Search Results (104)

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

The large portion of the eukaryotic genomes was considered non-functional and called the “dark matter” of the genome, now appearing as regulatory hubs coding for RNAs without the potential for making proteins, known as non-coding RNA. Long non-coding RNA (lncRNA) is defined as functional RNA molecules having lengths larger than 200 nucleotides without the potential for coding for proteins. Thousands of lncRNAs are identified in different plants and animals. LncRNAs are characterized by a low abundance, fewer exons than mRNA, tissue-specific expression, and low sequence conservation compared to protein-coding genes (PCGs). LncRNAs, like PCGs, are regulated by promoters and enhancers with characteristic chromatin signatures, DNA methylation, multiple exons, introns, and alternate splicing. LncRNAs interact with DNA, mRNA, microRNA, and proteins, including chromatin/histone modifiers, transcription factors/repressors, epigenetic regulators, spliceosomal, and RNA-binding proteins. Recent observations indicate that lncRNAs code for small peptides, also called micropeptides (<100 amino acids), and are involved in the development and growth of plants, suggesting the bi-functional activities of lncRNAs. LncRNAs have emerged as the major regulators of diverse functions, principally by altering the transcription of target genes. LncRNAs are involved in plant growth, development, immune responses, and various physiological processes. Abiotic, biotic, nutrient, and other environmental stresses alter the expressions of numerous lncRNAs. Understanding the mechanisms of actions of lncRNAs opens up the possibility of improving agronomic traits by manipulating lncRNAs. However, further studies are required in order to find the interactions among the deregulated lncRNAs and validate the findings from high-throughput studies to harness their potential in crop improvement. Full article

In this study, we investigated the functional role of Peroxiredoxin 5 (SmPrx5) in the cuttlefish Sepiella maindroni. The full-length SmPrx5 cDNA is 934 base pairs (bp) in length, comprising a 31 bp 5′ untranslated region (UTR), a 330 bp 3′ UTR, and an open reading frame (ORF) of 573 bp that encodes a polypeptide consisting of 190 amino acids. Sequence analysis revealed the presence of a conserved peroxidase catalytic motif VPGAFTPGCSQTHLPG and the signature domain DGTGLTCSL, indicating that SmPrx5 belongs to the 2-Cys Prx subfamily. Quantitative real-time PCR (RT-qPCR) analysis demonstrated that SmPrx5 is broadly expressed across various tissues in S. maindroni, with particularly high expression levels observed in the testes, hemocytes, liver, and ovaries. Upon challenge with Vibrio alginolyticus, SmPrx5 expression was significantly upregulated in both the liver and hemocytes, peaking at 24 h post-infection and gradually returning to baseline levels within 48 h. Furthermore, the recombinant SmPrx5 protein exhibited notable antioxidant activity in vitro, suggesting its involvement in the oxidative stress response. These findings enhance our understanding of the molecular mechanisms underlying immune defense in marine cephalopods and highlight the potential role of Prx5 in host immunity. Full article

KRAS is a pivotal oncoprotein that regulates cell proliferation and survival through interactions with downstream effectors such as RAF1. Despite significant advances in understanding KRAS biology, the structural and dynamic mechanisms of KRAS allostery remain poorly understood. In this study, we employ microsecond molecular dynamics simulations, mutational scanning, and binding free energy calculations together with dynamic network modeling to dissect how engineered DARPin proteins K27, K55, K13, and K19 engage KRAS through diverse molecular mechanisms ranging from effector mimicry to conformational restriction and allosteric modulation. Mutational scanning across all four DARPin systems identifies a core set of evolutionarily constrained residues that function as universal hotspots in KRAS recognition. KRAS residues I36, Y40, M67, and H95 consistently emerge as critical contributors to binding stability. Binding free energy computations show that, despite similar binding modes, K27 relies heavily on electrostatic contributions from major binding hotspots while K55 exploits a dense hydrophobic cluster enhancing its effector-mimetic signature. The allosteric binders K13 and K19, by contrast, stabilize a KRAS-specific pocket in the α3–loop–α4 motif, introducing new hinges and bottlenecks that rewire the communication architecture of KRAS without full immobilization. Network-based analysis reveals a strikingly consistent theme: despite their distinct mechanisms of recognition, all systems engage a unifying allosteric architecture that spans multiple functional motifs. This architecture is not only preserved across complexes but also mirrors the intrinsic communication framework of KRAS itself, where specific residues function as central hubs transmitting conformational changes across the protein. By integrating dynamic profiling, energetic mapping, and network modeling, our study provides a multi-scale mechanistic roadmap for targeting KRAS, revealing how engineered proteins can exploit both conserved motifs and isoform-specific features to enable precision modulation of KRAS signaling in oncogenic contexts. Full article

The family Alphaflexiviridae comprises plant- and fungus-infecting viruses with single-stranded, positive-sense RNA genomes ranging from 5.4 to 9 kb. Their virions are flexuous and filamentous, measuring 470–800 nm in length and 12–13 nm in diameter. The family includes 72 recognized species, classified into six genera: Allexivirus, Lolavirus, Platypuvirus, Potexvirus (plant-infecting), and Botrexvirus and Sclerodarnavirus (fungus-infecting). The genus Potexvirus is the largest, with 52 species, including Potexvirus ecspotati (potato virus X), an important crop pathogen and plant virology model. The genera are distinguished by genome organization and host range, while species differentiation relies on nucleotide and protein sequence identity thresholds. In this review, we summarize the current knowledge on the genomic structure, conserved genes, and phylogenetic relationships within Alphaflexiviridae, with a particular focus on the replicase and coat protein genes as signature markers. Additionally, we update the model of cellular remodeling driven by the triple gene block proteins, which are essential for virus movement, among other viral functions. Beyond their biological significance, alphaflexiviruses serve as valuable models for studying virus–host dynamics and hold potential applications in plant disease control and biotechnology. This review provides an updated framework for understanding Alphaflexiviridae and their broader impact on plant virology. Full article

The Russian Black Pied (RBP) is one of Russia’s most popular dairy cattle breeds. It was developed in the USSR during the 1930s by crossing Russian native cattle with Dutch cattle. Since the mid-1970s, the RBP has been cross-bred with Holstein cattle to enhance milk production. To trace haplotypes and selection signatures in the RBP genomes formed during cross-breeding and selection, we conducted local ancestry inference and scans for selection signatures in 12 resequenced RBP animals. Scans for selection using hapFLK and window-based F_ST identified 65 putatively selected regions across the genome, with 4 identified by both methods. Candidate missense variants were detected within these regions, including KIAA1755, CNBD1, and MPZL3, which may be related to milk traits, pathogen resistance, and climate adaptation. Local ancestry inference and functional annotation analyses suggest that regions with a higher fraction of native haplotypes are enriched for genes associated with keratin filaments, antimicrobial defence, the immune system, protein digestion, and amino acid transporters. The results of our study could be utilised for conservation purposes and the improvement of this and other breeds. Full article

Accumulating evidence suggests that the plasma membrane-localized phosphate transporter 1 (PHT1) family plays a fundamental role in the absorption, translocation, and re-mobilization of phosphorus in plants. Buckwheat (Fagopyrum spp.) exhibits high efficiency in phosphate uptake and wide adaptability to grow in under-fertilized soils. Despite their physiological importance, a systematic analysis of PHT1 genes in buckwheat has not been conducted yet. In this study, we performed a genome-wide identification and expression profile of the PHT1 gene family in Tartary buckwheat (Fagopyrum tataricum Gaertn). A total of eleven putative PHT1 genes (FtPHT1;1 to 1;11) were identified with an uneven distribution on all the F. tataricum chromosomes except for chromosomes 2, 3, and 5. All the FtPHT1s share the conserved domain GGDYPLSATIxSE, a typical signature of PHT1 transporters. A phylogenetic analysis indicated that FtPHT1 proteins could be clustered into four distinct subgroups, well supported by the exon–intron structure, consensus motifs, and the domain architecture. A gene duplication analysis suggested that tandem duplication may largely contribute to the expansion of the FtPHT1 gene family members. In silico predictions of cis-acting elements revealed that low-phosphate-responsive elements, such as W-box, P1BS, and MBS, were enriched in the promoter regions of FtPHT1 genes. Quantitative real-time PCR assays showed differential but partially overlapping expression patterns of some FtPHT1 genes in various organs under limited Pi supply and hormone stimuli, implying that these FtPHT1 transporters may be essential for Pi uptake, translocation, and re-mobilization, possibly through signaling cross-talk between the low phosphate and hormones. These observations provide molecular insights into the FtPHT1 gene family, which paves the way to a functional analysis of FtPHT1 members in the future. Full article

The trichomes of mustard leaves have significance due to their ability to combat unfavorable external conditions and enhance disease resistance. It was demonstrated that the MYB-bHLH-WD40 (MBW) ternary complex consists of MYB, basic Helix-Loop-Helix (bHLH), and WD40-repeat (WD40) family proteins and plays a key role in regulating trichome formation and density. The bHLH gene family, particularly the Myelocytomatosis (MYC) proteins that possess the structural bHLH domain (termed bHLH-MYC), are crucial to the formation and development of leaf trichomes in plants. bHLH constitutes one of the largest families of transcription factors in eukaryotes, of which MYC is a subfamily member. However, studies on bHLH-MYC transcription factors in mustard have yet to be reported. In this study, a total of 45 bHLH-MYC transcription factors were identified within the Brassica juncea genome, and a comprehensive series of bioinformatic analyses were conducted on their structures and properties: an examination of protein physicochemical properties, an exploration of conserved structural domains, an assessment of chromosomal positional distributions, an analysis of the conserved motifs, an evaluation of the gene structures, microsynteny analyses, three-dimensional structure prediction, and an analysis of sequence signatures. Finally, transcriptome analyses and a subcellular localization examination were performed. The results revealed that these transcription factors were unevenly distributed across 18 chromosomes, showing relatively consistent conserved motifs and gene structures and high homology. The final results of the transcriptome analysis and gene annotation showed a high degree of variability in the expression of bHLH-MYC transcription factors. Five genes that may be associated with trichome development (BjuVA09G22490, BjuVA09G13750, BjuVB04G14560, BjuVA05G24810, and BjuVA06G44820) were identified. The subcellular localization results indicated that the transcription and translation products of these five genes were expressed in the same organelle: the nucleus. This finding provides a basis for elucidating the roles of bHLH-MYC family members in plant growth and development, and the molecular mechanisms underlying trichome development in mustard leaves. Full article

Understanding the genetic characteristics of indigenous goat breeds is vital for their conservation and breeding. Haimen goats, native to China’s Yangtze River Delta, possess distinctive traits such as white hair, moderate growth rate, high-quality meat, and small body size. However, knowledge regarding the genetic structure and germplasm characteristics of Haimen goats remains limited. In this study, we performed 20× whole-genome resequencing of 90 goats (60 Haimen goats and 30 Boer goats) to identify single-nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) associated with growth traits. Here, we analyzed population genetic structure and genome-wide selection signatures between the Haimen and Boer goats based on whole-genome resequencing data. The principal component analysis (PCA) and neighbor-joining (N-J) tree results demonstrated significant genetic differentiation between the Haimen and Boer goats. The nucleotide diversity (Pi) and linkage disequilibrium (LD) decay results indicated higher genomic diversity in the Haimen goat population. Furthermore, selective sweep analysis identified candidate genes associated with growth traits. These genes exhibited strong selection signatures and were related to body size (DONSON, BMPR1B, and EPHA5), muscle development (GART, VGLL3, MYH15), and fat metabolism (ADAMTS5, LRP6, XDH, CPT1A, and GPD1). We also identified growth-related candidate genes (NCOR1, DPP6, NOTCH2, and FGGY) specific to Haimen goats. Among these genes, pancreatic lipase-related protein 1 (PNLIPRP1) emerged as the primary candidate gene influencing growth phenotypes. Further analysis revealed that a 26 bp Indel in PNLIPRP1 increased its gene expression, suggesting that this Indel could serve as a molecular marker for early marker-assisted selection, potentially enhancing early growth in goats. These findings provide valuable molecular markers and candidate genes for improving growth traits in Haimen goat breeding. Full article

Sexually propagated scleractinian corals are in high demand for coral reef restoration. However, for threatened reef-building corals, many of the molecular mechanisms related to their reproduction remain largely unknown, which forms a major bottleneck in the large-scale cultivation of sexually reproducing corals. In this study, we analyzed the proteomic signatures of red eggs and spermatozoa from the ecologically significant coral Galaxea fascicularis, using a data-independent acquisition mass spectrometry (DIA-MS) method. A total of 7741 and 7279 proteins from mature red eggs and spermatozoa were identified, respectively. Among these proteins, 596 proteins were spermatozoa-specific and 1056 were egg-specific. Additionally, a total of 4413 differentially abundant proteins (DAPs) were identified, among which 3121 proteins were up-regulated in red eggs and 1292 proteins were up-regulated in spermatozoa. Furthermore, anenrichment analyses showed that DAPs identified in red eggs were mainly involved in the progesterone-mediated oocyte maturation pathway and lectin pathway; and DAPs detected in spermatozoa were mainly involved in the insulin secretion pathway and metabolic pathways for the generation of energy. This result will contribute to the discovery of the intrinsic regulation pathway of gamete maturation and fertilization. Furthermore, at least 57 proteins associated with gamete maturation and reproduction were identified, including the red fluorescent protein (RFP), vitellogenin proteins (VG), the egg protein (EP), the testis-specific serine/threonine-protein kinase family (TSSKs), and the EF-hand Ca²⁺-binding protein family (EFHC1 and EFHC2). Particularly, the third yolk protein EUPHY was reported for the first time in G. fascicularis. In conclusion, this study unveiled groundbreaking molecular insights into coral sexual reproduction, paving the way for more effective conservation and sustainable development of coral reef ecosystems Full article

Plant cation–chloride cotransporters (CCCs) are proposed to be Na⁺-K⁺-2Cl⁻ transporting membrane proteins, although evolutionarily, they associate more closely with K⁺-Cl⁻ cotransporters (KCCs). Here, we investigated grapevine (Vitis vinifera L.) VvCCC using 3D protein modeling, bioinformatics, and electrophysiology with a heterologously expressed protein. The 3D protein modeling revealed that the signatures of ion binding sites in plant CCCs resembled those of animal KCCs, which was supported by phylogenomic analyses and ancestral sequence reconstruction. The conserved features of plant CCCs and animal KCCs included predicted K⁺ and Cl⁻-binding sites and the absence of a Na⁺-binding site. Measurements with VvCCC-injected Xenopus laevis oocytes with VvCCC localizing to plasma membranes indicated that the oocytes had depleted intracellular Cl⁻ and net ⁸⁶Rb fluxes, which agreed with thermodynamic predictions for KCC cotransport. The ⁸⁶Rb uptake by VvCCC-injected oocytes was Cl⁻-dependent, did not require external Na⁺, and was partially inhibited by the non-specific CCC-blocker bumetanide, implying that these properties are typical of KCC transporters. A loop diuretic-insensitive Na⁺ conductance in VvCCC-injected oocytes may account for earlier observations of Na⁺ uptake by plant CCC proteins expressed in oocytes. Our data suggest plant CCC membrane proteins are likely to function as K⁺-Cl⁻ cotransporters, which opens the avenues to define their biophysical properties and roles in plant physiology. Full article

For the first time, we describe phylogenomic signatures of an epidemic lineage of vesicular stomatitis Indiana virus (VSIV). We applied multiple evolutionary analyses to a dataset of 87 full-length genome sequences representing the circulation of an epidemic VSIV lineage in the US between 2019 and 2020. Based on phylogenetic analyses, we predicted the ancestral relationship of this lineage with a specific group of isolates circulating in the endemic zone of Chiapas, Mexico. Subsequently, our findings indicate that the lineage diversified into at least four different subpopulations during its circulation in the US. We identified single nucleotide polymorphisms (SNPs) that differentiate viral subpopulations and assessed their potential relevance using comparative phylogenetic methods, highlighting the preponderance of synonymous mutations during the differentiation of these populations. Purifying selection was the main evolutionary force favoring the conservation of this epidemic phenotype, with P and G genes as the main drivers of the evolution of this lineage. Our analyses identified multiple codon sites under positive selection and the association of these sites with specific functional domains at P, M, G, and L proteins. Based on ancestral reconstruction analyses, we showed the potential relevance of some of the sites identified under positive selection to the adaptation of the epidemic lineage at the population level. Finally, using a representative group of viruses from Colorado, we established a positive correlation between genetic and geographical distances, suggesting that positive selection on specific codon positions might have favored the adaptation of different subpopulations to circulation in specific geographical settings. Collectively, our study reveals the complex dynamics that accompany the evolution of an epidemic lineage of VSIV in nature. Our analytical framework provides a model for conducting future evolutionary analyses. The ultimate goal is to support the implementation of an early warning system for vesicular stomatitis virus in the US, enabling early detection of epidemic precursors from Mexico. Full article

Perlucin is a shell matrix protein that plays a significant role in regulating shell biomineralization. This study aimed to isolate and characterize the perlucin gene and analyze its expression to explore its role in shell formation, regeneration, and responses to thermal stress and starvation in Pacific abalone. The isolated full-length cDNA sequence of Hdh-Perlucin is 1002 bp long, encoding a 163-amino-acid polypeptide with a signal peptide. The mature peptide of Hdh-Perlucin contains a C-type lectin domain with signature motif and six conserved cysteine residues. Gene Ontology analysis suggests that Hdh-Perlucin exhibits carbohydrate-binding activity. Significantly higher expression of Hdh-Perlucin was observed during the juvenile, veliger, and trochophore stages, compared with cell division stage during early development. Upregulated expression was recorded from slow to rapid growth phases and during shell biomineralization, while downregulated expression was noted during starvation. Under thermal stress, expression peaked at 30 °C and 25 °C for 6 and 12 h, respectively, while consistently higher levels were observed at 15 °C throughout the experiment. This study provides the first comprehensive structural and expression analysis of Hdh-Perlucin, highlighting its roles in metamorphosis, shell formation and regeneration, and responses to heat stress and starvation in abalone. Full article

Background: Transactivation Response Element RNA-binding Protein (TRBP2) is a double-stranded RNA-binding protein widely known for its critical contribution to RNA interference (RNAi), a conserved mechanism of gene-expression regulation mediated through small non-coding RNA moieties (ncRNAs). Nevertheless, TRBP2 has also proved to be involved in other molecular pathways and biological processes, such as cell growth, organism development, spermatogenesis, and stress response. Mutations or aberrant expression of TRBP2 have been previously associated with diverse human pathologies, including Alzheimer’s disease, cardiomyopathy, and cancer, with TRBP2 playing an essential role(s) in proliferation, invasion, and metastasis of tumor cells. Methods: Hence, the present study aims to investigate, via employment of advanced flow cytometry, immunofluorescence, cell transgenesis and bioinformatics technologies, new, still elusive, functions and properties of TRBP2, particularly regarding its cell cycle-specific control during cancer cell division. Results: We have identified a novel, mitosis-dependent regulation of TRBP2 protein expression, as clearly evidenced by the lack of its immunofluorescence-facilitated detection during mitotic phases, in several human cancer cell lines of different tissue origin. Notably, the obtained TRBP2-downregulation patterns seem to derive from molecular mechanisms that act independently of oncogenic activities (e.g., malignancy grade), metastatic capacities (e.g., low versus high), and mutational signatures (e.g., p53^−/− or p53^ΔΥ126) of cancer cells. Conclusions: Taken together, we herein propose that TRBP2 serves as a novel cell cycle-dependent regulator, likely exerting mitosis-suppression functions, and, thus, its mitosis-specific downregulation can hold strong promise to be exploited for the efficient and successful prognosis, diagnosis, and (radio-/chemo-)therapy of diverse human malignancies, in the clinic. Full article

Crohn’s disease is a chronic, debilitating, inflammatory bowel disease. Here, we report a critical role of phospholipase C-β3 (PLC-β3) in intestinal homeostasis. In PLC-β3-deficient mice, exposure to oral dextran sodium sulfate induced lethality and severe inflammation in the small intestine. The lethality was due to PLC-β3 deficiency in multiple non-hematopoietic cell types. PLC-β3 deficiency resulted in reduced Wnt/β-catenin signaling, which is essential for homeostasis and the regeneration of the intestinal epithelium. PLC-β3 regulated the Wnt/β-catenin pathway in small intestinal epithelial cells (IECs) at transcriptional, epigenetic, and, potentially, protein–protein interaction levels. PLC-β3-deficient IECs were unable to respond to stimulation by R-spondin 1, an enhancer of Wnt/β-catenin signaling. Reduced expression of PLC-β3 and its signature genes was found in biopsies of patients with ileal Crohn’s disease. PLC-β regulation of Wnt signaling was evolutionally conserved in Drosophila. Our data indicate that a reduction in PLC-β3-mediated Wnt/β-catenin signaling contributes to the pathogenesis of ileal Crohn’s disease. Full article