Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology最新文献

Disulfiram (DSF) and its oxidized derivatives (DSFoxy) are currently being investigated as potential anticancer agents. We previously found that DSFoxy initiate the paraptosis-like death of tumor cells, which is of potential interest for the treatment of tumors resistant to the initiation of apoptosis. Based on bioinformatics analysis of mass spectrometric data on protein ubiquitination, we formulated a conception about the important role of disruption of the retrograde transport of damaged proteins from the endoplasmic reticulum to the cytosol in the mechanism of initiation of paraptosis-like cell death. In the present work, it has been found that DSFoxy, in the process of initiating paraptosis-like death of human adenocarcinoma HEp-2 cells, also enhances the ubiquitination of histones and histone code enzymes. In particular, this applies to the ubiquitination of histone H2BC12, histone methyltransferases responsible for transcription and repair of damaged DNA, as well as acetylating and ubiquitin-conjugating proteins. Bioinformatics analysis of changes in ubiquitination of cell nuclear proteins using the STRING database revealed during this process an increase in the occurrence of ubiquitinated proteins (functional enrichment) of cell cycle regulation, cell response to DNA damage and DNA repair, the regulation of which also depends on the histone code. This directly indicates damage to the cell nucleus and is consistent with confocal microscopy data. These results indicate that paraptosis-like death initiated by DSFoxy is accompanied, along with impairment of retrograde transport and ER stress, also by a change in regulation of the histone code, which points to a pleiotropic mechanism of cell death induction.

Calcium signaling is a principal method of signal transduction in cells of non-excitable tissues. In both mouse and human sperm, it can be induced in response to progesterone, manifesting as oscillations or single peaks and followed by the acrosomal reaction. However, the molecular mechanisms of progesterone activation may vary between species. In this study, we aim to compare the calcium signaling mechanisms in human and mouse spermatozoa. We investigated the calcium response in mouse sperm activated by progesterone. We employed spectrofluorometry to quantify the rise in calcium concentration in response to progesterone in Fura-2 loaded mouse sperm cells in suspension. Our experiments demonstrated that mouse sperm cells respond to 50 μM progesterone with a peak 120 ± 35 s wide and 0.8 ± 0.3 μM high. Based on literature data, a scheme for the induction of calcium signaling was constructed, suggesting an intermediate stage with the synthesis of a certain prostanoid (possibly PGE2) and activation of mouse sperm by this prostanoid through a G-protein-coupled receptor. Based on the obtained reaction scheme, two computational models were developed: a point model and a three-dimensional model. As with human sperm, the point model provided only a qualitative description of calcium responses, whereas the three-dimensional model produced the shape of the calcium peak and the frequency of calcium oscillations in response to progesterone that were similar to the experimentally obtained values. Using in silico analysis, it was shown that in mouse sperm, the spatial distribution of signaling enzymes regulates the type and form of the calcium response. We conclude that the presence of time delays due to the diffusion and spatial distribution of calcium signaling enzymes regulates the calcium response in both human and mouse sperm.

The study of the permeability to water and urea of plasma membranes of endothelial cells of the normal cornea and cornea with a pronounced form of keratoconus was carried out. Human corneal endothelial cells were obtained from surgical material. Determination of osmotic water permeability (P_f) of normal and keratoconus endothelial cells did not reveal significant differences in the value of this parameter in both groups, where cells had a similar osmotic water permeability (control P_f = 0.53 ± 0.045 cm/s; keratoconus P_f = 0.63 ± 0.041 cm/s) (n = 25; p ≥ 0.05). The urea permeability coefficients (P_u) in both groups also had no statistically significant differences (control P_u = 0.049 ± 0.003 cm/s; keratoconus P_u = 0.056 ± 0.003 cm/s) (n = 25; p ≥ 0.05). Analysis of cell volume dynamics based on exponential approximation showed that corneal endothelial cells with keratoconus reduced their volume in a hypertonic medium to a greater extent compared to cells of a healthy cornea. An increase in cell volume as a result of isotonic urea inflow in a hypertonic medium also occurred to significantly higher values compared to normal cells. The conclusion was made about significant changes in the mechanism of regulation of the volume of corneal endothelial cells in keratoconus.

The effect of ionizing radiation (IR) on plants is mainly realized by altering the status of signaling systems and modifying stress signals. Variation potential (VP) is one of the types of electrical signals in plants. IR contributes to an increase in the amplitude of the VP, but the mechanisms of such influence are practically unknown. A possible way to implement changes arising from the action of IR is the regulation of gene expression. In the present work, the changes in the gene expression of participants in the generation and propagation of VP in irradiated plants are investigated. The experiments were performed on 14–15-day-old wheat plants (Triticum aestivum L.) grown under chronic irradiation (source ⁹⁰Sr–⁹⁰Y) with a dose rate of 31.3 μGy/h. The maximum accumulated dose was about 11.3 mGy. The irradiated plants showed no changes in the expression of calcium (TPC1), anionic (ALMT1 and CLC1), potassium (AKT1) channels, H⁺-ATPase (HA1), and NADPH oxidase (RBOHs) genes. A decrease in the expression of the SKOR potassium channel gene was revealed. The potassium channel blocker, tetraethylammonium chloride, caused an increase in response amplitude in control plants comparable to the increase in amplitude in the irradiated group. The obtained results indicate that one of the ways IR influences the electrical signals of plants is to inhibit the expression of the potassium channel.

Attachment to the vascular endothelium is the first step of the release of mature neutrophil granulocytes from the bone marrow into the blood and subsequent migration to the inflammatory center. Disturbance of neutrophil adhesiveness is critical for many diseases with inflammatory components. Endo- and exogenous factors modify the cell ability to adhere via different receptors, including nicotinic acetylcholine receptors (nAChRs). However, the involvement of nAChRs in the regulation of bone marrow (BM) granulocyte adhesion and the role of signaling components in the action of nicotine are poorly understood. In this work the role of different types of nAChRs in the regulation of murine BM granulocyte adhesion during acute inflammation was studied. The work was performed on BM granulocytes of the BALB/c mouse strain using static adhesion assay, confocal microscopy, inhibitor assay, and reverse transcription PCR (RT-PCR). The role of nAChR types was assessed applying selective antagonists: 10 nM α-CTX (α7), 10 nM GIC and 5 nM MII (α3β2), 200 nM MII (α3β2 and α7), RgIA and Vc1.1 (α9α10). The number of attached BM granulocytes did not differ between animals with and without inflammation. Nicotine (0.01–100 µM, 30 min) significantly increased cell adhesion in both groups. Toxins (α-CTX, RgIA, Vc1.1) enhanced cell adhesion in both groups, as 200 nM MII did in controls. Fluorescence labelling assays showed expression of α7 and α10 nAChR subunits on cytoplasmic membrane of native BM granulocytes. Using inhibitors, we showed that the effect of nicotine on BM granulocyte adhesion was mediated by heterotrimeric G-proteins, PKC, PI3K, and ROCK both normally and in the presence of inflammation. α7 and α9α10 nAChRs were predominantly involved in regulation of BM granulocyte adhesion, and participation of α3β2/α3α6*β2 was negligible, possibly due to low expression of α3/α6* subunits. In the regulation of cell adhesion by nicotine, the development of inflammation in the body enhanced the role of α7 nAChRs, which are conventionally expressed on the membrane of BM granulocytes.

Lipid droplets are organelles responsible for the accumulation and breakdown of neutral fats in the human body. Lipid droplets have a monolayer shell of phospholipids, which prevents their spontaneous fusion. The fusion of lipid droplets is carried out by specialized fusion proteins and is regulated by the lipid composition of the monolayer membrane. The efficiency of fusion is determined by the energy needed for the local approach of lipid droplets and the topological rearrangement of their monolayers. In this work, the fusion of monolayers is modeled within the framework of the theory of membrane elasticity. The energy barrier for fusion is calculated under various conditions simulating possible compositions of monolayers, as well as the possible effects of proteins. The calculation results show that the height of the barrier is most dependent on the distance between lipid droplets, which is determined by the fusion proteins. Lipid composition also affects the fusion efficiency and can change it several tens of times, which is consistent with previously obtained data on bilayer fusion.

Proteins of the anoctamine family (ANO) form calcium-activated chloride channels (CaCC) and phospholipid scramblases. The ANO6 (TMEM16F) protein, which combines the functions of a calcium-dependent scramblase and those of an ion channel, is considered as a molecular target for the treatment of blood clotting disorders, COVID-19-associated pneumonia, neurodegenerative diseases, and other pathologies. CaCCinh-A01, which is a channel blocker of the ANO family, is studied as a potential pharmacological drug. Previously, the effect of this inhibitor was studied using methods representing the integral ion current through the membrane, which does not allow the properties of single channels to be distinguished. Therefore, it remains unknown which characteristics of single channels are sensitive to the blocker: the channel open probability, the current amplitude, or the dwelling time of the channel open state. By registration of single ANO6 channels in HEK293 cells, we showed that the action of the inhibitor is due to a decrease in both the current amplitude and the dwelling time of the single ANO6 channels open state, which, in turn, leads to a decrease in their open state probability. Thus, we have characterized the mechanism of current reduction through ANO6 channels by the inhibitor CaCCinh A01.

The thickness of the lipid membrane is its substantial characteristics. Usually, the thickness of a lipid bilayer is experimentally determined by measuring its electrical capacitance in the approximation of a plane-parallel capacitor. However, membranes formed from a mixture of lipids or containing membrane-deforming inclusions are laterally inhomogeneous, and for them the plane-parallel capacitor approximation generally does not hold. In this work, corrections to the electrical capacitance resulting from deformation of the lipid membrane were numerically calculated. It is shown that the model of a planar capacitor (or their parallel connections), in the general case, does not quantitatively describe these corrections due to the non-zero tangential component of the electric field strength. It is shown that the relative deviation of corrections to the electrical capacitance calculated in various simplified models from the exact solution can reach 50%.

The protein endophilin A, which in the mammalian genome is encoded by three genes, endophilin A1, A2, and A3, regulates the synaptic vesicle cycle during exo- and endocytosis, and it is present in the reserve pool of synaptic vesicles (SVs), where its function is unknown. In vitro experiments suggest that endophilin, via its SH3 domain interactions, incorporates several components into the protein liquid phase that organizes SVs in the reserve pool. We investigated the effect of deletion of the genes encoding endophilin and one of its binding partners, dynamin, on the organization of SVs in living synapses formed by cortical neurons in culture. Our experiments showed that deletion of endophilin genes does not change the density of SVs in the reserve pool. At the same time, the deletion of dynamin 1 and dynamin 3 genes leads to a significant increase in the vesicle density. We suggest that other SH3-domain-containing proteins, which are components of the protein liquid phase, complement the function of endophilin in the SV reserve pool.

It is known that the molecules of innate and adaptive immunity can influence the synaptic activity of the central nervous system (CNS). Interferons (IFNs) are commonly used to treat viral and oncological diseases, although they are classified as ototoxic substances and their impact on the synaptic activity of the inner ear is not yet fully understood. In this study, the effect of interferon α2b (IFN-α2b) on the function of afferent glutamatergic synapse in the presence of drug application to the synaptic zone has been analyzed. The study was performed on the isolated vestibular apparatus of a frog (Rana temporaria) using the multiunit recording. Results showed that IFN-α2b (0.2–40 ng/mL) caused an increase in the background pulse activity of afferent fibers, followed by a decrease in the frequency of discharges, especially at high concentrations of IFN-α2b. Additionally, IFN-α2b reduced the glutamate (L-Glu) evoked response and influenced the level of afferent fiber activity restored by L-Glu when the glutamate release from hair cells was blocked in hyper-Mg²⁺ and hypo-Ca²⁺ solution. This indicates that IFN-α2b has a postsynaptic effect. Overall, the findings suggest that IFN has a neuromodulating effect on the synaptic activity of the afferent synapse of the vestibular apparatus.