Biochemistry (Moscow)最新文献

Air pollution remains a major environmental challenge, largely driven by urban dust composed of suspended solid particles of diverse origins and chemical compositions. Particulate matter (PM_2.5) and ultrafine urban dust nanoparticles (NPs), with diameters smaller than 2.5 μm and 100 nm, respectively, pose a particular threat to human health. In this study, we present the first evidence that NPs induce pro-inflammatory activation of human bronchial epithelial cells. Exposure to non-cytotoxic concentrations of NPs led to a significant increase in the mRNA levels of pro-inflammatory markers IL-8, IL-1β, IL-6, and ICAM-1, accompanied by increased secretion of the cytokines IL-8 and IL-6. Heat treatment of NPs, which removed their organic components, completely abolished their ability to stimulate cytokine secretion. NP-induced upregulation of pro-inflammatory gene expression depended on both surface-adsorbed organic compounds and inorganic particle constituents.

The PI3K/AKT signaling pathway is one of the most critical intracellular pathways, regulating cell proliferation, survival, and migration. Mutations in PI3K/AKT pathway components are frequently associated with progression and metastasis of malignant tumors. The initial stage of metastasis is epithelial-mesenchymal transition (EMT), during which tumor cells acquire the migratory capacity. The data on the impact of PI3K/AKT driver mutations on tumor cell motility are contradictory. We investigated EMT and changes in the motility of breast epithelial cells carrying four driver mutations commonly identified in malignant tumors and affecting different components of the PI3K/AKT pathway. Analysis of cell motility, expression of EMT markers, and morphology of adherens junctions (AJs) revealed that all these mutations induced partial EMT, as E-cadherin expression was preserved in all studied cell lines and cells maintained AJs. Analysis of EMT types induced by different mutations revealed that the increased cell motility did not correlate with the degree of EMT progression toward the mesenchymal phenotype. The greatest increase in the cell migratory capacity was observed for cells carrying the PIK3CA H1047R mutation, which induced the most pronounced mesenchymal phenotype, as well as for PTEN–/– cells, which retained the most epithelial phenotype. Our analysis showed that mutations indirectly affecting the MAPK/ERK pathway and promoting ERK activation have the greatest impact on EMT and cell motility.

Decreased insulin-mediated glucose uptake by skeletal muscle, which accounts for one-third of total body mass, plays a crucial role in the development of type 2 diabetes (T2D). However, the molecular mechanisms underlying this disorder remain poorly understood. In contrast to previous studies examining changes in phosphorylation of individual sites or global phosphoproteome profile of human skeletal muscle in response to insulin (euglycemic-hyperinsulinemic clamp test), the present study is the first to investigate changes in skeletal muscle signaling in response to a mixed meal normalized to body mass (a model of physiological postprandial response). Using mass spectrometry-based phosphoproteomics, postprandial changes across 4205 phosphorylation sites in 1208 proteins/protein groups were analyzed in healthy individuals (n = 8) and patients with obesity and T2D (n = 8) and key kinases associated with these changes were identified. Food intake altered phosphorylation levels of 70 sites in healthy individuals and 36 sites in patients. However, postprandial phosphorylation of canonical insulin cascade proteins was comparable between the groups, which might be attributed to significantly elevated postprandial blood insulin levels in the patients caused by a reduced insulin-dependent glucose uptake by tissues and a greater food intake compared to healthy individuals. Only healthy individuals exhibited changes in postprandial phosphorylation levels of several proteins regulating the translocation and/or exposure of GLUT4-containing vesicles (SRBS1, CIP4/2, ABI1, SVIL, CPZIP, PLEC, and COBL), suggesting that impaired insulin-dependent glucose uptake in skeletal muscle in patients with obesity and T2D is primarily due to impaired regulation of GLUT4-containing vesicles trafficking.

Patients with coronary and cerebral atherosclerosis are characterized by increased levels of total serum calcium, ionized calcium, and phosphate, against a background of reduced levels of total serum protein and albumin. Here we aimed to develop a rapid diagnostic assay for mineral homeostasis disorders, based on assessing capacity of the acidic plasma proteins to bind excess calcium and phosphate ions. Plasma from bony fish, amphibians, reptiles, birds, mice, and patients with myocardial infarction was incubated with excess concentrations of calcium and phosphate at 37°C for varying time periods. The following assay readouts were defined: (i) plasma optical density after supersaturation with calcium and phosphate ions, reflecting excessive formation of calciprotein particles (CPPs); and (ii) CPP concentration in plasma. CPPs were formed in all vertebrates. The most pronounced plasma calcification propensity was observed in the human and mouse plasma, suggesting an evolutionary significance of CPP formation as a mechanism for clearance of excess circulating calcium and phosphate ions in mammals. Among the 11 protocols of supersaturation with calcium and phosphate ions, stable increase in plasma optical density at 620 nm wavelength (normalized OD₆₂₀, a measure of plasma calcification propensity) was achieved by adding solutions of CaCl₂ (+2 mmol/L, +50 µL), Na₂HPO₄∙12H₂O (+2 mmol/L, +50 µL), and NaCl (+15.4 mmol/L, +20 µL) to plasma (80 µL). Increase in the normalized OD₆₂₀ was consistently detected within 10 min from the reaction onset during incubation in a microplate shaker (37°C), with mild-to-moderate variability across the parallel or sequential measurements and between the different operators. These results support relevance of validating the developed diagnostic assay for assessing mineral homeostasis disorders in the expanded cohorts of patients with myocardial infarction and ischemic stroke.

This review has considered the mechanisms of intracellular and intercellular signaling regulation by myeloperoxidase (MPO), an enzyme in neutrophil azurophilic granules, during oxidative/halogenative stress and inflammation development. The stages of enzyme functioning and the formation of reactive halogen species are described. The functioning of MPO and production of reactive halogen species are shown to depend on the activity of NADPH oxidase; the role of NADPH oxidase and reactive oxygen species in the regulation of MPO function is discussed. Particular attention is focused on the role of biological molecules modified by reactive halogen species in modulating NADPH oxidase activity, exocytosis of granular proteins, NETosis, and other neutrophil functions, based on the principle of positive feedback. A special feature of the review is the discussion of the non-canonical function of MPO, namely its signaling role in the regulation of cellular processes, which is not associated with the catalytic activity of the enzyme.

Gradient hydrogels represent a unique class of biomaterials capable of mimicking the spatial heterogeneity of native tissues and providing targeted effects on cells through mechanical, chemical, and biophysical gradients. In recent years, numerous fabrication strategies have been developed to generate gradient hydrogels, including layer-by-layer formation, photopolymerization, microfluidic techniques, and 3D/4D printing. This review summarizes current methodologies for the characterization of gradient hydrogels and highlights their emerging biomedical applications, such as controlled drug delivery, tissue engineering, regenerative medicine, organ-on-chip systems, and soft bioelectronic devices. Furthermore, the review discusses critical challenges related to the protocol standardization, manufacturing scalability, integration with additive manufacturing technologies, and potential regulatory barriers.

Cancer-testis antigens are expressed in the germ cells of the testes, but can also be produced in some types of cancer cells, thus representing an important protein group in oncoimmunology. They include sperm-specific proteins of glycolysis, in particular, sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDHS). This isoform differs from somatic isoform in a number of properties. Normally expressed in spermatids, GAPDHS is also found in uveal and skin melanoma cells. Because GAPDHS is a glycolytic protein and glycolysis is a key component of energy metabolism in a growing tumor, the review summarizes the data on the functional and structural features of GAPDHS and its role in the regulation of glycolysis in melanoma cells.

In recent years, targeted proteolysis systems have emerged as powerful tools for directed degradation of pathogenic proteins, offering novel therapeutic strategies for cancer, neurodegenerative disorders, and infectious diseases. This review systematizes key mechanisms and recent advances in inducible targeted proteolysis, including targeted proteasomal degradation (PROTACs, AbTACs, molecular glues), lysosome-mediated degradation (LYTACs, AUTACs, ATTECs) via endocytosis or autophagy, and targeted proteolysis in bacteria (BacPROTACs), which extends degradation technologies to prokaryotic systems. The structural features, advantages, and limitations of each platform are discussed in detail, along with key publications demonstrating their preclinical and clinical efficacy. Special attention is given to the prospects for translating these technologies into therapeutics, including overcoming challenges such as selectivity and in vivo delivery.

Pyridoxal-5′-phosphate (PLP)-dependent D-amino acid transaminases (DATAs) catalyze stereoselective transfer of an amino group from a D-amino acid to an α-keto acid to form new D-amino acid and α-keto acid. These enzymes are found in bacteria and plants; they are responsible for the synthesis of D-amino acids and are incorporated into the nitrogen cycle. In general, the mechanism of D-transamination is similar to the known mechanism of transamination for aspartate aminotransferase: D-transamination reaction consists of two half-reactions with intermediate transfer of the amino group to the cofactor and formation of its reduced form, pyridoxamine-5′-phosphate. DATAs are characterized by broad substrate specificity and an open active site, which, however, does not affect their high stereoselectivity: no side L-products is detected in the DATA-catalyzed D-transamination. As in other PLP-dependent fold type IV transaminases, the functional unit of DATAs is a dimer. The active site is formed by amino acid residues of both subunits and binding of α-carboxylate group is crucial for proper substrate coordination. DATAs with promiscuous activity towards substrates without an α-carboxylate group, primary (R)-amines, have also been discovered and characterized. The promiscuous activity is achieved through the mobility of certain residues in the active site of DATAs. High stereoselectivity and stability of DATAs make then promising candidates for multienzyme cascade processes as biocatalysts of the (R)-stereoselective amination stage. Open configuration of active site makes binding and conversion of bulk non-natural substrates possible. The review describes in detail properties, structure, and relationships of DATAs from two currently known groups differing in organization of their active sites. The prospects for biotechnological applications of DATAs are discussed as well.

The mechanism of selective specificity of oxidoreductases to NAD⁺ or NADP⁺ and the ability to change the coenzyme specificity of these enzymes are some of the most important fundamental and applied problems. The first work on the switch in the coenzyme specificity from NADP⁺ to NAD⁺ was performed in 1990 for glutathione reductase. In 1993, formate dehydrogenase (FDH, EC 1.2.1.2) from the methylotrophic bacterium Pseudomonas sp. 101 (PseFDH) became the first oxidoreductase whose coenzyme specificity was changed in the opposite direction – from NAD⁺ to NADP⁺. Mutant NADP⁺-specific FDHs are extensively used in fine organic synthesis (including production of chiral compounds). The switch in the coenzyme specificity from NAD⁺ to NADP⁺ in FDHs is achieved by substituting amino acids at positions 198, 221, 222, 260, 379, and 380 (numbering according to PseFDH); however available data do not allow the interpretation of the exact role of each individual substitution. Since 2010, five natural NADP⁺-dependent FDHs have been found. In 2015-2024, three 3D structures for two natural and four 3D structures for two mutant NADP⁺-specific FDHs have appeared in the Protein Data Bank (PDB). In this review, we briefly discussed the general principles of coenzyme specificity based on the experimental and modeled FDH structures and performed a detailed analysis of the type and arrangement of residues at positions corresponding to His379 and Ser380 in PseFDH, whose role in NADP⁺ binding is still debated.