Search Results (939)

TasA gene, encoding a functional amyloid protein critical for biofilm formation and antimicrobial activity, was cloned from the endophytic strain Bacillus amyloliquefaciens BS-3, isolated from rubber tree roots. This study identified the shortest functional TasA variant (483 bp, 160 aa) reported to date, featuring unique amino acid substitutions in conserved domains. Bioinformatics analysis predicted a signal peptide (1–27 aa) and transmembrane domain (7–29 aa), which were truncated to optimize heterologous expression. Two prokaryotic vectors (pET28a and pCZN1) were constructed, with pCZN1-TasA expressed solubly in Escherichia coli Arctic Express at 15 °C, while pET28a-TasA formed inclusion bodies at 37 °C. Purified recombinant TasA exhibited potent antifungal activity, achieving 98.6% ± 1.09 inhibition against Colletotrichum acutatum, 64.77% ± 1.34 against Alternaria heveae. Notably, TasA completely suppressed spore germination in C. acutatum and Oidium heveae Steinmannat 60 μg/mL. Structural analysis via AlphaFold3 revealed that truncation enhanced protein stability. These findings highlight BS-3-derived TasA as a promising biocontrol agent, providing molecular insights for developing protein-based biopesticides against rubber tree pathogens. Full article

Low-temperature stress severely limits plant growth and reduces agricultural productivity. Calmodulin-like (CML) proteins are crucial calcium sensors in plant cold responses. Transcriptome analysis of cold-stressed Saussurea involucrata identified seven differentially expressed CML genes. qRT-PCR confirmed that SiCML6 was strongly induced at 4 °C and −2 °C. Bioinformatics analysis showed that SiCML6 encodes a transmembrane protein containing an EF-hand domain. This protein carries a signal peptide and shows the closest phylogenetic relationship to Helianthus annuus CML3. Its promoter contains ABA, methyl jasmonate (MeJA), and cold-response elements. Arabidopsis plants overexpressing SiCML6 showed significantly higher survival rates at −2 °C than wild-type plants. Under freezing stress, SiCML6-overexpressing lines exhibited reduced malondialdehyde content, relative electrolyte leakage, and ROS accumulation (H₂O₂ and O₂⁻), along with increased proline, soluble sugars, soluble proteins, and total antioxidant capacity (T-AOC). SiCML6 elevated the expression of cold-responsive genes CBF3 and COR15a under normal conditions and further upregulated CBF1/2/3 and COR15a at 4 °C. Thus, low temperatures induced SiCML6 expression, which was potentially regulated by ABA/MeJA. SiCML6 enhances freezing tolerance by mitigating oxidative damage through boosted T-AOC and osmoprotectant accumulation while activating the CBF-COR signaling pathway. This gene is a novel target for improving crop cold resistance. Full article

The expanding distribution and geographic range of mosquitoes have potentially contributed to increased flaviviral dissemination and transmission. Despite the growing burden of flaviviral infections, there are no effective antiviral treatments or vaccines, highlighting the need for novel therapeutic targets. Tetraspanins, a superfamily of transmembrane domain glycoproteins involved in cellular organization, signaling, and protein–protein interactions have been recognized as potential mediators of flaviviral infection and transmission. While their roles in vertebrate hosts have been explored, their involvement in flaviviral replication and dissemination within medically important vectors remains poorly understood. In this study, we investigated the role of arthropod tetraspanins in mosquito cells and extracellular vesicles (EVs) derived from cells infected with Zika virus (ZIKV) and dengue virus (serotype 2; DENV2). Among several of the tetraspanins analyzed, only CD151 was significantly upregulated in both mosquito cells and in EVs derived from ZIKV/DENV2-infected cells. RNAi-mediated silencing of CD151 led to a marked reduction in viral burden, suggesting its crucial role in flavivirus replication. Inhibition of EV biogenesis using GW4869 further demonstrated that EV-mediated viral transmission contributes to flavivirus propagation. Additionally, co-immunoprecipitation and immunofluorescence analyses revealed direct interactions between CD151 and ZIKV NS2B and DENV2 capsid proteins. Overall, our findings highlight the functional importance of mosquito CD151 in the replication and transmission of ZIKV and DENV2. This study provides new insights into the molecular mechanisms of flaviviral infection in mosquitoes and suggests that targeting vector tetraspanins may offer a potential approach to controlling mosquito-borne flaviviruses. Full article

Heparan sulfate proteoglycans (HSPGs) within the neuronal niche are expressed during brain development, contributing to multiple aspects of neurogenesis, yet their roles in glial lineage commitment remain elusive. This study utilised three human cell models expanded under basal culture conditions followed by media-induced lineage induction to identify a reproducible and robust model of gliogenesis. SH-SY5Y human neuroblastoma cells (neuronal control), ReNcell CX human neural progenitor cells (astrocyte inductive) and ReNcell VM human neural progenitor (mixed neural induction) models were examined. The cultures were characterised during basal and inductive states via Q-PCR, Western Blotting, immunocytochemistry (ICC) and calcium signalling activity analyses. While the ReNcell lines did not produce fully mature or homogeneous astrocyte cultures, the ReNcell CX cultures most closely resembled an astrocytic phenotype with ReNcell VM cells treated with platelet-derived growth factor (PDGF) biased toward an oligodendrocyte lineage. The glycated variant of surface-bound glypican-2 (GPC2) was found to be associated with lineage commitment, with GPC6 and 6-O HS sulfation upregulated in astrocyte lineage cultures. Syndecan-3 (SDC3) emerged as a lineage-sensitive proteoglycan, with its cytoplasmic domain enriched in progenitor-like states and lost upon differentiation, supporting a role in maintaining neural plasticity. Conversely, the persistence of transmembrane-bound SDC3 in astrocyte cultures suggest continued involvement in extracellular signalling and proteoglycan secretion, demonstrated by increased membrane-bound HS aggregates. This data supports HSPGs and HS GAGs as human neural lineage differentiation and specification markers that may enable better isolation of human neural lineage-specific cell populations and improve our understanding of human neurogenesis. Full article

A novel barna-like virus was found to be associated with field-collected Afrina sporoboliae plant-parasitic nematodes. The positive-sense, single-stranded RNA genome of this virus, named Afrina barna-like virus (AfBLV), comprises 4020 nucleotides encoding four open reading frames (ORFs). ORF 1 encodes a protein product spanning a transmembrane, a peptidase, and VPg domains, whereas an overlapping ORF 2 encodes an RNA-dependent RNA polymerase (RdRP). ORF2 may be expressed via a −1 translational frameshift. In phylogenetic reconstructions, the RdRP of AfBLV was placed inside a separate clade of barna and barna-like viruses related to but distinct from the genera in the Solemoviridae and Alvernaviridae families, within the overall lineage of Sobelivirales. ORF 3 of AfBLV encodes a protein product of 206 amino acids (aa) long with homology to a putative protein encoded by a similarly positioned gene of an uncharacterized virus sequence identified previously as Barnaviridae sp. ORF 4 encodes a 161 aa protein with no significant similarities to sequences in the GenBank databases. AfBLV is the first barnavirus found in a nematode. Sequence comparisons of the AfBLV genome and genomes of other barna-like viruses suggested that a recombination event was involved in the evolution of AfBLV. Analyses of the phylogeny of RdRPs and genome organizations of barna-like and solemo-like viruses support the re-classification of Barnavirus and Dinornavirus genera as members of the Solemoviridae family. Full article

Background/Objectives: P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, plays a key role in multidrug resistance by actively exporting chemotherapeutic agents and xenobiotics from cells. Overexpression of P-gp significantly reduces intracellular drug accumulation and compromises treatment efficacy. Despite extensive research, clinically approved P-gp inhibitors remain elusive due to toxicity, poor specificity, and limited efficacy. This study investigates DMH1, a selective type I BMP receptor inhibitor, as a novel P-gp inhibitor. Methods: DMH1 cytotoxicity was assessed in P-gp-overexpressing (PC3-TxR, K562/Dox) and P-gp-deficient (PC3) cell lines using MTT assays. P-gp inhibition was evaluated using calcein AM retention and daunorubicin (DNR) accumulation assays. Kinetic analysis determined DMH1’s effect on P-gp-mediated transport (Vmax and Km). ATPase activity assays were performed to assess DMH1’s impact on ATP hydrolysis. Preliminary molecular docking (CB-Dock2) was used to predict DMH1’s binding site on the human P-gp structure (PDB ID: 6QEX). Results: DMH1 showed no cytotoxicity in P-gp-overexpressing or deficient cells. It significantly enhanced intracellular accumulation of Calcein AM and DNR, indicating effective inhibition of P-gp function. Kinetic data revealed that DMH1 reduced Vmax without affecting Km, consistent with noncompetitive, allosteric inhibition. DMH1 also inhibited ATPase activity in a dose-dependent manner. Docking analysis suggested DMH1 may bind to an allosteric site in the transmembrane domain, potentially stabilizing the inward-facing conformation. Conclusions: DMH1 is a promising noncompetitive, allosteric P-gp inhibitor that enhances intracellular drug retention without cytotoxicity, supporting its potential as a lead compound to overcome multidrug resistance and improve chemotherapeutic efficacy. 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

As a plant-specific peroxidase family, class III peroxidase (PRX) plays an important role in plant growth, development, and stress response. In this study, a preliminary functional analysis of CqPRX9L1 was conducted. Bioinformatics analysis revealed that CqPRX9L1 encodes a 349-amino acid protein belonging to the plant-peroxidase-like superfamily, featuring a transmembrane domain and cytoplasmic localization. The promoter region of CqPRX9L1 harbors various cis-acting elements associated with stress responses, hormone signaling, light regulation, and meristem-specific expression. The tissue-specific expression pattern of the CqPRX9L1 gene and its characteristics in response to different stresses were explored using subcellular localization, quantitative real-time PCR (qRT-PCR), and heterologous transformation into Arabidopsis thaliana. The results showed that CqPRX9L1, with a transmembrane structure, was localized in the cytoplasm, which encodes 349 amino acids and belongs to the plant-peroxisome-like superfamily. The promoter region contains stress-response elements, hormone-response elements, light-response elements, and meristem expression-related elements. The expression of CqPRX9L1 was relatively higher in ears and roots at the panicle stage than in stems and leaves. CqPRX9L1 showed a dynamic expression pattern of first decreasing and then increasing under abiotic stresses such as 15% PEG 6000, low temperature, and salt damage, with differences in response time and degree. CqPRX9L1 plays an important role in response to abiotic stress by affecting the activity of antioxidant enzymes such as superoxide dismutase (SOD) and peroxidase (POD), as well as the synthesis and decomposition of proline (Pro). CqPRX9L1 also affects plant bolting and flowering by regulating key flowering genes (such as FT and AP1) and gibberellin (GA)-related pathways. The results establish a foundation for revealing the functions and molecular mechanisms of the CqPRX9L1 gene. Full article

Background/Objectives: Bacteriophages infecting the genus Streptococcus play a crucial role in microbial ecology and have potential applications in biotechnology and medicine. Despite their importance, significant gaps remain in our understanding of their lysis modules. This study aims to address these deficiencies by analyzing the genomic diversity and lysis module organization in Streptococcus phages. Methods: A search was conducted in the NCBI RefSeq database to identify phage genomes infecting Streptococcus. A representative panel was selected based on taxonomic diversity. Lysis modules were annotated and visualized, functional domains in endolysins were identified, and holins were characterized. Results: A total of 205 phage genomes were retrieved from the NCBI RefSeq database, of which 185 complete genomes were analyzed. A subset of 34 phages was selected for in-depth analysis, ensuring the representation of taxonomic diversity. The lysis modules were annotated and visualized, revealing five distinct organizations. Among the 256 identified endolysins, 25 distinct architectural organizations were observed, with amidase activity being the most prevalent. Holins were classified into 9 of the 74 families listed in the Transporter Classification Database, exhibiting one to three transmembrane domains. Conclusions: This study provides insights into the structural diversity of lysis modules in Streptococcus phages, paving the way for future research and potential biotechnological applications. Full article

Tumor necrosis factor alpha (TNFα) is a central pro-inflammatory cytokine that mediates host immune responses during infection. In this study, we identified and characterized the TNFα gene in the starry flounder (Platichthys stellatus) through transcriptomic analysis. The deduced protein contained a conserved TNF domain and transmembrane region, and phylogenetic analysis confirmed its homology with other teleost TNFα proteins. Tissue-specific expression profiling revealed high baseline expression in immune-related peripheral organs and a distinct temporal modulation in response to Streptococcus parauberis infection. Recombinant TNFα (rTNFα), produced using a cell-free expression system, significantly enhanced phagocytic activity in peripheral and kidney-derived leukocytes in a dose-dependent manner. Peak activity was observed at 150–200 μg/mL, while a decline at higher concentrations suggested a threshold for immune stimulation. Importantly, hemolysis assays confirmed the safety of rTNFα even at the highest tested concentrations. These results demonstrate the immunomodulatory potential of TNFα as a molecular adjuvant in aquaculture vaccines and underscore its potential utility in immune-enhancing strategies for sustainable aquaculture. Full article

Despite current vaccines and therapeutics targeting SARS-CoV-2, the causative agent of the COVID-19 pandemic, cases remain high causing a burden on health care systems. Spike-protein mediated membrane fusion of SARS-CoV-2 is a critical step in viral entry. Herein, we describe entry inhibitors identified by first screening a library of about 160 compounds and then analogue synthesis. Specifically, compound 261 was found to inhibit SARS-CoV-2 infection in a tissue model with IC₅₀ of 0.3 µM. Using NMR, we found that 261 interacts with key residues in the aromatic-rich region of the spike protein directly next to the transmembrane domain. Molecular dynamic simulations of the 261 binding pocket in the spike protein was also mapped to the transmembrane domain, consistent with NMR findings. The amino acids in the binding site are conserved among different coronaviruses known to infect humans; therefore, inhibitors targeting this conserved binding site could be a useful addition to current therapeutics and may have pan-coronavirus antiviral activities. Full article

The broad-spectrum antiviral functions of interferon-inducible transmembrane 1 (IFITM1) rely on S-palmitoylation post-translational modification. α/β-hydrolase domain-containing 17A (ABHD17A) has been reported to be responsible for protein depalmitoylation over the past decade, but whether and how ABHD17A regulates the dynamic S-palmitoylation modification of IFITM1 remains unknown. Here, we demonstrated that ABHD17A physically interacts with IFITM1 and increases the S-palmitoylation level of IFITM1. Sequence alignment revealed that ABHD17A lacked the DHHC motif, which is capable of catalyzing the S-palmitoylation modification. Thus, we screened multiple candidate palmitoylating and depalmitoylating enzymes that may contribute to ABHD17A-induced upregulation of IFITM1 S-palmitoylation. The recently discovered depalmitoylase ABHD16A was significantly downregulated by ABHD17A, which counteracted the palmitate-removing reactions of ABHD16A on IFITM1 and subsequently upregulated the S-palmitoylation level and antiviral activity of IFITM1. Our work therefore elucidated the unconventional role of depalmitoylase ABHD17A in elevating the S-palmitoylation modification, expanded the biological functions of ABHD17A in innate immunity, and provided potential targets for viral disease therapy. Full article

The Colorado potato beetle (Leptinotarsa decemlineata, CPB) is a major pest in potato crops, notorious for its rapid dispersal and insecticide resistance, which are enabled by its robust elytra and flight-capable hindwings. The Miniature (Mi) gene, encoding a protein with a zona pellucida (ZP) domain, is involved in wing development and cuticle integrity, yet its functional role in beetles remains underexplored. In this study, we cloned and characterized the LdMi gene in the CPB and investigated its function using RNA interference (RNAi), morphological analyses, and spectroscopy. LdMi encodes a 146.35 kDa transmembrane protein with a conserved ZP domain, clusters with coleopteran homologs, and exhibits relative conservation across insect species. Expression profiling showed high LdMi transcript levels in the hindwings, the elytra, and the pupal stages. RNAi knockdown in fourth-instar larvae resulted in severe eclosion defects, including malformed wings and reduced adult weight. Scanning electron microscopy (SEM) revealed disrupted elytral patterns and deformed hindwing veins in knockdown individuals. Spectroscopic analyses using Fourier-transform infrared (FTIR) and Raman spectroscopy indicated a reduction in protein–chitin crosslinking and diminished hydrogen bonding, suggesting compromised cuticular integrity. These results highlight the essential role of LdMi in cuticle formation and the surface morphology of the elytra and hindwings, offering new insights into ZP domain proteins in insects. Full article

Aquaporins in rice (Oryza sativa L.) represent a pivotal class of transmembrane channel proteins that mediate the bidirectional transport of water and small solutes, which have critical functions in cellular osmoregulation and ion homeostasis maintenance. Their evolutionary diversity and functional plasticity constitute fundamental mechanisms underlying the adaptive responses to diversified environmental challenges. This review systematically summarizes rice AQPs’ evolutionary origins, structural characteristics, and spatiotemporal expression patterns under both physiological and stress conditions, highlighting the high conservation of their key functional domains across evolution and their environment-driven functional diversification. The molecular mechanisms governing AQPs in water utilization, nutrient uptake, and stress responses are unraveled. Furthermore, the potential of precision gene editing and multi-omics integration is discussed to decipher the intricate relationships between AQP evolutionary history, environmental adaptability, and functional specialization, thereby providing a theoretical basis for advancing crop stress resistance and high-quality breeding. Full article

Transmembrane channel-like (TMC) proteins are essential for hearing and balance; however, the molecular mechanisms that regulate their proper folding and membrane targeting remain poorly understood. Here, we establish Caenorhabditis elegans as a genetically tractable model to dissect TMC-1 trafficking by combining CRISPR knock-in strains, super-resolution microscopy, and genome-wide forward genetic screening. We show that TMC-1 robustly localizes to the plasma membrane in both neurons and muscle cells and identify a conserved valine (V803) in transmembrane domain 9 (TM9) as critical for its biogenesis and trafficking. Structural analyses guided by AlphaMissense and AlphaFold uncover two evolutionarily conserved functional hotspots, one in the extracellular loop adjacent to TM9 and the other in the TMC signature motif, which are interconnected by an evolutionarily conserved disulfide bond. Disrupting this bond in worm TMC-1 abolishes its cell-surface localization and destabilizes the mechanotransduction channel complex. Together, these findings provide a structural framework for interpreting deafness-causing mutations in human TMC1 and highlight disulfide-bond-linked hotspots as key molecular determinants of TMC protein biogenesis and trafficking. Full article