Acta Naturae最新文献

The ProTx-I toxin from Thrixopelma pruriens tarantula venom inhibits voltage-gated sodium (NaV), potassium, and calcium channels, as well as the chemosensitive TRPA1 ion channel, affecting the activating processes of these channels. Due to its action at the NaV1.7, NaV1.8, and TRPA1 channels involved in pain perception and propagation, ProTx-I may be used as a model for the development of next-generation analgesics. ProTx-I consists of 35 amino acid residues, with three disulfide bonds forming an inhibitor cystine knot (ICK) motif, which challenges its heterologous production. An efficient ProTx-I production system is necessary to study, at the molecular level, the mechanism by which the toxin acts. In this study, we tested several approaches for bacterial production of disulfide-containing toxins. Cytoplasmic expression of ProTx-I fused with either thioredoxin or glutathione-S-transferase failed to yield a correctly folded toxin. However, the natively folded ProTx-I was successfully obtained by "direct" expression in the form of cytoplasmic inclusion bodies, followed by renaturation, as well as by secretion into the periplasmic space via fusion with maltose-binding protein. The activity of the recombinant ProTx-I was studied by electrophysiology in X. laevis oocytes expressing rat and human TRPA1 channels. The toxin proved to be more active on the rat channel than on the human channel (IC₅₀ = 250 ± 85 and 840 ± 190 nM, respectively). The presence of an additional N-terminal methionine residue in the toxin obtained through "direct" expression significantly attenuated its activity.

Sorafenib is a multiple tyrosine kinase inhibitor that is used in the treatment of liver and renal cancers. We synthesized the hydroxamic derivatives of sorafenib bearing the pharmacophore elements of zinc-dependent histone deacetylase inhibitors. We uncovered that suppression of cancer cell proliferation by the synthesized hybrid inhibitors critically depends on the structure of the "deacetylase" element.

MicroRNAs are endogenous, small non-coding RNAs that regulate gene expression at the post-transcriptional level by cleaving target mRNAs. Mature microRNAs are products of the processing of their primary transcripts (pri-miRNAs). Now, it has been discovered that the products of the translation of some plant pri-miRNAs are peptide molecules (miPEP). These peptides have the capacity to physically interact with their open reading frames (ORFs) in the transcribed pri-miRNAs and, thus, positively regulate the accumulation of these RNAs and the corresponding mature microRNAs. Most conserved microRNAs play an important role in plants development and their response to stress. In this work, we obtained transgenic Physcomitrium patens moss plants containing Brassica oleracea miPEP156a ORF in the genome under the control of a strong 35S cauliflower mosaic virus promoter and analyzed the effect of the exogenous peptide on the transcription of this ORF in the protonemata of two transgenic moss lines. It turned out that the chemically synthesized peptide miPEP156a increases the accumulation of its own mRNA during moss culture growth, as was previously shown in studies by foreign researchers and in our own work for a number of peptides in monocotyledonous and dicotyledonous plants. These findings confirm that pri-miRNA regions that are located outside the coding region of the peptide are not required for transcriptional activation. Moreover, we have also succeeded in showing that the presence of a specific promoter of the microRNA gene does not affect the phenomenon of transcription activation; this phenomenon per se is not species-specific and is observed in transgenic plants, regardless of the origin of the miPEP.

OrthopoxVac, a fourth-generation smallpox vaccine, was the first of its kind registered worldwide in 2022, and it has been shown to be both safe and to induce only a mild reaction. A six-month clinical study confirmed its immunogenicity as compared to the first-generation live smallpox vaccine. Our study aimed to determine the levels of specific humoral and T-cell immune responses in volunteers following intradermal OrthopoxVac vaccine administration either in a single dose of 107 PoFU or in two doses of 106 PoFU, at 1.5, 3, and 5 years after initial vaccination. Following the immunization of volunteers with the OrthopoxVac vaccine at a dosage of 107 PoFU, the T-helper response remained at a relatively high level for three years, before it significantly dropped. Administration of the same vaccine twice at a dose of 106 PoFU resulted in a considerable decrease in the level of T-helpers, after 1.5 years. Additionally, some patients exhibited a reduction in viral neutralizing antibody (VNA) titers after 1.5 years of OrthopoxVac vaccine administration. When OrthopoxVac was administered at a dosage of 107 PoFU, no substantial differences were noted between groups at the 1.5-, 3-, and 5-year marks. In contrast, in the groups receiving two doses of 106 PoFU, VNA titers showed a significant reduction after 1.5 years. These findings indicate that a single intradermal dose of 107 PoFU of the OrthopoxVac vaccine elicits a significant and lasting immune response involving both antibodies and T-cells for a minimum of three years.

This paper presents a new bioinformatics tool to meet the needs of researchers in the search for short (≥ 3) amino acid subsequences in protein sequences annotated in public databases (UniprotKB, SwissProt) and illustrates its efficacy with the example of a search for the EVHH tetrapeptide in the human proteome, which is a molecular determinant of amyloid beta and is involved in interactions that are crucial in Alzheimer's disease pathogenesis. The topicality of developing such a tool is, on the one hand, supported by experimental data on the role of short tetrapeptide motifs in the architecture of intermolecular interfaces. On the other hand, there are currently no software products for efficient search for short (≥3) amino acid sequences in public databases, which drastically limits researchers' ability to identify proteins with exact matches of short subsequences. This tool (PepString server, http://pepstring.eimb.ru/) allows one to use intuitive queries to retrieve information about all the proteins that contain sequences of interest, as well as their combinations.

Immunoproteasomes, a unique type of proteasome complex, play a critical role in antigen presentation and cellular homeostasis. Unlike the constitutive 20S proteasome, the catalytic subunits β1, β2, and β5 in the immunoproteasome are replaced by inducible isoforms: β1i (LMP2), β2i (MECL-1), and β5i (LMP7). The expression of the genes encoding these subunits (Psmb9, Psmb10, and Psmb8) is activated by cytokines, primarily interferon-γ (IFNγ). Although it has been demonstrated more and more convincingly that immunoproteasomes are expressed in embryonic stem cells (ESCs), their involvement in maintaining pluripotency, promoting self-renewal, and regulating differentiation processes remains unexplored. This study implemented CRISPR/Cas9 technology to generate a Psmb9 gene knockout (Psmb9KO) mouse ESC line. The resulting cells exhibited a normal karyotype and morphology, maintained normal proliferation rates, and retained the capacity to form teratomas containing derivatives of all three germ layers. However, the differentiation induced by retinoic acid (RA) and IFNγ caused an accumulation of Mecl-1 precursors in Psmb9KO cells, suggesting modifications in immunoproteasome assembly. Furthermore, an increase in the caspase-like activity of immunoproteasomes was detected, suggesting the integration of a constitutive β1-subunit into the complex in place of Lmp2. The findings underscore the adaptability of the ubiquitin-proteasome system in maintaining cellular proteostasis by compensatory mechanisms that counteract the lack of Lmp2. The Psmb9KO line can serve as a valuable model for examining the function of immunoproteasomes in proteostasis regulation during early mammalian embryogenesis differentiation.

The advent of the T-cell engineering technology using chimeric antigen receptors (CARs) has revolutionized the treatment of hematologic malignancies and reoriented the direction of research in the field of immune cell engineering and immunotherapy. Regrettably, the effectiveness of CAR T-cell therapy in specific instances of hematologic malignancies and solid tumors is limited by a number of factors. These include (1) an excessive or insufficient CAR T-cell response, possibly a result of both resistance within the tumor cells or the microenvironment and the suboptimal structural and functional organization of the chimeric receptor; (2) a less-than-optimal functional phenotype of the final CAR T-cell product, which is a direct consequence of the manufacturing and expansion processes used to produce CAR T-cells; and (3) the lack of an adequate CAR T-cell control system post-administration to the patient. Consequently, current research efforts focus on optimizing the CAR structure, improving production technologies, and further developing CAR T-cell modifications. Optimizing the CAR structure to enhance the function of modified cells is a primary strategy in improving the efficacy of CAR T-cell therapy. Since the emergence of the first CAR T-cells, five generations of CARs have been developed, employing both novel combinations of signaling and structural domains within a single molecule and new systems of multiple chimeric molecules presented simultaneously on the T-cell surface. A well thought-out combination of CAR components should ensure high receptor sensitivity to the antigen, the formation of a stable immune synapse (IS), effective costimulation, and productive CAR T-cell activation. Integrating cutting-edge technologies - specifically machine learning that helps predict the structure and properties of a three-dimensional biopolymer, combined with high-throughput sequencing and omics approaches - offers new possibilities for the targeted modification of the CAR structure. Of crucial importance is the selection of specific modifications and combinations of costimulatory and signaling domains to enhance CAR T-cell cytotoxicity, proliferation, and persistence. This review provides insights into recent advancements in CAR optimization, with particular emphasis on modifications designed to enhance the therapeutic functionality of CAR T-cells.

The disruption of epigenetic regulation and the development of abnormal DNA methylation patterns are crucial steps in the pathogenesis of neurodegenerative diseases. Methylation alterations in multiple sclerosis (MS) patients may contribute to the dysregulation of gene expression linked to the regulation of inflammation, myelin production, and the preservation of the integrity of the myelin sheath. The possibility that epigenetic alterations could be reversed provides a rationale for studying their mechanisms. In this study, we evaluated the methylation status of LINE-1 retrotransposons in the peripheral blood cells of patients with MS and healthy controls. In healthy individuals, LINE-1 methylation levels were observed to decrease with advancing age. MS patients exhibited a positive correlation between LINE-1 methylation and MS duration. The study indicates that the level of LINE-1 methylation is notably higher in progressive MS compared to the remitting type. LINE-1 methylation variations in MS patients were observed to be associated with the serum levels of homocysteine and vitamin B9, and dependent on the genotype for the C677T polymorphism of the MTHFR gene as well. The data obtained point to the contribution of the C677T polymorphism to the appearance of epigenetic disorders in MS development and suggest that hypermethylation may be mediated by disruptions in the folate metabolism that accompany MS.

Calcium signaling ensures efficient cellular functioning; calcium homeostasis disruption leaves behind detrimental sequelae for the cell both under calcium excess and deficiency conditions. Malignant transformation is accompanied by significant alterations in the expression of the proteins critical for store-operated calcium entry, resulting in the dysregulation of calcium signaling. It is plausible that a remodeling of intracellular signal transduction pathways in cancer cells is required in order to accelerate metabolic processes, as well as fuel further tumor growth and invasion. Meanwhile, fine-tuning of calcium signaling is observed under both normal and pathological conditions. In this context, research into the changes accompanying signal transduction within the tumor microenvironment is a key aspect of the investigation of the role of calcium signaling in tumor development. Factors characteristic of the tumor microenvironment were shown to have a significant effect on the function of calcium channels and the proteins that regulate calcium signaling. Major, adverse microenvironmental factors, such as acidification, elevated levels of reactive oxygen species and hypoxia, have a bearing on the store-operated calcium entry. It is crucial to understand whether changes in the expression of the key SOCE components represent an adaptation to the microenvironment or a result of carcinogenesis.

Boron neutron capture therapy (BNCT) is a rapidly developing field of radiation therapy for cancer that is based on the accumulation of the radiosensitive 10B isotope in cancer cells, followed by tumor irradiation with thermal neutrons. Widespread use of BNCT in clinical practice remains limited because of the poor accumulation of boron-containing (10B) drugs in the tumor or their high toxicity to the body. This study focuses on the engineering of tumor-specific liposomes loaded with 4-L-boronophenylalanine (4-L-10BPA) for application in boron neutron capture therapy. According to the spectrophotometry and ICP-mass spectroscopy data, the 4-L-10BPA-to-liposome molar ratio is ~ 120,000. Liposomal targeting of human epidermal growth factor receptor 2 (HER2) was determined by HER2-specific designed ankyrin repeat protein (DARPin)_9-29 on the outer surface of liposomes. DARPin-modified liposomes were found to bind to HER2-overexpressing cells and be effectively internalized into the cytoplasm. The ability of DARPin-functionalized liposomes to precision-deliver large quantities of 4-L-10BPA into cancer cells may open up new prospects for BNCT.