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

Here we explore capabilities of size exclusion chromatography small-angle X-ray scattering method in case of horse spleen apoferritin solution as an example of a system with high oligomerization heterogeneity. For interpretation of the experimental data, we considered models of polydisperse solution containing monomers, dimers, and trimers of apoferritin globules. Dimers were defined only by intercenter distance L, while trimers were characterized by both L and apical corner value φ. We showed that a reasonable description of the major part of the chromatogram could be reached using the model containing monomers, dimers and one type of trimers with φ about 110°–120°. In the case of two types of trimers in the model, optimal φ values were 110°–140° and 90°–110°; however, the approximation results were considered as ambiguous. Validation of the preferred model (L = 12.5 nm, φ = 120°) showed insufficient description of the experimental data for the left part of the chromatogram (elution volume <12.2 mL) indicating the presence of aggregates in these fractions. Application of the method provided the opportunity to calculate concentrations for oligomers consisting of less than four apoferritin globules, and select averaging range for different fractions properly.

This paper presents a statistical analysis and comparison of all existing as of November 20, 2024 membrane protein structures that are represented in the most widely used databases mpstruc, Orientations of Proteins in Membranes (OPM), SCOP and PDBTM, with the total number of structures now reaching 13956. We evaluated the resolution dependencies of structure-derived membrane proteins on their size-related physical characteristics, such as molecular weight, radius and size of the hydrophilic part. Based on the obtained dependencies, we compared the resolving methods of X-ray diffraction and electron microscopy structures, and also compared the methods of protein crystallization in surfo and in meso. The change over time in the number of membrane protein structures obtained by different methods was also examined to analyze trends in structural biology. In 2024, cryo-electron microscopy has emerged as the dominant method, contributing to nearly half of the solved structures, now representing 49.6% of the total. The obtained results demonstrate that the resolution of structures obtained by X-ray diffraction on protein crystals, on average, tends to worsen with increasing protein size, while in electron microscopy, which has recently gained enormous popularity, there is no such trend, but the average resolution is worse. It was also shown that the character of the dependence of resolution on protein size is the same (within error) for the two most common methods of membrane proteins crystallization: in lipid cubic phases (in meso) and in detergent micelles (in surfo). Thus, statistically, neither of these two methods can be considered a priori preferable for achieving better resolution.

In this study, we investigated thermosensitive histidine kinase DesK from Bacillus subtilis with the aim of obtaining a monodisperse protein preparation suitable for further structural studies. A homogeneous sample, both in terms of oligomeric state and ligand binding, is favorable for electron microscopy or X-ray diffraction applications. The binding of ATP is required for autophosphorylation activity of DesK, thus we aimed at developing heterologous expression techniques that would yield ATP-bound protein. We focused on two critical factors that may influence ATP binding: the nutrient composition of the growth medium and the expression temperature. Protein characterization was performed using size-exclusion chromatography, with absorbance monitored at both 280 and 260 nm to assess protein homogeneity and nucleotide binding. Our results indicate that while nutrient composition had a negligible effect on the relative amount of ATP-bound protein, expression temperature played a significant role. Specifically, lowering the expression temperature from 37°C to 20°C at the time of induction markedly increased the proportion of ATP-bound DesK oligomers.

Bacterial biosensors based on the Escherichia coli and Bacillus subtilis whole cells are used to study toxicological characteristics of biologically active compounds and toxicants, and are applicable for ecological studies. This work describes the trials of lux-biosensors in the marine research expedition in the northern seas: Barents, Kara, and Laptev. The presence of toxicants in samples of bottom sediments and tissues of marine invertebrates was estimated by the activation of stress promoters in biosensor cells. It was shown that the collected samples generally did not show toxicological characteristics in terms of the ability to activate oxidative stress and SOS response. However, several samples were able to activate transcription of the DNA glycosylase promoter in biosensor bacterial cells. This effect was shown for some samples of bottom sediments and arthropod tissues from Mesidothea sabini var. sabini., Pycnogonida and some Amphipoda.

In this work we investigated the rearrangements in the cristae structure and possible connection of these changes with the level of MICOS proteins in rat liver mitochondria in experimentally induced hyperthyroidism. A model of hyperthyroidism (HT) was induced by chronic administration of thyroxine. A change in the structure of liver mitochondria of hyperthyroid rats (HR) was found to consist of swollen organelles with a slightly altered number of cristae. A part of mitochondria (13%) in HR had vacuolized matrix with simultaneously reduced number of cristae. An increase in the protein levels of MIC 60, MIC 25, and MIC 19 was shown in the HT condition, while the quantity of MIC 10 was unchanged. In turn, the levels of SAMM 50 and OPA1 were reduced in HR. The data obtained indicate that excess thyroid hormones cause partial changes in the structure of liver mitochondria and reduce the content of SAMM50 and OPA1 proteins, which can be considered as possible targets for investigating therapeutic strategies for metabolic disorders associated with mitochondrial dysfunction.

The trigeminovascular system plays a pivotal role in the generation of nociceptive activity, which underlies pain in migraine and includes trigeminal nerve afferents, dura mater vessels, and mast cells. Adenosine triphosphate (ATP) is one of the main mediators of the interaction between vessels, nerve endings, and mast cells. ATP activates P2X3 receptors of trigeminal nerve afferents and P2X7 receptors of mast cells. 24-Hydroxycholesterol is the primary brain cholesterol metabolite synthesized by neurons and is capable of freely entering the bloodstream by crossing the blood–brain barrier. The purpose of this study was to examine the impact of 24-hydroxycholesterol on the electrical activity of trigeminal nerve afferents triggered by ATP application and on the mast cells degranulation in rat brain meninges. The electrical activity of the trigeminal nerve was recorded using an extracellular electrode in a rat hemiskull preparation, mast cells were stained with toluidine blue. 24-Hydroxycholesterol had no direct effect on the frequency of action potentials of trigeminal nerve but suppressed the response triggered by ATP application. Furthermore, incubation of meninges in 24-hydroxycholesterol resulted in a reduction in mast cell degranulation in response to ATP. Therefore, 24-hydroxycholesterol prevented the activation of trigeminal afferents either directly by reducing the activity of ionotropic ATP receptors or indirectly by stabilizing mast cells.

Erythropoietin (EPO) is a hormone that plays a dual role: it regulates erythropoiesis and works as a neuroprotector, the synthesis of which is activated by brain cells in response to hypoxia. Several clinical trials showed that administration of EPO against anemia caused the improvement of cognitive brain functions, however the mechanisms of this action have not yet been characterized. Recently, the usage of Carbamylated darbepoetin (CdEpo), which is neuroprotective but lacks hematopoietic activity and has a more prolonged half-life, has become an attractive tool. The influence of administration of 100 ng/mL CdEpo on the activity and ultrastructural organization of mature hippocampal cultures in normoxic conditions was assessed. It was found that the application of CdEpo in normoxic conditions causes a negative neurotropic influence on neuronal activity that was measured via calcium imaging. Calcium imaging data revealed that CdEpo caused a decrease in the frequency of calcium oscillations; however, the duration of calcium events slightly increased. The decrease in neuronal activity upon administration of CdEpo coincided with compartment-dependent ultrastructural changes. In axons, there was an increase in total mitochondrial area with a decrease in mitochondrial and endoplasmic reticulum contact surface, and in neuronal soma, there was an increase in the surface of intermitochondrial contacts.

Formation of through conducting defects—pores—in the lipid bilayer affects many processes in living cells and can lead to strong changes in cellular metabolism. Pore formation is a complex topological rearrangement and occurs in several stages: first, a hydrophobic through pore is formed, then it is reconstructed into a hydrophilic pore with a curved edge, the expansion of which leads to membrane rupture. Pore formation does not occur spontaneously, since it requires significant energy costs associated with membrane deformation. The evolution of the system is associated with overcoming one or two energy barriers, the ratio of their heights affects the stability of the pore and the probability of its formation. We study the effect of membrane curvature on the height of the energy barrier for the transition of a pore to a metastable hydrophilic state. We apply the theory of elasticity of lipid membranes and generalize the model of pore formation in flat membranes to the case of arbitrary curvature. We show that the barrier for pore formation decreases by 8k_BT when the radius of curvature decreases from 1000 to 10 nm, which facilitates the formation of a metastable pore. Our results are consistent with experimental data and can be used to model complex processes occurring in curved regions of living cell membranes.

Progesterone regulates reproductive processes and affects many functions of various non-reproductive organs. Its effects in mammals and humans are mediated by nuclear (nPRs) and membrane progesterone receptors (mPRs). The action of progesterone through different types of receptors differs significantly and also has tissue-specific features. The expression of known types and subtypes of progesterone receptors in tissues of male and female rats has not been sufficiently studied. The aim of this work was to investigate the expression of five mPRs genes and nPRs gene, as well as membrane component of progesterone receptor PGRMC1 in reproductive organs and in 17 non-reproductive tissues of male and female rats by reverse transcription followed by real-time PCR. A high level of nPRs gene expression has been found not only in reproductive organs of female rats (uterus, ovary, mammary glands), but also in seminal vesicles of male rats, in the brain and trachea of both sexes, in blood vessels and in the pancreas of females. The highest level of expression of mPRs genes of all subtypes was in the testes, while expression of the gene encoding nPRs was practically undetectable in them. Expression of genes encoding mPRs was also detected in the liver and spleen of male and female rats, while expression of the gene encoding nPRs was at the background level. No expression of nPRs, mPRs and membrane component of progesterone receptor (PGRMC1) genes was detected in muscle, and its level was very low in heart in animals of both sexes. Nuclear and membrane receptor mRNA expression in non-reproductive tissues in rats has been shown to be sex-dependent. Transcription of nPRs and three subtypes of mPRs (α, β, δ) was predominant in females, and two subtypes of mPRs (γ, ε) were predominant in males. Evidence for the presence of progesterone receptors in tissues not involved in reproduction supports the action of progesterone on these organs. The high mRNA levels of various progesterone receptors in male rat tissues such as pancreas, lung, kidney, and trachea suggest an important physiological role of progestins not only in females but also in males, which is still poorly understood. The paper also discusses the known functions of progesterone receptors in the tissues studied.