BACKGROUND:Previously, we proved that tetratricopeptide repeat domain 4 (TTC4) protected against inflammatory injury in allergic rhinitis (AR). This study further explored whether HECT, UBA, and WWE domain containing protein 1 (HUWE1) functions as an E3 ubiquitin ligase targeting TTC4 to participate in AR pathogenesis. METHODS:HNEpCs were stimulated with IL-13 in vitro, and mice were sensitized with ovalbumin to establish AR model. Cell viability measured by CCK-8. Intracellular and mitochondrial oxidant productions and mitochondrial membrane potential were detected with DCFH-DA, MitoSOX Red, and JC-1 staining, respectively. The proportions of Th17/Treg cells were analyzed with flow cytometry. RT-qPCR, western blot, immunofluorescence, immunohistochemistry, and ELISA were conducted to examine mRNA and protein levels. Nasal histopathology was assessed by H&E and Giemsa staining. Co-immunoprecipitation and ubiquitination assay dissect the molecular mechanism. RESULTS:HUWE1 was increased in AR mice and IL-13-stimulated HNEpCs. HUWE1 silencing stabilized TTC4 via inhibiting its ubiquitination-dependent degradation. HUWE1 knockdown diminished NLRP3 inflammasome activation and mitochondrial oxidative damage, and enhanced mitophagy under IL-13 condition, which were abolished by TTC4 silencing. Mitophagy inhibitor 3-MA/Mdivi-1 counteracted the beneficial effects of HUWE1 depletion/TTC4 overexpression on IL-13-induced HNEpCs injury. In AR mice, HUWE1 depletion relieved nasal mucosal thickening, inflammatory response and NLRP3 activation, as well as restored Treg/Th17 balance and inactivated FK506-binding protein 5 (FKBP5)/NF-κB pathway, which were abrogated upon TTC4 knockdown. CONCLUSION:HUWE1 silencing reduced TTC4 ubiquitination-dependent degradation, which enhanced mitophagy to relieve mitochondrial oxidative damage and mitigatedFKBP5/NF-κB-triggered Treg/Th17 imbalance, thus improving AR. This study provided promising therapeutic targets for AR.
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.
