Publications by authors named "Oxana V Galzitskaya"

97 Publications

Myosin Binding Protein-C Forms Amyloid-Like Aggregates In Vitro.

Int J Mol Sci 2021 Jan 13;22(2). Epub 2021 Jan 13.

Laboratory of the Structure and Functions of Muscle Proteins, Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, 142290 Pushchino, Russia.

This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5-10 min) formation of large (>2 μm) aggregates. sMyBP-C oligomers formed both at the initial 5-10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7-10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-β quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (<26%), alternating ordered/disordered regions in the protein molecule, and S-S bonds providing for general stability.
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http://dx.doi.org/10.3390/ijms22020731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828380PMC
January 2021

Bioinformatics Analysis of Actin Molecules: Why Quantity Does Not Translate Into Quality?

Front Genet 2020 10;11:617763. Epub 2020 Dec 10.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Russia.

It is time to review all the available data and find the distinctive characteristics of actin that make it such an important cell molecule. The presented double-stranded organization of filamentous actin cannot explain the strong polymorphism of actin fibrils. In this work, we performed bioinformatics analysis of a set of 296 amino acid actin sequences from representatives of different classes of the Chordate type. Based on the results of the analysis, the degree of conservatism of the primary structure of this protein in representatives of the Chordate type was determined. In addition, 155 structures of rabbit actin obtained using X-ray diffraction analysis and electron microscopy have been analyzed over the past 30 years. From pairwise alignments and the calculation of root-mean-square deviations (RMSDs) for these structures, it follows that they are very similar to each other without correlation with the structure resolution and the reconstruction method: the RMSDs for 11,781 pairs did not exceed 3 Å. It turned out that in rabbit actin most of the charged amino acid residues are located inside the protein, which is not typical for the protein structure. We found that two of six exon regions correspond to structural subdomains. To test the double-stranded organization of the actin structure, it is necessary to use new approaches and new techniques, taking into account our new data obtained from the structural analysis of actin.
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http://dx.doi.org/10.3389/fgene.2020.617763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758494PMC
December 2020

Antimicrobial and Amyloidogenic Activity of Peptides. Can Antimicrobial Peptides Be Used against SARS-CoV-2?

Int J Mol Sci 2020 Dec 15;21(24). Epub 2020 Dec 15.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Russia.

At present, much attention is paid to the use of antimicrobial peptides (AMPs) of natural and artificial origin to combat pathogens. AMPs have several points that determine their biological activity. We analyzed the structural properties of AMPs, as well as described their mechanism of action and impact on pathogenic bacteria and viruses. Recently published data on the development of new AMP drugs based on a combination of molecular design and genetic engineering approaches are presented. In this article, we have focused on information on the amyloidogenic properties of AMP. This review examines AMP development strategies from the perspective of the current high prevalence of antibiotic-resistant bacteria, and the potential prospects and challenges of using AMPs against infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
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http://dx.doi.org/10.3390/ijms21249552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765370PMC
December 2020

MIRRAGGE - Minimum Information Required for Reproducible AGGregation Experiments.

Front Mol Neurosci 2020 27;13:582488. Epub 2020 Nov 27.

Instituto de Biologia Molecular e Celular and Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.

Reports on phase separation and amyloid formation for multiple proteins and aggregation-prone peptides are recurrently used to explore the molecular mechanisms associated with several human diseases. The information conveyed by these reports can be used directly in translational investigation, e.g., for the design of better drug screening strategies, or be compiled in databases for benchmarking novel aggregation-predicting algorithms. Given that minute protocol variations determine different outcomes of protein aggregation assays, there is a strong urge for standardized descriptions of the different types of aggregates and the detailed methods used in their production. In an attempt to address this need, we assembled the Minimum Information Required for Reproducible Aggregation Experiments (MIRRAGGE) guidelines, considering first-principles and the established literature on protein self-assembly and aggregation. This consensus information aims to cover the major and subtle determinants of experimental reproducibility while avoiding excessive technical details that are of limited practical interest for non-specialized users. The MIRRAGGE table (template available in Supplementary Information) is useful as a guide for the design of new studies and as a checklist during submission of experimental reports for publication. Full disclosure of relevant information also enables other researchers to reproduce results correctly and facilitates systematic data deposition into curated databases.
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http://dx.doi.org/10.3389/fnmol.2020.582488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729192PMC
November 2020

New Model for Stacking Monomers in Filamentous Actin from Skeletal Muscles of .

Int J Mol Sci 2020 Nov 6;21(21). Epub 2020 Nov 6.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

To date, some scientific evidence (limited proteolysis, mass spectrometry analysis, electron microscopy (EM)) has accumulated, which indicates that the generally accepted model of double-stranded of filamentous actin (F-actin) organization in eukaryotic cells is not the only one. This entails an ambiguous understanding of many of the key cellular processes in which F-actin is involved. For a detailed understanding of the mechanism of F-actin assembly and actin interaction with its partners, it is necessary to take into account the polymorphism of the structural organization of F-actin at the molecular level. Using electron microscopy, limited proteolysis, mass spectrometry, X-ray diffraction, and structural modeling we demonstrated that F-actin presented in the EM images has no double-stranded organization, the regions of protease resistance are accessible for action of proteases in F-actin models. Based on all data, a new spatial model of filamentous actin is proposed, and the F-actin polymorphism is discussed.
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http://dx.doi.org/10.3390/ijms21218319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664232PMC
November 2020

Comparative Analysis of Proteomes of a Number of Nosocomial Pathogens by KEGG Modules and KEGG Pathways.

Int J Mol Sci 2020 Oct 22;21(21). Epub 2020 Oct 22.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

Nosocomial (hospital-acquired) infections remain a serious challenge for health systems. The reason for this lies not only in the local imperfection of medical practices and protocols. The frequency of infection with antibiotic-resistant strains of bacteria is growing every year, both in developed and developing countries. In this work, a pangenome and comparative analysis of 201 genomes of , spp., , and spp. was performed on the basis of high-level functional annotations-KEGG pathways and KEGG modules. The first three organisms are serious nosocomial pathogens, often exhibiting multidrug resistance. Analysis of KEGG modules revealed methicillin resistance in 25% of strains and resistance to carbapenems in 21% of spp. strains. has a wide range of unique efflux systems. One hundred percent of the analyzed strains have at least two drug resistance systems, and 75% of the strains have seven. Each of the organisms has a characteristic set of metabolic features, whose impact on drug resistance can be considered in future studies. Comparing the genomes of nosocomial pathogens with each other and with genomes can expand our understanding of the versatility of certain metabolic features and mechanisms of drug resistance.
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http://dx.doi.org/10.3390/ijms21217839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660090PMC
October 2020

Antimicrobial and Amyloidogenic Activity of Peptides Synthesized on the Basis of the Ribosomal S1 Protein from Thermus Thermophilus.

Int J Mol Sci 2020 09 2;21(17). Epub 2020 Sep 2.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

Controlling the aggregation of vital bacterial proteins could be one of the new research directions and form the basis for the search and development of antibacterial drugs with targeted action. Such approach may be considered as an alternative one to antibiotics. Amyloidogenic regions can, like antibacterial peptides, interact with the "parent" protein, for example, ribosomal S1 protein (specific only for bacteria), and interfere with its functioning. The aim of the work was to search for peptides based on the ribosomal S1 protein from , exhibiting both aggregation and antibacterial properties. The biological system of the response of Gram-negative bacteria to the action of peptides was characterized. Among the seven studied peptides, designed based on the S1 protein sequence, the R23I (modified by the addition of HIV transcription factor fragment for bacterial cell penetration), R23T (modified), and V10I (unmodified) peptides have biological activity that inhibits the growth of cells, that is, they have antimicrobial activity. But, only the R23I peptide had the most pronounced activity comparable with the commercial antibiotics. We have compared the proteome of peptide-treated and intact cells. These important data indicate a decrease in the level of energy metabolism and anabolic processes, including the processes of biosynthesis of proteins and nucleic acids. Under the action of 20 and 50 μg/mL R23I, a decrease in the number of proteins in cells was observed and S1 ribosomal protein was absent. The obtained results are important for understanding the mechanism of amyloidogenic peptides with antimicrobial activity and can be used to develop new and improved analogues.
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http://dx.doi.org/10.3390/ijms21176382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504387PMC
September 2020

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

Int J Mol Sci 2020 Jul 22;21(15). Epub 2020 Jul 22.

Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Moscow Region, Russia.

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 and 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.
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http://dx.doi.org/10.3390/ijms21155199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432502PMC
July 2020

Determination of amyloid core regions of insulin analogues fibrils.

Prion 2020 01;14(1):149-162

Institute of Protein Research, Russian Academy of Sciences , Pushchino, Russian Federation.

A rapid-acting insulin lispro and long-acting insulin glargine are commonly used for the treatment of diabetes. Clinical cases have described the formation of injectable amyloidosis with these insulin analogues, but their amyloid core regions of fibrils were unknown. To reveal these regions, we have analysed the hydrolyzates of insulin fibrils and its analogues using high-performance liquid chromatography and mass spectrometry methods and found that insulin and its analogues have almost identical amyloid core regions that intersect with the predicted amyloidogenic regions. The obtained results can be used to create new insulin analogues with a low ability to form fibrils.

Abbreviations: a.a., amino acid residues; HPLC-MS, high-performance liquid chromatography/mass spectrometry; m/z, mass-to-charge ratio; TEM, transmission electron microscopy.
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http://dx.doi.org/10.1080/19336896.2020.1776062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518739PMC
January 2020

Structural and Functional Peculiarities of α-Crystallin.

Biology (Basel) 2020 Apr 23;9(4). Epub 2020 Apr 23.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

α-Crystallin is the major protein of the eye lens and a member of the family of small heat-shock proteins. Its concentration in the human eye lens is extremely high (about 450 mg/mL). Three-dimensional structure of native α-crystallin is unknown. First of all, this is the result of the highly heterogeneous nature of α-crystallin, which hampers obtaining it in a crystalline form. The modeling based on the electron microscopy (EM) analysis of α-crystallin preparations shows that the main population of the α-crystallin polydisperse complex is represented by oligomeric particles of rounded, slightly ellipsoidal shape with the diameter of about 13.5 nm. These complexes have molecular mass of about 700 kDa. In our opinion, the heterogeneity of the α-crystallin complex makes it impossible to obtain a reliable 3D model. In the literature, there is evidence of an enhanced chaperone function of α-crystallin during its dissociation into smaller components. This may indirectly indicate that the formation of heterogeneous complexes is probably necessary to preserve α-crystallin in a state inactive before stressful conditions. Then, not only the heterogeneity of the α-crystallin complex is an evolutionary adaptation that protects α-crystallin from crystallization but also the enhancement of the function of α-crystallin during its dissociation is also an evolutionary acquisition. An analysis of the literature on the study of α-crystallin in vitro led us to the assumption that, of the two α-crystallin isoforms (αA- and αB-crystallins), it is αA-crystallin that plays the role of a special chaperone for αB-crystallin. In addition, our data on X-ray diffraction analysis of α-crystallin at the sample concentration of about 170-190 mg/mL allowed us to assume that, at a high concentration, the eye lens α-crystallin can be in a gel-like stage. Finally, we conclude that, since all the accumulated data on structural-functional studies of α-crystallin were carried out under conditions far from native, they cannot adequately reflect the features of the functioning of α-crystallin in vivo.
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http://dx.doi.org/10.3390/biology9040085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235859PMC
April 2020

Disordered Residues and Patterns in the Protein Data Bank.

Molecules 2020 Mar 27;25(7). Epub 2020 Mar 27.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, 142290 Moscow, Russia.

We created a new library of disordered patterns and disordered residues in the Protein Data Bank (PDB). To obtain such datasets, we clustered the PDB and obtained the groups of chains with different identities and marked disordered residues. We elaborated a new procedure for finding disordered patterns and created a new version of the library. This library includes three sets of patterns: unique patterns, patterns consisting of two kinds of amino acids, and homo-repeats. Using this database, the user can: (1) find homologues in the entire Protein Data Bank; (2) perform a statistical analysis of disordered residues in protein structures; (3) search for disordered patterns and homo-repeats; (4) search for disordered regions in different chains of the same protein; (5) download clusters of protein chains with different identity from our database and library of disordered patterns; and (6) observe 3D structure interactively using MView. A new library of disordered patterns will help improve the accuracy of predictions for residues that will be structured or unstructured in a given region.
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http://dx.doi.org/10.3390/molecules25071522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180803PMC
March 2020

Oligomers Are Promising Targets for Drug Development in the Treatment of Proteinopathies.

Front Mol Neurosci 2019 31;12:319. Epub 2020 Jan 31.

Laboratory of Bioinformatics and Proteomics, Institute of Protein Research, Russian Academy of Sciences, Pushchino, Russia.

Currently, there is no effective treatment of proteinopathies, as well as their diagnosis in the early stages of the disease until the first clinical symptoms appear. The proposed model of fibrillation of the Aβ peptide and its fragments not only describes molecular rearrangements, but also offers models of processes that occur during the formation of amyloid aggregates. Since this model is also characteristic of other proteins and peptides, a new potential target for drug development in the treatment of Alzheimer's disease (AD) and other proteinopathies is proposed on the basis of this model. In our opinion, it is oligomers that are promising targets for innovative developments in the treatment of these diseases.
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http://dx.doi.org/10.3389/fnmol.2019.00319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7006448PMC
January 2020

How Quickly Do Proteins Fold and Unfold, and What Structural Parameters Correlate with These Values?

Biomolecules 2020 01 29;10(2). Epub 2020 Jan 29.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.

The correlations between the logarithm of the unfolding rate of 108 proteins and their structural parameters were calculated. We showed that there is a good correlation between the logarithm of folding and unfolding rates (0.79) and protein stability and unfolding rate (0.79). Thus, the faster the protein folds, the faster it unfolds. Folding and unfolding rates are higher for the proteins with two-state kinetics, in comparison with the proteins with multi-state kinetics. At the same time, two-state bacterial proteins folds and unfolds two orders of magnitude faster than two-state eukaryotic proteins, and multi-state bacterial proteins folds and unfolds slower than multi-state eukaryotic proteins. Despite the fact that the folding rates of thermophilic and mesophilic proteins are close, the unfolding rates of thermophilic proteins is about two orders of magnitude lower than for mesophilic proteins. The correlation between unfolding rate and stability of thermophilic proteins is high (0.90). We also found that the unfolding rate correlates with such structural parameters as: size of the protein, radius of the cross-section, logarithm of absolute contact order, and radius of gyration. This information will be useful for engineering and designing new proteins with desired properties.
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http://dx.doi.org/10.3390/biom10020197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072309PMC
January 2020

Investigation of the Relationship between the S1 Domain and Its Molecular Functions Derived from Studies of the Tertiary Structure.

Molecules 2019 Oct 13;24(20). Epub 2019 Oct 13.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

S1 domain, a structural variant of one of the "oldest" OB-folds (oligonucleotide/oligosaccharide-binding fold), is widespread in various proteins in three domains of life: Bacteria, Eukaryotes, and Archaea. In this study, it was shown that S1 domains of bacterial, eukaryotic, and archaeal proteins have a low percentage of identity, which indicates the uniqueness of the scaffold and is associated with protein functions. Assessment of the predisposition of tertiary flexibility of S1 domains using computational and statistical tools showed similar structural features and revealed functional flexible regions that are potentially involved in the interaction of natural binding partners. In addition, we analyzed the relative number and distribution of S1 domains in all domains of life and established specific features based on sequences and structures associated with molecular functions. The results correlate with the presence of repeats of the S1 domain in proteins containing the S1 domain in the range from one (bacterial and archaeal) to 15 (eukaryotic) and, apparently, are associated with the need for individual proteins to increase the affinity and specificity of protein binding to ligands.
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http://dx.doi.org/10.3390/molecules24203681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832287PMC
October 2019

The number of domains in the ribosomal protein S1 as a hallmark of the phylogenetic grouping of bacteria.

PLoS One 2019 22;14(8):e0221370. Epub 2019 Aug 22.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.

The family of ribosomal proteins S1 contains about 20% of all bacterial proteins including the S1 domain. An important feature of this family is multiple copies of structural domains in bacteria, the number of which changes in a strictly limited range from one to six. In this study, the automated exhaustive analysis of 1453 sequences of S1 allowed us to demonstrate that the number of domains in S1 is a distinctive characteristic for phylogenetic bacterial grouping in main phyla. 1453 sequences of S1 were identified in 25 out of 30 different phyla according to the List of Prokaryotic Names with Standing in Nomenclature. About 62% of all records are identified as six-domain S1 proteins, which belong to phylum Proteobacteria. Four-domain S1 are identified mainly in proteins from phylum Firmicutes and Actinobacteria. Records belonging to these phyla are 33% of all records. The least represented two-domain S1 are about 0.6% of all records. The third and fourth domains for the most representative four- and six-domain S1 have the highest percentage of identity with the S1 domain from polynucleotide phosphorylase and S1 domains from one-domain S1. In addition, for these groups, the central part of S1 (the third domain) is more conserved than the terminal domains.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221370PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705787PMC
March 2020

Is there codon usage bias for poly-Q stretches in the human proteome?

J Bioinform Comput Biol 2019 02;17(1):1950010

* Institute of Protein Research, Russian Academy of Sciences, Institutskaya Str., 4, Pushchino, Moscow Region 142290, Russia.

We have analyzed codon usage for poly-Q stretches of different lengths for the human proteome. First, we have obtained that all long poly-Q stretches in Protein Data Bank (PDB) belong to the disordered regions. Second, we have found the bias for codon usage for glutamine homo-repeats in the human proteome. In the cases when the same codon is used for poly-Q stretches only CAG triplets are found. Similar results are obtained for human proteins with glutamine homo-repeats associated with diseases. Moreover, for proteins associated with diseases (from the HraDis database), the fraction of proteins for which the same codon is used for glutamine homo-repeats is less (22%) than for proteins from the human proteome (26%). We have demonstrated for poly-Q stretches in the human proteome that in some cases (28) the splicing sites correspond to the homo-repeats and in 11 cases, these sites appear at the -terminal part of the homo-repeats with statistical significance 10 .
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http://dx.doi.org/10.1142/S0219720019500100DOI Listing
February 2019

Ile351, Leu355 and Ile461 residues are essential for catalytic activity of bovine cytochrome P450scc (CYP11A1).

Steroids 2019 03 11;143:80-90. Epub 2019 Jan 11.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia. Electronic address:

Cytochrome P450scc (CYP11A1) is a mammalian mitochondrial enzyme which catalyzes cholesterol side chain cleavage to form pregnenolone. Along with cholesterol, some other steroids including sterols with a branched side chain like β-sitosterol are the substrates for the enzyme, but the activity towards β-sitosterol is rather low. Modification of the catalytic site conformation could provide more effective β-sitosterol bioconversion by the enzyme. This study was aimed to find out the amino acid residues substitution of which could modify the conformation of the active site providing possible higher enzyme activity towards β-sitosterol. After structural and bioinformatics analysis three amino acid residues I351, L355, I461 were chosen. Molecular dynamics simulations of P450scc evidenced the stability of the wild type, double (I351A/L355A) and triple (I351A/L355A/I461A) mutants. Mutant variants of cDNA encoding P450scc with the single, double and triple mutations were obtained by site-directed mutagenesis. However, the experimental data indicate that the introduced single mutations Ile351A, Leu355A and Ile461A dramatically decrease the target catalytic activity of CYP11A1, and no activity was observed for double and triple mutants obtained. Therefore, isoleucine residues 351 and 461, and leucine residue 355 are important for the cytochrome P450scc functioning towards sterols both with unbranched (cholesterol) and branched (sitosterol) side chains.
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http://dx.doi.org/10.1016/j.steroids.2019.01.002DOI Listing
March 2019

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

J Alzheimers Dis Rep 2018 Oct 24;2(1):181-199. Epub 2018 Oct 24.

Institute of Protein Research, Russian Academy of Science, Pushchino, Moscow Region, Russia.

Aβ and Aβ peptides are believed to be associated with Alzheimer's disease. Aggregates (plaques) of Aβ fibrils are found in the brains of humans affected with this disease. The mechanism of formation of Aβ 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 Aβ and Aβ 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.
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http://dx.doi.org/10.3233/ADR-180063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218156PMC
October 2018

Proteome-scale understanding of relationship between homo-repeat enrichments and protein aggregation properties.

PLoS One 2018 6;13(11):e0206941. Epub 2018 Nov 6.

Group of Bioinformatics, Institute of Protein Research, Russian Academy of Science, Pushchino, Moscow Region, Russia.

Expansion of homo-repeats is a molecular basis for human neurological diseases. We are the first who studied the influence of homo-repeats with lengths larger than four amino acid residues on the aggregation properties of 1449683 proteins across 122 eukaryotic and bacterial proteomes. Only 15% of proteins (215481) include homo-repeats of such length. We demonstrated that RNA-binding proteins with a prion-like domain are enriched with homo-repeats in comparison with other non-redundant protein sequences and those in the PDB. We performed a bioinformatics analysis for these proteins and found that proteins with homo-repeats are on average two times longer than those in the whole database. Moreover, we are first to discover that as a rule, homo-repeats appear in proteins not alone but in pairs: hydrophobic and aromatic homo-repeats appear with similar ones, while homo-repeats with small, polar and charged amino acids appear together with different preferences. We elaborated a new complementary approach to demonstrate the influence of homo-repeats on their host protein aggregation properties. We have shown that addition of artificial homo-repeats to natural and random proteins results in intensification of aggregation properties of the proteins. The maximal effect is observed for the insertion of artificial homo-repeats with 5-6 residues, which is consistent with the minimal length of an amyloidogenic region. We have also demonstrated that the ability of proteins with homo-repeats to aggregate cannot be explained only by the presence of long homo-repeats in them. There should be other characteristics of proteins intensifying the aggregation property including such as the appearance of homo-repeats in pairs in the same protein. We are the first who elaborated a new approach to study the influence of homo-repeats present in proteins on their aggregation properties and performed an appropriate analysis of the large number of proteomes and proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206941PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219797PMC
April 2019

What is Responsible for Atypical Dependence of the Rate of Amyloid Formation on Protein Concentration: Fibril-Catalyzed Initiation of New Fibrils or Competition with Oligomers?

J Phys Chem Lett 2018 Mar 12;9(5):1002-1006. Epub 2018 Feb 12.

Laboratory of Protein Physics and ‡Bioinformatics Group, Institute of Protein Research, Russian Academy of Sciences , Pushchino, 142290, Moscow Region, Russian Federation.

An abnormal dependence of the rate of amyloid formation on protein concentration has been recently observed by Meisl et al. for Aβ40 peptides associated with Alzheimer's disease. To explain this effect, Meisl et al. proposed a novel mechanism of fibril growth: the fibril-catalyzed initiation of fibril formation. In this paper we offer an alternative explanation of the observed anomalous kinetics: formation of metastable oligomers competing with fibril formation by decreasing the concentration of the fibril-forming free monomers. Here we show that the oligomer sizes resulting from the anomalous dependence of the fibril growth rate on protein concentration are close to the sizes of oligomers observed by electron microscopy.
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http://dx.doi.org/10.1021/acs.jpclett.7b03442DOI Listing
March 2018

To Be Fibrils or To Be Nanofilms? Oligomers Are Building Blocks for Fibril and Nanofilm Formation of Fragments of Aβ Peptide.

Langmuir 2018 02 30;34(6):2332-2343. Epub 2018 Jan 30.

Institute of Protein Research, Russian Academy of Sciences , Pushchino 142290, Russia.

To identify the key stages in the amyloid fibril formation we studied the aggregation of amyloidogenic fragments of Aβ peptide, Aβ(16-25), Aβ(31-40), and Aβ(33-42), using the methods of electron microscopy, X-ray analysis, mass spectrometry, and structural modeling. We have found that fragments Aβ(31-40) and Aβ(33-42) form amyloid fibrils in the shape of bundles and ribbons, while fragment Aβ(16-25) forms only nanofilms. We are the first who performed 2D reconstruction of amyloid fibrils by the Markham rotation technique on electron micrographs of negatively stained fragments of Aβ peptide. Combined analysis of the data allows us to speculate that both the fibrils and the films are formed via association of ring-shaped oligomers with the external diameter of about 6 to 7 nm, the internal diameter of 2 to 3 nm, and the height of ∼3 nm. We conclude that such oligomers are the main building blocks in fibrils of any morphology. The interaction of ring oligomers with each other in different ways makes it possible to explain their polymorphism. The new mechanism of polymerization of amyloidogenic proteins and peptides, described here, could stimulate new approaches in the development of future therapeutics for the treatment of amyloid-related diseases.
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http://dx.doi.org/10.1021/acs.langmuir.7b03393DOI Listing
February 2018

Comparative mechanical unfolding studies of spectrin domains R15, R16 and R17.

J Struct Biol 2018 02 5;201(2):162-170. Epub 2017 Dec 5.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia. Electronic address:

Spectrins belong to repetitive three-helix bundle proteins that have vital functions in multicellular organisms and are of potential value in nanotechnology. To reveal the unique physical features of repeat proteins we have studied the structural and mechanical properties of three repeats of chicken brain α-spectrin (R15, R16 and R17) at the atomic level under stretching at constant velocities (0.01, 0.05 and 0.1 Å·ps) and constant forces (700 and 900 pN) using molecular dynamics (MD) simulations at T = 300 K. 114 independent MD simulations were performed and their analysis has been done. Despite structural similarity of these domains we have found that R15 is less mechanically stable than R16, which is less stable than R17. This result is in agreement with the thermal unfolding rates. Moreover, we have observed the relationship between mechanical stability, flexibility of the domains and the number of aromatic residues involved in aromatic clusters.
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http://dx.doi.org/10.1016/j.jsb.2017.12.003DOI Listing
February 2018

Hepatic and Aortic Arch Expression and Serum Levels of Syndecan-1 in ApoE Mice.

Open Biochem J 2017 21;11:77-93. Epub 2017 Sep 21.

Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Moscow Region, Pushchino, 142290, Russia.

Background: Heparan sulfate proteoglycan (HSPG) syndecan-1 (Sdc1) acts as a receptor for triglyceride-rich lipoproteins (TRLs), growth factors, chemokines and enzymes. Due to the disordered structure, its function is as diverse as its ligands. In this paper, we have analyzed hepatic and aortic arch expression of Sdc1 in ApoE mice and examined their association with biochemical changes in plasma during the atheroma formation.

Methods: ApoE knockout (ApoE) mice as a model of atherosclerosis were used. Plasma chemistry parameters were estimated by automatic biochemical analyzer. The ELISA test was used to detect soluble Sdc1. The mRNA level of syndecan-1 in liver cells and aortic arch was determined by real time PCR.

Results: The Sdc1 mRNA level in liver cells was 1.5-2.5 times higher in ApoE mice compared to the wild-type species and increased with age, whereas it remained at the same level in wild-type mice upon aging. Furthermore, the plasma cholesterol level was 4-6 times higher in ApoE mice compared to the wild type; in contrast, triglyceride (TG) remained at the same level. Simultaneously, the expression of Sdc1 in the aortic arch of ApoE mice decreases with age; however, it increases in wild-type mice of the same age. We determined that the Sdc1 mRNA expression in liver cells is significantly higher compared to the cells of aortic arch. In addition, our research demonstrated that the level of soluble Sdc1 slightly increased with age and did not depend on mouse genotype; yet, the total amount of soluble Sdc1 was higher in ApoE mice.

Conclusion: Our data suggest that the level of soluble Sdc1 in serum of mice can be associated with chronic inflammation. In addition, we hypothesized that a compensatory increase in the Sdc1 expression in ApoE mice may prevent accumulation of triglycerides in serum, yet having no effect on cholesterol accumulation.
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http://dx.doi.org/10.2174/1874091X01711010077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5676011PMC
September 2017

Rosetta Stone for Amyloid Fibrils: The Key Role of Ring-Like Oligomers in Amyloidogenesis.

J Alzheimers Dis 2017 ;59(3):785-795

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Russia.

Deeper understanding of processes of protein misfolding, aggregation, formation of oligomers, protofibrils, and fibrils is crucial for the development of future medicine in treatment of amyloid-related diseases. While numerous reports illuminate the field, the above processes are extremely complex, as they depend on many varying parameters, such as the peptide concentration, temperature, pH, presence of metal ions, lipids, and organic solvents. Different mechanisms of amyloid fibril formation have been proposed, but the process of the oligomer-to-fibril transition is the least agreed upon. Our studies of a number of amyloidogenic proteins and peptides (insulin, Aβ peptides, the Bgl2 protein from the yeast cell wall), as well as their amyloidogenic fragments, have allowed us to propose a model of the fibril structure generation. We have found that the main building block of fibrils of any morphology is a ring-like oligomer. The varying models of interaction of ring oligomers with each other revealed in our studies make it possible to explain their polymorphism. Crucially, the amino acid sequence determines the oligomer structure for the given protein/peptide.
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http://dx.doi.org/10.3233/JAD-170230DOI Listing
April 2018

There and back again: Two views on the protein folding puzzle.

Phys Life Rev 2017 07 27;21:56-71. Epub 2017 Jan 27.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russian Federation.

The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured folding times of single-domain globular proteins range from microseconds to hours: the difference (10-11 orders of magnitude) is the same as that between the life span of a mosquito and the age of the universe. This review describes physical theories of rates of overcoming the free-energy barrier separating the natively folded (N) and unfolded (U) states of protein chains in both directions: "U-to-N" and "N-to-U". In the theory of protein folding rates a special role is played by the point of thermodynamic (and kinetic) equilibrium between the native and unfolded state of the chain; here, the theory obtains the simplest form. Paradoxically, a theoretical estimate of the folding time is easier to get from consideration of protein unfolding (the "N-to-U" transition) rather than folding, because it is easier to outline a good unfolding pathway of any structure than a good folding pathway that leads to the stable fold, which is yet unknown to the folding protein chain. And since the rates of direct and reverse reactions are equal at the equilibrium point (as follows from the physical "detailed balance" principle), the estimated folding time can be derived from the estimated unfolding time. Theoretical analysis of the "N-to-U" transition outlines the range of protein folding rates in a good agreement with experiment. Theoretical analysis of folding (the "U-to-N" transition), performed at the level of formation and assembly of protein secondary structures, outlines the upper limit of protein folding times (i.e., of the time of search for the most stable fold). Both theories come to essentially the same results; this is not a surprise, because they describe overcoming one and the same free-energy barrier, although the way to the top of this barrier from the side of the unfolded state is very different from the way from the side of the native state; and both theories agree with experiment. In addition, they predict the maximal size of protein domains that fold under solely thermodynamic (rather than kinetic) control and explain the observed maximal size of the "foldable" protein domains.
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http://dx.doi.org/10.1016/j.plrev.2017.01.025DOI Listing
July 2017

Proteome Evolution of Deep-Sea Hydrothermal Vent Alvinellid Polychaetes Supports the Ancestry of Thermophily and Subsequent Adaptation to Cold in Some Lineages.

Genome Biol Evol 2017 02;9(2):279-296

Sorbonne Universités, UPMC Univ. Paris 06, CNRS UMR 7144, Adaptation et Diversité en Milieu Marin, Equipe ABICE, Station Biologique de Roscoff, 29688 Roscoff, France.

Temperature, perhaps more than any other environmental factor, is likely to influence the evolution of all organisms. It is also a very interesting factor to understand how genomes are shaped by selection over evolutionary timescales, as it potentially affects the whole genome. Among thermophilic prokaryotes, temperature affects both codon usage and protein composition to increase the stability of the transcriptional/translational machinery, and the resulting proteins need to be functional at high temperatures. Among eukaryotes less is known about genome evolution, and the tube-dwelling worms of the family Alvinellidae represent an excellent opportunity to test hypotheses about the emergence of thermophily in ectothermic metazoans. The Alvinellidae are a group of worms that experience varying thermal regimes, presumably having evolved into these niches over evolutionary times. Here we analyzed 423 putative orthologous loci derived from 6 alvinellid species including the thermophilic Alvinella pompejana and Paralvinella sulfincola. This comparative approach allowed us to assess amino acid composition, codon usage, divergence, direction of residue changes and the strength of selection along the alvinellid phylogeny, and to design a new eukaryotic thermophilic criterion based on significant differences in the residue composition of proteins. Contrary to expectations, the alvinellid ancestor of all present-day species seems to have been thermophilic, a trait subsequently maintained by purifying selection in lineages that still inhabit higher temperature environments. In contrast, lineages currently living in colder habitats likely evolved under selective relaxation, with some degree of positive selection for low-temperature adaptation at the protein level.
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http://dx.doi.org/10.1093/gbe/evw298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381640PMC
February 2017

Taxonomic distribution, repeats, and functions of the S1 domain-containing proteins as members of the OB-fold family.

Proteins 2017 04 3;85(4):602-613. Epub 2017 Feb 3.

Group of Bioinformatics, Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.

Proteins of the nucleic acid-binding proteins superfamily perform such functions as processing, transport, storage, stretching, translation, and degradation of RNA. It is one of the 16 superfamilies containing the OB-fold in protein structures. Here, we have analyzed the superfamily of nucleic acid-binding proteins (the number of sequences exceeds 200,000) and obtained that this superfamily prevalently consists of proteins containing the cold shock DNA-binding domain (ca. 131,000 protein sequences). Proteins containing the S1 domain compose 57% from the cold shock DNA-binding domain family. Furthermore, we have found that the S1 domain was identified mainly in the bacterial proteins (ca. 83%) compared to the eukaryotic and archaeal proteins, which are available in the UniProt database. We have found that the number of multiple repeats of S1 domain in the S1 domain-containing proteins depends on the taxonomic affiliation. All archaeal proteins contain one copy of the S1 domain, while the number of repeats in the eukaryotic proteins varies between 1 and 15 and correlates with the protein size. In the bacterial proteins, the number of repeats is no more than 6, regardless of the protein size. The large variation of the repeat number of S1 domain as one of the structural variants of the OB-fold is a distinctive feature of S1 domain-containing proteins. Proteins from the other families and superfamilies have either one OB-fold or change slightly the repeat numbers. On the whole, it can be supposed that the repeat number is a vital for multifunctional activity of the S1 domain-containing proteins. Proteins 2017; 85:602-613. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/prot.25237DOI Listing
April 2017

Dataset of the molecular dynamics simulations of bilayers consisting of short amyloidogenic peptide VDSWNVLVAG from Bgl2p-glucantransferase of cell wall.

Data Brief 2016 Dec 3;9:597-601. Epub 2016 Oct 3.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

The amyloidogenic peptide VSWNVLVAG from Bgl2p-glucantransferase of cell wall and its modifying analog VSWNVLVAG were taken for the construction of four types of bilayers which differ by orientation of the peptides in the layers and of the layers relative to each other. These bilayers were used as starting models for the molecular dynamics (MD) at three charge states (neutral, pH3, and pH5). The changes of the fraction of secondary structure during 1 ns simulations were received for 96 MD trajectories. The data article contains the necessary information for the construction of models of β-strands organization in the oligomer structure. These results were used in the associated research article "Structural model of amyloid fibrils for amyloidogenic peptide from Bgl2p-glucantransferase of cell wall and its modifying analog. New morphology of amyloid fibrils" (Selivanova et al., 2016) [1].
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http://dx.doi.org/10.1016/j.dib.2016.09.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065633PMC
December 2016

The Mechanism Underlying Amyloid Polymorphism is Opened for Alzheimer's Disease Amyloid-β Peptide.

J Alzheimers Dis 2016 09;54(2):821-30

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.

It has been demonstrated using Aβ40 and Aβ42 recombinant and synthetic peptides that their fibrils are formed of complete oligomer ring structures. Such ring structures have a diameter of about 8-9 nm, an oligomer height of about 2- 4 nm, and an internal diameter of the ring of about 3-4 nm. Oligomers associate in a fibril in such a way that they interact with each other, overlapping slightly. There are differences in the packing of oligomers in fibrils of recombinant and synthetic Aβ peptides. The principal difference is in the degree of orderliness of ring-like oligomers that leads to generation of morphologically different fibrils. Most ordered association of ring-like structured oligomers is observed for a recombinant Aβ40 peptide. Less ordered fibrils are observed with the synthetic Aβ42 peptide. Fragments of fibrils the most protected from the action of proteases have been determined by tandem mass spectrometry. It was shown that unlike Aβ40, fibrils of Aβ42 are more protected, showing less ordered organization compared to that of Aβ40 fibrils. Thus, the mass spectrometry data agree with the electron microscopy data and structural models presented here.
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http://dx.doi.org/10.3233/JAD-160405DOI Listing
September 2016

Structural model of amyloid fibrils for amyloidogenic peptide from Bgl2p-glucantransferase of S. cerevisiae cell wall and its modifying analog. New morphology of amyloid fibrils.

Biochim Biophys Acta 2016 11 6;1864(11):1489-99. Epub 2016 Aug 6.

Institute of Protein Research, Russian Academy of Science, 142290 Pushchino, Moscow Region, Russia. Electronic address:

We performed a comparative study of the process of amyloid formation by short homologous peptides with a substitution of aspartate for glutamate in position 2 - VDSWNVLVAG (AspNB) and VESWNVLVAG (GluNB) - with unblocked termini. Peptide AspNB (residues 166-175) corresponded to the predicted amyloidogenic region of the protein glucantransferase Bgl2 from the Saccharomyces cerevisiae cell wall. The process of amyloid formation was monitored by fluorescence spectroscopy (FS), electron microscopy (EM), tandem mass spectrometry (TMS), and X-ray diffraction (XD) methods. The experimental study at pH3.0 revealed formation of amyloid fibrils with similar morphology for both peptides. Moreover, we found that the morphology of fibrils made of untreated ammonia peptide is not mentioned in the literature. This morphology resembles snakes lying side by side in the form of a wave without intertwining. Irrespective of the way of the peptide preparation, the rate of fibril formation is higher for AspNB than for GluNB. However, preliminary treatment with ammonia highly affected fibril morphology especially for AspNB. Such treatment allowed us to obtain a lag period during the process of amyloid formation. It showed that the process was nucleation-dependent. With or without treatment, amyloid fibrils consisted of ring-like oligomers with the diameter of about 6nm packed either directly ring-to-ring or ring-on-ring with a slight shift. We also proposed the molecular structure of amyloid fibrils for two studied peptides.
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http://dx.doi.org/10.1016/j.bbapap.2016.08.002DOI Listing
November 2016