Search Results (160)

Rice seed storage proteins (SSPs) play a critical role in determining the nutritional quality, cooking properties, and digestibility of rice. To enhance seed quality, CRISPR/Cas9 genome editing was applied to modify SSP composition by targeting genes encoding 13 kDa prolamins and type A glutelins. Three CRISPR/Cas9 constructs were designed: one specific to the 13 kDa prolamin subfamily and two targeting conserved GluA glutelin regions. Edited T₀ and T₁ lines were generated and analyzed using InDel analysis, SDS-PAGE, Bradford assay, and RP-HPLC. Insertions were more frequent than deletions, accounting for 56% and 74% of mutations in prolamin and glutelin genes, respectively. Editing efficiency varied between sgRNAs. All lines with altered protein profiles contained InDels in target genes. SDS-PAGE confirmed the absence or reduction in bands corresponding to 13 kDa prolamins or GluA subunits, showing consistent profiles among lines carrying the same construct. Quantification revealed significant shifts in SSP composition, including increased albumin and globulin content. Prolamin-deficient lines showed reduced prolamins, while GluA-deficient lines exhibited increased prolamins. Total protein content was significantly elevated in all edited lines, suggesting enrichment in lysine-rich fractions. These findings demonstrate that CRISPR/Cas9-mediated editing of SSP genes can effectively reconfigure the rice protein profile and enhance its nutritional value. Full article

This study integrated non-targeted metabolomics and transcriptomics to investigate dynamic changes in Antheraea pernyi pupae across five developmental stages. Metabolomic analysis identified 1246 metabolites, primarily organic acids, lipids, heterocyclic compounds, and oxygen-containing organics. Principal component analysis revealed stage-specific metabolic profiles: amino acid derivatives (pyruvate, proline, lysine) declined, while pyrimidines (cytidine, uridine, β-alanine) and monosaccharides (glucose, mannose) increased. 18β-glycyrrhetinic and ursolic acids accumulated significantly in the middle and late stages. Transcriptomic analysis identified 7230 differentially expressed genes (DEGs), with 366, 1705, and 5159 significantly differentially expressed genes in the T1, T3, and T5 comparison groups, respectively. KEGG enrichment highlighted ABC transporters, amino acid/pyrimidine metabolism, and tyrosine pathways as developmentally critical, with aminoacyl-tRNA biosynthesis upregulated in later phases. Integrated multi-omics analysis revealed coordinated shifts in metabolites and genes across developmental phases, reflecting dynamic nutrient remodeling during pupal maturation. This study systematically delineates the molecular transitions driving pupal development in Antheraea pernyi pupae, uncovering conserved pathway interactions and mechanistic insights into nutrient metabolism. These findings provide a scientific foundation for leveraging pupal resources in functional food innovation and bioactive compound discovery for pharmaceutical applications. Full article

The H1N1 swine influenza viruses CQ91 and CQ445, isolated from pigs in China, exhibited distinct virulence in mice despite sharing similar genomic constellations. CQ91 demonstrated higher pathogenicity (MLD₅₀: 5.4 log₁₀ EID₅₀) and replication efficiency in mice compared to CQ445 (MLD₅₀: 6.6 log₁₀ EID₅₀). Through reverse genetics, we found that the attenuation of CQ445 was due to a single substitution of glutamic acid (E) with lysine (K) at position 343 in the PB2 protein. Introducing the CQ445-PB2 (343K) into CQ91 significantly reduced viral replication and pathogenicity in mice, while replacing CQ445-PB2 with CQ91-PB2 (343E) restored virulence. In vitro studies showed that the K343E mutation impaired viral replication in MDCK and A549 cells and reduced polymerase activity in minigenome assays. Mechanistically, the amino acid at position 343 in the PB2 affects the transcription stage of the viral replication process. Structural modeling indicated that the charge reversal caused by E343K altered local electrostatic interactions without major conformational changes. Phylogenetic analysis revealed that PB2-343E is highly conserved (>99.9%) in human and swine H1/H3 influenza viruses, suggesting that PB2-343E confers an adaptive advantage. This study identifies PB2-343E as a critical determinant of influenza virus pathogenicity in mammals, highlighting its role in host adaptation. Full article

Yellow mealworm (Tenebrio molitor) meal is a promising sustainable protein for pet food, yet its effect on nutrient balance and granule texture is incompletely defined. Five dry dog food formulas containing 25%, 30%, 35%, 40%, and 45% of T. molitor meal were extruded and analyzed for proximate composition, fatty-acid and amino-acid profiles, and compressive mechanical properties. Crude-protein concentration remained stable, whereas fat and ash increased (p < 0.01) and carbohydrates decreased as the inclusion level rose. Tenebrio molitor meal enriches granules in oleic, linoleic, and α-linolenic acids, improving the ω-6:ω-3 ratio from 8.0 to 5.4. Essential amino acid levels were conserved, although lysine and methionine fell modestly (≤11%). Mechanical testing showed a linear decline in hardness (331 → 300 N) and stiffness (46 → 41 N mm⁻¹), indicating softer, more deformable granules at higher inclusion levels. The inclusion of up to 45% T. molitor meal can be incorporated without compromising protein quality while enhancing unsaturated fat content. Minor lysine/methionine dilution can be offset by targeted supplementation. These data support wider adoption of insect protein in hypoallergenic and eco-friendly canine diets. Full article

The ReAV enzyme from Rhizobium etli, a representative of Class 3 L-asparaginases, is sequentially and structurally different from other known L-asparaginases. This distinctiveness makes ReAV a candidate for novel antileukemic therapies. ReAV is a homodimeric protein, with each subunit containing a highly specific zinc-binding site created by two cysteines, a lysine, and a water molecule. Two Ser-Lys tandems (Ser48-Lys51, Ser80-Lys263) are located in the close proximity of the metal binding site, with Ser48 hypothesized to be the catalytic nucleophile. To further investigate the catalytic process of ReAV, site-directed mutagenesis was employed to introduce alanine substitutions at residues from the Ser-Lys tandems and at Arg47, located near the Ser48-Lys51 tandem. These mutational studies, along with enzymatic assays and X-ray structure determinations, demonstrated that substitution of each of these highly conserved residues abolished the catalytic activity, confirming their essential role in enzyme mechanism. Full article

Background: Malaria remains a significant global health burden, particularly in sub-Saharan Africa, accounting for high rates of illness and death. The growing resistance to frontline antimalarial therapies underscores the urgent need for novel drug targets and therapeutics. Bromodomain-containing proteins, which regulate gene expression through chromatin remodeling, have gained attention as potential targets. Plasmodium falciparum bromodomain protein 1 (PfBDP1), a 55 kDa nuclear protein, plays a key role in recognizing acetylated lysine residues and facilitating transcription during parasite development. Methods: This study investigated ex vivo PfBDP1 gene mutations and identified potential small molecule inhibitors using computational approaches. Malaria-positive blood samples were collected. Genomic DNA was extracted, assessed for quality, and amplified using PfBDP1-specific primers. DNA sequencing and alignment were performed to determine single-nucleotide polymorphism (SNP). Structural modeling used the PfBDP1 crystal structure (PDB ID: 7M97), and active site identification was conducted using CASTp 3.0. Virtual screening and pharmacophore modeling were performed using Pharmit and AutoDock Vina, followed by ADME/toxicity evaluations with SwissADME, OSIRIS, and Discovery Studio. GROMACS was used for 100 ns molecular dynamics simulations. Results: The malaria prevalence rate stood at 12.24%, and the sample size was 165. Sequencing results revealed conserved PfBDP1 gene sequences compared to the 3D7 reference strain. Virtual screening identified nine lead compounds with binding affinities ranging from −9.8 to −10.7 kcal/mol. Of these, CHEMBL2216838 had a binding affinity of −9.9 kcal/mol, with post-screening predictions of favorable drug-likeness (8.60), a high drug score (0.78), superior pharmacokinetics, and a low toxicity profile compared to chloroquine. Molecular dynamics simulations confirmed its stable interaction within the PfBDP1 active site. Conclusions: Overall, this study makes a significant contribution to the ongoing search for novel antimalarial drug targets by providing both molecular and computational evidence for PfBDP1 as a promising therapeutic target. The prediction of CHEMBL2216838 as a lead compound with favorable binding affinity, drug-likeness, and safety profile, surpassing those of existing drugs like chloroquine, sets the stage for preclinical validation and further structure-based drug design efforts. These findings are supported by prior experimental evidence showing significant parasite inhibition and gene suppression capability of predicted hits. Full article

This study explored the biosynthetic mechanisms and structural diversity of pyoverdines (PVDs) produced by Pseudomonas fulva. Genomic analysis using antiSMASH identified the PVD biosynthetic gene cluster, although the C-terminal peptide sequence could not be predicted. Subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis revealed the full peptide structure, including modified residues, such as N-acetylhydroxyornithine and cyclohydroxyornithine, and confirmed the presence of several PVD isoforms with different chromophore side chains. Comparative LC-MS analysis across Pseudomonas species demonstrated that P. fulva produces unique PVD molecular mass patterns. The bioinformatic and structural modeling of non-ribosomal peptide synthetase PvdL open reading frame 3 revealed that the A2 and A3 adenylation domains are lysine selective. Although their sequences differ from known lysine-specific signatures, AlphaFold3-based structural prediction revealed conserved substrate-binding configurations, suggesting that similar substrate-binding features may have arisen independently. Notably, Thr²⁹⁷, a unique residue in the non-ribosomal code, likely plays a key role in lysine recognition. The high degree of sequence similarity between the A2 and A3 domains may reflect domain duplication and could be involved in the diversification of the PVD structure. Further functional and ecological studies are required to assess the physiological significance of P. fulva PVDs in microbial iron acquisition. Full article

Tomato brown rugose fruit virus (ToBRFV) is an important emerging virus that poses a serious threat to the global agricultural economy. Ubiquitination is one of the key post-translational protein modification types in plant responses to biotic stress, but the extent to which ToBRFV infection alters the overall ubiquitination status has not been reported. This study conducted integrated ubiquitome and proteome analyses of Nicotiana benthamiana leaves infected with ToBRFV and identified differentially ubiquitinated proteins. A total of 346 lysine sites on 302 identified proteins were found to be affected, with 260 sites exhibiting upregulated ubiquitination levels in 224 proteins and 86 sites showing downregulated ubiquitination levels in 80 proteins. The differentially ubiquitinated proteins were primarily localized in the cytoplasm (29%), nucleus (18%), plasma membrane (8.9%), mitochondria (5.1%), and chloroplasts (4.6%). Fourteen conserved ubiquitination motifs, including ENNNK, ENNK, SK, and KNG, were identified. Furthermore, enrichment analysis indicated that ToBRFV infection induces an increase in the ubiquitination levels of proteins associated with ion transport, MAPK signaling pathways, and plant hormone signal transduction, while the ubiquitination levels of proteins related to carbon metabolism and secondary metabolite synthesis decreased. Functional analysis of the three differentially ubiquitinated proteins revealed that a RING/U-box superfamily protein negatively regulates ToBRFV infection. Our work provides the first systematic analysis of the ubiquitination profile in N. benthamiana leaves following ToBRFV infection, providing important resources for further studies on the regulatory mechanisms of ubiquitination in plant responses to ToBRFV. Full article

Fusarium verticillioides is a globally prevalent phytopathogenic fungus responsible for multiple diseases in maize and a major producer of the mycotoxin fumonisin B1 (FB1), a highly toxic fungal secondary metabolite (FSM). The histone code, which includes reversible modifications such as acetylation and methylation, plays a critical role in regulating chromatin structure and gene expression. In fungi, di- and tri-methylation of histone H3 at lysine 9 (H3K9me2/3) serves as a key epigenetic mark associated with heterochromatin formation and transcriptional repression. In this study, we identified and characterized a putative heterochromatin protein 1 (HP1) family member in F. verticillioides, designated FvHP1, based on conserved domain architecture and phylogenetic analyses. FvHP1 retains essential residues required for H3K9me2/3 recognition, supporting its functional conservation within the HP1 protein family. Phenotypic analysis of the ΔFvHP1 mutant revealed impaired vegetative growth, reduced conidiation and virulence, and altered FB1 mycotoxin production. Additionally, the accumulation of red pigment in the mutant was linked to the deregulation of secondary metabolism, specifically the overproduction of fusarubin-type naphthoquinones, such as 8-O-methylnectriafurone. These results support the role of FvHP1 in facultative heterochromatin-mediated repression of sub-telomeric biosynthetic gene clusters, including the pigment-associated PGL1 cluster. Our findings provide new insights into the epigenetic regulation of fungal pathogenicity and metabolite production, as well as the first evidence of a functional HP1 homolog in F. verticillioides. Full article

Hendra virus (HeV) is a highly pathogenic member of the Henipavirus genus (family Paramyxoviridae, order Mononegavirales), for which all basic replication processes are located in the cytoplasm. The HeV matrix (M) protein plays essential roles in viral assembly and budding at the plasma membrane, but also undergoes dynamic nuclear and nucleolar trafficking, accumulating in nucleoli early in infection, before relocalising to the plasma membrane. We previously showed that M targets sub-nucleolar compartments—the fibrillar centre (FC) and dense fibrillar component (DFC)—to modulate rRNA biogenesis by mimicking a process occurring during a nucleolar DNA-damage response (DDR). Here, we show that M protein sub-nucleolar localisation is regulated by ubiquitination, which controls its redistribution between the FC-DFC and granular component (GC). The mutagenesis of a conserved lysine (K258) reported to undergo ubiquitination, combined with the pharmacological modulation of ubiquitination, indicated that a positive charge at K258 is required for M localisation to the FC-DFC, while ubiquitination regulates subsequent egress from the FC-DFC to the GC. M proteins from multiple Henipaviruses exhibited similar ubiquitin-dependent sub-nucleolar trafficking, indicating a conserved mechanism. These findings reveal a novel mechanism regulating viral protein transport between phase-separated sub-nucleolar compartments and highlight ubiquitination as a key modulator of intra-nucleolar trafficking. Full article

Histone 3 lysine 4 methylation (H3K4me) is an evolutionarily conserved epigenetic marker associated with transcriptional activation, playing a crucial role in growth and development. In yeast, all forms of H3K4 methylation are catalyzed by the COMPASS complex. However, purifying endogenous COMPASS remains challenging due to its low abundance, compositional complexity, and structural instability, resulting in low yield, poor purity, and heterogeneity in isolated complexes. These technical limitations have impeded the structural elucidation of the intact COMPASS complex and contributed to inconsistencies in reported in vitro enzymatic activity, thereby limiting a comprehensive understanding of its functions. Here, we present an optimized tandem affinity purification strategy that enables the high-yield isolation of native COMPASS from Saccharomyces cerevisiae with >99% purity and intact subunit composition, as validated by biochemical analyses. Using recombinant nucleosomes as substrates, we systematically characterized its catalytic properties and found that endogenously purified COMPASS exhibited strict dependence on H2B ubiquitination for catalyzing H3K4 methylation. This work establishes an efficient purification strategy for future structural and functional studies of COMPASS and provides critical insights into its catalytic properties. Full article

This study investigates nitrogen metabolism during the middle of the second fermentation in stopped bottles of sparkling wine, focusing on two flor Saccharomyces cerevisiae yeast strains (G1 and N62) isolated from the velum of biologically aged wine. Nitrogen compounds, including amino acids, biogenic amines, and ammonium chloride, were quantified, revealing strain-specific differences in nitrogen utilization and production. Proteomic analysis identified 1053 proteins, with 127 showing significant differences between strains. Strain G1 demonstrated enhanced cell wall remodeling and prioritized nitrogen conservation via arginine and lysine biosynthesis, while strain N62 exhibited increased translational activity and alternative carbon utilization pathways. Notably, strain N62 produced higher concentrations of biogenic amines (putrescine and tyramine), likely due to its greater decarboxylation capacity. Principal Component Analysis (PCA) highlighted clear differentiation in the nitrogen compound profiles across the base wine and wines inoculated with the two strains. The proteome of strain N62 showed increased mitochondrial activity and TCA cycle involvement, facilitating faster fermentation (27 days vs. 52 days for G1), growth (46 × 10⁶ cells/mL vs. 21 × 10⁶ cells/mL for G1) and cell viability (4 × 10⁶ cells/mL vs. 0.7 × 10⁶ cells/mL for G1). These findings suggest that yeast strain selection significantly influences nitrogen metabolism and potentially aroma profiles and and fermentation dynamics in sparkling wine production. Understanding these metabolic adaptations provides valuable insights for optimizing yeast performance to enhance wine quality and preserve regional characteristics. Full article

In the mid-1970s, it was revealed that the 5′ end of the RNA genome of poliovirus (PV) was covalently linked to a peptide called VPg (viral protein, genome-linked). Subsequently, VPgs have been found attached to many other viruses and even phages. This review summarizes the patterns of physicochemical properties that are conserved within the VPgs of plus-strand RNA viruses where short-peptide VPgs have been identified. Mutagenesis and structural data indicate the importance of a 5 aa conserved motif at the N-termini of picornaviral VPgs (around the tyrosine 3 residue, which forms a covalent bond to UMP and the RNA). Hidden Markov models have been used to find motifs and VPgs in additional genera of picornaviruses, as well as dicistroviruses in insects and comoviruses in plants. These latter VPgs are bound to the RNA termina through linkages to serine or threonine. The role of free VPg and VPgpU needs clarification, especially in light of multiple genome copies in many of the viruses. Lysine and other positively charged side chains are hallmarks of VPgs. These may contribute to interactions with the viral RNA, polymerase, membranes and cellular proteins. The larger protein VPgs from potyviruses and noroviruses/caliciviruses may also show some areas of similar properties to these small peptides. Full article

Arabinogalactan proteins (AGPs) constitute a diverse class of hydroxyproline-rich glycoproteins implicated in various aspects of plant growth and development. However, their functional characterization in cotton (Gossypium spp.) remains limited. As a globally significant economic crop, cotton serves as the primary source of natural fiber, making it essential to understand the genetic mechanisms underlying its growth and development. This study aims to perform a comprehensive genome-wide identification and characterization of the AGP gene family in Gossypium spp., with a particular focus on elucidating their structural features, evolutionary relationships, and functional roles. A genome-wide analysis was conducted to identify AGP genes in Gossypium spp., followed by classification into distinct subfamilies based on sequence characteristics. Protein motif composition, gene structure, and phylogenetic relationships were examined to infer potential functional diversification. Subcellular localization of a key candidate gene, GhAGP50, was determined using fluorescent protein tagging, while gene expression patterns were assessed through β-glucuronidase (GUS) reporter assays. Additionally, hormonal regulation of GhAGP50 was investigated via treatments with methyl jasmonate (MeJA), abscisic acid (ABA), indole-3-acetic acid (IAA), and gibberellin (GA). A total of 220 AGP genes were identified in Gossypium spp., comprising 19 classical AGPs, 28 lysine-rich AGPs, 55 AG peptides, and 118 fasciclin-like AGPs (FLAs). Structural and functional analyses revealed significant variation in gene organization and conserved motifs across subfamilies. Functional characterization of GhAGP50, an ortholog of AGP18 in Arabidopsis thaliana, demonstrated its role in promoting epidermal hair formation in leaves and stalks. Subcellular localization studies indicated that GhAGP50 is targeted to the nucleus and plasma membrane. GUS staining assays revealed broad expression across multiple tissues, including leaves, inflorescences, roots, and stems. Furthermore, hormonal treatment experiments showed that GhAGP50 expression is modulated by MeJA, ABA, IAA, and GA, suggesting its involvement in hormone-mediated developmental processes. This study presents a comprehensive genome-wide analysis of the AGP gene family in cotton, providing new insights into their structural diversity and functional significance. The identification and characterization of GhAGP50 highlight its potential role in epidermal hair formation and hormonal regulation, contributing to a deeper understanding of AGP functions in cotton development. These findings offer a valuable genetic resource for future research aimed at improving cotton growth and fiber quality through targeted genetic manipulation. Full article

Alternative splicing is a critical post-transcriptional regulatory mechanism in eukaryotes. While infection with Ralstonia solanacearum GMI1000 significantly alters plant alternative splicing patterns, the underlying molecular mechanisms remain unclear. Herein, the effect of the GMI1000 Type III secretion system effectors on alternative splicing in the tomato cultivar Heinz 1706 was investigated. The RNA-seq analysis confirmed genome-wide alternative splicing changes induced by the Type III secretion system in tomato, including 1386 differential alternatively spliced events across 1023 genes, many of which are associated with plant defense. Seven nucleus-localized Type III effectors were transiently expressed in an RLPK splicing reporter system transgenic tobacco, identifying RipP2 as an effector that modulates alternative splicing levels. Sequence analysis, protein–protein interaction assays, and AlphaFold2 structural predictions revealed that RipP2 interacted with the tomato splicing factor SR34a. Furthermore, RipP2 acetylated a conserved lysine at position 132 within the SWQDLKD motif of SR34a, regulating its splicing pattern in defense-related genes and modulating plant immunity. This study elucidates how the “RipP2-SR34a module” influences plant immune responses by regulating the alternative splicing of immune-related genes, providing new insights into pathogen–plant interactions and splicing regulation. Full article