Ubiquitination Degradation Research Articles

CircXPO6 promotes breast cancer progression through competitively inhibiting the ubiquitination degradation of c-Myc.

The number of breast cancer (BC) patients is increasing year by year, which is severely endangering to human life and health. c-Myc is a transcription factor, studies have shown that it is a very significant factor in tumor progression, but how it is regulated in BC is still not well understood. Here, we used the RIP microarray sequencing to confirm circXPO6, which had a high affinity with c-Myc and highly expressed in triple-negative breast cancer (TNBC) tissues and cells. CircXPO6 overexpression promoted tumor growth in vivo and in vitro. Furthermore, circXPO6 largely promoted the expression of genes related to glucose metabolism, such as GLUT1, HK2, and MCT4 in TNBC cells. Finally, high levels of circXPO6 expression were found to be closely associated with malignant pathological factors, such as tumor size, lymph node metastasis, TNM staging, and histopathological grading of TNBC. Mechanistically, circXPO6 interacted with c-Myc to prevent speckle-type POZ-mediated c-Myc ubiquitination and degradation, thus promoting TNBC progression. Through the regulation of c-Myc-mediated signal transduction, circXPO6 plays a key role in TNBC progresses. This discovery can provide new ideas for TNBC molecular targeted therapy.

The Ubiquitin E3 Ligase FBXO33 Suppresses Stem Cell-Like Properties and Metastasis in Non-Small-Cell Lung Cancer by Promoting Ubiquitination and Degradation of Myc.

Non-small cell lung cancer (NSCLC) is a malignant form of lung cancer, and its prognosis could be improved by identifying key therapeutic targets. Thus, this study investigates the potential role of F-box Only Protein 33 (FBXO33) in NSCLC. The expression levels of FBXO33 in NSCLC were determined using University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) prediction, and its correlation with overall survival (OS) was analyzed via Kaplan-Meier survival analysis. These results were validated through quantitative polymerase chain reaction (qPCR), western blot (WB), and immunofluorescence (IF). We modulated FBXO33 expression by overexpression or knockdown and analyzed its effects on cell growth, proliferation, migration, invasion, and stemness characteristics in NSCLC cell lines. Additionally, the interaction between FBXO33 and Myelocytomatosis (Myc) and its impact on Myc ubiquitination were examined. An in vivo NSCLC xenograft model was used to corroborate the in vivo experimental results. The study found an inverse correlation between FBXO33 expression in NSCLC and OS. Lower FBXO33 expression enhanced the growth, proliferation, migration, invasion, and stemness characteristics of NSCLC cell lines. FBXO33 interacted with Myc to promote its ubiquitination and subsequent degradation, which suppressed NSCLC development. FBXO33 is expressed at low levels in NSCLC and correlates with lower OS. Overexpression of FBXO33 promotes Myc ubiquitination and degradation and inhibits tumor cell proliferation, migration and stemness characteristics, thereby impeding NSCLC progression.

BRD4-specific PROTAC inhibits basal-like breast cancer partially through downregulating KLF5 expression

Interest in the use of proteolysis-targeting chimeras (PROTACs) in cancer therapy has increased in recent years. Targeting bromodomain and extra terminal domain (BET) proteins, especially bromodomain-containing protein 4 (BRD4), has shown inhibitory effects on basal-like breast cancer (BLBC). However, the bioavailability of BRD4 PROTACs is restricted by their non-selective biodegradability and low tumor-targeting ability. We demonstrated that 6b (BRD4 PROTAC) suppresses BLBC cell growth by targeting BRD4, but not BRD2 and BRD3, for cereblon (CRBN)-mediated ubiquitination and proteasomal degradation. Compound 6b also inhibited expression of Krüppel-like factor 5 (KLF5) transcription factor, a key oncoprotein in BLBC, controlled by BRD4-mediated super-enhancers. Moreover, 6b inhibited HCC1806 tumor growth in a xenograft mouse model. The combination of 6b and KLF5 inhibitors showed additive effects on BLBC. These results suggest that BRD4-specific PROTAC can effectively inhibit BLBC by downregulating KLF5, and that 6b has potential as a novel therapeutic drug for BLBC.

GJB2 Promotes HCC Progression by Activating Glycolysis Through Cytoplasmic Translocation and Generating a Suppressive Tumor Microenvironment Based on Single Cell RNA Sequencing.

Despite substantial breakthroughs in the treatment of hepatocellular carcinoma (HCC) in recent years, many patients are diagnosed in the middle or late stages, denying them the option for surgical excision. Therefore, it is of great importance to find effective therapeutic targets of HCC. In this study, it is found that Gap junction protein beta-2 (GJB2) is highly enriched in malignant cells based on single-cell RNA sequencing and higher expression of GJB2 indicates a worse prognosis. The localization of GJB2 in HCC cancer cells is changed compared with normal liver tissue. In cancer cells, GJB2 tends to be located in the cytoplasm and nucleus, while in normal tissues, GJB2 is mainly located on the cell membrane. GJB2 is related to glycolysis, promoting NF-κB pathway via inducing the ubiquitination degradation of IκBa, and activating HIF-1α/GLUT-1/PD-L1 pathway. In addition, GJB2 knockdown reshapes tumor immune microenvironment and Salvianolic acid B inhibits the activity of GJB2. In conclusion, GJB2 promotes HCC progression by activating glycolysis through cytoplasmic translocation and generating a suppressive tumor microenvironment. Salvianolic acid B inhibits the expression of GJB2 and enhances the sensitivity of anti-PD1 therapy, which may provide insights into the development of novel combination therapeutic strategies for HCC.

The deubiquitinase USP15 drives malignant progression of gastric cancer through glucose metabolism remodeling

BackgroundUbiquitin-specific protease 15 (USP15) exhibits amplifications in various tumors, including gastric cancer (GC), yet its biological function and mechanisms in GC progression remain elusive.MethodsHere, we established stable USP15 knockdown or overexpression GC cell lines and explored the potential mechanism of USP15 in GC. Besides, we also identified interacting targets of USP15.ResultsUSP15 knockdown significantly impeded cell proliferation, invasion, epithelial-mesenchymal transition, and distal colonization in xenograft models, while enhancing oxaliplatin's antitumor effect. USP15 was involved in ubiquitination modification of glycolytic regulators. Silencing of USP15 suppressed glycolytic activity and impaired mitochondrial functions. Interference with USP15 expression reversed tumor progression and distal colonization in vivo. HKDC1 and IGF2BP3 were found as core interacting targets of USP15, and HKDC1 was identified as a substrate for ubiquitination modification by USP15, whereby USP15 regulated glucose metabolism activity by inhibiting the ubiquitination degradation of HKDC1.ConclusionsOur study unveiled aberrantly high expression of USP15 in GC tissues, correlating with malignant progression and nonresponse to neoadjuvant therapy. USP15 inhibitors, if developed, could be effective in promoting chemotherapy through glucose metabolism remodeling.

USP11 promotes renal tubular cell pyroptosis and fibrosis in UUO mice via inhibiting KLF4 ubiquitin degradation.

The pyroptosis of renal tubular epithelial cells leads to tubular loss and inflammation and then promotes renal fibrosis. The transcription factor Krüppel-like factor 4 (KLF4) can bidirectionally regulate the transcription of target genes. Our previous study revealed that sustained elevation of KLF4 is responsible for the transition of acute kidney injury (AKI) into chronic kidney disease (CKD) and renal fibrosis. In this study, we explored the upstream mechanisms of renal tubular epithelial cell pyroptosis from the perspective of posttranslational regulation and focused on the transcription factor KLF4. Mice were subjected to unilateral ureteral obstruction (UUO) surgery and euthanized on D7 or D14 for renal tissue harvesting. We showed that the pyroptosis of renal tubular epithelial cells mediated by both the Caspase-1/GSDMD and Caspase-3/GSDME pathways was time-dependently increased in UUO mouse kidneys. Furthermore, we found that the expression of the transcription factor KLF4 was also upregulated in a time-dependent manner in UUO mouse kidneys. Tubular epithelial cell-specific Klf4 knockout alleviated UUO-induced pyroptosis and renal fibrosis. In Ang II-treated mouse renal proximal tubular epithelial cells (MTECs), we demonstrated that KLF4 bound to the promoter regions of Caspase-3 and Caspase-1 and directly increased their transcription. In addition, we found that ubiquitin-specific protease 11 (USP11) was increased in UUO mouse kidneys. USP11 deubiquitinated KLF4. Knockout of Usp11 or pretreatment with the USP11 inhibitor mitoxantrone (3 mg/kg, i.p., twice a week for two weeks before UUO surgery) significantly alleviated the increases in KLF4 expression, pyroptosis and renal fibrosis. These results demonstrated that the increased expression of USP11 in renal tubular cells prevents the ubiquitin degradation of KLF4 and that elevated KLF4 promotes inflammation and renal fibrosis by initiating tubular cell pyroptosis.

Berbamine promotes ferroptosis of esophageal squamous cell carcinoma by facilitating USP51-mediated GPX4 ubiquitination and degradation

Esophageal cancer ranks among the most prevalent malignant tumors globally. The prognosis for esophageal squamous cell carcinoma remains poor, with a 5-year survival rate below 20 % due to limited advances in therapy. Ferroptosis, a novel form of iron-dependent lipid peroxidation-driven regulated cell death (RCD), shows significant promise in cancer treatment. Berbamine (BBM), a natural bisbenzylisoquinoline alkaloid derived from Berberis amurensis, exhibits anti-tumor effects against various cancers, yet its impact on esophageal cancer remains to be elucidated. This study aimed to explore the role of BBM in inducing ferroptosis in the treatment of esophageal cancer, focusing on its molecular mechanisms. Gene set enrichment analysis(GSEA) analysis highlighted the potential of BBM as an anti-cancer agent through ferroptosis induction. We found that BBM inhibited growth and epithelial-mesenchymal transition (EMT) in esophageal cancer cell lines, promoting Fe accumulation, ROS, and malondialdehyde (MDA) production, thereby triggering cell death. These suppressive effects were successfully reversed by Ferrostatin-1 (Fer-1). Mechanistically, BBM decreased deubiquitination enzyme USP51 levels, leading to ubiquitin degradation and glutathione peroxidase 4(GPX4) instability, and it stimulated ferroptosis. The Overexpression of USP51 mitigated the downregulation of GPX4 induced by BBM.BBM significantly inhibited tumor xenograft growth in nude mice. This discovery positions BBM as a promising therapeutic candidate for the treatment of esophageal cancer.

CLG promotes mTOR/ULK1 pathway-mediated autophagy to inhibit OS development by inhibiting TRAF6-mediated FLT3 ubiquitination.

Corilagin (CLG) has antitumor activities in certain human malignant cancers. Herein, the effects and mechanisms of CLG on osteosarcoma (OS) were investigated. OS cell viability and proliferation were detected by MTT and colony formation assay. Cell cycle and apoptosis were examined using flow cytometry. The interaction between TRAF6 and FLT3 was investigated using a co-immunoprecipitation assay. Results demonstrated that CLG treatment inhibited OS cell viability and proliferation but promoted OS cell autophagy and apoptosis in a concentration-dependent manner. Mechanically, CLG inhibited TRAF6-mediated FLT3 ubiquitination degradation. TRAF6 overexpression abolished the effects of CLG on OS cell proliferation, autophagy, and apoptosis. Finally, CLG administration inhibited OS tumor growth in mice by inducing autophagy-dependent apoptosis. Taken together, CLG inhibited OS progression by facilitating mTOR/ULK1 pathway-mediated autophagy through inhibiting TRAF6-mediated FLT3 ubiquitination, which indicated that CLG was a promising candidate for the treatment of OS.

MicroRNA-128-3p Affects Neuronal Apoptosis and Neurobehavior in Cerebral Palsy Rats by Targeting E3 Ubiquitin-Linking Enzyme Smurf2 and Regulating YY1 Expression.

This study was dedicated to investigating the effects of microRNA-128-3p (miR-128-3p) on neuronal apoptosis and neurobehavior in cerebral palsy (CP) rats via the Smurf2/YY1 axis.In vivo modeling of hypoxic-ischemic (HI) CP was established in neonatal rats. Neurobehavioral tests (geotaxis reflex, cliff avoidance reaction, and grip test) were measured after HI induction. The HI-induced neurological injury was evaluated by HE staining, Nissl staining, TUNEL staining, immunohistochemical staining, and RT-qPCR. The expression of miR-128-3p, Smurf2, and YY1 was determined by RT-qPCR and western blot techniques. Moreover, primary cortical neurons were used to establish the oxygen and glucose deprivation (OGD) model in vitro, cell viability was detected by CCK-8 assay, neuronal apoptosis was assessed by flow cytometry and western blot, and the underlying mechanism between miR-128-3p, Smurf2 and YY1 was verified by bioinformatics analysis, dual luciferase reporter assay, RIP, Co-IP, ubiquitination assay, western blot, and RT-qPCR.In vivo, miR-128-3p and YY1 expression was elevated, and Smurf2 expression was decreased in brain tissues of hypoxic-ischemic CP rats. Downregulation of miR-128-3p or overexpression of Smurf2 improved neurobehavioral performance, reduced neuronal apoptosis, and elevated Nestin and NGF expression in hypoxic-ischemic CP rats, and downregulation of Smurf2 reversed the effects of downregulation of miR-128-3p on neurobehavioral performance, neuronal apoptosis, and Nestin and NGF expression in hypoxic-ischemic CP rats, while overexpression of YY1 reversed the effects of Smurf2 on neurobehavioral performance, neuronal apoptosis, and Nestin and NGF expression in hypoxic-ischemic CP rats. In vitro, downregulation of miR-128-3p effectively promoted the neuronal survival, reduced the apoptosis rate, and decreased caspase3 protein expression after OGD, and overexpression of YY1 reversed the ameliorative effect of downregulation of miR-128-3p on OGD-induced neuronal injury. miR-128-3p targeted to suppress Smurf2 to lower YY1 ubiquitination degradation and decrease its expression.Inhibition of miR-128-3p improves neuronal apoptosis and neurobehavioral changes in hypoxic-ischemic CP rats by promoting Smurf2 to promote YY1 ubiquitination degradation and reduce YY1 expression.

Momordica charantia L.-derived exosome-like nanovesicles stabilize p62 expression to ameliorate doxorubicin cardiotoxicity

BackgroundDoxorubicin (DOX) is a first-line chemotherapeutic drug for various malignancies that causes cardiotoxicity. Plant-derived exosome-like nanovesicles (P-ELNs) are growing as novel therapeutic agents. Here, we investigated the protective effects in DOX cardiotoxicity of ELNs from Momordica charantia L. (MC-ELNs), a medicinal plant with antioxidant activity.ResultsWe isolated MC-ELNs using ultracentrifugation and characterized them with canonical mammalian extracellular vesicles features. In vivo studies proved that MC-ELNs ameliorated DOX cardiotoxicity with enhanced cardiac function and myocardial structure. In vitro assays revealed that MC-ELNs promoted cell survival, diminished reactive oxygen species, and protected mitochondrial integrity in DOX-treated H9c2 cells. We found that DOX treatment decreased the protein level of p62 through ubiquitin-dependent degradation pathway in H9c2 and NRVM cells. However, MC-ELNs suppressed DOX-induced p62 ubiquitination degradation, and the recovered p62 bound with Keap1 promoting Nrf2 nuclear translocation and the expressions of downstream gene HO-1. Furthermore, both the knockdown of Nrf2 and the inhibition of p62-Keap1 interaction abrogated the cardioprotective effect of MC-ELNs.ConclusionsOur findings demonstrated the therapeutic beneficials of MC-ELNs via increasing p62 protein stability, shedding light on preventive approaches for DOX cardiotoxicity.

METTL3-mediated m6A modification of LINC00520 confers glycolysis and chemoresistance in osteosarcoma via suppressing ubiquitination of ENO1

Chemoresistance remains the main obstacle limiting the treatment of osteosarcoma, seriously affecting the prognosis of adolescent patients with osteosarcoma. Recently, long non-coding RNAs (lncRNAs) were reported to be involved in chemoresistance, while the mechanisms of lncRNAs underlying osteosarcoma resistance to chemotherapy remain elusive. Here, LINC00520 was identified as a novel cisplatin resistance-related lncRNA in osteosarcoma, and its high expression was associated with poor prognosis of osteosarcoma patients. Functionally, LINC00520 could potentiate osteosarcoma resistance to cisplatin in vitro and in vivo. Mechanistically, LINC00520 bound to ENO1 and upregulated ENO1 protein expression by blocking FBXW7-mediated ENO1 ubiquitination and proteasomal degradation, thereby promoting glycolysis and ultimately inducing cisplatin resistance in osteosarcoma. Furthermore, METTL3 could stabilize and upregulate LINC00520 in an m6A-YTHDF2-dependent manner in osteosarcoma. This study proposes a novel lncRNA-driven mechanism for cisplatin resistance in osteosarcoma, and offers a promising therapeutic strategy for reversing chemoresistance in osteosarcoma by targeting the METTL3/LINC00520/ENO1/glycolysis axis.

Curcumol promotes ferroptosis of colon cancer by targeting the ubiquitination and degradation of GPX4

Background and aimColon cancer (CC) is one of the common malignant tumors in the digestive tract, the prognosis of CC patients has never been satisfying. A Ferrous-dependent form that regulates cell death, plays a key role in cancer development. As a core regulator of ferroptosis, GPX4 has become a potential molecular target for the development of antitumor drugs. Curcumol (Cur), a sesquiterpene natural product, it has significant anti-tumor effect. However, whether Cur mediates ferroptosis in colon cancer and its mechanism are still unclear. This study aimed to investigate the underlying mechanisms of Cur anti-tumor. Experimental procedureBy investigating the Cancer Genome Atlas (TCGA) database and tissue immunofluorescence staining was also used to detect the levels of GPX4 protein in CC and matching paracancerous tissues. The anti-CC and pro-ferroptosis effects of Cur were detected in the vivo and vitro experiment. The interaction between Cur and GPX4 was predicted. In addition, the potential mechanism of Cur anti-CC was further discussed. Co-immunoprecipitation was used to confirm Cur-mediated GPX4 ubiquitination. Results and conclusionGPX4 was upregulated in CC tissue and was correlated with poor survival of patients. Cur inhibited the proliferation of CC cells, accompanied by regulating Fe2+ overload, reactive oxygen species (ROS) formation, malondialdehyde (MDA) production and superoxide dismutase (SOD) consumption. Furthermore, GPX4 was predicted and verified as the direct target of Cur by molecular docking and structure-based virtual prediction. Meanwhile, Cur could promote the ubiquitination-mediated degradation of GPX4, induce ferroptosis in CC cells and regulate the expression of ferroptosis-related protein FTH1 and TfR1. In addition, when GPX4 was overexpressed (GPX4-OE), the inhibitory effect of Cur on the expression of GPX4 and ferroptosis-related protein FTH1 and the promotion of TfR1 expression were abolished. Cur could inhibit CC by increasing the ubiquitination degradation level of GPX4 to induce ferroptosis in CC cells.

Gubi decoction mitigates knee osteoarthritis via promoting chondrocyte autophagy through METTL3-mediated ATG7 m6A methylation.

Knee osteoarthritis (KOA) is a chronic joint disease that significantly affects the health of the elderly. As an herbal remedy, Gubi decoction (GBD) has been traditionally used for the treatment of osteoarthritis-related syndromes. However, the anti-KOA efficacy and mechanism of GBD remain unclear. This study aimed to experimentally investigate the anti-KOA efficacy and the underlying mechanism of GBD. The medial meniscus (DMM) mice model and IL-1β-stimulated chondrocytes were, respectively, constructed as invivo and invitro models of KOA to evaluate the osteoprotective effect and molecular mechanism of GBD. The UPLC-MS/MS analysis showed that GBD mainly contained pinoresinol diglucoside, rehmannioside D, hesperidin, liquiritin, baohuoside I, glycyrrhizic acid, kaempferol and tangeretin. Animal experiment showed that GBD could alleviate articular cartilage destruction and recover histopathological alterations in DMM mice. In addition, GBD inhibited chondrocyte apoptosis and restored DMM-induced dysregulated autophagy evidenced by the upregulation of ATG7 and LC3 II/LC3 I but decreased P62 level. Mechanistically, METTL3-mediated m6A modification decreased the expression of ATG7 in DMM mice, as it could be significantly attenuated by GBD. METTL3 overexpression significantly counteracted the protective effect of GBD on chondrocyte autophagy. Further research showed that GBD promoted proteasome-mediated ubiquitination degradation of METLL3. Our findings suggest that GBD could act as a protective agent against KOA. The protective effect of GBD may result from its promotion on chondrocyte autophagy by suppressing METTL3-dependent ATG7 m6A methylation.

CHIP-mediated ubiquitin degradation of BCAT1 regulates glioma cell proliferation and temozolomide sensitivity

Glioma, a malignant and infiltrative neoplasm of the central nervous system, poses a significant threat due to its high mortality rates. Branched-chain amino acid transaminase 1 (BCAT1), a key enzyme in branched-chain amino acid (BCAA) catabolism, exhibits elevated expression in gliomas and correlates strongly with poor prognosis. Nonetheless, the regulatory mechanisms underlying this increased BCAT1 expression remains incompletely understood. In this study, we reveal that ubiquitination at Lys360 facilitates BCAT1 degradation, with low ubiquitination levels contributing to high BCAT1 expression in glioma cells. The Carboxyl terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase, interacts with BCAT1 via its coiled-coil (CC) domain, promoting its K48-linkage ubiquitin degradation through proteasomal pathway. Moreover, CHIP-mediated BCAT1 degradation induces metabolic reprogramming, and impedes glioma cell proliferation and tumor growth both in vitro and in vivo. Furthermore, a positive correlation is observed between low CHIP expression, elevated BCAT1 levels, and unfavorable prognosis among glioma patients. Additionally, we show that the CHIP/BCAT1 axis enhances glioma sensitivity to temozolomide by reducing glutathione (GSH) synthesis and increasing oxidative stress. These findings underscore the critical role of CHIP/BCAT1 axis in glioma cell proliferation and temozolomide sensitivity, highlighting its potential as a diagnostic marker and therapeutic target in glioma treatment.

PRMT6 facilitates EZH2 protein stability by inhibiting TRAF6-mediated ubiquitination degradation to promote glioblastoma cell invasion and migration

Invasion and migration are the key hallmarks of cancer, and aggressive growth is a major factor contributing to treatment failure and poor prognosis in glioblastoma. Protein arginine methyltransferase 6 (PRMT6), as an epigenetic regulator, has been confirmed to promote the malignant proliferation of glioblastoma cells in previous studies. However, the effects of PRMT6 on glioblastoma cell invasion and migration and its underlying mechanisms remain elusive. Here, we report that PRMT6 functions as a driver element for tumor cell invasion and migration in glioblastoma. Bioinformatics analysis and glioma sample detection results demonstrated that PRMT6 is highly expressed in mesenchymal subtype or invasive gliomas, and is significantly negatively correlated with their prognosis. Inhibition of PRMT6 (using PRMT6 shRNA or inhibitor EPZ020411) reduces glioblastoma cell invasion and migration in vitro, whereas overexpression of PRMT6 produces opposite effects. Then, we identified that PRMT6 maintains the protein stability of EZH2 by inhibiting the degradation of EZH2 protein, thereby mediating the invasion and migration of glioblastoma cells. Further mechanistic investigations found that PRMT6 inhibits the transcription of TRAF6 by activating the histone methylation mark (H3R2me2a), and reducing the interaction between TRAF6 and EZH2 to enhance the protein stability of EZH2 in glioblastoma cells. Xenograft tumor assay and HE staining results showed that the expression of PRMT6 could promote the invasion of glioblastoma cells in vivo, the immunohistochemical staining results of mouse brain tissue tumor sections also confirmed the regulatory relationship between PRMT6, TRAF6, and EZH2. Our findings illustrate that PRMT6 suppresses TRAF6 transcription via H3R2me2a to enhance the protein stability of EZH2 to facilitate glioblastoma cell invasion and migration. Blocking the PRMT6-TRAF6-EZH2 axis is a promising strategy for inhibiting glioblastoma cell invasion and migration.

MLN4924 alleviates autoimmune myocarditis by promoting Act1 degradation and blocking Act1-mediated mRNA stability

BackgroundProlonged exposure to interleukin-17A (IL-17A) can induce autoimmune myocarditis, and MLN4924, an inhibitor of NEDD8 activating enzyme (NAE), has been reported to effectively suppress various inflammatory reactions. However, the effects of MLN4924 in IL-17A-mediated inflammation associated with autoimmune myocarditis remain uncertain. MethodsAn experimental autoimmune myocarditis (EAM) model was established and treated with MLN4924. The inflammation degree of heart tissues was assessed histopathologically. The expression levels of inflammatory cytokines and chemokines were measured using ELISA and RT-qPCR, respectively. Additionally, the interaction of biomacromolecules was detected through co-immunoprecipitation (Co-IP) and RNA immunoprecipitation (RIP). ResultsMLN4924 could attenuate IL-17A-induced inflammation. In the in vivo studies, MLN4924 treatment improved inflammatory responses, diminished immune cell infiltration and tissue fibrosis, and reduced the secretion of various inflammatory cytokines in serum, including IL-1β, IL-6, TNF-α, and MCP-1. In vitro experiments further corroborated these findings, showing that MLN4924 treatment reduced the secretion and transcription of pro-inflammatory factors, particularly MCP-1. Mechanistically, we confirmed that MLN4924 promoted Act1 ubiquitination degradation and disrupted Act1′s interaction with IL-17R, thereby impeding the formation of the IL-17R/Act1/TRAF6 complex and subsequent activation of TAK1, c-Jun, and p65. Moreover, MLN4924 interfered with Act1′s binding to mRNA, resulting in mRNA instability. ConclusionsIn conclusion, MLN4924 effectively alleviated inflammatory symptoms in EAM by disrupting the interaction between IL and 17R and Act1, thereby reducing Act1-mediated mRNA stability and resulting in decreased expression of pro-inflammatory factors.

Cinnamaldehyde activates AMPK/PGC-1α pathway via targeting GRK2 to ameliorate heart failure

BackgroundAccording to recent research, treating heart failure (HF) by inhibiting G protein-coupled receptor kinase 2 (GRK2) to improve myocardial energy metabolism has been identified as a potential approach. Cinnamaldehyde (CIN), a phenylpropyl aldehyde compound, has been demonstrated to exhibit beneficial effects in cardiovascular diseases. However, whether CIN inhibits GRK2 to ameliorate myocardial energy metabolism in HF is still unclear. PurposeThis study examines the effects of CIN on GRK2 and myocardial energy metabolism to elucidate its underlying mechanism to treat HF. MethodsThe isoproterenol (ISO) induced HF model in vivo and in vitro were constructed using Sprague-Dawley (SD) rats and primary neonatal rat cardiomyocytes (NRCMs). Based on this, the effects of CIN on myocardial energy metabolism and GRK2 were investigated. Additionally, validation experiments were conducted after interfering and over-expressing GRK2 in ISO-induced NRCMs to verify the regulatory effect of CIN on GRK2. Furthermore, binding capacity between GRK2 and CIN was explored by Cellular Thermal Shift Assay (CETSA) and Microscale Thermophoresis (MST). ResultsIn vivo and in vitro, CIN significantly improved HF as demonstrated by reversing abnormal changes in myocardial injury markers, inhibiting myocardial hypertrophy and decreasing myocardial fibrosis. Additionally, CIN promoted myocardial fatty acid metabolism to ameliorate myocardial energy metabolism disorder by activating AMPK/PGC-1α signaling pathway. Moreover, CIN reversed the inhibition of myocardial fatty acid metabolism and AMPK/PGC-1α signaling pathway by GRK2 over-expression in ISO-induced NRCMs. Meanwhile, CIN had no better impact on the stimulation of cardiac fatty acid metabolism and the AMPK/PGC-1α signaling pathway in ISO-induced NRCMs when GRK2 was disrupted. Noticeably, CETSA and MST confirmed that CIN binds to GRK2 directly. The binding of CIN and GRK2 promoted the ubiquitination degradation of GRK2 mediated by murine double mimute 2. ConclusionThis study demonstrates that CIN exerts a protective intervention in HF by targeting GRK2 and promoting its ubiquitination degradation to activate AMPK/PGC-1α signaling pathway, ultimately improving myocardial fatty acid metabolism.

Deubiquitinating enzyme mutagenesis screens identify a USP43-dependent HIF-1 transcriptional response

The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.

PLUNC inhibits invasion and metastasis in nasopharyngeal carcinoma by inhibiting NLRP3 inflammasome activation

Nasopharyngeal carcinoma (NPC) is a malignant tumor that occurs in the nasopharynx. Palate, lung, and nasal epithelium clone (PLUNC) has been identified as an early secreted protein that is specifically expressed in the nasopharynx. The aim of this study was to determine the role and mechanism of PLUNC in NPC. We used mRNA sequencing (seq) combined with ribosome-nascent chain complex (RNC)-seq to determine the biological role of PLUNC. The expression of epithelial-to-mesenchymal transition (EMT)-related molecules was detected by western blotting. Then, cell migration and invasion were detected by wound healing and Transwell chamber assays. NPC cells were injected into the tail vein of nude mice to explore the biological role of PLUNC in vivo. The sequencing results showed that PLUNC inhibited the progression of NPC and its expression was correlated with that of NOD-like receptors. Experiments confirmed that PLUNC inhibited the invasion and metastasis of NPC cells by promoting the ubiquitination degradation of NLRP3. PLUNC overexpression in combination with the treatment by MCC950, an inhibitor of NLRP3 inflammasome activation, was most effective in inhibiting NPC invasion and metastasis. In vivo experiments also confirmed that the combination of PLUNC overexpression and MCC950 treatment effectively inhibited the lung metastasis of NPC cells. In summary, our research suggested that PLUNC inhibited the invasion and metastasis of NPC by inhibiting NLRP3 inflammasome activation, and targeting the PLUNC-NLRP3 inflammasome axis could provide a new strategy for the diagnosis and treatment of NPC patients.

SERPINH1 modulates apoptosis by inhibiting P62 ubiquitination degradation to promote bone metastasis of prostate cancer

SERPINH1 modulates apoptosis by inhibiting P62 ubiquitination degradation to promote bone metastasis of prostate cancer