Biochemistry (Moscow)最新文献

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.

The process of signal transmission and transformation in the central nervous system requires active energy metabolism with high consumption of glucose and oxygen. Reactive oxygen species (ROS) produced as a result of these processes participate in intracellular signaling, but their overproduction leads to oxidative stress. Oxidative stress and α-synuclein aggregation are recognized as activators of neuronal death in Parkinson’s disease. However, much less is known about the physiological role of monomeric synucleins. Using acute brain slices and primary co-cultures of cortical neurons and glial cells derived from transgenic animals with knockout of α-, β-, and γ-synuclein genes, we investigated the role of these proteins in ROS production and energy metabolism. We found that absence of synucleins leads to the reduced ROS production compared to the wild-type cells. The xanthine oxidase (XO) inhibitor led to the decrease in ROS production in the wild-type cells and the brain slices with β-synuclein knockout, whereas in the slices lacking α- or γ-synuclein, the XO inhibition was not observed, suggesting possible regulation of this enzyme by these proteins. Knockout of α- and γ-synucleins resulted in the decrease in mitochondrial membrane potential and reduction in energy capacity (in the form of ATP), which could be one of the mechanisms of XO regulation by synucleins.

Cysteine is an amino acid essential for normal functioning of living organisms. In bacteria and plants, the main mechanism of cysteine synthesis is the thiolation pathway, the second stage of which is catalyzed 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 cysteine synthesis makes them promising targets for antimicrobial agents and new herbicides, and well as possible components of industrial production of cysteine. In addition to their main functions, cysteine synthases show the antimicrobial and antibiofilm activities. The review discusses the physicochemical characteristics of CysK and CysM, their diversity, and potential applications in biotechnology and medicine.

Argonaute proteins are an evolutionarily conserved family of proteins capable of recognizing and cleaving specific nucleic acid sequences using complementary guide molecules. Eukaryotic Argonautes play a key role in RNA interference by utilizing short RNAs of various classes to recognize target mRNAs. Prokaryotic Argonautes are much more diverse and most of them recognize DNA targets. The search for new Argonautes that would be active under varying conditions is important for both understanding their functions and developing new tools for genetic technologies. Many previously studied Argonautes exhibit low activity at low and moderate temperatures. To overcome this limitation, we isolated and studied two Argonaute proteins from psychrotolerant cyanobacteria, CstAgo from Cyanobacterium stanieri and CspAgo from Calothrix sp. Both proteins use short DNA guides to recognize and cleave DNA targets. CstAgo displayed no specificity for the 5′-end structure of the guide, while CspAgo demonstrated a weak preference for the 5′-terminal nucleotide. CstAgo was highly active and capable of cleaving single-stranded DNA at temperatures from 10 to 50°C. CspAgo was more cold-sensitive but cleaved double-stranded plasmid DNA using specific guides. Therefore, the studied proteins can be potentially used for DNA manipulations under a wide range of conditions.

Mitochondrial translation is a highly specialized process of synthesizing mitochondrially encoded proteins, mainly the components of the oxidative phosphorylation system. It involves four key stages: initiation, elongation, termination, and recycling of mitochondrial ribosomes. Each of these stages is regulated by a specific set of translation factors, most of which are encoded by the nuclear genome and imported into mitochondria. The termination of mitochondrial translation in yeast (Saccharomyces cerevisiae) is carried out by the MRF1 release factor. This nuclear-encoded factor is crucial for ensuring accurate protein synthesis within the organelle, as it recognizes stop codons and facilitates the release of completed polypeptide chains from the ribosome. In addition to this main function, MRF1 participates in maintaining mitochondrial genome stability. The aim of this study was to investigate the capacity of human homologues, hMTRF1, hMTRF1A, and mitoribosome rescue factors hMTRFR and hMRPL58, to compensate for the absence of the yeast mitochondrial translation termination factor MRF1 in S. cerevisiae cells. The results obtained suggest that human orthologues of MRF1, such as hMTRF1 and hMTRF1A, can contribute to maintaining the integrity of the yeast mitochondrial genome. However, they do not fully replace the function of MRF1, as they do not restore normal respiration of the mutant yeast strains.

The article addresses a formal paradox related to the formation of molecular oxygen during photosynthesis. Following the studies of van Niel in the early 1930s, it has become clear that in the oxygenic photosynthesis, molecular oxygen originates from water rather than carbon dioxide. However, the overall equation of photosynthesis, nCO₂ + nH₂O → (CH₂O)_n + nO₂, suggests that the amount of oxygen produced exceeds what could be derived from the water molecules involved. This paradox can be resolved by analyzing the light and dark reactions of photosynthesis, which ultimately result in the incorporation of carbon from CO₂ into carbohydrates and production of molecular oxygen. Despite its simplicity, the solution is not immediately obvious. One reason is that in the scientific and educational literature, the dark reactions of photosynthesis are often depicted schematically, without precise specification of all components involved. The author argues that analyzing this paradox and underlying physicochemical principles of photosynthesis can be valuable for students specializing in biochemistry.

17β-Hydroxysteroid dehydrogenase (17β-HSD) is an enzyme used in biotechnology for producing testosterone from phytosterol. Heterologous 17β-HSD from the fungus Cochliobolus lunatus catalyzes NADPH-dependent reduction of the 17-oxo group of androstenedione/androstadienedione formed in mycolicibacterial cells as a result of the inherent polyenzymatic process of side chain oxidation of phytosterols, yielding testosterone/Δ¹-dehydrotestosterone, respectively. The object of this study was heterologous 17β-HSD from the fungus C. lunatus (17β-HSD_Cl) with a 6×His tag (6×His-17β-HSD_Cl), synthesized in the cells of actinobacteria Mycolicibacterium neoaurum. Isolation and purification of the recombinant enzyme were performed using affinity chromatography. The 6×His-17β-HSD_Cl enzyme preparation exhibited the highest activity toward androstenedione. Activity of the 6×His-17β-HSD_Cl depended on NADPH and was observed in the pH range from 6.0 to 9.0 with an optimum at pH 7.0. Analysis of kinetic characteristics showed that the properties of the heterologous enzyme 6×His-17β-HSD_Cl synthesized in M. neoaurum cells are comparable with those reported for the 17β-HSD enzyme isolated from the fungus C. lunatus, as well as for the recombinant 17β-HSD_Cl enzymes synthesized in Escherichia coli and Mycolicibacterium smegmatis cells. The results expand our knowledge on microbial 17β-HSDs and suggest potential for the use of the recombinant M. neoaurum strains expressing a codon-optimized cDNA sequence encoding 17β-HSD_Cl from the fungus C. lunatus for producing testosterone from phytosterol.

The cytokine TRAIL is distinguished by its remarkable ability to preferentially induce apoptosis in transformed, but not in normal, cells. The recombinant TRAIL extracellular domain and other first-generation agonists of DR4 and DR5 death receptors (DRs) have shown very limited antitumor activity in clinical trials. To enhance the antitumor effect, we developed the multitarget recombinant fusion protein SRH-DR5-B-p48 based on the DR5-selective TRAIL variant DR5-B to simultaneously affect tumor cells (DR5-B-mediated apoptosis) and tumor microenvironment, in particular, to suppress angiogenesis. For this purpose, we modeled and produced the recombinant SRH-DR5-B-p48 fusion protein containing antagonistic synthetic peptides (SRH and p48) to VEGFR2 and FGFR1 receptors, respectively. Analysis of molecular trajectories using molecular dynamics methods showed that the SRH and p48 peptides form non-specific temporary contacts with the DR5-B domain. Using enzyme-linked immunosorbent assay, we showed that SRH-DR5-B-p48 was similar to DR5-B in its affinity for the death receptor DR5 and demonstrated a high affinity for VEGFR2 and FGFR1 with nanomolar dissociation constants. SRH-DR5-B-p48 killed tumor cells of various origin more efficiently than DR5-B and destroyed tumor-like structures in 3D cell models, as well as inhibited FGF2-mediated stimulation of fibroblast proliferation. Therefore, the SRH-DR5-B-p48 fusion protein can be considered as a promising agent for the therapy of solid tumors of various origin.