Search Results (1,221)

Animal venoms are rich in bioactive molecules with promising biotechnological potential. They comprise both protein and non-protein toxins. Among the protein toxins are enzymes, such as phospholipases A₂, proteases and L-amino acid oxidases (LAAOs). LAAOs exhibit antimicrobial, antiparasitic, antiviral, and anticancer effects, making them potential candidates for biotechnological applications. These activities are linked to their ability to catalyze oxidative reactions that convert L-amino acids into α-keto acids, releasing ammonia and hydrogen peroxide, which contribute to the immune response, pathogen elimination, and oxidative stress. However, in snakes of the Micrurus genus, LAAOs generally represent a small portion of the venom (up to ~7%), which limits their isolation and study. To overcome this, the present study aimed to produce Ml-LAAO, the enzyme from Micrurus lemniscatus, through heterologous expression in mammalian cells. The gene sequence was inferred from its primary structure and synthesized into the pSecTag2B vector for expression in HEK293T cells. After purification using a His Trap-HP column, the presence of recombinant Ml-LAAO (Ml-LAAOrec) was confirmed by Western blot and mass spectrometry, validating its identity. These results support successful recombinant expression of Ml-LAAO and highlight its potential for scalable production and future biotechnological applications. Full article

Prostate stem cell antigen (PSCA) is a Ly6/uPAR protein that targets neuronal nicotinic acetylcholine receptors (nAChRs). It exists in membrane-tethered and soluble forms, with the latter upregulated in Alzheimer’s disease. We hypothesize that PSCA may be linked to a wider spectrum of neurological diseases and could induce neuroinflammation. Indeed, PSCA expression is significantly upregulated in the brain of patients with multiple sclerosis, Huntington’s disease, Down syndrome, bipolar disorder, and HIV-associated dementia. To investigate PSCA’s structure, pharmacology, and inflammatory function, we produced a correctly folded water-soluble recombinant analog (ws-PSCA). In primary hippocampal neurons and astrocytes, ws-PSCA differently regulates secretion of inflammatory factors and adhesion molecules and induces pro-inflammatory responses by increasing TNFβ secretion. Heteronuclear NMR and ¹⁵N relaxation measurements reveal a classical β-structural three-finger fold with conformationally disordered loops II and III. Positive charge clustering on the molecular surface suggests the functional importance of ionic interactions by these loops. Electrophysiological studies in Xenopus oocytes point on ws-PSCA inhibition of α3β2-, high-, and low-sensitive variants of α4β2- (IC₅₀ ~50, 27, and 15 μM, respectively) but not α4β4-nAChRs, suggesting targeting of the β2 subunit. Ensemble docking and molecular dynamics simulations predict PSCA binding to high-sensitive α4β2-nAChR at α4/β2 and β2/β2 interfaces. Complexes are stabilized by ionic and hydrogen bonds between PSCA’s loops II and III and the primary and complementary receptor subunits, including glycosyl groups. This study gives new structural and functional insights into PSCA’s interaction with molecular targets and provides clues to understand its role in the brain function and mental disorders. Full article

With increasing interest and demand for plant protein-based foods, the allergenicity of plant proteins has been placed in a very important position. Among plant food allergens, peanuts have been considered the most potent because peanuts often cause severe allergic reactions, even life-threatening anaphylaxis. It is well-known that allergenic proteins in peanuts trigger peanut-induced allergic reactions through binding to the immunoglobulin E (IgE) antibodies in the patients sensitive to peanuts. So far, eighteen peanut allergens have been identified. These allergens belong to different protein superfamilies with distinctive characteristics and allergenic potentials. Due to the rapid rise in peanut allergy prevalence in the past few decades, many studies have been conducted to reveal the effects of primary structures (epitopes) and conformational structures of peanut proteins on their allergenicity and to explore methods and strategies to mitigate peanut allergenicity and peanut allergy. This comprehensive review highlights the nutritional value of peanut protein related to its amino acid composition, the current prevalence of peanut allergies and its impacts on quality of life, the recent research findings on the characteristics and allergenic potential of individual peanut allergens, and the potential and challenges of different approaches and methods for mitigating peanut allergenicity. Full article

Advancements in Raman spectroscopy have broadened the utilization possibilities for food applications. The present review covers the working principle and methodology of the emerging technique in the context of wheat (flour) as a bakery ingredient. Special attention is paid to the primary constituents of wheat flour, starch and gluten proteins, both in their isolated forms and within complex matrices such as flour, dough, and various end products. This review examines how compositional and structural variations in these components are reflected in their Raman spectra and imaging characteristics and how this can be interpreted in terms of quality and functionality. The review concludes by outlining prospective research directions and future opportunities for advancing Raman-based analysis in cereal and bakery science. Full article

Background/Objectives: The increasing global population and rising demand for protein-rich foods present significant challenges for the agri-food system. Cultivated meat, produced through cellular agriculture, is emerging as a promising alternative to traditional livestock farming, offering potential environmental and ethical benefits. However, its adoption remains controversial due to concerns about sustainability, safety, and cultural acceptance. This study investigates Italian consumers’ perceptions, knowledge, and willingness to purchase cultivated meat, considering psychological, demographic, and social factors. Methods: A structured online survey was conducted involving 437 Italian meat consumers, integrating established psychometric scales to assess key attitudes. Logistic regression analysis was applied to identify determinants of consumer acceptance. Results: Findings reveal that while awareness of cultivated meat is relatively high (81.92%), willingness to purchase it is low, at just 35.47%. The main motivations for interest are environmental sustainability (54.61%) and innovation appeal (25.00%), while the primary barriers are health concerns (31.58%) and doubts about production processes (34.59%). The results also show that food neophobia, environmental awareness, and inclination toward food innovation significantly influence purchasing decisions. Additionally, demographic factors, such as age, gender, income, and household size, play a crucial role. Conclusions: This study provides insights into consumer behavior toward food innovations, informing policymakers and industry stakeholders on strategies to enhance acceptance and promote sustainable food alternatives. Full article

Given the limited knowledge of the effect of post-translational modifications (PTMs) on protein structure, in this study we investigated whether introducing one-to-three RA-related PTMs into the α-fibrin (617–631) peptide influences the conformation and structure of the peptide antigen that could be responsible for the autoantibody recognition. Ten peptides containing a different number of PTMs within their primary structure were synthesized and their recognition by sera from RA patients was analyzed. The conformation of the peptides was studied by circular dichroism (CD) and the structure of the most relevant antigenic peptides was determined by nuclear magnetic resonance (NMR) and enhanced-sampling molecular dynamics (MD). Although peptides containing citrulline (Cit) showed a higher degree of binding to AMPAs than peptides containing only homocitrulline and/or acetyl-lysine, the latter were able to bind to AMPAs in sera that showed a small response to peptides with Cit, with the response being different depending on the position of each PTM. CD and NMR analyses indicated a series of half-turn conformations in the Lys620-Arg630 region. MD simulations generated a set of conformations compatible with the NMR NOEs. The effect of the PTMs was observed in intra-molecular contacts, hydrogen bonds and van der Waals interactions, generating more collapsed conformations. Differences in autoantibody reactivity between peptides bearing different PTMs within their primary structures are noted. Peptides with PTMs adopt different conformations than unmodified peptides, probably due to the lower net charge of peptides with multiple PTMs, which may explain their recognition by autoantibodies. Full article

When iron oxide nanoparticles are incubated together with a biological broth, the biomolecules compete for the binding sites at the solid–liquid interface. At the same time, the biomass rearranges in suspension, building agglomerated structures. Despite general knowledge of the forces involved in bio–nano interactions, gaps remain in the understanding of how biomolecules organize themselves in solution and onto surfaces. This work examines biomolecule adsorption onto metal oxide surfaces with the goal of strengthening this understanding, essential in industrial and natural processes. We demonstrate nearly complete separation of proteins from a biotechnological suspension for non-oxidized and highly oxidized magnetic nanoparticles. Varying the nanoparticle-to-biomass ratio, we find, can lead to different separation patterns, i.e., that selectivity using bare, low-cost materials is possible. Furthermore, we explore how preliminary “passivation” with a biological corona only partially reduces the ability to separate total protein mass from a new suspension in subsequent incubation steps. The study underscores the crucial role of concentration gradients with regard to targets and binding sites as the primary determinant of separation capacity and of biomolecule behavior in solution, highlighting the potential for using bio–nano coronae as biomolecule carriers across diverse fields, including environmental, biomedical, pharmaceutical and nutritional applications. Full article

The interfacial and foam properties of proteins can be enhanced by altering the interactions between polyphenols and proteins. The aim of this study was to determine the influence of gallic acid (GA) on the structural properties of whey protein isolate (WPI), specifically focusing on particle size, potential, and surface hydrophobicity, as well as the subsequent alterations in its interfacial and foam properties when utilized as a foaming agent. An increase in turbidity and a decrease in particle size suggested the formation of a soluble complex between GA and WPI at a pH of 6. The results from fluorescence spectroscopy and surface hydrophobicity analyses indicated that the primary interactions between GA and WPI are characterized by hydrogen bonding and hydrophobic interactions. The reduction in particle size enhances the capacity of WPI/GA complexes to lower the surface pressure, thereby demonstrating significant efficacy at the macroscopic scale. Furthermore, the structural connectivity of GA facilitates the formation of a stable interfacial film at the air–water interface by WPI/GA, resulting in high foam stability at a macroscopic level. This research contributes to a deeper understanding of the application of protein–polyphenol complexes as surfactants and provides theoretical support for their use in food applications. Full article

Isoniazid (isonicotinic acid hydrazide, INH) is a key drug used to treat tuberculosis (TB), which continues to be the world’s most lethal infectious disease. Nevertheless, the efficacy of INH has diminished because of the emergence of Mycobacterium tuberculosis (Mtb) strains that are resistant to INH. Our goal in this study was to modify INH to reduce this significant resistance chemically. We synthesized INH-based hydrazones (IP1–IP13) through the reaction of INH with in-house obtained benzaldehydes carrying a piperidine or piperazine ring in refluxing ethanol. Upon confirmation of their proposed structures by various spectral techniques, IP1–IP13 were evaluated for their antimycobacterial capacity against Mtb H37Rv strain and INH-resistant clinical isolates with katG and inhA mutations using the Microplate Alamar Blue Assay (MABA). The compounds were additionally tested for their cytotoxicity. The obtained data indicated that the compounds with moderately increased lipophilicity compared to INH (IP7–IP13) were promising antitubercular drug candidates, exhibiting drug-like properties and negligible cytotoxicity. Out of these, IP11 (N′-(4-(4-cyclohexylpiperazin-1-yl)benzylidene)isonicotinohydrazide) emerged as the most promising derivative, demonstrating the lowest MIC values against all Mtb strains tested. Subsequently, the target molecules were evaluated for their capacity to inhibit enoyl acyl carrier protein reductase (InhA), the main target enzyme of INH. Except for IP11 demonstrating 81% InhA inhibition at a concentration of 50 μM, direct InhA inhibition was shown not to be the primary mechanism responsible for the antitubercular activity of the compounds. The binding mechanism of IP11 to InhA was analyzed through molecular docking and molecular dynamics simulations. Altogether, our research identified a novel approach to modify INH to address the challenges posed by the rising prevalence of drug-resistant Mtb strains. Full article

BAR domains are a superfamily of widely conserved membrane binding motifs. In fungi, Pil1 family proteins are BAR domain containing proteins involved in organizing the plasma membrane. S. pombe encodes a sporulation-specific Pil1 family protein, Meu14, which has a specialized role in shaping the forespore membrane during sporulation. The functional analog of Meu14 in S. cerevisiae is Ssp1. While Ssp1 has no primary sequence homology to Pil1 or Meu14, AlphaFold predicts that it contains a Pil1-related BAR domain. Consistent with this structural prediction, mutation of residues in the putative lipid binding face of Ssp1 or in a residue implicated in multimerization disrupt sporulation. Characterization of the mutant proteins indicates that the BAR domain is necessary for recruitment of Ssp1 to the highly curved leading edge of the prospore membrane and multimerization of Ssp1 at that location is required for assembly of the leading edge complex. The distribution of Pil1 family proteins across an evolutionary tree of Ascomycetes reveals that Meu14 and Ssp1 arose independently in the lineages leading to S. pombe and S. cerevisiae, respectively. Full article

Follicular development is recognized as a highly complex biological process regulated by multiple factors. Thyroid hormone (TH) is considered one of the key regulators of female reproduction, and its dysregulation can significantly impair follicular development. Epigallocatechin gallate (EGCG), the main active component of green tea, possesses strong antioxidant properties. Numerous studies have demonstrated that EGCG positively influences reproductive function in both humans and animals. However, whether EGCG directly affects follicular development under conditions of TH dysregulation remains poorly understood. The primary objective of this study was to investigate the impact of hyperthyroidism on ovarian development, examine whether EGCG could mitigate the adverse effects of TH dysregulation, and elucidate the underlying molecular mechanisms. In the T₄-induced hyperthyroidism rat model, ovarian tissues were serially sectioned for Hematoxylin-Eosin (HE) and Masson’s trichrome staining to assess morphological changes, and follicle numbers were quantified at each developmental stage. Granulosa cell (GC) viability, proliferation, and apoptosis induced by T₃ were evaluated using CCK8, EdU, and TUNEL assays, respectively. Antioxidant enzyme activity was measured, and the expression levels of related proteins were analyzed via Western blotting. Results showed that hyperthyroidism altered ovarian structure, significantly increasing the number of atretic follicles. Levels of antioxidant enzymes, including Superoxide Dismutase (SOD), Glutathione Peroxidase (GSH-PX), and Catalase (CAT), were markedly decreased, whereas the lipid peroxidation product malondialdehyde (MDA) was significantly elevated. Furthermore, all ERS-related proteins, phosphorylated Eukaryotic Initiation Factor 2 Alpha (p-eIF2α), Activating Transcription Factor 4 (ATF4), C/EBP homologous protein (CHOP), and Caspase-3, were upregulated, accompanied by decreased glucose-regulated protein 78 (GRP78) expression. Treatment with EGCG alleviated these detrimental effects of hyperthyroidism. At the cellular level, high concentrations of T₃ reduced GC viability and proliferation while increasing apoptosis. Reactive oxygen species levels were elevated, and GRP78 expression was decreased. Notably, all T₃-induced effects were reversed by EGCG treatment. In summary, this study demonstrates that hyperthyroidism induces oxidative stress in GCs, which triggers endoplasmic reticulum stress via the eIF2α/ATF4 pathway and leads to apoptosis. EGCG mitigates apoptosis by enhancing antioxidant capacity, thereby preserving ovarian function. These findings establish EGCG as a protective agent for maintaining ovarian health and fertility. Full article

Penicillin-binding proteins (PBPs) are essential enzymes involved in bacterial cell wall biosynthesis and represent the primary targets of β-lactam antibiotics. However, the efficacy of these agents is threatened by β-lactamase production and PBP alterations, prompting the search for alternative strategies. In this context, boronic acids, long established as potent inhibitors of serine β-lactamases (SBLs), have been proposed as scaffolds for PBP inhibition based on the shared structural and mechanistic features of these enzyme families. This perspective provides a literature-based survey with structural analysis to evaluate emerging evidence on the potential role of boronic acids as PBP-targeting agents, with a particular focus on peptidomimetic boronic acids, repurposed β-lactamase inhibitors, and novel scaffold architectures. While early work showed limited activity against low-molecular-mass PBPs, more recent compounds, particularly certain bicyclic boronates, have demonstrated potent binding and, in some cases, antibacterial activity. Structural analyses reveal diverse binding modes and underscore the role of conformational dynamics in modulating affinity. Despite these advances, significant challenges remain, including target selectivity, membrane permeability, and species-specific differences. Nevertheless, the direct inhibition of PBPs by boronic acids, while still in early development, may offer a viable complement or alternative to β-lactam therapy, warranting further exploration through structure-guided design and comprehensive biological evaluation. Here, we analyze the potential of boronic acid inhibitors (BAIs) to target PBP enzymes, considering their promise as non-β-lactam antimicrobial agents with possible clinical relevance. Full article

The BRAF protein regulates cell growth and division through key signaling pathways. Mutations in BRAF, particularly the V600E variant, are frequently observed in colorectal cancer (CRC) and are associated with poor prognosis and therapeutic challenges. Tumors harboring certain BRAF mutations often exhibit primary resistance to BRAF inhibitor monotherapies. Over time, these tumors can also develop acquired resistance, further complicating treatment. In this study, we employed replica exchange molecular dynamics simulations combined with machine learning techniques to investigate the structural alterations induced by BRAF mutations and their contribution to drug resistance. Our analyses revealed that conformational changes in mutant BRAF proteins associated with dabrafenib residues psi494, phi600, phi644, phi663, psi675, and phi677 were sufficient for classifying drug-resistant vs. drug-sensitive variants. Similarly, for vemurafenib, residues psi450, phi484, phi495, phi518, psi622, and phi622 were the key residues that influence drug binding and resistance mechanisms. These residues are located in the N-lobe of CR3, which is responsible for ATP binding and the regulation of BRAF kinase activity. These findings offer deeper insights into the molecular basis of BRAF-driven resistance and provide predictive models for phenotypic outcomes of various BRAF mutations. The study underscores the importance of targeting specific BRAF variants for more effective, personalized therapeutic strategies in drug-resistant CRC patients. Full article

Background: Premenopausal women typically exhibit superior glucose metabolism compared to males, but this metabolic advantage is lost after menopause. The primary cause is the sharp decline in estrogen levels post-menopause. Genistein, a natural compound predominantly derived from leguminous plants, possesses structural similarity to estrogen. This enables specific binding to estrogen receptors, allowing genistein to exert estrogen-mimicking effects under conditions of estrogen deficiency. The aim of this study was to investigate the effects and potential mechanisms of genistein on glucose metabolism in the liver and skeletal muscle of ovariectomized (OVX) mice fed a high-fat diet (HFD). Methods: Animal experiments were performed using 8-week-old mice that were OVX to construct a model of estrogen deficiency and impaired their glucose metabolism by a continuous HFD. Genistein was administered by gavage (50 mg/kg-day) for 10 weeks and 17β-estradiol was administered subcutaneously (50 μg/kg) every 4 days for 10 weeks as a positive control. Results: Genistein significantly improved glucose metabolism (including fasting glucose, postprandial glucose, serum glucose levels, and HOMA-IR index) but did not affect serum estrogen levels and uterine weights in OVX mice. Genistein promoted increased expression and translocation of glucose transporter 4 (GLUT4) in the gastrocnemius muscle, enhanced phosphorylation of the PI3K/AKT pathway, and upregulated expression of the G protein-coupled estrogen receptor (GPER). Concurrently, it stimulates hepatic glycogen accumulation and upregulates GLUT2 expression in the liver. Conclusions: GEN improves glucose metabolism in ovariectomized mice, and this improvement is primarily attributed to increased expression and membrane translocation of GLUT4 in the gastrocnemius muscle mediated by the GPER-PI3K/AKT pathway. Full article

Sindbis virus (SINV), a prototype of the Alphavirus genus (family Togaviridae), is a globally distributed arbovirus causing febrile rash and debilitating arthritis in humans. Viral structural proteins—capsid (C), E1, and E2—are fundamental to the virion’s architecture, mediating all stages from assembly to host cell entry and pathogenesis, thus representing critical targets for study. This review consolidates the historical and current understanding of SINV structural biology, tracing progress from early microscopy to recent high-resolution cryo-electron microscopy (cryo-EM) and X-ray crystallography. We detail the virion’s precise T = 4 icosahedral architecture, composed of a nucleocapsid core and an outer glycoprotein shell. Key functional roles tied to protein structure are examined: the capsid’s dual capacity as a serine protease and an RNA-packaging scaffold that interacts with the E2 cytoplasmic tail; the E1 glycoprotein’s function as a class II fusion protein driving membrane fusion; and the E2 glycoprotein’s primary role in receptor binding, which dictates cellular tropism and serves as the main antigenic target. Furthermore, we connect these molecular structures to viral evolution and disease, analyzing how genetic variation among SINV genotypes, particularly in the E2 gene, influences host adaptation, immune evasion, and the clinical expression of arthritogenic and neurovirulent disease. In conclusion, the wealth of structural data on SINV offers a powerful paradigm for understanding alphavirus biology. However, critical gaps persist, including the high-resolution visualization of dynamic conformational states during viral entry and the specific molecular determinants of chronic disease. Addressing these challenges through integrative structural and functional studies is paramount. Such knowledge will be indispensable for the rational design of next-generation antiviral therapies and broadly protective vaccines against the ongoing threat posed by SINV and related pathogenic alphaviruses. Full article