Calorimetric investigation of copper(II) binding to Aβ peptides: thermodynamics of coordination plasticity

Metal ions have been shown to play a critical role in β-amyloid (Aβ) neurotoxicity, thus prompting an intense investigation into the formation of metal–Aβ complexes. Isothermal titration calorimetry (ITC) has been widely used to determine binding constants (K) for a variety of metal–protein interactions, including those in metal–Aβ complexes. In this study, ITC was used to more fully quantify the thermodynamics (K, ΔG, ΔH, and TΔS) of Cu²⁺ binding to Aβ16, N-acetyl-Aβ16, Aβ28, N-acetyl-Aβ28, and Aβ28 variants (H6A, H13A, H14A) at pH 7.4 and 37 °C. After deconvolution of competing reactions, K for Aβ16 was found to be 1.1 (±0.13) × 10⁹ and is in strong agreement with literature values measured under similar conditions. Further, a similar K value was obtained at two additional concentrations of competing ligand, suggesting that ternary complex formation is not significant. The acetylated peptide analogs reveal a marked decrease in the overall free energy upon binding, which is the result of less favorable enthalpic and entropic contributions. Circular dichroism spectroscopy shows conformational changes that are consistent with these results. Most importantly, data for Aβ28 variants lacking a potential Cu²⁺-binding histidine residue reveal that the overall free energy of binding remains constant, which is the result of entropy/enthalpy compensation. These data provide fundamental thermodynamic evidence for coordination plasticity in Cu²⁺ binding to Aβ and other intrinsically disordered peptides.     

Role of N-terminal residues in Aβ interactions with integrin receptor and cell surface

beta-Amyloid (Aβ) is the primary protein component of senile plaques in Alzheimer's disease (AD) and is believed to play a role in its pathology. To date, the mechanism of action of Aβ in AD is unclear. We and others have observed that Aβ interacts either with or in the vicinity of the α6 sub-unit of integrin, and believe this may be important in its interaction with neuronal cells. In this study, we used confocal microscopy and flow cytometry to explore the residue specific interactions of Aβ40 with the cell surface and the α6 integrin receptor sub-unit. We probed the importance of the RHD sequence in Aβ40 and found that removal of the residues or their mutation using the Aβ8-40 or the D7N early onset AD sequence, respectively, led to a greater interaction between Aβ40 and an antibody bound to the α6-integrin sub-unit, as measured by fluorescence resonance energy transfer (FRET). These results suggest that the RHD sequence of Aβ40 does not mediate Aβ–α6 integrin interactions. However, the cyclic RGD mimicking peptide, Cilengitide, reduced the measured interaction between Aβ40 fibrils without the RHD sequence and an antibody bound to the α6-integrin sub-unit. We further probed the role of electrostatic forces on Aβ40–cell interactions and observed that the Aβ sequence that included the N-terminal segment of the peptide had reduced cellular binding at low salt concentrations, suggesting that its first 7 residues contribute to an electrostatic repulsion for the cell surface. These findings contribute to our understanding of Aβ–cell surface interactions and may provide insight into development of novel strategies to block Aβ–cell interactions that contribute to pathology in Alzheimer's disease.     

Apolipoprotein E and Alzheimer's disease: the influence of apolipoprotein E on amyloid-β and other amyloidogenic proteins: Thematic Review Series: ApoE and Lipid Homeostasis in Alzheimer's Disease

Alzheimer's disease (AD) is one of the fastest-growing causes of death and disability in persons 65 years of age or older, affecting more than 5 million Americans alone. Clinical manifestations of AD include progressive decline in memory, executive function, language, and other cognitive domains. Research efforts within the last three decades have identified APOE as the most significant genetic risk factor for late-onset AD, which accounts for >99% of cases. The apoE protein is hypothesized to affect AD pathogenesis through a variety of mechanisms, from its effects on the blood-brain barrier, the innate immune system, and synaptic function to the accumulation of amyloid-β (Aβ). Here, we discuss the role of apoE on the biophysical properties and metabolism of the Aβ peptide, the principal component of amyloid plaques and cerebral amyloid angiopathy (CAA). CAA is characterized by the deposition of amyloid proteins (including Aβ) in the leptomeningeal medium and small arteries, which is found in most AD cases but sometimes occurs as an independent entity. Accumulation of these pathologies in the brain is one of the pathological hallmarks of AD. Beyond Aβ, we will extend the discussion to the potential role of apoE on other amyloidogenic proteins found in AD, and also a number of diverse neurodegenerative diseases.     

Design,synthesis, and bioevaluation of benzamides: Novel acetylcholinesterase inhibitors with multi-functions on butylcholinesterase,Aβ aggregation,and β-secretase

Alzheimer’s disease (AD) is a multifactorial syndrome with several target proteins contributing to its etiology. In this study, we conducted a structure-based design and successfully produced a series of new multi-site AChE inhibitors with a novel framework. Compound 2e, characterized by a central benzamide moiety linked to an isoquinoline at one side and acetophenone at the other, was the most potent candidate with K_i of 6.47 nM against human AChE. Particularly, it showed simultaneous inhibitory effects against BChE, Aβ aggregation, and β-secretase. We therefore conclude that compound 2e is a very promising multi-function lead for the treatment of AD.     

Scrutiny of chain-length and N-terminal effects in α-helix folding: a molecular dynamics study on polyalanine peptides

Protein folding remains an unsolved problem as main-chain, side-chain, and solvent interactions remain entangled and have been hard to resolve. Polyalanines are promising models to analyze protein folding initiation and propagation structurally as well as energetically. In the present work, the effect of chain-length and N-terminal residue stereochemistry in polyalanine peptides are investigated for their role in the nucleation of α-helical conformation. The end-protected polyalanine peptides, tetra-alanine, Ac-^LAla₄-NHMe (Ia) and Ac-^DAla-^LAla₃-NHMe (Ib), hexa-alanine, Ac-^LAla₆-NHMe (IIa) and Ac-^DAla-^LAla₅-NHMe (IIb), and octa-alanine, Ac-^LAla₈-NHMe (IIIa) and Ac-^DAla-^LAla₇-NHMe (IIIb), are assessed as chain-length and stereochemical-structure perturbed models. The appreciable variations in the sampling of α-helical conformation, including a sampling of α-helix folds, due to the cooperative effect of chain-length and N-terminal residue stereochemistry have been noted. The electrostatics of α-helical conformation rather than the conformational entropy of the main-chain appear to be decisive in the initiation of α-helix folding. The results of the present work will enhance our understanding on the nucleation of α-helical conformation in short peptides and aid in the design of novel peptides with α-helical structure that can modulate disease-related protein–protein interactions.     

Preconcentration with Bacillus subtilis–Immobilized Amberlite XAD-16: Determination of Cu2+ and Ni2+ in River,Soil, and Vegetable Samples

Solid-phase extraction (SPE) method was developed for the preconcentration of Cu²⁺ and Ni²⁺ before their determination by inductively coupled plasma optical emission spectrometry (ICP-OES). Bacillus subtilis–immobilized Amberlite XAD-16 was used as biosorbent. Effects of critical parameters such as pH, flow rate of samples, amount of Amberlite XAD-16 and biosorbent, sample volume, eluent type, and volume and concentration of eluent on column preconcentration of Cu²⁺ and Ni²⁺ were optimized. Applicability of the method was validated through the analysis of the certified reference tea sample (NCS ZC73014). Sensitivity of ICP-OES was improved by 36.4-fold for Cu²⁺ and 38.0-fold for Ni²⁺ by SPE-ICP-OES method. Limit of quantitation (LOQ) was found to be 0.7 and 1.1 ng/ml for Cu²⁺ and Ni²⁺, respectively. Concentrations of Cu²⁺ and Ni²⁺ were determined by ICP-OES after application of developed method. Relative standard deviations (RSDs) were lower than 4.9% for Cu²⁺ and 7.9% for Ni²⁺. The Tigris River that irrigates a large agricultural part of Southeast Turkey is polluted by domestic and industrial wastes. Concentrations of Cu²⁺ and Ni²⁺ were determined in water, soil, and some edible vegetables as a biomonitor for heavy metal pollution.     

Aβ peptide interactions with isoflurane, propofol, thiopental and combined thiopental with halothane: A NMR study

Aβ peptide is the major component of senile plaques (SP) which accumulates in AD (Alzheimer's disease) brain. Reports from different laboratories indicate that anesthetics interact with Aβ peptide and induce Aβ oligomerization. The molecular mechanism of Aβ peptide interactions with these anesthetics was not determined. We report molecular details for the interactions of uniformly ¹⁵N labeled Aβ40 with different anesthetics using 2D nuclear magnetic resonance (NMR) experiments. At high concentrations both isoflurane and propofol perturb critical amino acid residues (G29, A30 and I31) of Aβ peptide located in the hinge region leading to Aβ oligomerization. In contrast, these three specific residues do not interact with thiopental and subsequently no Aβ oligomerization was observed. However, studies with combined anesthetics (thiopental and halothane), showed perturbation of these residues (G29, A30 and I31) and subsequently Aβ oligomerization was found. Perturbation of these specific Aβ residues (G29, A30 and I31) by different anesthetics could play an important role to induce Aβ oligomerization.     

Impact of fluorination on proteolytic stability of peptides: a case study with α-chymotrypsin and pepsin

Protease stability is a key consideration in the development of peptide-based drugs. A major approach to increase the bioavailability of pharmacologically active peptides is the incorporation of non-natural amino acids. Due to the unique properties of fluorine, fluorinated organic molecules have proven useful in the development of therapeutically active small molecules as well as in materials and crop science. This study presents data on the ability of fluorinated amino acids to influence proteolytic stability when present in peptide sequences that are based on ideal protease substrates. Different model peptides containing fluorinated amino acids or ethylglycine in the P2, P1′or P2′ positions were designed according to the specificities of the serine protease, α-chymotrypsin (EC 3.4.21.1) or the aspartic protease, pepsin (EC 3.4.23.1). The proteolytic stability of the peptides toward these enzymes was determined by an analytical RP-HPLC assay with fluorescence detection and compared to a control sequence. Molecular modeling was used to support the interpretation of the structure–activity relationship based on the analysis of potential ligand-enzyme interactions. Surprisingly, an increase in proteolytic stability was observed only in a few cases. Thus, this systematic study shows that the proteolytic stability of fluorinated peptides is not predictable, but rather is a very complex phenomenon that depends on the particular enzyme, the position of the substitution relative to the cleavage site and the fluorine content of the side chain.     

Trace metal contamination initiates the apparent auto-aggregation,amyloidosis, and oligomerization of Alzheimer’s Aβ peptides

Nucleation-dependent protein aggregation (seeding) and amyloid fibril-free formation of soluble SDS-resistant oligomers (oligomerization) by hydrophobic interaction is an in vitro model thought to propagate -amyloid (A) deposition, accumulation, and incur neurotoxicity and synaptotoxicity in Alzheimers disease (AD), and other amyloid-associated neurodegenerative diseases. However, A is a high-affinity metalloprotein that aggregates in the presence of biometals (zinc, copper, and iron), and neocortical A deposition is abolished by genetic ablation of synaptic zinc in transgenic mice. We now present in vitro evidence that trace (0.8 µM) levels of zinc, copper, and iron, present as common contaminants of laboratory buffers and culture media, are the actual initiators of the classic A1–42-mediated seeding process and A oligomerization. Replicating the experimental conditions of earlier workers, we found that the in vitro precipitation and amyloidosis of A1–40 (20 µM) initiated by A1–42 (2 µM) were abolished by chelation of trace metal contaminants. Further, metal chelation attenuated formation of soluble A oligomers from a cell-free culture medium. These data suggest that protein self-assembly and oligomerization are not spontaneous in this system as previously thought, and that there may be an obligatory role for metal ions in initiating A amyloidosis and oligomerization.     

Effect of aluminum combined with ApoEε4 on Tau phosphorylation and Aβ deposition

BackgroundAluminum is an environmental neurotoxin widely exposed to animals and humans. Studies have shown that Alzheimer's disease (AD) is characterized by abnormally phosphorylated tau and Aβ deposition, aluminum exposure can lead to abnormal phosphorylated tau and Aβ deposition. Numerous epidemiological data and studies have confirmed that ApoEε4 is a risk factor for AD. However, whether there is an interaction effect between aluminum and ApoEε4 has yet to be verified.MethodsSH-SY5Y cells were exposed with AlCl₃ and transfected with ApoEε4 respectively. The experimental groups included the blank control group, the low dose group (200 μM AlCl₃), the medium dose group (400 μM AlCl₃), the high dose group (800 μM AlCl₃), empty plasmid group, ApoEε4 group and 400 μM AlCl₃+ApoEε4 group. The cell viability was determined by CCK-8 kit after transfection for 48 h.The contents of total tau proteins, tau-181, tau-231, tau-262, tau-396 and Aβ42, were determined by ELISA kit. The interaction between AlCl₃ and ApoEε4 was analyzed by factorial design.ResultsWith the increase of aluminum exposure, SH-SY5Y cell viability decreased, and the expression of the total tau, tau-181, tau-231, tau-262, tau-396 and Aβ content increased. The viability of cells transfected with ApoEε4 is significantly lower than control group, and the expressions of total tau, tau-181, tau-231, tau-262, tau-396 and Aβ in ApoEε4 transfected cells were significantly higher than control group. The viability of cells treated with AlCl₃ plus ApoEε4 was lower than those treated with, either AlCl₃, or ApoEε4. The expression of total tau, tau-181, tau-231, tau-262, tau-396 and Aβ in the cells treated with AlCl₃ plus ApoEε4 were significantly higher than those in other groups (p < 0.05). Moreover, analyzing data based on the factorial design, there was existed an interaction between AlCl₃ and ApoEε4 (p < 0.05).ConclusionAl and ApoEε4 gene can cause morphological changes of SH-SY5Y cells, reduce cell activity, and have obvious cytotoxic effects, and increase the phosphorylation levels of tau and the deposition of Aβ increases. In the presence of both Al and ApoEε4 genes, the two factors interact with each other and show a synergistic effect.     

Functions of Aβ, sAPPα and sAPPβ : similarities and differences

Amyloid peptide (Aβ) is derived from the cleavage of amyloid precursor protein (APP), which also generates the soluble peptide APPβ (sAPPβ). An antagonist and major APP metabolic pathway involves cleavage by alpha secretase, which releases sAPPα. Although soluble Aβ oligomers are neurotoxic, Aβ monomers share similar properties with sAPPα. These include neurotrophic and neuroprotective effects, as well as stimulation of neural-progenitor proliferation. The properties of Aβ monomers and the neurotrophic capacity of sAPPβ to stimulate axonal outgrowth suggest that Aβ production is not deleterious per se. Consequently, therapeutic strategies for Alzheimer's disease that are targeted at Aβ-cleaving enzymes should modulate rather than inhibit Aβ generation. These strategies should focus on the factors that induce the conversion of Aβ monomers into toxic soluble oligomers. Another interesting therapeutic approach is to focus on the mechanisms of the different properties of sAPPα. Indeed, increasing sAPPα levels could shift proliferating cells towards tumorigenesis. In contrast to its neuroprotective effects, sAPPα is also able to activate microglia, leading to neurotoxicity. Understanding the mechanisms that underlie the different properties of sAPPα could therefore lead to the development of therapeutic strategies against Alzheimer's disease, which could be curative as well as preventive.     

Mechanism of action of thrombin on fibrinogen: Reaction of the N-terminal CNBr fragment from the Bβ chain of bovine fibrinogen with bovine thrombin

The N-terminal cyanogen bromide fragment from the Bβ chain of bovine fibrinogen was isolated, and its molecular weight was estimated to be approximately 14,000–15,500. The ratio of the Michaelis-Menten constants, $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, for its hydrolysis by bovine thrombin was found to be 3 × 10^?7 [(NIH unit/liter)s]^?1, indicating that the Bβ fragment is a poor substrate for thrombin compared to the corresponding Aα chain fragment. This value of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ is too small to account for the rate of release of fibrinopeptide B from fibrinogen by thrombin. It is suggested that, while the Aα chain contains all of the amino acid residues necessary to interact with thrombin, the Bβ chain does not; i.e., some of the binding sites that are used in the hydrolysis of the Bβ chain are assumed to be located on either the α or γ chains of fibrinogen. An alternative hypothesis is that, after the Bβ chain fragment is removed from the fibrinogen molecule, it does not have the proper conformation to be hydrolyzed by thrombin.     

Cell-penetrating fusion peptides OD1 and OD2 interact with Bcr–Abl and influence the growth and apoptosis of K562 cells

The Bcr–Abl oncoprotein is the cause of chronic myelogenous leukemia (CML). Crystal structure analysis suggests that Bcr_30–63 is the core of the Bcr–Abl oligomerization interface for aberrant kinase activity; however, the precise role of other residues of Bcr_1–72 excluding Bcr_30–63 have not been evaluated. In this study, Bcr_30–63 was named OD2 and other residues of Bcr_1–72 were named OD1. Cytoplasmic transduction peptide (CTP) was used to carry molecules into cytoplasm. CTP-OD1 and CTP-OD2 fusion peptides were expressed from a cold-inducible expression system. Our results demonstrated that both fusion peptides could localize into the cytoplasm, specifically interact with the Bcr–Abl protein and further inhibit growth, induce apoptosis, and decrease the phosphorylation of Bcr–Abl in K562 cell lines. However, the viability of THP-1, a Bcr–Abl negative cell line, was unaffected. These results suggested that CTP-OD1 and CTP-OD2 may be an attractive therapeutic option to inhibit the activation of Bcr–Abl kinase in CML.     

Impact of Ca2+ on structure of soybean CDPKβ and accessibility of the Tyr-24 autophosphorylation site

Several plant CDPKs were recently shown to be dual specificity kinases rather than Ser/Thr kinases as traditionally classified by sequence analysis. In the present study we confirm the autophosphorylation of recombinant soybean His₆-GmCDPKβ at the Tyr-24 site using sequence- and modification- specific antibodies. Homology modeling of soybean CDPKβ based on recent structures determined for several apicomplexan CDPKs suggested that phosphotyrosine-24 may be inaccessible to phosphatases. However, we report that dephosphorylation of CDPKβ by the protein tyrosine phosphatase 1B, PTP1B, was not restricted in the presence of calcium. Thus, despite conformational changes likely associated with calcium binding to the CDPKs, phosphotyrosine sites remain fully accessible to dephosphorylation suggesting the possibility of conformational breathing and flexing.     

Effect of Cu2+ on Complex Formation Between a Deoxyribozyme and Its Substrates†

Abstract

Effect of metal ions on secondary-structure formation of a deoxyribozyme-two substrates complex has been investigated by using surface plasmon resonance and secondary-structure predicting calculation. The result showed that Cu²⁺ not only acts on the ligation reaction but also plays the role of a promoter which makes an active conformation of the deoxyribozyme-substrate complex.