Vol 88, No 5 (2023)
Articles
Glucocorticoids orchestrate adult hippocampal plasticity: growing points and translational aspects
Abstract
The review analyzes modern concepts regarding the control of various mechanisms of the hippocampal neuroplasticity in adult mammals and humans by glucocorticoids. Glucocorticoid hormones ensure the coordinated functioning of key components and mechanisms of hippocampal plasticity: neurogenesis, glutamatergic neurotransmission, microglia and astrocytes, systems of neurotrophic factors, neuroinflammation, protease activities, metabolic hormones, neurosteroids. Regulatory mechanisms are diverse; along with the direct action of glucocorticoids through their receptors, there are indirect glucocorticoid-dependent effects, as well as numerous interactions between various systems and components. Despite the fact that many connections in this complex regulatory scheme have not yet been established, the study of the factors and mechanisms considered in the review forms growth points in the field of glucocorticoid-regulated processes in the brain and primarily in the hippocampus. These studies are fundamentally important for the translation into the clinic and the potential treatment/prevention of common diseases of the emotional and cognitive spheres and respective comorbid conditions.
Biochemistry. 2023;88(5):693-724
693-724
Recombinant human cyclophilin A in combination with adoptive T cell therapy improves the efficacy of cancer immunotherapy in experimental models in vivot;
Abstract
Adoptive T-cell therapy (ACT) is successfully applied in cancer treatment, however, its efficiency can be limited by a low viability, short persistence time, and loss of functional activity of T cells after adoptive transfer. The search for novel immunomodulators that can improve the viability, expansion, and functions of T cells after their infusion with the minimal side effects could contribute to the development of more efficient and safe ACT strategies. Recombinant human cyclophilin A (rhCypA) is of particular interest in this respect, as it exhibits pleiotropic immunomodulatory activity and stimulates both innate and adaptive anti-tumor immunity. Here, we evaluated the effect of rhCypA on the efficacy of ACT in the mouse EL4 lymphoma model. Lymphocytes from transgenic 1D1a mice with an inborn pool of EL4-specific T cells were used as a source of tumor-specific T cells for ACT. In models of immunocompetent and immunodeficient transgenic mice, the course (3 days) rhCypA administration was shown to significantly stimulate EL4 rejection and prolong the overall survival of tumor-bearing mice after adoptive transfer of lowered doses of transgenic 1D1a cells. Our studies showed that rhCypA significantly improved the efficacy of ACT by enhancing the effector functions of tumor-specific cytotoxic T cells. These findings open up the prospects for the development of innovative strategies of adoptive T-cell immunotherapy for cancer using rhCypA as an alternative to existing cytokine therapies.
Biochemistry. 2023;88(5):725-736
725-736
О-Аcetylhomoserine sulfhydrylase from Clostridioides difficile: the role of tyrosine residues of the active center
Abstract
O-acetylhomoserine sulfhydrylase is one of the key enzymes in the biosynthesis of methionine in Clostridioides difficile. The mechanism of the γ-substitution reaction of O-acetyl-L-homoserine catalyzed by this enzyme is the least studied among pyridoxal-5′-phosphate-dependent enzymes involved in the metabolism of cysteine and methionine. To clarify the role of the active site residues Tyr52 and Tyr107, four mutant forms of the enzyme with replacements for phenylalanine and alanine were obtained. The catalytic and spectral properties of mutant forms were investigated. The rate of the γ-substitution reaction catalyzed by mutant forms with the replacement of the Tyr52 residue decreased by more than three orders of magnitude compared to the wild-type enzyme. Tyr107Phe and Tyr107Ala mutant forms practically did not catalyze this reaction. Replacements of the residues Tyr52 and Tyr107 led to a decrease in the affinity of the apoenzyme to the coenzyme by three orders of magnitude and changes in the ionic state of the internal aldimine of the enzyme. The obtained results allowed us to assume that Tyr52 is involved in ensuring the optimal position of the catalytic coenzyme-binding lysine residue at the stages of C-α-proton elimination and elimination of the side group of the substrate. Tyr107 can act as a general acid catalyst at the stage of acetate elimination.
Biochemistry. 2023;88(5):737-747
737-747
Effect of neurodegenerative mutations in NEFL gene on thermal denaturation of the neurofilament light chain protein
Abstract
The effects of amino acid substitutions E90K, N98S and A149V in the light chain of neurofilaments (NFL) on the structure and thermal denaturation of the NFL molecule was investigated. By using the circular dichroism spectroscopy, it was shown that these substitutions do not lead to a changes in the α-helical structure of NFL, but they caused a noticeable effects on the stability of the molecule. We also identified calorimetric domains in the NFL structure by using the differential scanning calorimetry. It was shown that the E90K replacement lead to the disappearance of the low-temperature thermal transition (domain 1). The mutations lead to changes in the enthalpy of melting of NFL domains, as well as lead to significant changes in the melting temperatures (Tm) of some calorimetric domains. Thus, despite the fact that all these amino acid substitutions are associated with the development of Charcot-Marie-Tooth neuropathy, and two of them are even located very close to each other in the coiled-coil domain 1A, they differently effects on the structure and stability of the NFL molecule.
Biochemistry. 2023;88(5):748-760
748-760
Blood oxylipin profiles as a marker of the pathogenesis of oncological diseases
Abstract
Oxylipins are signal lipids derived from polyunsaturated fatty acids (PUFAs), which are formed by polyenzymatic multi-acid pathways of danger (cyclooxygenase, lipoxygenase, epoxygenase, anandamide), as well as non-enzymatically. These PUFA transformation pathways are activated in parallel, forming a mixture of active inclusions. Although the combination of oxylipins with tumor secretions was performed a long time ago, the methods of instrumental analysis have only recently been improved, measurements of oxylipins of different classes (profiles) have been compared. The review considers approaches to obtaining the profile of oxylipins by HPLC-MS/MS. The profiles of oxylipins in samples of patients with oncological diseases (breast cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer) were compared. The conclusion is made about the possible possibilities of using the profile of blood oxylipins as markers of oncological diseases. Understanding the high level of exceeding the level of PUFAs and the physiological activity of oxylipin mixtures will be accompanied by the perfection of early diagnosis and prediction of the course of tumor diseases.
Biochemistry. 2023;88(5):761-772
761-772
Prediction of adverse effects of drug-drug interactions on the cardiovascular system based on the analysis of structure-activity relationships
Abstract
The co-administration of drugs often leads to drug-drug interactions, which may be accompanied by various adverse drug reactions that pose a threat to the life and health of patients. The effect caused by adverse drug reactions from the cardiovascular system is one of the most significant manifestations of drug-drug interaction. Clinical assessment of adverse drug reactions resulting from drug-drug interaction between all drug pairs used in therapeutic practice is not possible. The purpose of this work was to build models using structure-activity analysis to predict the effects of adverse drug reactions on the cardiovascular system, mediated by interactions between drug pairs when they are taken together. Data on adverse effects resulting from drug-drug interaction were obtained from the DrugBank database. The data on drug pairs that do not cause the corresponding effects, necessary for building accurate structure-activity models, were obtained from the TwoSides database, which contains the results of the analysis of spontaneous reports. Two types of descriptors were used to describe a pair of drug structures: PoSMNA descriptors and probabilistic estimates of the prediction of biological activities obtained using the PASS program. Structure-activity relationships were established using the Random Forest method. Prediction accuracy was calculated by means of a five-fold cross-validation. The highest accuracy values were obtained using PASS probabilistic estimates as descriptors. The area under the ROC curve was 0.94 for bradycardia, 0.96 for tachycardia, 0.90 for arrhythmia, 0.90 for ECG QT prolongation, 0.91 for hypertension, 0.89 for hypotension.
Biochemistry. 2023;88(5):773-784
773-784
Nucleoid-associated proteins HU and IHF: oligomerization in solution and hydrodynamic properties
Abstract
The structure and function of the bacterial nucleoid is controlled by nucleoid-associated NAP proteins. In any phase of growth, various NAPs, acting sequentially, condense the nucleoid and provide its transcriptionally active structure. However, in the late stationary phase, only one of the NAPs, the Dps protein, is strongly expressed, and DNA-protein crystals are formed that transform the nucleoid into a static, transcriptionally inactive structure, effectively protected from external influences. The discovery of crystal structures in living cells and the association of this phenomenon with bacterial resistance to antibiotics has aroused great interest in studying this phenomenon. The aim of this work is to obtain and compare the structures of two related NAPs (HU and IHF), since they are the ones that accumulate in the cell at the late stationary stage of growth, which precedes the formation of the protective DNA-Dps crystalline complex. For structural studies, two complementary methods were used in the work: small-angle X-ray scattering (SAXS) as the main method for studying the structure of proteins in solution and dynamic light scattering as an additional one. Various approaches and computer programs were used to interpret the SAXS data, which made it possible to determine the macromolecular characteristics and obtain reliable structural 3D models of various oligomeric forms of the HU and IHF proteins. It was shown that these proteins oligomerize in solution to varying degrees, and IHF is characterized by the presence of large oligomers consisting of initial dimers arranged in a chain. It was suggested that just before Dps expression, it is this protein that forms the toroidal structures previously observed in vivo and prepares the platform for the formation of DNA-Dps crystals. The results obtained are necessary for further study of the phenomenon of biocrystal formation in bacterial cells and finding ways to overcome the resistance of various pathogens to external conditions.
Biochemistry. 2023;88(5):785-802
785-802
Biochemical features of X or Y chromosome bearing spermatozoa for sperm sexing
Abstract
This review presents information on the biochemical features of spermatozoa bearing an X or Y chromosome, enabling to obtain a sperm fraction with a pre-defined sex chromosome. Almost the only technology currently used for such separation (called sexing) is based on sperm fluorescence-activated cell sorting in regard to DNA content. Besides its applications, this technology made it possible to analyze the properties of isolated populations of spermatozoa bearing an X or Y chromosome. In recent years, a number of works have appeared proving the existence of differences between these populations at the transcriptome and proteome level. It is noteworthy that these differences are primarily related to energy metabolism and structural proteins of the flagellum. New methods of sperm enrichment with X or Y chromosome cells are based on the differences in motility between spermatozoa with different sex chromosomes. Sperm sexing is a widespread part of the protocol of artificial insemination of cows with cryopreserved semen, it allows to increase the proportion of offspring of the required sex. In addition, advances in the separation of X and Y spermatozoa may allow this approach to be applied in clinical practice to avoid sex-linked diseases.
Biochemistry. 2023;88(5):803-816
803-816
Glutaredoxin 1 from evolutionary ancient Hydra: characteristics of the enzyme and its possible functions in cell
Abstract
Glutaredoxin (Grx) is an antioxidant redox protein that uses glutathione (GSH) as an electron donor. Grx plays a crucial role in various cellular processes, such as antioxidant defense, control of cellular redox state, redox control of transcription, reversible S-glutathionylation of specific proteins, apoptosis, cell differentiation, etc. In the current study, we have isolated and characterized dithiol glutaredoxin from Hydra vulgaris Ind-Pune (HvGrx1). Sequence analysis showed that HvGrx1 belongs to the Grx family with the classical Grx motif (CPYC). Phylogenetic analysis and homology modeling revealed that HvGrx1 is closely related to Grx2 from zebrafish. HvGrx1 gene was cloned and expressed in Escherichia coli cells; the purified protein had a molecular weight of 11.82 kDa. HvGrx1 efficiently reduced β-hydroxyethyl disulfide (HED) with the temperature optimum of 25°C and pH optimum 8.0. HvGrx1 was ubiquitously expressed in all body parts of Hydra. Expression of HvGrx1 mRNA and enzymatic activity of HvGrx1 were significantly upregulated post H2O2 treatment. When expressed in human cells, HvGrx1 protected the cells from oxidative stress and enhanced cell proliferation and migration. Although Hydra is a simple invertebrate, HvGrx1 is evolutionary closer to its homologs from higher vertebrates (similar to many other Hydra proteins).
Biochemistry. 2023;88(5):817-831
817-831
Detection of specific RNA targets by multimerization
Abstract
Detection of specific RNA targets via amplification-mediated techniques is widely used in fundamental studies and medicine due to an essential role of RNA in realization of genetic information and diseases development. Here, we report on an approach for detection of RNA targets based on a particular type of isothermal amplification, namely, reaction of nucleic acid multimerization. The proposed technique requires only a single DNA polymerase possessing reverse transcriptase, DNA-dependent DNA polymerase and strand-displacement activities. Reaction conditions that lead to efficient detection of the target RNAs through multimerization mechanism were determined. The approach was approved using genetic material of SARS-CoV-2 coronavirus as a model viral RNA. Reaction of multimerization allowed to differentiate SARS-CoV-2 RNA-positive samples from SARS-CoV-2 negative samples with high reliability. The proposed technique determines detection of RNA even in samples, which undergone multiple freezing.
Biochemistry. 2023;88(5):832-840
832-840
Mechanism of D-cycloserine inhibition of D-amino acid transaminase from Haliscomenobacter hydrossis
Abstract
D-cycloserine inhibits pyridoxal-5′-phosphate (PLP)-dependent enzymes. The inhibition efficiency depends on the organization of their active center and the mechanism of the catalyzed reaction. D-cycloserine interacts with the PLP form of enzyme similarly to substrate amino donor, and the interaction is predominantly reversible. Inhibition products include hydroxyisoxazole-pyridoxamine-5′-phosphate, oxime between PLP and β-aminooxy-D-alanine, ketimine between pyridoxamine-5′-phosphate and cyclic or open forms of D-cycloserine, pyridoxamine-5′-phosphate, etc. For some enzymes the formation of a stable aromatic product - hydroxyisoxazole can lead to irreversible D-cycloserine inhibition at certain pH value. The aim of this work was to study the mechanism of D-cycloserine inhibition of PLP-dependent D-amino acid transaminase from the bacterium Haliscomenobacter hydrossis. Spectral methods revealed several products of the interaction of D-cycloserine with PLP in the active site of transaminase: oxime between PLP and β-aminooxy-D-alanine, ketimine between pyridoxamine-5′-phosphate and cyclic or open forms of D-cycloserine, pyridoxamine-5′-phosphate. The formation of hydroxyisoxazole-pyridoxamine-5′-phosphate was not observed. The 3D structure of the complex of transaminase with D-cycloserine was obtained by X-ray diffraction analysis. In the active site of transaminase, a ketimine adduct between pyridoxamine-5′-phosphate and D-cycloserine in the cyclic form was found; the ketimine occupied two positions and was coordinated via hydrogen bonds with different active site residues. Using kinetic and spectral methods we have shown that D-cycloserine inhibition is reversible, and the activity of transaminase from H. hydrossis can be restored by adding an excess of keto substrate as well as by adding an excess of cofactor. The results obtained confirm the reversibility of D-cycloserine inhibition and the conversion of various adducts of D-cycloserine and PLP into each other.
Biochemistry. 2023;88(5):841-853
841-853
Adsorption of acylhydroperoxy-derived phospholipids from biomembranes by blood plasma lipoproteins
Abstract
It has been established that acylhydroperoxy derivatives of phospholipids of oxidized rat liver mitochondria during co-incubation with blood plasma lipoproteins are captured predominantly by LDL particles but not by HDL, which refutes the previously stated hypothesis about the involvement of HDL in the reverse transport of oxidized phospholipids and confirms the possibility of different mechanisms of lipohydroperoxide accumulation in LDL during oxidative stress.
Biochemistry. 2023;88(5):854-860
854-860
Femtosecond exciton relaxation in chlorosomes of the photosynthetic green bacterium Chloroflexus aurantiacus
Abstract
In the green bacteria Chloroflexus (Cfx.) aurantiacus, the process of photosynthesis begins with the absorption of light by chlorosomes, peripheral antennas consisting of thousands of bacteriochlorophyll c (BChl c) molecules combined into oligomeric structures. In this case, excited states are formed in BChl c, the energy of which migrates along the chlorosome towards the baseplate and further to the reaction center, where the primary charge separation occurs. Energy migration is accompanied by nonradiative electronic transitions between numerous exciton states, that is exciton relaxation. In this work, we studied the dynamics of exciton relaxation in Cfx. aurantiacus chlorosomes using difference femtosecond spectroscopy at cryogenic temperature (80 K). Chlorosomes were excited by 20 fs light pulses at wavelengths from 660 to 750 nm, and the difference (light-dark) kinetics were measured at a wavelength of 755 nm. Mathematical analysis of the data obtained revealed the kinetic components with characteristic time constants of 140, 220, and 320 fs responsible for the exciton relaxation. As the excitation wavelength decreased, the number of components and their relative contribution increased. Theoretical modeling of the results obtained was carried out on the base of the cylindrical model of BChl c aggregates. Nonradiative transitions between groups of exciton bands were ascribed by the system of kinetic equations. The model in which the energetic and structural disorder was taken into account turned out to be the most adequate.
Biochemistry. 2023;88(5):861-874
861-874
Display of oligo-α-1,6-glycosidase from Exiguobacterium sibiricum at the surface of Escherichia coli cells
Abstract
Cell-surface display based on the use of anchor motifs of outer membrane proteins allows the exposure of target peptides and proteins on the surface of microbial cells. Previously, we obtained and characterized recombinant oligo-α-1,6-glycosidase of the psychrotrophic bacterium Exiguobacterium sibiricum (EsOgl) which demonstrated high catalytic activity. It has also been shown that the autotransporter AT877 from Psychrobacter cryohalolentis and its deletion variants effectively exhibit the 10th domain of type III fibronectin (10Fn3) on the surface of Escherichia coli cells. The aim of the work was to obtain an EsOgl display system on the surface of bacterial cells based on AT877. The genes of the hybrid autotransporter EsOgl877 and its deletion mutants EsOgl877Δ239 and EsOgl877Δ310 were constructed. The enzymatic activity of EsOgl877 was investigated and it was found that the cells expressing this protein retained about 90% of the maximum activity in the range of 15-35°C. It was shown that activity of the cells containing EsOgl877Δ239 and EsOgl877Δ310 was 2.7 and 2.4 times higher, respectively, than of the cells expressing full-sized AT. Analysis of cells expressing shortened variants of EsOgl877 after treatment with proteinase K showed that the passenger domain is also localized on the cell surface. The obtained results can be used for optimization of the display systems of oligo-α-1,6-glycosidase and other heterologous proteins on the surface of E. coli cells.
Biochemistry. 2023;88(5):875-883
875-883