Search Results (106)

Exosomes are important mediators of host–parasite communication and contain diverse molecules that may support the survival of Clonorchis sinensis in the biliary tract. To explore their biochemical properties, exosomes isolated from excretory–secretory products of Korean C. sinensis isolates were characterized through integrated morphological, proteomic, and gene ontology analyses. The vesicles exhibited typical exosomal size ranges and marker profiles, and their protein components were enriched for cytoskeletal, metabolic, and vesicle-trafficking components relevant to epithelial signaling and immune modulation. Among these proteins, sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) was examined in detail to obtain molecular evidence suggesting its role in sphingolipid metabolism in the parasite. The C. sinensis SMPDL3A (Cs_SMPDL3A) shared the overall structure and core catalytic residues with mammalian homologs, SMPDL3A and sphingomyelin phosphodiesterase 1 (SMPD1), a finding consistent with the possibility that Cs_SMPDL3A may retain authentic sphingomyelinase activity. Although lacking the saponin B domain of SMPD1, Cs_SMPDL3A carries a C-terminal transmembrane segment that may facilitate sphingomyelin access by positioning the enzyme on lipid bilayers. Collectively, these findings suggest that Cs_SMPDL3A participates in host sphingomyelin turnover, potentially generating ceramide for uptake by SMPD1-lacking C. sinensis or contributing to ceramide-associated immune responses in the biliary tract, offering new insight into lipid-centered host–parasite interactions during clonorchiasis. Full article

Annual wild soybean is characterized by a high protein content. To elucidate the genetic basis, this study utilized a chromosome segment substitution line population (177 lines) constructed with cultivated soybean NN1138-2 as the recipient and wild soybean N24852 as the donor. Phenotypic analyses across three environments revealed significant variation in protein content ranging from 42.86% to 49.08%, with a high heritability of 0.70, indicating strong genetic control. Through high-throughput sequencing, six wild segments associated with high protein content were detected on chromosomes 3, 6, 9, 15, and 20, with phenotypic variation explained (PVE) by individual segments ranged from 3.58% to 22.46%, with segments on chromosomes 9, 15, and 20 as large-effect segments with PVE > 10%. All wild segments exhibited positive additive effects (0.42–1.09%), consistent with the characteristic of a high protein content in wild soybean. Compared with previous studies, five segments overlapped with reported loci, while qPro6.1 on chromosome 6 was a novel discovery. Integration of genomic and transcriptomic data identified 10 genes involved in nucleic acid binding, transmembrane protein transport, and amino acid synthesis pathway, with homologs validated in soybean, rice, and rapeseed. This research deepens the understanding of wild soybean’s high protein and offers new gene resources for breeding high-protein cultivated soybean. Full article

Toll-like receptors (TLRs) are central to pathogen recognition in teleost innate immunity. In this study, we surveyed 41 genomes from four representative teleost orders (i.e., Cypriniformes, Siluriformes, Perciformes, and Pleuronectiformes) for 15 TLR genes (TLR1–9, 12, 13, 18, 20–22) revealed a conserved core (TLR2/3/7 in nearly all examined species) alongside lineage-specific losses (TLR4/9/18/20/21/22), indicating both strong conservation and dynamic diversification of the TLR repertoire. We further identified and characterized 12 TLR genes in economically important darkbarbel catfish (Pelteobagrus vachellii). Corresponding cDNAs span 2089–4456 bp and encode proteins of 789–1,087 aa, with canonical extracellular LRR arrays and C-terminal TIR domains but notable “non-classical” features (such as absence of signal peptides in TLR1/13; no transmembrane segment in TLR7; multiple transmembranes in TLR3/8/13/18/22), suggesting subcellular and functional heterogeneity of various TLR genes. Subsequent gene-structure comparisons uncovered gene-specific exon–intron organizations and variable UTR lengths, implicating differential post-transcriptional regulation. Predicted 3D structures retain the traditional hallmark LRR horseshoe fold with subtle variations potentially tuning ligand specificity. Genomic synteny with Pseudobagrus ussuriensi and Pangasianodon hypophthalmus reveals conserved chromosomal organization, and phylogeny construction resolves each TLR subtype into well-supported monophyletic clades, which underscore evolutionary stability. Functionally, exogenous Aeromonas hydrophila challenge triggered rapid, tissue-dependent TLR up-regulation in the kidney, liver, and especially gill (with some transcripts > 1000-fold), highlighting coordinated mucosal and systemic surveillance in darkbarbel catfish. Taken together, these valuable data provide a comprehensive framework for the structural, evolutionary, and inducible expression landscape of catfish TLRs and establish a foundation for in-depth studies on antibacterial immunity in diverse teleost species. Full article

Plant vacuolar-type Na⁺, K⁺/H⁺ antiporters (NHXs) play important roles in pH and K⁺ homeostasis and osmotic balance under normal physiological conditions. Under salt stress, vacuolar-type NHX enhances salt tolerance by compartmentalizing Na⁺ into vacuoles. However, the ion transport mechanism of vacuolar-type NHX remains poorly understood due to the absence of resolved protein crystal structures. To investigate the ion transport mechanism for vacuolar-type NHX, the three-dimensional structure of rice vacuolar-type NHX1 (OsNHX1) was established through homology modeling and AlphaFold3.0. The OsNHX1 model contains thirteen transmembrane segments according to hydrophobic characteristics and empirical and phylogenetic data. Furthermore, this study validated the OsNHX1 model via functional experiments, revealing a set of key charged amino acids essential for its activity. Mapping these amino acids onto the OsNHX1 model revealed that its pore domain exhibits a transmembrane charge-compensated pattern similar to that of NHE1 while also displaying a distinct charge distribution on either side of the pore domain. Comparative analysis of the key amino acid sites responsible for ion transport in the crystal structure of OsSOS1 and NHE1 revealed that OsNHX1 employs a unique ion transport mechanism. This study will enhance our understanding of the function and catalytic mechanism of OsNHX1 and other plant vacuolar-type NHXs. Full article

Since colonic dopamine levels are markedly reduced during inflammatory bowel disease (IBD), we investigated how dopamine affects regulatory T-cells (Treg), which critically limit gut inflammation. Previously, we showed that the stimulation of the high-affinity dopamine receptor D₃ (Drd3) impairs suppressive Treg activity and limits their recruitment into the colon upon gut inflammation. Here we study the role of the low-affinity dopamine receptor Drd2 in Treg. We find that mice harbouring Drd2-deficient T-cells developed more severe colitis induced by dextran sodium sulphate. The stimulation of Drd2 potentiated the suppressive Treg activity and increased their ability to reach the colonic tissue. A transcriptomic analysis of intestinal mucosa from IBD patients revealed an association with increased DRD3 and reduced DRD2 expression. Bioluminescence resonance energy transfer assays revealed that Drd2 and Drd3 form a heteromer. An in situ proximity ligation assay indicated that the Drd2:Drd3 heteromer was expressed on colonic Treg, and its expression was increased upon inflammation. Using peptides analogous to the transmembrane (TM) segments from Drd2 and Drd3 in bimolecular fluorescence complementation assays, we found TM peptides able to disassemble this heteromer. The heteromer disassembly dampened the suppressive Treg activity and impaired the recruitment of Treg into the colon upon inflammation. Our findings indicate that the Drd2:Drd3 heteromer constitutes a dopamine sensor that regulates suppressive Treg activity and their colonic recruitment. Full article

Sugars are key metabolites influencing the flavor and quality of kiwifruit, with their accumulation in fruit relying on sugar transporters. Recently identified sugar transporters known as SWEETs play significant roles in modulating plant growth, development, and fruit ripening. However, the characteristics of SWEET genes in Actinidia eriantha remain poorly understood. In this study, a total of 26 AeSWEET genes were identified across 17 chromosomes. These genes encoded proteins ranging from 198 to 305 amino acids in length and contained 5 to 7 transmembrane helices. Both interspecific and intraspecific phylogenetic trees categorized AeSWEET proteins into four distinct clades. The motif and domain structures were conserved within each clade, although variations were observed in exon-intron organizations. One tandem and fourteen segmental duplication events were identified as primary drivers of the AeSWEET family expansion. Comparative syntenic mapping showed a closer homology of the AeSWEET family with that of dicotyledons compared to monocotyledons. Promoter cis-element analysis indicated the potential responses of AeSWEET genes to five phytohormones and seven environmental stressors. Quantitative real-time PCR analysis revealed tissue-specific expression profiles of AeSWEET genes, with two AeSWEET11 genes (AeSWEET11a and AeSWEET11b) showing significantly higher expression levels in fruit tissues. Their expressions were positively correlated with sucrose, fructose, and glucose contents throughout fruit development and ripening. Transient transformation tests in tobacco leaves verified the predominant localization of AeSWEET11a and AeSWEET11b to the plasma membrane. Functional assays in yeast mutants revealed that AeSWEET11a and AeSWEET11b both possessed sucrose and hexose transport activities. These findings highlight the potential of targeting AeSWEET11a and AeSWEET11b to enhance sugar accumulation in the fruit of A. eriantha, thereby providing a foundation for improving the flavor profile of commercial cultivars. Full article

Contactin-associated protein-like 2 (CASPR2) is a transmembrane protein of the neurexin superfamily, essential for clustering voltage-gated potassium channels, particularly Kv1, at the juxtaparanodal regions of myelinated axons. This precise localisation is essential for maintaining normal axonal excitability and preventing aberrant signal propagation. Autoantibodies targeting CASPR2 have been associated with various neurological syndromes, notably peripheral nerve hyperexcitability (PNH), which presents clinically with neuromyotonia and myokymia. PNH is characterised by distinctive electrophysiological findings, including neuromyotonic discharges, myokymic discharges, and afterdischarges, which provide diagnostic value and insight into underlying pathophysiology. This review explores the mechanisms of anti-CASPR2-associated PNH, focusing on how antibody-mediated disruption of Kv1 channel clustering leads to altered axonal excitability. Current evidence suggests that both the distal and proximal segments of the axon are sites of pathological activity, where impairments in action potential termination and re-entry prevention result in spontaneous, repetitive discharges. While afterdischarges likely originate within the axon, the precise location—whether in the alpha-motoneuron soma or axon—is uncertain. The involvement of spinal inhibitory circuits has also been proposed, though it remains speculative. Understanding the neurophysiological features of anti-CASPR2-associated PNH is essential for improving diagnostic accuracy and guiding treatment strategies. Further research is needed to clarify the mechanisms of CASPR2-related hyperexcitability. Full article

The GLABROUS1 Enhancer Binding Protein (GeBP) gene family plays a crucial role in plant growth, development, and stress responses. In this study, 28 GeBP genes were identified in Brassica oleracea using HMMER and validated through multiple conserved domain databases. A phylogenetic tree was constructed based on the GeBP protein sequences from B. oleracea, Arabidopsis thaliana, Brassica rapa, and Brassica napus, dividing them into four evolutionary clades (A–D), which revealed a close evolutionary relationship within the genus Brassica. Conserved motif and gene structure analyses showed clade-specific features, while physicochemical property analysis indicated that most BoGeBP proteins are hydrophilic, nuclear-localized, and structurally diverse. Gene duplication and chromosomal localization analyses suggested that both segmental and tandem duplication events have contributed to the expansion of this gene family. Promoter cis-element analysis revealed a dominance of light-responsive and hormone-responsive elements, implying potential roles in photomorphogenesis and stress signaling pathways. Notably, the protein encoded by BolC01g019630.2J possesses both a transmembrane domain and characteristics of the Major Facilitator Superfamily (MFS) transporter family, and it is predicted to localize to the plasma membrane. This suggests that it may act as a molecular bridge between environmental signal perception and transcriptional regulation, potentially representing a novel signaling mechanism within the GeBP family. This unique feature implies its involvement in transmembrane signal perception and downstream transcriptional regulation under environmental stimuli, providing valuable insights for further investigation of its role in stress responses and metabolic regulation. Overall, this study provides a theoretical foundation for understanding the evolutionary patterns and functional diversity of the GeBP gene family in B. oleracea and lays a basis for future functional validation and breeding applications. Full article

Cold environments challenge the structural and functional integrity of membrane proteins, requiring specialized adaptations to maintain activity under low thermal energy. Here, we investigate the molecular basis of cold tolerance in the peptide transporter PepT1 from the Antarctic icefish (Chionodraco hamatus, ChPepT1) using molecular dynamics simulations, binding free energy calculations (MM/GBSA), and dynamic network analysis. We compare ChPepT1 to its human ortholog (hPepT1), a non-cold-adapted variant, to reveal key features enabling psychrophilic function. Our simulations show that ChPepT1 displays enhanced global flexibility, particularly in domains adjacent to the substrate-binding site and the C-terminal domain (CTD). While hPepT1 loses substrate binding affinity as temperature increases, ChPepT1 maintains stable peptide interactions across a broad thermal range. This thermodynamic buffering results from temperature-sensitive rearrangement of hydrogen bond networks and more dynamic lipid interactions. Importantly, we identify a temperature-responsive segment (TRS, residues 660–670) within the proximal CTD that undergoes an α-helix to coil transition, modulating long-range coupling with transmembrane helices. Dynamic cross-correlation analyses further suggest that ChPepT1, unlike hPepT1, reorganizes its interdomain communication in response to temperature shifts. Our findings suggest that cold tolerance in ChPepT1 arises from a combination of structural flexibility, resilient substrate binding, and temperature-sensitive interdomain dynamics. These results provide new mechanistic insight into thermal adaptation in membrane transporters and offer a framework for engineering proteins with enhanced functionality in extreme environments. Full article

Background/Objectives: The Suppressor of Female Function (SOFF) and Shy Girl (SyGI) gene families play vital roles in sex determination in dioecious plants. However, their evolutionary dynamics and functional characteristics remain largely unexplored. Methods: Through this study, a systematic bioinformatics analysis of SOFF and SyGI families was performed in plants to explore their evolutionary relationships, gene structures, motif synteny and functional predictions. Results: Phylogenetic analysis showed that the SOFF family expanded over time and was divided into two subfamilies and seven groups, while SyGI was a smaller family made of compact molecules with three groups. Synteny analysis revealed that 125 duplicated gene pairs were identified in Kiwifruit where WGD/segmental duplication played a major role in duplicating these events. Structural analysis predicted that SOFF genes have a DUF 247 domain with a transmembrane region, while SyGI sequences have an REC-like conserved domain, with a “barrel-shaped” structure consisting of five α-helices and five β-strands. Promoter region analysis highlighted their probable regulatory roles in plant development, hormone signaling and stress responses. Protein interaction analysis exhibited only four SOFF genes with a close interaction with other genes, while SyGI genes had extensive interactions, particularly with cytokinin signal transduction pathways. Conclusions: The current study offers a crucial understanding of the molecular evolution and functional characteristics of SOFF and SyGI gene families, providing a foundation for future functional validation and genetic studies on developmental regulation and sex determination in dioecious plants. Also, this research enhances our insight into plant reproductive biology and offers possible targets for breeding and genetic engineering approaches. Full article

Sugar-Will-Eventually-be-Exported Transporters (SWEETs) play a crucial role in sugar transport in plants, mediating both plant development and stress responses. Despite their importance, there has been limited research characterizing the functional characteristics of CnSWEET genes in coconut (Cocos nucifera). In this study, we conducted a systematic analysis of SWEET genes in coconut using bioinformatics, subcellular localization studies, in silico promoter analysis, and functional assays with yeast mutants. A total of 16 CnSWEET genes were identified and grouped into four clades. Clade I contained the highest number of genes (eight), derived from four pairs of duplicated genomic segments. In contrast, the other clades had fewer genes (one to four) compared to those in Arabidopsis and other species in the Arecaceae family. An extensive analysis of gene expansion using MSCanX indicated significant divergence in gene expansion patterns, both between and within monocots and dicots, as well as among closely related species within the same family. Notable variations in conserved protein motifs and the number of transmembrane helices (TMHs) were detected within Clade I compared to other clades, affecting the subcellular localization of CnSWEET proteins. Specifically, seven TMHs were associated with proteins located in the cell membrane, while CnSWEET2A, which had five TMHs, was found in both the cell membrane and cytosol. Promoter analysis revealed that some CnSWEET genes contained endosperm or seed specific motifs associated with specific endosperm expression, consistent with expression patterns observed in transcriptome data. Functional analysis of five CnSWEET genes, with transcript sequences supported by transcriptome data, was conducted using yeast mutant complementation assays. This analysis demonstrated diverse transport activities for sucrose, fructose, glucose, galactose, and mannose. Our findings provide valuable insights into the functional diversity of SWEET genes in coconuts and their potential roles in stress responses and plant development. Full article

Somatic mutations are common in cancer, with only a few driving the progression of the disease, while most are silent passengers. Some mutations may hinder or even reverse cancer progression. The voltage-gated proton channel (H_V1) plays a key role in cellular pH homeostasis and shows increased expression in several malignancies. Inhibiting H_V1 in cancer cells reduces invasion, migration, proton extrusion, and pH recovery, impacting tumor progression. Focusing on HVCN1, the gene coding for the human voltage-gated proton channel (hH_V1), 197 mutations were identified from three databases: 134 missense mutations, 51 sense mutations, and 12 introducing stop codons. These mutations cluster in two hotspots: the central region of the N-terminus and the region coding for the S4 transmembrane domain, which contains the channel’s voltage sensor. Five somatic mutations within the S4 segment (R205W, R208W, R208Q, G215E, and G215R) were selected for electrophysiological analysis and MD simulations. The findings reveal that while all mutants remain proton-selective, they all exhibit reduced effective charge displacement and proton conduction. The mutations differentially affect hH_V1 kinetics, with the most pronounced effects observed in the two Arg-to-Trp substitutions. Mutation of the first voltage-sensing arginine (R1) to tryptophan (R205W) causes proton leakage in the closed state, accelerates channel activation, and diminishes the voltage dependence of gating. Except for R205W, the mutations promote the deactivated channel configuration. Altogether, these data are consistent with impairment of hH_V1 function by mutations in the S4 transmembrane segment, potentially affecting pH homeostasis of tumor cells. Full article

Public acceptance of genetically modified crops engineered with Bacillus thuringiensis (Bt) insecticidal protein genes (BT-GMCs), which confer resistance to various lepidopteran insect pests, is generally lacking. As a major concern over BT-GMCs is the allergenicity of insecticidal proteins, alleviating safety concerns should help increase public acceptance. In this study, three lepidopteran-specific Bt toxins, Cry1Aa, Cy1Ab, and Cry1Ac, were treated with simulated digestive fluids under various conditions. Western blotting using antiserum raised against individual segments (α-helices of domain I and β-sheets of domains II and III) of Cry1Aa showed that digestion produces a variety of polypeptides. In particular, the transmembrane α4–α5 of domain I, which may retain the ability to form pores, was the most resistant to digestion. Intact Cry1A toxins and these digests were then applied to RBL-2H3 cultured rat mast cells to determine whether the toxins directly induce histamine release. However, fluorescence microscopy revealed no specific binding of Cry1A toxins to RBL-2H3 cultured rat mast cells. In addition, neither the OPA method nor HPLC analysis detected significant histamine release from mast cells treated with Cry1A toxins and these digests. Our results provide important data supporting the safety of Cry1A toxins and potentially BT-GMCs. Full article

The angiotensin-converting enzyme-2 (ACE2) is a transmembrane glycoprotein, consisting of two segments: a large carboxypeptidase catalytic domain and a small transmembrane collectrin-like segment. This protein plays an essential role in blood pressure regulation, transforming the peptides angiotensin-I and angiotensin-II (vasoconstrictors) into angiotensin-1-9 and angiotensin-1-7 (vasodilators). During the COVID-19 pandemic, ACE2 became best known as the receptor of the S-protein of SARS-CoV-2 coronavirus. The purpose of the following research is to reconstruct the 3D structure of the catalytic domain of the rabbit enzyme rACE2 using its primary amino acid sequence, and then to compare it with the human analog hACE2. For this purpose, we have calculated the electric properties and thermodynamic stability of the two protein globules employing computer programs for protein electrostatics. The analysis of the amino acid content and sequence demonstrates an 85% identity between the two polypeptide chains. The 3D alignment of the catalytic domains of the two enzymes shows coincidence of the α-helix segments, and a small difference in two unstructured segments of the chain. The electric charge of the catalytic domain of rACE2, determined by 70 positively chargeable amino acid residues, 114 negatively chargeable ones, and two positive charges of the Zn²⁺ atom in the active center exceeds that of hACE2 by one positively and four negatively chargeable groups; however, in 3D conformation, their isoelectric points pI 5.21 coincide. The surface electrostatic potential is similarly distributed on the surface of the two catalytic globules, but it strongly depends on the pH of the extracellular medium: it is almost positive at pH 5.0 but strongly negative at pH 7.4. The pH dependence of the electrostatic component of the free energy discloses that the 3D structure of the two enzymes is maximally stable at pH 6.5. The high similarity in the 3D structure, as well as in the electrostatic and thermodynamic properties, suggests that rabbit can be successfully used as an animal model to study blood pressure regulation and coronavirus infection, and the results can be extrapolated to humans. Full article

Inflammatory bowel diseases (IBDs) involve chronic inflammation of the gastrointestinal tract, where effector CD4⁺ T-cells play a central role. Thereby, the recruitment of T-cells into the colonic mucosa represents a key process in IBD. We recently found that CCR9 and DRD5 might form a heteromeric complex on the T-cell surface. The increase in CCL25 production and the reduction in dopamine levels associated with colonic inflammation represent a dual signal stimulating the CCR9:DRD5 heteromer, which promotes the recruitment of CD4⁺ T-cells into the colonic lamina propria. Here, we aimed to analyse the molecular requirements involved in the heteromer assembly as well as to determine the underlying cellular mechanisms involved in the colonic tropism given by the stimulation of the CCR9:DRD5 complex. The results show that dual stimulation of the CCR9:DRD5 heteromer potentiates the phosphorylation of the myosin light chain 2 (MLC2) and the migration speed in confined microchannels. Accordingly, disrupting the CCR9:DRD5 assembly induced a sharp reduction in the pMLC2 in vitro, decreased the migratory speed in confined microchannels, and dampened the recruitment of CD4⁺ T-cells into the inflamed colonic mucosa. Furthermore, in silico analysis confirmed that the interface of interaction of CCR9:DRD5 is formed by the transmembrane segments 5 and 6 from each protomer. Our findings demonstrated that the CCR9:DRD5 heteromeric complex plays a fundamental role in the migration of CD4⁺ T-cells into the colonic mucosa upon inflammation. Thereby, the present study encourages the design of strategies for disassembling the formation of the CCR9:DRD5 as a therapeutic opportunity to treat IBD. Full article