Search Results (4,547)

Ceramide kinase (CerK) catalyzes the phosphorylation of ceramide to ceramide-1-phosphate (C1P), a bioactive sphingolipid with diverse signaling roles. While CerK has been identified in several cellular compartments, its presence and functional significance in kidney proximal tubules remain unexplored. Herein, we report the first characterization of CerK activity in basolateral membranes (BLMs) from porcine proximal tubule cells. We demonstrate that BLM fractions contain neutral and acidic sphingomyelinases, providing local substrate for CerK, which efficiently generates C1P under physiological pH (6.5–7.2) and temperature (30–37 °C) conditions. Enzyme activity was stimulated by cAMP in a protein kinase A-dependent manner but was not affected by angiotensin II. Lipidomic analysis confirmed the presence of C1P in human proximal tubule (HK-2) cells under basal conditions and revealed changes during ischemic stress. Transcriptomic analysis of kidney biopsies from patients with chronic kidney disease (CKD) further uncovered coordinated remodeling of sphingolipid metabolism genes, with increased expression of ceramidases (ASAH1 and NAAA) and downregulation of ceramide synthases (CERS4, CERS5), consistent with adaptive regulation of the Cer/CerK/C1P axis. Together, these findings identify for the very first time CerK activity in renal BLM, establish its biochemical requirements, and highlight its potential role in modulating transporter function and sphingolipid signaling in physiology and kidney disease. Full article

This study presents a novel approach to intensifying the anaerobic digestion of sewage sludge through enzymatic pretreatment using hydrolytic enzymes—cellulase and lysozyme. It aims to determine how enzymatic activation affects the efficiency of methane fermentation, defined as the degree of organic matter decomposition and yield and composition of biogas. An experiment was carried out under mesophilic conditions over 20 days, analyzing the physicochemical properties of sludge, biogas production, methane content, and sanitary parameters. The addition of cellulase and lysozyme significantly enhanced process efficiency, increasing both the rate of organic matter degradation and biogas yield. The highest biogas production values (0.73 L·g⁻¹ d.m. for cellulase and 0.72 L·g⁻¹ d.m. for lysozyme) were obtained at a 4% (w/w) enzyme concentration, with a corresponding increase in the degree of organic matter decomposition to 78.7% and 80.0%, respectively. The produced biogas contained 58–61% methane, exceeding the values observed in the control sample, which indicates a positive effect of enzymatic activation on methane selectivity. Enhanced biogas production was attributed to improved hydrolysis of complex organic compounds, resulting in greater substrate bioavailability for methanogenic microorganisms. Moreover, methane fermentation led to the complete elimination of E. coli from all supernatants, confirming the hygienization potential of the process. The results of this study indicate that enzymatic pretreatment may serve as a viable strategy to improve both the energy efficiency and hygienic safety of anaerobic digestion processes, with relevance for future optimization and full-scale wastewater treatment applications. Full article

The growing consumption of oilseed-pressed cakes (OSCs), a largely underutilized feedstock, plays a significant role in animal feed. The study evaluates the use of three OSCs—flax (FSC), pumpkin (PSC), and hemp (HSC)—as substrates for Bacillus subtilis ATCC 6051a (BS) in a solid-state fermentation (SSF) to enhance enzyme production and probiotic viability. The SSF process was assessed to evaluate the microbial growth, sporulation efficiency, enzymatic activity (protease, cellulase, xylanase, and phytase), and in vitro digestibility of fermented substrates. The results indicate that bacterial growth and sporulation varied significantly among substrates (p < 0.05). FSC presents the highest spore resistance (86.52%), followed by PSC (82.87%) and HSC (81.29%). Notably, protease was highest in HSC (184.67 U/g), while FSC supported maximum cellulase activity. HSC exhibited superior xylanase (1.86 ± 0.043 U/g DW, p < 0.05) and phytase production, while pH analysis indicated a shift toward alkalinity in PSC and HSC due to proteolytic activity. FSC maintained the most stable bacterial population during digestion, suggesting its potential as a probiotic carrier. These findings highlight that fermentation of OSCs with BS improved their nutritional value and can be used as a sustainable solution in feeding programs for piglets. Full article

Genetic information, whether inside or outside the nucleus, is exposed to a variety of harmful physico-chemical factors. Although DNA damage repair systems have been extensively studied, little information about post-repair and non-genomic DNA damage metabolism is available in the literature. Adenosine deaminase (ADA) is an abundant enzyme found on both sides of the cell membrane that regulates the concentration of adenine derivatives. In this article, it has been shown that 7,8-dihydro-8-oxo-2′-deoxyadenosine (^OXOdAdo) and (5′R/S) 5′,8-cyclo-2′-deoxyadenosine ((5′R/S)cdAdo) are suitable substrates for ADA. For this purpose, theoretical Density Functional Tight Binding and RP-HPLC analyses were applied. The products of ADA activity, i.e., ^OXOdIno (7,8-dihydro-8-oxo-2′-deoxyinosine) and (5′R/S) cdIno ((5′R/S) 8-cyclo-2′-deoxyinosine), were identified and confirmed by high-resolution mass spectroscopy. Although the (5′R) and (5′S)cdAdo enzymatic deamination processes are much slower (34% and 32% after 168 h, respectively) than the process observed for dAdo, 5′,8-cyclo-2′-deoxyinosine should be considered when monitoring cyclopurine levels in physiological fluids. The same should be considered in the case of ^OXOdAdo, which is completely converted to ^OXOdIno within one minute and may therefore be less visible than ^OXOdGuo during mass spectroscopy analysis. Both these observations are important, given the abundance of 2′-deoxyadenosine on both sides of the cell membrane and its potential conversion into ^OXOdAdo and (5′R/S)cdAdo. They may also explain why the observed level of ^OXOdAdo is much lower than that of ^OXOdGuo in cells and physiological fluids, even though their difference in ionisation potential is only 0.25 eV. Future studies are needed to further investigate the metabolism of DNA damage and to identify the enzymes involved in nucleic acid biochemistry. Full article

Post-translational modifications (PTMs) expand the structural diversity of proteins beyond the standard amino acids, influencing protein-protein interactions. Protein methylation, a prevalent PTM, involves the transfer of methyl groups from S-adenosylmethionine (SAM) to lysine and arginine residues. Arginine methylation is catalyzed by the Protein Arginine Methyltransferase (PRMT) family to yield mono- and dimethylarginine forms. PRMT1, the isozyme responsible for the majority of asymmetric dimethylation (ADMA) is implicated in various diseases, including cancer. Here, we report the synthesis and screening of a second-generation peptide library to identify novel PRMT1 substrates. The library, based on histone peptides, incorporated varying sequences of amino acids, facilitating substrate specificity studies. Screening identified 7 peptide sequences as exceptional PRMT1 substrates, which were confirmed by kinetic analysis. Consensus sequences revealed key recognition elements for PRMT1 catalysis, suggesting roles for small non-polar side chains and specific residues near the substrate arginine. Furthermore, we developed a peptide-based PRMT1 inhibitor by substituting the substrate arginine with a chloroacetamidine warhead. The inhibitor exhibited sub-micromolar inhibitory potency against PRMT1, surpassing previous peptide-based inhibitors. Our findings contribute to understanding PRMT1 substrate specificity and provide a scaffold for developing potent inhibitors targeting PRMT1 in diseases, including cancer. Full article

An intracellular α-glucosidase was isolated and purified from a Pseudanabaena sp. cyanobacterial strain. Before the enzyme purification, the optimal cultural conditions were determined. Optimal culture conditions (15 g/L maltose, 2 g/L yeast extract, 23 ± 1 °C) yielded 3.3 g/L of biomass and 2186 U/L of α-glucosidase in a lab-scale bioreactor. The purified enzyme displayed a molecular mass of 52 kDa with optimum activity at 40 °C and pH 7.0, and maintained stability within an acidic and neutral range of pH 4.0 to 7.0. Enzyme activity was affected by both the concentration and interaction time of the metal ions and chelator. Kinetic constants of K_m, V_max, and k_cat for the hydrolysis of pNPG were determined as 2.0 Mm, 2.9 μmol min⁻¹, and 14.86 min⁻¹, respectively. The activation energy (E_a) was 24.2 kJ mol⁻¹ and the thermodynamic parameters of enthalpy (ΔH^*), entropy (ΔS^*) of activation, Gibbs free energy (ΔG^*), free energy of substrate binding (ΔG^*_E-S), and transition state formation (ΔG^*_Ε-Τ) were 21.6, −116, 57.8, −22.2, and −41.2 kJ mol⁻¹, respectively. Moreover, the thermodynamic parameters for thermal inactivation of the enzyme were ΔH^*= 131 kJ mol⁻¹, 105 ≤ ΔS^* ≤ 108 kJ mol⁻¹, and 96 ≤ ΔG^* ≤ 98 kJ mol⁻¹, while the thermal inactivation energy (E_(a)d) was determined to be 133 kJ mol⁻¹. This is the first detailed investigation concerning the characterization of α-glucosidase derived from cyanobacteria. The presented enzymatic characteristics provide a valuable predictive model for identifying suitable applications. Full article

Biliary tract carcinoma (BTC) is a group of highly heterogeneous malignancies arising from the biliary epithelium. Anatomically, BTC is categorized into gallbladder cancer (GBC) and cholangiocarcinoma (CCA), with the latter further subdivided into intrahepatic (iCCA), perihilar (pCCA), and distal cholangiocarcinoma (dCCA). Epidemiological studies reveal a dismal five-year survival rate of less than 20% for BTC patients, with limited responses to current chemotherapy regimens, underscoring the urgent need to unravel its complex molecular pathogenesis. Recent research has increasingly focused on the regulatory networks of post-translational modifications, particularly the ubiquitin-proteasome system (UPS), in tumorigenesis. As the largest subfamily of deubiquitinating enzymes (DUBs), ubiquitin-specific proteases (USPs) regulate the stability of key oncoproteins such as phosphatase and tensin homolog (PTEN) and c-Myc, playing pivotal roles in tumor cell proliferation, apoptosis evasion, invasion, and metastasis. This review systematically summarizes the differential expression profiles of USP family members (e.g., USP1, USP3, USP7, USP8, USP9X, USP21, and USP22) in BTC and their clinical significance, with a focus on elucidating how specific USPs regulate tumor progression through key substrates, including poly(ADP-ribose) polymerase 1 (PARP1), dynamin-1-like protein (DNM1L), and O-GlcNAc transferase (OGT). Furthermore, based on recent advances, we discuss the therapeutic potential of small-molecule USP inhibitors in BTC targeted therapy, providing a theoretical foundation for developing novel precision treatment strategies. Full article

Tiliacora triandra (Yanang) leaf contains polyphenols, flavonoids, and mucilage polysaccharides with antioxidant and prebiotic functions, making it a promising substrate for probiotic fermentation. This study aimed to optimize Yanang extraction and sterilization to preserve bioactive mucilage and support probiotic survivability during freeze-drying–based encapsulation, and evaluate antimicrobial activity against poultry pathogens. Yanang extract was prepared under different leaf processing conditions and used as a substrate for Pediococcus acidilactici V202, Lactiplantibacillus plantarum TISTR 926, Streptococcus thermophilus TISTR 894, Bacillus subtilis RP4-18, and Bacillus licheniformis 46-2. Fermentation at 37 °C for 24 h revealed that lactic acid bacteria (P. acidilactici V202, L. plantarum TISTR 926, S. thermophilus TISTR 894) reduced pH (<4.10, p < 0.001) while maintaining high viable counts (>8.67 log CFU/mL, p < 0.01), whereas Bacillus strains (B. subtilis RP4-18, B. licheniformis 46-2) retained a higher pH (>5.00) and lower viability (<8.50 log CFU/mL). Total soluble solids decreased across treatments, with the lowest observed for B. subtilis RP4-18 (1.97 °Brix, p = 0.007). Freeze-dried probiotics encapsulated in enzyme-extracted rice bran carriers had comparable physicochemical properties (p > 0.05), while compared with Bacillus strains (p < 0.01), lactic acid bacteria had superior tolerance to simulated gastrointestinal and thermal stress. Supernatant from Yanang extract inhibited B. cereus WU22001, S. aureus ATCC25923, Escherichia coli ATCC25922, and Salmonella typhimurium WU241001 (MIC/MBC 25–50% v/v). These results indicate that Yanang extract supports effective probiotic fermentation, and rice bran encapsulation enhances survivability and antimicrobial functionality for potential functional feed applications. Full article

Background/Objective: Oncogenic tyrosine kinases (TKs) such as ALK and SRC promote cancer progression, but their effects on metabolic enzymes are still not well understood. This study examines how TK signaling regulates inosine monophosphate dehydrogenase 2 (IMPDH2), a rate-limiting enzyme in purine biosynthesis, and assesses its potential as a therapeutic target. Methods: Phosphoproteomic screening and in vitro kinase assays were used to identify phosphorylation sites on IMPDH2. Lipid-binding assays explored the role of phosphatidylinositol 3-phosphate (PI3P) in IMPDH2 regulation. Structure-based virtual screening discovered small-molecule allosteric inhibitors, which were tested in lymphoma cell models, including ALK and BTK-inhibitor resistant lines. Results: Here, we identify Inosine monophosphate dehydrogenase-2 (IMPDH2), a rate-limiting enzyme in purine biosynthesis, as a novel substrate of ALK and SRC. We show that phosphorylation at the conserved Y233 residue within the allosteric domain enhances IMPDH2 activity, linking TK signaling to metabolic reprogramming in cancer cells. We further identify PI3P as a natural lipid inhibitor that binds IMPDH2 and suppresses its enzymatic function. Using structure-based virtual screening, we developed Comp-10, a first-in-class allosteric IMPDH inhibitor. Unlike classical active-site inhibitors such as mycophenolic acid (MPA), Comp-10 decreases IMPDH1/2 protein levels, blocks filament (rod/ring) formation, and inhibits the growth of ALK and BTK inhibitor-resistant lymphoma cells. Comp-10 acts post-transcriptionally and avoids compensatory IMPDH upregulation observed with MPA (rod/ring) formation, and inhibited growth in TKI-resistant lymphoma cells. Notably, Comp-10 avoided the compensatory IMPDH upregulation observed with MPA. Conclusion: These findings uncover a novel TK–IMPDH2 signaling axis and provide mechanistic and therapeutic insight into the allosteric regulation of IMPDH2. Comp-10 represents a promising therapeutic candidate for targeting metabolic vulnerabilities in tyrosine kinase driven cancers. Full article

The SARS-CoV-2 spike protein, through its receptor binding domain (S1-RBD), binds to the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell membrane, leading to viral infection. Several mutations in S1-RBD in SARS-CoV-2 variants are known to enhance infection through an increased affinity for ACE2. While many reports are available describing the SARS-CoV-2 infection mechanism, there is a dearth of studies towards understanding the initial interaction of the S1-RBD with ACE2 on living host cells and the role of endocytosis and cytoskeleton in the process. Here, we reconstituted the interaction between S1-RBD- and ACE2-expressing host cells in a hybrid live cell-supported lipid bilayer (SLB) platform enabling live monitoring of the interaction between S1-RBD on SLBs and the ACE2 receptor on living cells and showed that cells depleted Omicron S1-RBD from SLB corrals, likely through endocytosis. Specifically, interaction of living host cells with S1-RBD-functionalized SLB substrates resulted in the enrichment of S1-RBD and ACE2 at the cell–SLB interface. Interaction of host cells with wild type (WT), Omicron, and Omicron Revertant S1-RBD functionalized on micron-scale SLB corrals, which mimic viral membranes but are flat, also resulted in their enrichment. However, cells interacting with Omicron S1-RBD revealed a depletion of the protein from many corrals, which was generally not observed with the WT S1-RBD and was reduced with the Omicron Revertant, which contains the Q493R mutation reversion, S1-RBD. Further, S1-RBD depletion coincided with the localization of the early endosomal marker EEA1. Importantly, treatment of cells with the clathrin inhibitor, pitstop 2, but not the myosin II inhibitor, blebbistatin, significantly reduced Omicron S1-RBD depletion. Collectively, these observations suggest that the SARS-CoV-2 Omicron variant has evolved, through mutations in its S1-RBD, to take advantage of the cellular endocytic pathway for enhanced infection, which is not observed with the parental SARS-CoV-2 and appears to be lost in the Omicron Revertant variant. Additionally, these results underscore the significance of the hybrid live cell–SLB platform in studying SARS-CoV-2 S1-RBD-ACE2 interaction and the potential impact of mutations in the S1-RBD on adapting to a specific cellular entry mechanism. Full article

Cytochrome P450 enzymes (CYPs) are versatile biocatalysts capable of performing selective oxidation reactions valuable for industrial and pharmaceutical applications. However, their catalytic efficiency is often constrained by dependence on costly electron donors, the requirement for redox partners, and uncoupling reactions that divert reducing power toward reactive oxygen species. Improving electron transfer efficiency through optimized redox partner interactions is therefore critical for developing effective CYP-based biocatalysts. In this study, we investigated the interaction between CYP119, a thermophilic CYP from Sulfolobus acidocaldarius, and putidaredoxin (Pdx), the redox partner of P450cam. Using rational design and computational modeling with PyRosetta 3, 14 CYP119 variants were modeled and analyzed by docking simulations on the Rosie Docking Server. Structural analysis identified three key mutations (N34E, D77R, and N34E/D77R) for site-directed mutagenesis. These mutations (N34E, D77R, and N34E/D77R) enhanced Pdx binding affinity by 20-, 3-, and 12-fold, respectively, without affecting substrate binding. Catalytic assays using lauric acid and indirect assays to monitor electron transfer revealed that, despite improved complex formation, the N34E variant showed reduced electron transfer efficiency compared to D77R. These findings highlight the delicate balance between redox partner binding affinity and catalytic turnover, emphasizing that fine-tuning electron transfer interfaces are essential for engineering efficient CYP biocatalysts. Full article

Patchoulene, the characteristic sesquiterpene of patchouli essential oil, is highly valued in the perfume industry for its distinctive woody note and fixative properties. Beyond its olfactory applications, patchoulene has demonstrated promising biological activities, including anti-inflammatory, antimicrobial, and neuroprotective effects. Current production relies mainly on extraction from Pogostemon cablin plants, which requires long growth cycles (≥8 months), exhibits low yields, and imposes significant environmental constraints. To overcome these limitations, this study aimed to enhance the Whole-cell yield of patchoulene synthase (PcPTS) through structure-informed protein engineering. A semi-rational design approach was employed, combining homology modeling, molecular docking, evolutionary analysis, and molecular dynamics simulations to identify functional residues within the enzyme active site. Ala-scanning mutagenesis highlighted Thr532 as essential for catalytic activity, and coevolutionary analysis indicated synergistic effects between Phe456 and Thr532. Site-directed mutagenesis was conducted to generate single (F456M, T532Y) and double (F456M/T532Y, designated M2) mutants. The double mutant M2 showed a 3.62-fold increase in patchoulene production compared to the wild-type enzyme. In silico analyses suggested that the enhanced performance of M2 originates from improved substrate positioning, reduced structural flexibility, and strengthened molecular interactions, collectively contributing to a lower energy barrier for catalysis. This study provides an effective strategy for the rapid optimization of terpenoid synthases and facilitates the development of microbial cell factories for sustainable and high-yield production of plant-derived terpenoids. Full article

Hypersensitized P2X3 receptor signaling has been described to play a role in several disorders, including chronic cough. The goal of our in vitro and in vivo studies was to investigate the biotransformation and the influence of CYP3A4 inhibition on the pharmacokinetics of the selective P2X3 antagonist filapixant. Metabolic turnover of filapixant in human liver microsomes and hepatocytes was moderate to high, indicating a complex metabolic pattern with mainly oxidative biotransformation. In recombinant CYP enzymes, depletion of filapixant was observed mainly with CYP3A4 and, to a significantly lesser extent, with CYP1A1, 2D6, 2J2, and 3A5. Drug depletion of [³H]filapixant and metabolite formation in human liver microsomes was significantly inhibited in the presence of strong CYP3A4 inhibitors, whereas other CYP isoform–selective inhibitors showed no or very minor effects. Co-administration of multiple daily doses of 200 mg itraconazole with 80 mg filapixant in humans increased the AUC and C_max of filapixant to 4.01 and 1.89-fold, respectively, indicating that filapixant is a moderately sensitive CYP3A4 substrate. Co-administration of itraconazole also prolonged the half-life of filapixant from 12.1 h to 22.8 h. Overall, changes in AUC, C_max, and half-life indicate that both the bioavailability and elimination of filapixant were affected. Filapixant was well tolerated alone and in combination with itraconazole. Full article

The antimalarial combination of sulfadoxine–pyrimethamine is used as a preventive treatment in pregnant women and children in Africa. Sulfadoxine inhibits the Plasmodium falciparum dihydropteroate synthase (PfDHPS), but resistance has emerged through point mutations in this enzyme. In this study, we investigate the impact of mutations on the structural and dynamic properties of PfDHPS using molecular dynamics simulations. Our results show that PfDHPS maintains overall structural integrity across various combinations of resistance-associated mutations. However, significant differences emerge in ligand binding. Sulfadoxine binding is particularly impacted and shows reduced stability in the mutant systems compared to the wild-type enzyme, while the natural substrate generally maintains stable or even enhanced binding affinity. A key finding is the critical role of the D2 loop, whose conformational flexibility influences ligand retention. In mutant enzymes, the disruption of interactions between the D2 loop and the natural substrate correlates with decreased affinity. In contrast, specific mutations in the loop are associated with an increased affinity. Conversely, sulfadoxine binding is associated with an open D2 loop conformation, facilitating its release from the active site. Finally, the intrinsic flexibility of sulfadoxine emerges as an important determinant of this process. Together, these results provide molecular-level insights into the mechanisms of resistance in PfDHPS and establish a structural and dynamic framework for future investigations into its catalytic function and inhibitor design. Full article

Background: L-arginine is a conditionally essential amino acid that serves as a substrate for nitric oxide synthase and regulates energy metabolism. While its ergogenic effects have been proposed, the mechanisms underlying its anti-fatigue properties are not fully understood. Methods: Male ICR mice were orally administered L-arginine (300, 600, or 1200 mg/kg bw/day) for 28 days. Fatigue was chronically induced using twice-weekly forced swimming or treadmill running, and fatigue resistance was then assessed under these paradigms. Blood, skeletal muscle, and liver were analyzed for biomarkers including glucose, lactate, LDH, CPK, NEFA, ammonia, glycogen, nitric oxide, cortisol, and antioxidant enzymes. In parallel, C2C12 myoblasts were treated with L-arginine under proliferative and differentiated conditions to assess hexokinase (HK) activity, myogenin expression, and ROS generation. Results: In vivo, L-arginine decreased serum LDH, CPK, NEFA, ammonia, nitric oxide, and cortisol, while enhancing blood glucose and glycogen storage in both muscle and liver. Forced swimming reduced serum lactate, whereas treadmill exercise elevated intramuscular lactate, suggesting context-dependent lactate regulation. Importantly, L-arginine did not significantly improve forced-swimming immobility time, whereas treadmill time-to-exhaustion increased at the highest dose. Antioxidant responses were improved, as reflected by normalized hepatic catalase activity. In vitro, L-arginine increased HK activity, promoted myogenin expression, and reduced ROS levels, supporting improved glucose utilization, muscle differentiation, and oxidative stress resistance. Conclusions: These findings demonstrate that L-arginine supplementation under chronic fatigue-inducing paradigms improves endurance and alleviates fatigue by enhancing energy metabolism, preserving glycogen, reducing muscle injury, and attenuating oxidative stress. L-arginine shows potential as a functional ingredient for promoting exercise performance and recovery. Full article