Search Results (397)

The phosphorylation of Rab10 (pT73-Rab10) by LRRK2 promotes the establishment of epithelial cell polarity by controlling the trafficking to the primary cilia membrane of cilia-resident proteins and signaling proteins. Previous studies have identified a site in the globular tail domain of MYO5A that specifically binds to only the phosphorylated form of Rab10. In this work, we have demonstrated that pT73-Rab10 does not associate with the globular tail of MYO5B. We have mapped the putative binding site to a required three amino acids (MEN, 1473–1475) in the MYO5A globular tail domain that are not found in the MYO5B globular tail. Substitution of the MEN amino acid sequence found in MYO5A into the paralogous position in the MYO5B globular tail conferred the ability to associate with pT73-Rab10. The results demonstrate that the interactors with MYO5A and MYO5B are not completely overlapping and that the interaction of pT73-Rab10 is specific to the MYO5A globular tail domain. Full article

Infections with cytomegalovirus (CMV) can result in increased morbidity and mortality in immunocompromised patients. The pUL97 kinase is a critical enzyme in the regulation of CMV replication. Although it does not phosphorylate deoxynucleosides, this enzyme is involved in the first phosphorylation step of ganciclovir (GCV), a viral DNA polymerase inhibitor. In contrast, maribavir (MBV) is a specific inhibitor of pUL97 kinase activity. In this paper, we analyzed the already-reported amino acid changes, conferring resistance to MBV and cross-resistance to GCV, in the pUL97 protein structure, predicted with AlphaFold2. Docking experiments suggest that MBV is a dual-site inhibitor, targeting ATP binding and substrate phosphorylation. Substitutions that confer resistance to MBV only may directly or indirectly alter the shape of the cavity in the vicinity of the invariant K355 in the putative ATP binding site, without affecting the viral growth. The most frequently encountered T409M substitution may correspond to a gatekeeper mutation. Substitutions that induce cross-resistance to MBV and GCV may directly or indirectly affect the environment of D456 and N461 residues in the catalytic loop, with reduced viral replicative capacity. These results have implications for the clinical use of MBV as well as for the design of novel pUL97 kinase inhibitors. Full article

Introduction: Genetic plasticity and adaptive camouflage in critical pathogens have contributed to the global surge in multidrug-resistant (MDR) infections, posing a serious threat to public health and therapeutic efficacy. Antimicrobial resistance, now a leading cause of global mortality, demands urgent action through diagnostics, vaccines, and therapeutics. In India, the Indian Council of Medical Research’s surveillance network identifies Escherichia coli as a major cause of urinary tract infections, with increasing prevalence in human gut microbiomes, highlighting its significance across One Health domains. Methods: Whole-genome sequencing of E. coli strain ECG015, isolated from a human gut sample, was performed using the Illumina NextSeq platform. Results: Genomic analysis revealed multiple antibiotic resistance genes, virulence factors, and efflux pump components. Phylogenomic comparisons showed close relatedness to pathovars from both human and animal origins. Notably the genome encoded protein tyrosine kinases (Etk/Ptk and Wzc) and displayed variations in the envelope stress-responsive CpxAR two-component system. Promoter analysis identified putative CpxR-binding sites upstream of genes involved in resistance, efflux, protein kinases, and the MazEF toxin–antitoxin module, suggesting a potential regulatory role of CpxAR in stress response and persistence. Conclusions: This study presents a comprehensive genomic profile of E. coli ECG015, a gut-derived isolate exhibiting clinically significant resistance traits. For the first time, it implicates the CpxAR two-component system as a potential central regulator coordinating antimicrobial resistance, stress kinase signaling, and programmed cell death. These findings lay the groundwork for future functional studies aimed at targeting stress-response pathways as novel intervention strategies against antimicrobial resistance. Full article

Introduction: The interface between T cells and the tumor microenvironment, termed the ‘immunological synapse’, consists of multiple checkpoint protein pairs co-expressed on both sides of the synapse. mir-16, a microRNA from a widely known tumor-suppressor family of miRNAs, was previously shown by us to be downregulated in melanoma. As other miRNAs from this family have been shown to directly target checkpoint proteins, here we investigated whether miR-16 influences the expression patterns of checkpoint proteins in melanoma. Methods: Single-cell gene expression data from the melanoma microenvironment were retrieved from a public database. Melanoma cell lines were established from metastatic lesions and transiently transfected with an hsa-miR-16-5p-mimic RNA or a mir-16-expressing plasmid. The mRNA expression profiles were analyzed using an Affymetrix microarray. Direct targets of miR-16 were identified by luciferase reporter assays. Protein levels were assessed by Western blotting. Results: Bioinformatic analysis revealed that the expression levels of eight checkpoint mRNAs, known to be present on the melanoma side of the immunological synapse, were highly correlated. Four of these mRNAs contained putative binding sites for the miR-15/16 family. miR-16 expression was significantly reduced in melanoma cells, compared to normal melanocytes. Luciferase reporter assays demonstrated that miR-16 directly targets the 3′ untranslated regions (3′UTRs) of CD40, CD80. The mRNAs downregulated following miR-16 overexpression were highly enriched for genes involved in autophagy, vesicle-mediated transport, and the regulation of protein membrane localization. Among these, VTI1B and SMPD1 were confirmed to be direct targets of miR-16. Transient overexpression of miR-16 resulted in a significant reduction in SMPD1 and VTI1B levels in melanoma cell lines. Conclusions: Our findings suggest that miR-16 potentially modulates melanoma tumorigenesis, metastasis and immunogenicity by altering the composition of checkpoint proteins at the immunological synapse and by regulating cellular pathways associated with intracellular trafficking and transmembrane protein presentation. Full article

Background/Objectives: Cancer progression and the hemostatic system are closely linked. Coagulation factor V (FV) has a key function in coagulation, with both pro- and anticoagulant functions. FV gene (F5) expression and F5 variants have been linked to breast cancer progression. The direct impact of F5 variants on FV expression and functional effects in breast cancer are unknown. We aimed to investigate whether the F5 variants FV Leiden (F5 rs6025) and F5 rs6028 influenced FV expression, coagulant activity, and apoptosis in breast cancer cells. Methods: MDA-MB-231 cells were transfected with overexpression plasmids containing F5 wild type, F5 rs6025 or F5 rs6028. We investigated the functional impact of the F5 variants on F5 mRNA, FV protein, FV coagulant activity, and apoptosis in vitro, and examined the potential of the variants as transcriptional regulators of F5 expression in silico. Results: Increased F5 mRNA, FV protein, and apoptosis were observed in cells transfected with F5 wild-type overexpression plasmid compared to empty vector. F5 mRNA, protein, coagulant activity, and apoptosis were further increased with the F5 rs6025 and F5 rs6028 variants compared to F5 wild type. Cis-expression quantitative trait loci analyses indicated a regulatory effect of F5 rs6028, and putative transcription factor binding sites for several transcription factors overlapped with the position of F5 rs6025. Conclusions: Our study demonstrated that F5 rs6025 and F5 rs6028 have a regulatory effect on FV synthesis that might affect apoptosis in breast cancer. The F5 variants might therefore enhance the tumor suppressor function of FV in breast cancer. Full article

Bile acids (BAs) are synthesized in the liver from cholesterol and are subsequently conjugated with glycine and taurine. In the intestine, bile acids undergo various modifications, such as deconjugation, dehydrogenation, oxidation, and epimerization by the gut microbiota. These bile acids are absorbed in the intestine and transported to the liver as well as the systemic circulation. BAs can activate many types of receptors, including nuclear receptors and cell surface receptors. By activating these receptors, BAs can exert various effects on the metabolic, immune, and nervous systems. Recently, the detailed structure of TGR5, the major plasma membrane receptor for BAs, was elucidated, revealing a putative second BA binding site along with the orthosteric binding site. Furthermore, BAs act as ligands for bitter taste receptors and the Leukemia inhibitory factor receptor. In addition, the Mas-related, G-protein-coupled receptor X4 interacts with receptor activity-modifying proteins. Thus, a variety of cell surface receptors are associated with BAs, and BAs are thought to have very complex activities. This review focuses on recent advances regarding cell surface receptors for bile acids and the biological actions they mediate. Full article

Background/Objectives: PAX3 is a transcription factor that drives melanoma progression by promoting cell growth, migration, and survival, while inhibiting cellular terminal differentiation. However, known PAX3 target genes are limited and cannot fully explain the wide impact of PAX3 function. The PAX3 protein can regulate DNA through two separate binding domains, the Paired Domain (PD) and Homeodomain (HD), which bind different DNA motifs. It is not clear if these two domains bind and work together to regulate genes and if they promote all or only a subset of downstream cellular events. Methods: PAX3 direct downstream targets were identified using Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assays in SK-MEL-5 melanoma cells. PAX3-binding genomic regions were identified through MACS2 peak calling, and peaks were categorized based on the presence of PD and/or HD binding sites (or neither) through HOMER motif analysis. The peaks were further characterized as Active, Primed, Poised, Repressed, or Closed based on ATAC-seq data and CUT&RUN for histone Post-Translational Modifications H3K4me1, H3K4me3, H3K27me3, and H3K27Ac. Results: This analysis revealed that most of the PAX3 binding sites in the SK-MEL-5 cell line were primarily through the PD and connected to Active genes. Surprisingly, PAX3 does not commonly act as a repressor in SK-MEL-5 cells. Pathway analysis identified genes involved with transcription, RNA modification, and cell growth. Peaks located in distal enhancer elements were connected to genes involved in neuronal growth, function, and signaling. Conclusions: Our results reveal novel PAX3 regulatory regions and putative genes in a melanoma cell line, with a predominance of PAX3 PD binding on active sites. Full article

Antibiotic resistance has emerged as a critical global public health challenge, prompting increased interest in non-antibiotic antimicrobial strategies such as bacteriophage-derived endolysins. Although endolysins possess strong lytic potential, their application to Gram-negative bacteria remains limited due to the outer membrane barrier. PlyKp104 is a recently identified phage-derived endolysin that exhibits lytic activity against Gram-negative bacteria without the aid of membrane permeabilizers. In this study, the crystal structure of PlyKp104 was determined at a resolution of 1.85 Å. PlyKp104 consists solely of a catalytic SLT domain, and structure-based analysis revealed a putative active site and key structural features associated with substrate binding. Comparative analysis with homologous structures suggested that PlyKp104 belongs to lytic transglycosylase family 1. B-factor analysis and hydrophobic interaction mapping indicated that the domain exhibits high structural stability, supported by conserved hydrophobic residues clustered in motifs I and II. During structure determination, an unidentified electron density was consistently observed near a neutral, hydrophobic surface region. Its shape and environment suggest the presence of a lipid-like molecule, implying a potential lipid-binding site. These findings provide structural insight into PlyKp104 and contribute to the understanding of endolysin mechanisms against Gram-negative bacteria, with implications for future protein engineering efforts. Full article

Prolactin (PRL) regulates its own secretion by short-loop feedback to tuberoinfundibular dopaminergic (TIDA) neurons. PRL-induced cellular mechanisms in the regulation of tyrosine hydroxylase (TH) are not completely understood. The objectives were to (1) examine PRL-induced, time-dependent hypothalamic changes in JAK2-STAT5B signaling, TH activity, TH phosphorylation state and Th mRNA levels, and (2) evaluate direct influences of PRLR-STAT5B signaling on Th promoter activity. Ovariectomized rats were administered ovine PRL. JAK2 and STAT5 phosphorylation in the mediobasal hypothalamus peaked at 15 and 30–60 min, respectively. TH Ser40 phosphorylation in the median eminence was increased between 2 and 72 h, correlating with increased dihydroxyphenylalanine (DOPA) accumulation. Th mRNA levels in TIDA neurons were unchanged up to 72 h but elevated by 7 days. PRL did not alter Th promoter activity in CAD cells, and STAT5B did not bind three putative Gamma Interferon Activation Sites (GAS) elements. We conclude that PRL initiates an integrated cascade of cellular mechanisms in TIDA neurons, including JAK2-STAT5B activation, TH Ser40 phosphorylation coupled to increased TH activity, followed by a delayed rise in Th gene expression. PRL-induced changes in Th gene expression are not the result of STAT5-mediated transactivation but likely result from enduring changes in TIDA neuronal activity. Full article

Non-Small Cell Lung Cancer (NSCLC) is the most typical kind of lung cancer. Chemotherapy, radiation therapy, and other traditional cancer therapies are ineffective. Advancements in understanding cancer’s molecular causes have led to targeted therapies, such as those addressing NTRK gene fusions in NSCLC. Several machine-learning techniques were used in our work, including k-Nearest Neighbors (kNN), Support Vector Machine (SVM), Random Forest (RF), and Naive Bayes (NB). As a result, the RF model outperformed the other studied machine-learning methods, achieving an astonishing 93.12% accuracy for both training as well as testing datasets, and it was employed to screen 9000 chemicals, resulting in the discovery of 65 putative NTRK potential inhibitors. The active sites of NTRK proteins were then docked with these 65 active chemicals. Our findings show that Gancaonin X, 5-hydroxy-2-(4-methoxyphenyl)-8,8-dimethyl-2,3-dihydropyrano[2,3-h]chromen-4-one, (2S)-7-[[(2R)-3,3-dimethyloxiran-2-yl]methoxy]-5-hydroxy-2-phenyl-2,3-dihydrochromen-4-one, (2S)-5-hydroxy-2-(4-methoxyphenyl)-8,8-dimethyl-2,3-dihydropyrano[2,3-h]chromen-4-one, and methyl 2-(methylamino)-5-[(3S)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-3-yl]benzoate establish strong interactions inside the binding region of NTRK, as a result of which stable complexes are formed. This study employs 100 ns molecular dynamics simulations to investigate the dynamic behavior of phytochemical-NTRK complexes, revealing stable interactions through RMSD, RMSF, Rg, and SASA analyses. The detailed examination of protein–ligand interactions provides crucial atomic-level insights, enhancing our understanding of potential neurotrophic receptor kinase-targeted therapeutic strategies. This highlights their significant ability as NTRK antagonists, giving novel treatment options for NSCLC therapy. To summarize, the application of machine learning in combination with virtual screening in this study not only can discover new NSCLC therapeutics but also highlight new computer approaches in the field of drug discovery. Full article

RAS mutations occur in about 30% of human cancers, leading to enhanced RAS signaling and tumor growth. KRAS is the most commonly mutated oncogene in human tumors, especially lung, pancreatic, and colorectal cancers. Direct targeting of KRAS is difficult due to its highly conserved sequence; but, its complex with the guanine nucleotide exchange factor Son of Sevenless (SOS) 1 promises an attractive target for inhibiting RAS-mediated signaling. Here, we first revealed putative allosteric binding sites of the SOS1, KRASG12C-SOS1 complex, and the ternary KRASG13D-SOS1 complex structures using two network-based models, the essential site scanning analysis and the residue interaction network model. The results enabled us to identify two new putative allosteric pockets for the ternary KRASG13D-SOS1 complex. These were then screened together with the known ligand binding site against the natural compounds in the InterBioScreen (IBS) database using the Glide software package developed by Schrödinger, Inc. The docking poses of seven hit compounds were assessed using 400 ns long molecular dynamics (MD) simulations with two independent replicas using Desmond, coupled with thermal MM-GBSA calculations for the estimation of the binding free energy values. The structural skeleton of the seven proposed compounds consists of different functional groups and heterocyclic rings that possess anti-cancer activity and exhibit persistent interactions with key residues in binding pockets throughout the MD simulations. STOCK1N-09823 was determined as the most promising hit that promoted the disruption of the interactions R73 (chain A)/N879 and R73 (chain A)/Y884, which are key for SOS1-mediated KRAS activation. Full article

The European rabbit exists in the wild and has several highly bred domesticated forms. There are well-separated wild European rabbit populations, and intensive breeding has resulted in various forms and utilizations. In this work, we aimed to carry out an extended WGS-based population genomics study on several wild European rabbit populations and selected breeds. Utilizing multiple methods, we showed that although domestic and wild populations were clearly separated, there was evidence of admixture between them in France and Hungary. The populations showed various levels of inbreeding, with one of the Hungarian subpopulations having excess runs of homozygosity. We identified numerous variants fixed in either domestic or wild animals, two of which were found to be fixed at different alleles in the two populations. Some putatively selected regions did not overlap with any known genes in the rabbit genome, suggesting some importance to these intergenic sites. The enrichment of selected regions in certain types of transcription factor binding sites suggests a possible role for these regulatory elements during domestication. In addition, the new high-coverage rabbit whole-genome sequences may provide helpful material for further population genetics analyses. Full article

Glutathione S-transferases (GSTs) are promising pharmacological targets for developing antiparasitic agents against helminths, as they play a key role in detoxifying cytotoxic xenobiotics and managing oxidative stress. Inhibiting GST activity can compromise parasite viability. This study reports the successful identification of two selective inhibitors for the mu-class glutathione S-transferase of 25 kDa (Ts25GST) from Taenia solium, named i11 and i15, using a computationally guided approach. The workflow involved modeling and refining the 3D structure from the sequence using the AlphaFold algorithm and all-atom molecular dynamics simulations with an explicit solvent. Representative structures from these simulations and a putative binding site with low conservation relative to human GSTs, identified via the SILCS methodology, were employed for virtual screening through ensemble docking against a commercial compound library. The two compounds were found to reduce the enzyme’s activity by 50–70% under assay conditions, while showing a reduction of only 30–35% for human mu-class GSTM1, demonstrating selectivity for Ts25GST. Notable, i11 displayed competitive inhibition with CDNB, while i15 exhibited a non-competitive inhibition type. Full article

Quinoa (Chenopodium quinoa) is an Andean allotetraploid pseudocereal crop with higher protein content and balanced amino acid composition in the seeds. Ammonium (NH₄⁺), a direct source of organic nitrogen assimilation, mainly transported by specific transmembrane ammonium transporters (AMTs), plays important roles in the development, yield, and quality of crops. Many AMTs and their functions have been identified in major crops; however, no systematic analyses of AMTs and their regulatory networks, which is important to increase the yield and protein accumulation in the seeds of quinoa, have been performed to date. In this study, the CqAMTs were identified, followed by the quantification of the gene expression, while the regulatory networks were predicted based on weighted gene co-expression network analysis (WGCNA), with the putative transcriptional factors (TFs) having binding sites on the promoters of CqAMTs, nitrate transporters (CqNRTs), and glutamine-synthases (CqGSs), as well as the putative TF expression being correlated with the phenotypes and activities of GSs, glutamate synthase (GOGAT), nitrite reductase (NiR), and nitrate reductase (NR) of quinoa roots. The results showed a total of 12 members of the CqAMT family with varying expressions in different organs and in the same organs at different developmental stages. Complementation expression analyses in the triple mep1/2/3 mutant of yeast showed that except for CqAMT2.2b, 11/12 CqAMTs restored the uptake of NH₄⁺ in the host yeast. CqAMT1.2a was found to mainly locate on the cell membrane, while TFs (e.g., CqNLPs, CqG2Ls, B3 TFs, CqbHLHs, CqZFs, CqMYBs, CqNF-YA/YB/YC, CqNACs, and CqWRKY) were predicted to be predominantly involved in the regulation, transportation, and assimilation of nitrogen. These results provide the functions of CqAMTs and their possible regulatory networks, which will lead to improved nitrogen use efficiency (NUE) in quinoa as well as other major crops. Full article

The conserved role of juvenile hormone (JH) signals in preventing larvae from precocious metamorphosis has been confirmed in insects. Crustaceans have different metamorphosis types from insects; we previously proved that methyl farnesoate (MF) can prohibit larvae metamorphosis in mud crabs, but the molecular signal of this process still needs to be elucidated. In this study, methoprene-tolerant (Met) of Scylla paramamosain was obtained and characterized, which we named Sp-Met. Sp-Met contains a 3360 bp ORF that encodes 1119 amino acids; the predicted protein sequences of Sp-Met include one bHLH, two PAS domains, one PAC domain, and several long unusual Gln repeats at the C-terminal. AlphaFold2 was used to predict the 3D structure of Sp-Met and the JH binding domain of Met. Furthermore, the binding properties between Sp-Met and MF were analyzed using CD-DOCK2, revealing a putative high affinity between the receptor and ligand. In silico site-directed mutagenesis suggested that insect Mets may have evolved to exhibit a higher affinity for both MF or JH III compared to the Mets of crustaceans. In addition, we found that the expression of Sp-Met was significantly higher in female reproductive tissues than in males but lower in most of the other examined tissues. During larval development, the expression variation in Sp-Met and Sp-Kr-h1 was consistent with the immersion effect of MF. The most interesting finding is that knockdown of Sp-Met blocked the inhibitory effect of MF on metamorphosis in the fifth zoea stage and induced pre-metamorphosis phenotypes in the fourth zoea stage. The knockdown of Sp-Met significantly reduced the expression of Sp-Kr-h1 and two ecdysone signaling genes, Sp-EcR and Sp-E93. However, only the reduction in Sp-Kr-h1 could be rescued by MF treatment. In summary, this study provides the first evidence that MF inhibits crustacean larval metamorphosis through Met and that the MF-Met→Kr-h1 signal pathway is conserved in mud crabs. Additionally, the crosstalk between MF and ecdysteroid signaling may have evolved differently in mud crabs compared to insects. Full article