Biochemistry (Moscow)最新文献

Methyltransferases that modify spliceosomal small nuclear RNAs (snRNAs) play a crucial role in the cell by ensuring proper maturation of snRNAs, which is essential for optimal function of spliceosome. In this study, we investigated the enzyme METTL4, which catalyzes N6-methylation of 2′-O-methyladenosine at position 30 of U2 snRNA. Function of both the protein and the modification in splicing remains unclear. We demonstrated that inactivation of the METTL4 gene in HeLa S3 cells leads to significant changes in alternative splicing, general slowdown in spliceosome activity, and intron accumulation. In the cells lacking METTL4, expression of the set of genes associated with ribosomal RNA maturation is reduced, and the number of coilin-positive structures, most likely Cajal bodies, is decreased in the nuclei of these cells.

This study investigated permeability of the apical and basolateral membranes of rat corneal endothelial cells to water and urea. We demonstrated 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 that of the apical membrane (1.21E−05 ± 1.03E−07 cm/s). Permeability of the basolateral membrane to urea (1.23E−04 ± 1.56E−06 cm/s) was statistically significantly higher than that of the apical membrane (9.52E−05 ± 1.02E−06 cm/s) by approximately 30%. We examined contribution of the phloretin-inhibited urea transport across the apical and basolateral membranes in these cells. Phloretin at concentration of 0.1 mM significantly reduced urea permeability by more than 20% through both the apical and basolateral membranes. The results suggest that the compositions of transporters involved in water transport in the apical and basolateral membranes differ significantly. It is hypothesized that high apparent water permeability of the basolateral membrane of endothelial cells is due to contribution of the concomitant water transport with ions involved in active transport processes. Presence of the phloretin-sensitive urea transporters in the plasma membrane of endothelial cells, likely involved in its transcellular transport, has been demonstrated. The results indicate potential significance of urea for corneal function.

The SFPQ (Splicing Factor Proline and Glutamine rich) protein, initially identified as a splicing factor, is a multifunctional nuclear protein involved in various cellular processes. Its main cellular partner is NONO (Non-POU domain-containing octamer-binding protein), with which SFPQ forms a heterodimer that is a crucial component of subnuclear structures called paraspeckles and located near nuclear speckles. However, SFPQ can also function independently in certain cellular processes and is essential for cell viability. There is substantial evidence of the involvement of SFPQ in the repair of double-strand DNA breaks (DSBs), but a definitive understanding of the mechanism of its participation in this critical cellular process is still lacking. In this review, we aim to summarize and systematize the existing data on the role of SFPQ and its complex with NONO in the repair of double-strand DNA breaks.

The effect of the C-terminal tail on the bioactivity of Agaricus bisporus mannose-binding protein (Abmb) was investigated. Based on the earlier obtained crystal structure of Abmb, it was suggested that the additional C-terminal tail can modulate the binding of sugars to the protein. According to glycan microarray, Abmb can bind β-Gal sugars, which contradicted the results of SPR analysis showing that Abmb only interacts with α-Man and not with α-Gal. Here, we used MCF-7 and MDA-MB-231 breast cancer cells to demonstrate that the presence of the C-terminal tail decreased the anti-proliferative activity of Abmb. Pre-incubating Abmb with α-Gal did not eliminate the anti-proliferative activity, while pre-incubation with α-Man attenuated it. At the same time, preincubation with a mixture of α-Gal and α-Man strongly promoted the anti-proliferative activity of Abmb. In silico analysis using molecular docking suggested the presence of a second functional sugar-binding site for Gal, which had not been identified previously. The study provides new insights into the structure of lectins and their interaction with sugars

Tumor-associated fibroblasts (TAFs) are a key cellular component of solid tumors, including gliomas. They 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 tumor cell secretome, including growth factors, chemokines, and extracellular vesicles. Communication between malignant cells and TAFs occurs through direct cell–cell contacts and exchange of secreted molecules and membrane vesicles. In this work, apoptotic bodies (apoBDs) were obtained from two types of glioma cells (T98g cell line and Gbl25 cells isolated from a glioblastoma biopsy) and characterized for surface markers. The surface of tumor apoBDs contained glioblastoma tumor-associated markers, such as GD2 ganglioside and A2B5 antigen. Glioma apoBDs contained lower levels of “don’t eat me” molecules and higher levels of “eat me” molecules compared to the original intact glioma cells. On one hand, glioma apoBDs reduced the viability of normal dermal fibroblasts in a dose-dependent manner; on the other hand, they initiated their transformation into the inflammatory subtype of TAFs (iTAFs). iTAFs obtained in this way demonstrated upregulated transcription of genes encoding cytokines, chemokines, and growth factors (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 support it. It was found that glioma apoBDs were able to transfer tumor-associated markers (GD2 ganglioside and A2B5 antigen) to normal fibroblasts. The assessment of the effects of anti-GD2 antibody–drug conjugates (ADCs) on TAFs suggests the possibility of development of targeted drugs effective not only against tumor cells but also against tumor stroma.

Ferroptosis is an iron-dependent form of regulated cell death induced by hyperoxidation of polyunsaturated fatty acids (PUFAs) in cytoplasmic membrane phospholipids. Recent research has identified four key regulatory pathways of this process, with glutathione pathway (SLC7A11/SLC3A2)/GSH/GPX4 being the most central and well-studied. Functioning of all ferroptosis control systems is supported by the multilevel network of protein-coding and regulatory genes, whose dysregulated expression could trigger tumor cell transformation. Ferroptosis, alongside with other types of programmed cell death, plays a pivotal role in pathogenesis of many cancers, including non-small cell lung cancer (NSCLC). This review provides a comprehensive overview of the molecular mechanisms of ferroptosis and summarizes experimental evidence demonstrating involvement of the ferroptosis-associated non-coding RNAs (microRNAs and long non-coding RNAs) in the development and progression of NSCLC. Special emphasis is placed on the potential application of anti-ferroptotic and pro-ferroptotic non-coding RNAs in NSCLC therapy, focusing on targeted modulation of their expression to induce ferroptosis in tumor cells.

The most critical problem in clinical oncology is the metastasis of malignant neoplasms. The survival and growth of metastases in a new microenvironment fundamentally depend on adaptations in the energy metabolism of metastasizing cells. However, these adaptations are far less studied compared to primary tumors. A promising method for assessing the metabolic status of cells is fluorescence lifetime imaging microscopy (FLIM), based on recording the decay parameters of cellular autofluorescence emitted by pyridine and flavin cofactors. This work aims to identify differences in the fluorescence decay kinetics of NAD(P)H between metastatic breast cancer cells and the primary tumor, as well as between metastatic cells and lymph node tissue in a 4T1 mouse model experiment. The study revealed a decrease in the relative fraction of the free form of NAD(P)H (a1, %), i.e., the form not bound to enzymes and associated with glycolysis, in metastases. This indicates a shift in the balance towards mitochondrial respiration. Furthermore, 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. Additionally, it was found that metastatic cells have a higher contribution of the free NAD(P)H form a1 to the fluorescence decay and, consequently, a shorter mean lifetime τm compared to lymphoid tissue cells (p < 0.001). Thus, this study uses FLIM to demonstrate, for the first time, differences in the temporal characteristics of NAD(P)H autofluorescence between breast cancer metastases and the primary tumor, and between metastases and lymph node tissue. These findings align with existing concepts about the oxidative metabolism of breast cancer metastases. The obtained data are of interest for searching for therapeutic targets in the energy metabolism pathways of metastases and for developing new diagnostic approaches using autofluorescence.

Butylated hydroxytoluene (BHT) is a well-known synthetic antioxidant and a commonly used synthetic food additive. It is prominently employed in pharmaceutical, rubber, oil, and petroleum industries. However, the evidence supporting its role in preventing skeletal muscle atrophy is lacking. In this study, the effect of BHT on the oxidative stress (100 µM H₂O₂)-induced atrophy was investigated in C2C12 myotubes. The antioxidative potential of BHT was compared to that of β-carotene. BHT demonstrated a superior free radical-scavenging ability compared to β-carotene in both DPPH and ABTS assays. Furthermore, pretreatment with 25 μg/ml BHT for 4 h preserved myotube morphology and membrane integrity and promoted creatine kinase activity in the oxidative stress-induced atrophy model. BHT also prevented the degradation of myosin heavy chain (a key structural protein) by downregulating the activity of calpain and suppressing expression of MuRF-1 mRNA (ubiquitin proteasome system), as well as reduced lipid peroxidation and ROS levels and increased lactate dehydrogenase activity, indicating improved cellular resilience. This study provides the first direct evidence of the protective effects of BHT against H₂O₂-induced atrophy in cultured myotubes and highlights a therapeutic potential of BHT in the mitigation of oxidative stress-related muscle atrophy.

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 of EVs has not been investigated. The aim of the study is to perform comparative analysis of three EV quantification methods: total protein content measurement, nanoparticle tracking analysis (NTA), and esterase activity assessment using commercial FluoroCet exosome quantitation kit in EVs isolated from various biological fluids: blood plasma, ascitic fluid, uterine aspirates, gastric juice, and medium conditioned by ovarian and non-small cell lung cancer cells. All three methods demonstrated strong correlation for the EV samples derived from the conditioned medium, supporting their validity for in vitro EV quantification in highly purified samples. In contrast, blood plasma, ascitic fluid, and uterine aspirates exhibited discrepancies between the methods, likely attributable to the presence of non-vesicular nanoparticles. Notably, the EVs from gastric juice demonstrated strong correlation between the protein content and esterase activity, indicating prevalence of the vesicle-associated proteins and, potentially, unique EV composition in this fluid. The findings underscore the necessity for 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.