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

One of the problems of modern neuropharmacology is the search for new structures that affect specific targets in the nervous system. Among such promising structures is diazepinobenzimidazole, which includes two privileged subclasses: diazepine and benzimidazole. One of its derivatives, DAB-19, demonstrates a wide range of activities and appears to be very promising. However, the targets of DAB-19 and the molecular mechanisms of its action remain poorly understood. DAB-19 inhibits AMPA- and NMDA-subtypes of glutamate receptors, blocks voltage-gated sodium channels, and enhances spontaneous synaptic release of glutamate. This complex action profile complicates the analysis of action mechanisms. In this study, we investigated the effects of several analogues of DAB-19 to separate the action on different targets by structure–function analysis. We found that the presence of a side chain in diazepinobenzimidazole derivatives is necessary for both stimulating spontaneous release and blocking sodium channels. The presence of a long side chain containing an aromatic ring is critical for stimulating spontaneous release. Compounds with a shorter radical with a saturated ring lose their ability to stimulate spontaneous release but retain their ability to block sodium channels. The results are important for the further targeted design of new compounds that modulate the functions of the central nervous system (CNS).

Piezo1 channels are mechanically activated (MA) cation channels that play an important role in skeletal muscle physiology. However, the specific mechanisms of Piezo1 regulation in skeletal muscle are not yet fully understood. At the same time, the activity of different ion channels is known to be dependent on lipid rafts, which are dynamic microdomains in the cell membrane enriched in sphingolipids and cholesterol. Therefore, the aim of this study was to evaluate the role of lipid rafts in the muscle-specific regulation of Piezo1 using a cholesterol-removing agent, methyl-beta-cyclodextrin (MβCD), and the sphingolipid sphingosine-1-phosphate (S1P) in a C2C12 myoblast model. Fluorescence labeling and intracellular calcium measurements demonstrated that the disruption of lipid rafts with MβCD resulted in disassembly of the initially present actin cytoskeleton in C2C12 cells and decreased Piezo1-mediated Ca²⁺ influx into the cells. In contrast, stimulation of myoblasts with S1P resulted in increased formation of lipid rafts and actin stress fibers, as well as enhanced Ca²⁺ influx through Piezo1 channels. These findings suggest the involvement of lipid rafts in the regulation of Piezo1 activity in C2C12 myoblasts, which may be mediated by lipid raft components themselves and/or by lipid raft-induced rearrangements of the actin cytoskeleton.

Injuries to the central and peripheral nervous systems are accompanied by complex cellular and molecular processes, including neuroinflammation, oxidative stress, and programmed cell death. Nitric oxide (NO) and hydrogen sulfide (H₂S) play a key role in these processes, demonstrating dual effects. Apoptosis is a key mechanism involved in the death of neurons and glial cells in neurotrauma. NO and H₂S can regulate the expression of anti- and pro-apoptotic genes through direct modification of DNA and RNA or through more complex epigenetic mechanisms involving the activation or inhibition of transcription factors. This review provides a detailed overview of NO- and H₂S-dependent signaling mechanisms that regulate the expression of anti- and pro-apoptotic genes in various types of neurotrauma, as well as the dual effects of these gas transmitters in pharmacological regulation.

Coronaviruses, including the pandemic SARS-CoV-2, encode 16 non-structural proteins that regulate various stages of virus reproduction inside the infected cell but are not part of the virion structure. Some of these proteins are responsible for the formation of viroplasm, which is necessary for the reproduction of the viral genome. A characteristic feature of viroplasm is the formation of double-membrane vesicles, the physicochemical mechanisms of which are still unclear. At the same time, it is known that the non-structural protein 3 (nsp-3) plays an important role in this process. In this work, we showed that the X macrodomain (X-domain) of the nsp-3 protein of SARS-CoV-2 is capable of binding only to membranes containing acidic lipids. This domain forms various membrane tubulations, from filamentous to spherical, depending on the cholesterol content. Thus, X-domain of the nsp-3 protein of SARS-CoV-2 can be the initiator of the formation of double-membrane vesicles due to predominantly electrostatic interactions with membranes of cellular organelles.

Muscle atrophy and decreased strength are observed under hypokinesia; a decrease in strength can occur at the earliest stages of functional unloading. To study the relationship between calcium-dependent processes in the fiber and changes in the contractile properties of m. soleus, a 3-day unloading was used in male Wistar rats that were injected with nifedipine, a dihydropyridine receptor (DHPR) blocker, or ouabain, a cardiac glycoside that binds to the α-subunit of Na⁺,K⁺-ATP-ase. In two series of experiments, 3 groups of rats were used (16 animals in each group): control (C), hindlimb suspension (3HS), and either hindlimb suspension + nifedipine (3HS + N) or hindlimb suspension + ouabain (3HS + Ou). It was found that functional unloading resulted in a decrease in the mass of m. soleus in all groups with functional unloading (3HS, 3HS + N, and 3HS + Ou) (p < 0.05). After 3-day hindlimb suspension without drugs, the cross-sectional area (CSA) of fast and slow muscle fibers (MF) decreased, the level of Ca²⁺ in the MF myoplasm increased, and the maximum strength of single and tetanic contractions of m. soleus decreased relative to the control (p < 0.05). Inhibition of DHPR during 3-day-long functional unloading prevented a decrease in CSA, a decrease in strength, an increase in the level of Ca²⁺ in the myoplasm of MF, and a decrease in the maximum strength of single and tetanic contraction of m. soleus. Upon the addition of ouabain, an increase in CSA and a decrease in the level of Ca²⁺ in the myoplasm were observed, while the specific (normalized by the physiological cross-section of the muscle) maximum force of single and tetanic contraction of m. soleus in animals decreased significantly compared to the group without drugs (3HS). However, ouabain significantly affected the passive mechanical properties of m. soleus, namely, the maximum peak force and maximum force at the end of the stretching test in muscles with the administration of the drug did not differ from the intact control group. Thus, the use of ouabain and nifedipine leads to the prevention of the accumulation of myoplasmic calcium under the functional unloading, which is accompanied by the preservation of specific stiffness and muscle strength, slowing the decrease in the cross-sectional area of MF and preventing a change in the percentage of MF towards fast-type fibers.

Myeloid-derived suppressor cells (MDSC) play a key role in the formation of immune tolerance, including during pregnancy, due to their ability to suppress the immune response using various mechanisms. One of the important regulators of the immune system during gestation is pregnancy specific glycoprotein (PSG), which has pronounced immunosuppressive properties. The aim of this study was to investigate the effect of native and recombinant PSG on the functional activity of MDSC obtained from the peripheral blood of healthy donors. For this purpose, CD11b+ cells were isolated by immunomagnetic separation and differentiated into MDSCs using GM-CSF, IL-1β, and lipopolysaccharide (LPS). Physiological concentrations of native (1, 10, 100 μg/mL) and recombinant (1, 10 μg/mL) PSG were used in the experiment. Cell phenotyping was performed by flow cytometry with determination of PD-L1 and CD73 membrane proteins, as well as measurement of inducible NO synthase (iNOS) levels and analysis of the cytokine profile (17 markers) using multiplex analysis. It was found that recombinant PSG at a concentration of 1 μg/mL significantly increased the density of PD-L1 protein on the surface of MDSC, and at a concentration of 10 μg/mL increased the level of CD73, while native PSG had no significant effect on these parameters. Both variants of PSG did not affect iNOS production, but recombinant PSG (10 μg/mL) reduced the level of MIP-1β chemokine without altering the production of other cytokines studied. The results obtained indicate that recombinant PSG can enhance the immunosuppressive potential of MDSC by increasing the membrane molecules PD-L1 and CD73, as well as suppressing MIP-1β production, which may be important for the development of new bio-pharmacological approaches to correcting the immune response in autoimmune diseases and transplantation. The structural features of recombinant PSG associated with post-translational modifications probably determine its selective effect on the functional properties of MDSC.

Under conditions of insufficient muscle activity (with their functional unloading), a number of pathological processes are observed, leading to deterioration of muscle functions. Some of these processes are based on changes in gene expression, which leads to the transformation of the phenotype of muscle fibers from “slow,” with a predominantly oxidative metabolism and resistant to fatigue, to “fast,” with a predominantly glycolytic metabolism and prone to fatigue. Based on the literature data, it can be assumed that CpG methylation of promoter regions of genes may be involved in regulating the expression of genes that implement the “slow” and “fast” phenotype of muscle fibers. A decrease in the expression of genes regulating mitochondrial biogenesis and muscle fiber phenotype under conditions of mechanical muscle unloading may be determined by a lack of alpha-ketoglutarate (a coenzyme of TET translocases demethylating CpG islands). To test this hypothesis, male Wistar rats were divided into three groups of 8 animals each: (1) C, vivarium control with daily intraperitoneal administration of placebo (saline solution); (2) 7HS, 7-day hind limb suspension with daily intraperitoneal administration of placebo (saline solution), and (3) 7HSD, 7-day hind limb suspension with daily intraperitoneal administration of 200 mg/kg dimethyl-2-ketoglutarate (precursor of alpha-ketoglutarate). Based on the analysis of experimental data, it was found that the administration of dimethyl-2-ketoglutarate to the suspended animals partially prevents the decrease in the accumulation of mRNA regulators of mitochondrial biogenesis and the content of mitochondrial DNA observed during 7-day hind limb suspension. This effect can be realized through the violation of the CpG methylation by the drug; however, in the 7HSD group, an increase in the phosphorylation of AMP-activated protein kinase was also found compared to the 7HS and C groups, which may explain the effect of dimethyl-2-ketoglutarate on the accumulation of mRNA regulators of mitochondrial biogenesis and the content of mitochondrial DNA upon suspension of the hindlimbs of rats.

Early changes in the myosin genes expression pattern lead to slow-to-fast fiber type shift, functional and metabolic alterations in skeletal muscles. A detailed understanding of myosin isoform transcriptional regulation could be the key for effective countermeasures against the unloading-induced negative changes. Slow-type myosin mRNA expression changes non-linearly in postural soleus muscle during rat hindlimb suspension, with a partial re-activation of slow-type myosin expression between the 2nd and the 4th day. Calcium ions accumulation is a well-studied activator of slow-type myosin expression, and after the second day of rat hindlimb suspension calcium was shown to increase in soleus muscle myoplasm. We suggested that calcium ions accumulation could contribute to the transient re-activation in slow-type myosin gene expression after the 2nd day of rat hindlimb suspension via activation of calcineurin/NFATc1 and/or blocking of HDAC4/MRF4 signaling pathways. If this hypothesis is correct, the prevention of the unloading-induced calcium accumulation by L-type channels inhibitor nifedipine would be expected to downregulate the expression of slow-type myosin. To test our hypothesis, the experiment with 3-day rat hindlimb suspension (HS) accompanied by nifedipine (L-type calcium channels blocker) administration was conducted. After 3 days of HS, we observed an increase in slow-type myosin mRNA expression and an increase in NFATc1 MCIP1.4 reporter activity, which was not observed in the group receiving nifedipine. In the group with 3-day rat hindlimb suspension and receiving nifedipine (group HS + Nifedipine), we also observed an accumulation of HDAC4 and MRF4 in the nucleus and a decrease in NFATc1 content in the nucleus compared to 3 days of HS. These data indicate that calcium ions accumulation after 3 days of HS contributes to the reactivation of slow myosin expression and activates both HDAC4-dependent and NFATc1-dependent signaling pathways during unloading.

Functional unloading of skeletal muscles is observed during space flight, prolonged bed rest, or limb immobilization. This leads to skeletal muscle atrophy, which has serious health consequences and significantly reduces quality of life. In addition, during functional unloading, mitochondrial dysfunction is observed and the release of reactive oxygen species (ROS) by mitochondria increases. It is known that some antioxidants can reduce the manifestation of atrophy during functional unloading. We hypothesized that the mitochondria-specific antioxidant Mito-TEMPO would block the accumulation of mitochondrial ROS, prevent the increase in mRNA expression of ubiquitin ligases and the decrease in anabolic parameters during 7 days of functional unloading, which together may reduce the degree of atrophy. In our study, animals receiving Mito-TEMPO during 7 days of hind limb suspension partially prevented the decrease in cross-sectional area of m. soleus muscle fibers, the increase in MuRF-1 and Atrogin mRNA expression, and the decrease in rRNA content. In addition, Mito-TEMPO prevented ROS-dependent tropomyosin oxidation during 7 days of suspension in the soleus muscle. Thus, the accumulation of mitochondrial ROS in the soleus muscle during 7 days of functional unloading affects both protein synthesis and degradation, which is reflected in a decrease in the cross-sectional area of muscle fibers in the rat soleus muscle.

The research described in this paper is based on a mathematical model for the proton-dependent regulation of electron and proton transport in chloroplasts. We have considered redox transformations of photosynthetic (PS) reaction centres PS1 (P₇₀₀) and PS2 (P₆₈₀) and of the mobile electron transport carriers ferredoxin (Fd), plastoquinone (PQ), and plastocyanin (Pc), as well as the transmembrane proton transport processes associated with electron transport and ATP synthesis by membrane ATP synthase. The simulation results are in good agreement with the literature data on pH measurements in the lumen (pH_in) and stroma (pH_out) of chloroplasts at different cytosol pH values (pH_cyt). The steady-state pH values established in these compartments when chloroplasts are illuminated under photophosphorylation conditions satisfy the following inequalities: pH_in ≈ 6.2–6.4 < pH_cyt ≈ 7.2–7.4 < pH_out ≈ 7.8–8.0. Variation of the pH_cyt model parameter affects the electron flows along different electron transport pathways, such as non-cyclic electron transport from PS2 to PS1 and further into the Calvin–Benson cycle, cyclic electron transport around PS1, and pseudocyclic electron transport involving molecular oxygen (the ‘water–water’ cycle). It has also been shown that sufficiently strong acidification of the cytosol (pH_cyt < 7) reduces electron efflux from the acceptor side of PS1 (noncyclic and pseudocyclin electron transport) and stimulates cyclic electron transport around PS1, and also decreases the rate of pH-dependent ATP synthesis.