Bioorganičeskaâ himiâ

Plant regulatory peptides represent a novel class of signaling molecules that play a central role in the regulation of plant growth, development, and stress responses. Owing to their high biological activity at low concentrations, they are considered promising biostimulants for environmentally sustainable agriculture. This review summarizes key theoretical approaches and experimental methods used for the discovery and identification of these peptides, including mass spectrometry, bioinformatics, bioassays, and in silico screening. An overview is provided of the major peptides identified to date – such as systemin, PSK, PSY, AtPep1, CLV3, TDIF, CEP, and CIF – along with the methods used for their isolation, chemical synthesis, and functional validation. Special attention is given to model systems based on cell cultures and seedlings, which are commonly employed to screen peptide activity, as well as to strategies for identifying their corresponding receptors. The review highlights the critical role of bioassays as a final and indispensable stage in peptide discovery pipelines, enabling the functional evaluation of both identified and putative peptides.

Bacteria utilize a wide range of regulatory systems to adapt to life in various environmental conditions. Among these regulators, small non-coding RNAs (ncRNAs) play a particularly important role. Acting primarily at the post-transcriptional level, small ncRNAs enable bacteria to rapidly adjust expression of genes in response to external stimuli. They participate in the regulation of virtually all cellular processes, including replication, transcription, translation, energy and general metabolism, antibiotic resistance, bacterial virulence, as well as mechanisms associated with bacterial pathogenesis. Bacterial small ncRNAs are capable of mediating interactions between the bacterium and the host organism, directly modulating the expression of eukaryotic genes (most often those related to the immune response). Thus, ncRNAs serve as universal and powerful regulatory elements that ensure the survival and active functioning of bacteria under any adverse conditions.

8-Oxo-2'-deoxyguanosine is a well-known marker of oxidative stress. Research over the past decade suggests that this compound is probably not a byproduct of oxidative DNA damage, but an important bioregulator driving the cellular response to stress. This review collected and analyzed data on the participation of 8-oxo-2'-deoxyguanosine in the processes of mutagenesis, DNA repair and regulation of gene expression, inflammatory responses, adaptive response to stress, apoptosis and cell transformation. Particular attention is paid to the potential of 8-oxo-2'-deoxyguanosine as a therapeutic agent for inflammatory, autoimmune, degenerative and oncological diseases, as well as traumatic and toxic injuries.

Three-finger toxins (TFTs) form one of the most abundant families of toxins in snake venoms. TFTs are common for most Elapid venoms, but are almost never found in viper venoms. Using the venom glands of the vipers Vipera nikolskii and V. berus, 21 cDNA clones encoding this group of toxins were obtained. The amino acid sequences of 9 TFTs were deduced from the obtained cDNA sequences. The identified sequences have signal peptides containing 19-21 amino acid residues, followed by a mature protein consisting of 67 residues. All viper TFTs belong to the group of non-conventional toxins, and their sequences contain 9 cysteine residues. The TFT encoded by one of the transcripts was obtained by heterologous expression in E. coli cells as a fusion protein with the plant partner protein SUMO, followed by cleavage with the specific plant protease BdSENP1 and chromatographic purification. The structure of the obtained protein was confirmed by mass spectrometry. Analysis of its biological activity showed that this toxin is a weak antagonist of nicotinic acetylcholine receptors of the neuronal α7 and α3β2 subtypes. Using the fusion protein with SUMO, we also attempted to obtain the TFT Aze-2 of the viper Azemiops feae, the amino acid sequence of which was previously established by us as a result of transcriptome analysis of the venom gland of A. feae, and the protein itself was identified in minimal quantities in the venom of this snake. However, a toxin exactly corresponding in mass to Aze-2 could not be obtained using this approach. Thus, as a result of the work, the amino acid sequences of 9 viper TFTs were established, one of which was obtained by gene expression in E. coli cells and showed the ability to interact with nicotinic acetylcholine receptors of the neuronal α7 and α3β2 subtypes.

The visualization of macromolecular complexes is an important task in modern bioorganic chemistry, which can be addressed using various methodological approaches. The most widely used techniques involve radioactive- and fluorescent-labeled ligands. In this study, molecular probes were developed in the form of hybrid constructs combining one of three snake toxins (α-bungarotoxin, α-cobratoxin, or neurotoxin NT-II) with the red fluorescent protein mKate2. These chimeric proteins were produced in a bacterial expression system and purified via gel filtration. Using competitive radioligand binding assay with radiolabeled α-bungarotoxin, it was demonstrated that the obtained probes exhibit high affinity for the nicotinic acetylcholine receptor of the electric organ from orpedo californicaT, with half-maximal inhibitory concentration values in the nanomolar range. The fluorescent probes were successfully employed for the visualization of acetylcholine receptors on the surface of SH-SYSY cells.

Zyxin is a cytoskeletal protein that plays a crucial role in the assembly and restoration of actin filaments. Research conducted in our laboratory utilizing a model of Xenopus laevis embryos has demonstrated that Zyxin is significantly involved in gene expression regulation and the process of cell differentiation. In recent years, we have acquired compelling data that suggest the capacity of this mechanosensitive protein to participate in mechanisms that link morphogenetic movements to the expression of genes responsible for the formation of axial structures and the maintenance of stem cell status during the intricate process of embryogenesis, which is pivotal in the life cycle of any organism. In this article, we present the latest findings from our investigation into the genes, signaling pathways, and biological processes that are regulated in conjunction with the activity of Zyxin. To conduct this study, high-throughput sequencing of mRNA pools from X. laevis embryonic cells at the neurula stage was performed. This analysis included samples exhibiting normal Zyxin function, as well as those with increased or suppressed Zyxin function, induced by morpholino oligonucleotides. The application of bioinformatics analysis enabled us to identify Zyxin-dependent signaling pathways and biological processes that are essential for embryogenesis from a comprehensive dataset of genes. Our results indicate that the suppression of Zyxin expression leads to alterations in the expression profiles of genes involved in more than 16 distinct signaling cascades and impacts 27 biological processes. Notably, the most pronounced effects were observed in processes associated with morphogenesis and gene expression. The findings of this study hold significant fundamental implications. Investigating Zyxin role in transducing mechanical stimuli to the gene expression machinery is vital for understanding the coordination between biomechanics and differentiation during embryogenesis. Furthermore, this research may pave the way for utilizing Zyxin as a potential diagnostic marker for various diseases.

Development of an orthotopic intracranial model based on a human glioblastoma cell culture in immunodeficient mice is an important task both for studying the invasiveness and aggressiveness of tumor cell behavior, and for creating a reliable model to evaluate the efficacy of new drugs for glioblastoma therapy. In this study, a comparative analysis was conducted using a glioblastoma cell line (U87MG) and primary glioblastoma cells obtained from a patient (culture 022), following subcutaneous and orthotopic intracranial xenotransplantation into NSG immunodeficient mice. It was shown that in both groups of animals with orthotopic xenografts, the tumors grew both deep into the brain tissue and along the brain surface, while in the case of the primary culture 022, growth toward the ventricles was also observed. The non-cell-line-derived (primary) cells exhibited an epithelioid morphology, whereas U87MG cells showed a more sarcomatoid appearance. The U87MG cell line was tumorigenic in both locations. However, the primary culture 022 formed tumors only following intracranial, but not subcutaneous, xenotransplantation, indicating the neuro-specificity of this model. Therefore, it may serve as a more relevant glioblastoma model compared to the U87MG cell line-based model.

The properties of the voltage-gated potassium Kv1.1 channel, formed in Neuro-2a cells from dimers of the human α-subunits Kv1.1 linked by the Lys-Leu dipeptide and fused at the N-terminus with the mKate2 fluorescent protein (mKate2-(Kv1.1)₂), were studied. It was found that the linking of α-subunits Kv1.1 into a dimer did not affect the membrane expression of the channel and the features of its cellular distribution compared with monomers mKate2-Kv1.1. No differences were found between channels based on mKate2-(Kv1.1)₂ dimers and mKate2-Kv1.1 monomers in the half-activation potential, channel activation constants, the magnitude and nature of potassium ion currents. The data obtained suggest the possibility of creating bioengineered protein structures by linking two different α-subunits in a similar way, which would form fluorescent heterotetrameric voltage-gated potassium channels with an α-subunit stoichiometry of 2 : 2 in mammalian cells.

Cellular heterogeneity is a feature of glioblastoma, a malignant and aggressive brain tumor, and one of the reasons for the ineffectiveness of standard treatment methods, probably arising from the differentiation of tumor stem cells. The causes of increased adaptive abilities and resistance of glioblastoma cells to drugs and stress arise at the level of molecular processes. For this reason, there were evaluated changes in the expression of the genes of transcription factors MYCN, MYCC, PHOX2A and PHOX2B, which are involved in the regulation of the cell cycle and differentiation, using real-time PCR in response to changes in cultivating conditions in this work. As a result, it was shown that primary cultures in an environment with fetal bovine serum acquire morphology and gene expression similar to cell lines. In addition, it was shown for the first time that all cell lines and primary cultures of glioblastoma differ in the expression profiles of the studied genes, however, in response to changes in cultivating conditions, all of them demonstrate a multiple increase in MYCN expression, as well as the opposite response of PHOX2A and PHOX2B, indicating a possible role of these genes in glioblastoma resistance to stress. The obtained data should be taken into account while selecting individual treatment for patients, as well as when developing therapeutic agents and further investigating molecular processes in glioblastoma cells.

Lung adenocarcinoma is a malignant tumor, which is the most common type of non-small cell lung cancer. Low efficiency of standard methods of treatment of lung adenocarcinoma with mutation of the Stk11 tumor suppressor gene is a serious problem in clinical practice. Search and improvement of new therapeutic approaches to this disease remains an urgent task of modern biomedicine. The aim of the work was to create an in vitro model of lung cancer based on the LLC1 cell line with knockout of the Stk11 gene to assess the sensitivity of mutant cells to various types of radiation therapy, including irradiation with photons, protons and neutrons. The main methods used were CRISPR/Cas9 genome editing technologies to obtain mutant clones, laser cell sorting, PCR analysis to confirm the deletion, as well as assessment of viability, proliferation (metabolic tests, Mki67 marker expression), apoptosis induction (annexin V-PI method) and Pten gene expression after cell irradiation with a dose of 2 Gy. As a result, heterozygous mutant lines LLC1-STK11-Mut were obtained. Cell irradiation revealed that in Stk11 mutant cells, radio-induced growth stimulation persisted longer than in wild-type cells, and a significant increase in the proportion of late apoptotic and necrotic cells was observed. At the same time, Mki67 expression temporarily decreased after irradiation, but quickly recovered in mutant cells, which indicates their higher radioresistance. Unlike wild-type cells, the expression level of the Pten gene in mutant cells did not change significantly after irradiation. Thus, the Stk11 mutation contributes to the formation of radioresistance in tumor cells by triggering various adaptation mechanisms. The obtained in vitro model can be used for further study of radioresistance and development of new approaches to the therapy of tumors with STK11 mutation.

Conventional and multifunctional cationic liposomes that efficiently deliver plasmid DNA (pDNA) were obtained. Partial inhibition of receptor-mediated endocytosis of pDNA complexes with multifunctional cationic liposomes containing folate lipids was shown in the presence of free folic acid in the cellular medium. Additional formation of pDNA complexes with the nuclear localization signal (NLS) peptide allowed increasing the efficiency of green fluorescent protein expression by 1.5–2 times using conventional and multifunctional cationic liposomes. Addition of the NLS peptide to pDNA and subsequent formation of complexes with cationic liposomes can be used to solve the problem of efficient pDNA delivery into eukaryotic cells.