Search Results (2,593)

Background/Objectives: Antibiotic resistance in bacteria poses a major health problem worldwide. Therefore, to counteract this life-threatening problem, we sought to investigate in the present study the possible potentiation of the efficacy of amoxicillin (AMX) and clavulanic acid (CA) by 1,8-cineole (CN), a candidate resistance-modulating agent. The approach seeks to investigate, in vitro and in silico, the interactions among these three molecules. Methods: The antibacterial activity was determined against resistant Escherichia coli (E. coli) using microdilution methods, synergy tests, and time-kill assays for AMX, CA, and CN, used either separately or in combinations: AMX–CA, AMX–CN, CA–CN, and AMX–CA–CN. Furthermore, an in silico drug design methodology was employed, utilizing an integrated workflow that combines Density Functional Theory (DFT) for ligand optimization with molecular docking simulations to evaluate binding energies and interactions between the penicillin-binding protein (PBP) and ligands. Results: In vitro synergy experiments and time-kill assays revealed substantial antibacterial efficacy of the AMX–CA–CN combination. In silico analyses, performed under the simplifying assumption of a pre-assembled multi-ligand entity, were consistent with these findings: within our docking model, the AMX–CA–CN combination exhibited the most favorable computed binding affinity to a representative penicillin-binding protein (PBP3, PDB 7ONW). Conclusions: 1,8-cineole can potentiate the antibacterial effects of AMX–CA, indicating that the AMX–CA–CN combination warrants further evaluation as a candidate adjunctive strategy against ESBL-producing E. coli. Full article

Background: Polymorphonuclear (PMN) leukocyte recruitment to activated pulmonary endothelium is a central mechanism in acute respiratory distress syndrome (ARDS). This process is mediated by selectins and their counter-ligand, P-selectin glycoprotein ligand-1 (PSGL-1), encoded by SELPLG. Genetic variation in SELPLG has been associated with ARDS susceptibility, while disruption of PSGL-1/P-selectin interactions attenuates lung injury in preclinical models. Because inflammatory stimuli increase both SELPLG expression and circulating PSGL-1 levels, PSGL-1 represents a promising biomarker and therapeutic target. We sought to define the genetic determinants of plasma PSGL-1 levels and evaluate their causal relationships with key inflammatory and endothelial biomarkers. Methods: Genome-wide association study (GWAS) summary statistics for plasma PSGL-1 levels were obtained from the UK Biobank Pharma Proteomics Project (n = 35,571) and the SCALLOP consortium (n = 21,758 across 13 cohorts). Associated variants underwent functional annotation and in silico analyses to identify potential effects on protein structure and gene regulation. Bidirectional Mendelian randomization (MR) was performed using GWAS summary statistics for C-reactive protein (CRP), E-selectin, GlycA, and soluble intercellular adhesion molecule-1 (sICAM-1) to assess potential causal relationships with PSGL-1 levels. Results: Multiple cis- and trans-acting loci were significantly associated with plasma PSGL-1 concentrations. Three coding SELPLG variants (rs201689859, rs74792300, and rs139943851) were predicted to alter PSGL-1 protein structure and were associated with lower circulating PSGL-1 levels. Four promoter variants (rs1420663, rs1833245, rs1420664, and rs8179110) were linked to altered transcriptional activity, including a potential effect of rs1420664 on hypoxia-inducible factor binding. Bidirectional MR demonstrated that genetically predicted CRP, E-selectin, GlycA, and sICAM-1 levels were associated with increased plasma PSGL-1 concentrations. Additional loci implicated pathways related to immune signaling, cell adhesion, and protein stability. Conclusions: Large-scale GWAS and Mendelian randomization analyses identified genetic variants that regulate plasma PSGL-1 levels and demonstrated causal links between inflammatory and endothelial biomarkers and PSGL-1 expression. These findings provide new insights into the genetic regulation of leukocyte trafficking pathways and support a role for PSGL-1 in inflammatory diseases, including ARDS, sepsis, and cardiovascular disorders. Full article

Early childhood exposure to bisphenol A (BPA) is closely associated with autism spectrum disorder (ASD), though the precise molecular mechanisms remain unclear. To investigate this, we employed an integrated computational approach combining network toxicology, molecular docking, and molecular dynamics simulation. Potential targets of BPA and ASD-related genes were collected from multiple databases, identifying 57 overlapping targets. Protein–protein interaction network analysis highlighted 16 core targets among them. Gene Ontology and KEGG pathway enrichment analyses indicated these targets are primarily involved in synaptic transmission, GABAergic signaling, and neuroactive ligand–receptor interactions. Molecular docking demonstrated potential binding between BPA and several core targets, including estrogen receptor 1 (ESR1), gamma-aminobutyric acid type A receptor subunit beta-2 (GABRB2), and amyloid precursor protein (APP), with binding energies below −5 kcal/mol. The stability of the BPA-ESR1 complex was further supported through 100 ns molecular dynamics simulation. These results suggest that BPA may contribute to ASD by disrupting neuroendocrine pathways and synaptic function via interactions with key targets such as ESR1. Full article

Heme is a crucial cofactor involved in various biological processes, such as electron transport, catalysis, and oxygen binding. Understanding the binding of heme to heme-binding proteins (HBPs) is essential for clarifying their functions and molecular mechanisms and for applications in enzyme engineering and therapeutic development. AlphaFold3 (AF3), an artificial intelligence (AI)-based macromolecular prediction tool, has been applied to the structural modeling of various proteins, including HBPs. Nevertheless, whether AF3 provides reliable structural information for HBPs has not yet been investigated. To determine the AF3 predictions for HBPs, the apo and holo states of four human HBPs, including the cytochrome b5 domain of sulfite oxidase (SO-b5), the cytochrome b5 domain of NADH cytochrome b5 oxidoreductase (Ncb5or-b5), cytochrome b5 type B (CYB5B), and neuroglobin (NGB), generated by AF3, were examined and compared with experimental HBP structures. The overall positions of the heme molecules were well docked into the heme-binding pockets of HBPs; however, there were differences in the heme-binding configurations, including pocket geometry and coordination environment, which are crucial for functional interpretation. The experimental NGB structure contains a disulfide bond near the heme-binding region, whereas the AF3-predicted model lacks this bond, causing differences in local folding that affect the heme-binding environment. Molecular dynamics simulations demonstrated that the AF3-predicted NGB structure exhibited distinct molecular conformations and flexibility compared with the experimental structure. These data indicate both the potential and the limitations of using AF3-predicted structures to model the heme-binding states of HBPs. Full article

Factor Xa (FXa) remains an important target in the development of anticoagulant and antithrombotic agents. In this study, twenty isosteviol-derived oxime ether analogs previously reported as FXa inhibitors were used to develop a predictive QSAR model. The compounds were geometry-optimized at the B3LYP/6-311++G(d,p) level, and Dragon molecular descriptors were calculated from the optimized structures. After descriptor filtering, Random Forest-based supervised preselection and correlation-based pruning were applied. Several models of increasing complexity were evaluated, including multiple linear regression, additive MARSplines, and constrained second-order MARSplines models. The final model employed four active basis functions involving R6p+, C-025, ATSC7e, and Mor31p and demonstrated excellent calibration and cross-validated predictive ability (R² = 0.929 and Q²_LOO = 0.865). Based on this model, twenty new isosteviol-derived analogs were designed and their activities were predicted after DFT optimization and descriptor calculation. Eight representative compounds were subsequently subjected to molecular docking against human factor Xa (PDB ID: 2P16), molecular dynamics simulations, MM/GBSA binding free energy calculations, and preliminary SwissADME/pkCSM profiling. Docking protocol validation yielded a redocking RMSD of 0.953 Å. Although ISV-M20 was the highest-ranked compound according to the QSAR model, subsequent receptor-based analyses identified ISV-M19, ISV-M04, and ISV-M06 as the derivatives with the most favorable combination of structural stability, persistent protein–ligand interactions, and binding free energies. The ADMET/toxicity screen further refined this prioritization: ISV-M19, ISV-M04, and ISV-M06 showed the most favorable preliminary toxicity balance among the prioritized derivatives, whereas ISV-M20 displayed additional developability liabilities, including very high lipophilicity, poor predicted solubility, P-gp substrate status, and a predicted hERG II alert. Overall, the results demonstrate that integrating interpretable QSAR modeling with receptor-based simulations and early ADMET/toxicity filtering provides a more balanced strategy for the rational design and prioritization of novel isosteviol-derived FXa inhibitors than any single computational approach alone. Full article

Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) is a nitramine explosive widely used in military and industrial fields. While emerging evidence suggests the neurotoxicity of HMX, the mechanisms underlying central nervous system (CNS) damage remain largely unknown. In the present study, we established a mouse model of 28-day subacute HMX exposure to explore HMX-induced neurotoxicity and underlying mechanisms in vivo. Behavioral assessments revealed that HMX increased spontaneous locomotor activity and central exploration in the open field test, and reduced immobility time in the forced swimming test, indicating abnormal emotional regulation. The Morris water maze further demonstrated impaired hippocampus-dependent spatial learning and memory in HMX-treated mice, as evidenced by prolonged platform latency. Histopathological analysis showed hippocampal demyelination in HMX-treated mice, accompanied by downregulation of myelin structural proteins (MBP, PLP1) and oligodendrocyte lineage proteins (OLIG2, CNPase). Additionally, proteomic analysis identified 173 differentially expressed proteins in the HMX-exposed hippocampus, which were enriched in myelination, synaptic transmission and neuroactive ligand–receptor interaction pathways. Collectively, our findings demonstrate that subacute HMX exposure induces behavioral deficits and demyelination in mice hippocampus, providing a novel mechanistic insight into HMX neurotoxicity and a theoretical basis for occupational health protection against HMX exposure. Full article

This study investigates the synergistic therapeutic potential of Rivastigmine (RVG) and Epigallocatechin (EGC) in Alzheimer’s disease (AD), a multifactorial neurodegenerative disorder characterized by neuroinflammation, oxidative stress, and dysregulated signaling pathways. Conventional therapies primarily provide symptomatic relief and target limited pathways, highlighting the need for multi-target strategies with improved efficacy and safety. An integrated in silico approach combining pharmacokinetic evaluation, network pharmacology, molecular docking, and molecular dynamics simulations is used to determine the synergistic potential of RVG and EGC. Pharmacokinetic analysis indicates favorable drug-likeness and acceptable ADME/Tox profiles for both compounds. Network pharmacology identified 146 overlapping targets associated with AD, highlighting key hub genes including NFKB1, MAPK1, STAT1, PRKACA, GRB2, LYN, and PTPN11, which are involved in neuroinflammation, synaptic signaling, and neuronal survival. Functional enrichment analysis indicated significant involvement of MAPK/ERK signaling and immune-regulatory pathways. Importantly, the PD-1/PD-L1 signaling pathway is identified as a novel mechanism connecting neuroimmune modulation with intracellular kinase-driven neurodegeneration. Molecular docking studies showed strong binding affinities of RVG and EGC toward key AD-related targets, particularly MAPK1, supported by stable hydrogen bonding and interaction profiles. Molecular dynamics simulations confirmed stable protein-ligand interactions, with EGC contributing structural stability and RVG exhibiting adaptive flexibility within the binding pocket. These results suggest that the RVG-EGC combination exhibits synergistic potential by simultaneously modulating neuroinflammatory, oxidative stress, and kinase-mediated signaling pathways. The integration of PD-1/PD-L1 and MAPK/ERK signaling provides a novel mechanistic pathway for multi-target therapeutic intervention in AD. Full article

S-adenosyl-L-homocysteine hydrolase (SAHH) is an evolutionarily conserved enzyme present in eukaryotes, bacteria, and archaea. As the rate-limiting enzyme in the methionine cycle, it catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine (SAH) to adenosine and homocysteine, thereby modulating the S-adenosylmethionine/SAH ratio and cellular methylation potential. Dysregulation of SAHH activity is causally linked to cancer, cardiovascular disorders, and neurodegenerative conditions. This review systematically examines the biological distribution, catalytic mechanisms, structural architecture, and regulation of SAHH across diverse species. We highlight lineage-specific adaptations—including C-terminal truncation, a 40-residue substrate-binding-domain insertion, and a His-Phe molecular gate—that fine-tune substrate preference, cofactor affinity, and thermostability, with metal ions and NAD⁺ serving as key modulators of activity and conformational dynamics. These variations exemplify an evolutionary trade-off between catalytic efficiency and structural rigidity, particularly pronounced in archaeal and thermophilic orthologs. Collectively, these insights underpin the enzyme’s multifaceted translational value: SAHH serves as a therapeutic target for diverse diseases (e.g., cancer, viral infections, tuberculosis), a source of diagnostic/prognostic biomarkers (e.g., plasma homocysteine and SAH/SAM ratio), and a versatile biocatalyst for synthesizing pharmaceutical-grade adenosine and its derivatives. By integrating mechanistic, structural, and evolutionary perspectives, this review establishes a unified framework that explains these functional adaptations and their translational implications. This framework guides the rational development of SAHH-targeted inhibitors, diagnostic tools, and engineered biocatalysts, with broad applications in precision medicine and biotechnology. Full article

Objectives: Malaria remains a major global health burden, particularly in endemic regions such as Djibouti, where Plasmodium falciparum and Plasmodium vivax co-circulate, complicating disease control strategies. Increasing resistance to current antimalarial drugs reduces treatment effectiveness and highlights the urgent need for new, safe, and affordable therapeutic agents. This study aimed to identify potential inhibitors from Djiboutian medicinal plants using an integrated in silico approach targeting key proteins from both parasite species. Methods: A library of 222 phytoconstituents was screened against Plasmodium vivax FK506-binding protein 35 (PDB ID: 3IHZ) and Plasmodium vivax dihydrofolate reductase–thymidylate synthase (PDB ID: 1J3K) using molecular docking. Top-ranked compounds were further analyzed for binding interactions and evaluated for drug-likeness and pharmacokinetic properties using QikProp in Maestro v11.5. Selected protein–ligand complexes were subjected to 100 ns molecular dynamics simulations, and their stability was assessed using multiple descriptors, including structural deviation, flexibility, compactness, solvent exposure, and hydrogen bond persistence. Results: Several phytoconstituents exhibited strong binding affinities, with docking scores ranging from −6.09 to −7.54 kcal/mol, outperforming the reference drug artemisinin. Interaction analysis revealed key hydrogen bonds and hydrophobic contacts with essential active-site residues. ADMET predictions indicated favorable pharmacokinetic profiles, including high oral absorption, good membrane permeability, and low predicted toxicity. Molecular dynamics simulations demonstrated stable behavior for most complexes, with compound 121 showing enhanced stability in the 1J3K system and compound 123 exhibiting consistent dynamic stability in the 3IHZ system. In contrast, compound 82 displayed greater structural fluctuations despite maintaining stable hydrogen bond interactions. Conclusions: The integration of molecular docking, ADMET prediction, and molecular dynamics simulations identified compounds 121 and 123 as the most promising antimalarial candidates, exhibiting an optimal balance of binding affinity, favorable pharmacokinetic properties, and dynamic stability. These findings highlight the potential of Djiboutian medicinal plants as a valuable source of novel antimalarial agents and provide a strong computational foundation for future experimental validation. Full article

Pneumolysin (PLY), a cholesterol-dependent cytolysin produced by Streptococcus pneumoniae, plays a key role in the pathogenesis of bacterial pneumonia. The present study aimed to identify potential phytocompound inhibitors of pneumolysin and evaluate their protective effects against toxin-induced cytotoxicity using combined in silico and in vitro approaches. A ligand library comprising 200 phytocompounds was constructed using three-dimensional structures obtained from the PubChem database, while the PLY protein structure was retrieved from the Protein Data Bank. Molecular docking was performed to analyse protein-ligand interactions, followed by visualization using BIOVIA Discovery Studio Visualizer. Top-ranked phytocompounds from docking were further screened by computational ADME analysis, followed by molecular dynamics (MD) simulations for stability analysis. Selected compounds were then validated using RAW 264.7 macrophages, and cytotoxicity was assessed by flow cytometry. Among the screened compounds, three showed high binding affinity, with oridonin exhibiting the most favourable interaction profile (−7.906 kcal/mol). MD simulation confirmed the stability of the pneumolysin–oridonin complex. In vitro results demonstrated that pneumolysin induced significant, concentration-dependent cytotoxicity, whereas pre-incubation with oridonin significantly reduced cell death. To provide direct functional evidence of pneumolysin inhibition, haemolysis inhibition assay using sheep erythrocytes was performed where reduction of pneumolysin-mediated haemolysis by oridonin in a concentration-dependent manner was demonstrated. These findings suggest that oridonin may serve as a potential inhibitor of pneumolysin-mediated cytotoxicity and pneumococcal virulence. Full article

Background: Diabetic cardiomyopathy (DCM) is characterized by metabolic dysfunction, inflammation, extracellular matrix (ECM) remodeling, and myocardial fibrosis. Increasing evidence suggests that ferroptosis-associated oxidative injury may contribute to cardiac remodeling; however, the interaction between ferroptosis-related pathways and fibrosis-associated molecular networks remains incompletely understood. This study explored the ferroptosis–fibrosis axis using an integrative transcriptomic and systems pharmacology framework. Methods: Differentially expressed genes were identified from the GSE5406 myocardial transcriptomic dataset comparing nonfailing donor hearts with ischemic and idiopathic cardiomyopathy samples and analyzed using functional enrichment, protein–protein interaction, and disease-association approaches. Cross-dataset comparison and exploratory sample-level external evaluation were performed using the independent GSE263297 DCM-related dataset. Candidate genes were further evaluated by receiver operating characteristic (ROC) analysis and machine learning-based feature selection using least absolute shrinkage and selection operator (LASSO), random forest, and support vector machine-recursive feature elimination (SVM-RFE). Representative compounds associated with fibrosis-, oxidative stress-, inflammation-, and ferroptosis-related pathways were subsequently assessed by molecular docking against TGFBR1, STAT3, GPX4, AKT1, SMAD3, and ACSL4. Results: Transcriptomic analyses highlighted ECM organization, collagen-containing ECM, and fibrosis-related pathways as dominant biological themes. Cross-dataset comparison showed partial preservation of transcriptional patterns between independent myocardial cohorts, with 20 of 51 evaluated genes demonstrating concordant expression direction across datasets. ROC analysis identified LUM and ASPN as having the highest area under the curve (AUC) values among candidate genes, whereas COL1A1, COL1A2, and COL3A1 also showed elevated AUC values. Machine learning analyses identified FCN3, HOPX, CNN1, and GLUL as the core signature consistently prioritized across all three algorithms, whereas LUM was additionally identified by two of three algorithms. Internal validation yielded a cross-validated AUC of 0.934 (95% CI: 0.820–1.000), and exploratory sample-level external evaluation of the four-gene signature in GSE263297 yielded an AUC of 0.673 (95% CI: 0.380–0.967). Exploratory docking analyses suggested potential structural compatibility between several candidate compounds and fibrosis-, inflammation-, and ferroptosis-associated targets, with comparatively lower predicted binding-energy values observed for selected ligand–target combinations. Conclusions: The findings are consistent with a fibrosis-dominant remodeling signature and suggest potential network-level links between ferroptosis-associated processes and cardiac fibrosis. These observations should be regarded as exploratory and hypothesis-generating and require validation in independent cohorts and experimental studies. Full article

Carbohydrate-active enzymes (CAZymes) encoded by the human gut microbiome are central mediators of dietary glycan metabolism and host–microbe biochemical homeostasis. Among these, β-glucuronidases represent functionally pivotal hydrolases implicated in metabolism, intestinal physiology, and therapeutic modulation. The present study performs an integrative in silico structural and functional interrogation of β-glucuronidases derived from Acidobacterium capsulatum (3VNY), Bacteroides ovatus (6D8K), and Faecalibacterium prausnitzii (6ED2). An integrated computational framework encompassing physicochemical parameters profiling, hierarchical structural prediction, tertiary-structure validation, salt-bridge energetics, functional domain and motif annotation, protein–protein interaction reconstruction, ligand-binding thermodynamics via molecular docking, and residue-resolved non-covalent interaction network mapping using the Protein Contacts Atlas (PCA) was employed. Physicochemical analyses indicated that all enzymes are thermostable, intracellular, and hydrophilic, while secondary-structure organization revealed a functional balance between helix-mediated rigidity and coil-driven flexibility. Structural validation metrics identified 6ED2 as the most conformationally stable architecture, whereas 6D8K displayed enhanced functional complexity, including enriched motif composition, membrane-associated features, and superior ligand-binding affinity. Docking simulations highlighted castanospermine and calcium saccharate as the most favorable interacting ligands across enzyme variants. Importantly, PCA-based interaction analysis revealed distinct ligand-centered atomic contact networks, with immediate contact counts of 57 (3VNY), 32 (6D8K), and 41 (6ED2), providing residue-level insight into stabilization mechanisms and interaction topology beyond conventional docking metrics. Collectively, these findings establish a multidimensional computational framework linking structural stability, functional diversification, ligand recognition, and atomic interaction networks in gut microbial β-glucuronidases, thereby supporting future biochemical validation, microbiome-targeted therapeutics, and biotechnological or cosmeceutical applications. Full article

The divalent metal transporter DMT1 (SLC11A2) is implicated in diverse human pathologies including cancers, inflammatory and degenerative diseases. Small molecules targeting this membrane protein are actively searched. Following the identification of the pyranocoumarin praeruptorin A as an inhibitor of ferroptosis that is able to bind to DMT1, we have investigated the interaction of related natural products with DMT1 using molecular modeling to determine structure-binding relationships. Two series of compounds were tested: praeruptorins A-H and qianhucoumarins A-J, all isolated previously from the roots of the Chinese medicinal plant Peucedanum praeruptorum Dunn (Bai-Hua Qian-Hu). The antitumor compound praeruptorin C was identified as the best DMT1 ligand in the series, with a binding capacity largely superior to that of praeruptorin A and also well superior to the reference organoselenium product ebselen, at least from an in silico perspective. Praeruptorin C, and to a lower extent praeruptorins F and H, can form stable complexes with DMT1 upon binding close to the ebselen binding site. Qianhucoumarins C and I were also identified as potential binders. Altogether, the analysis of the 18 natural products enabled identification of structural elements implicated in the target binding process. The curvature of the tricyclic pyranocoumarin scaffold and the angeloyl side chain at position 9 seem to contribute importantly to the protein interaction. An experimental validation is required but the docking study paves the way to the discovery and design of tricyclic coumarin derivatives targeting DMT1. Full article

In triple-negative breast cancer (TNBC), immune checkpoint pathways play a central role in tumor immune evasion. Programmed death protein 1 (PD-1) is an inhibitory receptor expressed on T cells, while its ligand, programmed death-ligand 1 (PD-L1), is commonly expressed on tumor cells and cells within the tumor microenvironment. Their interaction suppresses T-cell activation and promotes immune escape. In this study, we evaluated the potential of small interfering RNA (siRNA) to silence PD-L1 expression in TNBC. Transcriptomic analysis of GEO datasets revealed consistent upregulation of CD274 (PD-L1) in TNBC samples. Three siRNA candidates were designed and evaluated in MDA-MB-231 cells. All siRNAs significantly reduced CD274 expression (>70%), as determined by RT-qPCR. Immunofluorescence analysis confirmed a reduction in PD-L1 protein levels (54.3 vs. 98.7 a.u.), while MTT assays demonstrated preserved cell viability at the working concentration (100 pM), supporting a non-cytotoxic and specific gene-silencing effect. These findings highlight PD-L1 as a viable molecular target and support siRNA-mediated silencing as a promising therapeutic strategy in TNBC. Full article

The Zona Pellucida (ZP) and its structural analogs are evolutionarily ancient extracellular matrix components. These are essential for oocyte protection, species-specific gamete recognition, and prevention of polyspermy across Metazoa. Defined by the conserved ZP-domain—comprising ZP-N and ZP-C subdomains—these glycoproteins self-assemble into fibrillar matrices through tightly regulated polymerization. Mechanisms of the regulated polymerization involve furin cleavage, disulfide bonding, and hydrophobic interactions. Once considered a vertebrate innovation, the canonical ZP-domain—defined by its bipartite ZP-N/ZP-C architecture, eight conserved cysteine residues, and capacity for matrix polymerization—is now recognized as an ancient metazoan extracellular module, with homologs identified in basal lineages including Porifera, Cnidaria, and Placozoa. While ZP-like sequences have been reported in choanoflagellates such as Salpingoeca rosetta, these lack the complete canonical features and are considered distant structural relatives rather than true ZP-modules. There they function in cell adhesion and tissue integrity, suggesting an origin predating the evolution of specialized reproductive coats. Previous phylogenetic analyses across 97 metazoan species have revealed that vertebrate ZP genes arose from ancestral duplications of the canonical ZP-module. Accordingly, they give rise to eight subfamilies (ZP1–ZP4, ZPD, ZPAX, ZPX, ZPY), with lineage-specific expansions, losses, and pseudogenization reflecting adaptations to diverse reproductive strategies. Positive selection in sperm-binding regions of ZP2 and ZP3 drives a rapid adaptive evolution. It underscores coevolutionary arms races with sperm ligands, contributing to reproductive isolation and speciation. In invertebrates such as abalone and insects, ZP-domain proteins mediate analogous functions through lineage-specific elaborations, including tandem repeats and domain shuffling. Post-translational modifications, particularly glycosylation, fine-tune sperm receptor specificity and matrix stability. The functional transition from a general protective barrier in early metazoans to a sophisticated gamete recognition interface in vertebrates exemplifies modular evolution. This synthesis highlights the domain-level deep homology of ZP-domain proteins as a foundational element of metazoan extracellular matrices, repurposed through gene duplication, neofunctionalization, and selection to meet the demands of evolving reproductive modes. These insights bridge evolutionary biology, reproductive medicine, and developmental genetics. However, major gaps remain, including unresolved orthology between vertebrate and invertebrate ZP genes, the relative contribution of glycans versus protein backbone in sperm recognition, and the lack of functional evidence for canonical ZP-domain proteins in insects. Future studies integrating glycoproteomics, single-cell transcriptomics, and CRISPR-based models are needed to resolve these questions. Full article