Search Results (958)

Background and objectives: Adducins are cytoskeletal proteins essential for membrane stability, actin–spectrin network organization, and cell signaling. Mutations in the genes ADD1, ADD2, and ADD3 have been linked to hypertension, neurodevelopmental disorders, and cancer. However, no comprehensive in silico saturation mutagenesis study has systematically evaluated the pathogenic potential and structural consequences of all possible missense mutations in adducins. This study aimed to identify high-risk variants and their potential impact on protein stability and function. Methods: We performed computational saturation mutagenesis for all possible single amino acid substitutions across the adducin proteins family. Pathogenicity predictions were conducted using four independent tools: AlphaMissense, Rhapsody, PolyPhen-2, and PMut. Predictions were validated against UniProt-annotated pathogenic variants. Predictive performance was assessed using Cohen’s Kappa, sensitivity, and precision. Mutations with a prediction probability ≥ 0.8 were further analyzed for structural stability using mCSM, DynaMut2, MutPred2, and Missense3D, with particular focus on functionally relevant domains such as phosphorylation and calmodulin-binding sites. Results: PMut identified the highest number of pathogenic mutations, while PolyPhen-2 yielded more conservative predictions. Several high-risk mutations clustered in known regulatory and binding regions. Substitutions involving glycine were consistently among the most destabilizing due to increased backbone flexibility. Validated variants showed strong agreement across multiple tools, supporting the robustness of the analysis. Conclusions: This study highlights the utility of multi-tool bioinformatic strategies for comprehensive mutation profiling. The results provide a prioritized list of high-impact adducin variants for future experimental validation and offer insights into potential therapeutic targets for disorders involving ADD1, ADD2, and ADD3 mutations. Full article

Sorghum is an important cereal crop. The maintenance of leaf color significantly influences sorghum growth and development. Although the mechanisms of leaf color mutation have been well studied in many plants, those in sorghum remain largely unclear. Here, we identified a sorghum gradient-pale-green leaf mutant (sbgpgl1) from the ethyl methanesulfonate (EMS) mutagenesis mutant library. Phenotypic, photosynthesis-related parameter, ion content, transcriptome, and metabolome analyses were performed on wild-type BTx623 and the sbgpgl1 mutant at the heading stage, revealing changes in several agronomic traits and physiological indicators. Compared with BTx623, sbgpgl1 showed less height, with a smaller length and width of leaf and panicle. The overall Chl a and Chl b contents in sbgpgl1 were lower than those in BTx623. The net photosynthetic rate, stomatal conductance, and transpiration rate were significantly reduced in sbgpgl1 compared to BTx623. The content of copper (Cu), zinc (Zn), and manganese (Mn) was considerably lower in sbgpgl1 leaves than in BTx623. A total of 4469 differentially expressed genes (DEGs) and 775 differentially accumulated metabolites (DAMs) were identified by RNA-seq and UPLC-MS/MS. The results showed that sbgpgl1 primarily influenced sorghum metabolism by regulating metabolic pathways and the biosynthesis of secondary metabolites, especially flavonoids and phenolic acids, resulting in the gradient-pale-green leaf phenotype. These findings reveal key genes and metabolites involved on a molecular basis in physiological variations of the sorghum leaf color mutant. Full article

The rise of multidrug-resistant Pseudomonas aeruginosa underscores the need for novel therapeutic targets beyond conventional peptidoglycan biosynthesis. Some bacterial strains bypass MurA inhibition by fosfomycin via a cell wall salvage pathway. This study targeted P. aeruginosa AmgK (PaAmgK) and MurU (PaMurU) to identify inhibitors that could complement fosfomycin therapy. A malachite-green-based dual-enzyme assay enabled efficient activity measurements and high-throughput chemical screening. Screening 232 compounds identified Congo red and CTAB as potent PaMurU inhibitors. A targeted mass spectrometric analysis confirmed the selective inhibition of PaMurU relative to that of PaAmgK. Molecular docking simulations indicate that Congo red preferentially interacts with PaMurU through electrostatic contacts, primarily involving the residues Arg28 and Arg202. The binding of Congo red to PaMurU was corroborated further using SUPR-differential scanning fluorimetry (SUPR-DSF), which revealed ligand-induced thermal destabilization. Ongoing X-ray crystallographic studies, in conjunction with site-directed mutagenesis and enzyme kinetic analyses, aim to elucidate the binding mode at an atomic resolution. Full article

Background: Protein kinase R (PKR) inhibits general mRNA translation by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). PKR also modulates NF-κB signaling during viral infections, but comparative studies of PKR-mediated NF-κB responses across mammalian species and their regulation by viral inhibitors remain largely unexplored. This study aimed to characterize the conserved antiviral and inflammatory roles of mammalian PKR orthologs and investigate their modulation by poxviral inhibitors. Methods: Using reporter gene assays and quantitative RT-PCR, we assessed the impact of 17 mammalian PKR orthologs on general translation inhibition, stress-responsive translation, and NF-κB-dependent induction of target genes. Congenic human and rabbit cell lines infected with a myxoma virus strain lacking PKR inhibitors were used to compare the effects of human and rabbit PKR on viral replication and inflammatory responses. Site-directed mutagenesis was employed to determine key residues responsible for differential sensitivity to the viral inhibitor M156. Results: All 17 mammalian PKR orthologs significantly inhibited general translation, strongly activated stress-responsive ATF4 translation, and robustly induced NF-κB target genes. Inhibition of these responses was specifically mediated by poxviral K3 orthologs that effectively suppressed PKR activation. Comparative analyses showed human and rabbit PKRs similarly inhibited virus replication and induced cytokine transcripts. Amino acid swaps between rabbit PKRs reversed their sensitivity to viral inhibitor M156 and NF-κB activation. Conclusions: Our data show that the tested PKR orthologs exhibit conserved dual antiviral and inflammatory regulatory roles, which can be antagonized by poxviral K3 orthologs that exploit eIF2α mimicry to modulate the PKR-NF-κB axis. Full article

Equine lutropin hormone/choriogonadotropin receptor (LH/CGR) is a G protein-coupled receptor that binds to both luteinizing hormone and choriogonadotropin, with multiple potential N-linked glycosylation sites in the long extracellular domain region. The roles of these glycosylation sites in hormone binding have been widely studied; however, their relationships with cyclic adenosine monophosphate (cAMP) activation, loss of cell surface receptors, and phosphorylated extracellular signal-regulated kinases1/2 (pERK1/2) expression are unknown. We used site-directed mutagenesis with the substitution of Asn for Gln to alter the consensus sequences for N-linked glycosylation, and cAMP signaling was analyzed in the mutants. Specifically, the N174Q and N195Q mutants exhibited markedly reduced expression levels, reaching approximately 15.3% and 2.5%, respectively, of that observed for wild-type equine LH/CGR. Correspondingly, the cAMP EC₅₀ values were decreased by 7.6-fold and 5.6-fold, respectively. Notably, the N195Q mutant displayed an almost complete loss of cAMP activity, even at high concentrations of recombinant eCG, suggesting a critical role for this glycosylation site in receptor function. Despite these alterations, Western blot analysis revealed that pERK1/2 phosphorylation peaked at 5 min following agonist stimulation across all mutants, indicating that the ERK1/2 signaling pathway remains functionally intact. This study demonstrates that the specific N-linked glycosylation site, N195, in equine LH/CGR is indispensable for cAMP activity but is normally processed in pERK1/2 signaling. Thus, we suggest that in equine LH/CGR, agonist treatment induces biased signaling, differentially activating cAMP signaling and the pERK1/2 pathway. Full article

Background: The amylin receptor is a receptor for the peptide hormone amylin, and its activation is known to reduce body weight. The amylin receptor functions as a heterodimer complex that consists of the calcitonin receptor for peptide hormone calcitonin and an accessary protein. Although the structural information of amylin receptors is currently available, receptor–ligand binding studies that support the peptide binding mode for amylin receptors remain incomplete. Methods: Here, we introduced mutagenesis to the amylin receptor 1 extracellular domain and examined mutational effects on peptide binding affinity. We focused on several residues mainly from the peptide-binding pocket (D97, D101, E123, N124, and N135 of the calcitonin receptor). Two well-known peptide ligands for amylin receptors were used for this study: a salmon calcitonin fragment and an antagonist amylin analog AC413 fragment with Y25P mutation. Results: Among the introduced mutations, D101A and N135A mutations abolished peptide ligand binding, suggesting that these residues are critical for peptide interaction. The N124A mutation also significantly decreased the peptide binding affinity by more than 8-fold. Intriguingly, the N124D mutation restored the decreased affinity of the salmon calcitonin fragment, while it failed to restore the decreased affinity of the AC413 fragment. Structural analyses suggested that there was a potential role of salmon calcitonin serine 29 in the interaction with aspartate of the N124D mutation. Conclusions: This study validates the critical residues of the amylin receptor 1 extracellular domain for the interaction with C-terminal fragments of peptide ligands. This study also suggests that modulating receptor–ligand interaction is feasible by the modification of receptor amino acids near an interacting peptide ligand. Full article

Rhamnose is a natural sugar found in glycoproteins and structural polysaccharides of plants, fungi, and bacteria. Its incorporation into glycoconjugates is mediated by rhamnosyltransferases (RHTs), key enzymes for biomolecular stability and function. While rhamnose biosynthesis has been studied in certain fungal genera, the evolutionary history and distribution of RHTs across the fungal kingdom remain largely unknown. In this study, 351 fungal species were found to encode putative RHTs. Phylogenetic and structural analyses revealed conserved patterns and similarities with previously characterized RHTs. Molecular docking predicted a high affinity of these proteins for UDP-L-rhamnose, and in silico mutagenesis identified key residues potentially involved in substrate binding. Carbohydrate profiling confirmed the presence of rhamnose in the cell walls of multiple fungi, including Aspergillus, Madurella, Metarhizium, and Trichoderma species. Enzymatic assays further supported rhamnose transfer activity. These findings provide the first comprehensive in silico characterization of fungal RHTs, uncovering conserved sequence motifs despite overall diversity, which may be linked to functional adaptation in different fungal lineages. Full article

Phage display has advanced the discovery of peptides that selectively bind to a wide variety of cell surface molecules, offering new modalities to modulate disease-related protein–protein interactions (PPIs). These cell-binding peptides occupy a unique pharmaceutical space between small molecules and large biologics, and their growing popularity has opened up new avenues for targeting cell surface proteins that were previously considered undruggable. This work provides an overview of methods for identifying cell-selective peptides using phage display combinatorial libraries, covering in vitro, ex vivo, and in vivo biopanning approaches. It addresses key considerations in library design, including the peptide conformation (linear vs. cyclic) and length, and highlights examples of clinically approved peptides developed through phage display. It also discusses the on-phage chemical cyclization of peptides to overcome the limitations of genetically encoded disulfide bridges and emphasizes advances in combining next-generation sequencing (NGS) with phage display to improve peptide selection and analysis workflows. Furthermore, due to the often suboptimal binding affinity of peptides identified in phage display selections, this article discusses affinity maturation techniques, including random mutagenesis and rational design through structure–activity relationship (SAR) studies to optimize initial peptide candidates. By integrating these developments, this review outlines practical strategies and future directions for harnessing phage display in targeting challenging cell surface proteins. Full article

Background: Treatment of metastatic cancer remains a challenge, because cancer cells acquire resistance even to the most contemporary therapies. This study analyzed the role of the phosphoprotein Stathmin 1 (STMN1) in regulating cancer cell growth and metastatic potential. Methods: Public datasets with metastatic castration-resistant prostate cancer (mCRPC) and breast cancer (BC) were analyzed to determine the interrelationship between STMN1, hepatocyte growth factor (HGF) and MET proto-oncogene (MET) expression, overall survival, and response to chemotherapy. Site-directed mutagenesis, cell cycle analysis, proliferation, and migration and invasion assays determined the impact of STMN1 phosphorylation on proliferation and metastatic potential. Results: Increased STMN1 associates with HGF and MET gene expression in mCRPC, and taxane chemotherapy further increases HGF expression. STMN1 and HGF are highest, and overall survival is poorest in mCRPC in the liver compared to other sites, implying the metastatic site influences their expression levels and potentially the pattern of metastatic spread. Increased STMN1 and MET also predict taxane responsiveness in BC patients. Analysis of STMN1 serine (S)16, 25, 38, and 63 determined that total (t) STMN1 and STMN1 S16 phosphorylation (pSTMN1^S16) are co-regulated by HGF/MET during cell cycle progression, pSTMN1^S16 alone can promote cell proliferation, and pSTMN1^S16 shortens the cell cycle similar to HGF treatment, while STMN1^S16 dephosphorylation lengthens the cell cycle to arrest cell growth in G2/M, similar to HGF plus the MET inhibitor AMG337. Importantly, STMN1^S16 does not promote metastasis. Conclusions: Selectively inhibiting STMN1^S16 phosphorylation may provide an alternative strategy for inhibiting MET-mediated cell growth to eliminate metastatic cancer cells and inhibit further metastasis. Full article

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea. Preventative vaccines or novel treatments based on a better understanding of the molecular basis of N. gonorrhoeae infection are required as resistance to current antibiotics is widespread. Toxin–antitoxin (TA) systems modulate bacterial physiology by interfering with vital cellular processes; type II TA systems, where both toxin and antitoxin are proteins, are the best-studied. Bioinformatics analysis revealed genes encoding an uncharacterized type II HicAB TA system in the N. gonorrhoeae strain FA1090 chromosome, which were also present in >83% of the other gonococcal genome sequences examined. Gonococcal HicA overproduction inhibited bacterial growth in Escherichia coli, an effect that could be counteracted by the co-expression of HicB. Kill/rescue assays showed that this effect was bacteriostatic rather than bactericidal. The site-directed mutagenesis of key histidine and glycine residues (Gly22, His24, His29) abolished HicA-mediated growth arrest. N. gonorrhoeae FA1090∆hicAB and complemented derivatives that expressed IPTG-inducible hicA, hicB, or hicAB, respectively, grew as wild type, except for IPTG-induced FA1090∆hicAB::hicA. RT-PCR demonstrated that hicAB are transcribed in vitro under the culture conditions used. The deletion of hicAB had no effect on biofilm formation. Our study describes the first characterization of a HicAB TA system in N. gonorrhoeae. Full article

TP53 is the most frequently mutated gene in human cancers. Many studies have reported oncogenic gain of function by mutant p53 and suggested that mutant p53 is a potential therapeutic target. In striking contrast, a recent approach relying on CRISPR-mediated mutagenesis led to the conclusion that mutant p53 removal in tumors had no therapeutic value. However, experimental limitations likely affected this study, including the difficulty of recapitulating the events leading to mutant p53 gain of function in cancer cell lines. Furthermore, a low statistical power may have masked the impact of mutant p53 removal in organoid-derived tumors. Independently, two studies focusing on the human hotspot mutant TP53^Y220C and its murine homolog Trp53^Y217C recently provided compelling evidence that mutant p53 can be a valid therapeutic target. Drugs designed to stabilize the mutant protein and restore wild-type p53 functions, or to inhibit the inflammatory effects associated with mutant p53, appear particularly promising. Full article

The immune response triggered when plant cell surface receptors recognize pathogen-associated molecular patterns (PAMPs) is known as PAMP-triggered immunity (PTI). Several studies have demonstrated that extracellular plant ferredoxin-like protein (PFLP) can enhance PTI signaling, thereby conferring resistance to bacterial diseases in various plants. The C-terminal casein kinase II (CK2) phosphorylation region of PFLP is essential for strengthening PTI. However, whether phosphorylation at this site directly enhances PTI signaling and consequently increases plant disease resistance remains unclear. To investigate this, site-directed mutagenesis was used to generate PFLPT90A, a non-phosphorylatable mutant, and PFLPT90D, a phospho-mimetic mutant, for functional analysis. Based on the experimental results, none of the recombinant proteins were able to enhance the hypersensitive response induced by the HrpN protein or increase resistance to the soft rot pathogen Pectobacterium carotovorum subsp. carotovorum ECC17. These findings suggest that phosphorylation at the T90 residue might be essential for PFLP-mediated enhancement of plant immune responses, implying that this post-translational modification is likely required for its disease resistance function in planta. To further explore the relationship between PFLP phosphorylation and endogenous CK2, the Arabidopsis insertion mutant cka2 and the complemented line CKA2R were analyzed under treatment with flg22_Pst from Pseudomonas syringae pv. tomato. The effects of PFLP on the hypersensitive response, rapid oxidative burst, callose deposition, and susceptibility to soft rot confirmed that CK2 is required for these immune responses. Furthermore, expression analysis of PTI-related genes FRK1 and WRKY22/29 in the mitogen-activated protein kinase (MAPK) signaling pathway demonstrated that CK2 is necessary for PFLP to enhance flg22_Pst-induced immune signaling. Taken together, these findings suggest that PFLP enhances A. thaliana resistance to bacterial soft rot primarily by promoting the MAPK signaling pathway triggered by PAMP recognition, with CK2-mediated phosphorylation being essential for its function. Full article

Tea has become one of the most popular drinks worldwide thanks to its pleasant sensory attributes and diverse health benefits. However, tannin-rich compositions have several negative effects and significantly impact the quality of tea beverages. Among various detannification methods, tannase treatment appears to be the most secure and environmentally friendly strategy. Although numerous microbial tannases have been identified and used in food processing, they are predominantly mesophilic with compromised heat tolerance, which limit their application in high-temperature tea extraction processing. Computer-assisted rational design and site-directed mutagenesis has emerged as a promising strategy in enzyme engineering to improve the thermostability of industrial enzymes. Nevertheless, relevant studies for tannase thermostability improvement remain lacking. In the present study, a novel thermophilic tannase called TanPL1 from marine fungus Penicillium longicatenatum strain SM102 was expressed in the food-grade host Yarrowia lipolytica. After purification and characterization, the thermostability of this enzyme was improved through site-directed mutagenesis guided by computer-aided rational design and molecular dynamics simulations. Then the thermostable mutant MuTanPL1 was applied in green tea processing for both polyphenol extraction and ester catechin hydrolysis. The tannase yield and specific activity values of 166.4 U/mL and 1059.3 U/mg, respectively, were achieved. The optimum pH and temperature of recombinant TanPL1 were determined to be 5.5 and 55 °C, respectively, and the enzyme exhibited high activity toward various gallic acid ester substrates. The site-directed mutagenesis method successfully generated a single-point mutant, MuTanPL1, with significantly enhanced thermostability and a higher optimum temperature of 60 °C. After 2 h of detannification by MuTanPL1, nearly all gallated catechins in green tea infusion were biotransformed. This resulted in a 202.4% and 12.1-fold increase in non-ester catechins and gallic acid levels, respectively. Meanwhile, the quality of the tea infusion was also markedly improved. Sensory evaluation and antioxidant activity assays revealed notable enhancements in these properties, while turbidity was reduced considerably. Additionally, the α-amylase inhibition activity of the tannase-treated tea infusion declined from 50.49% to 8.56%, revealing a significantly lower anti-nutritional effect. These findings suggest that the thermostable tannase MuTanPL1 holds strong application prospects in tea beverage processing. Full article

The Fabaceae family, the third-largest among flowering plants, is nutritionally vital, providing rich sources of protein, dietary fiber, vitamins, and minerals. Leguminous plants, such as soybeans, peas, and chickpeas, typically contain two to three times more protein than cereals like wheat and rice, with low fat content (primarily unsaturated fats) and no cholesterol, making them essential for cardiovascular health and blood sugar management. Since the release of the soybean genome in 2010, genomic research in Fabaceae has advanced dramatically. High-quality reference genomes have been assembled for key species, including soybeans (Glycine max), common beans (Phaseolus vulgaris), chickpeas (Cicer arietinum), and model legumes like Medicago truncatula and Lotus japonicus, leveraging long-read sequencing, single-cell technologies, and improved assembly algorithms. These advancements have enabled telomere-to-telomere (T2T) assemblies, pan-genome constructions, and the identification of structural variants (SVs) and presence/absence variations (PAVs), enriching our understanding of genetic diversity and domestication history. Functional genomic tools, such as CRISPR-Cas9 gene editing, mutagenesis, and high-throughput omics (transcriptomics, metabolomics), have elucidated regulatory networks controlling critical traits like photoperiod sensitivity (e.g., E1 and Tof16 genes in soybeans), seed development (GmSWEET39 for oil/protein transport), nitrogen fixation efficiency, and stress resilience (e.g., Rpp3 for rust resistance). Genome-wide association studies (GWAS) and comparative genomics have further linked genetic variants to agronomic traits, such as pod size in peanuts (PSW1) and flowering time in common beans (COL2). This review synthesizes recent breakthroughs in legume genomics, highlighting the integration of multi-omic approaches to accelerate gene cloning and functional confirmation of the genes cloned. Full article

Barley, rich in beneficial ingredients, has been recognised as a healthy food and is widely used in the production of healthy foods for humans. The current study identified a new barley mutant with the SSIIa, SSIIIa, SBEIIa, and SBEIIb genes inactivated in the genome-edited offspring of targeted mutagenesis of starch synthetic genes using multiplex genome editing. The grain compositions and starch properties of the ssIIa/ssIIIa/sbeIIa/sbeIIb mutant were analysed and compared with the corresponding parameters of ssIIa, ssIIIa, sbeIIa/sbeIIb, ssIIa/sbeIIa/sbeIIb, and non-genome-edited lines (NE), respectively. ssIIa/ssIIIa/sbeIIa/sbeIIb exhibited the highest contents of β-glucan and amylose content among all mutants and NE, but not the most prominent in resistant starch, fructan, and fibre contents. The loss of SSIIa, SSIIIa, SBEIIa, and SBEIIb genes also resulted in significant changes in starch properties. This study enriched the genotypes of healthy barley and provided a theoretical basis for improving barley quality. Full article