Search Results (300)

Acute kidney injury (AKI) is a frequent and severe complication in trauma patients, affecting up to 28% of intensive care unit (ICU) admissions and contributing significantly to morbidity, mortality, and long-term renal impairment. Trauma-related AKI (TRAKI) arises from diverse mechanisms, including hemorrhagic shock, ischemia–reperfusion injury, systemic inflammation, rhabdomyolysis, nephrotoxicity, and complex organ crosstalk involving the brain, lungs, and abdomen. Pathophysiologically, TRAKI involves early disruption of the glomerular filtration barrier, tubular epithelial injury, and renal microvascular dysfunction. Inflammatory cascades, oxidative stress, immune thrombosis, and maladaptive repair mechanisms mediate these injuries. Trauma-related rhabdomyolysis and exposure to contrast agents or nephrotoxic drugs further exacerbate renal stress, particularly in patients with pre-existing comorbidities. Traditional markers such as serum creatinine (sCr) are late indicators of kidney damage and lack specificity. Emerging structural and stress response biomarkers—such as neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), liver-type fatty acid-binding protein (L-FABP), interleukin-18 (IL-18), C-C motif chemokine ligand 14 (CCL14), Dickkopf-3 (DKK3), and the U.S. Food and Drug Administration (FDA)-approved tissue inhibitor of metalloproteinases-2 × insulin-like growth factor-binding protein 7 (TIMP-2 × IGFBP-7)—allow earlier detection of subclinical AKI and better predict progression and the need for renal replacement therapy. Together, functional indices like urinary sodium and fractional potassium excretion reflect early microcirculatory stress and add clinical value. In parallel, risk stratification tools, including the Renal Angina Index (RAI), the McMahon score, and the Haines model, enable the early identification of high-risk patients and help tailor nephroprotective strategies. Together, these biomarkers and risk models shift from passive AKI recognition to proactive, personalized management. A new paradigm that integrates biomarker-guided diagnostics and dynamic clinical scoring into trauma care promises to reduce AKI burden and improve renal outcomes in this critically ill population. Full article

Adeno-associated virus (AAV) vectors are the preferred gene delivery tool in gene therapy owing to their safety, long-term gene expression, broad tissue tropism, and low immunogenicity. Affinity ligands that can bind multiple AAV serotypes endure harsh clean-in-place (CIP) conditions and are critical for industrial-scale purification. However, current ligands lack broad serotype recognition and adequate alkaline stability, which limits their reusability in large-scale manufacturing. In this study, we employed a competitive biopanning strategy to isolate a single-domain antibody (VHH) that simultaneously binds AAV2, AAV8, and AAV9. The VHH retained structural integrity and binding activity after exposure to 0.1 M NaOH, demonstrating robust alkaline stability. Structural modeling revealed that the VHH primarily recognizes the DE loop region of the VP3 capsid protein across the three serotypes, explaining its cross-serotype reactivity. Affinity chromatography using the VHH yielded infectious AAV particles, confirming its potential for downstream processing. This strategy provides a versatile platform for developing high-performance AAV affinity ligands and may be extended to other viral vector systems. Full article

The ultimate function of a protein is a summation of the activities of all its modules or domains. A major mechanism for regulating protein activity, besides modulation of its levels through translation or degradation, is covalent post-translational modification (PTM) of these modules, including phosphorylation and dephosphorylation of tyrosine, threonine, and/or serine residues. Phosphorylation is a fast, reversible, and highly specific mode of regulating protein function. Unlike proteins that are marked with other PTMs, phosphorylated proteins orchestrate an extensive network of protein interactions because of their ability to bind many protein partners. Protein phosphorylation is crucial for many cellular processes—signaling, transcription, and metabolism—because it precisely controls these processes in time and space. In this review, we will focus on signaling coordinated by tyrosine phosphorylation–dephosphorylation, specifically structural insights that govern the mechanism of recognition of phosphotyrosine (pY)-containing ligands by Src homology 2 (SH2) domains. We update the approaches used to target the SH2 domains and techniques applied in drug discovery, highlighting inhibitors that have reached clinical development. Full article

Endolysins, phage-derived enzymes capable of lysing bacterial cell walls, hold significant promise as novel antimicrobials against resistant Gram-positive and Gram-negative pathogens. In this study, we undertook an integrative approach combining extensive in silico analyses and experimental validation to characterize the novel endolysin LysPALS22. Initially, sixteen endolysin sequences were selected based on documented lytic activity and enzymatic diversity, and subjected to multiple sequence alignment and phylogenetic analysis, which revealed highly conserved catalytic and binding domains, particularly localized to the N-terminal region, underscoring their functional importance. Building upon these sequence insights, we generated three-dimensional structural models using Swiss-Model, EBI-EMBL, and AlphaFold Colab, where comparative evaluation via Ramachandran plots and ERRAT scores identified the Swiss-Model prediction as the highest quality structure, featuring over 90% residues in favored conformations and superior atomic interaction profiles. Leveraging this validated model, molecular docking studies were conducted in PyRx with AutoDock Vina, performing blind docking of key peptidoglycan-derived ligands such as N-Acetylmuramic Acid-L-Alanine, which exhibited the strongest binding affinity (−7.3 kcal/mol), with stable hydrogen bonding to catalytic residues ASP46 and TYR61, indicating precise substrate recognition. Visualization of docking poses using Discovery Studio further confirmed critical hydrophobic and polar interactions stabilizing ligand binding. Subsequent molecular dynamics simulations validated the stability of the LysPALS22–NAM-LA complex, showing minimal structural fluctuations, persistent hydrogen bonding, and favorable interaction energies throughout the 100 ns trajectory. Parallel to computational analyses, LysPALS22 was heterologously expressed in Escherichia coli (E. coli) and Pichia pastoris (P. pastoris), where SDS-PAGE and bicinchoninic acid assays validated successful protein production; notably, the P. pastoris-expressed enzyme displayed an increased molecular weight (~45 kDa) consistent with glycosylation, and achieved higher volumetric yields (1.56 ± 0.31 mg/mL) compared to E. coli (1.31 ± 0.16 mg/mL), reflecting advantages of yeast expression for large-scale production. Collectively, these findings provide a robust structural and functional foundation for LysPALS22, highlighting its conserved enzymatic features, specific ligand interactions, and successful recombinant expression, thereby setting the stage for future in vivo antimicrobial efficacy studies and rational engineering efforts aimed at combating multidrug-resistant Gram-negative infections. Full article

The interactions of leukocyte glycoproteins with adhesion and signaling molecules through glycan recognition are not well understood. We previously demonstrated that galectin-8, a tandem-repeat lectin with N- and C-terminal carbohydrate binding domains which is highly expressed in endothelial and epithelial cells, can bind to activated neutrophils to induce surface exposure of phosphatidylserine (PS) without DNA fragmentation or apoptosis, in a process termed preaparesis. However, the receptors for Gal-8 on leukocytes have not been identified. Here we report our results using both proteomics and affinity chromatography with both full-length Gal-8 and the separate Gal-8 C-terminal and N-terminal domains to identify glycoprotein ligands in HL-60 cells for Gal-8. Two of the major ligands for Gal-8 are CD45RA and CD45RC (Protein Tyrosine Phosphatase, PTP) and basigin (CD147). Both CD45 and basigin are integral membrane glycoproteins that carry poly-N-acetyllactosamine modifications on N- and/or O-glycans, required for Gal-8 binding. Inhibition of the phosphatase activity of CD45 reduced Gal-8-induced PS exposure, indicating a possible role of CD45 in Gal-8 signaling of preaparesis in human leukocytes. These results demonstrate unique glycoprotein recognition by Gal-8 involved in cell recognition and signaling. Full article

We previously developed SA-conf, a method designed to quantify backbone structural variability in protein targets. This approach is based on the HMM-SA structural alphabet, which enables efficient and rapid comparison of local backbone conformations across multiple structures of a given target. In this study, SA-conf (version for python2.7) was applied to a dataset of 130 crystallographic chains of Bcl-xL, a protein involved in promoting cell survival. SA-conf quantified and mapped backbone structural variability, revealing the protein’s capacity for conformational rearrangement. Our results showed that while most mutations had minimal impact on backbone conformation, some were associated with long-range structural effects. By jointly analyzing residue flexibility and backbone rearrangements across apo and holo structures, SA-conf identified key regions where the backbone undergoes structural adjustments upon ligand binding. Notably, the $\alpha 2$–$\alpha 3$ region was shown to be a hotspot of structural plasticity, exhibiting ligand-specific conformational signatures. Furthermore, SA-conf enabled the construction of a structural map of the binding site, distinguishing a conserved anchoring core from flexible peripheral regions that contribute to ligand specificity. Overall, this study highlights SA-conf’s capacity to detect conformational changes in protein backbones upon ligand binding and to uncover structural determinants of selective ligand recognition. Full article

The tomato leafminer (Tuta absoluta), a globally invasive pest, poses a major economic threat to tomato production. Although chemical control remains the primary management method, sustainable alternatives are urgently needed. Sex pheromone communication is critical for moth courtship and mating, with pheromone-binding proteins (PBPs) playing a key role in this process. In this study, we identified a PBP gene, TabsPBP2, from the T. absoluta transcriptome. Real-time quantitative PCR (RT-qPCR) revealed that TabsPBP2 is highly expressed in the antennae, with a strong male-biased expression pattern. Ligand-binding assays demonstrated that TabsPBP2 has the highest affinity for the sex pheromones (3E, 8Z, 11Z)-tetradecatrienyl acetate (TDTA) and (3E, 8Z)-tetradecadienyl acetate (TDDA). It also demonstrated a moderate-to-strong binding affinity to several tomato volatiles, including 2-carene, myrcene, α-pinene, cis-3-hexen-l-ol, methyl salicylate, sabinene, and α-terpinene. Molecular docking suggested that hydrophobic interactions predominantly stabilize the TabsPBP2–ligand complexes, with PHE118, PHE12, LEU90, LEU68, and ALA73 identified as key interacting residues. Electroantennogram (EAG) and Y-tube olfactometer assays confirmed that TDTA and TDDA act as strong attractants for male T. absoluta. This study enhances our understanding of the pheromone recognition in T. absoluta and provides a foundation for developing novel, pheromone-based pest control strategies. Full article

Background/Objective: Programmed cell death ligand-1 (PD-L1), which is overexpressed in certain tumors, inhibits the body’s natural immune response by providing an “off” signal that enables cancer cells to evade the immune system. It has been demonstrated that [¹⁷⁷Lu]Lu-DOTA-iPD-L1 (PD-L1 inhibitor cyclic peptide) promotes immune responses. This study aimed to synthesize and evaluate [¹⁸F]AlF-NOTA-iPD-L1 as a novel radiotracer for PD-L1 positron emission tomography (PET) imaging and as a potential theranostic pair for [¹⁷⁷Lu]Lu-DOTA-iPD-L1. Methods: The NOTA-iPD-L1 peptide conjugate was synthesized and characterized by U.V.-vis, I.R.-FT, and UPLC-mass spectroscopies. Radiolabeling was performed using [¹⁸F]AlF as the precursor, and the radiochemical purity (HPLC), partition coefficient, and serum stability were assessed. Cellular uptake and internalization (in 4T1 triple-negative breast cancer cells), binding competition, immunofluorescence, and Western blot assays were applied for the radiotracer in vitro characterization. Biodistribution in mice bearing 4T1 tumors was performed, and molecular imaging (Cerenkov images) of [¹⁸F]AlF-NOTA-iPD-L1 and [¹⁷⁷Lu]Lu-DOTA-iPD-L1 in the same mouse was obtained. Results: [¹⁸F]AlF-NOTA-iPD-L1 was prepared with a radiochemical purity greater than 97%, and it demonstrated high in vitro and in vivo stability, as well as specific recognition by the PD-L1 protein (IC₅₀ = 9.27 ± 2.69 nM). Biodistribution studies indicated a tumor uptake of 6.4% ± 0.9% ID/g at 1-hour post-administration, and Cerenkov images showed a high tumor uptake of both [¹⁸F]AlF-NOTA-iPD-L1 and ¹⁷⁷Lu-iPD-L1 in the same mouse. Conclusions: These results warrant further studies to evaluate the clinical usefulness of [¹⁸F]AlF-NOTA-iPD-L1/[¹⁷⁷Lu]Lu-DOTA-iPD-L1 as a radiotheranostic pair in combination with anti-PD-L1/PD1 immunotherapy. Full article

The Campylobacter genus includes many pathogenic species, with Campylobacter hepaticus primarily implicated in spotty liver disease in poultry. Chemotaxis is one of the well-established mechanisms of pathogenesis of Campylobacter. The chemoreceptor Tlp3, previously studied in C. jejuni, mediates responses to diverse ligands. Differences between the ligand-binding pockets of Tlp3s in C. hepaticus and C. jejuni may influence ligand specificity and niche adaptation. Here, we report a method for production of the ligand-binding domain of C. hepaticus Tlp3 (Ch Tlp3-LBD) in Escherichia coli inclusion bodies that yields crystallisable protein. Size-exclusion chromatography analysis showed Ch Tlp3-LBD is a monomer in solution. Ch Tlp3-LBD was crystallised using PEG 6000 and LiCl as the precipitants. The crystal lattice symmetry was P222₁, with unit cell geometry of a = 82.0, b = 137.7, c = 56.1 Å, and α = β = γ = 90°. X-ray diffraction data have been acquired to 1.6 Å resolution using synchrotron radiation. Estimation of the Matthews coefficient (V_M = 2.8 ų Da⁻¹) and the outcome of molecular replacement suggested the asymmetric unit is composed of two protein molecules. This work lays the foundation for studies towards understanding the structural basis of ligand recognition by C. hepaticus Tlp3 and its role in pathogenesis. Full article

The SARS-CoV-2 main protease (Mpro), essential for viral replication, remains a prime target for antiviral drug design against COVID-19 and related coronaviruses. In this study, we present a systematic investigation into the molecular determinants of Mpro inhibition using an integrated approach combining large-scale data mining, cheminformatics, and quantum chemical calculations. A curated dataset comprising 963 high-resolution structures of Mpro–ligand complexes—348 covalent and 615 non-covalent inhibitors—was mined from the Protein Data Bank. Cheminformatics analysis revealed distinct physicochemical profiles for each inhibitor class: covalent inhibitors tend to exhibit higher hydrogen bonding capacity and sp³ character, while non-covalent inhibitors are enriched in aromatic rings and exhibit greater aromaticity and lipophilicity. A novel descriptor, Weighted Hydrogen Bond Count (WHBC), normalized for molecular size, revealed a notable inverse correlation with aromatic ring count, suggesting a compensatory relationship between hydrogen bonding and π-mediated interactions. To elucidate the energetic underpinnings of molecular recognition, 40 representative inhibitors (20 covalent, 20 non-covalent) were selected based on principal component analysis and aromatic ring content. Quantum mechanical calculations at the double-hybrid B2PLYP/def2-QZVP level quantified non-bonded interaction energies, revealing that covalent inhibitors derive binding strength primarily through hydrogen bonding (~63.8%), whereas non-covalent inhibitors depend predominantly on π–π stacking and CH–π interactions (~62.8%). Representative binding pocket analyses further substantiate these findings: the covalent inhibitor F2F-2020198-00X exhibited strong hydrogen bonds with residues such as Glu166 and His163, while the non-covalent inhibitor EDG-MED-10fcb19e-1 engaged in extensive π-mediated interactions with residues like His41, Met49, and Met165. The distinct interaction patterns led to the establishment of pharmacophore models, highlighting key recognition motifs for both covalent and non-covalent inhibitors. Our findings underscore the critical role of aromaticity and non-bonded π interactions in driving binding affinity, complementing or, in some cases, substituting for hydrogen bonding, and offer a robust framework for the rational design of next-generation Mpro inhibitors with improved selectivity and resistance profiles. Full article

Aging and aging-related neurodegenerative disorders, such as Alzheimer’s disease, are characterized by chronic inflammation that progressively damages nervous tissue within the central nervous system (CNS). In addition to cytokines, lectin-like carbohydrate recognition molecules play a critical role in modifying cellular communication during inflammation. Among these, galectins—particularly anti-inflammatory galectin-1 and pro-inflammatory galectin-3—stand out due to their immunological functions and specificity for N-acetyllactosamine structures. Almost every cell type within the CNS can express and recognize galectins, influencing various essential cellular functions. N-acetyllactosamines, the ligand structures recognized by galectins, are found beneath sialylated termini in protein-linked oligosaccharides. During aging, protein-linked oligosaccharide structures become shorter, exposing N-acetyllactosamines on protein surfaces, which enhances their availability as binding sites for galectins. Genomic studies indicate that the number of galectin-1- and galectin-3-expressing microglial cells increases with age- or age-related disease (Alzheimer’s disease), reflecting an aging-associated rise in galectin concentrations within the CNS. This increase parallels a rise in free N-acetyllactosamine-like ligands, suggesting that galectin-N-acetyllactosamine interactions gain prominence and play a more significant role in aging-related CNS disorders. Understanding these interactions and their molecular implications offers potential avenues for targeted therapeutic strategies in combating aging-related CNS inflammation and neurodegeneration. Full article

Accurately predicting protein–ligand binding affinity is an important step in the drug discovery process. Deep learning (DL) methods have improved binding affinity prediction by using diverse categories of molecular data. However, many models rely heavily on interaction and sequence data, which impedes proper learning and limits performance in de novo applications. To address these limitations, we developed a novel graph neural network model, called StructureNet (structure-based graph neural network), to predict protein–ligand binding affinity. StructureNet improves existing DL methods by focusing entirely on structural descriptors to mitigate data memorization issues introduced by sequence and interaction data. StructureNet represents the protein and ligand structures as graphs, which are processed using a GNN-based ensemble deep learning model. StructureNet achieved a PCC of 0.68 and an AUC of 0.75 on the PDBBind v.2020 Refined Set, outperforming similar structure-based models. External validation on the DUDE-Z dataset showed that StructureNet can effectively distinguish between active and decoy ligands. Further testing on a small subset of well-known drugs indicates that StructureNet has high potential for rapid virtual screening applications. We also hybridized StructureNet with interaction- and sequence-based models to investigate their impact on testing accuracy and found minimal difference (0.01 PCC) between merged models and StructureNet as a standalone model. An ablation study found that geometric descriptors were the key drivers of model performance, with their removal leading to a PCC decrease of over 15.7%. Lastly, we tested StructureNet on ensembles of binding complex conformers generated using molecular dynamics (MD) simulations and found that incorporating multiple conformations of the same complex often improves model accuracy by capturing binding site flexibility. Overall, the results show that structural data alone are sufficient for binding affinity predictions and can address pattern recognition challenges introduced by sequence and interaction features. Additionally, structural representations of protein–ligand complexes can be considerably improved using geometric and topological descriptors. We made StructureNet GUI interface freely available online. Full article

Insect odorant-binding proteins (OBPs) are promising molecular targets for developing novel pest management strategies by modulating chemoreception-driven behaviors. The tea gray geometrid Ectropis grisescens (Lepidoptera, Geometridae) is a major pest in tea plantations, causing substantial economic losses in China. In this study, we identified 18 OBPs from E. grisescens antennal transcriptome. All of the encoded proteins possessed N-terminal signal peptides and conserved cysteine residues, behaviors which are characteristic of insect OBPs. Phylogenetic analysis categorized these proteins into plus-C, minus-C, and classic OBP subfamilies. MEME motif analysis identified conserved sequence features potentially involved in odor detection. Tissue- and sex-specific expression profiling showed that EgriGOBP1-2, OBP3, OBP8, and OBP13 were highly expressed in the antennae of both sexes, suggesting roles in olfactory communication. Among them, EgriGOBP1-2, OBP3, and OBP13 exhibited similar expression levels between males and females, while other EgriOBPs were predominantly expressed in the legs, wings, or other tissues, indicating additional physiological functions beyond chemoreception. To investigate functional specificity, we selected antenna-enriched EgriGOBP2 for ligand-binding analysis. Fluorescence binding assays demonstrated that EgriGOBP2 exhibited broad binding affinity toward 8 of 12 host volatiles and 11 of 12 plant essential oil-derived volatiles. These combined findings lay the foundation for mechanistic studies of chemical recognition in E. grisescens and provide insights into the development of ecologically friendly pest control alternatives. Full article

PD-L1 (programmed cell death ligand-1) is a protein located on the surface of regulatory cells. It has an immunosuppressive role as it binds specifically to the protein programmed cell death-1 (PD-1), a checkpoint glycoprotein, present on the surface of immune cells such as T and B lymphocytes. Many tumor cells block the immune response by overexpressing PD-L1 on their surface; therefore, targeting PD-L1 represents a powerful strategy that allows tumor localization. To determine the presence of PD-L1 in cells, the use of ad hoc functionalized peptides that bind to PD-L1 can be exploited. One of them is the peptide CLP002 (Trp-His-Arg-Ser-Tyr-Tyr-Thr-Trp-Asn-Leu-Asn-Thr), which, bound to surface-enhanced Raman scattering (SERS) gold nanostructures via a suitable linker, was shown to be highly effective in recognizing MDA-MB-231 breast cancer cells and, importantly, this recognition can be measured by SERS experiments. To characterize, on a molecular scale, the interaction between PD-L1 and peptide functionalized nanostructures, we performed molecular dynamics (MDs) simulations, studying the features of peptide monolayers bound on gold surfaces in the absence and presence of PD-L1. The results obtained allow us to explain why the nature of the linker plays a fundamental role in the binding and why a peptide carrying the same amino acids as CPL002 but with a different sequence (scrambled) is much less active than CLP002. These results open the way to an in silico evaluation of the key parameters that regulate the binding of PD-L1 useful for cancer recognition. Full article

Comparative studies using lysophosphatidic acid (LPA) and the synthetic agonist, oleoyl-methoxy glycerophosphothionate (OMPT), in cells expressing the LPA₃ receptor revealed differences in the action of these agents. The possibility that more than one recognition cavity might exist for these ligands in the LPA₃ receptor was considered. We performed agonist docking studies exploring the whole protein to obtain tridimensional details of the ligand–receptor interaction. Functional in cellulo experiments using mutants were also executed. Our work includes blind docking using the unrefined and refined proteins subjected to hot spot predictions. Distinct ligand protonation (charge −1 and −2) states were evaluated. One LPA recognition cavity is located near the lower surface of the receptor close to the cytoplasm (Lower Cavity). OMPT displayed an affinity for an additional identification cavity detected in the transmembrane and extracellular regions (Upper Cavity). Docking targeted to Trp102 favored binding of both ligands in the transmembrane domain near the extracellular areas (Upper Cavity), but the associating amino acids were not identical due to close sub-cavities. A receptor model was generated using AlphaFold3, which properly identified the transmembrane regions of the sequence and co-modeled the lipid environment accordingly. These two models independently generated (with and without the membrane) and adopted essentially the same conformation, validating the data obtained. A DeepSite analysis of the model predicted two main binding pockets, providing additional confidence in the predicted ligand-binding regions and support for the relevance of the docking-based interaction models. In addition, mutagenesis was performed of the amino acids of the two detected cavities. In the in cellulo studies, LPA action was much less affected by the distinct mutations than that of OMPT (which was almost abolished). Therefore, docking and functional data indicate the presence of distinct agonist binding cavities in the LPA₃ receptor. Full article