Ubiquitin-proteasome Degradation Research Articles

HRD1-mediated ubiquitination of HDAC2 regulates PPARα-mediated autophagy and alleviates metabolic-associated fatty liver disease

BackgroundMetabolic-associated fatty liver disease (MAFLD) is a leading cause of chronic liver disease worldwide. Autophagy plays a pivotal role in lipid metabolism; however, the mechanism underlying the reduced autophagic activity in MAFLD remains elusive. MethodsAutophagy was monitored by TUNEL assay and immunofluorescence staining of LC3. The expression of autophagy-related proteins, PPARα, HDAC2, and HRD1 was detected by Western blot. The association between HDAC2 and PPARα promoter was assessed by chromatin immunoprecipitation (ChIP) and dual-luciferase assays, and the HRD1-mediated ubiquitin-proteasomal degradation of HDAC2 was detected by co-immunoprecipitation (co-IP). The in vitro findings were validated in a hypoxia-induced MAFLD mouse model. Histological changes, fibrosis, and apoptosis in liver tissues were detected by hematoxylin and eosin staining, Masson's trichrome staining, and TUNEL assay. The immunoreactivities of key molecules were examined by IHC analysis. ResultsHypoxia-suppressed autophagy in hepatocytes. Hypoxic exposure downregulated HRD1 and PPARα, while upregulating HDAC2 in hepatocytes. Overexpression of PPARα promoted hepatic autophagy, while knocking down HDAC2 or overexpressing HRD1 reduced hypoxia-suppressed autophagy in hepatocytes. Mechanistically, HDAC2 acted as a transcriptional repressor of PPARα, and HRD1 mediated the degradation of HDAC2 through the ubiquitin-proteasome pathway. Functional studies further showed that hypoxia-suppressed hepatic autophagy via the HRD1/HDAC2/PPARα axis in vitro and in vivo. ConclusionHRD1-mediated ubiquitination of HDAC2 regulates PPARα-mediated autophagy and ameliorates hypoxia-induced MAFLD.

BRD4 promotes gouty arthritis through MDM2-mediated PPARγ degradation and pyroptosis

BackgroundGouty arthritis (GA) is characterized by monosodium urate (MSU) crystal accumulation that instigates NLRP3-mediated pyroptosis; however, the underlying regulatory mechanisms have yet to be fully elucidated. The present research endeavors to elucidate the regulatory mechanisms underpinning this MSU-induced pyroptotic cascade in GA.MethodsJ774 cells were exposed to lipopolysaccharide and MSU crystals to establish in vitro GA models, whereas C57BL/6 J male mice received MSU crystal injections to mimic in vivo GA conditions. Gene and protein expression levels were evaluated using real-time quantitative PCR, Western blotting, and immunohistochemical assays. Inflammatory markers were quantified via enzyme-linked immunosorbent assays. Pyroptosis was evaluated using immunofluorescence staining for caspase-1 and flow cytometry with caspase-1/propidium iodide staining. The interaction between MDM2 and PPARγ was analyzed through co-immunoprecipitation assays, whereas the interaction between BRD4 and the MDM2 promoter was examined using chromatin immunoprecipitation and dual-luciferase reporter assays. Mouse joint tissues were histopathologically evaluated using hematoxylin and eosin staining.ResultsIn GA, PPARγ was downregulated, whereas its overexpression mitigated NLRP3 inflammasome activation and pyroptosis. MDM2, which was upregulated in GA, destabilized PPARγ through the ubiquitin–proteasome degradation pathway, whereas its silencing attenuated NLRP3 activation by elevating PPARγ levels. Concurrently, BRD4 was elevated in GA and exacerbated NLRP3 activation and pyroptosis by transcriptionally upregulating MDM2, thereby promoting PPARγ degradation. In vivo experiments showed that BRD4 silencing ameliorated GA through this MDM2–PPARγ–pyroptosis axis.ConclusionBRD4 promotes inflammation and pyroptosis in GA through MDM2-mediated PPARγ degradation, underscoring the therapeutic potential of targeting this pathway in GA management.

Selection and characterization of aptamers targeting the Vif-CBFβ-ELOB-ELOC-CUL5 complex.

The viral infectivity factor (Vif) of human immunodeficiency virus 1 forms a complex with host proteins, designated as Vif-CBFβ-ELOB-ELOC-CUL5 (VβBCC), initiating the ubiquitination and subsequent proteasomal degradation of the human antiviral protein APOBEC3G (A3G), thereby negating its antiviral function. While recent cryo-electron microscopy (cryo-EM) studies have implicated RNA molecules in the Vif-A3G interaction that leads to A3G ubiquitination, our findings indicated that the VβBCC complex can also directly impede A3G-mediated DNA deamination, bypassing the proteasomal degradation pathway. Employing the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we have identified RNA aptamers with high affinity for the VβBCC complex. These aptamers not only bind to the VβBCC complex but also reinstate A3G's DNA deamination activity by inhibiting the complex's function. Moreover, we delineated the sequences and secondary structures of these aptamers, providing insights into the mechanistic aspects of A3G inhibition by the VβBCC complex. Analysis using selected aptamers will enhance our understanding of the inhibition of A3G by the VβBCC complex, offering potential avenues for therapeutic intervention.

Acetylation- and ubiquitination-regulated SFMBT2 acts as a tumor suppressor in clear cell renal cell carcinoma

BackgroundClear cell renal cell carcinoma (RCC) is the most common kidney tumor. The analysis from medical database showed that Scm-like with four MBT domains protein 2 (SFMBT2) was decreased in advanced clear cell RCC cases, and its downregulation was associated with the poor prognosis. This study aims to investigate the role of SFMBT2 in clear cell RCC.MethodsThe expression of SFMBT2 in clear cell RCC specimens were determined by immunohistochemistry staining and western blot. The overexpression and knockdown of SFMBT2 was realized by infection of lentivirus loaded with SFMBT2 coding sequence or silencing fragment in 786-O and 769-P cells, and its effects on proliferation and metastasis were assessed by MTT, colony formation, flow cytometry, wound healing, transwell assay, xenograft and metastasis experiments in nude mice. The interaction of SFMBT2 with histone deacetylase 3 (HDAC3) and seven in absentia homolog 1 (SIAH1) was confirmed by co-immunoprecipitation.ResultsIn our study, SFMBT2 exhibited lower expression in clear cell RCC specimens with advanced stages than those with early stages. Overexpression of SFMBT2 inhibited the growth and metastasis of clear cell RCC cells, 786-O and 769-P, in vitro and in vivo, and its silencing displayed opposites effects. HDAC3 led to deacetylation of SFMBT2, and the HDAC3 inhibitor-induced acetylation prevented SFMBT2 from SIAH1-mediated ubiquitination modification and proteasome degradation. K687 in SFMBT2 protein molecule may be the key site for acetylation and ubiquitination.ConclusionsSFMBT2 exerted an anti-tumor role in clear cell RCC cells, and HDAC3-mediated deacetylation promoted SIAH1-controlled ubiquitination of SFMBT2. SFMBT2 may be considered as a novel clinical diagnostic marker and/or therapeutic target of clear cell RCC, and crosstalk between its post-translational modifications may provide novel insights for agent development.

UFM1 inhibits hypoxia-induced angiogenesis via promoting proteasome degradation of HIF-1α.

Angiogenesis is crucial for blood flow recovery and ischemic tissue repair of peripheral artery disease (PAD). Exploration of new mechanisms underlying angiogenesis will shed light on the treatment of PAD. Ubiquitin-fold modifier 1 (UFM1), a newly identified ubiquitin-like molecule, has been discovered to be involved in various pathophysiological processes. However, the role of UFM1 in the pathogenesis of PAD, especially in endothelial angiogenesis remains obscure, and we aimed to clarify this issue in this study. We initially found UFM1 was significantly upregulated in gastrocnemius muscles of PAD patients and hind limb ischemia mice. And UFM1 was mainly colocalized with endothelial cells in ischemic muscle tissues. Further, elevated expression of UFM1 was observed in hypoxic endothelial cells. Subsequent genetic inhibition of UFM1 dramatically enhanced migration, invasion, adhesion, and tube formation of endothelial cells under hypoxia. Mechanistically, UFM1 reduced the stability of hypoxia-inducible factor-1α (HIF-1α) and promoted the von Hippel-Lindau-mediated K48-linked ubiquitin-proteasome degradation of HIF-1α, which in turn decreased angiogenic factor VEGFA expression and suppressed VEGFA related signaling pathway. Consistently, overexpression of UFM1 inhibited the angiogenesis of endothelial cells under hypoxic conditions, whereas overexpression of HIF-1α reversed this effect. Collectively, our data reveal that UFM1 inhibits the angiogenesis of endothelial cells under hypoxia through promoting ubiquitin-proteasome degradation of HIF-1α, suggesting UFM1 might serve as a potential therapeutic target for PAD.

FBXL19 Targeted STK11 Degradation Enhances Cigarette Smoke-Induced Airway Epithelial Cell Cytotoxicity

Objective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11’s role in CS-induced airway epithelial cell cytotoxicity.Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs–human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits.Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner.Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death. HIGHLIGHTS Lung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression. STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity. STK11 depletion augments CS-induced airway epithelial cell cytotoxicity. CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.

Tripartite motif 2b (trim2b) restricts spring viremia of carp virus by degrading viral proteins and negative regulators NLRP12-like receptors.

Tripartite motif (TRIM) proteins are involved in different cellular functions, including regulating virus infection. In teleosts, two orthologous genes of mammalian TRIM2 are identified. However, the functions and molecular mechanisms of piscine TRIM2 remain unclear. Here, we show that trim2b-knockout zebrafish are more susceptible to spring viremia of carp virus (SVCV) infection than wild-type zebrafish. Transcriptomic analysis demonstrates that NOD-like receptor (NLR), but not RIG-I-like receptor (RLR), signaling pathway is significantly enriched in the trim2b-knockout zebrafish. In vitro, overexpression of Trim2b fails to degrade RLRs and those key proteins involved in the RLR signaling pathway but does for negative regulators NLRP12-like proteins. Zebrafish Trim2b degrades NLRP12-like proteins through its NHL_TRIM2_like and IG_FLMN domains in a ubiquitin-proteasome degradation pathway. SVCV-N and SVCV-G proteins are also degraded by NHL_TRIM2_like domains, and the degradation pathway is an autophagy lysosomal pathway. Moreover, zebrafish Trim2b can interfere with the binding between NLRP12-like protein and SVCV viral RNA and can completely block the negative regulation of NLRP12-like protein on SVCV infection. Taken together, our data demonstrate that the mechanism of action of zebrafish trim2b against SVCV infection is through targeting the degradation of host-negative regulators NLRP12-like receptors and viral SVCV-N/SVCV-G genes.IMPORTANCESpring viremia of carp virus (SVCV) is a lethal freshwater pathogen that causes high mortality in cyprinid fish. In the present study, we identified zebrafish trim2b, NLRP12-L1, and NLRP12-L2 as potential pattern recognition receptors (PRRs) for sensing and binding viral RNA. Zebrafish trim2b functions as a positive regulator; however, NLRP12-L1 and NLRP12-L2 function as negative regulators during SVCV infection. Furthermore, we find that zebrafish trim2b decreases host lethality in two manners. First, zebrafish Trim2b promotes protein degradations of negative regulators NLRP12-L1 and NLRP12-L2 by enhancing K48-linked ubiquitination and decreasing K63-linked ubiquitination. Second, zebrafish trim2b targets viral RNAs for degradation. Therefore, this study reveals a special antiviral mechanism in lower vertebrates.

Transcriptional Changes and Preservation of Muscle Protein Content in Hibernating Black Bears

Physical inactivity decreases mechanical load on the skeleton, which, when prolonged, leads to loss of muscle mass and strength in most mammalian species. In contrast, hibernating bears demonstrate limited loss of muscle mass and protein content over the prolonged periods (5-6 months) of inactivity and fasting during winter. This suggests that bears have natural adaptive mechanisms preserving muscle mass and functionality. To identify transcriptional changes that underlie molecular mechanisms attenuating muscle loss, we conducted a large-scale gene expression profiling (14,194 genes) in quadriceps muscle of adult black bear males, comparing hibernating animals (n=5) sampled in March, about 1 month before expected emergence, and summer active animals (n=5) using next generation sequencing (RNA-seq) of the transcriptome. In total, 336 differentially expressed (FDR<0.05) genes were up-regulated and 677 genes were down-regulated in muscle of hibernating bears. Gene set enrichment analysis with Reactome database showed significantly elevated proportion of overexpressed genes involved in eukaryotic translation initiation (FDR<0.0001) and elongation (FDR<0.0001) as well as in the mTORC1 mediated signaling pathway (FDR=0.021). In contrast, down-regulated genes were overrepresented among genes involved in catabolism of branched chain amino acid (BCAA: leucine, isoleucine and valine, FDR<0.0001) that suggests preservation of BCAA. These findings imply induction of protein biosynthesis through the mTORC1 signaling positively activated by availability of leucine in muscle during hibernation. Support for this conclusion comes from significant overexpression of Ras-related GTP-binding protein D (RRAGD, FC=2.12, FDR<0.0001), crucial regulator of the mTORC1 response to leucine availability, and up-regulation of eukaryotic translation initiation factor 4B (EIF4B, FC=1.95, FDR=0.012), downstream target of the mTORC1 mediated signaling. In addition to regulation of protein biosynthesis, activation of the mTOR signaling suppresses autophagy-dependent protein degradation. Consistent with reduction of autophagy, microtubule associated protein 1 light chain (MAP1LC3A, FC=-2.46, FDR<0.0001) and unc-51 like autophagy activating kinase (ULK1, FC=-1.69, FDR=0.052) were down-regulated in hibernating muscle. No expression differences were detected for two muscle atrophy markers, key members of the ubiquitin proteasome degradation, FBXO32 (Atrogin-1, MAFBX, FDR=0.85) and TRIM63 (MURF-1, FDR=0.63). The induction of protein biosynthesis and decrease in protein catabolism through the mTORC1 mediated signaling as response to BCAA availability likely contribute to the maintenance of muscle protein content through prolonged periods of immobility and fasting during hibernation. The follow up studies need to include metabolomic quantification of BCAA, phospho-proteomic assessment of key genes (RPS6KB1, RPS6) involved in the mTORC1 signaling and identification of upstream negative regulators suppressing BCAA catabolism in hibernating muscle. The work was supported by NIH COBRE under grant number [P20GM130443]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glutamine Mitigates Oxidative Stress-Induced Matrix Degradation, Ferroptosis, and Pyroptosis in Nucleus Pulposus Cells via Deubiquitinating and Stabilizing Nrf2.

Aims: Intervertebral disc degeneration (IDD) is closely related to low back pain, which is a prevalent age-related problem worldwide; however, the mechanism underlying IDD is unknown. Glutamine, a free amino acid prevalent in plasma, is recognized for its anti-inflammatory and antioxidant properties in various diseases, and the current study aims to clarify the effect and mechanism of glutamine in IDD. Results: A synergistic interplay was observed between pyroptosis and ferroptosis within degenerated human disc specimens. Glutamine significantly mitigated IDD in both ex vivo and invivo experimental models. Moreover, glutamine protected nucleus pulposus (NP) cells after tert-butyl hydroperoxide (TBHP)-induced pyroptosis, ferroptosis, and extracellular matrix (ECM) degradation in vitro. Glutamine protected NP cells from TBHP-induced ferroptosis by promoting the nuclear factor erythroid 2-related factor 2 (Nrf2) accumulation by inhibiting its ubiquitin-proteasome degradation and inhibiting lipid oxidation. Innovation and Conclusions: A direct correlation is evident in the progression of IDD between the processes of pyroptosis and ferroptosis. Glutamine suppressed oxidative stress-induced cellular processes, including pyroptosis, ferroptosis, and ECM degradation through deubiquitinating Nrf2 and inhibiting lipid oxidation in NP cells. Glutamine is a promising novel therapeutic target for the management of IDD.

STK16 promoted colorectal cancer progress in a c-MYC signaling-dependent manner

BackgroundColorectal cancer standed as a global health challenge, ranking third in cancer incidence and second in cancer-related deaths worldwide. A deeper understanding of the intricate mechanisms driving colorectal cancer development was pressing need. STK16 had garnered attention in recent researches, while its involvement in cancer had been minimally explored. c-MYC had emerged as a key player in cancer biology. Due to its complex structure, multifunctionality, and intricate interactions, directly inhibiting the activity of c-MYC proves to be challenging. Hence, current research was directing efforts towards modulating c-MYC expression levels.MethodsImmunoblot, Immunohistochemistry and immunoprecipitation assays were conducted to assess the indicated protein expression levels. RT-PCR was performed to detect the corresponding mRNA expression levels. The proliferation, migration, invasion, and colony formation abilities of the specified cancer cells were investigated using CCK8 assays, Brdu assays, transwell assays, and colony formation assays, respectively. Cellular and animal experiments were performed to investigate the correlation between STK16 signaling and c-MYC signaling.ResultsSTK16 plays a positive regulatory role in the progression of colorectal cancer. Delving into the molecular mechanisms, we unveiled that STK16 phosphorylated c-MYC at serine 452, a pivotal event hindering the ubiquitin-proteasome pathway degradation of c-MYC. Importantly, colorectal cancer proliferation mediated by STK16 was found to be dependent on the phosphorylation of c-MYC at S452. Furthermore, the researchers demonstrated that STK16 knockout or pharmacological inhibition significantly curtailed colorectal cancer proliferation and c-MYC expression in in vivo animal models.ConclusionWe discovered that STK16 phosphorylates c-MYC at serine 452, hindering its degradation via the ubiquitin-proteasome pathway. STK16 inhibition, either genetically or pharmacologically, effectively curtails cancer growth and c-MYC expression in vivo. These findings highlight STK16 as a potential therapeutic target for colorectal cancer.

RNF31 alleviates liver steatosis by promoting p53/BNIP3-related mitophagy in hepatocytes

Background & aimsNon-alcoholic fatty liver disease (NAFLD) is one of the liver illnesses that may be affected by mitophagy, which is the selective removal of damaged mitochondria. RNF31, an E3 ubiquitin ligase, is carcinogenic in many malignancies. However, the influence of RNF31 on mitochondrial homeostasis and NAFLD development remains unknown. MethodsOleic-palmitic acid treated hepatocytes and high-fat diet (HFD)-fed mice were established to observe the effect of RNF31 on hepatocyte mitophagy and steatosis. Mitophagy processes were comprehensively assessed by mt-Keima fluorescence imaging, while global changes in hepatic gene expression were measured by RNA-seq. ResultsThe present study discovered a reduction in RNF31 expression in lipotoxic hepatocytes with mitochondrial dysfunction. The observed decrease in RNF31 expression was associated with reduced mitochondrial membrane potential, disturbed mitophagy, and increased steatosis. Additionally, the findings indicated that RNF31 is a pivotal factor in the initiation of mitophagy and the facilitation of mitochondrial homeostasis, resulting in a decrease in steatosis in lipotoxic hepatocytes. Mechanistically, RNF31 enhanced p53 ubiquitination and subsequent proteasomal degradation. Down-regulation of p53 led to increased expression of the mitophagy receptor protein BCL2 and adenovirus E1B 19 kDa-interacting protein 3 (BNIP3), thereby promoting mitophagy in hepatocytes. Furthermore, it was demonstrated that the transportation of RNF31 via small extracellular vesicles derived from mesenchymal stem cells (referred to as sEV) had a substantial influence on reducing hepatic steatosis and restoring liver function in HFD-fed mice. ConclusionsThe findings highlight RNF31's essential role in the regulation of mitochondrial homeostasis in hepatocytes, emphasizing its potential as a therapeutic target for NAFLD.

FBXL8 inhibits post-myocardial infarction cardiac fibrosis by targeting Snail1 for ubiquitin-proteasome degradation

Abnormal cardiac fibrosis is the main pathological change of post-myocardial infarction (MI) heart failure. Although the E3 ubiquitin ligase FBXL8 is a key regulator in the cell cycle, cell proliferation, and inflammation, its role in post-MI ventricular fibrosis and heart failure remains unknown. FBXL8 was primarily expressed in cardiac fibroblasts (CFs) and remarkably decreased in CFs treated by TGFβ and heart subjected to MI. The echocardiography and histology data suggested that adeno-associated viruses (AAV9)-mediated FBXL8 overexpression had improved cardiac function and ameliorated post-MI cardiac fibrosis. In vitro, FBXL8 overexpression prevented TGFβ-induced proliferation, migration, contraction, and collagen secretion in CFs, while knockdown of FBXL8 demonstrated opposite effects. Mechanistically, FBXL8 interacted with Snail1 to promote Snail1 degradation through the ubiquitin–proteasome system and decreased the activation of RhoA. Moreover, the FBXL8ΔC3 binding domain was indispensable for Snail1 interaction and degradation. Ectopic Snail1 expression partly abolished the effects mediated by FBXL8 overexpression in CFs treated by TGFβ. These results characterized the role of FBXL8 in regulating the ubiquitin-mediated degradation of Snail1 and revealed the underlying molecular mechanism of how MI up-regulated the myofibroblasts differentiation-inducer Snail1 and suggested that FBXL8 may be a potential curative target for improving post-MI cardiac function.

Tyrosine-phosphorylated DNER sensitizes insulin signaling in hepatic gluconeogenesis by inducing proteasomal degradation of TRB3

ObjectiveHepatic insulin resistance, which leads to increased hepatic gluconeogenesis, is a major contributor to fasting hyperglycemia in type 2 diabetes mellitus (T2DM). However, the mechanism of impaired insulin-dependent suppression of hepatic gluconeogenesis remains elusive. Delta/Notch-like epidermal growth factor (EGF)-related receptor (DNER), firstly described as a neuron-specific Notch ligand, has been recently identified as a susceptibility gene for T2DM through genome-wide association studies. We herein investigated whether DNER regulates hepatic gluconeogenesis and whether this is mediated by enhanced insulin signaling. MethodsThe association between DNER, tribbles homolog 3 (TRB3) and Akt signaling was evaluated in C57BL/6J, ob/ob and db/db mice by western blot analysis. DNER loss-of-function and gain-of-function in hepatic gluconeogenesis were analyzed by western blot analysis, quantitative real-time PCR, glucose uptake and output assay in AML-12 cells and partially validated in primary mouse hepatocytes. Hepatic DNER knockdown mice were generated by tail vein injection of adenovirus to confirm the effects of DNER in vivo. The interaction between DNER and TRB3 was investigated by rescue experiments, cycloheximide chase analysis, co-immunoprecipitation and immunofluorescence. The potential insulin-stimulated phosphorylation sites of DNER were determined by co-immunoprecipitation, LC-MS/MS analysis and site-specific mutagenesis. ResultsHere we show that DNER enhanced hepatic insulin signaling in gluconeogenesis by inhibiting TRB3, an endogenous Akt inhibitor, through the ubiquitin-proteasome degradation pathway. In AML-12 hepatocytes, insulin-stimulated activation of Akt and suppression of gluconeogenesis are attenuated by DNER knockdown, but potentiated by DNER over-expression. In C57BL/6J mice, hepatic DNER knockdown is accompanied by impaired glucose and pyruvate tolerance. Furthermore, the in vitro effects of DNER knockdown or over-expression on both Akt activity and hepatic gluconeogenesis can be rescued by TRB3 knockdown or over-expression, respectively. In response to insulin stimulation, DNER interacted directly with insulin receptor and was phosphorylated at Tyr677. This site-specific phosphorylation is essential for DNER to upregulate Akt activity and then downregulate G6Pase and PEPCK expression, by interacting with TRB3 directly and inducing TRB3 proteasome-dependent degradation. ConclusionsTaken together, the crosstalk between insulin-Akt and DNER-TRB3 pathways represents a previously unrecognized mechanism by which insulin regulates hepatic gluconeogenesis.

Abstract 4409: FBXW7 and BCL9L altered pathways as hallmarks of early-onset colorectal cancer

Abstract Background: The incidence of early-onset colorectal cancer (EO-CRC) has been alarmingly increasing, particularly among non-Hispanic Whites (NHWs), resulting in similar rates to ones in African Americans (AAs). Overall, the median age of diagnosis has decreased from 72 to 66 years. The molecular features that could potentially distinguish this phenotype are still not well-established. This study aimed to identify genetic, epigenetic, and transcriptomic factors associated with EO-CRC. Methods: 510 patients with microsatellite-stable tumors analyzed for somatic mutations, targeting 20 cancer driver genes. We included a cohort of 175 AAs from the Yale New Haven Health System, a previously studied cohort of 51 AAs from the Chicago Colorectal Cancer Consortium, and the publicly available molecular data from TCGA COAD-READ PanCancer cohort (50 AAs and 229 NHWs). We analyzed the transcriptomic and methylation profiles from the Yale cohort using Tempo-SeqTM and Illumina EPIC array, respectively. Results: Of the 510 patients, 18% were diagnosed with EO-CRC. Using a multiple variant analysis, adjusting for sex, race, tumor location, and stage, EO-CRC patients were less likely to be male (OR = 0.59, 95% CI: 0.37-0.94, p-value = 0.03), their tumors lacked mutations in APC (OR = 0.37, 95% CI: 0.22-0.63, p-value = 0.0002), and they were more likely to have mutations in FBXW7 (OR = 3.1, 95% CI: 1.60-6.00, p-value = 0.0007) and BCL9L (OR = 3.1, 95% CI: 1.00-8.72, p-value = 0.04). We compared tumors with and without mutations in FBXW7 or BCL9L in two independently analyses. Combining transcriptomics and methylation data, we identified 10 candidate genes that showed significant gene downregulation with hypermethylation or upregulation with hypomethylation. Among FBXW7 mutant tumors, we found the Cartilage oligomeric matrix protein (COMP) gene was significantly downregulated and hypermethylated. In CRC, COMP has been associated with a crosstalk between the TGF-β pathway and immune cell infiltration. Moreover, inactivation of FBXW7 resulted in the accumulation of phosphorylated TGIF1 molecules and repression of TGFβ-dependent transcription in cancer cell lines. Deficiency of FBXW7 is a key element in the phosphorylation-dependent ubiquitination and subsequent proteasome degradation of oncoproteins, such as c-MYC, NOTCH, and Cyclin E. Among BCL9L mutant tumors, keratin 20 (KRT20), the intermediate filament protein and marker of intestinal differentiation in CRC, was downregulated and hypermethylated. BCL9L plays an important role in tumorigenesis induced by aberrant Wnt signaling or aneuploidy tolerance. Conclusion: Our findings further demonstrate the importance of specific etiologic mechanisms in EO-CRC at the level of somatic alterations, providing new ways to understand the biology of EO-CRC and prospects for the development of targeted therapies. Citation Format: Reger Mikaeel, Dante Bellomo, Seeta Rajpara, David Buckley, Mary Yagle, Baris Kerimoglu, Curtis Thorne, Megha Padi, Bodour Salhia, Nathan A. Ellis, Xavier Llor, Rosa Xicola. FBXW7 and BCL9L altered pathways as hallmarks of early-onset colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4409.

Abstract 4503: Preclinical characterization of PRT7732: A highly potent, selective, and orally bioavailable targeted protein degrader of SMARCA2

Abstract SWI/SNF (BAF) complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core catalytic subunits of the SWI/SNF complexes, containing an ATPase domain and a bromodomain. SMARCA4 protein expression is lost in some cancers due to loss-of-function (LOF) mutations and homozygous deletions, and SMARCA4-deleted cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. Therefore, targeting SMARCA2 in SMARCA4-deficient cancers using selective SMARCA2 degraders induces synthetic lethality while sparing SMARCA4 wild type (WT) normal cells. We have recently identified a series of orally bioavailable SMARCA2 selective degraders that demonstrate robust efficacy in pre-clinical animal models with favorable pharmacokinetic properties and safety profiles. Our nomination candidate PRT7732 exhibits >1000x selectivity for SMARCA2 over SMARCA4 in cell-based assays, with DC50 values in cancer cell lines in the low nanomolar range. The PRT7732-induced SMARCA2 degradation was rescued by a proteasome inhibitor and neddylation inhibitor, indicating ubiquitin-proteasome dependent degradation. Furthermore, PRT7732 does not change levels of known CRBN ligand neo-substrates such as IKZF2/3, GSPT1 and SALL4. PRT7732 inhibits only SMARCA4-deficient cancer cell proliferation with IC50 values ranging from 5.0-50 nM, but not SMARCA4 WT cells in vitro. Oral administration of PRT7732 resulted in near-total degradation of SMARCA2 protein with complete selectivity over SMARCA4 protein in a SMARCA4 WT lung cancer model in mice, consistent with the SMARCA2 degradation kinetics-based pharmacodynamic prediction model. PRT7732 oral daily administration showed significant tumor growth inhibition of SMARCA4-deficient lung cancer xenograft models at well tolerated doses. The treated tumor tissues show robust SMARCA2 protein reduction for 24h post dosing. In summary, our orally bioavailable SMARCA2 degraders induce synthetic lethality in SMARCA4-deficient cancers in vitro and in vivo. Efforts to further evaluate these compounds in additional models and in combination with other agents are ongoing. Citation Format: Artem Shvartsbart, Koichi Ito, Joseph Rager, Michael Hulse, Anjana Agarwal, Komali Vykuntam, Jessica Burtell, Min Wang, Justin Kurian, Miles Cowart, Joy Cote, Nick Stahl, Monisha Sivakumar, Anthony Reichelderfer, Jack Carter, Alexander Grego, Andrew Moore, Neha Bhagwat, Ross Kuskovsky, Shanthi Ganesan, Stefan Ruepp, Tom Emm, Philip Pitis, Corey Basch, Klare Bersch, Yongchun Pan, Song Mei, Raul Leal, John Rose, Dani Roth, Chaoyi Xu, Ganfeng Cao, Kris Vaddi, Sang Hyun Lee, Sandy Geeganage, Andrew Combs, Peggy Scherle. Preclinical characterization of PRT7732: A highly potent, selective, and orally bioavailable targeted protein degrader of SMARCA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4503.

Qixian granule inhibits ferroptosis in vascular endothelial cells by modulating TRPML1 in the lysosome to prevent postmenopausal atherosclerosis

Ethnopharmacological relevanceQiXian Granule (QXG) is an integrated traditional Chinese medicine formula used to treat postmenopausal atherosclerotic (AS) cardiovascular diseases. The previous studies have found that QXG inhibited isoproterenol (ISO)-induced myocardial remodeling. And its active ingredient, Icraiin, can inhibit ferroptosis by promoting oxidized low-density lipoprotein (xo-LDL)-induced vascular endothelial cell injury and autophagy in atherosclerotic mice. Another active ingredient, Salvianolic Acid B, can suppress ferroptosis and apoptosis during myocardial ischemia/reperfusion injury by reducing ubiquitin-proteasome degradation of Glutathione Peroxidase 4 (GPX4) and down-regulating the reactive oxygen species (ROS)- c-Jun N-terminal kinases (JNK)/mitogen-activated protein kinase (MAPK) pathway. Aim of the studyThe objective of this research was to assess the possible impact of QXG on atherosclerosis in postmenopausal individuals and investigate its underlying mechanisms. Materials and methodsFemale ApoE−/− mice underwent ovariectomy and were subjected to a high-fat diet (HFD) to establish a postmenopausal atherosclerosis model. The therapeutic effects of QXG were observed in vivo and in vitro through intraperitoneal injection of erastin, G-protein Coupled Estrogen Receptor (GPER) inhibitor (G15), and silent Mucolipin Transient Receptor Potential Channel 1 (TRPML1) adenovirus injection via tail vein. UPLC-MS and molecular docking techniques identified and evaluated major QXG components, contributing to the investigation of QXG's anti-postmenopausal atherosclerotic effects. ResultsQXG increased serum Estradiol levels, decreased follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, which indicated QXG had estrogen-like effects in Ovx/ApoE−/− mice. Furthermore, QXG demonstrated the potential to impede the progression of AS in Ovx/ApoE−/− mice, as evidenced by reductions in serum triglycerides (TG), total cholesterol (TC), and low-density lipoprotein-cholesterol (LDL-C) levels. Additionally, QXG inhibited ferroptosis in Ovx/ApoE−/− mice. Notably, UPLC-MS analysis identified a total of 106 active components in QXG. The results of molecular docking analysis demonstrated that Epmedin B, Astragaloside II, and Orientin exhibit strong binding affinity towards TRPML1. QXG alleviates the progression of atherosclerosis by activating TRPML1 through the GPER pathway or directly activating TRPML1, thereby inhibiting GPX4 and ferritin heavy chain (FTH1)-mediated iron pendant disease. In vitro, QXG-treated serum suppressed proliferation, migration, and ox-LDL-induced MMP and ROS elevation in HAECs. ConclusionQXG inhibited GPX4 and FTH1-mediated ferroptosis in vascular endothelial cells through up-regulating GPER/TRPML1 signaling, providing a potential therapeutic option for postmenopausal females seeking a safe and effective medication to prevent atherosclerosis. The study highlights QXG's estrogenic properties and its promising role in combating postmenopausal atherosclerosis.

Membrane-associated RING-CH 7 inhibits stem-like capacities of bladder cancer cells by interacting with nucleotide-binding oligomerization domain containing 1

BackgroundCancer stem-like capacities are major factors contributing to unfavorable prognosis. However, the associated molecular mechanisms underlying cancer stem-like cells (CSCs) maintain remain unclear. This study aimed to investigate the role of the ubiquitin E3 ligase membrane-associated RING-CH 7 (MARCH7) in bladder cancer cell CSCs.MethodsMale BALB/c nude mice aged 4–5 weeks were utilized to generate bladder xenograft model. The expression levels of MARCHs were checked in online databases and our collected bladder tumors by quantitative real-time PCR (q-PCR) and immunohistochemistry (IHC). Next, we evaluated the stem-like capacities of bladder cancer cells with knockdown or overexpression of MARCH7 by assessing their spheroid-forming ability and spheroid size. Additionally, we conducted proliferation, colony formation, and transwell assays to validate the effects of MARCH7 on bladder cancer CSCs. The detailed molecular mechanism of MARCH7/NOD1 was validated by immunoprecipitation, dual luciferase, and in vitro ubiquitination assays. Co-immunoprecipitation experiments revealed that nucleotide-binding oligomerization domain-containing 1 (NOD1) is a substrate of MARCH7.ResultsWe found that MARCH7 interacts with NOD1, leading to the ubiquitin–proteasome degradation of NOD1. Furthermore, our data suggest that NOD1 significantly enhances stem-like capacities such as proliferation and invasion abilities. The overexpressed MARCH7 counteracts the effects of NOD1 on bladder cancer CSCs in both in vivo and in vitro models.ConclusionOur findings indicate that MARCH7 functions as a tumor suppressor and inhibits the stem-like capacities of bladder tumor cells by promoting the ubiquitin–proteasome degradation of NOD1. Targeting the MARCH7/NOD1 pathway could be a promising therapeutic strategy for bladder cancer patients.

Expansion of targeted degradation by Gilteritinib-Warheaded PROTACs to ALK fusion proteins

Proteolysis targeting chimeras (PROTACs) induce the ubiquitination and subsequent proteasomal degradation of targeted proteins. Numerous PROTACs have emerged as promising drug candidates for various disease-related proteins. This study investigates PROTACs targeted to degrade anaplastic lymphoma kinase (ALK) fusion proteins, which are implicated in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. We recently reported the development of a gilteritinib-warheaded PROTAC to target and degrade the Fms-like tyrosine kinase 3 (FLT3) protein. Gilteritinib is a tyrosine kinase inhibitor that targets FLT3, and recent studies have revealed that it also functions as an ALK inhibitor. We conducted a structure–activity relationship (SAR) study and expanded the range of target proteins for gilteritinib-warheaded PROTACs to include echinoderm microtubule-associated protein-like 4 (EML4)-ALK and nucleophosmin (NPM)-ALK, in addition to FLT3. Our SAR study utilized three types of ligands for E3 ligase— inhibitor of apoptosis protein (IAP), cereblon (CRBN), and von Hippel-Lindau (VHL)— in the PROTAC designs and we observed varied efficacy in the degradation of target proteins. The CRBN-based PROTAC effectively reduced the protein expression of FLT3, EML4-ALK, and NPM-ALK. The IAP-based PROTAC reduced expression of both FLT3 and EML4-ALK proteins but not that of NPM-ALK, while the VHL-based PROTAC was ineffective against all target proteins. Several ALK-targeted PROTACs have already been developed using CRBN or VHL as E3 ligase, but this is the first report of an IAP-based ALK degrader. The length of the linker structure utilized in PROTAC also had a significant effect on their efficacy and activity. PROTACs formed with shorter linkers demonstrated an enhanced degradation activity to target proteins compared with those formed with longer linkers. These findings provide valuable insight for the development of effective PROTACs to target and degrade ALK fusion proteins.

GSTP1 is a negative regulator of MAVS in the antiviral signaling against SVCV infection

Glutathione S-transferase P1 (GSTP1), the most ubiquitous member of the GST superfamily, plays vital roles in the detoxification, antioxidant defense, and modulation of inflammatory responses. However, limited studies have been conducted on the function of GSTP1 in antiviral innate immunity. In this study, we have cloned the homolog of GSTP1 in triploid hybrid crucian carp (3nGSTP1) and investigated its regulatory role in the interferon signaling pathway. The open reading frame of 3nGSTP1 is composed of 627 nucleotides, encoding 209 amino acids. In response to spring viremia of carp virus (SVCV) infection, the mRNA level of 3nGSTP1 was up-regulated in the liver, kidney, and caudal fin cell lines (3 nF C) of triploid fish. The knockdown of 3nGSTP1 in 3 nF C improved host cell's antiviral capacity and attenuated SVCV replication. Additionally, overexpression of 3nGSTP1 inhibited the activation of IFN promoters induced by SVCV infection, poly (I:C) stimulation, or the RLR signaling factors. The co-immunoprecipitation assays further revealed that 3nGSTP1 interacts with 3nMAVS. In addition, 3nGSTP1 dose-dependently inhibited 3nMAVS-mediated antiviral activity and reduced 3nMAVS protein level. Mechanistically, 3nGSTP1 promoted ubiquitin-proteasome degradation of MAVS by promoting its K48-linked polyubiquitination. To conclude, our results indicate that GSTP1 acts as a novel inhibitor of MAVS, which negatively regulates the IFN signaling.

EFHD2 cooperates with E3 ubiquitin ligase Smurf1 to facilitate virus infection by promoting the degradation of TRAF6 in teleost fish.

The ubiquitin-proteasome system is one of the most important protein stability regulation systems. It can precisely regulate host immune responses by targeting signaling proteins. TRAF6 is a crucial E3 ubiquitin ligase in host antiviral signaling pathway. Here, we discovered that EF-hand domain-containing protein D2 (EFHD2) collaborated with the E3 ubiquitin ligase Smurf1 to potentiate the degradation of TRAF6, hence facilitating RNA virus Siniperca chuatsi rhabdovirus infection. The mechanism analysis revealed that EFHD2 interacted with Smurf1 and enhanced its protein stability by impairing K48-linked polyubiquitination of Smurf1, thereby promoting Smurf1-catalyzed degradation of TRAF6. This study initially demonstrated a novel mechanism by which viruses utilize host EFHD2 to achieve immune escape and provided a new perspective on the exploration of mammalian innate immunity.IMPORTANCEViruses induce host cells to activate several antiviral signaling pathways. TNF receptor-associated factor 6 (TRAF6) plays an essential role in these pathways. Numerous studies have been done on the mechanisms of TRAF6-mediated resistance to viral invasion. However, little is known about the strategies that viruses employ to antagonize TRAF6-mediated antiviral signaling pathway. Here, we discovered that EFHD2 functions as a host factor to promote viral replication. Mechanistically, EFHD2 potentiates Smurf1 to catalyze the ubiquitin-proteasomal degradation of TRAF6 by promoting the deubiquitination and stability of Smurf1, which in turn inhibits the production of proinflammatory cytokines and interferons. Our study also provides a new perspective on mammalian resistance to viral invasion.