Rare UBAP2L variants have been linked to neurodevelopmental disorders (NDDs), such as autism spectrum disorder (ASD), but the role of UBAP2L in brain development remains unclear. Here, we generated neural-specific conditional knockout mice for Ubap2l using Nestin-Cre system, including heterozygous (cHET) and homozygous (cKO) mutants, to investigate the role of UBAP2L in neurodevelopment. Histological analyses revealed that adult cKO mice exhibited reduced body weight and fewer mature cortical neurons. At embryonic day 18.5 (E18.5), these mutant mice showed abnormal cortical lamination. Additionally, at E14.5, the abundance of cortical neural progenitor cells was reduced, and their proliferation was impaired. Ubap2l cKO mice exhibited impaired social interaction and deficits in learning and memory, whereas cHET mice showed milder behavioral and cognitive impairments. Transcriptomic profiling of the E14.5 cortex showed that differentially expressed genes were enriched in transcriptional regulation, neuronal differentiation, and cell proliferation, with proliferation-related genes predominantly downregulated. Pathway analysis further revealed dysregulation of the PI3K-AKT, MAPK, and Wnt signaling pathways. Cell-cycle analysis in the E14.5 cortex, together with flow cytometric analysis of UBAP2L-deficient cells, demonstrated that Ubap2l loss causes cell-cycle defects, namely S-phase shortening and G2/M arrest. Re-expression of wild-type UBAP2L rescued these defects, whereas NDD-associated UBAP2L variants failed to rescue cell-cycle progression. Collectively, these findings indicate that UBAP2L is required for cortical development and that its deficiency contributes to ASD-related phenotypes by disrupting neural progenitor proliferation and cell-cycle progression.
Hepatocellular carcinoma (HCC) is a highly aggressive cancer with a poor prognosis. CIAP2 is overexpressed in HCC and functions as an oncogene; however, the mechanisms regulating CIAP2 expression in HCC remain incompletely understood. In this study, we screened ubiquitin-specific proteases (USPs) that may regulate CIAP2 expression and identified USP45 as a key regulator that enhances CIAP2 levels. Bioinformatic analysis revealed that USP45 is overexpressed in HCC and is associated with worse patient survival. Knockdown of USP45 in two HCC cell lines resulted in reduced cell viability, impaired colony formation, cell cycle arrest, increased apoptosis, attenuated migration and invasion, and compromised sphere formation. Furthermore, USP45 deficiency suppressed the NF-κB signaling pathway and reduced the expression of key transcription factors involved in cancer cell stemness. Mechanistically, USP45 interacts with CIAP2 in HCC cells, removes its ubiquitination, and enhances its stability. Ubiquitin mutant assays revealed that USP45 specifically targets K48-linked polyubiquitin chains on CIAP2, while endogenous co-immunoprecipitation confirmed the physiological interaction between USP45 and CIAP2. Overexpression of CIAP2 significantly restored the malignant traits of USP45-deficient HCC cells. In an HCC xenograft mouse model, USP45 depletion also inhibited tumor growth and disrupted various malignant characteristics. Collectively, these findings demonstrate that USP45 acts as an oncogene in HCC by regulating the ubiquitination and stability of CIAP2.
Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by dopaminergic neuronal loss and α-synuclein (α-syn) aggregation. The human-specific GLUD2 T1492G variant has been associated with an earlier onset of PD, but its pathogenic mechanism remains unclear. In this study, we generated A53T α-syn transgenic mice with astrocyte-specific expression of GLUD2 T1492G and investigated its effects on PD-related pathology. GLUD2 T1492G exacerbated α-syn pathology, nigrostriatal dopaminergic neurodegeneration, and motor impairment. Transcriptomic analysis identified complement-related inflammatory signaling as a major pathway altered by GLUD2 T1492G, whereas metabolomic profiling revealed disrupted metabolic homeostasis. Mechanistically, GLUD2 T1492G enhanced astrocytic C3 induction, promoted microglial C3aR/NF-κB-associated inflammatory activation, and amplified neuroinflammatory responses. Conditioned-medium transfer experiments, together with astrocyte-specific C3 knockdown and pharmacological inhibition of C3aR, supported the functional involvement of the astrocyte–microglia C3–C3aR signaling axis in GLUD2 T1492G-associated pathology. Collectively, our findings suggest that activation of the astrocyte–microglia C3–C3aR signaling axis contributes to GLUD2 T1492G-mediated exacerbation of PD pathology and highlight this pathway as a potential therapeutic target for PD.
Endothelial dysfunction is a major contributor to diabetic vascular complications, yet the mechanisms linking hyperglycemia to impaired endothelial homeostasis remain insufficiently defined. This study identifies calpains as key regulators of endothelial autophagy and mitophagy under diabetic conditions. In HUVECs and primary lung endothelial cells, hyperglycemia activated calpains and induced autophagic flux blockade, characterized by SQSTM1 accumulation, reduced LC3B expression, and impaired mitophagy associated with mitochondrial fragmentation. Pharmacological calpain inhibition or genetic calpastatin overexpression restored autophagic flux and preserved mitochondrial network architecture. Transcriptomic profiling revealed that calpain inhibition counteracted major hyperglycemia‑driven pathways, including oxidative phosphorylation, ROS signaling, and the unfolded protein response. Collectively, our findings demonstrate that calpain overactivation disrupts endothelial autophagy and mitophagy in diabetes, contributing to mitochondrial dysfunction and altered vascular reactivity. Restoring autophagic flux through calpain inhibition emerges as a promising strategy to preserve endothelial homeostasis and limit diabetic vascular complications.
Bone metastasis is a major cause of disease progression and cancer-related death in advanced prostate cancer (PC). Although spindle and centriole associated protein 1 (SPICE1) has been implicated in tumor biology, its role in PC metastasis and its post-translational regulation remain largely unknown. SPICE1 expression was assessed in PC tissues and bone metastatic lesions, and its clinicopathological relevance was analyzed. functional assays were performed in PC3 and C42B cells to evaluate cell proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT). Subcutaneous xenograft and intra-arterial bone metastasis models were used to determine the in vivo effects of SPICE1 silencing. Co-immunoprecipitation, proximity ligation assay, mutagenesis, cycloheximide chase, and ubiquitination/SUMOylation assays were employed to define the underlying mechanism. SPICE1 was markedly upregulated in PC tissues and was further increased in bone metastatic lesions. High SPICE1 expression was associated with aggressive clinicopathological features and poor prognosis. Functionally, SPICE1 knockdown suppressed PC cell proliferation, migration, invasion, and EMT in vitro, and attenuated tumor growth and bone metastasis in vivo. Mechanistically, AURKA directly interacted with SPICE1 and phosphorylated SPICE1 at serine 811, thereby enhancing SPICE1 stability. This phosphorylation event promoted the recruitment of the SUMO E3 ligase TRIM28 and facilitated TRIM28-dependent SUMOylation of SPICE1 at lysine 838, which antagonized ubiquitination-mediated degradation. Restoration of AURKA or TRIM28 partially rescued the inhibitory phenotypes induced by SPICE1 silencing. At the downstream level, SPICE1 stabilization was accompanied by increased FASN expression and activation of PI3K/AKT signaling. These findings identify SPICE1 as a pro-metastatic effector in PC and establish the AURKA/TRIM28/SPICE1 axis as a central post-translational mechanism driving bone metastasis. AURKA-mediated phosphorylation licenses TRIM28-dependent SUMOylation, suppresses ubiquitin-mediated degradation, and stabilizes SPICE1. Downstream changes in FASN/PI3K/AKT signaling likely contribute to the malignant phenotype but are secondary to this core regulatory axis.
Neuropeptide Y (NPY) is a key regulator of neuroimmune homeostasis. However, its role in the intrahepatic immune microenvironment and hepatic fibrosis remains unclear. We aimed to elucidate whether NPY participates in hepatic fibrogenesis via neuroimmune crosstalk and to dissect the underlying molecular mechanisms. Liver tissues from cirrhotic and non-cirrhotic human subjects were collected. Hepatic fibrosis was induced in mice via intraperitoneal carbon tetrachloride (CCl₄) injection or the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. The distribution of NPY and its receptors was detected in human and mouse hepatic tissues. NPY-knockout mice were used to evaluate the impact of NPY deletion on hepatic fibrosis, inflammation, and macrophage polarization. Hepatic NPY receptors were silenced in vivo, and the corresponding receptors or downstream kinases were blocked in vitro, to define the underlying signaling mechanisms. NPY was elevated in fibrotic human and mouse livers and primarily secreted by intrahepatic sympathetic nerves. NPY1R and NPY2R (but not NPY4R/NPY5R) were increased and localized in hepatic macrophages. NPY deletion mitigated liver injury, collagen deposition, and inflammation, and suppressed both M1 and M2 macrophage polarization. In vivo knockdown of NPY1R or NPY2R recapitulated these protective effects. In vitro, NPY enhanced M1 polarization via NPY1R/NPY2R-coupled PI3K–PAK1–c-Jun signaling, whereas it promoted M2 polarization through the PI3K–PAK1–RSK cascade. NPY drives both M1 and M2 macrophage polarization and accelerates hepatic fibrogenesis through NPY1R/NPY2R–PI3K–MAPK-dependent neuroimmune signaling. These findings identify NPY and its receptors as potential therapeutic targets for hepatic fibrosis. Intrahepatic sympathetic nerve-derived NPY promotes M1 and M2 macrophage polarization through NPY1R/NPY2R–PI3K–PAK1 cascades (c-Jun for M1, RSK for M2), driving hepatic stellate cell activation via profibrotic cytokines (TNF-α, IL-1β, TGF-β, and PDGF-BB). Solid lines: experimentally validated pathways. Dashed lines: putative unvalidated intercellular interactions (direct NPY–hepatic stellate cell axis and macrophage–hepatic stellate cell crosstalk)
Radiotherapy-induced B cell lymphopenia and hematopoietic suppression represent major complications of cancer therapy. The mechanisms that initiate B cell lineage regeneration from its source have yet to be elucidated, and targeted interventions are currently lacking. Here, we demonstrate that radiation exposure induces transcriptional dysregulation of histone deacetylases (HDACs) in splenic B cells. The pan-HDAC inhibitor panobinostat (PANO) specifically counteracts this radiation-induced immune injury. Building on this effect, PANO significantly restored hematopoietic progenitor cells (HPCs) in the bone marrow. In the spleen, it selectively reconstituted mature B cells and the follicular B cell (FoB) compartment without disrupting T cell homeostasis. This cellular specificity underscores PANO’s capacity to promote B cell regeneration across both central and peripheral compartments. Mechanistically, transcriptomic analysis revealed that PANO treatment was associated with the expression of genes in the IL-17 signaling pathway, as evidenced by upregulating critical mediators (ACT1/Traf3ip2, Traf5, Traf6) and remodeling the chaperone network (Stip1, Dnaja1, Hsp90). This coordinated enhancement of key B cell transcription factors (Pax5, Ebf1, Myc), coupled with suppression of pro-inflammatory mediators (S100a8/a9), promoted B cell regeneration. Our study demonstrates that PANO enhances in situ proliferation of splenic mature B cells after radiation, accompanied by transcriptomic alterations in IL-17 signaling and HSP90 chaperone networks, providing hypothetical regulatory clues for B cell radioprotection.
Ferroptosis, a key mechanism in acute kidney injury (AKI), is centrally regulated by solute carrier family 7 member 11 (SLC7A11). Studies have shown that the splicing factor LUC7-like 2 (LUC7L2) modulates SLC7A11 in the context of glucose metabolism in cancer cells; however, the potential involvement of this regulatory axis in ferroptosis or renal injury remains unknown. Given the well-established roles of SLC7A11 in ferroptosis, we hypothesize that LUC7L2 may influence renal ferroptosis through SLC7A11 in AKI. Integrated in vitro and in vivo models of cisplatin-induced AKI were established. LUC7L2 expression was modulated by siRNA, plasmid overexpression, and adeno-associated virus 9-mediated knockdown. Ferroptosis was evaluated by cell viability, lipid peroxidation, iron accumulation, glutathione levels, and GPX4 expression. Mechanistic studies included exon-specific splicing assays and RNA immunoprecipitation. Public single-cell RNA sequencing datasets were reanalyzed to define cell–type–specific expression patterns, and the findings were validated in human kidney biopsy specimens and urine samples. The results demonstrate that LUC7L2 expression is upregulated in cisplatin-induced models of AKI. Functionally, it promotes ferroptosis by regulating SLC7A11, thereby reducing glutathione synthesis and exacerbating oxidative stress. We identified a dual-pathway mechanism in which cisplatin-activated p53 represses transcription of SLC7A11, while LUC7L2 post-transcriptionally controls pre-mRNA splicing of SLC7A11 via direct binding and is associated with increased exon skipping. Under cisplatin stress, LUC7L2 is associated with non-productive SLC7A11 splicing and reduced functional SLC7A11, thereby promoting ferroptotic susceptibility. In functional assays, we found that renal ferroptotic injury is attenuated by LUC7L2 knockdown but exacerbated by its overexpression. Taken together, these findings identify LUC7L2 as a contributor to cisplatin-induced AKI through splicing-dependent control of SLC7A11. This study reveals a previously unrecognized post-transcriptional mechanism governing ferroptosis in the kidney and highlights LUC7L2 as a potential diagnostic biomarker and therapeutic target.
TDP-43 has many functions, including the regulation of autophagy and stress granule dynamics, and mutations in TDP-43 are causative of amyotrophic lateral sclerosis (ALS). Among these, the G376D mutation is associated with TDP-43 cytoplasmic mislocalization and mitochondrial and lysosomal dysfunctions. We discovered that upon TDP-43 silencing, the abundance of RILP, a RAB7A effector, decreases. Similarly, expression of the ALS-causative G376D mutant protein reduces RILP intracellular levels, compromising the autophagic flux. RILP overexpression restores the autophagic flux and reduces stress granule marker levels, demonstrating that RILP can overcome TDP-43 malfunctioning. Mechanistically, we show that TDP-43 controls RILP expression mainly at a translational level. L-leucine restored RILP levels in TDP-43-depleted and TDP-43G376D-expressing cells, whereas inhibition of mTOR signaling or protein synthesis abolished this effect. Polysome profiling further demonstrated that TDP-43 depletion reduces the association of RILP mRNA with heavy polysomes, supporting impaired translational efficiency. RILP downregulation and defective autophagic flux were confirmed in iPSC-derived TDP-43G376D motor neurons, where RILP overexpression improved autophagy and cell viability. In addition, TDP-43 co-immunoprecipitates with both RILP and RAB7A, although only RILP interacts directly with TDP-43. The interaction occurs between the RILP C-terminus and amino acids 320–346 of TDP-43. Interestingly, the TDP-43 mutant displays a stronger interaction with RILP, and its expression reduces RILP-RAB7A interaction, suggesting that pathogenic TDP-43 perturbs the functional coupling between them. These data identify RILP as a new TDP-43 interactor and reveal that disruption of the TDP-43/RILP/RAB7A axis, induced by TDP-43G376D, contributes to defective autophagy that characterizes ALS and possibly other neurodegenerative disorders.
Left-sided obstructive lesions (LSOLs), represented by coarctation of the aorta (CoA), cause persistent vascular pathology and pressure overload that can lead to pathological cardiac remodeling. However, the epigenetic changes associated with local vascular abnormalities and ventricular remodeling remain incompletely understood. We performed H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) in paired aortic tissues from patients with CoA to characterize region-associated super-enhancer (SE) landscapes and transcriptional alterations. Integrated multi-omics analysis, together with single-cell RNA sequencing (scRNA-seq) from LSOLs patients, identified neuropeptide W (NPW) as a SE-associated candidate. c-JUN CUT RUN-qPCR showed AP-1 enrichment at successfully amplified NPW-associated SE regions (Peak1). In primary smooth muscle cells (SMCs), broad BET inhibition or NPW silencing was associated with reduced contractile-marker expression, whereas exogenous NPW attenuated angiotensin II-induced cardiomyocyte hypertrophy in vitro. Plasma NPW levels were lower in patients with CoA and were associated with echocardiographic indices of remodeling. These findings identify NPW as a SE-associated candidate pathway in CoA and provide supportive evidence for broader relevance to LSOLs-associated cardiac remodeling.
Signalling via the epidermal growth factor receptor (EGFR) is indispensable for morphogenesis and tissue homeostasis. It is activated by extracellular ligands, typically released from transmembrane precursors by proteolysis. Ligand shedding activity is provided by the conserved rhomboid intramembrane serine proteases in Drosophila, but by the unrelated ADAM family metalloproteases in mammals, leaving the functions of mammalian non-mitochondrial rhomboids underexplored. Using quantitative proteomics, we show that EGFR is the main endogenous substrate of the human rhomboid protease RHBDL2 in keratinocytes. By shedding the EGFR ectodomain, thus producing a decoy receptor, RHBDL2 suppresses EGFR signalling, limiting cell migration and invasion. Conspicuously, RHBDL2 activity is upregulated by elevated intracellular calcium concentration, a condition typical of keratinocyte differentiation. These effects are recapitulated in primary human keratinocytes, and human skin equivalents deficient in RHBDL2 display incomplete differentiation and are morphologically disordered compared to wild-type cells. We propose that context-specific fine-tuning of EGFR signalling and sensitivity to cross-talk from other signalling pathways could be important and hitherto overlooked roles of rhomboid proteases in mammals.
Stress granules (SGs) are dynamic organelles that form under cellular stress and are generally regarded as protective entities. However, their role in pathogenesis is increasingly recognized, yet the underlying mechanisms remain elusive due to the diversity of stress types and biological contexts. Here, we investigate how different regimes of glycolytic inhibition influence SG dynamics and transcriptomic partitioning. We subjected cells to glucose depletion (GD), 2-deoxy-D-glucose addition (2DG), or their combination (GD+2DG). We show that SGs formed under single assaults dissipate during prolonged treatment and correlate with integrated stress response (ISR), whereas SGs induced by GD+2DG persist and are associated with mitochondrial dysfunction and increased apoptosis. Using G3BP1-APEX2 proximity labeling, we find that GD+2DG uniquely enriches oxidative phosphorylation (OXPHOS) transcripts in the G3BP1-associated transcriptome. We demonstrate that mitochondrial inhibition renders SGs formed under single assaults persistent. We propose a model in which two counteracting feedback loops determine SG fates under glycolytic stress: a negative ISR-mediated loop promotes SG recovery, while a positive loop involving G3BP1-partitioned OXPHOS transcripts drives SG persistence and metabolic collapse. This framework provides mechanistic insights into the paradoxical duality of SGs in stress adaptation and disease.
Cardiovascular disease is the leading cause of mortality in patients with hemodialysis. Coronary artery calcification represents a major pathological substrate and an independent risk factor for adverse cardiovascular outcomes. Although endothelial-smooth muscle cell crosstalk is critical in vascular calcification and extracellular vesicles (EVs) are key intercellular mediators, their role in coronary artery calcification remains unexplored. Additionally, the mechanisms linking α-1,6-fucosyltransferase (FUT8) upregulation in calcified vascular smooth muscle cells to endothelial EV signaling are unknown. High phosphorus and high calcium conditions were applied to coronary artery endothelial cells to induce EV secretion. The EVs were isolated and characterized, and their uptake by coronary artery smooth muscle cells was examined. Molecular analyses assessed the effects of EV-derived miR-122b-3p on FOXP1 repression, FUT8 expression, integrin αV (ITGAV) core fucosylation, and autophagy suppression. Endothelial EVs were highly enriched with miR-122b-3p under calcifying conditions and were internalized by smooth muscle cells, leading to FOXP1 repression, FUT8 upregulation, enhanced ITGAV core fucosylation, autophagy suppression, and accelerated coronary artery calcification. In uremic rats, EVs overexpressing miR-122b-3p aggravated coronary artery calcification by modulating the FOXP1/ FUT8/ITGAV signaling pathway. Preliminary analysis of serum EVs from healthy controls and patients with CKD/uremia further showed increased serum EV miR-122b-3p levels and associations with renal dysfunction and mineral metabolism-related parameters, supporting the potential clinical relevance of this EV-miRNA axis. This study identifies a novel extracellular vesicle-dependent intercellular communication mechanism linking FUT8-mediated core fucosylation to autophagy dysfunction in coronary artery calcification.
Type I interferon (IFN-I) is widely used for the treatment of hepatitis B virus (HBV) infection, yet its therapeutic efficacy remains limited. Here, we identify guanylate-binding protein 2 (GBP2), an interferon-stimulated gene, as a host factor that paradoxically promotes HBV replication. GBP2 overexpression stabilized the viral regulatory protein HBx and markedly prolonged its half-life, whereas GBP2 knockdown facilitated HBx degradation. Mechanistically, GBP2 recruited the E3 ligase HUWE1 to induce K29-linked ubiquitination of HBx at lysine 95, which in turn suppressed K48-linked ubiquitination and proteasomal degradation. Functionally, GBP2-mediated HBx stabilization enhanced HBV replication and promoted hepatoma cell migration and invasion. Importantly, GBP2 expression was elevated in PBMCs of HBV-infected patients and further induced by IFN-I treatment, while HBV delayed early IFN-mediated GBP2 induction in HBV-containing hepatocyte-derived cells. Together, these findings reveal a novel mechanism by which GBP2 antagonizes the antiviral activity of IFN-I by stabilizing HBx, thereby facilitating HBV replication and potentially contributing to hepatocarcinogenesis. In brief, IFN activates ISGF3 via the JAK-STAT signaling pathway to produce the IFN-stimulating gene, GBP2, which promotes the K29 chain ubiquitination of HBx protein on the K95 site, which in turn competitively inhibits the K48 chain ubiquitination of HBx, thereby inhibiting HBx protein degradation. In brief, IFN activates ISGF3 via the JAK-STAT signaling pathway to produce the IFN-stimulating gene, GBP2, which promotes the K29 chain ubiquitination of HBx protein on the K95 site, which in turn competitively inhibits the K48 chain ubiquitination of HBx, thereby inhibiting HBx protein degradation.
Cancer cells possess unlimited proliferative potential, and the high expression of lncRNAs widely promotes cancer progression. Mammalian early embryos share similar characteristics, where numerous endogenous retroviruses become transcriptionally active during zygotic genome activation (ZGA). However, whether cancer-promoting lncRNAs can also facilitate early embryonic development remains unclear. In this study, we demonstrated that TUG1, which is highly expressed in tumors, regulates early embryonic development in mice. TUG1 deficiency results in the arrest of mouse embryos at the two-cell stage, accompanied by disruption of the MAPK signaling pathway, and consequently leads to the failure of ZGA. Mechanistically, we uncover a TUG1-hnRNPA1 ribonucleoprotein complex that actively licenses ZGA by promoting the transcription of MAPK8, thereby activating the essential MAPK signaling pathway. This study transforms our understanding of TUG1 from a cancer-specific effector to a critical regulator of the ZGA, raising the possibility that related regulatory principles may operate in other biological contexts.
Integrins are cell-surface adhesion receptors that mediate bidirectional signaling governing migration, proliferation, and survival. Emerging evidence reveals that integrin function is finely controlled by an interconnected network of post-translational modifications (PTMs), spanning classical modifications such as glycosylation and phosphorylation, as well as novel metabolite-driven alterations including carbamoylation and cysteine carboxyethylation. These PTMs collectively orchestrate integrin stability, conformation, trafficking, and downstream signaling, with profound implications for tumor progression, metastasis, fibrosis, and therapy resistance. We further highlight critical cross‑talk among distinct modification types and demonstrate how environmental cues (e.g., metabolic byproducts, gut microbiota metabolites) directly modify integrins. Finally, we explore emerging therapeutic strategies inspired by these insights, including targeted enzyme inhibitors, proteolysis targeting chimeras (PROTACs), deubiquitinase-targeting chimeras (DUBTACs), and PTM-editing tools such as dCasRx-based m6A editors. Deciphering the integrin PTM code transforms our understanding of cell adhesion biology and opens new frontiers for diagnostic biomarkers and precision therapy.
Abstract The SM-protein Munc18 and its binding partner syntaxin are crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the release site, and priming reactions that prepare docked granules for Ca 2+ dependent fusion. Both proteins cluster simultaneously at the docking site seconds after a granule arrives at the plasma membrane. To study the mechanisms of these separate Munc18 functions, we generated a Munc18-1 knockout (1KO) and a double knockout of both Munc18-1 and Munc18-2 (DKO) in insulin-secreting cells, and quantified granule docking, exocytosis, and protein clustering at the docking site by TIRF imaging and capacitance measurements. In the 1KO, priming and exocytosis were lost, but granule docking remained intact. In the DKO, both priming and granule docking were impaired. Expression of either Munc18-1 or -2 rescued these defects, as did expression of mutants with weakened binding to syntaxin (EA and EK mutants). Munc18 clustered at granule docking sites, with apparent affinities decreasing in the order Munc18-1 > Munc18-1EA > Munc18-1EK > Munc18-2. Clustering of syntaxin-1 and − 3 at granules was impaired in both knockout lines. Finally, single molecule and imaging fluorescence recovery after photobleaching (FRAP) revealed that Syntaxin and Munc18 molecules were bound to each other, regardless of whether the proteins were clustered at a granule or not. We conclude that that Munc18-1 and − 2 molecules co-cluster with syntaxin at the release site and support docking and priming, and different affinities of the isoforms for the granule docking site contribute to the observed functional preferences.
African swine fever (ASF), caused by the ASF virus (ASFV), is a fatal hemorrhagic disease of domestic pigs that causes significant economic losses in the global pig industry. Systemic hemorrhages and abnormal coagulation of multiple tissues and organs are the typical lesions of ASFV infection, but the mechanism is still unknown. The exaggerated inflammation caused by ASFV infection is believed to contribute to endothelial injury and coagulopathy. Here, we demonstrate that circulating endothelial cells are significantly elevated in ASFV-infected pigs. In vitro infection assay shows that both primary and immortalized endothelial cells are permissive for ASFV replication, and ASFV infection triggers apoptosis of infected cells. ASFV disrupts redox homeostasis by negatively regulating the NRF2 pathway, which in turn leads to oxidative stress and ROS-mediated endothelial cell apoptosis. Mechanistically, ASFV D117L suppresses NRF2 signaling by interacting with KEAP1 and promoting the ubiquitination of NRF2. Functional analyses reveal that KEAP1 acts as a proviral factor for ASFV infection in endothelial cells, whereas its substrate, NRF2, suppresses viral replication. Further, pharmacological inhibition of KEAP1 by 4-Octyl itaconate (4-OI) and Sulforaphane (SFN) inhibits ASFV replication and ROS-dependent cell death. Our findings uncover a potential mechanism for endothelial dysfunction during ASFV infection and suggest that targeting KEAP1 to maintain endothelial redox homeostasis could be a promising approach for ASF treatment.
Interferon-stimulated gene 15 (ISG15) is an essential biomarker in several cancers. However, the molecular mechanism of ISG15 regulating thyroid carcinoma (THCA) has rarely been studied. In our study, papillary thyroid carcinoma cell lines of TPC-1 and IHH-4, clinical samples of THCA, and nude mice were employed. The expression levels of genes were tested by real-time quantitative PCR and western blotting analyses. Cell activity was assessed using cell counting kit-8 assay and colony formation assays. Flow cytometry measured the ROS level and mitochondrial membrane potential. To induce mitophagy, cells were treated with 10µM CCCP (Carbonyl Cyanide m-Chlorophenylhydrazone) for 6 h. The level of mitophagy was assessed by transmission electron microscopy, LC3B-TOMM20 co-localization, mitochondrial Parkin level, p-S65-Ub and related protein expression. Co-immunoprecipitation assays determined the binding relationship between ISG15 and VCP. Chromatin immunoprecipitation, electrophoretic mobility shift and dual-luciferase reporter assay measured the targeting of PEG3 on the ISG15 promoter. Our results showed that ISG15 was highly expressed in THCA. ISG15 overexpression promoted cell proliferation and mitophagy. Mechanistically, ISG15 bound to VCP and promoted its stability. VCP integrated with ISG15 to promote cell viability, and mitophagy. Further experiments revealed that PEG3, which acted as a transcriptional inhibitor of ISG15, exerted an effect opposing that of ISG15. The role of ISG15 and PEG3 were also confirmed in xenograft mice model. In conclusion, our present study explored the molecular mechanism of ISG15 on THCA and found that PEG3 transcriptionally regulated ISG15 and promoted the proliferation and mitophagy of papillary thyroid carcinoma cells through VCP. Our present study revealed a novel mechanism of ISG15 on THCA, which might be a promising therapeutic target for THCA.
The interplay between triggering receptor expressed on myeloid cells 2 (TREM2) and low-density lipoprotein receptor-related protein 1 (LRP1) is critical for amyloid-beta (Aβ) clearance in Alzheimer’s disease (AD). Current therapeutic strategies targeting this axis can be broadly divided into two pharmacological approaches: indirect induction via metabolic modulation (e.g., ACAT1 inhibition) and direct functional restoration of LRP1. While ACAT1 inhibition effectively upregulates LRP1, recent findings suggest that this effect is mechanistically coupled to ADAM10/17-mediated shedding of TREM2. This raises a critical pharmacological dilemma: balancing the benefit of LRP1 upregulation against the reduction of cell-surface TREM2. While TREM2 shedding may represent a functional shift from membrane-bound sensing to soluble Aβ clearance rather than a simple loss of function, cell-surface TREM2 signaling remains essential for Aβ sensing and microglial fitness in the long term. Conversely, strategies focusing on the direct restoration of LRP1 transport achieve robust Aβ clearance but face challenges regarding delivery specificity. Integrating emerging evidence on lipid droplet–driven microglial dysfunction and membrane fluidity, this article discusses the trade-offs of these strategies and proposes that preserving surface TREM2 signaling while enhancing LRP1 activity is key to long-term therapeutic efficacy. Clinical evidence from the INVOKE-2 trial further supports the need for combination strategies targeting both TREM2 signaling and LRP1-mediated clearance.
