[Direct Molecular Fishing of Zinc-Dependent Protein Partners of Amyloid-beta 1-16 with the Taiwan (D7H) Mutation and Phosphorylated Ser8 Residue].

We previously showed that the metal-binding domain 1-16 of intact amyloid-beta (Aß) is involved in interactions with a number of proteins from the cytosolic fraction of SK-N-SH human neuroblastoma cells in a zinc-dependent manner only. It is known that hereditary mutations in the Aß metal-binding domain (Aß(1-16)), which accelerate the development of Alzheimer's disease and post-translational modifications of amino acid residues, can significantly affect the domain's structure in the presence of zinc ions. In this work, using the molecular fishing methodology for Aß(l-16) isoforms with the Taiwanese mutation (D7H) and a phosphorylated Ser8 residue, proteins from the cytosol of SK-N-SH cells were found that are able to form zinc-dependent non-covalent complexes with these domains. The partner proteins identified for these isoforms differed from those for intact Aß(1-16). In contrast, the Aß(1-16) isoform with the English mutation (H6R) and the Aß(1-16) isoform containing both an isomerized Asp7 residue and phosphorylated Ser8 residue did not interact with cytosolic proteins. The results are useful for developing methods for rational modulation of protein-protein interactions involving natural isoforms of beta-amyloid, and also indicate the possible role of beta-amyloid with phosphorylated Ser8 as a molecule involved in normal physiological processes.

[The Convergence of Alzheimer's Disease Pathogenesis Concepts].

Advances in the research of molecular factors involved in the onset and progression of Alzheimer's disease, have led to the creation of several pathogenesis concepts of the most common neurodegenerative disease in the world, and amyloid, cholinergic, and neuroinflammatory hypotheses became leading. Over past twenty years, based on these hypotheses, hundreds of drug prototypes were developed, but none of them were able to stop the development of Alzheimer's disease. In this review, based on the latest experimental data on structure-function properties of chemically modified amyloid-beta isoforms, the concept of the origin and the mechanism of action of amyloid-beta with isomerized Asp7 residue, as a molecular agent of Alzheimer's disease pathogenesis, is presented. This concept makes it possible not only to combine the most important aspects of existing hypotheses but also indicates ways of creating agents for fighting Alzheimer's disease with a principally new mechanism of action, "disease-modifying drugs."

[Effects of the H6R and D7H Mutations on the Heparin-Dependent Modulation of Zinc-Induced Aggregation of Amyloid ß].

Zinc ions and glycosaminoglycans (GAGs) are found in amyloid deposits and are known to modulate the ß-amyloid peptide (Aß) aggregation, which is thought to be a key event in the pathogenesis of Alzheimer's disease (AD). Correlation spectroscopy was used to study how the H6R and D7H mutations of the metal-binding domain (MBD) of Aß42 affect the modulation of its zinc-induced aggregation by the model GAG heparin. The H6R mutation was shown to decrease and the D7H mutation to increase the Aß42 propensity to aggregate in the presence of zinc ions. In addition, H6R diminished and D7H enhanced the modulating effect of heparin. The difference in the heparin-dependent modulation was associated with coordination of zinc ions within the MBDs of the mutant peptides. The findings indicate that anion-binding sites formed by complexes of zinc ions with the Aß MBD play an essential role in the interaction of zinc-induced Aß aggregates with heparin.

Anti-amyloid Therapy of Alzheimer's Disease: Current State and Prospects.

Drug development for the treatment of Alzheimer's disease (AD) has been for a long time focused on agents that were expected to support endogenous ß-amyloid (Aß) in a monomeric state and destroy soluble Aß oligomers and insoluble Aß aggregates. However, this strategy has failed over the last 20 years and was eventually abandoned. In this review, we propose a new approach to the anti-amyloid AD therapy based on the latest achievements in understanding molecular causes of cerebral amyloidosis in AD animal models.

[Enalaprilat Inhibits Zinc-Dependent Oligomerization of Metal-Binding Domain of Amyloid-beta Isoforms and Protects Human Neuroblastoma Cells from Toxic Action of these Isoforms].

Intact amyloid-ß peptides (Aß) may undergo prion-like aggregation when they interact with chemically or structurally modified variants of Aß present in extracellular pathohistological inclusions (amyloid plaques). This aggregation is regarded as one of the key molecular mechanisms of Alzheimer's disease (AD) pathogenesis. Zinc ions are involved in the pathological dimerization and oligomerization of natural Aß isoforms, and zinc-induced oligomers can also initiate the pathological aggregation of Aß. Based on the earlier found molecular mechanism of zinc-dependent oligomerization of Aß, it has been suggested that the targeted inhibition of the 11EVHH14 site in one Aß molecule from zinc-mediated interactions with the same site of another Aß molecule can effectively inhibit the oligomerization and aggregation of Aß. Taking into account the similarity in the structural organization of zinc-binding sites within Aß and angiotensin-converting enzyme (ACE), we hypothesized that inhibitors of the ACE active sites could specifically interact with the 11EVHH14 site of Aß. Using a surface plasmon resonance biosensor and nuclear magnetic resonance spectroscopy, we have found that the ACE inhibitor enalaprilat effectively inhibits zinc-dependent dimerization of the metal-binding domains of intact Aß and Aß with isomerized Asp7 (isoAß). We have also found that enalaprilat protects SH-SY5Y human neuroblastoma cells from the toxic effects of Aß(1-42) and isoAß(1-42), which are among the most common components of amyloid plaques. The results confirm the role of zincdependent oligomerization of Aß in AD pathogenesis and make it possible one to consider enalaprilat as a prototype of antiaggregation agents for treating AD.

Amyloid-ß with isomerized Asp7 cytotoxicity is coupled to protein phosphorylation.

Neuronal dysfunction and loss associated with the accumulation of amyloid-ß (Aß) in the form of extracellular amyloid plaques and hyperphosphorylated tau in the form of intraneuronal neurofibrillary tangles represent key features of Alzheimer's disease (AD). Amyloid plaques found in the brains of AD patients are predominantly composed of Aß42 and its multiple chemically or structurally modified isoforms. Recently, we demonstrated that Aß42 with isomerised Asp7 (isoAß42) which is one of the most abundant Aß isoform in plaques, exhibited high neurotoxicity in human neuronal cells. Here, we show that, in SH-SY5Y neuroblastoma cells, the administration of synthetic isoAß42 rather than intact Aß42 resulted in a significantly higher level of protein phosphorylation, especially the phosphorylation of tau, tubulins, and matrin 3. IsoAß42 induced a drastic reduction of tau protein levels. Our data demonstrate, for the first time, that isoAß42, being to date the only known synthetic Aß species to cause AD-like amyloidogenesis in an animal AD model, induced cell death by disabling structural proteins in a manner characteristic of that observed in the neurons of AD patients. The data emphasize an important role of isoAß42 in AD progression and provide possible neurotoxicity paths for this particular isoform.

[Secondary Structure of Aß(1-16) Complexes with Zinc: A Study in the Gas Phase Using Deuterium/Hydrogen Exchange and Ultra-High-Resolution Mass Spectrometry].

Complexes of peptide fragment 1-16 of beta-amyloid with transition metals play an important role in the development of a broad class of neurodegenerative diseases, which determines the interest in investigating the structures of these complexes. In this work, we have applied the method of the deuterium/hydrogen exchange in combination with ultra-high-resolution mass spectrometry to study conformational changes in (1-16) beta-amyloid peptide induced by binding of zinc(II) atoms. The efficiency of the deuterium/hydrogen exchange depended on the number of zinc atoms bound to the peptide and on the temperature of the ionization source region. Deuterium/hydrogen exchange reactions have been performed directly in the ionization source. The number of exchanges decreased considerably with an increasing numbers of zinc atoms. The relationship has been described with a damped exponential curve, which indicated that the binding of zinc atoms altered the conformation of the peptide ion by making it less open, which limits the access to inner areas of the molecule.

[Zinc-induced interactions of the metal-binding domain of beta-amyloid with nucleic acids and glycosaminoglycans].

Zinc ions form complexes with ß-amyloid peptides and play an important role in Alzheimer's disease pathogenesis. It has been demonstrated by turbidimetry and correlation spectroscopy that synthetic peptide Aß16 representing the metal-binding domain of ß-amyloid is able to interact with nucleic acids, chondroitin polysulfate, and dextran sulfates in the presence of zinc ions. The amino acid D7H substitution enhanced the peptide binding to polyanions, whereas the H6R and H6A-H13A substitutions abolished this interaction. It is suggested that the metal-binding domain may serve as a zinc-dependent site of ß-amyloid interaction with biological polyanions including DNA, RNA, and glycosaminoglycans.

[The role of zinc ions and structural polymorphism of ß-amyloid in the Alzheimer's disease initiation].

Aggregation of ß-amyloid peptide (Aß) underlies the development of Alzheimer's disease. Here we review the main stages of Aß formation and aggregation. We highlight the importance of interaction of zinc ions with the metal-binding domain 1-16 of Aß as a molecular mechanism that leads to Aß aggregation. We analyze recent studies of the native modifications of the Aß metal-binding domain that determine its structural polymorphism. The prospects for further studies of these modifications aimed at revealing the pathogenic mechanism of Aß aggregation are discussed.

[Effect of mutations and modifications of amino acid residues on zinc-induced interaction of the metal-binding domain of ß-amyloid with DNA].

Interaction of intranuclear ß-amyloid with DNA is considered to be a plausible mechanism of Alzheimer's disease pathogenesis. The interaction of single- and double-stranded DNA with synthetic peptides was analyzed using surface plasmon resonance. The peptides represent the metal-binding domain of ß-amyloid (amino acids 1-16) and its variants with chemical modifications and point substitutions of amino acid residues which are associated with enhanced neurotoxicity of ß-amyloid in cell tests. It has been shown that the presence of zinc ions is necessary for the interaction of the peptides with DNA in solution. H6R substitution has remarkably reduced the ability of domain 1-16 to bind DNA. This is in accordance with the supposition that the coordination of a zinc ion by amino acid residues His6, Glu11, His13, and His14 of the ß-amyloid metal-binding domain results in the occurrence of an anion-binding site responsible for the interaction of the domain with DNA. Zinc-induced dimerization and oligomerization of domain 1-16 associated with phosphorylation of Ser8 and the presence of unblocked amino- and carboxy-terminal groups have resulted in a decrease of peptide concentrations required for detection of the peptide-DNA interaction. The presence of multiple anion-binding sites on the dimers and oligomers is responsible for the enhancement of the peptide-DNA interaction. A substitution of the negatively charged residue Asp7 for the neutral residue Asn in close proximity to the anion-binding site of the domain 1-16 of Aß facilitates the electrostatic interaction between this site and phosphates of a polynucleotide chain, which enhances zinc-induced binding to DNA.

[Physico-chemical methods for studing ß-amyloid aggregation].

Alzheimer's disease is the most prevalent neurodegenerative pathology. According to the amyloid cascade hypothesis, a key event of the Alzheimer's disease pathogenesis is a transition of the ß-amyloid peptide (Аß) from the monomeric form to the aggregated state. The mechanism of Ðß aggregation is intensively studied in vitro, by means of synthetic peptides and various physico-chemical methods allowing evaluation of size, molecular structure, and morphology of the formed aggregates. The paper reviews both the well-known and recently introduced physico-chemical methods for analysis of Ðß aggregation, including microscopу, optical and fluorescent methods, method of electron paramagnetic resonance, electrochemical and electrophoretic methods, gel-filtration, and mass spectrometric methods. Merits and drawbacks of the methods are discussed. The unique possibility to simultaneously observe Ðß monomers as well oligomers and large aggregates by means of atomic force microscopy or fluorescence correlation spectroscopy is emphasized. The high detection sensitivity of the latter method, monitoring the aggregation process in Ðß solutions at low peptide concentrations is underlined. Among mass spectrometric methods, the ion mobility mass spectrometry is marked out as a method enabling to obtain information about both the spectrum of Ðß oligomers and their structure. It is pointed out that the use of several methods giving the complementary data about Ðß aggregates is the best experimental approach to studying the process of b-amyloid peptide aggregation in vitro.

[New biomarkers and drug targets for diagnosis and therapy of Alzheimer's disease (molecular determinants of zinc-dependent oligomerization of Β-amyloid)].

In view of the amyloid hypothesis of Alzheimer's disease (AD), the key molecular event is the structural transition of Β-amyloid from the physiologically normal monomer state to soluble neurotoxic oligomers accumulating in the form of insoluble extracellular aggregates (amyloid plaques) in brain tissues. Zinc ions are known to play a crucial role in the formation of these pathological aggregates. The authors and collaborators have identified that the certain chemical modification and point amino acid substitutions in the metal-binding domain play a critical role in the formation of neurotoxic zinc-dependent oligomers and induce the development of cerebral amyloidosis and other pathological processes characteristic of AD. The results allow to use these forms of Β-amyloid as potential biomarkers of early diagnosis of AD. Zinc-dependent dimerization and oligomerization of Β-amyloid can be used as drug target for treatment of AD.

The English (H6R) familial Alzheimer's disease mutation facilitates zinc-induced dimerization of the amyloid-ß metal-binding domain.

Interaction of Zn(2+) with the metal-binding domain of the English (H6R) amyloid-ß mutant results in the formation of peptide dimers. The mutation causes the exclusion of His6 from the zinc chelation pattern observed in the intact domain and triggers the assembly of the dimers via zinc ions coordinated by (11)EVHH(14) fragments.

[Estimation of phosphorilation level of amyloid beta, extracted from human blood plasma. Ultra high resolution mass spectrometry].

Recently it has been shown that phosphorylation of the Ser8 residue in amyloid-beta (pS8-Abeta) is tightly involved in the pathogenesis ofAlzheimer's disease. Since this modification occurs in the key metal-binding domain of amyloid-beta, and thus should seriously affect the interaction of pS8-Abeta with zinc ions, this isoform might be a potential precursor of pathogenic oligomeric forms of amyloid beta. Hence the level of pS8-Abeta in human biological fluids (such as blood, urine, cerebral spinal fluid) might resemble the different stages of the pathogenesis of Alzhe- imer's disease. The aim of this workwas to develop a prototype of an analytical method for quantitative determination of the level of pS8-Abeta isoform in binary mixtures with native amyloid-beta in order to further use it to determine the levels of phosphorylated amyloid-beta in blood plasma samples of patients with Alzheimer's disease.

[Identification of the minimal zinc-binding center in natural isoforms of amyloid-beta domain 1-16 using ESI-MS].

Alzheimer's disease, is a lethal neurodegenerative pathology, characterized by the formation of soluble neurotoxic oligomers of the human amyloid-beta peptide Abeta which get accumulated forming polymeric extracellular aggregates (so-called amyloid plaques). The isomerized at aspartate 7 isoform of the human Abeta (isoAbeta) is the main component of these plaques and is considered as the potential pathogenic agent of AD. Besides this, there is a possible generation mechanism for this isoform from a genetically deficient D7N Abeta variant (Tottori mutation). On the contrary the rodent Abeta (rat Abeta), which has three amino acid substitutions in its metal-binding domain, is not susceptible to pathogenic aggregation in vivo, unlike the other known natural isoforms of Abeta. Interactions with zinc ions play a crucial role in the aggregation of monomeric human Abeta in vitro and in vivo. In the presented article using high resolution ESI-MS methods it was shown that domains 1-16 of isoAbeta and D7NAbeta bind zinc ions in the exactly the same manner as the normal human Abeta1-16, whereas ratAbeta has significant differences in structure of its minimal zinc binding center. These results confirm the overall interaction mechanism between zinc ions and the humanAbeta isoforms and allows to suppose that perhaps modulation of the structure of region 6-14 of Abeta can be used as a promising therapeutic approach to AD treatment.

Peripherally applied synthetic peptide isoAsp7-Aß(1-42) triggers cerebral ß-amyloidosis.

Intracerebral and intraperitoneal inoculation with ß-amyloid-rich brain extracts originating from patients with Alzheimer's disease as well as intracerebral injection of aggregates composed of synthetic Aß can induce cerebral ß-amyloidosis, and associated cognitive dysfunctions in susceptible animal hosts. We have found that repetitive intravenous administration of 100 µg of synthetic peptide corresponding to isoAsp7-containing Aß(1-42), an abundant age-dependent Aß isoform present both in the pathological brain and in synthetic Aß preparations, robustly accelerates formation of classic dense-core congophilic amyloid plaques in the brain of ß-amyloid precursor protein transgenic mice. Our findings indicate this peptide as an inductive agent of cerebral ß-amyloidosis in vivo.

[Identification of transthyretin posttranslational modifications 1n human blood using mass-spectrometric methods].

Transthyretin, one of the major plasma proteins, has a number of posttranslational modifications and mutations, some of which are associated with the development of severe diseases, for instance, familial amyloid neuropathy and Alzheimer's disease. In order to investigate the role of modified forms in the development of these diseases a complex analytical platform, based on two mass-spectrometric approaches (bottom-up and op-down) has been developed. The high efficiency of this method was shown using 10 plasma samples obtained from patients with Alzheimer's disease and healthy individuals.

[Optimization of the methods for small peptide solution structure determination by NMR spectroscopy].

NMR spectroscopy was recognized as a method of protein structure determination in solution. However, determination of the conformation of small peptides, which undergo fast molecular motions, remains a challenge. This is mainly caused by impossibility to collect required quantity of the distance and dihedral angle restraints from NMR spectra. At the same time, short charged peptides play an important role in a number of biological processes, in particular in pathogenesis of neurodegenerative diseases including Alzheimer's disease. Therefore development of a method for structure calculation of small peptides in a water environment using the most realistic force fields seems to be of current importance. Such algorithm has been developed using the Amber-03 force field and software package Gromacs after updating its program code. The algorithm of calculation has been verified on a model peptide for which the solution structure is known, and on the metal binding fragment of rat beta-amyloid for which structure has been determined by alternative methods. The developed algorithm substantially increases quality of structures, in particular Ramachandran plot statistics, and decreases RMSD of coordinates of atoms inside calculated family. The described protocol of calculation can be used for determination of conformation of short peptides, and also for structure optimization of larger proteins containing poorly structured fragments.