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Vol 90, No 10 (2025)
Articles
CYSTEINE SYNTHASE: CHARACTERISTICS OF THE MAIN ENZYME IN THE CYSTEINE SYNTHETIC PATHWAY
Abstract
Cysteine is an extremely important amino acid for the normal functioning of living organisms. In bacteria and plants, the main route of cysteine synthesis is the thioylation pathway, the second stage of which is provided by either cysteine synthase A (CysK) if the substrate is inorganic sulfide, or cysteine synthase B (CysM) if the substrate is thiosulfate. The crucial role of these enzymes in the synthesis of cysteine makes them promising targets of antimicrobial agents for the treatment of infectious diseases, the creation of new herbicides, as well as in the industrial production of cysteine. However, besides of the main functions, the additional activity of these enzymes is of interest, such as their use as antimicrobial or antibiotic film agents. This review examines the physico-chemical characteristics of CysK and CysM, discusses their diversity and potential applications in biotechnology and medicine.
Biochemistry. 2025;90(10):1403-1424
1403-1424
MULTIFUNCTIONAL SFPQ PROTEIN: ROLE IN DOUBLE-STRANDED DNA BREAK REPAIR
Abstract
The SFPQ (Splicing Factor Proline and Glutamine rich) protein, initially discovered as a splicing factor, is a multifunctional nuclear protein that participates in various cellular processes. Its main partner in the cell is the NONO (Non-POU domain-containing octamer-binding protein) protein. SFPQ forms with NONO a heterodimer, which is an important component of subnuclear compartments called paraspeckles and located near such nuclear structures as splicing speckles. However, SFPQ can participate in some cellular processes independently, so it is SFPQ, but not NONO, that is apparently necessary for cell viability. There is a lot of data on the involvement of SFPQ in the repair of double-stranded DNA breaks, yet the precise mechanism of its involvement in this important cellular process remains unclear. In this review, we have tried to summarize and systematize the existing data on the role of SFPQ and its complex with NONO in the repair of double-stranded DNA breaks.
Biochemistry. 2025;90(10):1425-1438
1425-1438
FERROPTOSIS MECHANISMS AND NON-CODING RNAs IN NON-SMALL CELL LUNG CANCER
Abstract
Ferroptosis is an iron-dependent type of regulated cell death induced by hyperoxidation of polyunsaturated fatty acids within the phospholipids of the cytoplasmic membrane. According to recent studies, four key regulatory pathways of this process have been identified, with the glutathione pathway (SLC7A11/SLC3A2)/GSH/GPX4) being central and the most extensively studied. The functioning of all ferroptosis control systems is ensured by a multilayered network of protein-coding and regulatory genes, and disturbances in their expression may serve as a trigger for tumor cell transformation. Ferroptosis, along with other types of programmed cell death, plays a key role in the pathogenesis of many cancers, including non-small cell lung cancer (NSCLC). This review provides a detailed overview of the key molecular mechanisms of ferroptosis and summarizes the results of experimental studies demonstrating the involvement of ferroptosis-associated non-coding RNAs (microRNAs and long non-coding RNAs) in the development and progression of NSCLC. Special attention is given to the prospects of using anti-ferroptotic and pro-ferroptotic ncRNAs in NSCLC therapy, based on targeted modulation of their expression levels to induce ferroptosis in tumor cells.
Biochemistry. 2025;90(10):1439-1462
1439-1462
TRANSMEMBRANE TRANSPORT OF WATER AND UREA IN RAT CORNEAL ENDOTHELIAL CELLS
Abstract
The permeability of the apical and basolateral membrane of rat corneal endothelial cells to water and urea was studied. It was shown that the apparent water permeability of the basolateral membrane of endothelial cells (4.43E-05 ± 7.57E-07 cm/s) is more than three times higher than the permeability of the apical membrane (1.21E-05 ± 1.03E-07 cm/s). The permeability of the basolateral membrane of endothelial cells to urea (1.23E-04 ± 1.56E-06 cm/s) was higher than the permeability of the apical membrane (9.52E-05 ± 1.02E-06 cm/s) by 30%. The contribution of phloretin-inhibited urea transport across the apical and basolateral membrane in these cells was studied. Phloretin at a concentration of 0.1 mM reduced the urea permeability by more than 20% through both the apical and basolateral membranes. The results suggest that the compositions of the transporters involved in water transport in the apical and basolateral membranes differ significantly. It is suggested that the high apparent water permeability of the basolateral membrane of endothelial cells could be explained by the contribution of concomitant water transport with ions involved in active transport processes. The presence of phloretin-sensitive urea transporters in the plasma membrane of endothelial cells that could take a part in urea transcellular movement, has been shown. The results indicate the possible importance of urea for corneal function.
Biochemistry. 2025;90(10):1463-1473
1463-1473
COMPARISON OF METHODS FOR ASSESSING THE CONCENTRATION OF EXTRACELLULAR VESICLES ISOLATED FROM DIFFERENT BIOLOGICAL FLUIDS
Abstract
Accurate quantification of extracellular vesicles (EVs) remains a significant challenge in biomedical research. Although various analytical methods have been developed, their reliability is often limited by the presence of non-vesicular nanoparticles and biological contaminants, particularly in biological fluids. Moreover, for some sources of EVs, such as uterine aspirates and gastric juice, quantitative evaluation has been scarcely addressed. The aim of the study is to perform a comparative analysis of three EV quantification methods: total protein content measurement, nanoparticle tracking analysis (NTA), and esterase activity assessment using the commercial FluoroCet exosome quantitation kit in EVs isolated from various biological fluids: blood plasma, ascitic fluid, uterine aspirates, gastric juice, and conditioned medium of ovarian and non-small cell lung cancer cell lines. For EV samples derived from conditioned medium, all three methods demonstrated strong correlation, supporting their validity for in vitro EV quantification in highly purified samples. In contrast, plasma, ascitic fluid, and uterine aspirates exhibited discrepancies between methods, likely attributable to the presence of non-vesicular nanoparticles. Notably, EVs from gastric juice demonstrated a strong correlation between protein content and esterase activity, indicating a prevalence of vesicle-associated proteins and a potentially unique EV composition in this fluid. The findings underscore the necessity for a multifactorial approach to EV quantification, taking into account factors such as sample origin and limitations inherent to the specific method employed. These results may serve as a basis for the development of standardized protocols for EV quantification, which is particularly relevant for clinical sample analysis.
Biochemistry. 2025;90(10):1474-1486
1474-1486
ALPHA- AND GAMMA-SYNUCLEINS CONTROL ENERGY METABOLISM AND ACTIVITY OF XANTHIN OXIDASE IN BRAIN CELLS
Abstract
The process of transmission and transformation of signal in the central nervous system causes large energy expenditures in brain cells, which leads to active energy metabolism with high consumption of glucose and oxygen. Production of reactive oxygen species (ROS) as a result of these processes participate in signal transduction in the cell, but overproduction of ROS leads to the development of oxidative stress. Oxidative stress and aggregation of the α-synuclein is a hallmark in the mechanism of neuronal loss in Parkinson's disease. However, much less is known about the physiological role of monomeric synucleins. Using acute brain slices and primary neuroglial co-cultures prepared from transgenic mice with knockout of α-, β-, and γ-synuclein genes, we studied the role of these proteins in ROS production and energy metabolism. We found that synuclein knockout results in reduced ROS production compared to wild-type cells. Xanthine oxidase (XO) inhibitor oxypurinol reduced ROS production in wild-type cells and β-synuclein knockout brain slices, while XO-dependent ROS was not inhibited in α- or γ-synuclein knockout slices, suggesting that these proteins may regulate this enzyme. Knockout of α- and γ-synucleins resulted in a decrease in mitochondrial membrane potential and a reduction in energy capacity (in the form of ATP), which may be one of the mechanisms of XO regulation by synucleins.
Biochemistry. 2025;90(10):1487-1496
1487-1496
INTERROGATION OF METABOLIC CHARACTERISTICS OF BREAST CANCER METASTASES USING FLUORESCENCE LIFETIME IMAGING
Abstract
The most important problem of clinical oncology is tumor metastasis. Metabolic switches in tumor cells make a difference for metastases' survival and growth in conditions of a new microenvironment. However, they are less explored in comparison with primary tumors' metabolism. Fluorescence lifetime imaging microscopy (FLIM), based on recording the decay parameters of cellular autofluorescence emitted by pyridine and flavin cofactors, is considered a promising method for assessing the metabolic status of cells. This study aims to identify differences in the kinetics of NAD(P)H fluorescence decay between metastatic breast cancer cells and the primary tumor, as well as between metastatic cells and lymph node tissue in a 4T1 mouse model. The study revealed a decrease in the relative fraction of free NAD(P)H (a1, %), i.e., the fraction not bound to enzymes and associated with glycolysis, in metastases, indicating a shift in the balance toward mitochondrial respiration. Moreover, metastases were metabolically more heterogeneous at the cellular level than primary tumors, as evidenced by a higher dispersion of the mean NAD(P)H fluorescence lifetime τm. Furthermore, metastatic cells were found to have a higher contribution of free NAD(P)H a1 to fluorescence decay, resulting in a shorter mean lifetime τm compared to lymphoid tissue cells (p < 0,001). Thus, this study, using FLIM, demonstrated for the first time differences in the time-resolved characteristics of NAD(P)H autofluorescence between breast cancer metastases and the primary tumor, and between metastases and lymph node tissue. These differences are consistent with existing concepts of the oxidative metabolism of breast cancer metastases. The obtained data are of interest for the search for therapeutic targets in the pathways of energy metabolism of metastases and the development of new approaches to their diagnosis using autofluorescence.
Biochemistry. 2025;90(10):1497-1510
1497-1510
INDUCTION OF TUMOR-ASSOCIATED PHENOTYPE IN NORMAL FIBROBLASTS BY GLIOMA CELL APOPTOTIC BODIES
Abstract
The key cellular component of solid tumors, including gliomas, are tumor-associated fibroblasts (TAFs), which support the growth of malignant cells, stimulate their invasion and metastasis, induce chemoresistance and suppress the antitumor immune response. TAFs are formed from resident stromal cells under the influence of the tumor cell secretome, including growth factors, chemokines, and extracellular vesicles. Communication between malignant cells and TAFs occurs through direct cell-cell contacts and by mutual exchange of secreted molecules and membrane vesicles. In this work, apoptotic bodies (apoBD) were obtained from two types of glioma cells (T98g cell line and cells isolated from glioblastoma patient biopsy) and characterized by surface markers. The surface of tumor apoBD contains glioblastoma tumor-associated markers such as ganglioside GD2 and antigen A2B5. It was demonstrated that glioma apoBDs have decreased levels of "don't eat me" molecules and increased level of "eat me" signal compared to the original intact glioma cells. On the one hand, glioma apoBDs reduced the viability of normal dermal fibroblasts in a dose-dependent manner, but on the other hand, induced their transformation into the inflammatory subtype of TAFs. The iTAFs obtained in this way demonstrated enhanced transcription of genes encoding cytokines, chemokines and growth factors, including IL17A, IL18, IL33, IFN-γ, CCL3, CCL5, CXCL1, CXCL5, CXCL10, CXCL12, TGFB1, and TNF, responsible for maintaining both tumorigenesis itself and the ability of fibroblasts to maintain it. It was found that glioma apoBDs are able to transfer tumor-associated markers, ganglioside GD2 and antigen A2B5, into normal fibroblasts, and the effects of anti-GD2 ADC on TAFs were investigated. This result may become a prerequisite for the development of targeted drugs that are effective not only against tumor cells but also against tumor stroma.
Biochemistry. 2025;90(10):1511-1531
1511-1531
Erratum
Biochemistry. 2025;90(10):1532
1532