Post-traumatic stress disorder (PTSD) is a refractory psychiatric disorder with limited effective treatments. Emerging evidence indicates that abnormal microglial activation and pyroptosis in the hippocampus trigger persistent neuroinflammation, neuronal synaptic damage and progressive behavioral disorders. MicroRNA-124 (miR-124) is a brain-enriched miRNA with anti-inflammatory and neuroprotective functions. In the present study, we identified P2X7R as a direct target of miR-124 via bioinformatics prediction and dual-luciferase reporter assay. Using an mSPS-established rat PTSD model, we found that hippocampal miR-124 was markedly decreased in model animals, accompanied by upregulated P2X7R, enhanced NLRP3 inflammasome activation and excessive microglial pyroptosis. Lentivirus-mediated miR-124 overexpression downregulated P2X7R, reduced the levels of pyroptosis-related proteins and pro-inflammatory cytokines, and effectively rescued multiple PTSD-like behavioral phenotypes. Pharmacological blockade of P2X7R with A438079 recapitulated the protective effects of miR-124. Further in vitro co-culture experiments demonstrated that P2X7R-mediated microglial pyroptosis impaired neuronal synaptic function, while suppressing P2X7R restored the expression of synaptic proteins. This study demonstrates that miR-124 modulates microglial pyroptosis and hippocampal neuroinflammation through directly targeting P2X7R, thereby improving neuronal synaptic function and alleviating PTSD symptoms. Our findings provide novel mechanistic insights into PTSD pathogenesis and offer potential molecular targets for clinical intervention.
BACKGROUND AND OBJECTIVE:Knee osteoarthritis (KOA) is a degenerative joint disease characterized by progressive cartilage degradation, synovial inflammation, and subchondral bone remodeling. This study aimed to investigate the role of CSNK2A2 in KOA pathogenesis and its regulatory effects on the Wnt/β-catenin and nuclear factor (NF)-κB pathways. METHODS:Differentially expressed genes were identified from the GSE117999 dataset. A monosodium iodoacetate (MIA)-induced rat KOA model was established. Synovial tissues were collected for histological, micro-CT, and molecular analyses. Rat synovial cells (RSCs) were stimulated with interleukin (IL)-1β to mimic an in vitro KOA model. CSNK2A2 was knocked down using shRNA, and the β-catenin pathway was activated with C91. Synovial tissues from KOA patients and healthy controls were also collected to validate CSNK2A2 expression. Gene and protein expression levels were assessed by reverse transcription-quantitative polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, immunofluorescence, and immunohistochemistry. Joint damage was scored using the OARSI system. RESULTS:CSNK2A2 was upregulated in both in vivo and in vitro KOA models and in the synovial tissues of KOA patients. Knockdown of CSNK2A2 attenuated cartilage destruction, reduced pro-inflammatory cytokine production, preserved proteoglycan content, and improved subchondral bone microstructure. In RSCs, CSNK2A2 silencing suppressed inflammation, extracellular matrix degradation, and apoptosis induced by IL-1β. Mechanistically, CSNK2A2 knockdown increased GSK3β and decreased β-catenin and p-p65 expression, indicating inhibition of both Wnt/β-catenin and NF-κB signaling, and immunofluorescence confirmed that CSNK2A2 knockdown reduced p65 nuclear translocation, which was restored by C91. Conversely, activation of β-catenin with C91 reversed the protective effects of CSNK2A2 knockdown in both cellular and animal models. CONCLUSION:CSNK2A2 promoted KOA progression by activating the Wnt/β-catenin and NF-κB pathways, thereby driving inflammation, matrix degradation, and chondrocyte apoptosis.
BACKGROUND:Vault RNAs (vtRNAs), a group of small non-coding RNAs, are recognized to regulate host immune responses, mainly through the NF-κB/PKR signaling pathway. Viral recruitment of vtRNAs has been described in numerous infections, but their function in COVID-19 is still unclear. OBJECTIVE:To examine the expression profiles of vtRNAs (vtRNA1-1, vtRNA1-2, vtRNA1-3, and vtRNA2-1) in individuals with severe COVID-19 and evaluate their potential clinical implications. METHODS:Peripheral blood mononuclear cells were isolated from 50 patients diagnosed with severe COVID-19 and 50 matched healthy controls. Expression levels of vtRNAs were quantified using real-time PCR, normalized to ACTB, and analyzed through non-parametric statistical analyses. Associations with demographic and clinical features were calculated. Diagnostic performance was evaluated using ROC curve analysis. RESULTS:Expression of vtRNA1-2, vtRNA1-3, and vtRNA2-1 was significantly upregulated in COVID-19 patients compared with controls (p < 0.05). vtRNA1-1 expression showed no meaningful difference. Notably, vtRNA2-1 expression correlated with specific blood groups. ROC curve analysis showed statistically significant but modest discriminatory performance for vtRNA1-2 (AUC = 0.615), vtRNA1-3 (AUC = 0.622), and vtRNA2-1 (AUC = 0.673), indicating limited diagnostic utility when considered as individual markers. CONCLUSION:This study provides preliminary evidence that vtRNAs are dysregulated in PBMCs from patients with severe COVID-19. The observed upregulation of vtRNA1-2, vtRNA1-3, and vtRNA2-1 suggests that these non-coding RNAs may be involved in host molecular responses associated with severe SARS-CoV-2 infection. However, their diagnostic and mechanistic relevance requires validation in larger cohorts, including patients with different disease severities and appropriate disease-control groups.
Bioactive vitamin D3, 1α,25-(OH)2 D3, is responsible for an array of homeostatic processes, including bone remodeling and calcium and phosphate maintenance. Despite its importance in human health, very little is known regarding vitamin D3 recognition by its metabolic cytochrome P450 (CYP) enzymes. Herein, we utilize absorbance and NMR spectroscopies combined with molecular modeling, docking, and reconstituted functional assays to characterize a member of the CYP107 family (CYP107-sb3a) from the rare soil actinomycete Sebekia benihana, which reportedly functions as a 25 and 1α vitamin D3 hydroxylase. We report that the purified enzyme is a C25-hydroxylase, with no 1α-hydroxylation detected in vitro. However, CYP107-sb3a has a broad substrate specificity and binds to various vitamin D3 analogs and intermediates, vitamin D2, and variable length fatty acids. This study also reveals two key sites that are involved in recognition of vitamin D3: Q79 on the B’ helix, and S383 on the C-terminal loop. Functional data indicates that these residues play a role in substrate recognition as well as in the final bound orientation, with their mutagenesis to alanine resulting in enhanced C25 hydroxylase activity. These findings are discussed regarding their insight toward understanding vitamin D3 recognition in mammalian CYP enzymes.
Streptococcus anginosus, a gram-positive anaerobe found in the oral cavity and gastrointestinal tract, causes serious purulent abscesses in various tissues and is also associated with the occurrence of esophageal and gastric tumors. Mechanisms underlying the highly evolved acid tolerance are vital for bacterial growth and survival under acidic environments in the upper gastrointestinal tract or dental plaque. We previously observed that proton-pumping F-type ATPase (F-ATPase) of S. mutans plays a vital role in acid tolerance. Unlike S. mutans, S. anginosus possesses A-type ATPase (A-ATPase) in addition to F-ATPase. In other organisms, A-ATPases function as either proton- or sodium-translocating enzymes. In the present study, we investigated which ATPases are involved in the acid tolerance mechanism of S. anginosus and found that both ATPases were expressed in S. anginosus cells, with an increased expression level of F-ATPase under acidic conditions. Knockout cells of the F-ATPase catalytic β subunit gene demonstrated no growth at pH 5.30, whereas growth was detected at pH 7.40. Moreover, the colony-forming ability of the β-knockout cells significantly decreased at pH < 4.70 compared with that of wild-type cells, but it was similar at pH 7.40. These defects of the β-knockout cells in growth and survival under acidic conditions were recovered in the knockout cells expressing the β subunit. Conversely, knockout cells of the A-ATPase catalytic B subunit gene exhibited similar growth and survival under acidic conditions as those of wild-type cells. These results demonstrate that F-ATPase but not A-ATPase is essential for the acid tolerance mechanism of S. anginosus.
BACKGROUND:Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by epidermal barrier dysfunction and persistent pruritus. However, the molecular mechanisms linking non-coding RNA regulation to ferroptosis and inflammatory signaling in AD remain poorly understood. METHODS:A 2,4-dinitrochlorobenzene (DNCB)-induced mouse model of AD and a TNF-α + IFN-γ-induced inflammatory keratinocyte model were established. Gain- and loss-of-function experiments were performed using transfection approaches. Molecular interactions were validated by dual-luciferase reporter, co-immunoprecipitation, and deubiquitination assays. Inflammatory responses and ferroptosis were evaluated by Western blotting, ELISA, and biochemical analyses. RESULTS:The lncRNA 051252/miR-141-5p/USP14 regulatory axis was significantly dysregulated in AD-like skin lesions and inflammatory keratinocytes. Silencing lncRNA 051252 or overexpressing miR-141-5p markedly attenuated inflammatory responses and ferroptosis both in vivo and in vitro. Mechanistically, lncRNA 051252 functioned as a competing endogenous RNA by sponging miR-141-5p, thereby relieving its inhibitory effect on USP14. miR-141-5p directly targeted USP14, whereas USP14 stabilized cGAS by removing K48-linked ubiquitin chains, resulting in activation of the STING signaling pathway and promotion of ferroptosis. Furthermore, pharmacological inhibition of cGAS-STING signaling recapitulated the protective effects of lncRNA 051252 silencing and miR-141-5p overexpression. CONCLUSIONS:These findings identify a previously unrecognized lncRNA 051252/miR-141-5p/USP14/cGAS-STING signaling axis that drives inflammatory responses and ferroptosis in AD, providing new mechanistic insights into disease pathogenesis and highlighting potential therapeutic targets.
Ferroptosis is closely associated with the pathogenesis of osteoarthritis (OA), though the underlying molecular mechanisms remain unclear. This study aims at exploring the role of CDO1 in chondrocyte ferroptosis. In this study, cell viability was assessed using the CCK8 assay. ROS levels were measured via flow cytometry. Lipid peroxidation was evaluated using the C11 BODIPY 581/591 fluorescent probe, and mitochondrial morphology was examined by transmission electron microscopy. Our results demonstrated that CDO1 was upregulated in cartilage tissues from patients with severe knee OA. IL-1β stimulation elevated ferroptosis levels in SW1353 cells, as indicated by reduced cell viability, extracellular matrix (ECM) accumulation and GSH levels, increased oxidative stress, lipid peroxidation and MDA content, and impaired mitochondrial morphology. These effects were reversed by the ferroptosis inhibitor Fer-1. Overexpression of CDO1 reduced intracellular cysteine levels and enhanced ferroptosis, whereas CDO1 knockdown produced the opposite effects. We further identified VHL as a binding partner of CDO1 and showed that VHL promoted CDO1 protein degradation. The ferroptosis and ECM degradation induced by CDO1 overexpression were rescued by VHL co-overexpression. Importantly, cysteine deficiency enhanced VHL activity, promoted CDO1-VHL interaction, and accelerated CDO1 degradation, thereby may form a VHL/CDO1/cysteine negative feedback loop. The function of CDO1/VHL axis was further validated in a rat OA model, where CDO1 exacerbated OA progression and VHL alleviated it. In summary, our findings reveal that the VHL/CDO1 axis regulates intracellular cysteine levels, mediates chondrocyte ferroptosis and OA progression.
Copper nitrite reductases catalyse the reduction of nitrite to nitric oxide in the periplasm of Gram-negative bacteria as the second step of the denitrification pathway. Although these enzymes have been extensively studied, limited data are available regarding their thermal stability. The thermal unfolding mechanism of the copper nitrite reductase from the pathogenic bacterium Neisseria gonorrhoeae and the contribution of its copper centres to thermostability were investigated using differential scanning calorimetry and spectroscopic techniques. The results show that enzyme unfolding is irreversible and kinetically controlled and is best described by a three-state Lumry-Eyring model involving a reversible intermediate followed by an irreversible transition to an aggregated stated. Visible spectroscopy revealed that type-1 copper centre exhibits remarkable local stability, remaining spectroscopically intact up to 80 °C and undergoing irreversible disruption only at higher temperatures. In contrast, removal of the copper centres does not significantly alter the secondary structure, but reduces thermal stability by 30 °C, highlighting their critical role in stabilizing the protein. Together, these findings provided new insight into the thermal unfolding mechanism of a copper nitrite reductase from a pathogen and demonstrate the important contribution of the metal cofactors to the protein exceptional thermostability. These features likely contribute to enzyme robustness under the dynamic conditions encountered during host-pathogen interactions.
The ketogenic diet is increasingly used for metabolic and neurologic indications, yet its impact on hepatic mitochondrial function and xenobiotic metabolism remains incompletely defined. Cytochrome P450 2E1 (CYP2E1) is induced by the ketone body acetone and contributes to oxidative and carbonyl stress, but prior studies examining CYP2E1 regulation during ketosis have yielded conflicting results. Here, we investigated the effects of an 8-week medium chain triglyceride ketogenic diet (MCT-KD) on liver mitochondrial respiratory chain activity and CYP2E1 expression in young and aged Fisher 344 × Brown Norway F1 rats. In young animals, MCT-KD significantly reduced mitochondrial complex I activity without significant changes in complexes II, III, or IV. These changes occurred without altered citrate synthase activity, suggesting comparable mitochondrial content. In parallel, MCT-KD robustly increased hepatic CYP2E1 protein levels and activity in young and aged animals and upregulated its electron donor, P450 oxidoreductase (POR), particularly in young rats. Despite robust induction of the acetone-CYP2E1 pathway, methylglyoxal-derived protein adducts did not accumulate, even though hepatic GLO1 expression was reduced. Together, these findings demonstrate that long-term MCT-KD induces coordinated adaptations in hepatic mitochondrial function and the CYP2E1-POR pathway without increasing methylglyoxal-derived protein damage.
The mitochondrial ATP synthase catalyzes the formation of ATP from ADP and Pi. In the colorless alga Polytomella parva, this enzyme displays an atypical composition in the subunits that build the peripheral arm and in the ones involved in its dimerization. In addition to ten extra subunits (Asa1-10) apparently absent from other ATP synthases, the catalytic α and β subunits possess amino-acid extensions in their N- and C-terminal regions, respectively. The δ subunit-homologous to the bacterial ε subunit and responsible for linking the hydrophilic and hydrophobic sectors of the enzyme-also contains an atypical N-terminal extension. The ATP synthase of P. parva appears to lack an IF1 peptide, the natural inhibitor of ATP hydrolysis. Due to the potential closeness of the δ subunit N-terminal extension to the DELSEED region of the catalytical core of the enzyme, we hypothesized that the δ subunit could regulate the hydrolytic activity of the algal ATP synthase and evaluated this possibility experimentally. We also investigated whether the S. cerevisiae IF1 polypeptide could exert a cross-species inhibitory effect on the algal enzyme. Biochemical results, complexome profiling analysis, and 3D-structural data indicate the absence of a peptide with inhibitory capabilities in the algal ATP synthase and suggest that the enzyme hydrolytic activity may be regulated by ADP levels.
The development of isoform-selective inhibitors for homologous cysteine proteases remains challenging due to structural similarity and overlapping specificities. Metabolites isolated from Tabebuia aurea were investigated as modulators of Cathepsins B and L. Bioassay-guided fractionation yielded three compounds: 2,3-DH-5-HMB (1), veratric acid (2), and iridoid specioside (3). Enzymatic assays identified veratric acid as the most active compound (IC50 = 4.23 ± 0.17 μM for Cathepsin B and 1.28 ± 0.03 μM for Cathepsin L). Steady-state kinetic analyses revealed marked mechanistic divergence between isoforms. Against Cathepsin B, compounds 1 and 2 acted as competitive inhibitors with strong positive cooperativity, quantitatively captured by the cooperativity factor β (0.00041 and 0.0129, respectively), corresponding to 2400-fold and 78-fold increases in affinity upon binding of a second inhibitor molecule. For compound 2, where extreme cooperativity precluded independent estimation of microscopic parameters, the quadratic cooperativity parameter γ (0.273 μM-2) enabled robust quantification of the cooperative effect. In contrast, all three compounds inhibited Cathepsin L via a simple linear non-competitive mechanism (α ≈ 1), reflecting its more accessible active-site architecture. Cellular assays confirmed selective antiproliferative activity of veratric acid in K562 leukemia cells (EC50 = 12.47 μM) while sparing non-tumor HaCaT cells. Molecular docking and molecular dynamics simulations of 1:1 complexes provided structural context for the observed isoform-specific binding modes. These results demonstrate that structurally simple phenolic compounds can induce fundamentally different inhibition mechanisms in closely related enzymes, establishing a structure-mechanism framework for isoform-selective modulation of cysteine proteases.
1-Naphthol-2-hydroxylase (1NH, group-A flavoprotein monooxygenase) from Carbaryl-degrading Pseudomonas sp. C5pp catalyzes ortho-hydroxylation of 1-naphthol to 1,2-dihydroxynaphthalene. Homology model-guided site-directed-mutagenesis identified residues involved in FAD-binding, substrate-entry, catalysis and dimerization. Alanine substitution of R55, Q128, W298 and D318 caused activity loss with decreased flavin content, indicating roles in FAD-binding. Bulky substitutions in tunnel lumen (G207F, M260F, M380F and M383F) abolished activity, supporting its role in substrate-entry. Mutations at the active-site showed that H57 forms principal substrate-binding residue which is oriented by N125, while L229 and P325 contribute to substrate positioning. The apparent coupling efficiency (hydroxylation efficiency) of mutants remained comparable to that of wildtype enzyme, indicating that these substitutions affected activity without altering coupling between NADH oxidation and product formation. The C-terminal domain was crucial for folding and dimerization, while H541A/Y542A led to monomeric 1NH with ∼52% activity. These findings provide insights into role of domains and the catalytic mechanism of 1NH.
Elucidating novel signaling pathways that drive myotube hypertrophy may provide new insights into the mechanisms regulating skeletal muscle growth. We previously reported that 5-hydroxy-7-methoxyflavone (HMF) induces hypertrophy of murine C2C12 myotubes; however, the underlying molecular mechanism remains unclear. Here, loss- and gain-of-function analyses revealed that GPR97 positively regulates myotube size. Furthermore, knockdown and rescue experiments demonstrated that GPR97 is required for HMF-induced myotube hypertrophy. HMF activated mTORC1 signaling in myotubes, and intramuscular administration of HMF also activated mTORC1 signaling in mouse skeletal muscle in vivo. Gpr97 knockdown abolished HMF-induced mTORC1 signaling and protein synthesis. Furthermore, depletion of Gna12 and expression of dominant-negative Gα12 abolished HMF-induced myotube hypertrophy. Gna12 depletion also suppressed HMF-induced mTORC1 signaling and protein synthesis. Beclomethasone dipropionate, a reported GPR97 ligand, suppressed HMF-induced hypertrophy and mTORC1 signaling, while further enhancing HMF-induced serum response factor (SRF)-dependent transcription. Although dominant-negative RhoA inhibited HMF-induced SRF-dependent transcriptional activity, it did not affect myotube hypertrophy. These findings indicate that GPR97-Gα12-mTORC1 signaling promotes hypertrophy independent of the Gα12-RhoA-SRF pathway. GPR97 was predominantly expressed on the myotube surface as a C-terminal fragment generated by N-terminal fragment (NTF) cleavage, and HMF reduced its surface expression. HMF induced hypertrophy in myotubes expressing an NTF cleavage-resistant GPR97 mutant, but not in those expressing an NTF-deleted mutant. These results indicate that NTF cleavage is dispensable and suggest that NTF contributes to GPR97-mediated hypertrophic signaling. These findings identify a novel GPR97-Gα12-mTORC1 signaling axis that mediates HMF-induced myotube hypertrophy.
Background Diabetic foot ulcer (DFU) lacks effective early diagnostic and therapeutic targets. Objective To explore the expression, clinical value and mechanism of miR-543 in DFU. Methods A total of 80 healthy population, 83 T2DM patients without foot ulcer, and 124 DFU patients were enrolled. Serum miR-543 and NFAT5 levels were measured by qPCR. Correlation, ROC, and logistic regression analyses were conducted. HUVECswere treated with high glucose (HG) to establish an injury model. Cell function assays, dual-luciferase reporter assay and rescue experiments were performed. Results miR-543 was markedly downregulated and NFAT5 increased in DFU patients, with a strong negative correlation between the two. miR-543 showed good diagnostic value for DFU and was correlated with ulcer severity and ABI. In HG-induced HUVECs, miR-543 overexpression improved proliferation, migration and angiogenesis, and reduced inflammation. NFAT5 was verified as a direct target of miR-543, and its overexpression reversed the protective effects of miR-543. Conclusion miR-543 inhibits endothelial dysfunction by targeting NFAT5, thereby hindering the progression of DFU.
