ISSN (print): 0132-3423, ISSN (online): 1998-2860 Founders: Russian Academy of Sciences (RAS), Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry of RAS Editor-in-chief: Kochetkov Sergey Nikolaevich, academician RAS, Doctor of Sc., Full Professor Frequency / Access: 6 issues per year / Subscription Included in: White list (2nd level), Higher Attestation Commission list, RISC
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PREDISLOVIE K SPETsIAL'NOMU VYPUSKU ZhURNALA, POSVYaShchENNOMU 50-LETIYu KAFEDRY BIOORGANIChESKOY KhIMII BIOLOGIChESKOGO FAKUL'TETA MOSKOVSKOGO GOSUDARSTVENNOGO UNIVERSITETA IMENI M.V. LOMONOSOVA
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Modern Laboratory Test-Systems as Platforms for Validation of Clinically Promising T-Cell Receptors
The development of therapeutic antigen-specific T-cell receptors (TCRs) requires comprehensive preclinical validation of their functional activity. One of the approaches in the development of new drugs for cell therapy based on antigen-specific T lymphocytes is the modification of autologous T lymphocytes with endogenous T-cell receptors. The present work reviews modern laboratory platforms used to assess key TCR characteristics: immunological synapse formation, specificity and affinity of antigen binding, activation of signalling pathways, cytokine production and cytotoxic potential. Particular attention is paid to the creation of model T-cell lines expressing transgenic TCRs, optimisation of HLA context of target cells and application of multiparametric technologies for immune response analysis. The prospects of using 3D organoid models for validation of functional activity of transgenic TCRs under conditions close to physiological ones, as well as for predicting their clinical efficacy are discussed. The presented approaches form the basis for rational selection of candidate receptors for their subsequent application in immunotherapy of tumours and chronic infections.
Discovery and Identification of Plant Regulatory Peptides
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.
RNA-Interference as a Method for Validation of Pharmacological Targets in Fibrosis Treatment
RNA interference (RNAi) is an evolutionarily conserved mechanism of gene expression silencing based on the degradation of mRNA by small interfering RNAs (siRNAs). The discovery of this mechanism has not only become a powerful tool for fundamental research in biology, but has also opened up new perspectives for therapeutic medicine. In terms of efficacy and safety, siRNA therapy represents a promising alternative to traditional pharmaceutical approaches. Unlike traditional pharmacological approaches, which are often characterized by systemic toxicity and low specificity, siRNA-based therapy allows for the selective suppression of genes associated with pathologies, providing highly precise action and low toxicity. The use of siRNA to modulate the activity of macrophages, key effectors of innate immunity that play a central role in the development of liver fibrosis, represents particular interest. Due to their high plasticity, macrophages are able to polarize into proinflammatory (M1) or anti-inflammatory (M2) phenotypes, which determines their contribution to the progression or regression of fibrosis. Epigenetic modifications and suppression of key polarization regulators (such as EGR2, IRF5, IRF3, TLR4, HAS2) using siRNA allow targeted changes in their functional state. This review systematizes current data on the role of macrophages in the pathogenesis of liver fibrosis and the prospects for using siRNA therapy to control their activity. Strategies for precision targeting of key molecular targets are discussed, opening up new possibilities for the development of pathogenetically justified treatment methods.
Small Non-Coding RNAs of Bacteria are Global Regulators of the Bacterial Life Cycle
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.
Non-Classical Cannabinoid Receptors: Modern Concepts of Signal Transmission Functions and Mechanisms
The objective of this review is to summarize current understanding of non-classical cannabinoid receptors, their signaling mechanisms, and roles in physiological and pathological processes. Non-classical receptors (GPR55, GPR18, GPR119, etc.) demonstrate tissue-specific expression, interaction with multiple G proteins, and ligand-dependent activation of signaling pathways. GPR55 is involved in the regulation of pain, angiogenesis, and oncogenesis; GPR18 modulates inflammation and metabolism; GPR119 is promising for diabetes therapy. Their ability to heterodimerize has been identified, which complicates their pharmacological profile. Non-classical cannabinoid receptors represent promising targets for the treatment of socially significant diseases (cancer, diabetes, neurodegeneration). However, their context-dependent activity requires in-depth study to develop selective drugs.
8-Oxo-2'-deoxyguanosine – Oxidative Stress Control
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.
Proinflammatory Phenotype of CD161+ Double-Negative T Cells
Double-negative (DN) CD3+CD4-CD8- T lymphocytes represent a subgroup of unconventional peripheral T cells. Although their presence in peripheral blood is relatively low (3–5%), they play an important role in the development of inflammatory responses, oncological, autoimmune diseases, and allergic asthma. In the present study, we characterized the cytokine profile of CD3+CD4-CD8- DN T cells using multiplex technology with a wide panel of analytes. Our results demonstrate that the cytokine profile of proinflammatory αβTCR+CD161+ DN T cells includes the cytokines TNFα, IFNγ, IL-3, IL-13, and IL-27; the chemokines IL-8/CXCL8, MIG/CXCL9, MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5; as well as the growth factors M-CSF and GM-CSF.
Three-Finger Viper Toxins – cDNA Cloning and Expression in E. coli Using a Chimeric (Hybrid) Construction with a Partner Protein SUMO
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.
Improved Efficiency of OX40L-Based Gene Therapy Using a Non-Viral Delivery System in Fibroblast-Enriched Mouse Tumor Models
Malignant tumors, during their progression, are capable of forming a permissive microenvironment that influences their further growth and development. Tumor-associated fibroblasts (TAFs) play a significant role in this process. In the present study, we generated subcutaneous murine tumors by inoculating a co-culture of cancer cells and fibroblasts to create tumors enriched with microenvironmental cells. Once the tumor nodule had formed, an intratumoral injection was performed using a formulation containing a plasmid encoding the ligand for immune checkpoint receptors –OX40L – under the control of a CMV promoter. For efficient cellular delivery, the plasmid was encapsulated in a polymer shell based on PEG-PEI-TAT. We evaluated the impact of this treatment on tumor growth. In this experimental model, fibroblasts were artificially introduced into the tumor to partially simulate a developed tumor microenvironment. These tumors demonstrated an increased proliferation rate. However, intratumoral administration of the non-viral OX40L-encoding agent into fibroblast-enriched tumors resulted in a notable increase in the rate of complete tumor regression, reaching up to 25%. It is hypothesized that introduced fibroblasts may perform antigen-presenting functions and/or serve as an additional source of signals that activate the immune system.
Molecular Probes for Visualization of Nicotinic Acetylcholine Receptors Based on Snake Three-Finger Toxins and a Red Fluorescent Protein
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.
Cytoskeletal Regulator Zyxin Stimulates Translocations of YAP into Xenopus laevis Embryo Cell Nuclei
There is limited data in the scientific literature regarding the relationship between mechanotransduction associated with changes in Zyxin levels and biochemical signaling of the transcription factor YAP. Research in this area suggests that stress-induced reorganization of the actin cytoskeleton mediated by Zyxin may play a key role in YAP's mechanotransduction. However, the results of these studies do not provide clear outcomes concerning the effect of Zyxin on YAP distribution between the nucleus and the cytoplasm and its activity regulation. Here, we investigated the effects of Zyxin on the nuclear translocation of the Hippo-signaling pathway effector, YAP, in the early embryo of the clawed frog Xenopus laevis. Analysis of the nuclear-cytoplasmic distribution of YAP by immunoblotting and immunohistochemical staining, combined with the suppression of translation of endogenous Zyxin mRNA by morpholino antisense oligonucleotides and overexpression of synthetic Zyxin mRNA, revealed a stimulatory effect of Zyxin on the nuclear translocation of YAP in gastrula-stage embryos, but not in blastula and neurula stages. A similar conclusion was reached by analyzing the effect of the same changes in Zyxin concentration on the expression of a YAP-dependent luciferase reporter. Based on the results of our study, and taking into account the known role of Zyxin as one of the mechanotransducers, it can be assumed that this protein is involved in the mechano-dependent regulation of the Hippo signaling pathway in embryonic development at the gastrulation stage.
Transcriptome Analysis of Zyxin Cytoskeletal Protein Levels Influence on Metabolism and Signaling Pathways in a Model of Xenopus laevis Embryos
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.
Targeted Nanoliposomes Loaded with IR780 Dye as a Multifunctional Nanoplatform for Photothermal and Photodynamic Cancer Therapy
During cancer treatment the combined effect of photothermal and photodynamic therapy has unique advantages over each of these methods alone. In this study, a multifunctional targeted nanoplatform for simultaneous combined photothermal and photodynamic therapy under 808 nm infrared laser irradiation was developed. The developed system consists of ~140 nm liposomes specific to the tumor-associated HER2 antigen and loaded with the near-infrared heptamethincyanine dye IR780. The targeting of liposomes to the HER2 is determined by the HER2-specific scaffold protein DARPin_9-29 located on the outer surface of liposomes. It has been established that IR-780, loaded in liposomes, retains photothermal and photodynamic properties: upon irradiation, the temperature of IR780-loaded liposome solution rapidly increases (up to 60°C within 60 s), and the production of reactive oxygen species is also detected. In vitro experiments have shown that HER2-specific liposomes containing IR780 have photoinduced cytotoxicity against HER2-overexpressing cells, causing the death of 50% of the cell population at a concentration of 2.85 μM. The results of the study suggest that HER2-specific liposomes containing IR780 have excellent targeted characteristics, and IR780 can be used as an active substance for simultaneous photothermal and photodynamic therapy.
Development of an Experimental Intracranial PDX Model of Human Glioblastoma in NSG Mice
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.
Development of a Bacterial Expression System for Producing 15N/13C-Labeled Neuroglobin and Cytochrome C
An effective system for producing isotopically labeled heme-containing proteins of human neuroglobin and cytochrome c has been developed. The corresponding producer strains have been obtained, and optimal cultivation conditions have been selected: temperature and duration of incubation, composition of media, and concentration of expression inducer. Using far- and near-UV-vis CD spectroscopy, it was shown that the secondary structure composition of 15N-neuroglobin is close to the calculated one, and the orientation of the heme in the molecules is predominantly canonical. According to two-dimensional 1H/15N-HSQC NMR spectra of human neuroglobin and cytochrome c, the proteins are folded into a native conformation with a predominance of the α-helical structure. The resulting production system can be used to produce highly purified preparations of totally 15N/13C-labeled proteins for structural-dynamic studies using modern high-resolution NMR spectroscopy methods.
Properties of Potassium Channel Kv1.1 on the Basis of Fluorescent Dimer of Alpha-Subunits mKate2-Kv1.1-Kv1.1 in Neuro-2a Cells
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.
Study of the Antiproliferative Activity of a Humanized Antibody to the Cancer-Testis Antigen PRAME
The PRAME antigen (Preferentially Expressed Antigen in Melanoma), which belongs to the group of cancer-testis antigens and is expressed in various tumor types, represents an attractive target for targeted cancer therapy. In this study, a humanized antibody (6H8Hu) was developed based on the monoclonal antibody 6H8 specific to the PRAME protein. The antibody was produced in CHO cells. It was shown that the humanized antibody retains the high affinity of the parental mAb to the antigen (1.2 nM), binds to both recombinant and native PRAME protein, inhibits the proliferation of PRAME-positive human melanoma cell line Mel Ibr, and suppresses tumor nodule growth in vivo to a degree comparable to that of the murine antibody 6H8 and the cytostatic agent melphalan.
Influence of the Cultivation Conditions of Glioblastoma Cells on the Expression of Transcription Factors Genes
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.
Oral Indole-3-acetate Supplementation Increases the Abundance of Bifidobacterium pseudolongum and Akkermansia muciniphila in the Intestine of Mice on a High-Fat Diet
It is known that even a short-term high-fat diet has a negative effect on the metabolic health of the organism. However, under the influence of diet, first of all, the intestinal microbiota undergoes changes. The type of diet, dietary supplements and drugs affect both the taxonomic diversity of the microbiota and its functional state. It is known that with the participation of the intestinal microbiota, tryptophan is converted into indole and its various derivatives. The leading role of indoles in the regulation of the expression of tight junction proteins, and accordingly the regulation of intestinal permeability, has also been established. The aim of our study was to assess the effect of indole-3-acetate on the taxonomic diversity of the microbiota of the small and large intestines, as well as to establish the potential prebiotic value of this indole derivative under conditions of short-term use of a high-fat diet. C57/black6 SPF mice aged 4-5 weeks (n=60, females) were randomly divided into six groups. A high-fat diet was achieved by feeding laboratory animals a high-fat diet of animal origin, providing up to 30% of the total calories. Indole-3-acetate was administered together with a standard or high-fat diet via an atraumatic intragastric tube at a single dose of 0.1392 mg per mouse for 28 days. In our study, we showed for the first time that in C57/black6 SPF mice on a short-term high-fat diet, indole-3-acetate increases the representation of Bifidobacterium pseudolongum in the microbial community of both the small intestine and the colon. Whereas, the increase in Akkermansia muciniphila was only in the microbial community of the colon. Indole-3-acetate intake provides normoglycemia in animals on a short-term high-fat diet. The use of indole-3-acetate in various metabolic diseases associated with high-fat diet and dysbacteriosis may be a promising therapeutic approach to correct metabolic disorders through modulation of the microbiotic community.
Effect of STK11 Mutation in the LLC1 Mouse Lewis Lung Adenocarcinoma Line on Sensitivity to Particle Radiotherapy
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.
Changes in the Protein Composition of the Exiguobacterium sibiricum Membrane with Decreasing Temperature: a Proteomic Analysis
For the first time, the proteins of the membrane fraction isolated from the psychrotrophic bacterium Exiguobacterium sibiricum were analyzed using the label-free quantitative chromatography-mass spectrometry. Comparison of the samples from cells cultured at 10°C and at room temperature revealed significant differences in the content of many proteins involved in important physiological processes, including DNA and RNA binding proteins, membrane transporters, proteins providing protection against osmotic and oxidative stress, and others. The results of the work contribute to understanding of mechanisms of the processes occurring in the cell membranes of psychrotrophic bacteria at low temperatures, and complement existing ideas about the adaptation strategies of microorganisms living in permafrost deposits. The data obtained can also be used to search for potential biocatalysts with activity at low temperatures.
NLS Peptide Improves the Efficiency of pDNA Delivery into Eukaryotic Cells by Cationic Liposomes
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.
Construction of a Producer Strain of the Fibrinolitic Enzyme PAPC Based on Pichia pastoris Yeast
Yeast Pichia pastoris (Komagataella phaffii) is widely used in food and pharmaceutical industries as microbial cell factories of recombinant proteins due to its ability to heterologously overexpress many target proteins, including proteolytic enzymes. Protease-activator of protein C (PAPC) of blood plasma from micromycete Aspergillus ochraceus VKM F-4104D can potentially be introduced into therapeutic practice as a fibrinolytic drug and into diagnostic systems for blood coagulation analysis as the main component that activates protein C. To solve problems of using protease-activator of protein C in medicine and veterinary science, it is important to have a reliable system to produce the recombinant enzyme. Such a production system can be created on the basis of yeast. The aim of this work was to construct a PAPC-producing strain based on P. pastoris and to demonstrate effective production and secretion of the recombinant enzyme into the culture fluid. We assembled a vector carrying the papc gene. This vector was used to transform P. pastoris yeast, and transformants were selected on a zeocin-containing medium. The clones most effectively producing the target enzyme were selected using agar medium with casein and analysis of the culture fluid by SDS-PAGE. The dynamics of accumulation of the active form of PAPC in the culture fluid after induction of protein synthesis during submerged cultivation of the producing clone in a flask was studied. LC-MS analysis confirmed the presence of the enzyme in the culture medium and demonstrated that accumulation occurs in the mature active form. The obtained strain can be used for further production of experimental industrial batches of the enzyme in biotechnological production facilities that support yeast fermentation.
Ionizable Cationic Lipid for Intracellular RNA Delivery
Ionizable lipids are a key component of the lipid nanoparticle platform for RNA therapy. They ensure the efficient assembly of a lipid–nucleic acid complex, protect it from premature degradation, and after endocytosis, promote the release of RNA into the cytoplasm for further processing. The paper describes the synthesis of a new ionizable cationic lipid that is a diglyceride derivative of tertiary alkylamine – [5-[1,2-di(decanoyloxy)propane-3-iloxy]pentyl-(4-hydroxybutyl)-amino]pentoxy]-2-decanoyloxypropyl] decanoate (An-1). The synthesis is characterized by the simplicity and cost-effectiveness of reagents. Comparative experiments with the well-known ionizable lipid ALC-0315 have shown that An-1 lipid forms lipid nanoparticles (LNPs) similar in size and incorporation efficiency of the model mRNA. LNPs with mRNA of the green fluorescent protein formulated with An-1 lipid transfected cells in culture more efficiently than LNPs formulated with ALC-0315 lipid. It is assumed that the new ionizable lipid can be used in the production of mRNA vaccines.