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Post-translational modifications of beta-amyloid (Aβ) play an important role in the pathogenesis of Alzheimer’s disease (AD). Aβ modifications such as Ser8 phosphorylation (pS8-Aβ42) and Asp7 isomerization (iso-Aβ42) can significantly alter the properties of Aβ and have been detected in vivo. One of the reasons for the different pathogenicity of Aβ isoforms may be the activation of different signaling cascades leading to changes in the mechanical properties of cells. In this paper, we used correlative scanning ion-conductance microscopy (SICM) and Pt-nanoelectrodes to compare the effects of Aβ isoforms on the Young’s modulus of SH-SY5Y cells and the level of ROS. It was found that unmodified Aβ42 resulted in the largest increase in cell Young’s modulus of all isoforms after 4 h of incubation, while pS8-Aβ42 induced the greatest increase in stiffness and ROS levels after 24 h of incubation. Analysis of signaling proteins involved in the regulation of the actin cytoskeleton showed that Aβ42, pS8-Aβ42 and iso-Aβ42 have different effects on cofilin, GSK3β, LIMK, ERK and p38. This indicates that post-translational modifications of Aβ modulate its effect on neuronal cells through the activation of various signaling cascades, which affects the mechanical properties of cells.

Background/Objectivities: The presence of beta-amyloid plaques is a part of the pathogenesis of Alzheimer’s disease, but there is currently no universally accepted method for magnetic resonance (MR) imaging of the disease. However, it is known that magnetic nanoparticles (MNPs) can improve the T2 contrast in MR images of various targets. Methods: We used cubic MNPs, which were produced by thermal decomposition and then it was covalently bonded to a modified fluorescently labeled tetrapeptide, HAEE-Cy5, for visualizing beta-amyloid plaques. The interaction of MNPs-HAEE-Cy5 and beta-amyloid was determinate by confocal microscopy using SH-SY5Y cell line. Results: MNPs exhibit relatively high relaxivity (approximately 200 mM−1s−1), which is crucial for enhancing target visibility in MR imaging. HAEE provides targeted delivery of MNPs by specifically interacting with beta-amyloid, while the fluorescent label Cy5 enables monitoring the efficacy of the interaction through confocal microscopy. Conclusions: The MNPs modified with HAEE-Cy5 demonstrated excellent binding to beta-amyloid plaques in vitro, as shown by experiments on the SH-SY5Y cell line. These results suggest that the proposed method has potential for use in future MR imaging studies of Alzheimer’s disease.

Objective: Multidrug-resistant, highly pathogenic Escherichia coli strains are the primary causative agents of intestinal and extraintestinal human diseases. The extensive utilization of antibiotics for farm animals has been identified as a contributing factor to the emergence and dissemination of E. coli strains that exhibit multidrug resistance and possess high pathogenic potential. Consequently, a significant research objective is to examine the genetic diversity of pathogenic E. coli strains and to identify those that may pose a threat to human health. Methods: In this study, we present the results of genome sequencing and analysis, as well as the physiological characterization of E. coli strain APEC 36, which was isolated from the liver of a broiler chicken with generalized colibacillosis. Results: We found that APEC 36 possess a number of mechanisms of antibiotic resistance, including antibiotic efflux, antibiotic inactivation, and antibiotic target alteration/replacement/protection. The most widely represented group among these mechanisms was that of antibiotic efflux. This finding is consistent with the strain’s documented resistance to multiple antibiotics. APEC 36 has an extremely rare variant of the beta-lactamase CTX-M-169. Notwithstanding the multitude of systems for interfering with foreign DNA present in the strain, seven plasmids have been identified, three of which may possess novel replication origins. Additionally, qnrS1, which confers resistance to fluoroquinolones, was found to be encoded in the genome rather than in the plasmid. This suggests that the determinants of antibiotic resistance may be captured in the genome and stably transmitted from generation to generation. Conclusions: The APEC 36 strain has genes for toxins, adhesins, protectins, and an iron uptake system. The obtained set of genetic and physiological characteristics allowed us to assume that this strain has a high pathogenic potential for humans.

Background/Objectives: The development of blood tests for the early detection of individual predisposition to socially significant diseases remains a pressing issue. Methods: In this pilot study, multiple reaction monitoring mass spectrometry (MRM-MS) with a BAK-270 assay was applied for protein concentrations analysis in blood plasma from 21 healthy volunteers of the European cohort. Results: The levels of 138 plasma proteins were reliably and precisely quantified in no less than 50% of samples. The quantified proteins included 66 FDA-approved markers of cardiovascular diseases (CVD), and other potential biomarkers of pathologies such as cancer, diabetes mellitus, and Alzheimer’s disease. The analysis of individual variations of the plasma proteins revealed significant differences between the male (11) and female (10) groups. In total, fifteen proteins had a significantly different concentration in plasma; this included four proteins that exhibited changes greater than ±1.5-fold, three proteins (RBP4, APCS, and TTR) with higher levels in males, and one (SHBG) elevated in females. The obtained results demonstrated considerable agreement with the data collected from 20 samples of a North American cohort, which were analyzed with the similar MRM assay. The most significant differences between the cohorts of the two continents were observed in the level of 42 plasma proteins (including 24 FDA markers), of which 17 proteins showed a ≥1.5-fold change, and included proteins increased in North Americans (APOB, CRTAC1, C1QB, C1QC, C9, CRP, HP, IGHG1, IGKV4-1, SERPING1, RBP4, and AZGP1), as well as those elevated in Europeans (APOF, CD5L, HBG2, SELPLG, and TNA). Conclusions: The results suggest a different contribution of specific (patho)physiological pathways (e.g., immune system and blood coagulation) to the development of socially significant diseases in Europeans and North Americans, and they should be taken into account when refining diagnostic panels.

One of the hallmarks of Alzheimer's disease (AD) is the accumulation of beta-amyloid peptide (Aβ) leading to formation of soluble neurotoxic Aβ oligomers and insoluble amyloid plaques in various parts of the brain. Aβ undergoes post-translational modifications that alter its pathogenic properties. Aβ is produced not only in brain, but also in the peripheral tissues. Such Aβ, including its post-translationally modified forms, can enter the brain from circulation by binding to RAGE and contribute to the pathology of AD. However, the transport of modified forms of Aβ across the blood–brain barrier (BBB) has not been investigated. Here, we used a transwell BBB model as a controlled environment for permeability studies. We found that Aβ42 containing isomerized Asp7 residue (iso-Aβ42) and Aβ42 containing phosphorylated Ser8 residue (pS8-Aβ42) crossed the BBB better than unmodified Aβ42, which correlated with different contribution of endocytosis mechanisms to the transport of these isoforms. Using microscale thermophoresis, we observed that RAGE binds to iso-Aβ42 an order of magnitude weaker than to Aβ42. Thus, post-translational modifications of Aβ increase the rate of its transport across the BBB and modify the mechanisms of the transport, which may be important for AD pathology and treatment.

Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown.This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O2 saturation to 50% or less observed in healthy humans while at high altitude, or in red blood cell suspensions results in rising of the intraerythrocytic GSH level that is proportional to the reduction in Hb O2 saturation. This effect was not caused by the stimulation of GSH de novo synthesis or its release during deglutathionylation of Hb's cysteines. Using isothermal titration calorimetry and in silico modeling, we observed the non-covalent binding of four molecules of GSH to oxy-Hb and the release of two of them upon deoxygenation. Localization of the GSH binding sites within the Hb molecule was identified. Oxygen-dependent binding of GSH to oxy-Hb and its release upon deoxygenation occurred reciprocally to the binding and release of 2,3-bisphosphoglycerate. Furthermore, noncovalent binding of GSH to Hb moderately increased Hb oxygen affinity. Taken together, our findings have identified an adaptive mechanism by which red blood cells may provide an advanced antioxidant defense to respond to oxidative challenges immediately upon deoxygenation.

The biogenic polyamines, spermidine (Spd) and spermine (Spm), are present at millimolar concentrations in all eukaryotic cells, where they participate in the regulation of vitally important cellular functions. Polyamine analogs and derivatives are a traditional and important instrument for the investigation of the cellular functions of polyamines, enzymes of their metabolism, and the regulation of the biosynthesis of antizyme—a key downregulator of polyamine homeostasis. Here, we describe convenient gram-scale syntheses of a set of C-methylated analogs of Spd. The biochemical properties of these compounds and the possibility for the regulation of their activity by moving a methyl group along the polyamine backbone and by changing the stereochemistry of the chiral center(s) are discussed.

Bacterial cystathionine γ-lyase (bCSE) is the main producer of H2S in pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, etc. The suppression of bCSE activity considerably enhances the sensitivity of bacteria to antibiotics. Convenient methods for the efficient synthesis of gram quantities of two selective indole-based bCSE inhibitors, namely (2-(6-bromo-1H-indol-1-yl)acetyl)glycine (NL1), 5-((6-bromo-1H-indol-1-yl)methyl)- 2-methylfuran-3-carboxylic acid (NL2), as well as a synthetic method for preparation 3-((6-(7-chlorobenzo[b]thiophen-2-yl)-1H-indol-1-yl)methyl)- 1H-pyrazole-5-carboxylic acid (NL3), have been developed. The syntheses are based on the use of 6-bromoindole as the main building block for all three inhibitors (NL1, NL2, and NL3), and the designed residues are assembled at the nitrogen atom of the 6-bromoindole core or by the substitution of the bromine atom in the case of NL3 using Pd-catalyzed cross-coupling. The developed and refined synthetic methods would be significant for the further biological screening of NL-series bCSE inhibitors and their derivatives.

Abstract Background Beta-amyloid peptide (Aβ) is the key protein in the pathogenesis of Alzheimer’s disease, the most common age-related neurodegenerative disorder in humans. Aβ peptide induced pathological phenotypes in different model organisms include neurodegeneration and lifespan decrease. However, recent experimental evidence suggests that Aβ may utilize oligomerization and fibrillization to function as an antimicrobial peptide (AMP), and protect the host from infections. We used the power of Drosophila model to study mechanisms underlying a dual role for Aβ peptides. Results We investigated the effects of Drosophila treatment with three Aβ42 peptide isoforms, which differ in their ability to form oligomers and aggregates on the lifespan, locomotor activity and AMP genes expression. Aβ42 slightly decreased female’s median lifespan (by 4.5%), but the effect was not related to the toxicity of peptide isoform. The lifespan and relative levels of AMP gene expression in male flies as well as locomotor activity in both sexes were largely unaffected by Aβ42 peptide treatment. Regardless of the effects on lifespan, Aβ42 peptide treatment induced decrease in AMP genes expression in females, but the effects were not robust. Conclusions The results demonstrate that chronic treatment with Aβ42 peptides does not drastically affect fly aging or immunity.

Abstract Background S-glutathionylation is the formation of disulfide bonds between the tripeptide glutathione and cysteine residues of the protein, protecting them from irreversible oxidation and in some cases causing change in their functions. Regulatory glutathionylation of proteins is a controllable and reversible process associated with cell response to the changing redox status. Prediction of cysteine residues that undergo glutathionylation allows us to find new target proteins, which function can be altered in pathologies associated with impaired redox status. We set out to analyze this issue and create new tool for predicting S-glutathionylated cysteine residues. Results One hundred forty proteins with experimentally proven S-glutathionylated cysteine residues were found in the literature and the RedoxDB database. These proteins contain 1018 non-S-glutathionylated cysteines and 235 S-glutathionylated ones. Based on 235 S-glutathionylated cysteines, non-redundant positive dataset of 221 heptapeptide sequences of S-glutathionylated cysteines was made. Based on 221 heptapeptide sequences, a position-specific matrix was created by analyzing the protein sequence near the cysteine residue (three amino acid residues before and three after the cysteine). We propose the method for calculating the glutathionylation propensity score, which utilizes the position-specific matrix and a criterion for predicting glutathionylated peptides. Conclusion Non-S-glutathionylated sites were enriched by cysteines in − 3 and + 3 positions. The proposed prediction method demonstrates 76.6% of correct predictions of S-glutathionylated cysteines. This method can be used for detecting new glutathionylation sites, especially in proteins with an unknown structure.

Phosphorylation of beta-amyloid peptide (Aβ) at the Ser8 residue affects its neurotoxicity, metal-dependent oligomerisation, amyloidogenicity, and other pathogenic properties. Phosphorylated Aβ (pS8-Aβ) was detected in vivo in AD model mice and in the brains of patients with AD. However, the pS8-Aβ production and the regulation of its levels have not been previously studied in detail. In this paper, immunochemical methods together with radioactive labelling were used to study the Aβ phosphorylation by intracellular and surface protein kinases of HEK293 cells and brain endothelial cells (bEnd.3). It was found that HEK293 robustly phosphorylated Aβ, likely with contribution from casein kinase 2 (CK2), whereas in bEnd.3, the activity of Aβ phosphorylation was relatively low. Further, the study showed that both HEK293 and bEnd.3 could dephosphorylate pS8-Aβ, mainly due to the activity of protein phosphatases PP1 and PP2A. The Aβ dephosphorylation efficiency in bEnd.3 was three times higher than in HEK293, which correlated with the reduced abundance of pS8-Aβ in vascular amyloid deposits of patients with AD compared to senile plaques. These data suggest an important role of CK2, PP1, and PP2A as regulators of Aβ phosphorylation, and point to the involvement of the blood–brain barrier in the control of Aβ modification levels.

Heptose phosphates—unique linkers between endotoxic lipid A and O-antigen in the bacterial membrane—are pathogen-associated molecular patterns recognized by the receptors of the innate immune system. Understanding the mechanisms of immune system activation is important for the development of therapeutic agents to combat infectious diseases and overcome antibiotic resistance. However, in practice, it is difficult to obtain a substantial amount of heptose phosphates for biological studies due to the narrow scope of the reported synthetic procedures. We have optimized and developed an inexpensive and convenient synthesis for the first performed gram-scale production of 1-O-methyl d-glycero-α-d-gluco-heptoside 7-phosphate from readily available d-glucose. Scaling up to such amounts of the product, we have increased the efficiency of the synthesis and reduced the number of steps of the classical route through the direct phosphorylation of the O6,O7-unprotected heptose. The refined method could be of practical value for further biological screening of heptose phosphate derivatives.

Beta-amyloid (Aβ) 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 Aβ42 oligomers on Na,K-ATPase contributes to neuronal dysfunction in Alzheimer’s disease. Still, the physiological role of the monomeric form of Aβ42 interaction with Na,K-ATPase remains unclear. We report that Na,K-ATPase serves as a receptor for Aβ42 monomer, triggering Src kinase activation. The co-localization of Aβ42 with α1- and β1-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 Aβ42 causes Src kinase activation, but does not alter Na,K-ATPase transport activity. The interaction of Aβ42 with α1β1 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 Aβ-induced Src kinase activation. Stimulatory effect of Aβ42 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 Aβ42 receptor, thus opening a prospect on exploring the physiological and pathological Src kinase activation caused by Aβ42 in the nervous system.

Impaired lipopolysaccharide biosynthesis in Gram-negative bacteria results in the “deep rough” phenotype, which is characterized by increased sensitivity of cells to various hydrophobic compounds, including antibiotics novobiocin, actinomycin D, erythromycin, etc. The present study showed that E. coli mutants carrying deletions of the ADP-heptose biosynthesis genes became hypersensitive to a wide range of antibacterial drugs: DNA gyrase inhibitors, protein biosynthesis inhibitors (aminoglycosides, tetracycline), RNA polymerase inhibitors (rifampicin), and β-lactams (carbenicillin). In addition, it was found that inactivation of the gmhA, hldE, rfaD, and waaC genes led to dramatic changes in the redox status of cells: a decrease in the pool of reducing NADPH and ATP equivalents, the concentration of intracellular cysteine, a change in thiol homeostasis, and a deficiency in the formation of hydrogen sulfide. In “deep rough” mutants, intensive formation of reactive oxygen species was observed, which, along with a lack of reducing agents, such as reactive sulfur species or NADPH, leads to oxidative stress and an increase in the number of dead cells in the population. Within the framework of modern ideas about the role of oxidative stress as a universal mechanism of the bactericidal action of antibiotics, inhibition of the enzymes of ADP-heptose biosynthesis is a promising direction for increasing the effectiveness of existing antibiotics and solving the problem of multidrug resistance.

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 (Aβ) 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 Aβ42 with Na,K-ATPase and subsequent oligomerization leads to inhibition of the enzyme activity. In this study interaction interfaces between three common Aβ42 isoforms, and different conformations of human Na,K-ATPase (α1β1) have been obtained using molecular modeling, including docking and molecular dynamics (MD). Interaction sites of Na,K-ATPase with Aβ42 are localized between extracellular parts of α- and β- subunits and are practically identical for Na,K-ATPase at different conformations. Thermodynamic parameters for the formation of Na,K-ATPase:Aβ42 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 Aβ in all conformations allowed us to cross-screen potential inhibitors for this interaction and find pharmaceutical compounds that could block it.

Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD) and nucleotide binding domain (NBD) of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines’ thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG). Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459) were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor function of the Na,K-ATPase and alter responses of the enzyme to hypoxia or upon treatment with cardiotonic steroids.

One of the treatment strategies for Alzheimer’s disease (AD) is based on the use of pharmacological agents capable of binding to beta-amyloid (Aβ) 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 α4β2 nicotinic acetylcholine receptor and specifically binds to the 11–14 site of Aβ, 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 Aβ species directly in the brain.

The important role of miRNA in cell proliferation and differentiation has raised interest in exogenous ribonucleases (RNases) as tools to control tumour-associated intracellular and extracellular miRNAs. In this work, we evaluated the effects of the RNase binase from Bacillus pumilus on small non-coding regulatory RNAs in the context of mouse RLS40 lymphosarcoma inhibition. In vitro binase exhibited cytotoxicity towards RLS40 cells via apoptosis induction through caspase-3/caspase-7 activation and decreased the levels of miR-21a, let-7g, miR-31 and miR-155. Intraperitoneal injections of binase in RLS40-bearing mice resulted in the retardation of primary tumour growth by up to 60% and inhibition of metastasis in the liver by up to 86%, with a decrease in reactive inflammatory infiltration and mitosis in tumour tissue. In the blood serum of binase-treated mice, decreases in the levels of most studied miRNAs were observed, excluding let-7g, while in tumour tissue, the levels of oncomirs miR-21, miR-10b, miR-31 and miR-155, and the oncosuppressor let-7g, were upregulated. Analysis of binase-susceptible miRNAs and their regulatory networks showed that the main modulated events were transcription and translation control, the cell cycle, cell proliferation, adhesion and invasion, apoptosis and autophagy, as well as some other tumour-related cascades, with an impact on the observed antitumour effects.

The coordination of zinc ions by histidine residues of amyloid-beta peptide (Aβ) plays a critical role in the zinc-induced Aβ aggregation implicated in Alzheimer’s disease (AD) pathogenesis. The histidine to arginine substitution at position 6 of the Aβ sequence (H6R, English mutation) leads to an early onset of AD. Herein, we studied the effects of zinc ions on the aggregation of the Aβ42 peptide and its isoform carrying the H6R mutation (H6R-Aβ42) by circular dichroism spectroscopy, dynamic light scattering, turbidimetric and sedimentation methods, and bis-ANS and thioflavin T fluorescence assays. Zinc ions triggered the occurrence of amorphous aggregates for both Aβ42 and H6R-Aβ42 peptides but with distinct optical properties. The structural difference of the formed Aβ42 and H6R-Aβ42 zinc-induced amorphous aggregates was also supported by the results of the bis-ANS assay. Moreover, while the Aβ42 peptide demonstrated an increase in the random coil and β-sheet content upon complexing with zinc ions, the H6R-Aβ42 peptide showed no appreciable structural changes under the same conditions. These observations were ascribed to the impact of H6R mutation on a mode of zinc/peptide binding. The presented findings further advance the understanding of the pathological role of the H6R mutation and the role of H6 residue in the zinc-induced Aβ aggregation.

Cerebral β-amyloidosis, an accumulation in the patient’s brain of aggregated amyloid-β (Aβ) peptides abnormally saturated by divalent biometal ions, is one of the hallmarks of Alzheimer’s disease (AD). Earlier, we found that exogenously administrated synthetic Aβ with isomerized Asp7 (isoD7-Aβ) induces Aβ fibrillar aggregation in the transgenic mice model of AD. IsoD7-Aβ molecules have been implied to act as seeds enforcing endogenous Aβ to undergo pathological aggregation through zinc-mediated interactions. On the basis of our findings on zinc-induced oligomerization of the metal-binding domain of various Aβ species, we hypothesize that upon phosphorylation of Ser8, isoD7-Aβ loses its ability to form zinc-bound oligomeric seeds. In this work, we found that (i) in vitro isoD7-Aβ with phosphorylated Ser8 (isoD7-pS8-Aβ) is less prone to spontaneous and zinc-induced aggregation in comparison with isoD7-Aβ and intact Aβ as shown by thioflavin T fluorimetry and dynamic light scattering data, and (ii) intravenous injections of isoD7-pS8-Aβ significantly slow down the progression of institutional β-amyloidosis in AβPP/PS1 transgenic mice as shown by the reduction of the congophilic amyloid plaques’ number in the hippocampus. The results support the role of the zinc-mediated oligomerization of Aβ species in the modulation of cerebral β-amyloidosis and demonstrate that isoD7-pS8-Aβ can serve as a potential molecular tool to block the aggregation of endogenous Aβ in AD.

The triggers of late-onset sporadic Alzheimer’s disease (AD) are still poorly understood. Impairment of protein phosphorylation with age is well-known; however, the role of the phosphorylation in β-amyloid peptide (Aβ) is not studied sufficiently. Zinc-induced oligomerization of Aβ represents a potential seeding mechanism for the formation of neurotoxic Aβ oligomers and aggregates. Phosphorylation of Aβ by Ser8 (pS8-Aβ), localized inside the zinc-binding domain of the peptide, may significantly alter its zinc-induced oligomerization. Indeed, using dynamic light scattering, we have shown that phosphorylation by Ser8 dramatically reduces zinc-induced aggregation of Aβ, and moreover pS8-Aβ suppresses zinc-driven aggregation of non-modified Aβ in an equimolar mixture. We have further analyzed the effect of pS8-Aβ on the progression of cerebral amyloidosis with serial retro-orbital injections of the peptide in APPSwe/PSEN1dE9 murine model of AD, followed by histological analysis of amyloid burden in hippocampus. Unlike the non-modified Aβ that has no influence on the amyloidosis progression in murine models of AD, pS8-Aβ injections reduced the number of amyloid plaques in the hippocampus of mice by one-third. Recently shown inhibition of Na+,K+-ATPase activity by Aβ, which is thought to be a major contributor to neuronal dysfunction in AD, is completely reversed by phosphorylation of the peptide. Thus, several AD-associated pathogenic properties of Aβ are neutralized by its phosphorylation.

Cholinergic dysfunction in Alzheimer’s disease (AD) can be mediated by the neuronal α7 nicotinic acetylcholine receptor (α7nAChR). Beta-amyloid peptide (Aβ) binds to the α7nAChR, disrupting the receptor’s function and causing neurotoxicity. In vivo not only Aβ but also its modified forms can drive AD pathogenesis. One of these forms, iso-Aβ (containing an isomerized Asp7 residue), shows an increased neurotoxicity in vitro and stimulates amyloidogenesis in vivo. We suggested that such effects of iso-Aβ are α7nAChR-dependent. Here, using calcium imaging and electrophysiology, we found that iso-Aβ is a more potent inhibitor of the α7nAChR-mediated calcium current than unmodified Aβ. However, Asp7 isomerization eliminated the ability of Aβ to decrease the α7nAChR levels. These data indicate differences in the interaction of the peptides with the α7nAChR, which we demonstrated using computer modeling. Neither Aβ nor iso-Aβ competed with 125I-α-bungarotoxin for binding to the orthosteric site of the receptor, suggesting the allosteric binging mode of the peptides. Further we found that increased neurotoxicity of iso-Aβ was mediated by the α7nAChR. Thus, the isomerization of Asp7 enhances the inhibitory effect of Aβ on the functional activity of the α7nAChR, which may be an important factor in the disruption of the cholinergic system in AD.

Sodium-potassium adenosine triphosphatase (Na,K-ATPase) creates a gradient of sodium and potassium ions necessary for the viability of animal cells, and it is extremely sensitive to intracellular redox status. Earlier we found that regulatory glutathionylation determines Na,K-ATPase redox sensitivity but the role of basal glutathionylation and other redox modifications of cysteine residues is not clear. The purpose of this study was to detect oxidized, nitrosylated, or glutathionylated cysteine residues in Na,K-ATPase, evaluate the possibility of removing these modifications and assess their influence on the enzyme activity. To this aim, we have detected such modifications in the Na,K-ATPase α1-subunit purified from duck salt glands and tried to eliminate them by chemical reducing agents and the glutaredoxin1/glutathione reductase enzyme system. Detection of cysteine modifications was performed using mass spectrometry and Western blot analysis. We have found that purified Na,K-ATPase α1-subunit contains glutathionylated, nitrosylated, and oxidized cysteines. Chemical reducing agents partially eliminate these modifications that leads to the slight increase of the enzyme activity. Enzyme system glutaredoxin/glutathione reductase, unlike chemical reducing agents, produces significant increase of the enzyme activity. At the same time, the enzyme system deglutathionylates native Na,K-ATPase to a lesser degree than chemical reducing agents. This suggests that the enzymatic reducing system glutaredoxin/glutathione reductase specifically affects glutathionylation of the regulatory cysteine residues of Na,K-ATPase α1-subunit.