A series of thiadiazol–hydrazine derivatives (3a–3d) were rationally designed, synthesized, and characterized to explore their antimicrobial potential and possible interactions with dihydrofolate reductase (DHFR). The antimicrobial activity of the synthesized compounds was evaluated against representative Gram-positive bacteria (S. aureus and S. pyogenes), Gram-negative bacteria (E. coli and P. aeruginosa), and fungal strains (C. albicans, A. niger, and A. clavatus) using the minimum inhibitory concentration (MIC) method. Among the synthesized derivatives, compound 3b exhibited the most favorable overall antimicrobial profile, particularly against the tested bacterial strains, whereas the antifungal activity of the derivatives was comparatively limited. To investigate the possible molecular basis of the observed antimicrobial activity, molecular docking studies were performed against dihydrofolate reductase (DHFR; PDB ID: 1AOE). The synthesized derivatives exhibited favorable predicted binding within the DHFR active-site region, with compound 3b showing the most favorable docking score among the investigated compounds. The predicted binding mode of 3b was characterized by hydrogen-bonding and hydrophobic interactions with residues lining the DHFR binding pocket. Molecular dynamics simulations and interaction-energy analyses were subsequently employed to examine the stability and dynamic behavior of the predicted protein–ligand complexes. The simulation results supported the persistence of the predicted interactions and provided additional computational evidence for the favorable accommodation of 3b within the DHFR binding site. Density functional theory (DFT) calculations at the B3LYP-D3BJ/def2-TZVP level were performed to investigate the electronic properties of the synthesized derivatives. The calculated frontier molecular orbital characteristics and related electronic descriptors provided complementary insights into the influence of structural variation on molecular reactivity and the observed biological activity. Furthermore, haemolytic toxicity studies demonstrated low erythrocyte membrane disruption (< 3
Sodium dodecyl sulfate (SDS) is widely used in protein research. Micellar binding of SDS is accepted as a major mechanism of SDS binding to proteins. Here, we investigated SDS-protein interactions using near-UV circular dichroism (CD), fluorescence spectroscopy, and SDS-gel electrophoresis. Bovine serum albumin (BSA), IgG, and lysozyme showed unfolding by SDS without heating or disulfide reduction as determined by SDS-gel electrophoresis. Near-UV CD and fluorescence spectroscopy showed changes in local structures surrounding tryptophan residues upon addition of 0.05–1
The aim of this study was to develop a bioactive keratin-based nanocomposite for modulation of tyrosinase structure and function. Keratin nanoparticles (KNPs) were synthesized from ostrich feathers and functionalized with Diospyros lotus leaf extract (DLE). Physicochemical analyses (XRD, FT-IR, ζ-potential, DLS, TEM) confirmed a stable surface-functionalized nanocomposite, evidenced by a ζ-potential shift from − 16.9 mV to -40.9 mV and increased particle sizes (TEM: 87.1 to 113.3 nm; DLS: 275.3 to 325.2 nm). DLE-KNPs exhibited dose-dependent inhibition of mushroom tyrosinase with an IC50 of 0.077 mg/mL. Kinetic evaluation revealed a mixed-type inhibitory mechanism, supported by fluorescence quenching (KA=1.83 mL/mg). Far-UV CD spectroscopy showed a reduction in α-helical content (14.1
Gasdermin E (GSDME), a pivotal executor of pyroptosis, has emerged as a central regulator of immune responses across a spectrum of diseases. Initially identified as a tumor suppressor and a deafness-associated gene (DFNA5), GSDME is now recognized for its complex, context-dependent roles in health and pathology. Its activation, primarily via caspase-3 cleavage, converts apoptotic signals into a lytic, pro-inflammatory form of cell death, releasing damage-associated molecular patterns and cytokines that profoundly shape the immune microenvironment. This review synthesizes the current research landscape of GSDME, detailing its structural characteristics and canonical activation mechanisms, alongside recent discoveries of non-canonical, cleavage-independent pathways. We systematically dissect its dualistic functions in major disease categories—cancer, inflammatory/autoimmune disorders, neurodegenerative conditions, and organ injuries—highlighting the key cell-type-specific signaling pathways and their resultant immunomodulatory outcomes. Furthermore, we evaluate the burgeoning clinical potential of GSDME as a diagnostic/prognostic biomarker and a therapeutic target. We critically analyze the functional characteristics of emerging pharmacological strategies designed to either activate or inhibit the GSDME pathway, including small molecules, epigenetic modulators, and advanced nanoplatforms. Finally, we outline future research directions and propose a framework for the clinical translation of GSDME-targeted therapies, emphasizing the need for precision medicine approaches to harness its immunostimulatory potential in oncology while restraining its pathological role in inflammatory diseases.
Research publications on various aspects of functional genomics are constantly growing, providing an opportunity and challenge to mine for the information of interest, among them are the annotation of proteins into their specific functional class or the sheer degeneracy of their functions. While knowledge-driven approaches to functional annotations are based on mechanistic basis or data-driven predictive models based on deterministic features, they do not harness what is already reported in literature in different contexts. Natural language processing, combined with machine learning aims to bridge this gap. We have earlier developed a method to predict an intriguing protein functional property, called moonlighting in DNA-binding proteins using protein features with reasonable accuracy. However, the very development of training data and harnessing of available functional information from literature are the tasks, not addressed well so far for this problem. It may be noted that moonlighting being a problem of functional redundancy, literature mining may be a way to provide a cross-study perspective and hence a better prediction performance. Here we present an NLP-based model for literature mining and identifying moonlighting behaviour of proteins. A high-performing PubMed BERT model pre-trained on PubMed publications was further optimized through retraining on particular data sets, allowing accurate identification of moonlighting function in proteins. We show that this approach can identify moonlighting proteins with high accuracy and outperform first principle approaches reported earlier. The methods presented here are for moonlighting behaviour of proteins but are scalable to any literature-mining problem in biological domain. Data sets and codes used in this work are provided in GitHub repository https://github.com/Sciwhylab/moonlighting_nlp .
Neuroinflammation is a key process that shapes tumor progression and cellular stress responses. While pro-inflammatory signaling components amplify these responses, IL-37a functions as an endogenous suppressor that limits inflammation. Melittin is a bioactive peptide with cytotoxic and immunomodulatory properties; however, its effects on IL-37a-centered immune regulation in neuroblastoma cells remain unclear. SH-SY5Y neuroblastoma cells were treated in vitro with melittin at concentrations of 1–32 µM for 24 and 48 h. Cell viability was assessed using the MTT assay. Expression levels of selected genes related to inflammation and extracellular matrix were analyzed by qRT-PCR. Targeted gene expression data were further used for protein–protein interaction network analysis, pathway enrichment analyses, and multivariate statistical approaches. In addition, potential interactions between melittin and STAT3 or IL-1R8 were evaluated by molecular docking, and ADMET properties were predicted. Melittin induced dose- and time-dependent cytotoxicity in SH-SY5Y cells, with an IC50 value of 16 µM at 24 h. Melittin treatment led to a significant increase the expression of IL-37a, IL-18R1, and IL-1R8, while suppressing NF-κB, STAT3, MyD88, and Smad3. Caspase-1 expression was elevated, suggesting modulation of inflammation-associated cell death pathways. Extracellular matrix remodeling was differentially regulated, with increased MMP-2 and decreased MMP-9 expression. Multivariate analyses revealed a treatment-specific gene expression pattern. Molecular docking analyses suggested potential interactions between melittin and STAT3 or IL-1R8, while ADMET analysis indicated limited permeability and certain safety constraints. Melittin may emerge as a molecule that modulates inflammation-related gene expression in neuroblastoma cells and may be associated with IL-37a-related regulatory responses. These findings suggest that melittin could act as a potential modulator of inflammation-associated signaling pathways.
Enterotoxigenic Escherichia coli (ETEC) remains a major cause of diarrheal disease, and colonization factors together with enterotoxins are key targets for vaccine development. In this study, three ETEC-derived chimeric proteins, Cssa-Cssb-LTB, CfaB-ST-CfaE-LTB, and CooD-CotD, were produced and evaluated in free and chitosan nanoparticle-formulated forms. The recombinant proteins were expressed in E. coli BL21 (DE3) using the pET-28a vector, purified by Ni-NTA affinity chromatography, and confirmed by western blotting. Chitosan nanoparticles containing each recombinant protein were prepared and used for immunization of BALB/c mice. Antigen-specific antibody responses were assessed by ELISA. Cssa-Cssb-LTB was purified under soluble/native conditions, whereas CfaB-ST-CfaE-LTB and CooD-CotD were purified under denaturing conditions. The mean sizes of nanoparticles containing Cssa-Cssb-LTB, CfaB-ST-CfaE-LTB, and CooD-CotD were 121.5, 24.93, and 63.22 nm, respectively. Immunological evaluation showed that the cocktail formulation induced the strongest immune response, followed by the oral–injection regimen. The recombinant chimeric proteins induced marked humoral immune responses, and formulation in chitosan nanoparticles enhanced their immunogenic potential. These findings support the potential of chimeric antigen-loaded chitosan nanoparticles as a promising strategy for ETEC vaccine development.
A low-molecular-weight cytoplasmic protein called heart-type fatty acid-binding protein (H-FABP) is released by cardiac myocytes during an ischemic episode. The normal serum level of FABP is around 3.5 ± 0.4 ng/ml (males), 3.9 ± 0.4 ng/ml (females), while the cutoff value of H-FABP in case of acute myocardial infarction (AMI) is 21.85 ng/ml within 3 hours. In case of angina pectoris, ischemic patients can be distinguished from non-ischemic patients and treated appropriately by detecting the presence of serum H-FABP in a point-of-care (POC) diagnostic approach. As the mortality rate due to coronary heart disease is on the rise worldwide, serum H-FABP levels can act as a diagnostic marker to assist in the prediction of probable cardiac arrest. In the present work, a small peptide against H-FABP has been screened by phage DNA library screening, which can be used for the development of a rapid and accurate diagnostic system for serum H-FABP. The cloning, expression and purification of the H-FABP protein in bacteria and screening of a 12 amino-acid long screened peptide with moderate affinity for binding to H-FABP is reported. The interaction of the said peptide with H-FABP was confirmed by in-silico methods like molecular docking and molecular dynamics simulation. The affinity of the peptide binding to H-FABP was assessed using Isothermal Titration Calorimetry (ITC). A spontaneous process is indicated by ΔG (-7.98 kcal mol−1) values, but an endothermic interaction activity is implied by positive ΔH (3.11 kcal mol−1) values. In a futuristic approach, this peptide can be exploited for the development of an accurate and early detection technique for heart ischemia.
Bioactive peptides (BAPs) are short amino acid sequences released from precursor proteins through enzymatic hydrolysis, fermentation, or gastrointestinal digestion. Due to their diverse biological properties, BAPs have attracted significant attention for potential applications in functional foods, nutraceuticals, and therapeutic research. These peptides exhibit a wide range of activities, including antihypertensive, antioxidant, antimicrobial, anti-inflammatory, immunomodulatory, and anticancer effects, although the majority of current evidence remains derived from in vitro and preclinical studies. This comprehensive critical review summarises the major sources, production strategies, structural characteristics, and biological functions of food-derived bioactive peptides. Emphasis is placed on structure–activity relationships (SAR), residue-specific functional contributions, and molecular mechanisms underlying peptide bioactivity, including enzyme inhibition, membrane interaction, reactive oxygen species scavenging, and modulation of intracellular signalling pathways. In addition, the review critically evaluates translational and clinical limitations associated with bioactive peptides, including poor bioavailability, gastrointestinal instability, variability in peptide purification, and limited large-scale clinical validation. Comparative analytical synthesis further highlights the relationship between peptide sequence, structural features, and functional efficacy across different peptide classes. Overall, this comprehensive critical review provides an integrated synthesis of current evidence by combining structure–activity relationships (SAR) with mechanistic and translational insights, while highlighting major research gaps, clinical limitations, and future directions for peptide design, clinical evaluation, and functional food applications.
The persistent rise in demand for biocatalysts in industries has prompted the exploration of novel enzymes. Proteases are very important class of enzymes with many industrial applications. However, majority of the reported proteases are from mesophiles and exhibit low stability under harsh conditions limiting their potential applications. This study presents the expression and characterization of a novel thermostable recombinant subtilisin-like serine protease derived from Thermococcus onnurineus NA1. The subtilisin serine protease gene was successfully cloned and expressed in E. coli BL21. The recombinant protease was purified by affinity chromatography and has an estimated molecular mass of 29 kDa following SDS-PAGE analysis. It exhibited an optimal activity at a pH of 10 and temperature of 90 °C. The recombinant subtilisin-like serine protease maintained 96
Venom-derived peptides represent a promising new class of biotherapeutics for oral squamous cell carcinoma (OSCC), offering intrinsic tumor selectivity and multimodal anticancer activity that address key limitations of current chemoradiotherapy. By exploiting the aberrant biophysical features of OSCC cell membranes—including externalized anionic phospholipids, enhanced membrane fluidity, and microvilli-rich surface topology—cationic, amphipathic venom peptides preferentially bind to and disrupt malignant cells, triggering mitochondrial depolarization, reactive oxygen species generation, cytochrome‑c release, and caspase‑dependent apoptosis, alongside autophagy-associated death and regulated necrosis. Beyond direct cytotoxicity, these peptides modulate critical oncogenic pathways by inducing cell‑cycle arrest via cyclin-dependent kinase inhibition, targeting overexpressed ion channels, and suppressing epithelial–mesenchymal transition, extracellular matrix degradation, migration, and VEGF‑driven angiogenesis, thereby attenuating invasion and metastatic dissemination in OSCC models. Concurrently, selected venom peptides remodel the immunosuppressive tumor microenvironment through macrophage repolarization, dendritic-cell activation, enhancement of cytotoxic T‑cell responses, and interference with immune checkpoint signaling, linking rapid tumor debulking with more durable immune control. Recent advances in nanotechnology—including liposomes, polymeric and gold nanoparticles, chitosan-based systems, silica nanocarriers, and extracellular vesicles—have substantially improved venom peptide stability, pharmacokinetics, tumor-specific accumulation, and stimulus-responsive release, while mitigating systemic toxicity and hypersensitivity risks in preclinical OSCC models. However, translation to routine clinical practice still requires rigorous resolution of immunogenicity, off‑target effects, manufacturing scalability, and standardized dosing through well-designed, OSCC-focused preclinical studies and early-phase trials. Overall, this review positions venom-derived peptides not merely as cytolytic toxins but as a modular platform for precision, multi-targeted intervention in oral cancer, integrating direct membrane lysis, signaling reprogramming, anti-angiogenic and anti-metastatic effects, and onco-immunomodulation into a unified therapeutic strategy.
Procaspase-8 activation at the death-inducing signalling complex (DISC) is largely blocked by short c-FLIP isoforms (c-FLIPS). The interaction between c-FLIPS and procaspase-8 is mediated by their tandem death effector domains (tDED). The canonical model of tDED interaction suggests that c-FLIPS uses H1a/H4a α helices of DED1 to interact with the H2b/H5b α helices of procaspase-8 DED2. Here, based on the NMR chemical shift perturbation of c-FLIPS (F114G) resonances induced by titration with peptides derived from the helical regions of procaspase-8, a model structure of the c-FLIPS:procaspase-8 complex was constructed using HADDOK. The complex revealed that their interactions are mediated by the two binding clefts on DED1 of c-FLIPS and helices H1a/H4a of procaspase-8, the two clefts are formed by the α helices H1a, H5a, and H7a. The binding clefts on DED1 of c-FLIPS were also suggested to bind nuclear factor kappa B (NF-κB) essential modulator (NEMO) and initiate NF-κB pathway activation. To further investigate the interaction between c-FLIPS and NEMO, a c-FLIPS:NEMO complex model was also constructed. Two complex structural models revealed that c-FLIPS may interact with procaspase-8 and NEMO via a common binding surface. Furthermore, we hypothesized that c-FLIPS uses a similar binding surface to interact with both procaspase-8 and NEMO, thereby promoting cell survival by inhibiting caspase-8 activation and inducing NF-κB activity.
Attention is drawn to the availability of a version of the Scatchard expression that accommodates multivalence of the ligand. Its lack of application over the past four decades by biological researchers is surprising in view of the popularity of its univalent counterpart. This summary of its derivation and limited application presents published examples of situations where the curvilinearity of conventional Scatchard plots has been misinterpreted as signifying heterogeneity or negative cooperativity of binding sites on erythrocyte ghosts instead of tetravalence of glycolytic enzymes in their interaction with band 3 protein. Also presented is the rectangular hyperbolic relationship of which the generalized Scatchard expression is the linear transform.
Bioactive compounds, such as peptides, proteins, and protein complexes, including those with antioxidant properties, are abundant in marine organisms. However, the antioxidant properties of protein complexes from sea cucumbers are underexplored. In this study, we show for the first time that a stable multi-protein complex (SMPC) from the sea cucumber E. fraudatrix possesses oxidoreductase activities. The SMPC was obtained by sequential gel-filtration on Sepharose 4B and Sephacryl S-1000 sorbent. Using a spectrophotometric method and an in situ activity assay, showed that the SMPC catalyzed hydrogen peroxide decomposition (specific activity 85.1 ± 4.4 mM H2O2/min/mg) as well as 3,3′-diaminobenzidine (DAB) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidation both in the presence (84.2 ± 3.1 mM/min/mg; 285.1 ± 11.2 mM/min/mg) or absence (61.4 ± 1.8 mM/min/mg; 178.3 ± 5.0 mM/min/mg) of hydrogen peroxide respectively. The data obtained showed that the SMPC from E. fraudatrix possesses oxidoreductase activities and can be involved in reactive oxygen species level regulation. This study highlighted the need for further research on SMPCs and their potential role in marine organisms.
Cerium vanadate (CeVO4)–based materials have attracted attention due to their diverse physicochemical and biological properties; however, their interactions with detoxification-related enzymes remain insufficiently characterized. This study evaluated the in vitro effects of alkaline earth metal–doped CeVO4 derivatives (Be, Mg, Ca, Sr, and Ba) on human serum paraoxonase-1 (PON1) paraoxonase activity. PON1 was purified from human serum by hydrophobic interaction chromatography (Sepharose 4B–L-tyrosine–1-naphthylamine), and enzymatic activity was measured spectrophotometrically using paraoxon as the substrate. Inhibitory effects were assessed across multiple concentrations, and IC50 values were determined from concentration–response curves. All derivatives exhibited concentration-dependent inhibition of PON1 activity, with IC50 values ranging from 117 to 213 µM. Mg- and Ba-doped CeVO4 derivatives demonstrated the strongest inhibition at higher doping ratios, whereas Ca-doped derivatives showed reduced inhibitory potency with increasing substitution levels. These findings indicate that dopant identity and concentration significantly influence the interaction between CeVO4 derivatives and PON1. The study provides insight into metal oxide–enzyme interactions and highlights the importance of dopant-dependent effects in evaluating the biological and potential toxicological behavior of engineered vanadate-based materials.
Some proteins can be used as carriers for certain polyphenols (such as curcumin) to address the issue of their low bioavailability and to develop novel functional foods. As a result, one of the purposes of this review is to provide a brief overview of polyphenol carrier proteins. Bovine serum albumin (BSA), in particular, has the potential to become an ideal carrier material for curcumin encapsulation. Thus, for the possible use of curcumin-loaded BSA particles to reduce breast cancer incidence, an assessment of the impact of BSA particle size and curcumin concentration at pH 7 on curcumin encapsulation efficiency using BSA represents a valuable contribution to the BSA’s use as a delivery system for curcumin. This forms the main goal of this review. Hydrophobic interactions should play a relevant role in BSA-curcumin binding process such that, the main binding site on the BSA molecules for the curcumin molecules should be located inside a hydrophobic binding pocket of the BSA subdomain IIA near Trp213. On the other hand, when account is taken only of the impact of pH conditions on the BSA particle physicochemical characteristics, the largest size of BSA particles should correspond to BSA isoelectric point, and the larger the size of BSA particles, the higher the curcumin encapsulation efficiency. In addition to BSA particle size, curcumin concentration has a strong influence on the efficiency of its encapsulation. However, BSA particle size and curcumin-to-BSA ratio should be optimized for the possible use of curcumin-loaded BSA particles to reduce breast cancer incidence.
In this study, three types of fatty acid side chains were selected to conjugate with different insulin analogue precursors, leading to the design and synthesis of 11 novel insulin analogues. We selected three modifiers to modify the precursor of the insulin analogue. Liquid chromatography-mass spectrometry analysis confirmed that the actual molecular weight of the samples was consistent with the theoretical value. After confirming the structural integrity of the samples, their biological activities were evaluated to verify the necessity and functionality of the modifications. A preliminary assessment was made of the biological activities of all compounds on the HEK293/Luc/INSR cell line, successfully identifying candidate analogue 5. Further animal experiments were conducted on analogue 5. Through analysis of the body weight, blood drug concentration and AUC of diabetic rats, analogue 5 exhibited good hypoglycemic activity, effectively prolonging the drug action time and having an ideal application prospect.
