Comparison of deep learning models with simple method to assess the problem of antimicrobial peptides prediction.

Antibiotic-resistant strains are an emerging threat to public health. The usage of antimicrobial peptides (AMPs) is one of the promising approaches to solve this problem. For the development of new AMPs, it is necessary to have reliable prediction methods. Recently, deep learning approaches have been used to predict AMP. In this paper, we want to compare simple and complex methods for these purposes. We used the BERT transformer to create sequence embeddings and the multilayer perceptron (MLP) and light attention (LA) approaches for classification. One of them reached about 80 % accuracy and specificity in benchmark testing, which is on par with the best available methods. For comparison, we proposed a simple method using only the amino acid composition of proteins or peptides. This method has shown good results, at the level of the best methods. We have prepared a special server for predicting the ability of AMPs by amino acid composition: http://bioproteom.protres.ru/antimicrob/.

Influence of Chaperones on Amyloid Formation of Ðß Peptide.

BACKGROUND: An extensive study of the folding and stability of proteins and their complexes has revealed a number of problems and questions that need to be answered. One of them is the effect of chaperones on the process of fibrillation of various proteins and peptides. METHODS: We studied the effect of molecular chaperones, such as GroEL and α-crystallin, on the fibrillogenesis of the Aß(1-42) peptide using electron microscopy and surface plasmon resonance. RESULTS: Recombinant GroEL and Aß(1-42) were isolated and purified. It was shown that the assembly of GroEL occurs without the addition of magnesium and potassium ions, as is commonly believed. According to the electron microscopy results, GroEL insignificantly affects the fibrillogenesis of the Aß(1-42) peptide, while α-crystallin prevents the elongation of the Aß(1-42) peptide fibrils. We have demonstrated that GroEL interacts nonspecifically with Aß(1-42), while α-crystallin does not interact with Aß(1-42) at all using surface plasmon resonance. CONCLUSION: The data obtained will help us understand the process of amyloid formation and the effect of various components on it.

[Structural, Functional, and Evolutionary Characteristics of Proteins with Repeats].

The review summarizes and systematizes the data on the classification, taxonomic distribution, structural features, and functions of proteins with structural repeats. Modern approaches to the identification of structural repeats in proteins are considered. Features of specialized databases of protein domains are described. The review discusses the prospects of using repeat-containing proteins as scaffolds for drug design. The role of proteins with structural repeats in the pathogenesis of various diseases is considered. Various modern approaches to understanding the mechanisms of the evolutionary development of proteins with repeats are described and analyzed.

[Antibacterial effects of peptides synthesized based on the sequence of ribosome protein S1]. / Antibakterial'nye éffekty peptidov, sintezirovannykh na osnove posledovatel'nosti ribosomnogo belka S1.

Antibiotic resistance of bacteria is a topical problem on a global scale. Sometimes vigorous human activity leads to an increase in the number of bacteria carrying resistance genes in the environment. Antimicrobial peptides (AMPs) and similar compounds are potential candidates for combating antibiotic-resistant bacteria. Previously, we proposed and successfully tested on Thermus thermophilus a new mechanism of AMP action. This mechanism of directed coaggregation is based on the interaction of a peptide capable of forming fibrils with a target protein. In this work, we discuss the criteria for choosing a target for the targeted action of AMP, describe the features of the "parental" S1 ribosomal proteins T. thermophilus and Escherichia coli and the studied peptides using bioinformatic analysis methods, assess the antimicrobial effect of the synthesized peptides on a model organism of E. coli and cytotoxicity on cells of human fibroblasts. The obtained results will be important for the creation of new AMPs for pathogenic organisms.

[Changes in Titin Structure during Its Aggregation].

In this paper, the property of the muscle titin protein to form in vitro specific amyloid-like aggregates is discussed. The main difference from the known amyloid aggregates is the formation of a quaternary structure that resembles cross-ß, with no changes in the secondary structure. Based on the results obtained earlier, as well as the results of this study, we make assumptions about changes in the structure of titin that occur during the formation of amyloid-like aggregates. In particular, our X-ray diffraction data on the titin aggregates suggest that ß-strands in the aggregates of this protein are not located perpendicular to the fibril axis, as described for other amyloid proteins, but in parallel. The distance between the ß-sheets in the aggregates may vary, and the ß-sheets themselves are not strictly oriented along one of the axes, which can lead to the appearance of a diffuse ring reflection of ~8-12 Å. In this regard, the titin aggregates should not be called amyloid, but amyloid-like, with a quaternary structure that resembles cross-ß. It cannot be excluded that the formation of this quaternary structure can also occur due to the partial unfolding of titin domains, followed by the interaction of open ß-strands between neighboring domains and/or domains of neighboring molecules.

Comparative Analysis of Aggregation of Thermus thermophilus Ribosomal Protein bS1 and Its Stable Fragment.

Functionally important multidomain bacterial protein bS1 is the largest ribosomal protein of subunit 30S. It interacts with both mRNA and proteins and is prone to aggregation, although this process has not been studied in detail. Here, we obtained bacterial strains overproducing ribosomal bS1 protein from Thermus thermophilus and its stable fragment bS1(49) and purified these proteins. Using fluorescence spectroscopy, dynamic light scattering, and high-performance liquid chromatography combined with mass spectrometric analysis of products of protein limited proteolysis, we demonstrated that disordered regions at the N- and C-termini of bS1 can play a key role in the aggregation of this protein. The truncated fragment bS1(49) was less prone to aggregation compared to the full-size bS1. The revealed properties of the studied proteins can be used to obtain protein crystals for elucidating the structure of the bS1 stable fragment.

[An Overlap between Splicing Sites in RNA and Homo-Repeats in Human Proteins].

Proteins with homo-repeats of more than 4 amino acid residues in length were examined to understand whether some splicing sites in pre-mRNA may be attributed to homo-repeats in human proteins. The human proteome was found to contain a total of 404 proteins with homo-repeats that account for at least one splicing site in pre-mRNA. Pre-mRNA splicing sites were more often found in the C-terminal part (67%) than in the middle orN-terminal part of a homo-repeat. Ten homo-repeats were identified to have two splicing sites per repeat. The repeats were lysine homo-repeats in all but one case.

Identification of Amyloidogenic Regions in the Spine of Insulin Fibrils.

To reveal conformational changes resulting in the formation of insulin fibrils, it is necessary to identify amyloidogenic regions in the structure of protein monomers. Different models of insulin fibrillogenesis have been proposed previously. However, precise regions responsible for the formation of amyloid fibrils have not been identified. Using bioinformatics programs for predicting amyloidogenic regions, we have determined some common amyloidogenic sequences in the structure of insulin monomers. The use of limited proteolysis and mass spectrometry analysis of the obtained protein fragments resistant to the action of proteases allowed us to identify amino acid sequences in the insulin structure that can form the spine of the insulin fibrils. The obtained results are in agreement with the earlier proposed model of fibril formation from the ring-like oligomers and can be used for designing insulin analogs resistant to amyloidogenesis.

The binding of monomeric amyloid ß peptide to serum albumin is affected by major plasma unsaturated fatty acids.

Human serum albumin (HSA) serves as a natural depot of amyloid ß peptide (Aß). Improvement of Aß binding to HSA should impede Alzheimer's disease (AD). We developed a method for quantitation of the interaction between monomeric Aß40/42 and HSA using surface plasmon resonance spectroscopy. The dissociation constant of HSA complex with recombinant Aß40/42 is 0.2-0.3 µM. Flemish variant of Aß40 has 2.5-10-fold higher affinity to HSA. The parameters of the HSA-Aß interaction are selectively sensitive to HSA binding of major plasma unsaturated fatty acids and Cu2+. Linoleic and arachidonic acids promote the HSA-Aß42 interaction. The developed methodology for quantitation of HSA-Aß interaction may serve as a tool for search of compounds favoring HSA-Aß interaction, thereby preventing AD progression.

[Functionally Significant Amino Acid Motifs of Heat Shock Proteins: Structural and Bioinformatics Analyses of Hsp60/Hsp10 in Five Classes of Chordata].

The Hsp60/Hsp10 chaperonin system is one of the most studied systems of cell emergency responses to stresses associated with changes in environmental conditions. In this regard, we have performed a bioinformatics analysis of 164 amino acid sequences of Hsp60 and 125 amino acid sequences of Hsp10 in five classes of chordata. This enabled uncovering the relationship between the identity of the amino acid composition of Hsp60/Hsp10 and the evolutionary distance between classes of chordata. In the course of the study of the chaperonin crystal structure, potentially significant amino acid motifs responsible for the oligomerization of Hsp60 and Hsp10 monomers and the association/dissociation of the Hsp60 and Hsp10 heterodimer have been identified. In addition, we have established that Hsp60 and Hsp10 can form amyloid fibrils due to structural features through the alternative using of the oligomerization sites of monomers as well as association/dissociation sites.

Studies of the Process of Amyloid Formation by Aß Peptide.

Studies of the process of amyloid formation by Aß peptide have been topical due to the critical role of this peptide in the pathogenesis of Alzheimer's disease. Many articles devoted to this process are available in the literature; however, none of them gives a detailed description of the mechanism of the process of generation of amyloids. Moreover, there are no reliable data on the influence of modified forms of Aß peptide on its amyloid formation. To appreciate the role of Aß aggregation in the pathogenesis of Alzheimer's disease and to develop a strategy for its treatment, it is necessary to have a well-defined description of the molecular mechanism underlying the formation of amyloids as well as the contribution of each intermediate to this process. We are convinced that a combined analysis of theoretical and experimental methods is a way for understanding molecular mechanisms of numerous diseases. Based on our experimental data and molecular modeling, we have constructed a general model of the process of amyloid formation by Aß peptide. Using the data described in our previous publications, we propose a model of amyloid formation by this peptide that differs from the generally accepted model. Our model can be applied to other proteins and peptides as well. According to this model, the main building unit for the formation of amyloid fibrils is a ring-like oligomer. Upon interaction with each other, ring-like oligomers form long fibrils of different morphology. This mechanism of generation of amyloid fibrils may be common for other proteins and peptides.

Analysis of Insulin Analogs and the Strategy of Their Further Development.

We analyzed the structural properties of the peptide hormone insulin and described the mechanism of its physiological action, as well as effects of insulin in type 1 and 2 diabetes. Recently published data on the development of novel insulin preparations based on combining molecular design and genetic engineering approaches are presented. New strategies for creation of long-acting insulin analogs, the mechanisms of functioning of these analogs and their structure are discussed. Side effects of insulin preparations are described, including amyloidogenesis and possible mitogenic effect. The pathways for development of novel insulin analogs are outlined with regard to the current requirements for therapeutic preparations due to the wider occurrence of diabetes of both types.

[Search for Functionally Significant Motifs and Amino Acid Residues of Actin].

The scientific interest to the structural and functional properties of actin is determined by its abundance in cells. Being an important component of the cytoskeleton, actin is involved in many protein-protein interactions. Using crystal structures and molecular models, we have mapped the amino acid residues that are involved in these interactions and form the ATP-binding site of the actin monomer. Moreover, using mass spectrometry and high-performance liquid chromatography methods, we have discovered the regions of the amino acid sequence of actin that form the core of the actin fibril. According to the bioinformatic analysis, these regions are amyloidogenic and are located in the C-terminal region and in the hinge between the first and third subdomains. The data obtained are applicable to chordate actin, because multiple alignment revealed highly conserved amino acid sequences. In turn, the comparison of the chordate actin with the bacterial homologs showed the presence of numerous amino acid substitutions and insertions.

[Anomalous Kinetics of Amyloidogenesis Suggest a Competition between Oligomers and Fibrils].

Meisl et al. have recently observed an anomalous dependence of the amyloid formation rate on the protein concentration. A novel mechanism of fibril growth has been proposed by Meisl et al. to explain the abnormality; it consists in the fibril-catalyzed initiation of fibril formation with saturation of catalytic sites at high concentrations of substrates. Our article describes an alternative explanation of the anomalous kinetics, assuming that the formation of metastable oligomers competes with fibril formation by decreasing the concentration of free monomers. Oligomers are indeed observed in the course of amyloid formation, but are usually considered as seeds of amyloid fibrils rather as their competitors. However, the oligomers visually detectable by electron microscopy were shown to be close in size to those that can be derived from the anomalous dependence of the amyloid growth rate on the protein concentration, given that the anomaly results from competition between oligomer formation and amyloidogenesis.

[Molecular mechanism of amyloid formation by Ab peptide: review of own works]. / Molekuliarnyi mekhanizm amiloidoobrazovaniia Ab peptidom: obzor sobstvennykh rabot.

TA characteristic feature of amyloid structures is polymorphism. The study of amyloid structures and their formation process was carried out for synthetic and recombinant Ab(1-40) and Ab(1-42) peptide preparations. In the study of these peptides, we recognized fibrils of different morphologies. We observed fibrillar formations in the form of single fibrils, ribbons, bundles, bunches, and clusters. Polymorphism of fibrils was observed not only when the environmental conditions changed, but under the same conditions and this was a common characteristics of all amyloid formations. Fibrils of Ab(1-40) peptides tended to form aggregates of fibrils in the form of ribbons, while Ab(1-42) peptide under the same conditions polymerized in the form of rough fibrils of different diameters and tends to branch. We assume that the formation of fibrils of Ab(1-40) and Ab(1-42) peptides occurs according to a simplified scheme: a destabilized monomer ® a ring oligomer ® a mature fibril consisting of ring oligomers. Proceeding from the proposition that the ring oligomer is the main building block of amyloid fibril (similar to the cell in the body), it is easy to explain fibril polymorphism, as well as fragmentation of mature fibrils under various external influences, branching and irregularity of diameter (surface roughness) of fibrils. One aspect of the study of amyloidogenesis is the determination of the regions of the protein chain forming the core of the amyloid fibril. We theoretically predicted amyloidogenic regions for two isoforms of Ab peptides capable of forming an amyloid structure: 16-21 and 32-36 residues. Using the method of tandem mass spectrometry, these regions were determined experimentally. It was shown that the regions of Ab(1-40) peptide from 16 to 22 and from 28 to 40 residues were resistant to the action of proteases, i.e. its formed the core of the amyloid fibril. For Ab(1-42) peptide the whole sequence is not available for the action of proteases, which indicates a different way of associating ring oligomers in the formation of fibrils. Based on electron microscopy and mass spectrometry data we proposed a molecular model of the fibril formed by Ab(1-40) and Ab(1-42) peptides.

α-Crystallins Are Small Heat Shock Proteins: Functional and Structural Properties.

During its life cycle, a cell can be subjected to various external negative effects. Many proteins provide cell protection, including small heat shock proteins (sHsp) that have chaperone-like activity. These proteins have several important functions involving prevention of apoptosis and retention of cytoskeletal integrity; also, sHsp take part in the recovery of enzyme activity. The action mechanism of sHsp is based on the binding of hydrophobic regions exposed to the surface of a molten globule. α-Crystallins presented in chordate cells as two αA- and αB-isoforms are the most studied small heat shock proteins. In this review, we describe the main functions of α-crystallins, features of their secondary and tertiary structures, and examples of their partners in protein-protein interactions.

Amyloid Properties of Titin.

This review considers data on structural and functional features of titin, on the role of this protein in determination of mechanical properties of sarcomeres, and on specific features of regulation of the stiffness and elasticity of its molecules, amyloid aggregation of this protein in vitro, and possibilities of formation of intramolecular amyloid structure in vivo. Molecular mechanisms are described of protection of titin against aggregation in muscle cells. Based on the data analysis, it is supposed that titin and the formed by it elastic filaments have features of amyloid.

Peptide Aß(16-25) Forms Nanofilms in the Process of Its Aggregation.

A method for the synthesis and high purification of fragments of Aß(1-42) peptide has been elaborated. We have synthesized the amyloidogenic fragment Aß(16-25) predicted by us and studied the process of its aggregation by electron microscopy and X-ray analysis. Electron microscopy images show that the peptide forms a film, which is not characteristic of amyloid fibrils. At the same time, according to the X-ray diffraction data, its preparations display the presence of two main reflections (4.6-4.8 and 8-12 Å) characteristic of cross-ß structure of amyloid fibrils. Thus, the fragment Aß(16-25) that we predicted is a promising object not only for studying the process of polymerization of the peptides/proteins, but also for using it as a nanomaterial to study a number of biological processes.

Determination of Regions Involved in Amyloid Fibril Formation for Aß(1-40) Peptide.

The studies of amyloid structures and the process of their formation are important problems of biophysics. One of the aspects of such studies is to determine the amyloidogenic regions of a protein chain that form the core of an amyloid fibril. We have theoretically predicted the amyloidogenic regions of the Aß(1-40) peptide capable of forming an amyloid structure. These regions are from 16 to 21 and from 32 to 36 amino acid residues. In this work, we have attempted to identify these sites experimentally by the method of tandem mass spectrometry. As a result, we show that regions of the Aß(1-40) peptide from 16 to 22 and from 28 to 40 amino acid residues are resistant to proteases, i.e. they are included in the core of amyloid fibrils. Our results correlate with the results of the theoretical prediction.

Determination of Size of Folding Nuclei of Fibrils Formed from Recombinant Aß(1-40) Peptide.

We have developed a highly efficient method for purification of the recombinant product Aß(1-40) peptide. The concentration dependence of amyloid formation by recombinant Aß(1-40) peptide was studied using fluorescence spectroscopy and electron microscopy. We found that the process of amyloid formation is preceded by lag time, which indicates that the process is nucleation-dependent. Further exponential growth of amyloid fibrils is followed by branching scenarios. Based on the experimental data on the concentration dependence, the sizes of the folding nuclei of fibrils were calculated. It turned out that the size of the primary nucleus is one "monomer" and the size of the secondary nucleus is zero. This means that the nucleus for new aggregates can be a surface of the fibrils themselves. Using electron microscopy, we have demonstrated that fibrils of these peptides are formed by the association of rounded ring structures.