Abstract Background Parkinson’s disease (PD) is a progressive chronic neurodegenerative disease. The PARK2 gene encoding the Parkin protein accounts for approximately half of early-onset autosomal recessive PD cases in humans. Objective The aim of this work was to study the effect of the PARK2 gene knockout in mice on the dynamics of behavioral and biochemical parameters of PD. Methods The study was performed on C57BL/6-line mice aged from 4 months to 1.5 years: wild type ( park2 +/+ ), heterozygotes ( park2 +/- ) and homozygotes ( park2 -/- ) knocked out by the PARK2 using CRISPR-Cas9. The open field test, the Porsolt forced swimming test, the grid-walk test, the beam-walking test, the elevated plus maze test, the accelerating rotarod test were used to assess the behavioral phenotype. Measurement of the concentration of bioamines and their metabolites by HPLC and evaluation of the amount of tyrosine hydroxylase, BDNF and GDNF by Western Blot were used to study the biochemical signs of PD. Results Park2 -/- mice begin to show signs of decreased motor activity no earlier than at 4 months of life. At 12 months of life, it was shown only a decrease in the level of the mature isoform of GDNF and an increase in the number of immature isoforms in the frontal cortex and striatum were revealed. Conclusion The data obtained indicates a different age dynamic of the condition of mice associated with the PARK2 knockout. However, no pronounced specific manifestations of PD in human were found in park2 -/- mice.

Herein, we report a convenient synthetic approach and an in vitro evaluation of benzimidazole carbamide derivatives as potential autophagy activators. Two compounds exhibited autophagy-inducing activity at concentrations ≥100 μM. The most favorable activity-toxicity profile was observed for a benzimidazole derivative lacking a carbamide fragment. Mechanistic studies indicated that the compounds predominantly act via the AMPK pathway, while generally showing low selectivity toward individual components of this signaling cascade. Notably, the derivative bearing a pyridyl substituent in the carbamide moiety and a methylated benzimidazole core preferentially targeted AMPK within the signaling cascade. Overall, the synthesized benzimidazoles can be considered mild autophagy activators and relatively broad modulators of cellular signaling.

Colicin E3 (E3-rRNAse) abolishes protein biosynthesis in bacteria by cleaving 16S rRNA in the decoding centre. The RtcB2-PrfH 16S rRNA repair module prevents cellular death upon exposure to E3-rRNAse in E. coli. Upon overexpression, RtcB2 RNA ligase alone was capable of relieving E. coli growth, which was inhibited by E3-rRNAse. Using in vitro ribosome repair system based on recombinant components, we demonstrated that RtcB2 alone could repair E3-rRNAse cleaved 30S subunits and 70S ribosomes. The peptide chain release factor homolog (PrfH), which is able to hydrolyse peptidyl-tRNA bound by colicin-cleaved ribosomes, boosts RtcB2 ligation efficiency for damaged ribosomes engaged in translation.

The rapid increase in antimicrobial resistance underscores the urgent need for new antibacterial agents. One promising strategy involves designing novel compounds through targeted chemical modifications of existing antibiotics. Azithromycin (AZI), a widely used macrolide, has served as a versatile scaffold for developing numerous antibacterial candidates. However, the mechanistic consequences of such modifications remain largely unexplored. Here, we characterize the activity and mechanism of action of three AZI-benzoxaborole (AZI-BB) conjugates. We show that these compounds inhibit bacterial translation in vitro and remain active against a model Escherichia coli strain carrying an inducible ermCL-ermC operon, which confers resistance to macrolide antibiotics. Unlike erythromycin, these derivatives, along with AZI itself, exhibit minimal induction of ErmC expression. Structural analysis reveals that the benzoxaborole moiety of AZI-BB2 forms additional interactions with nucleotides C2441 and C2586 of 23S rRNA, likely contributing to premature ribosome stalling at the ermCL regulatory sequence and thereby preventing ErmC expression. Furthermore, high-throughput toeprinting analysis combined with deep sequencing (Toe-seq) demonstrates that AZI-BB2 exhibits reduced sequence specificity for canonical macrolide-sensitive stalling motifs. Altogether, these findings demonstrate that targeted chemical modification of AZI can reshape its context-specific interaction with the ribosome and attenuate the induction of macrolide resistance mechanisms.

Antimicrobial resistance threatens the long-standing efficacy of antibiotics and underscores the need to expand, refine, and diversify antimicrobial therapies. Translation is a uniquely druggable process: its machinery is essential, conserved in bacteria, and sufficiently divergent from the eukaryotic counterpart to enable selectivity. This review synthesizes recent progress on inhibitors of initiation, elongation, termination, and recycling. High-resolution structural and biophysical studies have resolved longstanding ambiguities, reassigned ribosomal binding sites, uncovered stage-specific activities in scaffolds previously thought to act elsewhere, and revealed multistage, context-dependent mechanisms. Beyond the canonical stages, quality-control pathways that offer orthogonal points of intervention were observed. Collectively, these advances support structure-guided, context-aware, and hybrid/combination strategies for antibiotic design and therapeutic development.

BackgroundParkin (PARK2) mutations are among the most common causes of autosomal recessive Parkinson’s disease (PD); however, the phenotypic manifestations of Parkin deficiency in mouse models remain inconsistent.ObjectiveThe aim of this work was to study the effect of the PARK2 gene knockout in mice on the dynamics of behavioral and biochemical parameters of PD.MethodsThe study was performed on C57BL/6-line mice aged from 4 months to 1.5 years: wild type (park2 +/+), heterozygotes (park2 +/-) and homozygotes (park2 -/-) knocked out by the PARK2 using CRISPR-Cas9. The open field test, the Porsolt forced swimming test, the grid-walk test, the beam-walking test, the elevated plus maze test, the accelerating rotarod test were used to assess the behavioral phenotype. Measurement of the concentration of bioamines and their metabolites by HPLC and evaluation of the amount of tyrosine hydroxylase, BDNF and GDNF by Western Blot were used to study the biochemical signs of PD.ResultsPark2 -/- mice begin to show signs of decreased motor activity no earlier than at 4 months of life. At 12 months of life, it was shown only a decrease in the level of the mature isoform of GDNF and an increase in the number of immature isoforms in the frontal cortex and striatum were revealed.ConclusionThese findings indicate that PARK2 knockout mice recapitulate specific functional aspects of Parkinson’s disease while lacking robust neurodegeneration, suggesting their utility as a model of early-stage functional impairment rather than progressive dopaminergic loss.

The sequence of messenger RNA (mRNA) not only determines the protein sequence synthesized by a ribosome but also defines the efficiency of this process. Many antibiotics lethal to bacteria inhibit various stages of translation by targeting ribosomal functional centers. Some antibiotics exhibit specificity not only for particular stages of the ribosomal working cycle but also for specific patterns within mRNA sequences. This review covers a broad range of approaches—including in vivo and in vitro methods, low- and high-throughput techniques such as reporter constructs, characterization of inhibitors of protein synthesis (ChIPS), toeprinting, cryogenic electron microscopy (cryo-EM), protein labeling, and those integrated with next-generation sequencing (NGS) like ribosome profiling with following NGS (Ribo-seq), inverse toeprinting coupled with NGS (iTP-seq), high-throughput toeprinting and NGS (Toe-seq), and ribosome display—used to study the sequence specificity of translation inhibitors, a rapidly evolving field crucial to molecular biology. It presents various methodologies, discusses their applications, and provides a comparative analysis. The fundamental research value of this review lies in establishing standardized experimental selection guidelines for scientists investigating ribosome stalling mechanisms, thereby minimizing trial-and-error costs. Equally important is its applied relevance. The review highlights its translational value in aiding the screening and mechanistic analysis of sequence-specific small-molecule inhibitors. Moreover, understanding the mechanisms underlying protein biosynthesis inhibition and their dependence on particular mRNA sequences could enable the development of selective agents that precisely suppress the synthesis of certain polypeptides, such as proteins from pathogenic bacteria or cancer-associated proteins.

Background Isotopically labelled S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH) are essential cofactors in methylation reactions. They serve as key tools for structural and mechanistic studies of methyltransferases and metabolic flux analysis using NMR spectroscopy and isotope-dilution mass spectrometry (MS). However, uniformly labelled SAM and SAH are not commercially available, and the existing methods of production (enzymatic, chemical or microbial) are costly, technically demanding, or yield mixtures of stereoisomers requiring extensive purification. Results We have developed a purification strategy that exploits the naturally high-affinity binding of SAM and SAH by selected methyltransferases. His-tagged methyltransferases were overexpressed in Escherichia coli grown on isotope-enriched minimal media, enabling the co-purification of the isotopically labelled cofactors to be co-purified by the standard metall chelate chromatography. Subsequent protein denaturation quantitatively released the bound ligand. Conclusions This work introduces a simple, robust, and widely applicable platform for the in-house production of isotopically labelled SAM and SAH using standard protein expression and purification equipment. By repurposing high-affinity methyltransferases as disposable affinity carriers, the method eliminates the need for expensive precursors, specialized enzymatic cascades, metabolic engineering, or preparative chromatography, while delivering products of exceptional stereochemical and isotopic purity. The approach is readily adaptable to different isotope-labeling schemes and can be generalized to other tightly bound metabolites, providing a versatile tool for structural biology, enzymology, and metabolic research.

The FLAD1 gene codes for flavin adenine dinucleotide (FAD) synthase. FAD is a cofactor for many redox enzymes involved in vital processes from respiration to signal transduction. In this work, we described a clinical case of 2 siblings carrying compound heterozygous mutations in the FLAD1 gene resulting in the substitutions A418V and R542* at the protein level. The patients demonstrate adrenal insufficiency, which has not previously been associated with FLAD1 protein defects. To verify that adrenal insufficiency is caused by FLAD1 mutations, we created a personalized mouse model carrying the mutations found in the patients. The mutation in the FLAD1 gene, leading to the A418V substitution, appeared viable in the homozygous state, with minimal difference from the WT. The FLAD1 gene mutation leading to the R542* truncation is lethal when homozygous. The mouse model of the compound heterozygous FLAD1 A418V/R542* mutations recapitulated the physiological, biochemical, and endocrine manifestations of FLAD1 mutations in patients. The mouse model created demonstrates the causal effect of FLAD1 mutations on the described pathology and potentially paves the way for understanding the disease’s molecular mechanism and developing better therapies.

Antimicrobial resistance is a major threat to modern society and healthcare, as it severely compromises the efficacy of standard antibiotic treatments. To meet the ever-increasing demand for novel antimicrobial drugs, it is crucial to develop new strategies for screening antimicrobial compounds and improve existing high-throughput techniques. Reporter systems that employ specific genetic markers are powerful tools not only for detecting antimicrobial activity of the substance being studied, but also for identifying the potential mechanism of its action. Among other metabolic pathways, RNA biosynthesis machinery is considered a promising molecular target as it remains underutilized in current antimicrobial therapy and therefore is rarely exposed to drug pressure. However, there is no suitable biomarker for identifying compounds that inhibit the transcription in Gram-negative bacteria. Combining bioinformatic search and RT-qPCR experimental validation, we have established the overexpression of the spermidine synthase gene (speE) as a biomarker associated with impaired transcription in Escherichia coli. Monitoring the expression level of speE in antibiotic-treated cells enables reliable detection of compounds that inhibit bacterial RNA-polymerase, such as rifampicin and fidaxomicin. Moreover, our screening system was successfully applied in practice to analyze chromatography fractions from fermentation broth of antibiotic producers, with compounds of the rifamycin family being identified as hits and isolated. The proposed method has the potential to be used in sequential screening procedures to reveal active antimicrobial compounds that inhibit bacterial transcription process, giving the world novel antimicrobials with minimal risk of resistance development.

Background/Objectives: Autophagy is an important cellular self-cleansing process whose normal functioning is essential for preventing many age-related diseases. The search for and study of new autophagy activators allows the proposal of novel therapeutic approaches for the treatment of age-related diseases. Medical plants are a rich source of bioactive compounds with variable functions. In this study, we propose an HR-LCMS/MS-based technique for identifying the principal autophagy activators in plant extracts. Methods: We performed a Western blot analysis of the autophagy-inducing activity of plant extract HPLC fractions on a model of SH-SY5Y cells. The composition of the fractions showing autophagy-activating potential was determined via HR-LCMS/MS. Results: We analyzed five plants known to produce autophagy activators and proved the ability of the method to detect the main bioactive compounds. Additional screening demonstrated for the first time that Astragalus dasyanthus is a producer of the autophagy-inducer glabrol. Conclusions: The described HR-LCMS/MS-based method for identifying autophagy activators in multicomponent plant extracts is effective and could be used for further high-throughput screening.

Diabetes and obesity are associated with poorer outcomes after ischemic stroke; however, it remains unclear whether this results from increased neuronal susceptibility to injury or from vascular dysfunction induced by metabolic syndrome. To minimize the contribution of vascular factors, we used a model of photoinduced thrombosis (PT) in cortical vessels, which generates lesions of reproducible size and is less dependent on collateral blood flow. PT was induced in wild-type (WT) mice, as well as in ob/ob (leptin-deficient) and db/db (leptin receptor-deficient) mice. Magnetic resonance imaging (MRI) revealed that PT produced comparable infarct volumes in all mouse groups. Several genes associated with inflammation and activation of microglia and macroglia in the peri-infarct area (Cst7, Ccl3, Tlr2, Gfap) exhibited similar expression patterns across all three mouse strains, while transcriptional response to cerebral ischemia of Tnfa, Cxcl9, Il6, Cox2, Mmp3, and Bdnf genes depended on the genotype. Overall, despite individual differences in the expression profiles of certain genes, disruption of leptin signaling (whether due to leptin deficiency or leptin receptor deficiency) caused no genotype-specific exacerbation of stroke-induced injury. Assessment of post-stroke neurological deficits revealed substantial differences in absolute scores between WT and ob/ob or db/db mice, attributable to baseline disparities in body weight and motor activity. In db/db mice, normalization of post-stroke neurological status scores to pre-injury values revealed a more pronounced relative functional decline compared to ob/ob mice, suggesting impairments in early compensatory mechanisms and an important role of leptin signaling in neuroplasticity rather than in the extent of acute neuronal damage. Thus, under conditions that minimize vascular complications, neither leptin deficiency nor leptin receptor deficiency exacerbated acute ischemic brain damage or neuroinflammation.

The dynamic equilibrium between synthesis and degradation of biomolecules is maintained by cells, however, with aging, this balance is disrupted, resulting in the onset of diseases, including diabetes and neurodegenerative diseases. A decrease in autophagy, a key cellular process that is involved in lysosome-mediated degradation of damaged or dysfunctional cellular components, may contribute to this imbalance. Autophagy is strictly regulated within the cell through multiple signaling pathways, e.g., through the AMPK-dependent pathway, which functions as a key sensor of cellular energy limitation. In this study, we assessed the autophagy/mitophagy activation ability of a small set of 1,3-diaza-2-oxophenoxazine derivatives and analogs using a fluorescent reporter assay and immunoblot analysis. The two lead compounds, AR493 and AR900, which exhibited the highest autophagy induction levels, were demonstrated to activate the AMPK-dependent pathway. The introduction of a 2'-hydroxyl group into AR493 had almost no influence on its activity, while subsequent attachment of a metabolizable masked phosphate group resulted in a notable increase in activity, although accompanied by substantial toxicity. When analyzing the specificity of the lead compounds to AMPK and its main upstream regulator SIRT1 on the corresponding knockout cell lines, AR493 demonstrated the greatest specificity of action to AMPK. Molecular docking revealed that AR493 binds to Site 2 of the AMPK γ-subunit, which may promote AMPK activation by two possible mechanisms: by preventing ATP binding to Site 3, thus favoring AMP binding; and by directly engaging the αRIM2 motif to stabilize its interaction with the γ-subunit.

The RNA methyltransferase NSUN7 has been reported to be involved in the regulation of longitudinal columns positioning in sperm flagella, but its catalytic mechanism remains unclear. In this study, we investigated the functional role of NSUN7's methylation in the longitudinal column positioning by generating a mouse strain with a substitution of the putative catalytic cysteine in motif IV to alanine (Nsun7C382A). Contrary to predictions based on the typical reaction mechanism of NOP2/Sun family methyltransferases, Nsun7C382A mice did not exhibit any phenotypical characteristics of knockouts and had normal fertility, sperm motility, and longitudinal column positioning, similar to wild-type mice. Structural modelling suggests that NSUN7 possesses an unusual catalytic site composition, including three highly conserved cysteines from motifs IV, VI and VIII. The distance between the canonical catalytic cysteines in motifs IV and VI is significantly greater than in related methyltransferases, while a cysteine from motif VIII is positioned closer to motif VI. These findings allow us to assume that NSUN7 may have an essential function in the spermatogenesis of mice independent on its methyltransferase activity.

Spermatozoid's flagella assemble in transcriptionally silent spermatids and thus depend on posttranscriptional regulation of gene expression. Mutations in Nsun7 gene are known to cause male infertility in human and mice. We identified m5C-specific NSUN7 RNA methyltransferase as a protein present in elongated spermatids and interacting with RNAs specific for this type of spermatozoid's precursor cells. Inactivation of the Nsun7 gene in mice leads to upregulation of its RNA interactors, thus indicating that NSUN7 downregulates a set of RNAs in the elongated spermatids. A physiologic consequence of Nsun7 gene knockout is male infertility, which is mechanistically explained by the observed mispositioning of longitudinal columns relative to the axonemal microtubular doublets leading to a motility defect.

The rise of antimicrobial resistance among pathogenic bacteria poses a critical challenge to modern medicine, highlighting an urgent need for novel therapeutic agents. Bottromycin A2 (BotA2) is a promising candidate for future drug development, demonstrating potent activity against clinically relevant pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and Mycoplasma species, although its molecular mechanism of action has remained unclear until now. Here, we demonstrate that BotA2 inhibits bacterial translation with unique context specificity determined by the mRNA coding sequence. Using high-throughput toe-printing coupled with deep sequencing (Toe-seq analysis), we show that BotA2 induces ribosome pausing predominantly when a glycine codon enters the A-site of the ribosome, regardless of the codon identities in the P- and E-sites. Our biochemical and biophysical data indicate that BotA2 specifically arrests glycine-delivering ternary complexes on the ribosome, thereby preventing full accommodation of incoming Gly-tRNAGly within the peptidyl transferase center. Altogether, our findings uncover a previously undescribed mechanism of translation inhibition, driven by the context-specific immobilization of ternary complexes on elongating ribosomes.

Methyltransferases that modify spliceosomal small nuclear RNAs (snRNAs) play a crucial role in the cell by ensuring proper maturation of snRNAs, which is essential for optimal function of spliceosome. In this study, we investigated the enzyme METTL4, which catalyzes N6-methylation of 2′-O-methyladenosine at position 30 of U2 snRNA. Function of both the protein and the modification in splicing remains unclear. We demonstrated that inactivation of the METTL4 gene in HeLa S3 cells leads to significant changes in alternative splicing, general slowdown in spliceosome activity, and intron accumulation. In the cells lacking METTL4, expression of the set of genes associated with ribosomal RNA maturation is reduced, and the number of coilin-positive structures, most likely Cajal bodies, is decreased in the nuclei of these cells.

Spermiogenesis requires extensive molecular and structural remodeling to produce motile sperm. Mutations in the testis-specific RNA methyltransferase NSUN7 are associated with defective fibrous sheath, impaired sperm motility, and male infertility. However, the underlying molecular mechanisms remain poorly understood. Here, we performed proteomic profiling of sorted, elongated, and round spermatids, as well as mature spermatozoa from Nsun7 knockout mice. We showed that NSUN7 is present at all stages of spermiogenesis and is most abundant in round spermatids, which corresponds to the formation of the flagellum and fibrous sheath assembly. Loss of NSUN7 altered the abundance of proteins essential for dynein arm assembly (PIH1D3, CCDC103, CCDC40), intraflagellar transport (IFT122), and fibrous sheath organization (AKAP3, AKAP4, ROPN1L). We also showed that the previously detected impaired retention of cytoplasm in elongated spermatids may be caused by plectin accumulation. Interestingly, no statistically significant changes were found in mature sperm proteomes upon Nsun7 inactivation. Our findings support a model in which NSUN7 primarily stabilizes protein complexes and coordinates flagellar assembly. This indicates that NSUN7 is a critical regulator of spermiogenesis, and its malfunction is a contributing factor to male infertility.

Mitoregulin (Mtln) is a small mitochondrial protein that was only recently identified. Despite this, a substantial number of studies on its function have already been published. Although sometimes contradictory, these studies have revealed the localization of Mtln, its protein and lipid partners, and its role in lipid homeostasis, energy metabolism, oxidative stress, and other aspects of mitochondrial functioning. Moreover, research using knockout and transgenic mouse models has revealed the important role of Mtln in mammalian physiology. Metabolic changes, along with muscle, kidney, and fat-related phenotypes, have been linked to Mtln dysfunction. In this review, we summarize a comprehensive set of published data on Mtln. While controversies remain, we seek to offer a unified view of its functions, spanning molecular mechanisms to organism-level effects.