Biochemistry (Moscow)最新文献

In this review, we analyze diversity of mitochondrial uncouplers, a class of compounds, which was in the focus of Vladimir Skulachev’s attention throughout his scientific career, starting from the basics of bioenergetics and validation of Mitchell’s chemiosmotic theory to the development of concepts of mild uncoupling and its therapeutic role. The review is the first to put forward the idea of classifying uncouplers by the type of a functional group that provides their protonophoric activity, i.e., the ability to transfer protons across the membrane, causing its depolarization and thereby uncoupling the ATP synthesis from the operation of proton pumps in the electron transport chain. In particular, it is shown that anionic and zwitterionic uncouplers can be divided into groups of OH-, NH-, SH-, and CH-acids. Of importance, here we consider metabolic transformations of mitochondrial uncouplers determining tissue-specificity of their action.

We have earlier begun to investigate the reaction of isolated solubilized dithionite-reduced cytochrome bd-I of Escherichia coli with cyanide [Borisov, V. B., and Arutyunyan, A. M. (2024) The fully reduced terminal oxidase bd-I isolated from Escherichia coli binds cyanide, J. Inorg. Biochem., 259, 112653]. The present work is a continuation of this study. Using absorption and CD spectroscopy, the following new results were obtained: (i) The membrane form of the fully reduced enzyme is also capable of binding cyanide. The apparent dissociation constant and second-order rate constant values are 81.1 ± 7.8 mM KCN and 0.11 ± 0.01 M⁻¹∙s⁻¹, respectively. This contradicts the data of other studies according to which the bd-I oxidase located in native membranes, in the fully reduced state does not bind cyanide. (ii) CO added to the cyano adduct of both membrane and isolated solubilized forms of the fully reduced cytochrome bd-I displaces cyanide, resulting in the formation of the CO/enzyme complex. This indicates the reversibility of cyanide binding to the protein. To saturate the oxidase binding site with CO in the presence of 100 mM KCN, much more CO is required than upon CO addition to the enzyme not pre-treated with cyanide. CO and cyanide compete for the binding to the same site in the oxidase (heme d²⁺). Being a stronger ligand, CO “wins” the competition with cyanide. (iii) The effect of cyanide on the optical activity of dithionite-reduced cytochrome bd-I was studied. The CD spectra of the enzyme obtained before and after cyanide treatment indicate that the formation of the cyano adduct of heme d²⁺ leads to a significant weakening of the excitonic interactions between heme d²⁺ and heme b₅₉₅²⁺. Schemes of the interaction of cyanide and CO in the presence of excess cyanide with the enzyme active site are proposed.

Micropeptides encoded by small open reading frames (sORFs) represent a novel, actively studied class of functional molecules regulating key cellular processes. Studying micropeptides is complicated by methodological challenges, in particular, their small size, low cellular abundance, and difficulty in generating specific antibodies. The review systematizes modern approaches to the identification and functional characterization of micropeptides. The main strategies for their discovery include the use of bioinformatic algorithms, global translation analysis via ribosome profiling, direct detection using mass spectrometry-based proteomics, and phenotypic screenings. The methods for confirming the functions of micropeptides and elucidating molecular mechanisms of their action genetic knockouts, affinity tagging for visualization, and investigation of protein-protein interactions. The review discusses key challenges and future prospects in the field, emphasizing the importance of an integrated multi-omics approach for the comprehensive micropeptidome mapping.

Cysteine cathepsins are a group of closely related proteolytic enzymes active at low pH. The most well-studied function of these enzymes is protein degradation within lysosomes. However, accumulating evidence suggests that cysteine cathepsins also function at physiological pH levels in other cellular compartments outside lysosomes, as well as in the extracellular space. Many of these extra-lysosomal functions of cysteine cathepsins are typically associated with pathological processes, contributing to conditions such as oncogenesis and metastasis, neurodegenerative diseases, cardiovascular disorders, and autoimmune and inflammatory processes. Consequently, cysteine cathepsins have been proposed as diagnostic and prognostic molecular markers, as well as pharmacological targets. Notably, the pathological processes involving these enzymes often operate independently of their classical lysosomal functions. This work aims to outline key questions, the answers to which could enhance our understanding of the fundamental mechanisms governing the extra-lysosomal functions of cysteine cathepsins. Addressing these questions is also critical for developing novel therapeutic strategies to treat diseases in which cysteine cathepsins play a pathogenic role.

In 80-100% of cases, transformation of human somatic cells into tumor cells is associated with the increased expression of the catalytic subunit of telomerase reverse transcriptase (hTERT). The hTERT gene transcription inhibition in tumor cells may become one of the approaches to antitumor therapy. The hTERT promoter contains a G-rich region with length of 68 nucleotides, which is capable of forming G-quadruplexes (G4) under certain conditions in vitro. It is known that G4s interfere with activity of the human RNA polymerases. Thus, the G4 structure stabilization in the promoter could be considered as a possible strategy to reduce hTERT expression. To prove G4 formation in the hTERT promoter G-rich sequence in the double-stranded supercoiling DNA, plasmid constructs based on the pRFPCER plasmid were obtained. The plasmids contained genes of fluorescent proteins (RFP and Cerulean) and sequence of the central G4 in the hTERT promoter region. G4 formation in the central hTERT promoter region in the obtained constructs was demonstrated with the DNA polymerase stop assay. The influence of G228A and G250A substitutions on G4 stability under physiological conditions was investigated. It was established that the low-molecular weight ligands BRACO19 and TMPyP4, the well-studied stabilizers of the G4 structure, can effectively interact with the hTERT promotor central G4 in the range of concentrations 5-25 μM.

Over 20 years of extensive studies on DNA barcoding of various types of multicellular organisms have resulted in the selection of specific markers for multiple taxonomic groups, development of primers for many selected markers, establishment of DNA barcodes for more than 400 thousand species, and creation of the BOLD database. Next-generation sequencing methods allow DNA barcodes to be obtained immediately for many samples, including those stored in museum collections. DNA barcode analysis has revealed many previously unknown and undescribed species in various animal groups. DNA barcoding has been successfully used in many practical applications. However, certain problems and controversial issues remain, primarily, regarding description of new species based on DNA barcodes and the accuracy of sample identification using reference libraries.

Adaptive laboratory evolution (ALE) is aimed at elucidating the molecular basis of adaptation and is widely employed as a tool for gaining deeper insight into genetic and/or metabolic pathways underlying evolutionary processes. One of the primary goals of experimental evolution is to predict mutations representing the key driving forces of adaptation. The use of whole-genome resequencing enables easy identification of mutations that arise during ALE, and consequently, biochemical alterations that occur in the experimental lineages. ALE has also proven highly relevant in practical applications, as it provides an innovative approach to the construction of evolved microbial strains with desirable performance, such as rapid growth, stress resistance, efficient utilization of diverse substrates, and production of compounds with a high added value (amino acids, ethanol, aromatic compounds, lipids, etc.). In this review, we analyzed the results of studies focused on the demonstration and explanation of relationships between mutations and resulting phenotypic and biochemical changes, as well as discussed a potential of microorganisms as model systems for ALE experiments and testing of various evolutionary hypotheses. We also described achievements reached by using ALE strategies, as well as the still unresolved issues and methodological limitations of this approach.

A rising global prevalence of psychoneurological and neurodegenerative disorders emphasizes the critical need for effective therapeutics and methods for early and highly sensitive diagnostics in order to ensure efficient and timely treatment of these disorders. Expanding the range of available biomarkers for better characterization of disease features and progression is a promising direction in modern diagnostics. The discovery of novel biomarkers depends on elucidating molecular mechanisms underlying disease development and pathogenesis. Numerous psychoneurological and neurodegenerative disorders are associated with the dysregulation of protein translation. The review summarizes information on the action mechanisms of translation factors DENR and eIF2D and evaluates their potential as diagnostic biomarkers for psychoneurological and neurodegenerative diseases.

The development of personalized medicine, including the treatment of hereditary diseases, requires translation of advances in biochemistry into medical practice. Our work is dedicated to solving this problem in a clinical case of hereditary Charcot–Marie–Tooth neuropathy type 2K (CMT2K), induced by the compound heterozygous mutations in the GDAP1 gene leading to the protein variants with the most common in Europe substitution L239F (inherited from the father) and previously uncharacterized substitution A175P (inherited from the mother). The ganglioside-induced differentiation-associated protein 1 (GDAP1) encoded by the GDAP1 gene is located in the outer mitochondrial membrane and belongs to the glutathione S-transferase superfamily. Our structure-function analysis of GDAP1 shows that dimerization of its monomers with either L239F or A175P substitutions, along with the half-of-the-sites reactivity of GDAP1 to hydrophobic ligands, may synergistically impair the binding due to the double amino acid substitution in one of the active sites. This mechanism explains the early disease onset and progress in the child, whose parents heterozygous by each of the mutations are asymptomatic. Published phenotypes of amino acid substitutions in the GDAP1 region comprising the binding site for hydrophobic compounds are analyzed, including phenotypes of the homozygous L239F substitution and its compound heterozygous combinations with other substitutions in this region. Based on the found association of these substitutions with the axonal form of Charcot–Marie–Tooth disease (CMT) and disturbances in the NAD⁺- and thiamine diphosphate (ThDP)-dependent mitochondrial metabolism, the therapeutic effect of nicotinamide riboside (NR) and thiamine (precursors of NAD⁺ and ThDP, respectively) in the patient is studied. Oral administration of thiamine and NR increases levels of ThDP and NAD⁺ in the patient’s blood, improves the hand grip strength, and, after a long-term administration, normalizes the ThDP-dependent metabolism. After the therapy, the diseased-altered activities of transketolase (TKT) and its apo-form, as well as the relationship between the activity of the TKT holoenzyme and ThDP and NAD⁺ levels in the patient’s blood, approach those of healthy women. Our results demonstrate the therapeutic potential of thiamine and NR in correcting metabolic dysregulation in CMT caused by mutations in GDAP1, suggesting the underlying molecular mechanisms. Genetic diagnostics and biochemical characterization of mechanisms involved in the pathogenicity of mutations in clinically asymptomatic patients or patients at the early CMT stages may increase the efficacy of therapy, as it is easier to protect from the accumulating metabolic damage than to reverse it.

Mitochondrial genomes of most animals contain the same set of genes, with all or many protein-coding genes (PCGs) arranged in the same order, forming conserved blocks termed syntenies. Some syntenies have been preserved for hundreds of millions of years and are found in both vertebrates and invertebrates. This evolutionary conservation indicates functional role for PCG arrangement; however, biochemical and/or physiological mechanisms by which the gene order in mtDNA affects viability are unknown. Among animals, there are taxa that have completely lost conserved syntenies in mtDNA. Canonical animal syntenies in mtDNA have not been reported in nematodes, until some were recently discovered in the previously unstudied nematode taxa, including the marine family Thoracostomopsidae (Nematoda, Enoplida). We sequenced the complete mitochondrial genomes of three thoracostomopsid species, determined gene order, and their expression levels from the RNA-seq data for all available family representatives. We found that six species of the Thoracostomopsidae there are three distinct patterns of PCG arrangement, and the relative mRNA levels correlate with the gene order rather than species phylogeny. We hypothesize that the influence of PCG translocations on their expression levels underlies the long-term preservation of mitochondrial syntenies among animals.