Sergey A. Kozin

Isomerization of the Asp7 Residue Results in Zinc-Induced Oligomerization of Alzheimer’s Disease Amyloid β(1–16) Peptide

Alzheimer’s disease (AD)—a fatal neurodegenerative disorder that primarily affects the elderly—is pathophysiologically characterized by the extracellular deposition of a 40/42-aminoacid-long protein, referred to as amyloid-b peptide (Ab), in the brains of AD victims. Although the molecular mechanism of AD onset is unknown, the transformation of Ab from its native monomer conformation via soluble dimers and higher oligomers into insoluble fibrillar b-sheet aggregates, which finally accumulate into the amyloid plaques, is believed to be a key event in AD pathogenesis. One plausible hypothesis suggests that the amyloid neuropathology of AD depends on zinc ions released during neurotransmission, and so it is assumed that binding of zinc to Ab might play an important role in initiating pathogenic amyloid deposition, as well as some additional still unidentified proteinaceous factors. The Ab molecules isolated from AD brain lesions have numerous endogenous post-translational modifications (PTMs), which should profoundly affect both the Ab conformation and its oligomeric state and make up a pool of potential pathogenic agents in AD. The most abundant PTM of Ab is isomerization of the Asp7 residue; this results in the formation of an lisoAsp7 isoform (isoaspartate). This nonenzymatic modification occurs spontaneously in polypeptides through an intramolecular rearrangement of aspartate or asparagine residues and is generally regarded as a degradation reaction that occurs in vivo during tissue ageing. In the case of isomerized Ab (isoAb) it is still unclear whether the isoaspartyl residues are the cause or the result of the pathological accumulation of Ab. Nevertheless, recent in vitro experimental evidence indicates that isoAb might potentially be involved in the onset of AD. To investigate the role of the Asp7 isomerization in zinc-induced oligomerization of Ab we have studied the thermodynamics of zinc binding and the oligomeric states of two synthetic model peptides that correspond to region 1–16 in Ab and in isoAb : Ab16 and isoAb16, respectively. Earlier, this region was identified as the zinc-binding domain of Ab, which binds Zn with 1:1 stoichiometry and a 6 mm dissociation constant. Both Ab16 and its complex with Zn 2+ were found to be monomeric under physiological conditions for at least six months over a wide concentration range, and so were used as monomer reference standards throughout this work. The isoAb16 was also shown to possess zinc-binding ability; [11] however, the properties of the Zn–isoAb16 complex have not been studied previously. To compare Zn binding to Ab16 and to isoAb16 (in 50 mm Tris buffer at pH 7.3), isothermal titration calorimetry (ITC) was used. The thermodynamic data demonstrate that Ab16 binds one zinc ion with an association constant of 1.7ACHTUNGTRENNUNG( 0.4)A10m 1 (Figure 1), which corresponds to previously published da ACHTUNGTRENNUNGta.

Zinc-induced dimerization of the amyloid-β metal-binding domain 1–16 is mediated by residues 11–14

Analysis of complex formation between amyloid-β fragments using surface plasmon resonance biosensing and electrospray mass spectrometry reveals that region 11-14 mediates zinc-induced dimerization of amyloid-β and may serve as a potential drug target for preventing development and progression of Alzheimers disease.

Capabilities of MS for analytical quantitative determination of the ratio of α- and βAsp7 isoforms of the amyloid-β peptide in binary mixtures.

There is strong evidence that the amyloid-β peptide (Aβ) plays a crucial role in the pathogenesis of Alzheimers disease (AD), a lethal neurodegenerative disorder of the elderly. During pathology development, the peptide as well as its various chemically modified isoforms is accumulated in specific brain tissues as characteristic proteinaceous deposits, the so-called amyloid plaques, which are the pathomorphological mark of AD, although the level of Αβ in the blood is the same for healthy individuals and for AD patients. Earlier, it has been shown that isomerization of aspartate 7, the most abundant post-translational modification of the Αβ peptide, is tightly involved in a set of molecular processes associated with AD progression. Therefore, the isoAsp 7-containing Αβ isomer (isoAβ) is assumed to be a potential biomarker of AD that can be identified in the blood. Here, we present an analytical mass spectrometric method for quantitative determination of the ratio of normal and isomerized Αβ fragments 1-16 in their binary mixtures, and all analytical capabilities, such as accuracy, detection limits, and sensitivity of the presented method, are determined and thoroughly discussed. On the basis of this method, an analytical approach for quantitative determination of this modification in the blood will be developed in further studies.

The Effects of Endogenous Non-Peptide Molecule Isatin and Hydrogen Peroxide on Proteomic Profiling of Rat Brain Amyloid-β Binding Proteins: Relevance to Alzheimer's Disease?

The amyloid-β peptide is considered as a key player in the development and progression of Alzheimer’s disease (AD). Although good evidence exists that amyloid-β accumulates inside cells, intracellular brain amyloid-binding proteins remain poorly characterized. Proteomic profiling of rat brain homogenates, performed in this study, resulted in identification of 89 individual intracellular amyloid-binding proteins, and approximately 25% of them were proteins that we had previously identified as specifically binding to isatin, an endogenous neuroprotector molecule. A significant proportion of the amyloid-binding proteins (more than 30%) are differentially expressed or altered/oxidatively modified in AD patients. Incubation of brain homogenates with 70 µM hydrogen peroxide significantly influenced the profile of amyloid-β binding proteins and 0.1 mM isatin decreased the number of identified amyloid-β binding proteins both in control and hydrogen peroxide treated brain homogenates. The effects of hydrogen peroxide and isatin have been confirmed in optical biosensor experiments with purified glyceraldehyde-3-phosphate dehydrogenase, one of the known crucial amyloid-β binding proteins (also identified in this study). Data obtained suggest that isatin protects crucial intracellular protein targets against amyloid binding, and possibly favors intracellular degradation of this protein via preventing formation of amyloid-β oligomers described in the literature for some isatin derivatives.

Zinc-induced heterodimer formation between metal-binding domains of intact and naturally modified amyloid-beta species: implication to amyloid seeding in Alzheimer’s disease?

Zinc ions and modified amyloid-beta peptides (Aβ) play a critical role in the pathological aggregation of endogenous Aβ in Alzheimer’s disease (AD). Zinc-induced Aβ oligomerization is mediated by the metal-binding domain (MBD) which includes N-terminal residues 1–16 (Aβ1–16). Earlier, it has been shown that Aβ1–16 as well as some of its naturally occurring variants undergoes zinc-induced homodimerization via the interface in which zinc ion is coordinated by Glu11 and His14 of the interacting subunits. In this study using surface plasmon resonance technique, we have found that in the presence of zinc ions Aβ1–16 forms heterodimers with MBDs of two Aβ species linked to AD: Aβ containing isoAsp7 (isoAβ) and Aβ containing phosphorylated Ser8 (pS8-Aβ). The heterodimers appear to possess the same interface as the homodimers. Simulation of 200 ns molecular dynamic trajectories in two constructed models of dimers ([Aβ1–16/Zn/Aβ1–16] and [isoAβ1–16/Zn/Aβ1–16]), has shown that conformational flexibility of the N-terminal fragments of the dimer subunits is controlled by the structure of corresponding sites 6–8. The data suggest that isoAβ and pS8-Aβ can be involved in the AD pathogenesis by means of their zinc-dependent interactions with endogenous Aβ resulting in the formation of heterodimeric seeds for amyloid aggregation.

Protein interactomics based on direct molecular fishing on paramagnetic particles: Practical realization and further SPR validation

There is increasing evidence that proteins function in the cell as integrated stable or temporally formed protein complexes, interactomes. Previously, using model systems we demonstrated applicability of direct molecular fishing on paramagnetic particles for protein interactomics (Ershov et al. Proteomics, 2012, 12, 3295). In the present study, we have used a combination of affinity‐based molecular fishing and subsequent MS for investigation of human liver proteins involved in interactions with immobilized microsomal cytochrome b5 (CYB5A), and also transthyretin and BSA as alternative affinity ligands (baits). The LC−MS/MS identification of prey proteins fished on these baits revealed three sets of proteins: 98, 120, and 220, respectively. Comparison analysis of these sets revealed only three proteins common for all the baits. In the case of paired analysis, the number of common proteins varied from 2 to 9. The binding capacity of some identified proteins has been validated by a SPR‐based biosensor. All the investigated proteins effectively interacted with the immobilized CYB5A (Kd values ranged from 0.07 to 1.1 μM). Results of this study suggest that direct molecular fishing is applicable for analysis of protein–protein interactions (PPI) under normal and pathological conditions, in which altered PPIs are especially important.

Chronic Administration of Dimebon does not Ameliorate Amyloid-β Pathology in 5xFAD Transgenic Mice

Dimebon has been tested as a potential modifier of Alzheimers disease (AD), resulting in mixed clinical trial outcomes. Originally utilized as an antihistamine, Dimebon was later found to ameliorate AD symptoms in initial human trials. Although subsequent trials have reportedly failed to replicate these finding, there is a growing body of evidence that Dimebon might be neuroprotective in certain models of neurodegeneration. The precise mechanism by which Dimebon is thought to act in AD is unclear, though changes in receptor activity, mitochondria function, and autophagy activity have been proposed. It is thus necessary to test Dimebon in transgenic animal model systems to determine if and how the drug affects development and manifestation of pathology, and which pathogenic processes are altered. In the present study we treated mice harboring five familial mutations associated with hereditary AD (5xFAD line) with a chronic regime of Dimebon. The compound was not found to improve the general health or motor behavior of these mice, nor prevent accumulation of Aβ peptides in the brain. Modest changes in response to an anxiogenic task were, however, detected, suggesting Dimebon might improve behavioral abnormalities and cognition in disease in a mechanism independent of protecting against amyloidosis.

A review of the biomedical innovations for healthy longevity

Keywords: longevity ; aging ; biomarkers ; geroprotectors ; epigenetics ; transcriptomics Reference EPFL-ARTICLE-227576doi:10.18632/aging.101163View record in Web of Science Record created on 2017-05-01, modified on 2017-05-26

Zinc-Induced Interaction of the Metal-Binding Domain of Amyloid-β Peptide with DNA

The interaction of the 16-mer synthetic peptide (Aβ16), which represents the metal-binding domain of the amyloid-β with DNA, was studied employing the surface plasmon resonance technique. It has been shown that Aβ16 binds to the duplex DNA in the presence of zinc ions and thus the metal-binding domain can serve as a zinc-dependent DNA-binding site of the amyloid-β. The interaction of Aβ16 with DNA most probably depends on oligomerization of the peptide and is dominated by interaction with phosphates of the DNA backbone.

Amyloid β Modification: A Key to the Sporadic Alzheimer's Disease?

Last year marked 25 years of research into the amyloid hypothesis of Alzheimer’s disease (AD) (Selkoe and Hardy, 2016). Over the last few years, studies on this subject have provided a number of insights into the pathology of the most widespread cognitive disorder of aging; however, a successful treatment strategy has yet to be developed. The amyloid hypothesis was on the edge of being discredited due to the indistinct correlation between β-amyloid (Aβ) deposition and neuronal loss (Holmes et al., 2008; Mullane and Williams, 2013). However, recent studies have defended the Aβ peptide as a causative factor in AD and have proved it to be necessary but not sufficient to explain the pathogenesis of the disease in full (Musiek and Holtzman, 2015). An updated hypothesis suggests that, Aβ accumulation is an essential trigger that initiates a pathological cascade implicating tau protein, synuclein, and other aggregation-prone proteins. The questions still to be answered are: which events pull the trigger on the Aβ aggregation cascade and how exactly does destabilization of amyloid proteostasis promote the downstream tau pathology. The answer to the first question is clear and transparent in familial AD (fAD) as it is induced by genetic aberrations. However, it remains a mystery in so-called sporadic AD (sAD), which accounts for more than 90% of the disease cases. Currently, sporadic AD is amajor subject of study with the primary focus being, to make it “less sporadic” by finding a genetic or aging-related basis for the disease. A possible insight into the problem of sADwas found within the amyloid plaques. An analysis of plaque composition has shown that aggregated β-amyloid peptides are modified in different ways, primarily by isomerization and truncation of Aβ (Roher et al., 1993). Subsequent in vitro and in vivo studies revealed that a plethora of modifications exhibit pathogenic features; these include: increased aggregation, neurotoxicity, amyloidogenicity, and an ability to suppress long-term potentiation in the hippocampus (Shimizu et al., 2002; Kumar, 2011; Al-Hilaly et al., 2013; Kozin S. et al., 2013; Mitkevich et al., 2013; Barykin et al., 2016). Hence, we propose a model in which, aberrant post-translational modification (PTM) of the amyloid β peptide increases amyloid neurotoxicity and facilitates its aggregation thus initiating or promoting progression of sAD.