Search Results (564)

DNMT1 variants are linked to complex neurodegenerative syndromes, yet their variant-specific functional and epigenomic consequences remain poorly defined. DNMT1 variants were identified in eight patients using gene-panel or whole-exome sequencing. Functional effects were assessed by site-directed mutagenesis and transient expression in HEK293T cells. Genome-wide methylation profiling of peripheral blood leukocyte DNA was performed using Nanopore sequencing, enabling direct quantification of 5-methylcytosine (5mC). CpG island-level differential methylation and gene set enrichment analysis (GSEA) were conducted. Variants in the replication foci targeting sequence (RFTS) domain (p.Y511H, p.Y540C, p.H569R) exhibited reduced DNMT1 protein expression, decreased enzymatic activity, and cytosolic aggregation. Variants in the C-terminal catalytic domain (p.A1334V and p.P1546S) showed reduced protein expression with relatively mild enzymatic impairment. Patients carrying the p.Y511H variant demonstrated a significant reduction in global 5mC levels compared with controls. Principal component analysis revealed distinct methylomic profiles separating most patients from controls, with marked intra- and inter-familial heterogeneity. CpG island-level analysis identified a single significantly hypomethylated region in p.Y511H carriers, and GSEA revealed differential enrichment of multiple Gene Ontology biological pathways. This study defines domain-dependent functional effects of DNMT1 variants and provides the first nanopore-based methylome analysis, revealing variant-specific and heterogeneous epigenomic alterations. Full article

Monoacylglycerols (MAGs) are significant intermediate byproducts in the hydrolysis of oils and fats. The accumulation of MAGs not only reduces the quality and purity of the final products in biodiesel production and edible oil refining but also poses challenges for downstream separation processes. Therefore, the development of efficient biocatalysts for the specific MAG conversion is of great industrial importance. The lipase from Aspergillus oryzae (AOL) has shown potential for lipid modification; however, the wild-type enzyme (WT) suffers from poor solubility, tendency to aggregate, and low specific activity towards MAGs in aqueous systems, which severely restricts its practical application. In this study, a combinatorial protein engineering strategy was employed to overcome these limitations. We integrated fusion protein technology with rational design to enhance both the functional expression and catalytic efficiency of AOL. Firstly, the superfolder green fluorescent protein (sfGFP) was fused to the N-terminus of AOL. The results indicated that the sfGFP fusion tag significantly improved the solubility and stability of the enzyme, preventing the formation of inclusion bodies. The fusion protein sfGFP-AOL exhibited a MAG conversion rate of approximately 65%, confirming the positive impact of the fusion tag on enzyme developability. To further boost catalytic performance, site-directed mutagenesis was performed based on structural analysis. Among the variants, the mutant sfGFP-Y92Q emerged as the most potent candidate. In the MAG conversion, sfGFP-Y92Q achieved a conversion rate of 98%, which was not only significantly higher than that of sfGFP-AOL but also outperformed the widely used commercial immobilized lipase, Novozym 435 (~54%). Structural modeling and docking analysis revealed that the Y92Q mutation optimized the geometry of the active site. The substitution of Tyrosine with Glutamine at position 92 likely enlarged the substrate-binding pocket and altered the local electrostatic environment, thereby relieving steric hindrance and facilitating the access of the bulky MAG substrate to the catalytic center. In conclusion, this work demonstrates that the synergistic application of sfGFP fusion and rational point mutation (Y92Q) can dramatically transform the catalytic properties of AOL. The engineered sfGFP-Y92Q variant serves as a robust and highly efficient biocatalyst for MAG degradation. Its superior performance compared to commercial standards suggests immense potential for cost-effective applications in the bio-manufacturing of high-purity fatty acids and biodiesel, offering a greener alternative to traditional chemical processes. Full article

Leukocyte-specific protein 1 (LSP1) is known as an endothelial gatekeeper because it controls endothelial permeability and transendothelial cell migration, including that of leukocytes and potentially metastatic cancer cells. In endothelial cells, LSP1 is predominantly in the nucleus under resting conditions but translocates to extranuclear compartments upon stimulation with TNF-α. The discrepancy between its predicted molecular weight (~37 kDa) and its observed migration on SDS-PAGE (≥52 kDa), along with its dynamic subcellular distribution, suggests a possible post-translational modification by SUMOylation. To investigate this, we examined endogenous LSP1 in murine primary endothelial cells and overexpressed recombinant LSP1 in murine endothelial (SVEC4-10EE2) and HEK293T cells. Our results demonstrate that LSP1 is SUMOylated by SUMO1, with Ubc9 serving as the conjugating enzyme and SENP1 as the deSUMOylating protease. Site-directed mutagenesis of lysines K270 and K318 abolished SUMOylation, resulting in a marked reduction in LSP1 steady-state levels. This reduction was attributed to enhanced ubiquitination and accelerated proteasomal degradation of LSP1 in the SUMOylation-deficient state. Furthermore, deSUMOylation impaired the TNF-α-induced translocation of LSP1 from the nucleus to extranuclear compartments, particularly the cytoskeleton. In summary, our findings establish that LSP1 is a SUMO1-modified protein. SUMOylation stabilizes LSP1 by preventing proteasomal degradation and is essential for its proper subcellular trafficking in endothelial cells in response to inflammatory stimuli. Full article

Tyrosinase (EC 1.14.18.1) is a binuclear copper enzyme responsible for the rate-limiting steps of melanogenesis, catalyzing the hydroxylation of L-tyrosine and oxidation of L-DOPA into o-quinones that polymerize melanin. Beyond its physiological role in pigmentation, tyrosinase is also implicated in food browning and oxidative stress–related disorders, making it a key target in cosmetic, food, and biomedical industries. This systematic review, conducted following PRISMA guidelines, aimed to identify and analyze microbial metabolites with tyrosinase inhibitory potential as sustainable alternatives to conventional inhibitors such as hydroquinone and kojic acid. Literature searches in Scopus and Web of Science (March 2025) yielded 156 records; after screening and applying inclusion criteria, 11 studies were retained for analysis. The inhibitors identified include indole derivatives, phenolic acids, peptides, and triterpenoids, mainly produced by fungi (e.g., Ganoderma lucidum, Trichoderma sp.), actinobacteria (Streptomyces, Massilia), and microalgae (Spirulina, Synechococcus). Reported IC₅₀ values ranged from micromolar to milli-molar levels, with methyl lucidenate F (32.23 µM) and p-coumaric acid (52.71 mM). Mechanisms involved competitive and non-competitive inhibition, as well as gene-level regulation. However, methodological heterogeneity, the predominance of mushroom tyrosinase assays, and limited human enzyme validation constrain translational relevance. Computational modeling, site-directed mutagenesis, and molecular dynamics are proposed to overcome these limitations. Overall, microbial metabolites exhibit promising efficacy, stability, and biocompatibility, positioning them as emerging preclinical candidates for the development of safer and more sustainable tyrosinase inhibitors. Full article

Based on PCR with complementary primer pairs and Pfu DNA polymerase, QuickChange site-directed mutagenesis has been widely employed, but its efficiency varies from mutation to mutation. An alternative strategy relies on partially overlapping primer pairs with 3′-overhangs, and this strategy has led to the recent development of P3a and P3b site-directed mutagenesis, in which the use of SuperFi II and Q5 polymerases raises the mutagenesis efficiency to ~100%. It is unclear whether these two DNA polymerases also improve the QuickChange method. Herein, we have evaluated this possibility by engineering 46 mutations on seven expression plasmids, two of which possess extremely GC-rich sequences. As Pfu DNA polymerase is a slow enzyme, its replacement with SuperFi II and Q5 polymerases reduced PCR length. Moreover, the average efficiency for each of the seven plasmids ranged from 48% to 69%, thereby outperforming the original QuickChange method. However, this efficiency is still lower than that from the P3a and P3b methods, supporting the superiority of primer pairs with 3′-overhangs. Analysis of the incorrect plasmids from the improved QuickChange method revealed frequent insertions at primer sites. The insertions were derived from primers and varied from mutation to mutation, with certain sites much more prone to such insertions. In comparison, these insertions occurred at a much lower frequency with the P3a and P3b methods, suggesting that primer pairs with 3′-overhangs enhance mutagenesis efficiency by reducing the likelihood to introduce insertions at primer sites. Thus, this study improves the QuickChange mutagenesis method, supports the superiority of the P3a and P3b methods, and uncovers a novel molecular mechanism by which the efficiency of PCR-based mutagenesis with completely overlapping primer pairs is negatively affected. Full article

Syringic acid (SA) is a natural derivative of syringaldehyde (SD), derived from lignin depolymerization. Its application in the food industry focuses on the properties of natural functional ingredients; it is mainly used as a food antioxidant and food preservative, but can also be used as an ingredient to enhance food flavor and functional foods. This compound exhibits a remarkable spectrum of biological activities, including potent antioxidant, anti-inflammatory, neuroprotective, hypoglycemic, detoxifying, and anti-cancer effects, positioning it as a highly promising candidate for pharmaceutical and nutraceutical applications. In this study, suitable sites were first screened through homologous sequence alignment, and a variant of aryl-alcohol oxidase (CgAAO) with high efficiency in catalyzing the conversion of SD to SA was obtained via site-directed mutagenesis. A deep eutectic solvent (DES) system based on choline chloride/urea (ChCl/UR) in water was developed to enhance SA production. Additionally, key parameters of the biological reaction were optimized, including temperature, pH, metal ions, as well as the type and dosage of DES. The optimal performance was achieved using recombinant E. coli pRSFDuet-CgAAO-Y335F whole-cell biocatalysts, yielding 75% and producing 0.75 g/L SA in 100 mM KPB buffer (pH 7.0) containing 5 wt% ChCl/UR and 1 mM Fe³⁺. This study established a novel biosynthetic pathway for SA that was efficient, mild, green, and environmentally friendly. Full article

The stable and safe integration of exogenous DNA into the genome is crucial to both genetic engineering and gene therapy. Traditional transgenesis approaches, such as those using retroviral vectors, result in random genomic integration, posing the risk of insertional mutagenesis and transcriptional dysregulation. Safe harbor sites (SHSs), genomic loci that support reliable transgene expression without compromising endogenous gene function, genomic integrity, or cellular physiology, have been identified and characterized across various model organisms. Well-established SHSs such as AAVS1, ROSA26, and CLYBL are routinely utilized for targeted transgene integration in human cells. Recent advances in genome architecture, gene regulation, and genome editing technologies are driving the discovery of novel SHSs for precise and safe genetic modification. This review aims to provide a comprehensive overview of SHSs and their applications that will guide investigators in the choice of SHS, especially when complementary sites are needed for more than one transgene integration. First, it outlines safety and functional criteria that qualify a genomic site as a safe harbor site. It then discusses the two primary strategies for identifying SHSs: i) traditional lentiviral-based random transgenesis, and ii) modern genome-wide in silico screening followed by CRISPR-based validation. This review also provides an updated catalogue of currently known SHSs in the human genome, detailing their characteristics, uses, and limitations. Additionally, it discusses the diverse applications of SHSs in basic research, gene therapy, CAR T cell-based therapy, and biotechnological production systems. Finally, it concludes by highlighting challenges in identifying universally applicable SHSs and outlines future directions for their refinement and validation across biological systems. Full article

GC-rich sequences affect DNA replication, recombination and repair, as well as RNA transcription in vivo. Such sequences may also impede site-directed mutagenesis in vitro. P3a site-directed mutagenesis is a highly efficient method, but it has not been tested with plasmids possessing GC-rich sequences. Here we report that it is very efficient with a BRPF3 expression vector but unsuccessful with that for KAT2B. Because two GC-rich regions located within the synthetic CAG promoter and the KAT2B coding region may form guanine (G)-quadruplexes and hinder plasmid denaturation during PCR, we developed P3b site-specific mutagenesis, achieving an average efficiency of 97.5% in engineering ten KAT2B mutants. Importantly, deletion mutagenesis revealed that either of the two GC-rich regions is sufficient for rendering the plasmid incompatible with P3a mutagenesis. Consistent with this, only P3b mutagenesis worked efficiently with several widely used sgRNA/Cas9 expression vectors, which contain the CAG promoter, and with an expression vector for CDK13, which possesses an intrinsically disordered domain encoded by a GC-rich DNA fragment. Thus, this study highlights serious challenges posed by GC-rich sequences to site-directed mutagenesis and provides an effective remedy to address such challenges. The findings support that G-quadruplex formation is one mechanism whereby such sequences impede regular PCR-based mutagenesis methods. Full article

In all arrestins, the gate loop is the central part of the lariat loop, which has an unusual shape and participates in maintaining the basal conformation. The gate loop supplies two out of five charges that constitute a stabilizing intramolecular interaction, aspartates in the polar core between the two domains. To elucidate the functional role of individual gate loop residues, we performed comprehensive site-directed mutagenesis and tested the effects of mutations on arrestin-1 binding to its preferred target, phosphorylated light-activated rhodopsin, and unphosphorylated activated form. Out of 34 mutations tested, 24 and 25 affected the binding to phosphorylated and unphosphorylated rhodopsin, respectively. Manipulation of residues following polar core aspartates reduced preference for phosphorylated over unphosphorylated light-activated rhodopsin as dramatically as replacing these negatively charged aspartates with positively charged arginine. The data show that numerous lariat loop residues play distinct roles in arrestin-1 binding and its exquisite preference for phosphorylated light-activated rhodopsin. Full article

Adzuki bean (Vigna angularis), a major grain legume in Asia, is susceptible to infection by cucumber mosaic virus (CMV), which threatens crop productivity. Here, we characterized the CMV-Pa3 isolate from adzuki bean and investigated the role of specific amino acid residues in the viral 2a protein influencing systemic infection of legumes. Phylogenetic analysis demonstrated that CMV-Pa3 is genetically distinct from other legume-infecting isolates. Inoculation assays revealed that CMV-Pa3 causes systemic infection in adzuki bean, cowpea, soybean, and pea, whereas the control isolate CMV-Rs1 is restricted to inducing local necrotic lesions in cowpea and adzuki bean. Site-directed mutagenesis targeted two conserved amino acid positions (631 and 641) in the 2a protein of CMV-Rs1. Functional analysis showed that residue 631 (Tyr) facilitates systemic infection across all tested legumes, while alteration at position 641 (Ser) alone enables systemic infection in cowpea and pea. These findings identify amino acid determinants in the CMV 2a protein critical for overcoming host restrictions and mediating systemic infection in various leguminous species. This work offers new insights into the molecular mechanisms underlying CMV pathogenicity and host specificity. Full article

Human growth hormone (GH) exerts its pleiotropic effects by binding to its receptor (GHR), leading to receptor dimerization and activation. We combined structural, evolutionary, and genetic analyses to elucidate the critical determinants of GH-GHR interaction and the impact of disease-causing mutations. Protein contact analysis revealed the specific amino acid residues involved in two distinct binding interfaces between GH and two chains of GHR. ConSurf analysis demonstrated significant sequence conservation in the receptor-binding regions of GH across species, highlighting their functional importance. A comprehensive list of known disease-causing mutations in GH was compiled and mapped to these binding interfaces and conserved regions. Computational site-directed mutagenesis (SDM) analysis predicted the impact of several mutations on protein stability, revealing both stabilizing and destabilizing effects. Sequence comparisons with orthologs from various species further supported the evolutionary conservation of key functional residues. Integrated analysis of contact residues between GH and GHR showed a strong correlation between receptor-binding residues, evolutionary conservation, and the occurrence of disease-associated mutations. These findings underscore the critical role of specific GH residues in mediating high-affinity interactions with its receptor and how mutations in these conserved contact points can disrupt binding affinity and/or protein stability, ultimately leading to growth disorders. This multi-faceted approach provides valuable insights into the molecular mechanisms underlying growth hormone deficiency and related syndromes. Full article

Background: Non-homologous end joining (NHEJ) is a crucial pathway for repairing DNA double-strand breaks and a key contributor to chemoresistance in cancer. The assembly of the DNA Ligase IV (LIG4)–XRCC4 complex is essential for NHEJ fidelity, however, the regulatory mechanisms governing this complex in cancer remain poorly understood. This study aims to investigate whether and how lactate, a key metabolic byproduct of the Warburg effect, regulates the XRCC4–LIG4 complex and influences chemoresistance. Methods: The functional role of lactate in NHEJ was assessed using DNA repair reporter assays in ovarian cancer cells. Protein–protein interactions were examined through co-immunoprecipitation and pull-down assays. The molecular mechanism of lactate’s action was delineated using a combination of site-directed mutagenesis, in vitro binding assays, and molecular docking. Finally, the physiological relevance of lactate-mediated NHEJ was validated in a preclinical ovarian cancer mouse model treated with cisplatin. Results: We demonstrated that lactate enhances NHEJ repair efficiency and confers resistance to DNA-damaging chemotherapeutics. Mechanistically, lactate directly binds to XRCC4 at key residues, including Y66, E55, and S110, thereby strengthening the XRCC4–LIG4 association. This interaction is independent of protein lactylation. In vivo studies confirmed that lactate-driven NHEJ promotes chemoresistance in ovarian cancer. Conclusions: Our findings reveal lactate as a novel metabolic regulator of the NHEJ pathway by directly allosterically modulating the XRCC4–LIG4 complex. This work establishes a direct molecular link between the Warburg effect and DNA repair-driven chemoresistance, offering new insights into potential therapeutic strategies for ovarian cancer. Full article

Euryhaline fishes provide excellent material for the theoretical study of the broad-spectrum adaptability of organisms and the use of low-salinity and even freshwater environments, or high-salinity and seawater environments, for the domestication of fishes. Here, we studied the molecular mechanisms of osmotic pressure regulation in a euryhaline fish, marine medaka (Oryzias melastigma). As the fish progressed from seawater to freshwater, the changes in stress indicators (cortisol—COR; malondialdehyde—MDA; reactive oxygen species—ROS; superoxide dismutase—SOD) indicated that they gradually adapted to the freshwater environment. The transcriptome analysis also showed that there were 6850 DEGs (differentially expressed genes) involved in the process. By analyzing these DEGs deeply, we screened and identified the Hnf1ba-slc12a1 signal pathway involved in osmotic pressure regulation. The results of a dual-luciferase reporter assay in HEK293T cells, as well as an overexpression experiment by in vitro cultured gill cells of O. melastigma, confirmed that Hnf1ba transcriptionally regulates the slc12a1 gene. Fragment deletion and site-directed mutagenesis assays revealed a Hnf1ba-binding sequence (GATTAATCATTTACT, located at −1877 to −1863) in the slc12a1 promoter. Based on this result, we conducted a targeted regulation experiment on the slc12a1 gene using the CRISPR-dCas9 & Sun-Tag system. The most effective activation of slc12a1 gene expression was observed in the sgRNA2 group. These results enhance our understanding of adaptation mechanisms in salt-tolerant fish and provide a reference for efficiently promoting the domestication of fish adaptive to salinity changes. Full article

Aspergillus flavus is both an agricultural and clinical pathogen, notable for its ability to contaminate crops with aflatoxins and cause invasive aspergillosis. The increasing emergence of azole resistance in A. flavus poses a serious challenge to food safety and human health. Although mutations in ergosterol biosynthesis genes have been reported in resistant isolates, their functional contributions remain largely unvalidated. In this study, we investigated the role of the CYP51A Y119F mutation in azole resistance. Site-directed mutants were generated using PCR-based gene editing, and their susceptibility to antifungal agents was assessed through Clinical and Laboratory Standards Institute broth microdilution and agar diffusion assays. The Y119F mutation reduced susceptibility specifically to voriconazole and isavuconazole, while susceptibility to itraconazole and posaconazole remained unchanged. To explore the structural basis of this phenotype, molecular dynamics simulations were performed. The mutant protein exhibited greater fluctuations and reduced conformational stability compared to the wild-type enzyme. Tunnel analysis further indicated that the Y119F substitution caused narrowing and shortening of the main access tunnels to the heme-binding pocket, likely impairing azole access and binding. The combined biochemical and structural analyses suggest that Y119F represents a primary resistance-conferring mutation that modifies the structural dynamics of CYP51A. Full article

Bactrocera dorsalis Hendel is a devastating invasive pest that costs billions of dollars in agricultural losses worldwide. Current control strategies rely heavily on male-specific attractants such as methyl eugenol, which are less effective against females, underscoring the need for female-targeted control approaches. Here, we investigated the molecular mechanisms underlying female attraction to cis-linalool oxide by functionally characterizing the odorant receptor OR45a, identifying it as a molecular target for female-oriented pest management. We conducted spatiotemporal expression analysis of OR45a in response to cis-linalool oxide, followed by RNAi and behavioral assays. Phylogenetic analysis of OR45a orthologs from 10 Dipteran species, combined with structural topology prediction and solvent-accessible surface area (ASA) analysis, helped identify functional domains and residues. Site-directed mutagenesis and two-electrode voltage clamp (TEVC) recordings validated receptor–ligand interactions. Results showed that OR45a was specifically upregulated in antennae, with peak expression at 10 days post-eclosion, coinciding with oviposition periods. RNAi significantly reduced OR45a transcript levels and female behavioral responses to cis-linalool oxide. Phylogenetic analysis showed that OR45a is highly conserved within Tephritidae but diverges from Drosophilidae, with closest similarity to Anastrepha ludens, indicating ecological specialization. Structural modeling predicted a canonical seven-transmembrane architecture with three extracellular loops forming the ligand-binding pocket. Among five key residues identified, Leu122 and Ile146 were essential for ligand recognition, while Tyr107 contributed to protein stability. These findings reveal a female-specific odorant receptor mechanism in B. dorsalis and provide molecular targets for OR45a-based attractants, addressing a critical gap in female-focused pest management. Full article