Structure Comparison of Beta Amyloid Peptide Aß 1-42 Isoforms. Molecular Dynamics Modeling.

Beta amyloid peptide Aß 1-42 (Aß42) has a unique dual role in the human organism, as both the peptide with an important physiological function and one of the most toxic biological compounds provoking Alzheimer's disease (AD). There are several known Aß42 isoforms that we discuss here that are highly neurotoxic and lead to the early onset of AD. Aß42 is an intrinsically disordered protein with no experimentally solved structure under physiological conditions. The objective of this research was to establish the appropriate molecular dynamics (MD) methodology and model a uniform set of structures for the Aß42 isoforms that form the core of this study. For that purpose, force field selection and verification including convergence testing for MD simulations was made. Replica exchange MD and conventional MD modeling of several Aß42 and Aß16 isoforms that have neurotoxic and amyloidogenic effects impacting the severity of Alzheimer's disease were carried out with the optimal force field and solvent parameters. A standardized ensemble of structures for the Aß42 and Aß16 isoforms covering 30-50% of the conformational ensembles extracted from the free energy minima was calculated from MD trajectories. The resulting data set of modeled structures includes Aß42 wild type, isoD7, pS8, D7H, and H6R-Aß42 and Aß16 wild type, isoD7, pS8, D7H, and H6R-Aß16. The representative structures are given in the Supporting Information; they are open for public access. In the study, we also evaluated the differences between the structures of Aß42 isoforms and speculate on their possible relevance to the known functions. Utilizing several representative structures for a single disordered protein for docking, with their subsequent averaging by conformations, would markedly increase the reliability of docking results.

Molecular Mechanism of Zinc-Dependent Oligomerization of Alzheimer's Amyloid-ß with Taiwan (D7H) Mutation.

Amyloid-ß (Aß) is a peptide formed by 39-43 amino acids, heterogenous by the length of its C-terminus. Aß constitutes a subnanomolar monomeric component of human biological fluids; however, in sporadic variants of Alzheimer's disease (AD), it forms soluble neurotoxic oligomers and accumulates as insoluble extracellular polymeric aggregates (amyloid plaques) in the brain tissues. The plaque formation is controlled by zinc ions; therefore, abnormal interactions between the ions and Aß seem to take part in the triggering of sporadic AD. The amyloid plaques contain various Aß isoforms, among which the most common is Aß with an isoaspartate in position 7 (isoD7). The spontaneous conversion of D7 to isoD7 is associated with Aß aging. Aß molecules with isoD7 (isoD7-Aß) easily undergo zinc-dependent oligomerization, and upon administration to transgenic animals (mice, nematodes) used for AD modeling, act as zinc-dependent seeds of the pathological aggregation of Aß. The formation of zinc-bound homo- and hetero-oligomers with the participation of isoD7-Aß is based on the rigidly structured segment 11-EVHH-14, located in the Aß metal binding domain (Aß16). Some hereditary variants of AD are associated with familial mutations within the domain. Among these, the most susceptible to zinc-dependent oligomerization is Aß with Taiwan (D7H) mutation (D7H-Aß). In this study, the D7H-Aß metal binding domain (D7H-Aß16) has been used as a model to establish the molecular mechanism of zinc-induced D7H-Aß oligomerization through turbidimetry, dynamic light scattering, isothermal titration calorimetry, mass spectrometry, and computer modelling. Additionally, the modeling data showed that a molecule of D7H-Aß, as well as isoD7-Aß in combination with two Aß molecules, renders a stable zinc-induced heterotrimer. The trimers are held together by intermolecular interfaces via zinc ions, with the primary interfaces formed by 11-EVHH-14 sites of the interacting trimer subunits. In summary, the obtained results confirm the role of the 11-EVHH-14 region as a structure and function determinant for the zinc-dependent oligomerization of all known Aß species (including various chemically modified isoforms and AD-associated mutants) and point at this region as a potent target for drugs aimed to stop amyloid plaque formation in both sporadic and hereditary variants of AD.

Docking and Molecular Dynamics-Based Identification of Interaction between Various Beta-Amyloid Isoforms and RAGE Receptor.

Beta-amyloid peptide (Aß) is a ligand associated with RAGE (Advanced glycosylation end product-specific receptor). Aß is translocated in complexes with RAGE from the blood to brain across the blood-brain barrier (BBB) by transcytosis. Aß and its isoforms are important factors in the Alzheimer's disease (AD) pathogenesis. However, interaction with RAGE was previously studied for Aß but not for its isoforms. The present study has been directed at identifying the key interaction interfaces between RAGE and Aß isoforms (Aß40, Aß42, phosphorylated and isomerized isoforms pS8-Aß42, isoD7-Aß42). Two interfaces have been identified by docking: they are represented by an extended area at the junction of RAGE domains V and C1 and a smaller area linking C1 and C2 domains. Molecular dynamics (MD) simulations have shown that all Aß isoforms form stable and tightly bound complexes. This indicates that all Aß isoforms potentially can be transported through the cell as part of a complex with RAGE. Modeling of RAGE interaction interfaces with Aß indicates which chemical compounds can potentially be capable of blocking this interaction, and impair the associated pathogenic cascades. The ability of three RAGE inhibitors (RAP, FPS-ZM1 and RP-1) to disrupt the RAGE:Aß interaction has been probed by docking and subsequently the complexes' stability verified by MD. The RP-1 and Aß interaction areas coincide and therefore this inhibitor is very promising for the RAGE:Aß interaction inhibition.

Na,K-ATPase Acts as a Beta-Amyloid Receptor Triggering Src Kinase Activation.

Beta-amyloid (Aß) has a dual role, both as an important factor in the pathology of Alzheimer's disease and as a regulator in brain physiology. The inhibitory effect of Aß42 oligomers on Na,K-ATPase contributes to neuronal dysfunction in Alzheimer's disease. Still, the physiological role of the monomeric form of Aß42 interaction with Na,K-ATPase remains unclear. We report that Na,K-ATPase serves as a receptor for Aß42 monomer, triggering Src kinase activation. The co-localization of Aß42 with α1- and ß1-subunits of Na,K-ATPase, and Na,K-ATPase with Src kinase in SH-SY5Y neuroblastoma cells, was observed. Treatment of cells with 100 nM Aß42 causes Src kinase activation, but does not alter Na,K-ATPase transport activity. The interaction of Aß42 with α1ß1 Na,K-ATPase isozyme leads to activation of Src kinase associated with the enzyme. Notably, prevention of Na,K-ATPase:Src kinase interaction by a specific inhibitor pNaKtide disrupts the Aß-induced Src kinase activation. Stimulatory effect of Aß42 on Src kinase was lost under hypoxic conditions, which was similar to the effect of specific Na,K-ATPase ligands, the cardiotonic steroids. Our findings identify Na,K-ATPase as a Aß42 receptor, thus opening a prospect on exploring the physiological and pathological Src kinase activation caused by Aß42 in the nervous system.

Interaction Interface of Aß42 with Human Na,K-ATPase Studied by MD and ITC and Inhibitor Screening by MD.

Alzheimer's disease (AD) is a neurodegenerative disease accompanied by progressive cognitive and memory dysfunction due to disruption of normal electrotonic properties of neurons and neuronal loss. The Na,K-ATPase interaction with beta amyloid (Aß) plays an important role in AD pathogenesis. It has been shown that Na,K-ATPase activity in the AD brain was significantly lower than those in age-matched control brain. The interaction of Aß42 with Na,K-ATPase and subsequent oligomerization leads to inhibition of the enzyme activity. In this study interaction interfaces between three common Aß42 isoforms, and different conformations of human Na,K-ATPase (α1ß1) have been obtained using molecular modeling, including docking and molecular dynamics (MD). Interaction sites of Na,K-ATPase with Aß42 are localized between extracellular parts of α- and ß- subunits and are practically identical for Na,K-ATPase at different conformations. Thermodynamic parameters for the formation of Na,K-ATPase:Aß42 complex at different conformations acquired by isothermal titration calorimetry (ITC) are similar, which is in line with the data of molecular modeling. Similarity of Na,K-ATPase interaction interfaces with Aß in all conformations allowed us to cross-screen potential inhibitors for this interaction and find pharmaceutical compounds that could block it.

Myeloperoxidase-induced fibrinogen unfolding and clotting.

Due to its unique properties and high biomedical relevance fibrinogen is a promising protein for the development of various matrixes and scaffolds for biotechnological applications. Fibrinogen molecules may form extensive clots either upon specific cleavage by thrombin or in thrombin-free environment, for example, in the presence of different salts. Here, we report the novel type of non-conventional fibrinogen clot formation, which is mediated by myeloperoxidase and takes place even at low fibrinogen concentrations (<0.1 mg/ml). We have revealed fibrillar nature of myeloperoxidase-mediated fibrinogen clots, which differ morphologically from fibrin clots. We have shown that fibrinogen clotting is mediated by direct interaction of myeloperoxidase molecules with the outer globular regions of fibrinogen molecules followed by fibrinogen unfolding from its natural trinodular to a fibrillar structure. We have demonstrated a major role of the Debye screening effect in regulating of myeloperoxidase-induced fibrinogen clotting, which is facilitated by small ionic strength. While fibrinogen in an aqueous solution with myeloperoxidase undergoes changes, the enzymatic activity of myeloperoxidase is not inhibited in excess of fibrinogen. The obtained results open new insights into fibrinogen clotting, give new possibilities for the development of fibrinogen-based functional biomaterials, and provide the novel concepts of protein unfolding.

Zinc Induced Aß16 Aggregation Modeled by Molecular Dynamics.

It is widely accepted that the addition of zinc leads to the formation of neurotoxic nonfibrillar aggregates of beta-amyloid peptides Aß40 and Aß42 and at the same time destabilizes amyloid fibrils. However, the mechanism of the effect of zinc on beta-amyloid is not fully understood. In this study, a fast zinc-induced aggregation of Aß16 (as compared to a system without zinc) via the formation of Aß16 dimers with one zinc ion coordinated in the metal-binding site 11EVHH14, followed by their polymerization, has been studied by molecular dynamics. The best aggregation was shown by the system composed of Aß16 dimers bound by one zinc ion, with no additional zinc in solution. The presence of Aß16 dimers was a major condition, sufficient for fast aggregation into larger complexes. It has been shown that the addition of zinc to a system with already formed dimers does not substantially affect the characteristics and rate of aggregation. At the same time, an excessive concentration of zinc at the early stages of the formation of conglomerates can negatively affect aggregation, since in systems where zinc ions occupied the 11EVHH14 coordination center and the His6 residue of every Aß16 monomer, the aggregation proceeded more slowly and the resulting complexes were not as large as in the zinc-free Aß system. Thus, this study has shown that the formation of Aß16 dimers bound through zinc ions at the 11EVHH14 sites of the peptides plays an important role in the formation of neurotoxic non-fibrillar aggregates of beta-amyloid peptide Aß16. The best energetically favorable structure has been obtained for the complex of two Aß16 dimers with two zinc ions.

Direct interaction between ABCA1 and HIV-1 Nef: Molecular modeling and virtual screening for inhibitors.

HIV-1 infection impairs cellular cholesterol efflux by downmodulating the cholesterol transporter ABCA1, leading to metabolic co-morbidities like cardio-vascular disease. The main mechanism of this effect is impairment by the HIV-1 protein Nef of the ABCA1 interaction with the endoplasmic reticulum chaperone calnexin, which leads to a block in ABCA1 maturation followed by its degradation. However, ABCA1 is also downmodulated by Nef delivered with the extracellular vesicles, suggesting involvement of a direct Nef:ABCA1 interaction at the plasma membrane. Here, we present an optimized model of the Nef:ABCA1 interaction, which identifies interaction sites and provides an opportunity to perform a virtual screening for potential inhibitors. Interestingly, the predicted sites on Nef involved in the ABCA1 interaction overlap with those involved in the interaction with calnexin. The compounds previously shown to block Nef:calnexin interaction were among the top ranking ligands in docking simulations with ABCA1-interacting sites on Nef, suggesting the possibility that both interactions can be inhibited by the same chemical compounds. This study identifies a series of compounds for potential development as inhibitors of Nef-mediated co-morbidities of HIV infection.

Pharmacokinetics and Molecular Modeling Indicate nAChRα4-Derived Peptide HAEE Goes through the Blood-Brain Barrier.

One of the treatment strategies for Alzheimer's disease (AD) is based on the use of pharmacological agents capable of binding to beta-amyloid (Aß) and blocking its aggregation in the brain. Previously, we found that intravenous administration of the synthetic tetrapeptide Acetyl-His-Ala-Glu-Glu-Amide (HAEE), which is an analogue of the 35-38 region of the α4 subunit of α4ß2 nicotinic acetylcholine receptor and specifically binds to the 11-14 site of Aß, reduced the development of cerebral amyloidogenesis in a mouse model of AD. In the current study on three types of laboratory animals, we determined the biodistribution and tissue localization patterns of HAEE peptide after single intravenous bolus administration. The pharmacokinetic parameters of HAEE were established using uniformly tritium-labeled HAEE. Pharmacokinetic data provided evidence that HAEE goes through the blood-brain barrier. Based on molecular modeling, a role of LRP1 in receptor-mediated transcytosis of HAEE was proposed. Altogether, the results obtained indicate that the anti-amyloid effect of HAEE, previously found in a mouse model of AD, most likely occurs due to its interaction with Aß species directly in the brain.

Molecular patterns of oligopeptide hydrocarbons on graphite.

Graphitic materials including graphene, carbon nanotubes and fullerenes, are promising for use in nanotechnology and biomedicine. Non-covalent functionalization by peptides and other organic molecules allows changing the properties of graphitic surfaces in a controlled manner and represents a big potential for fundamental research and applications. Recently described oligopeptide-hydrocarbon derivative N,N'-(decane-1,10-diyl)bis(tetraglycineamide) (GM) is highly prospective for the development of graphitic interfaces in biosensor application as well as in structural biology for improving the quality of high-resolution atomic force microscopy (AFM) visualization of individual biomacromolecules. However, molecular organization of GM on graphitic surfaces is still unknown. In this work, the molecular model of GM at the water/highly oriented pyrolytic graphite (HOPG) interface has been developed basing on the high-resolution AFM and full-atom molecular modeling data. This model explains two periodicities observed in AFM images by GM self-assembly on a HOPG surface with formation of the stacks with the lateral shifts. The obtained results reveal the particular patterns and dynamics of GM molecules adsorbed on graphite and unravel the puzzle of peptide self-assembly on graphitic surfaces.

Tetrapeptide Ac-HAEE-NH2 Protects α4ß2 nAChR from Inhibition by Aß.

The cholinergic deficit in Alzheimer's disease (AD) may arise from selective loss of cholinergic neurons caused by the binding of Aß peptide to nicotinic acetylcholine receptors (nAChRs). Thus, compounds preventing such an interaction are needed to address the cholinergic dysfunction. Recent findings suggest that the 11EVHH14 site in Aß peptide mediates its interaction with α4ß2 nAChR. This site contains several charged amino acid residues, hence we hypothesized that the formation of Aß-α4ß2 nAChR complex is based on the interaction of 11EVHH14 with its charge-complementary counterpart in α4ß2 nAChR. Indeed, we discovered a 35HAEE38 site in α4ß2 nAChR, which is charge-complementary to 11EVHH14, and molecular modeling showed that a stable Aß42-α4ß2 nAChR complex could be formed via the 11EVHH14:35HAEE38 interface. Using surface plasmon resonance and bioinformatics approaches, we further showed that a corresponding tetrapeptide Ac-HAEE-NH2 can bind to Aß via 11EVHH14 site. Finally, using two-electrode voltage clamp in Xenopus laevis oocytes, we showed that Ac-HAEE-NH2 tetrapeptide completely abolishes the Aß42-induced inhibition of α4ß2 nAChR. Thus, we suggest that 35HAEE38 is a potential binding site for Aß on α4ß2 nAChR and Ac-HAEE-NH2 tetrapeptide corresponding to this site is a potential therapeutic for the treatment of α4ß2 nAChR-dependent cholinergic dysfunction in AD.

Influence of pixelization on height measurement in atomic force microscopy.

Though AFM is capable of obtaining sub-angstrom resolution in z-direction, the accurate height measurement of protruding particles is hindered by raster nature of this technique. In this work using Monte Carlo simulations we have quantified the influence of pixelization on the mean AFM apparent height (hmean) of spheres and cylinders. We have demonstrated that for a zero size AFM probe hmean may be increasing, decreasing function of a pixel size, or has more complex character depending on the standard deviation of a particle size. Therefore, AFM pixelization effects may induce both under- and overestimation of the true diameter. The observed complex behavior of hmean is explained by interplay of two opposing factors: the mismatch of the position of the "highest" pixel to the real topographical maximum and higher registration probabilities of larger particles. Consideration of the AFM probe size results in even bigger pixelization induced drops of hmean, which may amount to ∼50% of the true value. The obtained results contribute to AFM data interpretation and methodological aspects of AFM operation in many fields of nanoscience. In particular, they may be used for estimation of true height of nanoparticles from their AFM images obtained with different (even low) pixel resolution.