Vol 50, No 3 (2024)

To date, polychaetes remain a poorly studied class of invertebrate animals in the context of clarification of their immune system functioning and, in particular, of antimicrobial peptides (AMPs) biodiversity. AMPs, also known as host defense peptides, play a key role in host protection from various pathogens and regulation of the species composition of symbiotic microbes. The study of biosynthesis of AMPs in polychaetes has revealed an interesting pattern, namely so-called BRICHOS domain in the precursor proteins of a number of such peptides. The conserved structure of this domain allows to perform a bioinformatic search for AMP precursors in polychaete transcriptomes. In this work, we found and studied a new BRICHOS-associated AMP from the lugworm Arenicola marina, which represents a structural family of defensin-like peptides stabilized by four disulfide bonds, not previously identified in marine worms. The peptide, designated as AmBRI-44a, contained 44 amino acid residues and was obtained by heterologous expression in Escherichia coli. AmBRI-44a was shown to have a specific activity against a narrow spectrum of Gram-positive bacteria and did not exhibit pronounced cytotoxic effects on eukaryotic cell line HEK293T. A potential mechanism of the antibacterial action of this peptide may be associated with inhibition of bacterial cell wall biosynthesis, as indicated by genetic and phenotypic analysis of selected AmBRI-44a-resistant bacteria Bacillus licheniformis B-511. The results obtained allow us to consider the novel peptide AmBRI-44a as a candidate compound for the development of an antibiotic agent that could potentially be effective in the treatment of infectious diseases mediated by multidrug-resistant Gram-positive bacteria.

Chimeric genes and transcripts can be biological markers as well as the reasons for tumor progression and development. Modern algorithms and high-throughput sequencing are the complementary clues to the question of the tumor origin and cancer detection as well as to the fundamental question of chimeric genes origin and their influence on molecular processes of the cell. A wide-range of algorithms for chimeric genes detection was developed, with various differences in computing speed, sensitivity, specificity, and focus on the experimental design. There exist three main types of bioinformatic approaches, which act according to the sequencing read length. Algorithms, which focus on short-read high-throughput sequencing (about 50–300 bр of read length) or long-read sequencing (about 5000–100000 bр of read length) exclusively or algorithms, which combine the results of both short and long-read sequencing. These algorithms are further subdivided into: 1) mapping-first approaches (STAR-Fusion, Arriba), which map reads to the genome or transcriptome directly and search the reads supporting the fused gene or transcript; 2) assembly-first approaches (Fusion-Bloom), which assemble the genome or transcriptome from the overlapping reads, and then compare the results to the reference transcriptome or genome to find transcripts or genes not present in the reference and therefore raising questions; 3) pseudoalignment approaches, which do not make local alignment, but just search for the closest transcript subsequence to the reads seed, following the precomputed index for all reference transcripts and provides the results. This article describes the main classes of available software tools for chimeric gene detection, provides the characteristics of these programs, their advantages and disadvantages. To date the most resource intensive and slowest are still assembly-first algorithms. Mapping-first approaches are quite fast and rather accurate at fusion detection, still the fastest and resource-saving are the pseudoalignment algorithms, but, worth noting, that the quick search is carried out at the expense of chimeras search quality decrease.

Wound healing is a complex process based on the regulation of proliferation and migration of epithelial cells. Chronic wounds are characterized by increased proliferation and lack of migration of epidermal cells. The secreted human protein SLURP-2 regulates the growth and differentiation of epithelial cells. It has previously been shown that the targets of SLURP-2 are various types of nicotinic acetylcholine receptors (nAChRs), as well as muscarinic acetylcholine receptors involved in the regulation of epithelial cell homeostasis. In this work, we found that the previously demonstrated acceleration of keratinocyte migration under the incubation with SLURP-2 is due to its interaction with α7 type nAChR. Using alanine scanning mutagenesis, we showed that the R20A mutation of the SLURP-2 molecule increases the inhibitory activity of SLURP-2 towards α7-nAChR and leads to an even greater stimulation of Het-1A keratinocyte migration, while, in contrast to SLURP-2, does not stimulate, but suppresses the proliferation of Het-1A cells. At the same time, other SLURP-2 mutations simultaneously lead to inhibition of α7-nAChR, proliferation and migration of keratinocytes. Thus, new information was obtained about the localization of regions of the SLURP-2 molecule, the replacement of which can lead to a targeted change in the biological activity of SLURP-2. Further research into the possibility of regulating the activity of SLURP-2 and the creation of targeted drugs based on it may be useful for the development of new drugs that stimulate wound healing.

Zyxin is a conserved mechanosensitive LIM-domain protein that regulates the assembly of F-actin filaments at cell junctions. At the same time, under mechanical stress, it can move from focal adhesions to stress fibrils and into the nucleus and affect gene expression. In Xenopus laevis embryonic cells, western blots with antibodies against Zyxin’s N-terminal and C-terminal LIM-domain regions revealed two full-length and two short isoforms. The intracellular localization of these isoforms and the number depending on the stage of embryo development were determined. According to our study, full forms with different electrophoretic mobility are localized differently in the cell, and the shortest isoform containing LIM-domains is stable during development, mainly located in the nucleus, and participates in gene expression regulation. This study may be of great value to understanding how the LIM-domain mechanotransducer proteins jointly influence morphogenesis and differentiation in vertebrates at early stages of development.

The genes of hybrid proteins including Exiguobacterium sibiricum proteorhodopsin (ESR) and various N-terminal soluble domains have been constructed. Effective synthesis in Escherichia coli cells was observed only in the case of hybrids with chaperone Caf1M and maltose-binding protein MBP expressed as precursors with their own signal sequences. The study of the isolated MBP-ESR protein in micelles and proteoliposomes demonstrated formation and decay of the main photocycle intermediates at pH > 8. The photoelectric response of the hybrid proteins Caf-ESR and MBP-ESR is comparable in amplitude to the wild-type ESR response, indicating their homogeneous orientation in the membrane. The obtained constructions can be used to create bacterial expression systems for various retinal proteins, ensuring their uniform incorporation into proteoliposomes.

Transmission electron microscopy (TEM) with contrast staining by uranyl acetate solution was used to study morphological differences between soil humic acids (HAs) and their A, B and C + D fractions obtained using coupling preparative low-pressure size exclusion chromatography and analytical polyacrylamide gel electrophoresis. The electrophoretic mobility of fractions varied in order C + D > B > A. The distribution of various morphological elements between fractions showed that large structures such as vesicle-like formations 70–150 nm long and 30–80 nm wide with clear edges were found exclusively in fraction A and occupied ~55% of the TEM image area. On the other hand, long fibrils with a length of 60–100 nm, a width of 4–6 nm and a thickness of 2–3 nm, as well as their bundles with a length of >150 nm and a diameter of 30–70 nm were identified only in the C + D fraction and occupied ~59 % area of TEM images. Smaller morphological elements such as point particles with a diameter of 2–3 nm, ring particles with a diameter of 4–6 nm, worm-shaped short particles with a length of 20–30 nm, and spheroids with a diameter of 10– 30 nm were observed in all samples, but in varying quantities. Significant morphological differences between the fractions can be explained by their composition, previously established by using a few physico-chemical methods. The ratio C_ar(165–108 ppm)/C_alk(108–0 ppm), or aromaticity index, calculated from ¹³C-NMR, could be one of the indicators of the various morphological structures formation. The obtained TEM results clearly confirm the supramolecular organization of soil HAs.

The three dimensional structure of DiB3-F53L/F74L/L129M – a triple mutant of the fluorescent non-covalent complex of a genetically engineered variant of the bacterial protein lipocalin Blc with the synthetic GFP-like chromophore M739, has been studied by molecular dynamics methods. It was found that the chromophore binding site in the complex is similar to DiB1 and differs from the alternative site in DiB3. The complex is characterized by increased fluorescence brightness compared to those with other genetically engineered variants of lipocalin, which makes it one of the promising markers of biological objects in cell biology.