Biochemistry (Moscow)最新文献

Inosine Monophosphate Dehydrogenase (IMPDH) catalyzes rate-limiting step of the reaction converting inosine monophosphate (IMP) to guanine nucleotides. IMPDH is up-regulated in the healthy proliferating cells and also in tumor cells to meet their elevated demand for guanine nucleotides. An exclusive regulatory mechanism for this enzyme is filamentation, through which IMPDH can resist allosteric inhibition by the end product, GTP. It has been proven that intracellular IMP, which rises during the proliferative state, potentially promotes IMPDH filamentation. On the other hand, interaction of IMPDH with ADP-ribosylation factor-like protein 13B (ARL13B) directs guanine biosynthesis toward the de novo pathway. However, ARL13B is not localized in the IMPDH-based cytoophidia, super structures composed of bundled IMPDH filaments and other proteins. Here, we hypothesized that ARL13B could increase availability of the de novo-produced IMP for IMPDH by interacting with the IMP-free IMPDH and microtubules adjacent to the purinosome. Following IMP-binding, IMPDH would be released from ARL13B and preferentially associated with its cytoophidia. Considering clinical side effects of catalytic inhibitors of IMPDH and their ability to induce IMPDH cytoophidia, we suggest that combination of proper doses of IMPDH catalytic inhibitors and inhibitors of the de novo IMP biosynthesis could be more effective in controlling cell proliferation.

Identification and analysis of repetitive elements (motifs) in DNA, RNA, and protein macromolecules is an important step in studying structure and functions of these biopolymers. Functional role of NA-BSE (non-adjacent base-stacking element, a widespread tertiary structure motif in various RNAs) in RNA–RNA interactions at various stages of the ribosome function during translation has been investigated in this work. Motifs of this type have been described to date that are reversibly formed during mRNA decoding, moving of the ribosome subunits relative to each other, and moving mRNA and tRNA along the ribosome during translocation. The EF-G-dependent NA-BSE formation involving 5S rRNA and 23S rRNA nucleotide residues is considered separately.

Hypoxia poses a serious challenge for all animals; however, certain animals exhibit a remarkable resilience in the case of prolonged and severe hypoxia. The Siberian wood frog Rana amurensis is a unique amphibian capable of surviving for up to several months at almost complete anoxia. We investigated changes in the metabolome of R. amurensis at the onset of hypoxia (day 1) and within 1 h of reoxygenation after a long-term hypoxia using ¹H NMR. We compared our results to the data obtained for animals exposed to 17 days of hypoxia and controls. Despite the differences between the samples analyzed in three different experimental series, we were able to obtain some interesting insights. In most studied vertebrates, succinate accumulates under hypoxic conditions and undergoes rapid conversion upon reoxygenation. We found that reoxygenation caused a decrease in the succinate content in the brain, but not in the liver, where it remained unchanged, suggesting an existence of a mechanism that inhibits succinate conversion. Furthermore, we observed intriguing differences in the behavior of two substances with unknown functions: glycerol and 2,3-butanediol. Glycerol exhibited rapid accumulation during hypoxia and equally rapid processing during reoxygenation. In contrast, 2,3-butanediol required an extended period of time to accumulate, yet persisted after reoxygenation. Overall, our data demonstrate rapid accumulation of most substances during exposure to hypoxia followed by their slower processing upon reoxygenation.

Nuclear export protein (NEP) of the influenza A virus, being one of the key components of the virus life cycle, is a promising model for studying characteristics of formation of amyloids by viral proteins. Using atomic force microscopy, comparative study of aggregation properties of the recombinant NEP variants, including the protein of natural structure, as well as modified variants with N- and C-terminal affinity His₆-tags, was carried out. All protein variants under physiological conditions are capable of forming aggregates of various morphologies: micelle-like nanoparticles, flexible protofibrils, rigid amyloid fibrils, etc. His₆-tag attached to the C-terminus has the greatest effect on aggregation kinetics and morphology of nanoparticles, which indicates important role of the C-terminal domain in the process of protein self-assembly. Molecular dynamics simulation did not reveal substantial influence of the His₆-containing fragments on the protein structure, but demonstrated some variations in the mobility of these fragments that may explain the observed differences in the aggregation kinetics of the different NEP variants. Hypothetical mechanisms for formation and interconversion of various aggregates are considered.

Food safety is one of the primary demands of modern society. Mycotoxins are toxic metabolites of food-contaminating fungi. Fungi enter the food chain by infecting crops and irreversibly contaminate them due to the structural stability of mycotoxins. Mycotoxins are stable even at extremely high temperatures; they do not lose their activity during food processing, thus posing a threat to human health. Therefore, it is crucial to detect mycotoxins in food crops during the planting process and at the beginning of the harvest, which requires a rapid and simple detection method. One of the current solutions for this problem is aptamer-based sensors. Here, we deciphered the structure of the binding site in the developed DNA aptamer against deoxynivalenol. The binding site is formed by short single-stranded sequences at the 5′- and 3′-ends of the hairpin, with the Cyanine 3 label at 3′-end. The shortest aptamer with the affinity for deoxynivalenol was used as a recognition element in the surface-enhanced Raman spectroscopy-based sensor to detect mycotoxins in wheat crops.

The auxin-inducible degron (AID) system is widely used to study function of various proteins. The plant hormone auxin is used as an inducer in this system, which easily penetrates into the cells and causes proteasomal degradation of the protein of interest fused to a small degron tag. It is often assumed that as a plant hormone, auxin does not significantly affect physiology of animal cells. In order to test how auxin affects gene expression in human and mouse cells, we collected a set of published data on the levels of gene expression in various experiments with the auxin degradation system of various proteins. The analysis showed that in human HCT116, DLD1, and HAP1 cell lines, as well as in mouse embryonic stem cell lines, auxin treatment leads to activation of aryl hydrocarbon receptor (AHR)-related genes. However, activation of AHR pathway genes does not occur upon auxin treatment in the human IMR32 cells and mouse G1E-ER4 cells, which are characterized by low AHR gene expression. To verify this observation, we conducted an experiment treating human U87, A549, and HCT116 cells with auxin and demonstrated activation of one of the main AHR pathway responders, the CYP1B1 gene. We believe that activation of the AHR pathway should be taken into account by those using the auxin degradation system in their studies.

Skeletal muscles account for ~30-40% of the total weight of human body and are responsible for its most important functions, including movement, respiration, thermogenesis, and glucose and protein metabolism. Skeletal muscle damage negatively impacts the whole-body functioning, leading to deterioration of the quality of life and, in severe cases, death. Therefore, timely diagnosis and therapy for skeletal muscle dysfunction are important goals of modern medicine. In this review, we focused on the skeletal troponins that are proteins in the thin filaments of muscle fibers. Skeletal troponins play a key role in regulation of muscle contraction. Biochemical properties of these proteins and their use as biomarkers of skeletal muscle damage are described in this review. One of the most convenient and sensitive methods of protein biomarker measurement in biological liquids is immunochemical analysis; hence, we examined the factors that influence immunochemical detection of skeletal troponins and should be taken into account when developing diagnostic test systems. Also, we reviewed the available data on the skeletal troponin mutations that are considered to be associated with pathologies leading to the development of diseases and discussed utilization of troponins as drug targets for treatment of the skeletal muscle disorders.

Bacterial and viral RNA polymerases are promising targets for the development of new transcription inhibitors. One of the potential blockers of RNA synthesis is 7,8-dihydro-8-oxo-1,N⁶-ethenoadenine (oxo-εA), a synthetic compound that combines two adenine modifications: 8-oxoadenine and 1,N⁶-ethenoadenine. In this study, we synthesized oxo-εA triphosphate (oxo-εATP) and showed that it could be incorporated by the RNA-dependent RNA polymerase of SARS-CoV-2 into synthesized RNA opposite template residues A and G in the presence of Mn²⁺ ions. Escherichia coli RNA polymerase incorporated oxo-εATP opposite A residues in the template DNA strand. The presence of oxo-εA instead of adenine in the template DNA strand completely stopped transcription at the modified nucleotide. At the same time, oxo-εATP did not suppress RNA synthesis by both RNA polymerases in the presence of unmodified nucleotides. Therefore, the oxo-εA modification significantly disrupts nucleotide base pairing during RNA synthesis by RNA polymerases of different classes, and the corresponding nucleotide derivatives cannot be used as potential antiviral or antibacterial transcription inhibitors.

Integration of various types of omics data is an important trend in contemporary molecular oncology. In this regard, high-throughput analysis of trace and essential elements in cancer biosamples is an emerging field that has not yet been sufficiently addressed. For the first time, we simultaneously obtained gene expression profiles (RNA sequencing) and essential and trace element profiles (inductively coupled plasma mass spectrometry) for a set of human cancer samples. The biosamples were formalin-fixed, paraffin-embedded primary tumor tissue blocks: 67 for colorectal cancer patients and 18 for other solid cancer types (16 types). Mass spectrometry profiles were obtained for 45 chemical elements: Ag, Al, As, Au, B, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, Ga, Ge, Hg, I, K, La, Li, Mg, Mn, Mo, Na, Ni, P, Pb, Pd, Pt, Rb, Sb, Sc, Se, Si, Sn, Sr, Te, Ti, Tl, Zn, U, V, W, and Zr. The expression levels were profiled for 36,596 known human genes, and the activation levels were assessed for 10,520 human intracellular molecular pathways. For the concentrations of essential elements Ca, Cu, Fe, K, Mg, Na, P, and Zn we detected statistically significant correlations on both gene expression and pathway activation levels for both colorectal cancer samples and at the pan-cancer level. In total, 222/137, 122/220, 1/0, 239/186, 71/44, 1/0, 354/294, 69/82 gene/pathway biomarkers were detected for Ca, Cu, Fe, K, Mg, Na, P, and Zn, respectively. We believe that this first-in-class database provided here will be valuable for multiomics cancer research.

Human carbonic anhydrase IX (CAIX) plays a key role in maintaining pH homeostasis of malignant neoplasms, thus creating a favorable microenvironment for the growth, invasion, and metastasis of tumor cells. Recent studies have established that inhibition of CAIX expressed on the surface of tumor cells significantly increases the efficacy of classical chemotherapeutic agents and makes it possible to suppress the resistance of tumor cells to chemotherapy, as well as to increase their sensitivity to drugs (in particular, to reduce the required dose of cytostatic agents). In this work, we studied the ability of new CAIX inhibitors based on substituted 1,2,4-oxadiazole-containing primary aromatic sulfonamides, to potentiate the cytostatic effect of gefitinib (selective inhibitor of epidermal growth factor receptor tyrosine kinase domain) under hypoxic conditions. We investigated a combined effect of gefitinib and CAIX inhibitors 4-(3-phenyl-1,2,4-oxadiazol-5-yl)thiophene-2-sulfonamide (1), 4-(5-(thiophene-3-yl)-1,2,4-oxadiazol-3-yl)benzenesulfonamide (2), 4-(3-(pyridin-2-yl)-1,2,4-oxadiazol-5-yl)thiophene-2-sulfonamide (3), and 4-(5-methyl-1,2,4-oxadiazol-3-yl)benzenesulfonamide (4) on gefitinib cytotoxicity, cell proliferation, activation of caspases-3/7, and cell cycle control in human lung adenocarcinoma A549 cells. It was found that the combinations of compounds 1 and 2 with gefitinib suppressed the invasive potential of A549 cells. Compound 1 had the greatest effect and can be considered as a promising candidate for further research.