The Strategies of Development of New Non-Toxic Inhibitors of Amyloid Formation.

In recent years, due to the aging of the population and the development of diagnostic medicine, the number of identified diseases associated with the accumulation of amyloid proteins has increased. Some of these proteins are known to cause a number of degenerative diseases in humans, such as amyloid-beta (Aß) in Alzheimer's disease (AD), α-synuclein in Parkinson's disease (PD), and insulin and its analogues in insulin-derived amyloidosis. In this regard, it is important to develop strategies for the search and development of effective inhibitors of amyloid formation. Many studies have been carried out aimed at elucidating the mechanisms of amyloid aggregation of proteins and peptides. This review focuses on three amyloidogenic peptides and proteins-Aß, α-synuclein, and insulin-for which we will consider amyloid fibril formation mechanisms and analyze existing and prospective strategies for the development of effective and non-toxic inhibitors of amyloid formation. The development of non-toxic inhibitors of amyloid will allow them to be used more effectively for the treatment of diseases associated with amyloid.

Determination of the Most Stable Packing of Peptides from Ribosomal S1 Protein, Protein Bgl2p, and Aß peptide in ß-layers During Molecular Dynamics Simulations.

Our task was to determine the most stable packing of peptides in ß-layers to construct an oligomer structure for fibril growth. The ß-layers consisting of eight short peptides with the amino acid sequences IVRGVVVAID, VDSWNVLVAG (VESWNVLVAG), KLVFFAEDVG, and IIGLMVGGVV were built. These sequences correspond to the amyloidogenic regions of ribosomal S1 protein from E. coli, protein glucantransferase Bgl2p from the yeast cell wall, and Aß peptide. First, the amyloidogenic regions were predicted theoretically, and then were confirmed experimentally. Four ß-layers with different orientation of the peptides in the layers and the layers relative to each other were constructed. To determine the most stable packing of ß-strands, the molecular dynamic (MD) simulations in explicit water were carried out. Two charge states (pH3 and pH5) for each ß-layer were considered. The fraction of the secondary structure was a measure of stability for ß-layers. ß-Layers, in which ß-strands are antiparallel relative to each other, were the most stable. Using this packing for ß-strands, we constructed the oligomer structures and also checked their stability by using MD simulations.

Amyloidogenic Propensities of Ribosomal S1 Proteins: Bioinformatics Screening and Experimental Checking.

Structural S1 domains belong to the superfamily of oligosaccharide/oligonucleotide-binding fold domains, which are highly conserved from prokaryotes to higher eukaryotes and able to function in RNA binding. An important feature of this family is the presence of several copies of the structural domain, the number of which is determined in a strictly limited range from one to six. Despite the strong tendency for the aggregation of several amyloidogenic regions in the family of the ribosomal S1 proteins, their fibril formation process is still poorly understood. Here, we combined computational and experimental approaches for studying some features of the amyloidogenic regions in this protein family. The FoldAmyloid, Waltz, PASTA 2.0 and Aggrescan programs were used to assess the amyloidogenic propensities in the ribosomal S1 proteins and to identify such regions in various structural domains. The thioflavin T fluorescence assay and electron microscopy were used to check the chosen amyloidogenic peptides' ability to form fibrils. The bioinformatics tools were used to study the amyloidogenic propensities in 1331 ribosomal S1 proteins. We found that amyloidogenicity decreases with increasing sizes of proteins. Inside one domain, the amyloidogenicity is higher in the terminal parts. We selected and synthesized 11 amyloidogenic peptides from the Escherichia coli and Thermus thermophilus ribosomal S1 proteins and checked their ability to form amyloids using the thioflavin T fluorescence assay and electron microscopy. All 11 amyloidogenic peptides form amyloid-like fibrils. The described specific amyloidogenic regions are actually responsible for the fibrillogenesis process and may be potential targets for modulating the amyloid properties of bacterial ribosomal S1 proteins.

Should the Treatment of Amyloidosis Be Personified? Molecular Mechanism of Amyloid Formation by Aß Peptide and Its Fragments.

Aß40 and Aß42 peptides are believed to be associated with Alzheimer's disease. Aggregates (plaques) of Aß fibrils are found in the brains of humans affected with this disease. The mechanism of formation of Aß fibrils has not been studied completely, which hinders the development of a correct strategy for therapeutic prevention of this neurodegenerative disorder. It has been found that the most toxic samples upon generation of fibrils are different oligomeric formations. Based on different research methods used for studying amyloidogenesis of Aß40 and Aß42 peptides and its amyloidogenic fragments, we have proposed a new mechanism of formation of amyloid fibrils. In accord with this mechanism, the main building unit for fibril generation is a ring-like oligomer. Association of ring-like oligomers results in the formation of fibrils of different morphologies. Our model implies that to prevent development of Alzheimer's disease a therapeutic intervention is required at the earliest stages of amyloidogenesis-at the stage of formation of ring-like oligomers. Therefore, the possibility of a personified approach for prevention not only of Alzheimer's disease development but also of other neurodegenerative diseases associated with the formation of fibrils is argued.

Nucleation-based prediction of the protein folding rate and its correlation with the folding nucleus size.

The problem of protein self-organization is in the focus of current molecular biology studies. Although the general principles are understood, many details remain unclear. Specifically, protein folding rates are of interest because they dictate the rate of protein aggregation which underlies many human diseases. Here we offer predictions of protein folding rates and their correlation with folding nucleus sizes. We calculated free energies of the transition state and sizes of folding nuclei for 84 proteins and peptides whose other parameters were measured at the point of thermodynamic equilibrium between their unfolded and native states. We used the dynamic programming method where each residue was considered to be either as folded as in its native state or completely disordered. The calculated and measured folding rates showed a good correlation at the temperature mid-transition point (the correlation coefficient was 0.75). Also, we pioneered in demonstrating a moderate (-0.57) correlation coefficient between the calculated sizes of folding nuclei and the folding rates. Predictions made by different methods were compared. The established good correlation between the estimated free energy barrier and the experimentally found folding rate of each studied protein/peptide indicates that our model gives reliable results for the considered data set.

Computer simulation of gas-phase neutralization of electrospray-generated protein macroions.

The process of neutralizing hydrated multicharged gas-phase protein ions with small counterions was simulated using a molecular dynamics (MD) technique. Hen egg white lysozyme (HEWL) molecules with different numbers of positive charges, both dry and solvated by up to 1500 water molecules, were first equilibrated. Simulations revealed that the hydration layer over a highly charged protein surface adapted a spiny structure with water protrusions composed of oriented water dipoles. MD simulations of the neutralization process showed that the impact of a small dehydrated single-charged counterion with a dehydrated HEWL ion bearing eight uncompensated charges resulted in a short local increase in temperature by 600-1000 K, which quickly (in 3-5 ps) dissipated over the whole protein molecule, increasing its average temperature by 20-25 K. When the protein ion was solvated, no drastic local increase in the temperature of the protein atoms was observed, because the impact energy was dissipated among the water molecules near the collision site.