P4HA2 promotes glioma cell proliferation by interacting with Hsp27 to activate the EGFR/ERK signaling pathway.

The role of ubiquitin in post-translational modifications is important for tumor progression, but how these mechanisms regulate bladder cancer (BLCA) is not completely known. The study demonstrated that FBXL6, a member of the F-box protein family, could drive oncogenesis in BLCA, as shown by integrative bioinformatic analysis and clinical sample validation. Experiments demonstrated that FBXL6 speeds up the in vitro growth and migration of BLCA cells and contributes to tumor development and metastasis in vivo. Mechanistically, transcriptomic and metabolic studies indicate that FBXL6 regulates the glycolytic pathway. Although FBXL6 knockdown has minimal impact on the mRNA levels of the key glycolytic enzyme ENO1, FBXL6 knockdown does alter ENO1 protein levels, suggesting post-translational regulation. Co-immunoprecipitation and GST pull-down assays validated the interaction between FBXL6 and ENO1, confirming that the LRR domain of FBXL6 and the C-terminal region of ENO1 are essential for their binding. Additionally, ubiquitination assays indicated that FBXL6 promotes K63-linked polyubiquitination of ENO1, which stabilizes it. Bringing back ENO1 expression partially offset the consequences of FBXL6 knockdown on proliferation and migration. In summary, our findings propose a new model where FBXL6 promotes BLCA progression by stabilizing ENO1 through K63 linkage, emphasizing its potential as a target for BLCA therapy.

Liver fibrosis caused by chronic inflammation remains the major driver of various liver diseases. However, limited effective therapies have been identified for liver fibrosis. Herein, we elucidated the complicated molecular mechanisms underlying liver fibrosis. Primary hepatocytes were co-cultured with JS-1 cells. Inflammatory cytokine levels were assessed by ELISA. Liver fibrosis markers and target molecular levels were detected by western blotting and immunohistochemical staining. Molecular mechanisms were analyzed by Acyl-biotin exchange (ABE) assay, Co-IP, proximity ligation assay, biotin pull-down, and GST pull-down assays. Co-localization and subcellular localization of molecules were observed by immunofluorescent staining. Liver fibrosis was induced by CCl4 in mice and determined by Masson Trichrome and Sirius Red staining. Liver injury was evaluated by HE staining, serum ALT, and AST levels. High mobility group box 1 (HMGB1) bound to toll-like receptor 4 (TLR4) to facilitate palmitoylation of stimulator of interferon genes (STING), which led to hepatocyte inflammation and JS-1 cell activation in vitro. Furthermore, cysteine-rich secreted protein LCCL domain protein 2 (CRISPLD2) blocked HMGB1/TLR4 axis-mediated palmitoylation of STING, and subsequent liver fibrosis. Mechanistically, CRISPLD2 recruited 78kDa glucose-regulated protein (GRP78) to trigger TLR4 degradation via an autophagic-lysosomal pathway. CRISPLD2 treatment alleviated CCl4-induced inflammation and liver fibrosis in mice via inactivation of the HMGB1/TLR4/STING pathway. CRISPLD2 mitigated hepatocyte inflammatory response and fibrosis via interaction with GRP78 to inactivate the HMGB1/TLR4 pathway and consequently restrain STING palmitoylation.

Glycolytic enzyme hexokinase 2 (HK2) plays a crucial role in the development of several human cancers, including nasopharyngeal carcinoma (NPC). This project is designed to explore the role and mechanism of HK2 in NPC progression. HK2 and membrane-associated RING-CH-type finger 3 (MARCH3) mRNA and protein levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Cell viability, proliferation, migration, invasion, and apoptosis were measured using Cell Counting Kit-8 (CCK-8), Colony formation, wound healing, transwell, and flow cytometry. Glycolysis level was detected via specific kits. The stability of HK2 was assessed by Cycloheximide (CHX) assay. After Ubibrowser online database prediction, the interaction between MARCH3 and HK2 was validated using Co-immunoprecipitation (Co-IP) assay and GST pull-down assay. The effect of MARCH3 on PTX sensitivity of NPC was analyzed using in vivo studies. HK2 level was increased in NPC tissues and cell lines, and MARCH3 expression was decreased. Furthermore, HK2 knockdown repressed NPC cell proliferation, migration, invasion, glycolysis, and improved cell sensitivity to PTX. At the molecular level, MARCH3 could promote HK2 ubiquitination and decrease its stability. In addition, overexpressing MARCH3 increased the PTX sensitivity of NPC in vivo. MARCH3 could block NPC progression and strengthen the sensitivity of NPC cells to PTX, possibly by advancing the ubiquitination degradation of HK2, providing a promising therapeutic target for NPC treatment.

As a member of the Betacoronavirus genus, bovine coronavirus (BCoV) is a major etiological agent in cattle, causing diarrhea, fever, and reduced milk production, thereby imposing a substantial economic burden on the livestock industry. However, the molecular mechanisms underlying BCoV pathogenesis remain incompletely defined. In this study, we report that BCoV infection of Madin-Darby bovine kidney (MDBK) cells activates ZBP1-PANoptosome-associated signaling and induces the host sialidase neuraminidase 1 (Neu1). Functional studies using CRISPR-Cas9-mediated knockdown revealed that Neu1 depletion attenuated ZBP1-PANoptosome activation and reduced viral replication in vitro. Co-immunoprecipitation and GST pull-down assays, together with molecular docking and molecular dynamics analyses, supported a physical association between Neu1 and ZBP1, suggesting a potentially stable interaction interface. In vivo, oral inoculation of BALB/c mice with BCoV resulted in increased Neu1 and ZBP1 signals with partial colocalization in brain and colonic tissues, accompanied by decreased serum sialic acid levels and elevated interleukin-1 beta (IL-1β), which was consistent with Neu1 activation and enhanced inflammatory responses. Histopathological examination further revealed progressive vascular congestion and epithelial injury, suggesting an association between Neu1-ZBP1 signaling and virus-induced tissue damage. Collectively, our findings suggest that Neu1 may contribute to ZBP1-associated PANoptosis during BCoV infection and link sialic acid metabolism to nucleic acid-sensing and inflammatory cell death responses. These results provide mechanistic insight into BCoV pathogenesis and suggest Neu1 as a potential target for future studies aimed at controlling coronavirus infections in cattle and possibly other species.

KAT2B inhibits cholangiocarcinoma proliferation by binding with histone demethylase KDM6B.

Prolactinomas are the most common functional pituitary adenomas, and dopamine agonists (DAs) are the first-line therapy; however, approximately 10-30% of patients develop resistance, highlighting the need for effective sensitization strategies. In clinical specimens, we observed reduced p300 expression in tumors with poor DA responsiveness, and p300 levels were inversely associated with DA dosage. In cellular and xenograft models, DAs decreased p300 by suppressing the cAMP/PKA/CREB pathway. We therefore tested whether upregulating or activating p300 could enhance DA efficacy and investigated the underlying mechanism using immunohistochemistry, immunofluorescence, Western blot, genetic manipulations, RNA sequencing, CUT&Tag, ChIP-qPCR, Seahorse metabolic assays, flow cytometry, co-immunoprecipitation, and GST pull-down assays. Augmenting p300 markedly potentiated DA-induced antitumor effects in vitro and in vivo, a process accompanied by the elevated histone H3K18 lactylation (H3K18la). Mechanistically, p300-dependent H3K18la promoted transcriptional upregulation of Ndufs7 and Washc1. NDUFS7 induction was associated with increased mitochondrial ROS, whereas WASH1 bound the ubiquitin-associated domain of p62, impairing recognition and clearance of damaged mitochondria, suppressing mitophagy, and thereby sustaining mitochondrial ROS accumulation and apoptosis. Moreover, YF-2, a p300 HAT-domain activator, synergized with DAs to inhibit tumor growth in MMQ and AtT-20 cells. Together, these data identify a p300-H3K18la-NDUFS7/WASH1 axis that links mitophagy inhibition to mitochondrial ROS accumulation and provide a mechanistic rationale for targeting p300 as an adjuvant approach to improve DAs efficacy in prolactinomas.

The YAP1-NPM1 nuclear complex regulates MYC and reveals a targetable oncogenic node.

Ginsenoside Rg5 targets the KAT8-CISD2 axis to maintain mitochondrial homeostasis and antagonize senescence.

Diffuse-type gastric cancer (DGC) is characterized by strong invasiveness and poor prognosis, frequently associated with peritoneal metastasis. Dysregulation of protein kinase A catalytic subunit beta (PRKACB) has been implicated in various cancers, but its role in DGC remains unclear. This study investigates the expression, function, and molecular mechanisms of PRKACB in DGC metastasis. PRKACB expression was analyzed by immunohistochemistry in tissue samples from DGC and intestinal-type gastric cancer (IGC) patients. Functional assays and a mouse peritoneal metastasis model were employed to evaluate the impact of PRKACB on metastasis. The interaction between PRKACB and RhoA was explored using co-immunoprecipitation, GST pull-down assays, and in situ proximity ligation assays. PRKACB expression was significantly lower in DGC tissues compared to IGC and adjacent non-tumor tissues. Multivariate Cox regression analysis identified low PRKACB expression as an independent prognostic factor for poor overall survival. In DGC cell lines, PRKACB knockdown enhanced cell migration, invasion, pseudopodia formation, and epithelial-mesenchymal transition (EMT), while PRKACB overexpression suppressed these activities. In vivo experiments demonstrated that PRKACB knockdown promoted earlier onset of peritoneal metastasis. Mechanistically, PRKACB interacted with RhoA and promoted its phosphorylation at S188, thereby inhibiting RhoA signaling and its downstream effectors ROCK1 and FAK. Common RhoA mutations in DGC (V38G and N41K) weakened its interaction with PRKACB, leading to reduced phosphorylation and enhanced metastatic potential. Importantly, RhoA inhibitor treatment reversed the pro-metastatic effects induced by low PRKACB expression. In conclusion, low PRKACB expression promotes DGC metastasis through activation of RhoA signaling, and PRKACB downregulation combined with functional RhoA mutations contributes to the aggressive phenotype of DGC. These findings provide new insights into DGC progression and suggest that PRKACB may serve as both a prognostic biomarker and a potential therapeutic target for DGC patients.

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with a poor prognosis, driven by metabolic reprogramming and immune evasion. The role of T-complex protein 1 subunit beta (CCT2) in HCC remains unclear. This study aimed to elucidate the function of CCT2 in HCC tumorigenesis. Bioinformatics analysis and Clinical samples investigation were integrated with in vitro and in vivo experiments to investigate CCT2's role in HCC metabolism and immune modulation. The glycolytic activity was assessed by measuring extracellular acidification rate, glucose uptake, lactate levels, and metabolomic profiles. Coimmunoprecipitation or GST pulldown assays confirmed CCT2 interactions with aldolase A (ALDOA) and glutathione S-transferase P (GSTP1), while THP1 co-culture assays evaluated tumor immune crosstalk. CCT2 directly interacts with and stabilizes the glycolytic enzyme ALDOA, as shown by co-immunoprecipitation and metabolic assays revealing increased extracellular acidification rate, glucose uptake, and lactate production in HCC cells. Genetic depletion of CCT2 suppresses tumor cell proliferation and migration in vitro and inhibits tumor growth in vivo. Furthermore, co-culture and exosome treatment experiments reveal that CCT2 promotes M2 macrophage polarization and establishes an immunosuppressive tumor microenvironment through coordinated metabolic and exosome-mediated mechanisms. In mouse models, CCT2 knockdown significantly enhances the antitumor efficacy of PD-1 blockade. CCT2 stabilizes ALDOA and facilitates exosome-mediated immunosuppressive signaling, thereby linking metabolic reprogramming to immune evasion in HCC and supporting its potential as a mechanistically informed therapeutic target.

Hepatocellular carcinoma (HCC) is a highly aggressive primary liver malignancy characterized by limited therapeutic options and poor prognosis. Within the tumor microenvironment (TME), tumor-associated macrophages (TAMs) predominantly exhibit an M2-like phenotype, contributing to immune escape and tumor progression. Zymogen granule protein 16 (ZG16) has been reported to be downregulated in HCC, but its precise biological function and molecular mechanisms remain poorly understood. Therefore, we aimed to investigate the impact of ZG16 on HCC cell metastasis and TAM infiltration, as well as to elucidate its molecular mechanism. Gain- and loss-of-function assays were used to verify the effect of ZG16 on HCC cell metastasis, as well as the recruitment and M2 polarization of TAMs. The underlying mechanism of ZG16 was explored by immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC/MS) analysis, co-immunoprecipitation (co-IP) assay, and GST pull-down assay. Our results demonstrated that ZG16 overexpression significantly inhibited metastasis of HCC cells while also suppressing the recruitment and M2 polarization of TAMs, suggesting its dual role in both tumor cell-intrinsic and microenvironmental regulation. Notably, sorting nexin 9 (SNX9), a facilitator of HCC, was identified as a downstream target of ZG16. Mechanistically, we uncovered that ZG16 physically interacted with SNX9 and promoted its protein degradation through the ubiquitin-proteasome pathway. Functional rescue experiments provided compelling evidence that SNX9 overexpression effectively counteracted ZG16-mediated suppression of both HCC progression and TAM M2 polarization. Further mechanism exploration confirmed that ZG16 promoted the ubiquitination and degradation of SNX9 by recruiting itchy E3 ubiquitin protein ligase (ITCH). Our findings certify that ZG16 suppresses tumor progression and M2 polarization of TAMs in HCC through ITCH-mediated ubiquitination and subsequent degradation of SNX9. The ZG16/ITCH/SNX9 axis may represent an important regulatory pathway and potential therapeutic target for HCC.

The aggregation of α-synuclein (α-syn) is a central event in Parkinson's disease (PD) pathogenesis. However, the cellular factors that initiate and accelerate the process are not fully understood. Synaptogyrin-3 (SYNGR3) is a synaptic vesicle protein whose role in α-syn pathology remains unexplored. This study investigated whether SYNGR3 is a key factor triggering the pathological process of PD. This study investigated the expression of SYNGR3 in the brains of transgenic A53T α-syn mutant mouse line M83 (TgA53T) PD model mice using Western blot. The direct interaction between SYNGR3 and α-syn was assessed by GST pull-down assays. This study examined the effect of SYNGR3 on α-syn aggregation kinetics and fibril stability invitro through the thioflavin T (Th T) assays and proteinase K (PK) digestion. By overexpressing or knocking down SYNGR3 in HEK-293 cells stably transfected with α-syn, primary neurons, and TgA53T mice, the effects of enhanced or deficient function of SYNGR3 on α-syn pathology, synaptic integrity, mitochondrial function, and motor behavior were evaluated. SYNGR3 levels were significantly elevated in an age-dependent manner in the striatum of TgA53T mice. The study found that SYNGR3 directly interacts with the central region of α-syn and accelerates its aggregation into fibrils that are more resistant to PK digestion. Overexpression of SYNGR3 exacerbated α-syn aggregation, synaptic protein loss, mitochondrial dysfunction, and apoptosis in cellular models. Invivo, SYNGR3 intensified α-syn pathology, dopaminergic neurodegeneration, and PD-like motor deficits. Conversely, knockdown of SYNGR3 effectively alleviated these pathological and behavioral impairments. This study identifies SYNGR3 as a novel and critical promoter of α-syn aggregation and neurotoxicity. These findings establish SYNGR3 as a key contributor to PD pathogenesis and highlight its potential as a therapeutic target for intervention.

RCN2 facilitates esophageal squamous cellular carcinoma metastasis and cisplatin resistance through UBR5-mediated PPP2CA ubiquitination and degradation.

Arthrinium phaeospermum is the primary pathogen responsible for shoot blight disease in Bambusa pervariabilis × Dendrocalamopsis grandis. Previous research identified ApCtf1β, a key gene involved in pathogenicity encoding a cutinase transcription factor involved in pathogenicity, as essential for infecting hybrid bamboo. However, the interacting target proteins and functions of ApCtf1β within the fungus remain unclear, limiting the comprehensive understanding of A. phaeospermum's pathogenic pathways. Therefore, this study employed yeast two-hybrid, luciferase protein complementation, and GST pull-down assays to detect and confirm ApCtf1β-interacting proteins BDPH1 and BDEUL12. Further bioinformatics analyses of these proteins were conducted. Using Agrobacterium-mediated fungal genetic transformation, we generated BDPH1 and BDEUL12 overexpression transformants, gene-silenced transformants, as well as Apctf1β-BDPH1 and Apctf1β-BDEUL12 co-expression transformants. Functional studies of these interacting proteins were performed at different developmental stages, examining gene expression, hyphal growth rate, spore production, chemical susceptibility, pathogenicity and ubiquitination function. The results indicated that BDPH1 and BDEUL12 are closely associated with the virulence of A. phaeospermum, with the BDEUL12 gene exerting a more potent effect on virulence. This study provides a foundation for further elucidating the molecular mechanisms underlying the pathogenicity of A. phaeospermum.

Promotion of hepatocellular carcinoma by small nuclear ribonucleoprotein polypeptide F through upregulation of transmembrane P24 trafficking protein 2 and triggering of the cGAS-STING pathway.

Metabolic‒epigenetic crosstalk critically orchestrates hepatocellular carcinoma (HCC) pathogenesis. Deciphering the precise mechanism underlying epigenetic remodeling and metabolic reprogramming in HCC may lead to novel treatment paradigms, however, the key mechanisms remain elusive. RT-qPCR, western blotting and tissue microarrary Immunohistochemistry were used to detect the expression of RasGEF domain family member 1B (RASGEF1B) in HCC and normal liver tissues. Transcriptome sequencing and high-resolution untargeted metabolomics were integrated to identify the downstream regulatory mechanism through which RASGEF1B inhibited the HCC progression. Epigenetic regulation was investigated using methylation-specific PCR and luciferase reporter assays. Bioinformatic prediction and molecular docking suggested a functional interplay among RASGEF1B, ALDH7A1, and BMI1, which was experimentally confirmed through coimmunoprecipitation, GST pull-down, and immunofluorescence assays. Protein stability and ubiquitination status of ALDH7A1 were examined using cycloheximide, immunoprecipitation assay, and an in vitro reconstituted ubiquitination system. In this study, the antitumor role of RASGEF1B was confirmed in vitro and in vivo. Transcriptomic profiling revealed that RASGEF1B overexpression significantly reduced the snail family transcriptional repressor 1 (SNAI1), a master regulator of the epithelial-mesenchymal transition. Untargeted metabolomics revealed that RASGEF1B promoted SNAI1 DNA methylation through Betaine-mediated methionine metabolic reprogramming. Further analysis confirmed that RASGEF1B competitively protected the ALDH7A1 protein from BMI1-dependent ubiquitination, thereby elevating cellular Betaine levels in HCC. This study revealed that RASGEF1B inhibited SNAI1 to suppress HCC through metabolite‒epigenetic crosstalk. Our findings potentially offer a new perspective on the classical RAS signaling framework, uncovering a metabolic‒epigenetic axis as an innovative therapeutic approach for improving clinical outcomes in patients with HCC.

A wide range of species contract a haemorrhagic disease caused by Bluetongue virus (BTV), which is transmitted by hematophagous midges of Culicoides species. The virus shows variation in its pathogenicity and virulence between the mammalian and insect hosts, implicating the role of host proteins in its life cycle. NS3, a versatile protein of BTV, performs a major role in viral egress by acting as perforin. Multifunctional roles of NS3 have also been reported in viral pathogenesis, but there are critical gaps in explaining the exact mechanisms of its crosstalk with the host, creating the space for NS3-host interaction studies. In this study, we report the screening of host proteins interacting with NS3 protein of BTV using yeast-two hybrid (Y2H) assay. An ovine cDNA library derived from sheep lung and the truncated NS3 (NS3 t ) bait were constructed and evaluated for competency parameters. Following stringent screening, 37 putative host interaction partners were identified, of which eight proteins were selected for validation using alternative yeast-based assays; seven of these interactions were confirmed as positive. NAP1L1/NS3 interaction was further verified with GST pull-down, co-immunoprecipitation and mammalian two-hybrid assays. The results indicate that NAP1L1, a protein involved in nucleosome assembly and cell cycle regulation, interacts with the BTV NS3 protein.

The aggressive cancer known as pancreatic ductal adenocarcinoma (PDAC) has a remarkably poor response to treatment, especially gemcitabine (GEM). As a member of the noncanonical IκB kinase family, inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBKE) is known to regulate tumor progression in multiple cancer types. However, its functional role in modulating chemosensitivity and its impact on cell death pathways in PDAC remain unclear. We examined IKBKE expression in patient tumor tissues and publicly available databases and assessed its prognostic value to investigate its function in PDAC chemoresistance. Loss-of-function approaches, including shRNA knockdown and pharmacological inhibition using amlexanox, were employed in vivo and in vitro. Furthermore, cytotoxicity along with cell death patterns induced by GEM were assessed through flow cytometry, electron microscopy, and CCK-8 assays. Co-IP and GST pull-down assays were applied to determine whether IKBKE interacts with gasdermin E (GSDME), the executioner protein of pyroptosis. Moreover, in vitro kinase assays, phosphorylation mass spectrometry, and site-specific gain-/loss-of-function mutant functional experiments were conducted to investigate the specific pathways via which IKBKE affects GSDME-mediated pyroptosis. The therapeutic potential of IKBKE targeting was further validated using patient-derived tumor organoids and xenograft models. IKBKE was significantly elevated in PDAC tissues and was closely linked to worse clinical outcomes. Functional studies of the signaling pathway showed that via the activation of the protein kinase B/glycogen synthase kinase-3β signaling pathway, IKBKE facilitates the aggressive phenotype of PDAC cells. IKBKE inhibition or silencing increased GEM sensitivity and caused caspase-3/GSDME-dependent pyroptosis in PDAC cells. Co-IP and GST pull-down analysis revealed IKBKE–GSDME interaction. Further in vitro cleavage and kinase assays, mass spectrometry analysis, and mutant functional studies showed that IKBKE inhibits pyroptosis by phosphorylating GSDME at Thr6, thereby hindering its cleavage by caspase-3. Treatment with amlexanox markedly suppressed tumor growth and synergized with GEM to enhance anti-tumor efficacy in organoid models by targeting IKBKE. Our findings identify IKBKE as a key regulator of chemotherapy-induced pyroptosis in PDAC. Specifically, IKBKE phosphorylates GSDME at Thr6, which alters its conformation, thereby impeding caspase-3–mediated cleavage, ultimately contributing to GEM chemoresistance. IKBKE targeting reverses GEM resistance and enhances tumor immunogenic cell death via the caspase-3/GSDME pathway, supporting IKBKE inhibition as a promising therapeutic strategy for PDAC.

Protein prenylation, a key post-translational modification which can be mediated by geranylgeranyltransferases (GGTs), has been implicated in plant stress responses, but its role and mechanism in cotton remain elusive. Here, we demonstrate that the β-subunit of type I protein geranylgeranyltransferase in Gossypium hirsutum (GhGGB) negatively regulates drought resistance. Virus-induced gene silencing (VIGS) of GhGGB significantly enhanced drought survival rates by 40% compared to controls, accompanied by improved physiological indices of drought tolerance. We identified two cotton Rho-related GTPases(ROP), GhROP1 and GhROP8, as interacting partners of GhGGB through Bimolecular Fluorescence Complementation (BiFC) and GST pull-down assays. The interaction affinity was determined by the C-terminal CaaL prenylation motif. Silencing GhROP1 or GhROP8 similarly enhanced drought tolerance, with enhanced survival rates of 30% and 37%, respectively. Mechanistically, silencing GhGGB, GhROP1, and GhROP8 altered the transcriptional landscape of hormonal pathways, characterized by the upregulation of key ABA biosynthesis and signaling genes alongside the downregulation of auxin-related biosynthetic and responsive genes. Our findings suggest that the GhGGB-GhROP1/8 module functions as a negative regulator of drought resistance, presumably through the prenylation of ROPs to coordinate the balance between ABA and auxin pathways at the transcriptional level. Taken together, this module presents a promising breeding strategy for developing high-yielding, drought-resilient cotton cultivars without growth penalties.