Search Results (1,585)

The alkaline pectate lyase A from Paenibacillus barcinonensis, encoded by pelA (GenBank accession no. CAB40884), is an enzyme with high activity on pectin and potential application in sustainable industrial biotechnology. In this study, pelA was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by pelA, and two of them in conserved regions of class III pectate lyases, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both standard and glycosylation deficient strains of S. cerevisiae. The correct targeting of the recombinant fusion proteins was confirmed by Western blot analysis using Pir-specific antibodies, whilst enzymatic activity on polygalacturonic acid was demonstrated on both plate assays and colorimetric assays. Maximum activities were over two and a half times higher when the enzyme was expressed in the glycosylation deficient strain, suggesting a better adaptation of this strain to the secretion of the functional enzyme. Full article

Myocardial fibrosis is a common pathological feature of multiple cardiovascular diseases, including heart failure, hypertension, and myocardial infarction, and is associated with poor prognosis. Despite extensive research, clinically validated molecular biomarkers for early diagnosis and reliable therapeutic targets for myocardial fibrosis remain limited. Post-translational modifications (PTMs), including phosphorylation, acetylation, ubiquitination, SUMOylation, and glycosylation, are critical regulators of fibrosis-related signaling pathways, yet a systematic bioinformatics-driven identification of PTM-related hub genes has not been performed. Three publicly available GEO datasets (GSE57345, GSE133054, GSE76314) comprising cardiac tissue from heart failure and control patients were integrated. Differentially expressed genes (DEGs) were identified using the limma package, then intersected with a curated PTM gene set derived from PhosphoSitePlus and UniProt databases. Weighted gene co-expression network analysis (WGCNA) identified fibrosis-associated modules, and protein–protein interaction (PPI) network analysis via STRING and CytoHubba pinpointed hub genes. Diagnostic performance was assessed by receiver operating characteristic (ROC) analysis across independent validation cohorts. Immune cell infiltration was estimated using CIBERSORT.Molecular docking with AutoDock Vina (version 1.2.3) was performed to evaluate binding affinity of FDA-approved cardiovascular drugs against identified hub protein targets. A total of 863 DEGs were identified in the training cohort (|log₂FC| > 1.0, adjusted p < 0.05), of which 138 overlapped with the PTM gene set. WGCNA revealed a turquoise module (r = 0.79, p < 0.001) most significantly correlated with fibrosis severity. PPI analysis identified five hub genes: SIRT3, SMAD3, NEDD4L, UBC9, and CAMK2D. ROC analysis demonstrated strong diagnostic performance (AUC range: 0.82–0.92) validated in independent cohorts. Hub genes showed significant correlations with M2 macrophage infiltration. Molecular docking identified spironolactone and finerenone as top-ranked ligands with binding energies of −8.7 and −8.4 kcal/mol against SMAD3 and SIRT3, respectively. This study, which is entirely in silico and based on publicly available transcriptomic datasets, systematically identifies five PTM-related hub genes as candidate diagnostic biomarkers and prioritised drug-repurposing targets in myocardial fibrosis. These findings are hypothesis-generating and require experimental validation (protein-level confirmation, cell- and animal-based functional assays, and biophysical binding studies) before any diagnostic or therapeutic claim can be made. Full article

Background/Objectives: Protein–carbohydrate interactions are implicated in amyloid aggregation pathways associated with Alzheimer’s disease (AD). Designing glycomimetics that modulate amyloid assembly represents a promising strategy. In addition, the interaction of Aβ_1–42 oligomers (Aβo) with prion protein (PrP^C) activates Fyn kinase and leads to Tau hyperphosphorylation, another process characterizing AD. Thus, we generated a library of phenyl 2-acetamidoselenogalactoside mimetics to evaluate their interactions with Aβo and disruption of Aβo–PrP^C binding, and consequently their potential to inhibit Fyn kinase activation. Methods: The synthetic approach comprised azidophenylselenylation, a modified one-pot Staudinger reduction–acylation, a selective α-glycosylation, and deacetylation. Structural diversity was achieved mainly via acylation or ureation. The compounds were screened for binding to Aβo using STD-NMR, ¹⁹F-NMR, and rapid equilibrium dialysis (RED). ADME properties were assessed through microsomal metabolism and solubility assays, while cytotoxicity was evaluated by MTT assays in human embryonic kidney (HEK) cells. Results: Several compounds bound Aβo in STD-NMR experiments, mainly through aromatic and anomeric protons, and phenyl 2-deoxy-2-phenylureido-1-seleno-α-d-galactopyranoside (34) showed the most consistent response, with >50% increase in relative binding signal in competition assays, demonstrating also some inhibition of Aβo–PrP^C interactions (12%). Selenium at the anomeric position enhanced binding compared to sulphur and oxygen analogs. RED experiments confirmed weak binding interactions, consistent with STD-NMR results. ADME revealed that acetylated compounds undergo microsomal metabolism, whereas deacetylated derivatives displayed high aqueous solubility (>100 μM) and showed no cytotoxicity. Conclusions: Phenyl 2-acetamidoselenogalactosides are a novel class of amyloid-binding glycomimetics. Among them, 34 emerges as the most promising compound, combining favorable solubility, metabolic stability, low toxicity, and measurable interference with Aβo and Aβo–PrP^C interactions, thus supporting further developments toward therapeutic applications in AD. Full article

ATP-binding cassette (ABC) transporters are one of the largest superfamilies of membrane proteins, but little is known about the structural and evolutionary features of their non-domain regions. To clarify the diversity of these non-canonical regions across evolutionary lineages, we performed an analysis of intrinsically disordered regions, site-specific selection and predicted post-translational modification (PTM) sites among five architectural classes involving 1581 prokaryotic and eukaryotic sequences. Linker and flanking regions were more disordered than transmembrane and nucleotide-binding domains in all architectures. Disorder fraction was significantly different between region types after phylogenetic correction (Pagel’s λ ≈ 0.97). Predicted PTM sites are enriched in disordered non-domain segments, with N-linked glycosylation and phosphoserine showing the strongest positive enrichment. A total of 140 sites satisfied a tiered conservation criterion (MusiteDeep score ≥ 0.5; cross-species conservancy ≥ 30%), including 40 high-confidence or moderate-confidence sites (conservancy ≥ 50%) as well as novel phosphotyrosine candidates in half transporters and NBD-only proteins. Site-specific selection analyses showed pervasive purifying selection across domain cores and architecture-dependent enrichment of episodic positive selection in non-domain regions, with significant non-domain enrichment in full reverse and half forward transporters (Fisher’s exact, BH-adjusted p < 0.05). In summary, these findings establish that non-canonical regions of ABC transporters are evolutionarily dynamic and contain conserved predicted modification sites, supporting the idea that these regions are evolutionary dynamic segments that deserve experimental characterization as candidate regulatory interfaces. Full article

Obesity is a major global health challenge characterized by chronic low-grade inflammation, oxidative stress, and an increased risk of cardiometabolic disorders. Although sex-related differences in inflammatory and redox biomarkers have been reported in obese populations, the molecular mechanisms underlying this heterogeneity remain incompletely understood. In this study, we applied a proteomics-based approach to investigate urinary extracellular vesicles from 45 obese individuals (BMI 30–40 kg/m²; age 50–70 years) in order to identify molecular signatures associated with metabolic dysregulation. Shotgun proteomics analysis performed by nanoLC–MS/MS enabled the identification of 3822 proteins. Hierarchical clustering of proteomic profiles revealed two distinct molecular groups, predominantly enriched in males (Group I) and females (Group II). Label-free quantitative analysis identified 466 differentially abundant proteins between the two clusters. Functional enrichment analysis highlighted pathways associated with immune response, metabolic regulation, and redox homeostasis, including glycolysis/gluconeogenesis, lysosome activity, leukocyte transendothelial migration, and glutathione, cysteine and methionine metabolism. Notably, proteins related to ferroptosis were enriched, suggesting the involvement of iron-dependent oxidative cell death mechanisms in the metabolic imbalance observed in a subset of subjects. Furthermore, the non-enzymatic glycosylation of urinary proteins was significantly higher in Group I compared with Group II (p = 0.0002), indicating increased formation of advanced glycation products in individuals with a more pronounced pro-oxidant state. Preliminary follow-up data suggested a higher incidence of pathological events, including cardiovascular complications, among individuals belonging to Group I. Overall, these findings demonstrate that urinary proteomic profiling can identify distinct molecular phenotypes among obese individuals and highlight oxidative stress, ferroptosis, and protein glycation as potential determinants of metabolic vulnerability, supporting the use of non-invasive proteomic approaches for improved risk stratification in obesity. Full article

Human metapneumovirus (HPMV) is a significant pathogen that causes lower respiratory tract infections. Given the weak immunogenicity thereof, and the few relevant studies, the utility of the viral membrane protein G as a vaccine remains controversial. In this study, the G extracellular domain (RMG) of HMPV was expressed either alone or fused with the cholera toxin B subunit (CTB) and “cross-reacting material 197” (CRM197) carrier proteins (giving G-CTB/G and CRM197), to enhance immunogenicity. The non-glycosylated G protein (REG) expressed in Escherichia coli served as a control. SDS-PAGE and anti-His tag Western blotting verified that each protein was successfully expressed and correctly identified. BALB/c mice were immunized with each protein and subjected to challenge with HMPV. The results showed that, although immunization with RMG alone failed to induce potent neutralizing antibodies, it modestly reduced viral loads in the lungs of mice. However, the pathological damage caused by lung inflammation was more aggravated than that of the control challenge group. The level of specific IgG antibody induced by the recombinant G-CTB was significantly higher than that elicited by RMG. Compared to the RMG group, the viral load in the lungs of the G-CTB group tended to be reduced. Also, the damage caused by lung inflammation was significantly alleviated. Our study proves that HMPV G may be a valuable antigen in terms of HMPV vaccine development and offers a promising strategy for modulating the immunogenicity and safety thereof. Full article

Cancer progression is closely linked to the enhanced uptake of extracellular amino acids, mediated by specific transporters that support biosynthesis, metabolic activity, and energy production through the tricarboxylic acid cycle. By increasing the expression of these transporters, tumor cells secure a continuous amino acid supply that sustains the proliferation, metabolic balance, and activation of major signaling pathways. While most studies have emphasized post-translational control of amino acid transporters, such as phosphorylation, ubiquitination, glycosylation, and palmitoylation, emerging evidence highlights regulatory crosstalk between these transporters and other membrane proteins, including G protein-coupled receptors and receptor tyrosine kinases. This review summarizes the current literature on the receptor-mediated mechanisms governing amino acid uptake and explores how interactions among families of membrane proteins contribute to the regulation of transporter activity. Full article

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and cognitive decline. Its main pathological features are extracellular plaques composed of aggregated amyloid-β (Aβ) peptides and intracellular neurofibrillary tangles formed by hyperphosphorylated tau. The Aβ hypothesis proposes that Aβ accumulation is a key driver of AD, influencing tau pathology, neuroinflammation, and neurodegeneration. However, therapies that reduce Aβ have shown limited clinical benefits. This suggests that the mechanisms underlying peptide-mediated modulation of AD pathology are much more complex. Both Aβ and tau undergo various post-translational modifications (PTMs) that affect their structure, aggregation, and toxicity. In addition, these abnormal proteins are not efficiently cleared in AD, indicating dysfunction of the protein quality control (PQC) system that maintains proteostasis. Such abnormal PTMs and impaired PQC likely work together to drive disease progression, which may explain the limited success of Aβ-reduction therapies. In this review, we describe how major PTMs, including phosphorylation, ubiquitination, acetylation, glycosylation, and oxidation, regulate the pathological behavior of Aβ and tau. We also discuss the role of the PQC systems in the pathology of AD. We propose that dysregulation of PTMs and PQC constitutes a convergent mechanism underlying AD pathogenesis. Therapeutic strategies targeting these processes may provide more effective and sustained disease modification than approaches focused solely on Aβ reduction. Full article

Silylation of phenyl 1-thio-α-d-mannopyranoside, ethyl 1-thio-α- and β-d-mannopyranosides under different conditions was studied. Low-temperature NMR analysis revealed that the silylated products typically exist as an equilibrium of two chair conformations with a predominance of the axially rich ¹C₄ conformation. The dependence of the ratio of conformers on the anomeric configuration, the type of silyl groups and the nature of the aglycone was established. The fully silylated phenyl 1-thio-α-d-mannopyranoside, ethyl 1-thio-α- and β-d-mannopyranosides were tested as glycosyl donors in the synthesis of di- and trisaccharides, including one-pot synthesis. In all cases, only α-linked oligosaccharides as mixtures of conformers were formed. The obtained deprotected Manp-(1→2)-α-d-Manp-(1→5)-α-d-Araf trisaccharide 2-azidoethyl glycoside, related to the non-reducing terminal fragment of the mannose-capped lipoarabinomannan (ManLAM) of Mycobacterium tuberculosis, can be used for further preparation of conjugates with proteins to provide antigens, which are important for development of new tuberculosis screening assays. Full article

The Svx protein is an established virulence factor in the phytopathogenic pectolytic bacterium Pectobacterium atrosepticum and is secreted into the host plant apoplast. However, its particular role has long remained enigmatic. In our recent studies, we showed that Svx proteins from pectolytic bacteria are metallopeptidases composed of two domains: peptidase and acyltransferase-like domains. Structural organization of the peptidase domain active site led us to hypothesize that its preferred substrates are extensins—hydroxyproline-rich glycoproteins of the plant cell wall. Nevertheless, direct experimental confirmation of extensin hydrolysis by Svx was lacking, and the precise role of the acyltransferase-like domain remained unclear. The present study aimed to address these issues. We showed that Svx indeed cleaves extensins while not degrading some other glycosylated and non-glycosylated proteins. The acyltransferase-like domain was shown to be critical for recognition of arabinan substituents in extensins, thereby providing optimal enzyme–substrate complementarity. Deletion of the acyltransferase-like domain abolished extensin hydrolysis by the truncated variant of Svx. Our study provides the first example of an apoplast-secreted protease from a phytopathogenic bacterium whose specificity toward specific target proteins (extensins) is achieved, at least in part, through structural elements that specifically recognize the distinctive glycosylation pattern of the target proteins. Full article

Environmental pollution from plastics is largely driven by inadequate waste management, particularly in food packaging that relies heavily on petroleum-derived materials. This study utilized gelatin capsule waste (GCW) as a sustainable biopolymer and incorporated yellow peacock flower extract (YPE), obtained via ultrasound-assisted extraction (UAE), at various concentrations (0–2%, w/v) to develop biodegradable films with enhanced functional and antioxidant properties. The main phenolic constituents of YPE were flavonoid aglycones and their glycosylated derivatives. YPE showed total phenolic content of 98.44–129.34 mg GAE/g dry extract, with ABTS, DPPH, and FRAP antioxidant activities ranging from 5.51 to 8.11, 3.17–7.63, and 3.86–5.82 mg TE/g dry extract, respectively. Incorporation of YPE into GCW films significantly improved light barrier properties, thermal stability, mechanical strength, and antioxidant activity, along with a reduction in water vapor permeability and an increase in contact angle, indicating enhanced film hydrophobicity. All films exhibited excellent biodegradability, with complete disintegration within 15 days under soil burial conditions. Films containing 2% YPE (GF4) showed significantly higher thickness, tensile strength, and thermal stability, along with increased opacity, compared with the control (GF0), indicating a reinforcing effect. FTIR analysis revealed the interaction between protein and phenolic compounds from YPE. In a food application model, GF4 film pouches (5 × 5 cm²) effectively delayed oxidative deterioration of dried shrimp during storage at 25 ± 2 °C for 15 days. These findings highlight YPE as a promising bioactive ingredient for biodegradable active packaging and demonstrate the feasibility of GCW as a sustainable biopolymer for eco-friendly films. Full article

Streptococcus agalactiae infections severely threaten global tilapia aquaculture, causing substantial mortality and economic damage. The “Zhuangluo 1” (ZL) strain, derived from the fast-growing GIFT Nile tilapia and refined through multiple generations of selection, uniquely combines robust resistance to S. agalactiae with improved growth traits. This study examined gene expression and regulation of gill mucus microbiota in ZL during experimental S. agalactiae challenge. 16S rRNA sequencing revealed Flavobacterium, Vogesella, Hydrogenophaga, Acidovorax, Rheinheimera, and Deinococcus as prominent genera in the gill mucus microbiome of ZL across time points. Transcriptome time-course analysis identified differentially expressed genes in gills of ZL that were predominantly enriched in cytoskeleton in muscle cells and motor protein pathways. Abundances of the dominant genera Flavobacterium and Hydrogenophaga showed significant correlations with genes regulating mucus secretion, mucin glycosylation, immune modulation, and oxidative stress response in ZL. Untargeted metabolomics of gill mucus revealed substantially higher levels of metabolites potentially linked to microbial metabolism and host–microbiota interactions in ZL. A complementary genome-wide association study for resistance in ZL further localized genes underlying these expression–microbiota associations. These findings elucidated microbiota–host interactions between ZL and gill mucus microbiota, and provide more insights into the role of mucus regulation in disease resistance. Full article

In this study, we investigated the effect of ultrasound-assisted Maillard glycosylation reaction time on the structural, physicochemical, and acid-induced gel properties of Zophobas morio protein–maltodextrin (ZMP–MD) conjugates. Ultrasound treatment up to 45 min (100 kHz, 450 W, 70 °C) significantly accelerated the conjugation efficiency (15.81%) compared to that of wet heating at 70 °C for 6 h (13.62%) (p < 0.05). Fourier transform infrared spectroscopy (FT-IR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses confirmed that both Maillard glycosylation methods formed ZMP–MD conjugates. In addition, the results for secondary structure, surface hydrophobicity, and zeta potential revealed that the ultrasound treatment promoted greater protein structural unfolding, decreasing α-helix while increasing β-sheet and random coil content compared to wet heating. These changes in structural and physicochemical properties of ZMP–MD conjugates impacted the glucono-δ-lactone (GDL)-based acid-induced gel properties. Even though Maillard glycosylation with MD weakened gel properties compared to native ZMP, the gel obtained after 45 min of ultrasound treatment exhibited a higher storage modulus, gel strength, and water-holding capacity than the wet-heated ZMP–MD gel. In conclusion, these findings suggest that properly controlled ultrasound-assisted Maillard glycosylation can modify protein structure, potentially improving its gel properties. Full article

Traditional edible plants such as quelites are an important component of the Mexican diet due to their nutritional and functional value; however, the effects of postharvest and culinary processing on their phytochemical composition remain poorly understood. This study evaluated the impact of oven-drying and freeze-drying, as well as thermal preparation (raw vs. boiled), on the proximal chemical composition, phenolic profile, and antioxidant capacity of leaves and inflorescences of Chenopodium berlandieri subsp. nuttalliae (huauzontle), using an integrated metabolomic approach. Proximal analysis showed that major macronutrients (protein, dietary fiber, lipids, and carbohydrates) were largely preserved across drying methods, whereas moisture and ash contents differed significantly among tissues and treatments (p < 0.05). Raw freeze-dried inflorescences exhibited the highest total phenolic content and antioxidant capacity. UPLC-DAD-ESI-QToF/MS enabled the identification and quantification of 26 phenolic compounds, predominantly glycosylated flavonols derived from quercetin, kaempferol, and isorhamnetin, while naringin was identified as the main flavanone glycoside present. Quercetin glucuronide was the most abundant compound, particularly in inflorescences. Multivariate analyses (principal component analysis [PCA], permutational multivariate analysis of variance [PERMANOVA], and partial least squares discriminant analysis [PLS-DA]) suggested that the drying method was a major source of variability, followed by thermal treatment and tissue type, although these patterns should be interpreted as indicative rather than conclusive. Overall, freeze-drying appeared to be the most effective method for preserving the phytochemical quality of huauzontle under the conditions evaluated, highlighting its potential as a valuable source of bioactive compounds within the genus Chenopodium. Full article

Non-glycosylated liver-derived acute-phase amyloid A1 and A2 proteins (SAA1 and SAA2, 104 amino acids), generated by two different genes in humans (SAA1/2) and other mammalian species, are considered the prime acute-phase reactants following inflammatory conditions during host defense in cells, tissues, and the circulation. While human SAA3 has been identified as a pseudogene, Saa3 genes in other mammalian species are coding for primarily extrahepatically expressed Saa3 proteins that also may act as suitable inflammatory markers. The discovery of SAA4 (112 amino acids, carrying an octapeptide insert) in humans and mice has paved a new avenue for the exploration of different functions of this so far unknown member of the SAA superfamily. SAA4 has originally been termed a “constitutively” expressed SAA protein, apparently due to its nature not to act as an inflammatory marker. The present overview aimed to cover possible functions—so far identified—for human SAA4 (following its expression in various diseases on mRNA and protein level) and to work out whether SAA4 might be considered—at least in part—an acute-phase protein. Alternatively, we are raising the question whether SAA4 may solely act as a bystander or even underdog within the whole SAA family, where SAA1 and SAA2 proteins (commonly termed acute-phase SAA) hold undoubtedly an eminent status during inflammatory conditions, not only as host defense reactants but also as long-lasting markers for chronic diseases and malignancies in humans. Full article