Search Results (3,738)

Vif and Vpr are HIV-1 accessory proteins that create optimal conditions for viral replication. They are considered as potential targets for the development of therapeutic agents. Natural amino acid substitutions in these proteins have previously been associated with disease progression. The aim of this study was to analyze the genetic diversity of Vif and Vpr in HIV-1 group M clades. A total of 5286 sequences were downloaded and analyzed. For 37 clades in group M, the consensus sequences, amino acid natural variation, and clade-specific amino acid residue substitutions (CSSs) were evaluated. Structural analysis and modeling of consensus sequences were performed for subtypes A1, B, C, and D. The average conservation degree in the HIV-1 group M was 86.4% for Vif and 91.3% for Vpr. In both proteins, the lowest amino acid diversity was observed in sub-subtype A6, and the highest in subtype B. In consensus sequences, the substitutions, which might influence pathogenesis, have been determined: in Vif—22H (11_cpx, 91_cpx) and 136P (A6, 01_AE, 15_01B, 59_01B, 89_BF1, 103_01B, 111_01C, 133_A6B), in Vpr—41N (06_cpx) and 55A (B, 07_BC, 35_01D, 56_cpx, 66_cpx, 66_BF1, 71_BF1, 85_BC, 137_0107). In functional motifs, CSSs associated with changes in the chemical properties of amino acid residues were noted. These findings could be taken into account for the development of therapeutic drugs in the future. No correlation was observed between the subtypes and the spatial organization of the oligomeric structures of Vif and Vpr. Using the structural analysis and modeling, it has been shown for the first time that Vif can interact with APOBEC3G as an oligomer. Full article

Mtx1 is a mosquitocidal protein that exhibits high toxicity toward Culex species. It is produced during the vegetative phase of Lysinibacillus sphaericus but at very low levels and is rapidly degraded. The low expression appears to result from a weak promoter and a potential regulatory stem-loop structure in the 5′ untranslated region. To investigate this regulation, promoter variants of mtx1 were constructed to disrupt stem-loop formation, and promoter activity was assessed using green fluorescent protein (GFP) as a reporter. Disruption of the inverted repeat resulted in approximately twofold higher fluorescence compared with the wild-type promoter in L. sphaericus 2297, indicating partial derepression of translation. To improve protein stability, Bacillus subtilis WB800N, a protease-deficient host, was employed for heterologous expression. Truncated Mtx1 (tMtx1) was secreted into the culture medium, and no obvious degradation products were detected by Western blot analysis under the conditions tested. Although the overall yield was low and not quantitatively determined, the secreted protein retained biological activity. Larvicidal assays showed elevated mortality in tMtx1-containing culture supernatants, with an estimated LC₅₀ at approximately a 1:83 dilution and detectable activity up to a 1:512 dilution relative to control cultures. These results demonstrate that the upstream inverted repeat contributes to partial repression of mtx1 expression in L. sphaericus and that protease-deficient B. subtilis can be used as a host for producing biologically active tMtx1, although further optimization will be required to improve yield. Full article

Cumulus cells (CCs) are a distinct population of granulosa cells (GCs) that surround the developing and ovulated mammalian oocyte. The features of their structural organization and the expression pattern of key genes significantly affect oocyte viability. Changes in the functional activity of the nucleus are often expressed in changes in the structure of nuclear bodies (NBs), including Cajal bodies (CBs). The diagnostic protein of CBs is coilin, which maintains their structural integrity. Using fluorescent and electron microscopy, we examined maternal aging-associated changes in coilin pattern in mouse CCs. We found that older mice had a decrease in the number of coilin-positive bodies, while external transcriptome data analysis revealed no significant changes in Coil and Smn1 gene expression. We hypothesized that the age-related dynamics of coilin-containing bodies are determined not by changes in the expression level of key components of these bodies, but by age-related changes in CC metabolism. Considering that CCs are a by-product of IVF protocols, making them available for analysis in sufficient quantities, age-related changes in the number and size of coilin-positive NBs in CCs may serve as a promising biomarker for assessing ovarian functional aging. Full article

Redox (reduction–oxidation) processes underlie all forms of life and are a universal regulatory mechanism that maintains homeostasis and adapts the organism to changes in the internal and external environments. From capturing solar energy in photosynthesis and oxygen generation to fine-tuning cellular metabolism, redox reactions are key determinants of life activity. Proteins containing sulfur- and selenium-containing amino acid residues play a crucial role in redox regulation. Their reversible oxidation by physiological oxidants, such as hydrogen peroxide (H₂O₂), plays the role of molecular switches that control enzymatic activity, protein structure, and signaling cascades. This enables rapid and flexible cellular responses to a wide range of stimuli—from growth factors and nutrient signals to toxins and stressors. Mitochondria, the main energy organelles and also the major sources of reactive oxygen species (ROS), play a special role in redox balance. On the one hand, mitochondrial ROS function as signaling molecules, regulating cellular processes, including proliferation, apoptosis, and immune response, while, on the other hand, their excessive accumulation leads to oxidative stress, damage to biomolecules, and the development of pathological processes. So, mitochondria act not only as a “generator” of redox signals but also as a central link in maintaining cellular and systemic redox homeostasis. Redox signaling forms a multi-layered cybernetic system, which includes signal perception, activation of signaling pathways, the initiation of physiological responses, and feedback regulatory mechanisms. At the molecular level, this is manifested by changes in the activity of redox-regulated proteins of which the redox proteome consists, thereby affecting the epigenetic landscape and gene expression. Physiological processes at all levels of biological organization—from subcellular to systemic—are controlled by redox mechanisms. Studying these processes opens a way to understanding the universal principles of life activity and identifying the biochemical mechanisms whose disruption causes the occurrence and development of pathological reactions. It is important to emphasize that new approaches to redox balance modulation are now actively developed, ranging from antioxidant therapy and targeted intervention on mitochondria to pharmacological and nutraceutical regulation of signaling pathways. This article analyzes the pivotal role of redox balance and its regulation at various levels of living organisms—from molecular and cellular to tissue, organ, and organismal levels—with a special emphasis on the role of mitochondria and modern strategies for influencing redox homeostasis. Full article

Saccharomyces cerevisiae was cultured under the influence of static magnetic fields (SMFs) to assess their impact on the biosynthesis of silver nanoparticles (AgNPs). Cell-free media derived from SMF-exposed cultures facilitated the formation of AgNPs, with a significant reduction in nanoparticle size observed at an optimal field strength of 7 mT. AgNPs synthesized under SMF conditions exhibited smaller crystalline structures than those produced in control media, as evidenced by dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. Over a 75-day period, SMF-exposed AgNPs demonstrated enhanced stability, as determined by DLS and polydispersity index (PDI) assessments. Further analysis through sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR) suggested the formation of a protein corona on the AgNPs in SMF-treated samples, which likely inhibits agglomeration and enhances long-term stability. These findings indicate that SMF-induced stress in S. cerevisiae triggers the secretion of specific proteins that contribute to the stabilization of AgNPs, providing a novel approach to controlling nanoparticle synthesis and stability through magnetic field exposure. Full article

The emergence of multidrug-resistant (MDR) bacterial pathogens has heightened the urgency for novel antibacterial agents. The bacterial cell wall usually comprises peptidoglycan, which presents a prime target for antibacterial drug development due to its indispensable role in maintaining cellular integrity. Conventional antibiotics such as β-lactams and glycopeptides hinder peptidoglycan synthesis through competitive binding of penicillin-binding proteins (PBPs) and sequestration of lipid-linked precursor molecules. Nevertheless, prevalent resistance mechanisms including target modification, β-lactamase hydrolysis, and multi-drug efflux pumps have limited their clinical utility. This comprehensive analysis explicates the molecular machinery underlying bacterial cell wall assembly, evaluates both explored and unexplored enzymatic nodes within this pathway, and highlights the transformative impact of high-resolution structural elucidation in accelerating structure-guided drug discovery. Novel targets such as GlmS, GlmM, GlmU, Mur ligases, D,L-transpeptidases are assessed for their inclusiveness for the discovery of next-generation antibiotics. Additionally, cell wall inhibitors are also examined for their mechanisms of action and evolutionary constraints on MDR development. High-resolution crystallographic data provide valuable insights into molecular blueprints for structure-guided optimization of pharmacophores, enhancing binding affinity and circumventing resistance determinants. This review proposes a roadmap for future innovation, advocating for the convergence of computational biology platforms, machine learning-driven compound screening, and nanoscale delivery systems to improve therapeutic efficacy and pharmacokinetics. The synergy of structural insights and cutting-edge technologies offers a multidisciplinary framework for revitalizing the antibacterial arsenal and combating MDR infections efficiently. Full article

Background: The apicomplexan parasite Toxoplasma gondii causes serious diseases in animals and humans. The in vitro efficacy of the antimicrobial peptide mixture tyrothricin, composed of tyrocidines and gramicidins, against T. gondii tachyzoites was investigated. Methods: Effects against T. gondii were determined by monitoring inhibition of tachyzoite proliferation and electron microscopy, host cell and splenocyte toxicity was measured by Alamar blue assay, and early embryo toxicity was assessed using zebrafish embryos. Differential affinity chromatography coupled to mass spectrometry and proteomics (DAC-MS-proteomics) was employed to identify potential molecular targets in T. gondii cell-free extracts. Results: Tyrothricin inhibited T. gondii proliferation at IC₅₀s < 100 nM, with tyrocidine A being the active and gramicidin A the inactive component. Tyrothricin also impaired fibroblast, T cell and zebrafish embryo viability at 1 µM. Electron microscopy carried out after 6 h of treatment revealed cytoplasmic vacuolization and structural alterations in the parasite mitochondrion, but these changes appeared only transiently, and tachyzoites recovered after 96 h. Tyrothricin also induced a reduction in the mitochondrial membrane potential. DAC-MS-proteomics identified 521 proteins binding only to tyrocidine A. No specific binding to gramicidin A was noted, and four proteins were common to both peptides. Among the proteins binding specifically to tyrocidine A were several SRS surface antigens and secretory proteins, mitochondrial inner and outer membrane proteins associated with the electron transfer chain and porin, and several calcium-binding proteins putatively involved in signaling. Discussion: These results suggest that tyrocidine A potentially affected multiple pathways important for parasite survival and development. Full article

In response to growing consumer interest in plant-based and eco-friendly diets, the food industry is seeking to enrich bakery products with functional plant proteins, which are highly nutritious and environmentally beneficial. This study aimed to evaluate the effect of incorporating pea protein isolate (PPI) and rice protein isolate (RPI) at levels of 5% and 15% on the structural and sensory quality and nutritional properties of vegan cookies. The addition of plant-derived proteins significantly influenced the colour and texture of the cookies. The addition of PPI increased lightness (L* up to 70.05 at 5%), whereas the addition of RPI caused pronounced browning (BI = 100.00 at 15%) and structural hardening at higher inclusion levels. Protein content increased with enrichment, reaching 9.49 g/100 g in the P15% sample (a 95.7% increase compared to the control sample). Total phenolic content increased markedly, particularly in the P15% sample, rising by 144%. However, this was not accompanied by a proportional increase in antioxidant activity, as determined by ABTS and DPPH assays. Sensory evaluation revealed that cookies enriched with 5% protein maintained high consumer acceptability (overall scores > 7.0), whereas higher levels of protein incorporation led to a significant reduction in sensory quality. The results indicate a trade-off between nutritional enhancement and sensory acceptability in vegan cookies enriched with pea and rice protein isolates, with 5% incorporation providing the most balanced outcome within the studied formulations. Full article

This study first adopted a liquid microbial-enzymatic co-fermentation process to enhance the nutritional value of walnut meal (WM) and sesame meal (SM), and systematically evaluated its effect on the nutrient digestibility of growing pigs. WM and SM are two underutilized high-protein by-products, whose application is hindered by anti-nutritional tannin and fiber. Optimal fermentation parameters were determined via single-factor experiments and response surface methodology, utilizing a consortium of Lactobacillus I, Candida utilis, and protease. Fermentation significantly reduced tannin (39.41% in WM) and crude fibre (28.79% in WM), reduced tannin (18.67% in SM) and crude fibre (4.00% in SM), while elevating crude protein (10.63% in WM, 7.47% in SM) and acid-soluble protein in both WM and SM. Results of the microstructure of fermented WM and SM revealed structural loosening, surface porosity, and polysaccharide degradation. Microbial community shifts highlighted the dominance of Lactobacillus and Bacillus in fermented substrates. In growing pigs, fermented WM and SM exhibited improved standardized ileal digestibility (SID) of key amino acids (threonine, tryptophan, valine; p < 0.05), alongside enhanced digestible energy (DE) and metabolizable energy (ME) for SM (p < 0.05). These findings demonstrate that liquid co-fermentation effectively degrades anti-nutritional factors, enhances nutrient bio-availability, and positions WM and SM as viable alternatives to conventional protein sources in swine diets, supporting strategies to reduce reliance on soybean meal. Full article

Necroptosis is an antiviral form of programmed cell death modulated by proteins that interact via RIP Homotypic Interaction Motifs (RHIMs). The result of the signaling pathways depends on which RHIM-containing proteins are involved: although both host and viral proteins contain RHIMs, virally encoded RHIM proteins, such as murine cytomegalovirus (MCMV)-encoded viral inhibitor of RIP activation (vIRA) serve to prevent cell death. Although every RHIM contains the same core four-amino-acid pattern, there are variations in individual sequences that we hypothesized would determine the differential outcomes in necroptotic signaling. As such, we replaced the RHIM in vIRA with the RHIMs from other proteins involved in the signaling cascade (RIPK1, RIPK3, ZBP1, ICP6) to assess the effect on necroptosis during MCMV infection. Although these RHIM-swap vIRA constructs remained able to bind to RIPK3, in the context of MCMV infection, they lost the ability to prevent necroptosis. These results are consistent with other studies that demonstrate that RHIM-containing proteins form amyloid fibrils unique to the proteins interfacing. Our results provide biological context for the growing model that the outcome of RHIM-based signaling is influenced by the specific amyloid fibril structures that are driven by the unique amino-acid sequences of each RHIM present. Full article

Clostridial neurotoxins, botulinum neurotoxins (BoNTs), and tetanus neurotoxin (TeNT) are potent toxins responsible for severe diseases, botulism and tetanus, respectively. BoNTs associate with non-toxic proteins (non-toxic non-hemagglutinin, hemagglutinins, and OrfXs), which protect BoNTs against acidic pH and protease degradation and facilitate BoNT passage through the intestinal barrier. TeNT enters motor neurons and undergoes a retrograde axonal transport until the target inhibitory interneurons in the central nervous system. BoNTs and TeNT recognize specific cell surface receptors which consist of complex sets of protein(s)-glycan-gangliosides and determine specific cell entry pathways. Recent data on structural and functional investigations of BoNT and TeNT receptors bring a better understanding of toxin trafficking in the host and entry into target neuronal cells, which is useful for the development of updated strategies of prevention and treatment of the corresponding diseases. Since clostridial neurotoxins, notably BoNTs, are important therapeutic tools, detailed knowledge of their activity opens the way of the development of engineered molecules for specific clinical applications. Full article

Vascular endothelial growth factor 165 (VEGF₁₆₅) stands out as a pivotal isoform of the VEGF-A protein and is critically involved in various angiogenesis-related diseases. Consequently, it has emerged as a promising target for diagnosing and treating such conditions. Structurally, VEGF₁₆₅ forms a homodimer, and each of its constituting monomers comprises a receptor-binding domain (RBD) and a heparin-binding domain (HBD). These two domains are linked by a flexible linker, and thus the overall structure of VEGF₁₆₅ remains incompletely understood. Aptamers are known as potent drugs that interact with VEGF₁₆₅, and dimeric aptamers that can simultaneously interact with two distant domains are frequently adopted to improve the potency. However, designing such aptamer dimers faces challenges in regard to determining the appropriate length of the linker connecting the two aptamer fragments. To gain insight into this distance information, we here employ biased molecular dynamics (MD) simulations with the umbrella sampling method, with the distance between the two HBDs serving as a reaction coordinate. Our simulations reveal an overall preference for compact conformations with HBD-HBD distances below 3 nm, with the minimum of the potential of mean force located at 1.1 nm. We find that VEGF₁₆₅ with the optimal HBD-HBD distance forms hydrogen bonds with its receptor VEGFR-2 that well match experimentally known key hydrogen bonds. We then try to computationally design aptamer homodimers consisting of two del5-1 aptamers connected by various linker lengths to target VEGF₁₆₅. Collectively, our findings may provide quantitative guidelines for rationally designing high-affinity aptamers for targeting VEGF₁₆₅. Full article

Slugs are significant agricultural pests and act as vectors for zoonotic parasites. However, current molluscicide options are limited and associated with substantial environmental risks. This study investigates the role of aldehyde dehydrogenase (ALDH) in the biosynthesis of farnesoic acid (FA), a key intermediate in the sesquiterpenoid hormone pathway, in two slug species: Philomycus bilineatus and Laevicaulis alte. Transcriptomic analysis revealed that both species possess conserved sesquiterpenoid biosynthetic pathways, yet they exhibit distinct levels of ALDH gene expression and differences in FA content. RNA interference (RNAi)-mediated gene silencing was employed to validate the potential of these candidate genes as targets for molluscicide development. Structural modeling of ALDH proteins using AlphaFold2 demonstrated notable divergence in the architecture of their active sites, suggesting species-specific enzymatic properties. Citral, a known inhibitor of ALDH, significantly reduced FA production in vivo and exhibited contact toxicity against both slug species. The lethal concentration 50 (LC₅₀) values were determined to be 378.2 g/L for P. bilineatus and 85.2 g/L for L. alte, respectively. Molecular docking analyses indicated that citral binds within the conserved substrate-binding tunnel of ALDH, potentially inhibiting the oxidation of farnesal. These findings establish ALDH as a critical enzymatic target for disrupting endogenous hormone biosynthesis in slugs and support the development of novel, eco-friendly molluscicides targeting the sesquiterpenoid pathway. Full article

RNA chaperones play a crucial role in the biogenesis and function of various RNAs in bacteria. They facilitate the interaction of small regulatory trans-encoded sRNAs with mRNAs, thereby significantly altering the pattern of gene expression in cells. This allows bacteria to respond quickly to changing environmental conditions, such as stress or adaptation to host organisms. Despite the identification of a large number of sRNAs in mycobacteria, none of the most common RNA chaperones have been found in their genomes. We determined the crystal structure of the cold shock protein CspB from Mycobacterium tuberculosis. It forms a dimer due to its elongated C-terminal region, which is a hairpin composed of two α-helices. It was also demonstrated that CspB from M. tuberculosis exhibits high affinity for MTS0997 sRNA and MTS1338 sRNA from the same organism, which is consistent with classical RNA chaperons such as Hfq and ProQ. Based on the putative RNA chaperone activity of bacterial proteins with cold-shock domains, we propose that CspB from M. tuberculosis may be involved in the regulation of mycobacterial pathogenesis through interaction with sRNAs. Full article

The prediction of polypeptide chain fragmentation during digestion (proteolysis) of protein substrates by trypsin was carried out for globular β-lactoglobulin (β-LG) and micellar β-casein (β-CN). Despite significant differences in the protein structures of these substrates, the concentrations of peptide fragments are calculated as functions of time or degree of hydrolysis using the same equations derived from the general proteolysis model. This model considers the opening of protein substrates in the course of proteolysis, the so-called demasking process, and the subsequent hydrolysis of specific peptide bonds at different rates determined by the amino acid sequence of hydrolyzed sites. The use of this model for in silico prediction of proteolysis is discussed. An algorithm for calculating demasking rate constants based on the experimental distribution of peptide fragments is presented. The calculated concentration dependence on the degree of hydrolysis of peptide bonds was compared with the experimental data for the intermediate and final peptide fragments of β-LG and β-CN. The predicted and experimental concentration curves for the final products were compared based on their curvatures. For both substrates, the predicted redistribution of peptide fragments in the course of proteolysis was found to be consistent with the experimental one. Full article