Biomedical Chemistry: Research and Methods

Efferocytosis as One of the Mechanisms for Realizing the Therapeutic Effects of Mesenchymal Stem Cells

G.A. Blinova — 2024-09-19

Mesenchymal stem cells (MSCs) stimulate regeneration and exhibit unique immunomodulatory properties, which makes them attractive for use in cell therapies of a wide range of pathologies. The clinical use of MSCs is hampered by the insufficiently clear understanding of their therapeutic action mechanisms. It has been reliably proven that MSCs after transplantation quickly die in the recipient's body by the mechanism of apoptosis and are cleared by professional, such as macrophages, and non-professional phagocytes, including endothelial cells, hepatocytes, resident stem cells of various tissues, including MSCs. The ingestion and processing of apoptotic cells by the phagocytes was named efferocytosis. Despite rapid elimination of transplanted cells, in most cases MSC transplantation leads to positive therapeutic effects. Clearance of apoptotic MSCs affects phagocytes, changing their phenotype, secretome, and further behavior. This review presents the basic molecular mechanisms of efferocytosis, examines the clearance of apoptotic MSCs and their therapeutic effects in various pathologies in the context of their efferocytosis by various types of phagocytes.

Strategy for Experimental Studies of Target Protein Interactomics

P.V. Ershov — 2024-10-01

It is known that intermolecular interactions of proteins and peptides play a critical role in life processes. Such interactions can be either directly related to the implementation of various functions or play the role of a regulator. Currently, there is no doubt that the majority of proteins function as part of various molecular complexes, the formation of which occurs due to protein-protein interactions (PPIs), the totality of which can be defined as the “protein interactome”. Protein subinteractome studies are critical for studying the functions and regulatory mechanisms of unknown or poorly annotated proteins, understanding the architecture of intracellular molecular machines, and the design of PPI modulators. Previously, we used combinations of experimental approaches, as well as analytical and preparative methods, to study the subinteractomes of functionally different cellular proteins, which allowed us to identify the protein subinteractomes of several clinically significant human proteins. The purpose of this work was to conceptualize the principles of the experimental platform we developed for studying protein subinteractomes and to describe its features in detail.

New Highly Sensitive Methods for Electroanalysis of the Catalytic Activity of Enzymes of Medical Significance

V.V. Shumyantseva — 2024-10-01

The review is devoted to new highly effective methods for analyzing the catalytic activity of enzymes of medical significance, such as cytochromes P450, trypsin, asparaginase, beta-lactamase, and nucleases. The methods are based on registration the specific activity of enzymes using electroanalytical methods. The review analyzes the experimental data obtained by the authors. Two platforms have been developed that allow quantitative measurement of catalytic activity based on the electrochemical properties of the enzyme (cytochrome P450, bactosomes, asparaginase) or substrate (trypsin, nucleases, restriction enzymes, beta-lactamase).

Prediction of Peptide Ion Distribution in Positive Electrospray Ionization

A.I. Voronina — 2024-09-19

We have investigated the possibility of predicting the distribution of ions of different charge during electrospray ionization of peptides in mass spectrometric experiments using neural networks. Three independent data sets obtained on the same equipment and deposited in ProteomeXchange (PXD032141, PXD051750, PXD019263) were used as training and test samples. A set of fractional values for 1+ to 5+ ions was calculated as predicted values for each of the newly identified peptides. Four different sets of peptide descriptions were used as independent variables, including both the spectrum of amino acid residues and the physicochemical properties of the amino acid residues. Sixty-four variants of neural networks were analyzed, varying the input description, number and type of layers, activation and loss functions. The coefficient of determination and a set of Euclidean, Sørensen, Chebyshev, and Cosine metrics were considered as measures of prediction quality. For the best selected variants, the error did not exceed 10% in 80% of the cases. This accuracy may be sufficient for a preliminary estimation of the probability of detecting a peptide ion of a given charge.

Bigdata of National Medicine Registers

P.I. Savosina — 2024-10-01

Currently, various databases of approved drugs are widely used in the development and application of computer-aided drug design for training sets and validating predicted models. Most of the freely available databases contain information on a limited number of drugs. This leads to reduction in the space of pharmacotherapeutic chemistry studied and used by researchers. Information on drugs developed and used locally, in one or two countries, can be obtained from relevant national medicine registries. We have identified and analyzed registries from more than 70 countries around the world that are accessible through open web resources on the Internet. In addition to lists of approved therapeutics, many web resources offer the opportunity to review official documents published by medical authorities after the approval process. These documents contain a wide range of information about drugs, including data on structural formulations, pharmacodynamics and pharmacokinetics, toxicity, etc. The compounds represented in the registries, both in terms of quantity and structural diversity, exceed the known widely used databases of approved drugs. Combined data from national drug registries represent an example of “Big Data” in the biomedical field, taking into account all the difficulties involved in their processing. Its use in computer-aided drug design will not only expand the pharmacotherapeutic chemical space studied, but also improve the quality of the original data.

Multi-directional Changes in 8-OHdG Concentration in Saliva during HER2 Expression in Breast Cancer

E.I. Dyachenko — 2024-10-01

The purpose of this work was to evaluate changes in the structure of 8-OHdG in saliva in patients with breast cancer depending on molecular biological subtypes. The findings show that decreased salivary 8-OHdG concentrations in the HER2-positive group rapidly correlated with decreased cell differentiation and high Ki-67 levels, reflecting the aggressive nature of the tumor disease and poor survival prognosis. Thus, the presence of high levels of 8-OHdG in the oral cavity may lead to the development of the role of a biological marker of the HER2-positive group with a poor prognosis of breast cancer and can be used as an additional method of diagnosis, patient condition and response to therapy.

Age-related Correlations of Telomere Length of Predator Fish Muscle Tissues with Potentially Different Ageing Mechanisms

M.V. Mikhailova — 2024-10-01

The mechanisms of aging differ and have their own features both mammals, and in different species groups of fish. Telomere length is an indicator of the theoretical number of cell cycles that cells of a particular tissue can go through; therefore, the age-related dynamics of telomere length characterizes changes in the tissue's ability to regenerate and is necessary to describe the mechanism of tissue aging. In this work, age-related linear regressions of the telomere lengths of muscle tissue of northern pike (Esox lucius) and zander (Sander lucioperca) were empirically obtained for the wide age groups of individuals of both sexes. The identified significant difference in the dependences on their slope values indicates different degrees of decrease in the ability to regenerate muscle tissue with age, which is consistent with the previously discovered physiological characteristics of the muscle tissue of pike. In both fish species studied, telomere length in females decreases with age much more slowly than in males, which is a common feature in the aging mechanisms of most vertebrates.

Recombinant Proteins Combining the Inter-domain Region of Pneumococcal Surface Antigen "A" and Adaptive W-type Polypeptide of Thermotoga Bacteria as a Potential Component Base for the Development of New Diagnostics and Genetically Engineered Subunit Vaccines against Pneumococcal Infection

O.K. Parfenova — 2024-10-01

The problems related to the development of pneumococcal vaccines require combination of traditional solutions and alternative systems optimizing this procedure. Recombinant subunit vaccines have undeniable advantages over inactivated and live-attenuated vaccines: they induce cell-mediated and humoral immunologic responses effectively and with high specificity, but without the risks associated with authentic pathogen processing. However, subunit vaccines require specific adjuvants to enhance the immune response or special fusion partners to improve solubility, expression and optimize subsequent fine purification of the protein of interest. In the framework of this work, a structurally conserved region of the most immunogenic region of the vaccine-valuable surface antigen PspA of Streptococcus pneumoniae was chosen as a model protein, and the adaptive polypeptide CheW from the hyperthermophilic microorganism Thermotoga petrophila was used as a promising fusion protein. Appropriate expression plasmid vectors were designed in silico and constructed in vitro. Efficient E. coli producer strains were obtained and appropriate conditions for heterologous production of chimeric proteins were selected. The fusion partner from T. petrophila positively influenced the properties of the resulting constructs such as thermostability, solubility, and homogeneity. During this work, the optimal pH and temperature ranges of the created proteins were determined, and the principles of low-stage purification were elaborated. We obtained and characterized new proteins, which were not previously found in nature in a similar bioconfiguration. The results indicate that the biotechnologically valuable characteristics of the fusion protein were more expressed when the adaptive CheW protein was combined with the N-terminus of the PspA antigen.

Modified Knottins as Potential Inhibitors of HCV NS3 Protease

A.V. Talanova — 2024-10-01

Knottins form a group of peptides containing approximately 30 amino acid residues. Their structures are stabilized by three disulfide bonds forming a characteristic “pseudo-knotted” structure. Several modifications in knottin from Momordica cochinchinensis were made to convert it to inhibitor of human hepatitis C (HCV) NS3 protease. These modifications of the knottin template included deletion of several residues from the N-terminus, replacement of residues in- and outside the inhibitor loop and replacement of certain L-amino acids by their D-stereoisomers. Binding energy values for protein-knottin complexes were estimated by MM-GBSA methods. Two designed knottins showed high stability in knottin-protease HCV complexes and values of binding energy comparable with known peptide inhibitors from crystal structures.

Molecular Profile of the HepG2 Tumor Cell Line

V.A. Arzumanian — 2024-10-01

Cell lines are widely used in scientific research due to their accessibility, stability, and functional similarity to the original cells. The HepG2 line, being the fourth most popular cell culture, is often used in toxicological and metabolic studies due to its partial retention of hepatocyte properties.In our study, the molecular portrait of the HepG2 cell culture was constructed for the first time. To build this portrait, we used previously obtained data for a single sample, including results of whole-genome sequencing (WGS), whole-genome bisulfite sequencing (WGBS), transcriptome (RNA-seq), translatome (Polysome-seq), and proteome (LC-MS/MS) profiling. For the assessment of HepG2 cell line heterogeneity, we analyzed whole-genome and transcriptome data published in the NCBI SRA database, as well as proteome research results available in the PRIDE resource.Our study showed that the HepG2 cell line generally demonstrates a high degree of stability at the genomic and transcriptomic levels; however, samples from China require closer attention when transferring the results of transcriptomic and proteomic experiments. The HepG2 genotype is characterized by stable chromosomal rearrangements, such as translocation between the short arms of chromosomes 1p and 21p, tetrasomy of chromosome 20, loss of the short arm of all SAT chromosomes, and the long arm of the Y chromosome. Despite the absence of 1216 protein-coding genes at the genomic level, 12,602 genes are expressed at the transcriptomic level, of which only 10,461 are detected at the translatome level, and only 1027 genes are identified at the proteome level, which is related to the limitations of mass spectrometry sensitivity. As a result of the omics data analysis, we presented a detailed molecular portrait of the HepG2 cell culture, illustrating the omics profile at various levels for each gene.

Immobilization of L-asparaginase on Oxidized Bacterial Cellulose to Improve the Thermal Stability of the Enzyme

A.N. Shishparenok — 2024-10-01

Bacterial cellulose (BC) membranes can be modified for covalent immobilization of macromolecules. One type of modification is oxidation, after which the oxidized BC membrane (OBC) could be used as a matrix for covalent immobilization of enzymes. In this work, the BC membrane was chemically oxidized with sodium periodate (NaIO4) to increase the stability of immobilized mesophilic L-asparaginase (L-ASNase) from Erwinia carotovora (EwA). IR spectroscopy confirmed the immobilization of L-ASNase EwA on OBC membranes. Immobilization of the enzyme increased its temperature optimum for its activity by 15°C and raised the inactivation temperature to 60°C. The OBC membrane could be used as a potential carrier for covalent immobilization of enzymes to improve their pharmacological properties by increasing their thermostability.

Cytochromes P450 as Tools for Electroenzymatic Synthesis

P.I. Koroleva — 2024-10-01

The electrocatalytic properties of cytochrome P450 2C9 and the cytochrome P450 2C9/FAD and cytochrome P450 2C9/FMN complexes have been studied using a two-electrode system. The system consisted of an enzymatic catalyst electrode modified by the membrane-like compound didodecyldimethylammonium bromide (SPE/DDAB) and a measuring electrode, modified with carbon nanotubes (SPE/CNT). To study the effectiveness of electroenzymatic reactions catalyzed by cytochrome P450 2C9, the nonsteroidal anti-inflammatory drug diclofenac was used as a substrate. Cytochrome P450 2C9 catalyzes the stereospecific hydroxylation reaction to form 4′-hydroxydiclofenac. The metabolite 4′-hydroxydiclofenac was recorded at a potential E=+0.12 (relative to Ag/AgCl).The use of FAD and FMN as low-molecular mediators made it possible to increase the efficiency of electrocatalysis of the SPE/DDAB/CYP2C9/FAD system to 148±10% and SPE/DDAB/CYP2C9/FMN to 113±6% compared to SPE/DDAB/CYP2C9 (100±5%), and also increase the rate of the enzymatic reaction by 1.5 and 1.13 times, respectively.

New Nitrogen-containing Androstane Derivatives Suppressing Prostate Carcinoma Cells Proliferation

A.S. Latysheva — 2024-10-01

Derivatives of 3β-hydroxyandrost-5,16-diene and 3β-hydroxyandrost-5-ene containing 2-oxazoline, 2-benzoxazole, and 2-benzimidazole substituents at C-17 position were synthesized. Docking of the synthesized compounds into the active site of human CYP17A1 predicted their high affinity for the enzyme. Of the 6 new compounds, 5 suppressed the proliferation of prostate carcinoma cells LNCaP and PC-3, and the activity of the oxazoline and benzimidazole derivatives of androsta-5,16-diene significantly exceeded the activity of the known anticancer agents abiraterone and galeterone.

L-asparaginase Conjugates of Rhodospirillum Rubrum with Polymers of Different Structures with Improved Biocatalytic Properties

N.V. Dobryakova — 2024-10-01

L-asparaginase from the mesophyllic bacterium Rhodospirillum rubrum shows promise as a potential biomedicine treatment for tumor diseases. To enhance the enzyme's catalytic parameters, we have synthesized covalent conjugates with chitosan-PEG, chitosan-glycol and polyethylenimine. Interestingly, binding to these polymers had minimal impact on the enzyme's substrate binding constant. The RrA-PEI conjugate showed the most significant changes in secondary structure content. The success of the previous modification was evaluated by IR spectroscopy, which indicated that the polycations used in the work have a positive effect on the catalytic activity of the enzyme. The RrA-chitosan-PEG conjugate demonstrated the highest enzymatic activity.