In prokaryotes, translation initiation orchestrates protein synthesis through a network of dynamic interactions among the ribosome, mRNA, initiator tRNAfMet, and initiation factors (IFs). Traditional approaches that rely on radioactive labeling or surface immobilization are hindered by inherent safety risks and methodological constraints. We present a fluorescence-based analytical platform that integrates microscale thermophoresis (MST) as a unified, multiparametric toolkit for comprehensive interrogation of bacterial translation initiation at the molecular level. By systematically applying MST to a panel of fluorescently labeled components—initiator tRNAfMet, mRNAs, and initiation factors—we quantify assembly pathways and equilibria as initiation progresses from simple bimolecular interactions to higher-order, multicomponent complexes. To broaden the fluorescence toolbox for ribosomal studies, we developed a robust BODIPY-labeling protocol for 70S ribosomes and confirmed preservation of structural integrity and function by nano differential scanning fluorimetry, stopped-flow kinetic assays, and peptide-synthesis activity tests. Our microscale fluorescent system facilitates probing initiation at a variety of steps, since the role of magnesium ions and initiation factors upon 30S initiation complex formation. The same platform can be applied to investigate the effects of different compounds on translation initiation, as demonstrated for a number of antibiotics, aptamers, and antimicrobial peptides. Using this approach, we determined the antibiotic streptomycin dissociation constant for both 30S and 70S ribosomes, which proved identical at 0.3 ± 0.1 μM, and demonstrated the effect of the antimicrobial peptide rumicidin-1 on translation initiation. Offering a cost-effective and high-sensitivity alternative to conventional methods, this approach advances mechanistic understanding of prokaryotic translation and provides a versatile framework for the discovery of novel protein synthesis inhibitors.

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.

RelA/SpoT homologue family enzymes participate in controlling the cellular levels of the alarmone (p)ppGpp, thereby activating the stringent response and promoting survival under stress conditions. These proteins contain an N-terminal catalytic domain and a C-terminal regulatory domain. They catalyze both the synthesis of ppGpp/pppGpp from ATP and GDP/GTP and their hydrolysis to GDP/GTP and pyrophosphate. Here, we report the crystal structure of the N-terminal domain of Rel from Streptococcus equisimilis in complex with pppGpp at 3.2 Å resolution. The asymmetric unit contains a dimer with asymmetric ligation: pppGpp occupies only the synthetase site in one monomer, whereas in the other monomer, it is bound in both the hydrolase and synthetase sites. The two monomers exhibit distinct conformational states, with pronounced rearrangements of the flexible loops surrounding the binding pockets, including the α2/α3 and α8/α9 loops that act as steric gates. Molecular dynamics simulations support the dual binding arrangement and reveal additional probable transient binding sites, including a region in the linker between hydrolase and synthetase subdomains. These findings provide a structural framework for understanding how pppGpp binding modulates the opposing catalytic activities of bifunctional Rel enzymes and suggest possible mechanisms for (p)ppGpp-mediated autoregulation.

The development of highly sensitive approaches for detecting tumor cells in biological samples remains a critical challenge in laboratory and clinical oncology. In this study, we investigated the structural and magnetic properties of iron oxide nanoparticles incorporated into cellulose microspheres of two size ranges (~100 and ~700 μm) and evaluated their potential for targeted tumor cell isolation. In the smaller microspheres, magnetite-based magnetic nanoparticles (MNPs) were synthesized in situ via co-precipitation, whereas pre-synthesized MNPs were embedded into the larger microspheres. The geometrical characteristics of the resulting magnetic cellulose microspheres (MSCMNs) were assessed by confocal microscopy. Transmission electron microscopy and X-ray diffraction analyses revealed an average magnetic core size of approximately 17 nm. Magnetic properties of the MNPs within MSCMNs were characterized using a highly sensitive nonlinear magnetic response technique, and their dynamic parameters were derived using a formalism based on the stochastic Hilbert-Landau-Lifshitz equation. To evaluate their applicability in cancer diagnostics and treatment monitoring, the MSCMNs were functionalized with a TKD peptide that selectively binds membrane-associated Hsp70 (mHsp70), yielding TKD@MSCMNs. Magnetic separation enabled the isolation of tumor cells from biological fluids. The specificity of TKD-mediated binding was confirmed using Flamma648-labeled Hsp70 and compared with control alloferone-conjugated microspheres (All@MSCMNs). The ability of TKD@MSCMNs to selectively extract mHsp70-positive tumor cells was validated using C6 glioma cells and mHsp70-negative FetMSCs controls. Following co-incubation, the extraction efficiency for C6 cells was 28 ± 14%, significantly higher than that for FetMSC (7 ± 7%, p < 0.05). These findings highlight the potential of TKD-functionalized magnetic cellulose microspheres as a sensitive platform for tumor cell detection and isolation.

Extracellular vesicles (EVs), nanoscale membrane-enclosed particles, are natural carriers of proteins and nucleic acids. Microalgae are widely used as a source of bioactive substances in the food and cosmetic industries and definitely have a potential to be used as the producers of EVs for biomedical applications. In this study, the extracellular vesicles isolated from the culture medium of two unicellular microalgae, Chlamydomonas reinhardtii (Chlamy-EVs) and Parachlorella kessleri (Chlore-EVs), were characterized by atomic force microscopy (AFM), cryo-electronic microscopy (cryo-EM), and nanoparticle tracking analysis (NTA). The biocompatibility with human cells in vitro (HEK-293T, DF-2 and A172) and biodistribution in mouse organs and tissues in vivo were tested for both microalgal EVs. An exogenous therapeutic protein, human heat shock protein 70 (HSP70), was successfully loaded to Chlamy- and Chlore-EVs, and its efficient delivery to human glioma and colon carcinoma cell lines has been confirmed. Additionally, in order to search for potential therapeutic biomolecules within the EVs, their proteomes have been characterized. A total of 105 proteins were identified for Chlamy-EVs and 33 for Chlore-EVs. The presence of superoxide dismutase and catalase in the Chlamy-EV constituents allows for considering them as antioxidant agents. The effective delivery of exogenous cargo to human cells and the possibility of the particle yield optimization by varying the microalgae growth conditions make them favorable producers of EVs for biotechnology and biomedical application.

Parkinson’s disease, associated with mutations in the GBA1 gene (GBA1-PD), is the most common genetic form of Parkinson’s disease (PD), marked by clinical heterogeneity influenced by mutation type. Extracellular vesicles (EVs), key mediators of intercellular communication, are implicated in PD pathogenesis through the transport of pathological proteins and lipids. In this study, we analyzed blood plasma-derived EVs from GBA1-PD patients carrying p.N370S and p.L444P mutations and from healthy controls using cryo-electron microscopy, lipidomics, and proteomics. EVs from GBA1-PD patients were significantly larger than those from controls, with the largest size and most multilayered vesicles observed in p.N370S carriers. Lipidomic profiling identified 237 lipid species; of these, 186 lipids were altered in p.N370S and 24 in p.L444P versus controls. Mutation carriers showed distinct lipid signatures, with p.L444P samples enriched predominantly in sphingolipids, while p.N370S carriers exhibited more extensive lipid remodeling across multiple classes, including triglycerides, cholesteryl esters, and phospholipids. Notably, Cer 23:0 was elevated across all GBA1-PD groups. Proteomic analysis revealed enrichment in pathways related to lipid transport, immune regulation, and vesicle-mediated processes. Overall, GBA1-PD patients share a distinct lipidomic EV signature, with mutation-specific patterns reflecting differing mechanisms of lysosomal dysfunction. These findings support the potential of EV profiling to unravel disease heterogeneity and identify biomarkers.

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.

Proline-rich antimicrobial peptides (PrAMPs) are promising compounds for overcoming antibiotic resistance, one of the global health threats, and stand out from other types of AMPs by their high safety profile. The main cellular target of PrAMPs, like most modern antibiotics, is the conservative cellular structure – the ribosome. PrAMPs bind in the ribosomal tunnel, forming multiple interactions with nucleotides of 23S rRNA, and are divided into two classes depending on their mechanism of action: inhibition of elongation or termination. The N-terminal part of the peptides, which is important for the activity of class I peptides, extends into the A-site pocket, preventing the binding of aminoacyl-tRNA. A new family of PrAMPs, rumicidins, was discovered using genomic search methods. Its representatives have the longest N-terminal part, as well as a unique pair of amino acids Trp23 and Phe24 at the C-terminus. The Trp-Phe dyad forms a spacer at the constriction site of the ribosomal tunnel, stabilizing the binding and leading to increased antibacterial activity. New structural studies of the class I peptide Bac5 have demonstrated its ability to disrupt the correct positioning of the CCA-end of the P-site tRNA in the peptidyltransferase center of the ribosome, which can affect the assembly of functional initiation complexes. Class II PrAMPs, according to new data, have additional binding sites on the ribosome and have a complex effect on the bacterial cell: they disrupt the termination of protein synthesis, block the cellular ribosome release system, prevent the correct assembly of the 50S ribosomal subunits, and, possibly, affect the first stage of translocation. Recent studies expand our understanding of the antimicrobial activity of PrAMPs and contribute to the creation of future therapeutic drugs based on AMPs.

Bacterial translation initiation factor IF3 is composed of two distinct domains, a well-characterized C-terminal domain (IF3C), which enhances the speed and fidelity of translation initiation, and a less understood N-terminal domain (IF3N). In this study, we developed an aptamer (Apt343) that targets IF3N with the goal of elucidating its contribution to translation initiation. Rapid kinetics assays revealed that Apt343 reduces the rate of IF3 association with the 30S ribosomal subunit by 13-fold, while inducing a pronounced rearrangement of both IF3 domains on the 30S. These changes compromise IF2-, mRNA-, and fMet-tRNAfMet-dependent movements of IF3, delaying 30S initiation complex (30S IC) formation by up to two orders of magnitude. Cryo-EM analysis suggests that Apt343 may sterically clash with fMet-tRNAfMet, thereby perturbing the canonical pathway by which the initiator tRNA is accommodated after IF2-dependent recruitment and prior to start codon decoding. However, once the 30S IC is formed, blocking IF3N does not prevent 50S subunit joining or 70S IC assembly. Collectively, these findings support a role for IF3N in enhancing an efficient path for fMet-tRNAfMet accommodation towards the 30S IC and promoting IF3C displacement to unlock 50S recruitment. Moreover, this aptamer-based strategy offers a valuable tool for dissecting domain-specific activities of multidomain factors within complex environments such as the initiating ribosome. ### Competing Interest Statement The aptamers described in this study are protected in Peru by patent 000424-2019/DIN. The authors Ana Sanchez-Castro, Katherin Penaranda, and Pohl Milon are the inventors of the aptamers discussed in this article.

Abstract Translation is a critical step in gene expression and a target for multiple antibiotics. Inhibition of protein synthesis by many antibiotics depends on mRNA context, making them selective towards particular mRNAs. Toe-printing is a standard low-throughput method to determine ribosome position along mRNA. Hereby we report the development and application of the Toe-seq method, combining toe-printing with next-generation sequencing. Toe-seq proved to be efficient to monitor translation and its inhibition by antibiotics for the library of over 37 000 mRNAs. Context-specificity of antibiotic action could be determined for different antibiotics via Toe-seq. Previously known sequence dependencies of translation inhibition were reproduced by Toe-seq. Data obtained by Toe-seq demonstrated context-specificity of etamycin A (EtaA) action whose binding site is located in the nascent peptide tunnel. Comparison of EtaA specificity with that of erythromycin (Ery) and tetracenomycin X (TcmX) revealed important differences and commonalities. Structural basis for the context-specificity of translation inhibition by EtaA has been deciphered by cryo-electron microscopy at 2.2 Å resolution. Toe-seq method allows one to monitor ribosome progression and stalling in vitro at a codon resolution, making it possible to assess antibiotics context specificity in a multiple parallel assay.

The search for peptides that can specifically bind to regulatory regions in DNA is a necessary step for creating drugs that can regulate gene expression. The work studies the peculiarities of binding of a model peptide, which carries an ionic self-complementary motif and is capable of forming amyloid-like fibrils, with model double-stranded DNA. The stoichiometric ratios of the components of the complex were found using the retardation method in agarose gel. Using microscale thermophoresis, it was shown that the peptide in the oligomeric amyloid-like state is capable of binding to model 45-bp double-stranded DNA, with a micromolar equilibrium dissociation constant. Using electron and atomic force microscopy, the morphology of peptide-DNA complexes was studied; using dynamic light scattering and nanoparticle tracking analysis, as well as small-angle neutron scattering, the spatial parameters of the resulting DNA-peptide complexes were characterized. Molecular dynamics simulations showed that the arginine side chains of the peptide are prone to interact with guanine nitrogenous bases. It was shown that the formation of peptide-dsDNA complexes interferes with the operation of restriction endonucleases that have guanine-cytosine pairs in the recognition center, which is consistent with the results of prediction of interaction sites obtained using computer modeling. The results of the work can be used in the development of peptides capable of interacting with functional regions of DNA, as well as in the development of new carriers for transfection of DNA constructs. ### Competing Interest Statement The authors have declared no competing interest.

The OCT4 transcription factor is necessary to maintain cell stemness in the early stages of embryogenesis and is involved in the formation of induced pluripotent stem cells, but its role in oncogenesis is not yet entirely clear. In this work, OCT4 expression was investigated in malignant gliomas. Twenty glioma cell lines and a sample of normal adult brain tissue were used. OCT4 expression was found in all studied glioma cell lines but was not detected in normal adult brain tissue. For one of these lines, OCT4 knockdown caused tumor cell death. By varying the culture conditions of these cells, we unexpectedly found that OCT4 expression increased when cells were incubated in serum-free medium, and this effect was significantly enhanced in serum-free and L-glutamine-free medium. L-glutamine and the Krebs cycle, which is slowed down in serum-free medium according to our NMR data, are sources of α-KG. Thus, our data indicate that OCT4 expression in gliomas may be regulated by the α-KG-dependent metabolic reprogramming of cells.

The sustained rise of antimicrobial resistance (AMR) causes a strong need to develop new antibacterial agents. One of the methods for addressing the problem of antibiotic resistance is through the design of hybrid antibiotics. In this work, we proposed a synthetic route for the conjugation of an azithromycin derivative with chloramphenicol and metronidazole hemisuccinates and synthesized two series of new hybrid molecules 4a–g and 5a–g. While a conjugation did not result in tangible synergy for wild-type bacterial strains, new compounds were able to overcome AMR associated with the inducible expression of the ermC gene on a model E. coli strain resistant to macrolide antibiotics. The newly developed hybrids demonstrated a tendency to induce premature ribosome stalling, which might be crucial since they will not induce a macrolide-resistant phenotype in a number of pathogenic bacterial strains. In summary, the designed structures are considered as a promising direction for the further development of hybrid molecules that can effectively circumvent AMR mechanisms to macrolide antibiotics.

The most extensively studied beta-d-galactosidases (EC3.2.1.23) belonging to four glycoside hydrolase (GH) families 1, 2, 35, and 42 are widely distributed among Bacteria, Archaea and Eukaryotes. Here, we report a novel GH35 family beta-galactosidase from the hyperthermophilic Thermoprotei archaeon Desulfurococcus amylolyticus (Da beta Gal). Unlike fungal monomeric six-domain beta-galactosidases, the Da beta Gal enzyme is a dimer; it has an extra jelly roll domain D7 and three composite domains (D4, D5, and D6) that are formed by the distantly located polypeptide chain regions. The enzyme possesses a high specificity for beta-d-galactopyranosides, and its distinguishing feature is the ability to cleave pNP-beta-d-fucopyranoside. Da beta Gal efficiently catalyzes the hydrolysis of lactose at high temperatures, remains stable and active at 65 degrees & Scy;, and retains activity at 95 degrees & Scy; with a half-life time value equal to 73 min. These properties make archaeal Da beta Gal a more attractive candidate for biotechnology than the widely used fungal beta-galactosidases.

The 26S proteasome is a unique multicatalytic proteinase complex, together with a ubiquitination system, providing controlled degradation of most intracellular eukaryotic proteins. The problem of studying the proteasome is the multiplicity of its intracellular forms, which are formed due to the modularity of the proteasome assembly process. In this study, using cryoelectron microscopy, we described for the first time the structure of the 26S human immunoproteasome in comparison with its constitutive form with a resolution of 3.6 Å. A detailed analysis of the structural features of the two complexes revealed the opening of the entrance in the outer heptameric 20S ring of the immunoproteasome subunit due to the separation of the N-terminal regions of the PSMA4 and PSMA5 subunits and the formation of a π–π stacking between the amino acid residues Tyr5 and Phe9 of the PSMA5 and PSMA6 subunits, respectively. The revealed removal of steric obstruction in the central channel of the 20S subunit may indicate the preactivation phenotype of the 26S human immunoproteasome, even in the absence of a bound substrate.

Background/Objectives: Stress protein HSP70 administered exogenously has demonstrated high potential as an efficient adjuvant in antitumor immune response. To enhance the antigen-presenting activity, bioavailability, and stability of exogenous recombinant human HSP70, we propose incorporating it into plant extracellular vesicles. Earlier, we found that grapefruit-derived extracellular vesicles (GEV) were able to store the protein with no loss of its major function, chaperone activity. Methods: In this study, we tested whether HSP70 loaded into GEV (GEV-HSP70) could elicit an antitumor immune response in cellular and animal models of colorectal cancer. Results: To test the hypothesis in vitro, human and mouse colorectal cancer cell lines were used. We have shown that the addition of HSP70, either in free form or as part of GEVs, increases the sensitivity of human (HCT-116, DLD1) or mouse (CT-26) colon cancer cells to mouse cytotoxic lymphocytes and human NK-92 cells. Moreover, the amount of protein in the form of GEV-HSP70 required to cause the same activation of antitumor immunity was 20 times less than when HSP70 was added in free form. In a colon carcinoma model in vivo, GEV-HSP70 were inoculated subcutaneously into BALB/c mice together with CT-26 cells to form a tumor node. As compared with the control groups, we observed an increase in the lifespan of animals and a decrease in the tumor size, as well as a decrease in the level of TGFB1 IL-10 factors in the blood plasma. In vitro analysis of the immunomodulatory activity of GEV-HSP70 showed that antitumor response in GEV-HSP70-treated mice was associated with the accumulation of CD8+ cells. Conclusions: These results demonstrate the high feasibility and efficacy of the new technique based on HSP70 encapsulated in plant vesicles in activation of the specific response to colon tumors.

This work describes a fast implementation of a software algorithm associated with determination of protein secondary structure based on the Define Secondary Structure of Proteins (DSSP) algorithm. This implementation is fully compatible with the DSSP v.4 and DSSP v.2 algorithms and implemented as a native GROMACS trajectory analysis module, which allows us to analyze molecular dynamics trajectories without any restrictions of the original DSSP implementation. This implementation works much faster than the original DSSP v.4 and DSSP v.2 algorithms.

The antimicrobial resistance crisis along with challenges of antimicrobial discovery revealed the vital necessity to develop new antibiotics. Many of the animal proline-rich antimicrobial peptides (PrAMPs) inhibit the process of bacterial translation. Genome projects allowed to identify immune-related genes encoding animal host defense peptides. Here, using genome mining approach, we discovered a family of proline-rich cathelicidins, named rumicidins. The genes encoding these peptides are widespread among ruminant mammals. Biochemical studies indicated that rumicidins effectively inhibited the elongation stage of bacterial translation. The cryo-EM structure of the Escherichia coli 70S ribosome in complex with one of the representatives of the family revealed that the binding site of rumicidins span the ribosomal A-site cleft and the nascent peptide exit tunnel interacting with its constriction point by the conservative Trp23-Phe24 dyad. Bacterial resistance to rumicidins is mediated by knockout of the SbmA transporter or modification of the MacAB-TolC efflux pump. A wide spectrum of antibacterial activity, a high efficacy in the animal infection model, and lack of adverse effects towards human cells in vitro make rumicidins promising molecular scaffolds for development of ribosome-targeting antibiotics. The antimicrobial resistance crisis calls for development of new classes of antibiotics. Here, authors use genome mining approach to discover a distinct family of ribosome-targeting proline rich antimicrobial peptides.

This work introduces a new faster implementation of the hydrogen bond network (complex arrangement of hydrogen bonds between or within molecules) search algorithm in biomacromolecules and their environment. Existing implementation of such an algorithm in GROMACS [Abraham et al. GROMACS 2024.2 Manual. 2024.] has limitations in the analysis of large structures and trajectories. The new implementation, in the form of a native GROMACS trajectory analysis module, allows for quick analysis of molecular dynamics trajectories without restrictions, thus overcoming the limitations of the original algorithm. The application of the developed method enabled the acquisition and analysis of hydrogen bond networks in the studied defensin-like protein Pentadiplandra brazzeana, as well as the study of hydrogen bond occupancies between the protein's residues and water molecules. The data obtained using the new implementation coincided with the experimental data.

The characteristics of six new glioblastoma cell lines obtained from tumor material from patients are presented. The studied glioblastomas do not have mutations in the genes IDH1 and IDH2, which indicated a poor prognosis for their therapy. Two glioblastomas carry the pathogenic mutation p.Arg110Pro in the gene TP53. All cell lines studied express RNA of tumor suppressor and oncogenic isoforms of the p73 protein. The glioblastomas responded differently to radiotherapy, with five of them being more resistant to γ irradiation than the standard A172 glioma line. All six cell lines express RNA genes for vascular endothelial growth factor and its receptor (VEGFR-1) in different ratios. Testing of an immunotherapeutic regimen with monoclonal antibodies to VEGFR-1 on one of the cell lines confirms that the studied glioblastomas are sensitive to blocking vascular growth factor and its receptor. Thus, these glioblastomas can become a promising model for studying the formation of tumor-cell resistance to radiotherapy and the effectiveness of immunotherapy that blocks growth factors and their receptors.