where concentrations of amyloid beta (Aβ) monomers and aggregates vary between neurons. Extracellular variations in these concentrations may arise due to limited diffusivity of Aβ monomers and a high rate of Aβ monomer production at lipid membranes, requiring a substantial concentration gradient for diffusion-driven transport of Aβ monomers. The dimensionless formulation of the model is presented, which identifies four key dimensionless parameters governing the solutions for Aβ monomer and aggregate concentrations, as well as the radius of a growing Aβ plaque within the control volume. These parameters include the dimensionless diffusivity of Aβ monomers, the dimensionless rate of Aβ monomer production, and the dimensionless half-lives of Aβ monomers and aggregates. A dimensionless parameter is then introduced to evaluate the validity of the lumped capacitance approximation. An approximate solution is derived for the scenario involving large diffusivity of Aβ monomers and dysfunctional protein degradation machinery, resulting in infinitely long half-lives for Aβ monomers and aggregates. In this scenario, the concentrations of Aβ aggregates and the radius of the Aβ plaque depend solely on a single dimensionless parameter that characterizes the rate of Aβ monomer production. According to the approximate solution, the concentration of Aβ aggregates is linearly dependent on the rate of monomer production, and the radius of an Aβ plaque is directly proportional to the cube root of the rate of monomer production. However, when departing from the conditions of the approximate solution (e.g., finite half-lives), the concentrations of Aβ monomers and aggregates, along with the plaque radius, exhibit complex dependencies on all four dimensionless parameters. For instance, under physiological half-life conditions, the plaque radius reaches a maximum value and stabilizes thereafter.

Rotating magnetic field inhibits Aβ protein aggregation and alleviates cognitive impairment in Alzheimer's disease mice.

Amyloid beta (Aβ) monomers aggregate to form fibrils and amyloid plaques, which are critical mechanisms in the pathogenesis of Alzheimer's disease (AD). Given the important role of Aβ1-42 aggregation in plaque formation, leading to brain lesions and cognitive impairment, numerous studies have aimed to reduce Aβ aggregation and slow AD progression. The diphenylalanine (FF) sequence is critical for amyloid aggregation, and magnetic fields can affect peptide alignment due to the diamagnetic anisotropy of aromatic rings. In this study, we examined the effects of a moderate-intensity rotating magnetic field (RMF) on Aβ aggregation and AD pathogenesis. Results indicated that the RMF directly inhibited Aβ amyloid fibril formation and reduced Aβ-induced cytotoxicity in neural cells in vitro. Using the AD mouse model APP/PS1, RMF restored motor abilities to healthy control levels and significantly alleviated cognitive impairments, including exploration and spatial and non-spatial memory abilities. Tissue examinations demonstrated that RMF reduced amyloid plaque accumulation, attenuated microglial activation, and reduced oxidative stress in the APP/PS1 mouse brain. These findings suggest that RMF holds considerable potential as a non-invasive, high-penetration physical approach for AD treatment.

The Roles of the Amyloid Beta Monomers in Physiological and Pathological Conditions.

Amyloid beta peptide is an important biomarker in Alzheimer's disease, with the amyloidogenic hypothesis as one of the central hypotheses trying to explain this type of dementia. Despite numerous studies, the etiology of Alzheimer's disease remains incompletely known, as the pathological accumulation of amyloid beta aggregates cannot fully explain the complex clinical picture of the disease. Or, for the development of effective therapies, it is mandatory to understand the roles of amyloid beta at the brain level, from its initial monomeric stage prior to aggregation in the form of senile plaques. In this sense, this review aims to bring new, clinically relevant data on a subject intensely debated in the literature in the last years. In the first part, the amyloidogenic cascade is reviewed and the possible subtypes of amyloid beta are differentiated. In the second part, the roles played by the amyloid beta monomers in physiological and pathological (neurodegenerative) conditions are illustrated based on the most relevant and recent studies published on this topic. Finally, considering the importance of amyloid beta monomers in the pathophysiology of Alzheimer's disease, new research directions with diagnostic and therapeutic impacts are suggested.

Calmodulin and Amyloid Beta as Coregulators of Critical Events during the Onset and Progression of Alzheimer's Disease.

Calmodulin (CaM) and a diversity of CaM-binding proteins (CaMBPs) are involved in the onset and progression of Alzheimer's disease (AD). In the amyloidogenic pathway, AβPP1, BACE1 and PSEN-1 are all calcium-dependent CaMBPs as are the risk factor proteins BIN1 and TREM2. Ca2+/CaM-dependent protein kinase II (CaMKII) and calcineurin (CaN) are classic CaMBPs involved in memory and plasticity, two events impacted by AD. Coupled with these events is the production of amyloid beta monomers (Aβ) and oligomers (Aβo). The recent revelations that Aβ and Aβo each bind to both CaM and to a host of Aβ receptors that are also CaMBPs adds a new level of complexity to our understanding of the onset and progression of AD. Multiple Aβ receptors that are proven CaMBPs (e.g., NMDAR, PMCA) are involved in calcium homeostasis an early event in AD and other neurodegenerative diseases. Other CaMBPs that are Aβ receptors are AD risk factors while still others are involved in the amyloidogenic pathway. Aβ binding to receptors not only serves to control CaM's ability to regulate critical proteins, but it is also implicated in Aβ turnover. The complexity of the Aβ/CaM/CaMBP interactions is analyzed using two events: Aβ generation and NMDAR function. The interactions between Aβ, CaM and CaMBPs reveals a new level of complexity to critical events associated with the onset and progression of AD and may help to explain the failure to develop successful therapeutic treatments for the disease.

Understanding the screening effect of aqueous DES on the IDPs: A molecular dynamics simulation study using amyloid [formula omitted] monomer

Deep eutectic solvents (DESs) have emerged as the promising replacement to the ionic liquids in solvent engineering for bio-compatibility. We aim to understand the effect of aqueous deep eutectic solvents on the conformation of intrinsically disordered proteins (IDPs). In this context, we have studied the effect on amyloid beta (Aβ42) monomer in the hydrated DES composed of tetrabutylammonium chloride and ethylene glycol in a 3:1 ratio using all-atom molecular dynamics simulations. DES is found to effectively screen the interaction of four zones of the amyloid beta monomer with water. Water molecules and the DES constituents modulate the local protein–solvent interactions, in the solvation shell of the protein. In addition, the aqueous DES medium conserves the secondary structure of the Aβ42 monomer by increasing the intramolecular hydrogen bonding and D23–K28 salt-bridge interactions when compared to the pure water medium. The current study provides insights into the impact of DES in stabilizing an IDP, at molecular level. We envisage the hindered aggregation of the amyloid beta structures in DES medium over the pure water medium due to the screening of hydrophobic intramolecular interactions.

Glaucoma and Alzheimer: Neurodegenerative disorders show an adrenergic dysbalance.

Glaucoma disease is characterized by an increased intraocular pressure (IOP), glaucomatous alterations of the optic disc and corresponding visual field defects. Even lowering the main risk factor IOP until an individual target level does not prevent this neurodegenerative disorder from proceeding. Several autoimmune mechanisms were discovered, partly showing a functionality. One of these autoimmune phenomena targets the ß2-adrenergic receptor (ß2-AR; i.e. agonistic autoantibodies; ß2-agAAb) and is linked to an elevated IOP and an impaired retinal microcirculation. As neurodegenerative disorder, Alzheimer’s Disease (AD) is postulated to share a common molecular mechanism with glaucoma. In the present study we investigated autoimmune phenomena targeting the ß2-AR in patients with AD. Sera of the patients were analyzed in a rat cardiomyocyte bioassay for the presence of functional autoantibodies against ß2-AR. In addition, different species of amyloid beta (Aß) monomers were tested (Aß1-14, Aß10-25, Aβ10–37 Aß1-40, Aß1-42, Aβ28–40, and Aß-[Pyr]3–43). Our results demonstrate that none of the short-chain Aß (Aß1-14, Aß10-25, or Aβ28–40) showed any agonistic or inhibitory effect on ß2-AR. Contrary, long-chain Aß-[Pyr]3–43, representing a major neurogenic plaque component, exerted an activation that after blocking by the ß2-AR antagonist ICI118.551, could be identified as that the effect was realized via the ß2-AR. Moreover, the long chain Aß1-40, Aβ1–42, and Aβ10–37, yet not the short-chain Aß peptides prevented the clenbuterol induced desensitization of the ß2-AR. In addition, we identified functional autoantibodies in the sera of AD patients, activating the ß2-AR, like the ß2-agAAb found in patients with glaucoma. As autoimmune mechanisms were reportedly involved in the pathogenesis of glaucoma and Alzheimer’s Disease, we postulate that overstimulation of the ß2-AR pathway can induce an adrenergic overdrive, that may play an important role in the multifactorial interplay of neurodegenerative disorders.

Chemoattractant releasing microneedles for enhanced DNA vaccination

Transcutaneous delivery of a DNA vaccine using microneedle (MN) has attracted much attention since there are a variety of immune cells underneath the skin. However, the clinical use of DNA vaccine is limited due to its poor cellular uptake of naked plasmid DNA (pDNA) and suboptimal presentation of an antigen by antigen presenting cells (APC). Herein, we designed a MN system for delivering polyplex-based DNA vaccine and recruiting APCs by a chemoattractant N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP)-releasing microspheres (fMLP-MS) using MN coated with pH-responsive polyelectrolyte multilayer assembly (PMA). The PMA can be rapidly disrupted upon the application of MN to the skin, leading to the release of polyplex and fMLP-MS. The polyplex would be taken up by the epidermal cells and resident APCs to express and secrete the antigen (amyloid beta monomer, Aβ1-42) encoded in the DNA vaccine. fMLP released from fMLP-MS could recruit APCs to the site of MN administration by chemotactic effects. The MN-based vaccination system was able to induce robust antigen-specific antibody production.

Detection limit enhancement of fiber optic localized surface plasmon resonance biosensor by increased scattering efficiency and reduced background signal

To reduce the limit of detection (LOD) in fiber optic localized surface plasmon resonance (FO LSPR) sensors, design strategies with high scattering efficiency and low background signal are proposed. Despite the cost effectiveness of fabrication, miniaturization capabilities, and high portability of optic fibers, the LOD is a limiting factor for fiber optic sensor applications. To overcome this limitation, we propose methods to increase the scattering efficiency and reduce background signal using gold capping and reactive-ion etching (RIE). Specifically, three strategies are implemented. First, the effective coverage for detection is selected by analyzing the sensor output according to the density of gold nanoparticles (AuNPs), which have been immobilized on the cross-section of the optical fiber. Next, the sensitivity is studied by examining changes in scattering efficiency of the AuNPs grown with different capping times. Third, RIE is performed on the fiber optic surface to reduce the reflectance in areas where the AuNPs are not fixed, thereby decreasing the background signal. Thyroglobulin, amyloid beta monomer, and oligomer are measured using the thus-fabricated FO LSPR sensor. The results are compared with those obtained with a sensor fabricated without using these strategies. The former showed the lower LOD and higher accuracy for biomarkers of the two. • The effective coverage of AuNPs is selected on the optical fiber. • For improvement of scattering efficiency, AuNPs are directly grown on fiber optic surface. • Surface etching is executed for reduction of background in bare surface. • The enhancement of the LODs is evaluated for several biomarkers.

Aβ Beyond the AD Pathology: Exploring the Structural Response of Membranes Exposed to Nascent Aβ Peptide.

The physiological and pathological roles of nascent amyloid beta (Aβ) monomers are still debated in the literature. Their involvement in the pathological route of Alzheimer’s Disease (AD) is currently considered to be the most relevant, triggered by their aggregation into structured oligomers, a toxic species. Recently, it has been suggested that nascent Aβ, out of the amyloidogenic pathway, plays a physiological and protective role, especially in the brain. In this emerging perspective, the study presented in this paper investigated whether the organization of model membranes is affected by contact with Aβ in the nascent state, as monomers. The outcome is that, notably, the rules of engagement and the resulting structural outcome are dictated by the composition and properties of the membrane, rather than by the Aβ variant. Interestingly, Aβ monomers are observed to favor the tightening of adjacent complex membranes, thereby affecting a basic structural event for cell-cell adhesion and cell motility.

Abstract C050: A novel, potent, and selective hypoxia-inducible factor (HIF)-2α antagonist

Abstract The microenvironment of solid tumors is known to be hypoxic and requires induction of genes associated with metabolism, growth, proliferation, and angiogenesis for tumor cells to survive and metastasize. The master transcriptional regulator of hypoxia-induced genes is the Hypoxia-Inducible Factor (HIF), consisting of an oxygen-regulated alpha monomer, of which there are three isoforms (HIF-1α, HIF-2α, and HIF-3α), that can heterodimerize with a constitutively-expressed beta monomer (HIF-1β/ARNT) using Per-ARNT-SIM (PAS) protein-protein interaction domains. In normoxia, proline residues present in the oxygen-dependent degradation domain of the HIF-α subunits are hydroxylated, allowing for recognition by the von Hippel-Lindau (pVHL) E3-ubiquitin ligase complex and subsequent proteasomal degradation. Upon exposure to low oxygen conditions or in the case of VHL mutation or silencing, HIF-α subunits accumulate in the cell and mediate transcription of various pro-tumorigenic gene sets. In patients, overexpression of HIF is associated with poor prognosis, and both preclinical and clinical evidence is mounting that suggests inhibiting HIF-2α is a valid approach to destroy tumor cells, particularly in clear cell renal carcinoma, warranting development of next-generation inhibitors. Using a suite of in vitro and in vivo assays designed to evaluate HIF-2α-specific effects, herein we describe pharmacological properties associated with novel, potent, and selective small-molecule antagonists of HIF-2α. These compounds inhibited HIF-dependent reporter gene transcription as well as VEGF protein secretion in a human renal cell adenocarcinoma line. Compounds that were confirmed to bind the HIF-2α PAS-B domain by Microscale thermophoresis (MST) and Thermal shift assay (TSA) also significantly inhibited HIF-2α, but not HIF-1α, target gene expression in a hepatocellular carcinoma cell line (P<0.05). Further, NanoString analyses revealed pathway signatures upregulated in hypoxic conditions, including angiogenesis, metabolism, and metastasis, that were significantly decreased with antagonist treatment. Selective antagonists were evaluated in vivo to determine efficacy and PK-PD relationships. Additionally, syngeneic tumor models were used to characterize the effects of HIF-2α inhibition (alone or in combination with anti-PD-1 and anti-adenosine agents) on tumor growth, immune cell infiltration, and expression of genes associated with adenosine production/signaling. Collectively, these data support the rationale to target HIF-2α for the treatment of cancers, particularly indications with a HIF-2α-driven hypoxia signature, and describe the properties of novel and selective HIF-2α antagonists. Citation Format: Kelsey E Sivick Gauthier, Kenneth V Lawson, Dana Piovesan, Matthew J Walters, Ada Chen, Xiaoning Zhao, Cesar Meleza, Nikki Kimura, Tim Park, Steve Young, Anh Tran, Samuel L Drew, Lixia Jin, Manmohan Leleti, Elaine Ginn, Jay P Powers. A novel, potent, and selective hypoxia-inducible factor (HIF)-2α antagonist [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C050. doi:10.1158/1535-7163.TARG-19-C050

Two instantaneous fluorogenic steps for detection of nanomolar amyloid beta monomer and its interaction with stoichiometric copper(II) ion

A small molecule SEA-SC2 exhibited fluorescence in response to 10–60 nM amyloid beta (Aβ)40 and Aβ42 monomers at pH 7.4, in the presence of gold nanoparticle (AuNP). Here we report the fluorogenic detection due to the response from low nanomolar level of Aβ peptide monomer. The small organic molecule or AuNP has been reported to show detection limit up to 200 nM Aβ peptide. Furthermore, fluorescence enhancement studies with different stoichiometries of Aβ:Cu(II) confirmed the initial Aβ:Cu(II) adduct at the nanomolar level. This methodology has been applied to the detection of nanomolar concentrations of Aβ monomer in artificial cerebrospinal fluid and human serum albumin.

Amyloid Beta monomers regulate cyclic adenosine monophosphate response element binding protein functions by activating type-1 insulin-like growth factor receptors in neuronal cells.

SummaryAlzheimer's disease (AD) is a progressive neurodegenerative disorder associated with synaptic dysfunction, pathological accumulation of β‐amyloid (Aβ), and neuronal loss. The self‐association of Aβ monomers into soluble oligomers seems to be crucial for the development of neurotoxicity (J. Neurochem., 00, 2007 and 1172). Aβ oligomers have been suggested to compromise neuronal functions in AD by reducing the expression levels of the CREB target gene and brain‐derived neurotrophic factor (BDNF) (J. Neurosci., 27, 2007 and 2628; Neurobiol. Aging, 36, 2015 and 20406 Mol. Neurodegener., 6, 2011 and 60). We previously reported a broad neuroprotective activity of physiological Aβ monomers, involving the activation of type‐1 insulin‐like growth factor receptors (IGF‐IRs) (J. Neurosci., 29, 2009 and 10582, Front Cell Neurosci., 9, 2015 and 297). We now provide evidence that Aβ monomers, by activating the IGF‐IR‐stimulated PI3‐K/AKT pathway, induce the activation of CREB in neurons and sustain BDNF transcription and release.

The effects of betulinic acid on neurobehavioral activity, electrophysiology and histological changes in an animal model of the Alzheimer’s disease

Alzheimer's disease (AD) is a common disorder characterized by aggregation and conversion of amyloid beta (Aβ) monomers to fibrils. Betulinic acid (BA) strongly accelerated this pathway through circumventing the oligomeric intermediate state. BA at doses of 0.2 and 0.4μM/10μl/rat (intra-hippocampal or i.h injection, vehicle: DMSO) was bilaterally administrated 180 and 10min before co-administration of Aβ (0.1μM/5μl/rat, i.h injection, vehicle: PBS) and Streptozotocin (STZ, 1.5mg/kg/10μl/rat, intracerebroventricular or i.c.v. injection, vehicle: aCSF). The behavioral assessments (spatial and passive avoidance memory, anxiety, locomotion, depression, and motor coordination), electrophysiological evaluations (hippocampal long- term potentiation (LTP)) as well as histological changes were evaluated 30days after injections. The indices of spatial and passive avoidance memory, anxiety/depression and LTP records were significantly impaired in AD rats in comparison with the sham. Pretreatment of BA (0.4μM) showed a more significant effect on memory, anxiety, all LTP parameters, and histological damage compared to a low dose in contrast to the AD group. Overall, BA pretreatment was able to prevent AD-induced neurobehavioral and LTP deficits in rats and the best effect was observed in molar ratio of 1:4 (Aβ to BA).

Monomeric Amyloid Beta Peptide in Hexafluoroisopropanol Detected by Small Angle Neutron Scattering.

Small proteins like amyloid beta (Aβ) monomers are related to neurodegenerative disorders by aggregation to insoluble fibrils. Small angle neutron scattering (SANS) is a nondestructive method to observe the aggregation process in solution. We show that SANS is able to resolve monomers of small molecular weight like Aβ for aggregation studies. We examine Aβ monomers after prolonged storing in d-hexafluoroisopropanol (dHFIP) by using SANS and dynamic light scattering (DLS). We determined the radius of gyration from SANS as 1.0±0.1 nm for Aβ1–40 and 1.6±0.1 nm for Aβ1–42 in agreement with 3D NMR structures in similar solvents suggesting a solvent surface layer with 5% increased density. After initial dissolution in dHFIP Aβ aggregates sediment with a major component of pure monomers showing a hydrodynamic radius of 1.8±0.3 nm for Aβ1–40 and 3.2±0.4 nm for Aβ1–42 including a surface layer of dHFIP solvent molecules.

Polyplex-releasing microneedles for enhanced cutaneous delivery of DNA vaccine

Microneedle (MN)-based DNA vaccines have many advantages over conventional vaccines administered by hypodermic needles. However, an efficient strategy for delivering DNA vaccines to intradermal cells has not yet been established. Here, we report a new approach for delivering polyplex-based DNA vaccines using MN arrays coated with a pH-responsive polyelectrolyte multilayer assembly (PMA). This approach enabled rapid release of polyplex upon application to the skin. In addition to the polyplex-releasing MNs, we attempted to further maximize the vaccination by developing a polymeric carrier that targeted resident antigen presenting cells (APCs) rich in the intradermal area, as well as a DNA vaccine encoding a secretable fusion protein containing amyloid beta monomer (Aβ1–42), an antigenic determinant. The resulting vaccination system was able to successfully induce a robust humoral immune response compared to conventional subcutaneous injection with hypodermal needles. In addition, antigen challenge after immunization elicited an immediate and strong recall immune response due to immunogenic memory. These results suggest the potential utility of MN-based polyplex delivery systems for enhanced DNA vaccination.

Amyloid Beta Misfolds and Increases its Affinity for Cell Membranes at the Initial Step of Aggregation

An amyloid protein changes its molecular conformation as it aggregates. However, this aspect of aggregation has remained difficult to measure. Alzheimer’s amyloid beta (Aβ) monomers are thought to be relatively unstructured, while the mature aggregates have a β-sheet-rich hairpin-like conformation where the two termini come close together. It is not clear at what stage of aggregation this change (‘misfolding’) takes place, and how this affects the properties of the aggregate, such as its stability, shape and toxicity. It is also not known why monomers, presumably present in healthy humans, are non-toxic, and how toxicity arises at some stage of aggregation. Here we use Fluorescence Correlation Spectroscopy, Forster Resonance Energy Transfer (FRET), fluorescence quenching, and vesicle and cell binding assays to address these questions in physiological buffer conditions. We show that the major conformational transition takes place right at the first step of aggregation, viz. with the formation of the small oligomers. This change predominantly involves the core region of the peptide, as the structures of the two termini remain relatively unchanged between the monomer and the oligomer. In both the species, the N-terminus is almost fully solvent exposed, while the C-terminus is much more shielded. This monomer to oligomer transition increases the affinity of Aβ for artificial lipid vesicles by at least an order of magnitude. Confocal microscopy shows that the oligomers at physiological concentrations have a strong affinity for the plasma membrane of living HEK 293T cells, while the monomers at the same concentration do not have any detectable affinity. Our results imply that the major change of molecular structure occurs at the initial step of aggregation, and suggest that this plays a major role in transforming nontoxic monomers into toxic cell-adherent oligomers.

Investigating How Peptide Length and a Pathogenic Mutation Modify the Structural Ensemble of Amyloid Beta Monomer

The aggregation of amyloid beta (Aβ) peptides plays an important role in the development of Alzheimer's disease. Despite extensive effort, it has been difficult to characterize the secondary and tertiary structure of the Aβ monomer, the starting point for aggregation, due to its hydrophobicity and high aggregation propensity. Here, we employ extensive molecular dynamics simulations with atomistic protein and water models to determine structural ensembles for Aβ42, Aβ40, and Aβ42-E22K (the Italian mutant) monomers in solution. Sampling of a total of >700 microseconds in all-atom detail with explicit solvent enables us to observe the effects of peptide length and a pathogenic mutation on the disordered Aβ monomer structural ensemble. Aβ42 and Aβ40 have crudely similar characteristics but reducing the peptide length from 42 to 40 residues reduces β-hairpin formation near the C-terminus. The pathogenic Italian E22K mutation induces helix formation in the region of residues 20–24. This structural alteration may increase helix-helix interactions between monomers, resulting in altered mechanism and kinetics of Aβ oligomerization.

Role of water in protein aggregation and amyloid polymorphism.

A variety of neurodegenerative diseases are associated with amyloid plaques, which begin as soluble protein oligomers but develop into amyloid fibrils. Our incomplete understanding of this process underscores the need to decipher the principles governing protein aggregation. Mechanisms of in vivo amyloid formation involve a number of coconspirators and complex interactions with membranes. Nevertheless, understanding the biophysical basis of simpler in vitro amyloid formation is considered important for discovering ligands that preferentially bind regions harboring amyloidogenic tendencies. The determination of the fibril structure of many peptides has set the stage for probing the dynamics of oligomer formation and amyloid growth through computer simulations. Most experimental and simulation studies, however, have been interpreted largely from the perspective of proteins: the role of solvent has been relatively overlooked in oligomer formation and assembly to protofilaments and amyloid fibrils. In this Account, we provide a perspective on how interactions with water affect folding landscapes of amyloid beta (Aβ) monomers, oligomer formation in the Aβ16-22 fragment, and protofilament formation in a peptide from yeast prion Sup35. Explicit molecular dynamics simulations illustrate how water controls the self-assembly of higher order structures, providing a structural basis for understanding the kinetics of oligomer and fibril growth. Simulations show that monomers of Aβ peptides sample a number of compact conformations. The formation of aggregation-prone structures (N*) with a salt bridge, strikingly similar to the structure in the fibril, requires overcoming a high desolvation barrier. In general, sequences for which N* structures are not significantly populated are unlikely to aggregate. Oligomers and fibrils generally form in two steps. First, water is expelled from the region between peptides rich in hydrophobic residues (for example, Aβ16-22), resulting in disordered oligomers. Then the peptides align along a preferred axis to form ordered structures with anti-parallel β-strand arrangement. The rate-limiting step in the ordered assembly is the rearrangement of the peptides within a confining volume. The mechanism of protofilament formation in a polar peptide fragment from the yeast prion, in which the two sheets are packed against each other and create a dry interface, illustrates that water dramatically slows self-assembly. As the sheets approach each other, two perfectly ordered one-dimensional water wires form. They are stabilized by hydrogen bonds to the amide groups of the polar side chains, resulting in the formation of long-lived metastable structures. Release of trapped water from the pore creates a helically twisted protofilament with a dry interface. Similarly, the driving force for addition of a solvated monomer to a preformed fibril is water release; the entropy gain and favorable interpeptide hydrogen bond formation compensate for entropy loss in the peptides. We conclude by offering evidence that a two-step model, similar to that postulated for protein crystallization, must also hold for higher order amyloid structure formation starting from N*. Distinct water-laden polymorphic structures result from multiple N* structures. Water plays multifarious roles in all of these protein aggregations. In predominantly hydrophobic sequences, water accelerates fibril formation. In contrast, water-stabilized metastable intermediates dramatically slow fibril growth rates in hydrophilic sequences.