Biochemistry (Moscow)最新文献

L36 is a structural protein of the large ribosomal subunit of bacterial, mitochondrial, and chloroplast ribosomes. L36 stabilizes the peptidyl transferase center and the L7/L12 stalk, which is a binding site for the elongation factors during the translation cycle. According to the cryoelectron microscopy data, L36 incorporates into the large ribosomal subunit in both bacteria and mitochondria at the final assembly step. Bacterial L36 is not an essential protein, since deletion of its gene in bacteria did not impair the colony growth or reduce the mRNA translation levels. Deletion of the RTC6 gene coding for the mitochondrial homologue of L36 (bL36m) in Saccharomyces cerevisiae, impeded yeast growth on the media with non-fermentable carbon sources. Our findings indicate that the mitochondrial dysfunction associated with the absence of bL36m was caused by a decreased activity of cytochrome c oxidase complex that resulted from the selective disruption of synthesis of its subunits encoded in the mitochondrial genome. Furthermore, selective inhibition of mitochondrial protein synthesis did not induce critical structural abnormalities of mitochondrial ribosomes or reduce their ability to bind mRNA. Furthermore, we demonstrated that in contrast to S. cerevisiae, the absence of bL36m protein in human cells had no substantial impact on the synthesis of mitochondrially encoded proteins or mitochondrial ribosome assembly. However, the observed reduction in the mitochondrial respiration in the bL36m-deficient cells may be indicative of disturbances in the respiratory chain organization not associated with the mitochondrial translation.

Early preclinical diagnostics of neurodegenerative diseases (synucleinopathies, Alzheimer’s disease, etc.) can be achieved through the detection of pathological amyloid aggregates in biological fluids. Protein amplification methods (PMCA, RT-QuIC) have become promising diagnostic tools by offering high sensitivity and specificity for detecting α-synuclein oligomers at the early disease stages and studying the fibril formation kinetics and mechanisms of amyloid aggregation. However, these methods have several significant limitations, as they are technically complex and time-consuming and lack standardized protocols, control samples, and substrates. Despite these challenges, protein amplification methods hold potential as the most productive, accessible, and standardizable diagnostic approaches in synucleinopathies. This review presents the results of bibliometric analysis of publications on the use of protein amplification methods in the diagnostics of synucleinopathies. We also provide a comparative analysis of common RT-QuIC protocols, with special focus on the method principles, approaches for its optimization, types of biological materials, key factors influencing the sensitivity and specificity of this technique, and areas for its improvement.

This review presents the data on structural diversity of rhamnans in the cell walls and S-layers of Gram-positive (monoderm) bacteria – representatives of the phyla Bacillota (“Firmicutes”) and Actinomycetota (“Actinobacteria”). Diversity of the rhamnan structures is determined by configuration and alternation of the (1→2)- and (1→3)-glycosidic bonds in the polymer core, as well as by the presence and composition of side glycosyl and other substituents. Structural features of rhamnans in several bacterial groups are noted: presence of single glycerol phosphate units in the side chains of polymers in Streptococcus, Lactococcus, and Ruminococcus (Mediterraneibacter); presence of rhamnans with l- and d-isomers of rhamnose in actinobacteria; and complexes of rhamnans and teichoic acids or glycosyl 1-phosphate polymers (two-component heteropolysaccharides) in Enterococcus and Lactobacillus. The review provides information on the structural features of rhamnans underlying serological classification of streptococci. The role of rhamnose and rhamnans in bacterial cell physiology (cell wall biogenesis and cell division, viability, adaptation to adverse conditions, antibiotic resistance), phage reception, adhesion, biofilm formation, and in the “bacterial–host” interaction (induction of inflammatory processes in tissues, immune system activation, and virulence manifestation) is discussed.

Filamin C is an adapter protein involved in the regulation of cytoskeleton; it interacts with more than 90 protein partners, including small heat shock proteins (sHsps). However, the details of filamin C interaction with sHsps remain poorly characterized. Here, we used immunochemistry methods, size-exclusion chromatography, native gel electrophoresis, and chemical crosslinking to investigate the interactions of a long C-terminal fragment of filamin C containing immunoglobulin (Ig)-like domains 19-24 (FLNC19-24) with sHsps. Out of five analyzed sHsps (HspB1, phosphorylation-mimicking 3D mutant of HspB1, HspB5, HspB6, HspB7, and HspB8), only HspB7 formed complexes with FLNC19-24. Taking into account that HspB7 interacted with the isolated Ig-like domain 24 and filamin fragments containing Ig-like domains 22-24 and 19-24, we concluded that HspB7 is a bona fide partner of filamin C. Selective binding of the α-crystallin domain of HspB7 with the Ig-like domain 24 induced dissociation of filamin dimers, which might promote filamin C translocation in the cell and facilitate the repairs of damaged contractile apparatus.

The influence of microRNAs (miRNAs) on the expression of conserved genes is well established now, but their role in regulating evolutionarily young genes remains largely unexplored. The lawc gene emerged de novo within the 5′-untranslated region (UTR) of the Trf2 gene. We used this gene pair as a model system to investigate whether miRNAs may act as evolutionary adaptation tools ensuring coordinated expression of overlapping genes. Transgenic Drosophila lines with the inducible expression of miRNA (miR-4968, miR-2491, miR-13b-1) precursors along with a GFP-based sensor were employed to visualize miRNA-mediated repression of the target sequences in vivo. Tissue-specific transgene activation was achieved using the yeast Gal4/UAS system. Luciferase assay in Drosophila S2 and human HEK293 cells confirmed the activity of a unique promoter predicted within the coding sequence (CDS) of the lawc gene and enabled assessment of the miRNA-mediated regulation of lawc expression in S2 cells. Additionally, we analyzed protein and mRNA levels and visualized activity of the GFP sensor in Drosophila tissues by immunoblotting, RT-PCR, and confocal microscopy, respectively. Our findings experimentally validate the functionality of the internal promoter within the lawc CDS and demonstrate the role of evolutionarily young miRNAs in modulating its expression. Using lawc as an example, we showed that CDS-located promoters are not exclusive to prokaryotes and yeast but can also occur in evolutionarily young genes of higher eukaryotes that arise within the regulatory regions of other genes. Notably, such genes rapidly become regulated by miRNAs of a similar evolutionary age, suggesting a co-evolutionary mechanism that underlies the formation of new regulatory networks and may facilitate adaptation of both genes to their overlapping configuration.

Elevated systemic production of interleukin-6 (IL-6) is associated with a broad spectrum of autoimmune and inflammatory diseases. Monoclonal antibody therapies targeting IL-6 or its receptor are widely used to neutralize the accompanying adverse effects. This study addresses the challenge of independently quantifying free and olokizumab (OKZ)-bound IL-6 in biological samples using a preclinical mouse model with regulated overexpression of human IL-6. We developed an enzyme-linked immunosorbent assay (ELISA), in which biotin-labeled OKZ served as a detecting agent, enabling in vivo tracking of dynamics of IL-6/OKZ immune complexes. OKZ effectively reduced free IL-6 levels in mice with cytokine overexpression for at least 7 days, while persisting in circulation as IL-6/OKZ complexes. Additionally, we assessed the kinetics of neutralizing anti-OKZ antibody formation in this preclinical model. The methods developed can be applied for clinical monitoring during anti-cytokine therapy and in preclinical studies using mouse models to evaluate the efficacy of such therapy in controlling IL-6-dependent inflammation in experimentally induced diseases.

Klebsiella grimontii is a recently described pathogen (former member of the Klebsiella oxytoca phylogroup K06), which has been elevated to the rank of a new species. The capsular polysaccharide (CPS) of K. grimontii is a principal virulence factor that significantly enhances the capacity of the bacterium to induce infections. The structure of CPS from K. grimontii strain K15g determined is this study was found to represent a novel type of polysaccharide consisting of branched hexasaccharide K-units, each composed of four monosaccharides of the main chain and a disaccharide side chain containing pyruvic acid 4,6-acetal residues. Since the CPS cleavage initiates the infection of bacterial cells by a phage, we investigated the depolymerase activity of the phage Silvester structural protein and demonstrated that the recombinant depolymerase of the phage Silvester cleaved K. grimontii K15g CPS at the β-D-GlcpA-(1→2)-α-L-Rhap linkage between the repeating units. The pathway for the synthesis of CPS of this type was proposed.

To some extent, fortune favored announcement of the ERP discovery: Zavoisky’s paper was published fairly promptly in both Russian and in English in 1945 and thus fortunately slipped through the tiny gap between the two epochs – just in time before the Iron Curtain descended across Europe. Thus, scientists beyond the borders of the USSR became aware of the discovery and were in fact the first to cite and acknowledge Zavoisky’s work. In 1944-early 1945, Zavoisky delivered his paper at a series of seminars attended by a number of renowned physicists, chemists, chemical physicists, biophysicists, and geophysicists from the USSR’s best scientific institutions. Nevertheless, for nearly a decade EPR had been of interest almost exclusively to physicists who belonged to Zavoisky’s school he established in Kazan. Beyond Kazan, A. I. Shalnikov, P. L. Kapitsa, and Ya. K. Syrkin appeared to have been the only scientists in the Soviet Union who immediately recognized promise of the EPR discovery. Moreover, there were the works of Syrkin’s student L. A. Blumenfeld and his friend V. V. Voevodsky that paved the way for the EPR method to spread beyond Kazan and physics, into chemistry and biology research all across the USSR. After late 1950s, the number of publications on EPR in Soviet journals grew exponentially. Research groups studying magnetic resonance phenomena were established in many other scientific institutions. In the present paper, those groups and their studies, as well as scientific instrumentation for EPR and NMR spectroscopy in the USSR are briefly discussed.

In this chapter, Zavoisky’ history of Nobel Prize nominations is discussed. Once his name became publicly known after a decade of obscurity due to his involvement in the Soviet nuclear program, Zavoisky began to be proposed for the Prize by his international peers. C. J. Gorter, Zavoisky’s competition in his search for EPR, was the first to nominate him, in 1958. On the Soviet side, the first nomination came from the physicist I. M. Frank, in 1959. In the next decade, Zavoisky’s most persistent nominee was Croatian-Swiss chemist L. Ružička. The period covered herein ends in 1966, as information for later years was not yet disclosed by the Nobel Organization at the time of writing the original publication.