Equol exerts anti-tumor effects on choriocarcinoma cells by promoting TRIM21-mediated ubiquitination of ANXA2.

Choriocarcinoma is a malignant cancer that belongs to gestational trophoblastic neoplasia (GTN). Herein, serum metabolomic analysis was performed on 29 GTN patients and 30 healthy individuals to characterize the metabolic variations during GTN progression. Ultimately 24 differential metabolites (DMs) were identified, of which, Equol was down-regulated in GTN patients, whose VIP score is the 3rd highest among the 24 DMs. As an intestinal metabolite of daidzein, the anticancer potential of Equol has been demonstrated in multiple cancers, but not choriocarcinoma. Hence, human choriocarcinoma cell lines JEG-3 and Bewo were used and JEG-3-derived subcutaneous xenograft models were developed to assess the effect of Equol on choriocarcinoma. The results suggested that Equol treatment effectively suppressed choriocarcinoma cell proliferation, induced cell apoptosis, and reduced tumorigenesis. Label-free quantitative proteomics showed that 136 proteins were significantly affected by Equol and 20 proteins were enriched in Gene Ontology terms linked to protein degradation. Tripartite motif containing 21 (TRIM21), a E3 ubiquitin ligase, was up-regulated by Equol. Equol-induced effects on choriocarcinoma cells could be reversed by TRIM21 inhibition. Annexin A2 (ANXA2) interacted with TRIM21 and its ubiquitination was modulated by TRIM21. We found that TRIM21 was responsible for proteasome-mediated degradation of ANXA2 induced by Equol, and the inhibitory effects of Equol on the malignant behaviors of choriocarcinoma cells were realized by TRIM21-mediated down-regulation of ANXA2. Moreover, ß-catenin activation was inhibited by Equol, which also depended on TRIM21-mediated down-regulation of ANXA2. Taken together, Equol may be a novel candidate for the treatment for choriocarcinoma.

L3MBTL1, a polycomb protein, promotes Osimertinib acquired resistance through epigenetic regulation of DNA damage response in lung adenocarcinoma.

Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR-TKI) approved for patients with EGFR T790M resistance mutations as first- or second-line treatment of EGFR-positive patients. Resistance to Osimertinib will inevitably develop, and the underlying mechanisms are largely unknown. In this study, we discovered that acquired resistance to Osimertinib is associated with abnormal DNA damage response (DDR) in lung adenocarcinoma cells. We discovered that the polycomb protein Lethal(3) Malignant Brain Tumor-Like Protein 1 (L3MBTL1) regulates chromatin structure, thereby contributing to DDR and Osimertinib resistance. EGFR oncogene inhibition reduced L3MBTL1 ubiquitination while stabilizing its expression in Osimertinib-resistant cells. L3MBTL1 reduction and treatment with Osimertinib significantly inhibited DDR and proliferation of Osimertinib-resistant lung cancer cells in vitro and in vivo. L3MBTL1 binds throughout the genome and plays an important role in EGFR-TKI resistance. It also competes with 53BP1 for H4K20Me2 and inhibits the development of drug resistance in Osimertinib-resistant lung cancer cells in vitro and in vivo. Our findings suggest that L3MBTL1 inhibition is a novel approach to overcoming EGFR-TKI-acquired resistance.

Lys-63-specific deubiquitinase BRCC36 enhances the sensitivity of multiple myeloma cells to lenalidomide by inhibiting lysosomal degradation of cereblon.

Multiple myeloma (MM) is the second most common hematological tumor in adults. Immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide (Len), are effective drugs for the treatment of multiple myeloma. Len can recruit IKZF1 and IKZF3 to cereblon (CRBN), a substrate receptor of the cullin 4-RING E3 ligase (CRL4), promote their ubiquitination and degradation, and finally inhibit the proliferation of myeloma cells. However, MM patients develop resistance to IMiDs over time, leading to disease recurrence and deterioration. To explore the possible approaches that may enhance the sensitivity of IMiDs to MM, in this study, we used the proximity labeling technique TurboID and quantitative proteomics to identify Lys-63-specific deubiquitinase BRCC36 as a CRBN-interacting protein. Biochemical experiments demonstrated that BRCC36 in the BRISC complex protects CRBN from lysosomal degradation by specifically cleaving the K63-linked polyubiquitin chain on CRBN. Further studies found that a small-molecule compound SHIN1, which binds to BRISC complex subunit SHMT2, can upregulate CRBN by elevating BRCC36. The combination of SHIN1 and Len can further increase the sensitivity of MM cells to IMiDs. Therefore, this study provides the basis for the exploration of a possible strategy for the SHIN1 and Len combination treatment for MM.

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.

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.

Exposure to BaA inhibits trophoblast cell invasion and induces miscarriage by regulating the DEC1/ARHGAP5 axis and promoting ubiquitination-mediated degradation of MMP2.

Benz[a]anthracene (BaA), a hazardous polycyclic aromatic hydrocarbon classified by the EPA, is a probable reproductive toxicant. Epidemiological studies suggest that BaA exposure may be a risk factor for recurrent miscarriage (RM). However, the underlying mechanisms are not well understood. This study identified DEC1 as a key gene through RNA-seq and single-cell RNA sequencing analysis. DEC1 expression was found to be downregulated in villous tissues from women with RM and in primary extravillous trophoblasts (EVTs) exposed to BaA. BaA suppressed DEC1 expression by promoting abnormal methylation patterns. Further analysis revealed that ARHGAP5 is a direct target of DEC1 in EVTs, where DEC1 inhibits trophoblast invasion by directly regulating ARHGAP5 transcription. Additionally, BaA destabilized matrix metalloproteinase 2 (MMP2) by activating the aryl hydrocarbon receptor (AhR) and promoting E3 ubiquitin ligase MID1-mediated degradation. In a mouse model, BaA induced miscarriage by modulating the DEC1/ARHGAP5 and MID1/MMP2 axes. Notably, BaA-induced miscarriage in mice was prevented by DEC1 overexpression or MID1 knockdown. These findings indicate that BaA exposure leads to miscarriage by suppressing the DEC1/ARHGAP5 pathway and enhancing the MID1/MMP2 pathway in human EVTs.

Micafungin protects mouse heart against doxorubicin-induced oxidative injury via suppressing MALT1-dependent k48-linked ubiquitination of Nrf2.

Oxidative stress contributes greatly to doxorubicin (DOX)-induced cardiotoxicity. Down-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) is a key factor in DOX-induced myocardial oxidative injury. Recently, we found that mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1)-dependent k48-linked ubiquitination was responsible for down-regulation of myocardial Nrf2 in DOX-treated mice. Micafungin, an antifungal drug, was identified as a potential MALT1 inhibitor. This study aims to explore whether micafungin can reduce DOX-induced myocardial oxidative injury and if its anti-oxidative effect involves a suppression of MALT1-dependent k48-linked ubiquitination of Nrf2. To establish the cardiotoxicity models in vivo and in vitro, mice were treated with a single dose of DOX (15 mg/kg, i.p.) and cardiomyocytes were incubated with DOX (1 µM) for 24 h, respectively. Using mouse model of DOX-induced cardiotoxicity, micafungin (10 or 20 mg/kg) was shown to improve cardiac function, concomitant with suppression of oxidative stress, mitochondrial dysfunction, and cell death in a dose-dependent manner. Similar protective roles of micafungin (1 or 5 µM) were observed in DOX-treated cardiomyocytes. Mechanistically, micafungin weakened the interaction between MALT1 and Nrf2, decreased the k48-linked ubiquitination of Nrf2 while elevated the protein levels of Nrf2 in both DOX-treated mice and cardiomyocytes. Furthermore, MALT1 overexpression counteracted the cardioprotective effects of micafungin. In conclusion, micafungin reduces DOX-induced myocardial oxidative injury via suppression of MALT1, which decreases the k48-linked ubiquitination of Nrf2 and elevates Nrf2 protein levels. Thus, micafungin may be repurposed for treating DOX-induced cardiotoxicity.

Proteostasis perturbation of N-Myc leveraging HSP70 mediated protein turnover improves treatment of neuroendocrine prostate cancer.

N-Myc is a key driver of neuroblastoma and neuroendocrine prostate cancer (NEPC). One potential way to circumvent the challenge of undruggable N-Myc is to target the protein homeostasis (proteostasis) system that maintains N-Myc levels. Here, we identify heat shock protein 70 (HSP70) as a top partner of N-Myc, which binds a conserved "SELILKR" motif and prevents the access of E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), possibly through steric hindrance. When HSP70's dwell time on N-Myc is increased by treatment with the HSP70 allosteric inhibitor, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites and forms polyubiquitination chains linked by the K11 and K63 sites. Notably, HSP70 inhibition significantly suppressed NEPC tumor growth, increased the efficacy of aurora kinase A (AURKA) inhibitors, and limited the expression of neuroendocrine-related pathways.

Inhibition of TOPORS ubiquitin ligase augments the efficacy of DNA hypomethylating agents through DNMT1 stabilization.

DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies, although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein (TOPORS), which encodes a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis, suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks, which undergo SUMOylation, followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. In TOPORS knockout cells, DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination, resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1, thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently, the ubiquitination inhibitor, TAK-243, and the SUMOylation inhibitor, TAK-981, show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks.

Glucose inhibits the inflammatory response in goose fatty liver by increasing the ubiquitination level of PKA.

Protein kinase A (PKA) plays an important role in cellular life activities. Recently, PKA was found to bind to the inhibitor of nuclear factor-kappaB (IκB), a key protein in the nuclear factor-kappaB (NF-κB) pathway, to form a complex involved in the regulation of inflammatory response. However, the role of PKA in the anti-inflammatory of goose fatty liver is still unclear. A total of 14 healthy 70-d-old male Lander geese were randomly divided into a control group and an overfeeding group. Inflammation level was analyzed by histopathological method in the liver. The mRNA and protein abundance of PKA and tumor necrosis factor-alpha (TNFα), as well as the ubiquitination level of PKA, were detected. Moreover, goose primary hepatocytes were cotreated with glucose, harringtonine, and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132). Finally, the co-immunoprecipitated samples of PKA from the control and overfeeding group were used for protein mass spectrometry. The results showed that no difference in PKA mRNA expression was observed (P > 0.05), while the PKA protein level in the overfed group was significantly reduced (P < 0.05) when compared with the control group. The ubiquitination level of PKA was higher than that of the control group in fatty liver. The mRNA expression of PKA was elevated but protein abundance was reduced in goose primary hepatocytes with 200 mmol/L glucose treatment (P < 0.05). The PKA protein abundance was dramatically reduced in hepatocytes treated with harringtonine (P < 0.01) when compared with the glucose-supplemented group. Nevertheless, MG132 tended to alleviate the inhibitory effect of harringtonine on PKA protein abundance (P = 0.081). There was no significant difference in TNFα protein level among glucose-treated groups and control (P > 0.05). Protein mass spectrometry analysis showed that 29 and 76 interacting proteins of PKA were screened in goose normal and fatty liver, respectively. Validation showed that PKA interacted with the E3 ubiquitination ligases ring finger protein 135 (RNF135) and potassium channel modulatory factor 1 (KCMF1). In summary, glucose may inhibit the inflammatory response in goose fatty liver by increasing the ubiquitination level of PKA. Additionally, RNF135 and KCMF1 may be involved in the regulation of PKA ubiquitination level as E3 ubiquitination ligases.

No obvious pathological symptoms such as inflammation were observed in fatty goose liver, suggesting that there is a unique mechanism to inhibit the development of inflammation during the goose fatty liver formation. Previous studies have shown that high glucose activated the ubiquitin­proteasome. Protein kinase A (PKA) can interact with a key protein in the nuclear factor-kappaB pathway to activate the pathway and trigger inflammatory response. To further understand how inflammation is suppressed during goose fatty liver formation. The present study showed that inflammation and PKA protein level were reduced in goose fatty liver. Meanwhile, PKA can be modified by ubiquitination in goose liver and hepatocytes. The result of the study implied that glucose deposited during goose fatty liver formation may reduce the PKA protein content by increasing the PKA ubiquitination level, thereby inhibiting the inflammatory response. Our study not only contributes to elucidate the new mechanism for suppressed inflammation in goose fatty liver but also provides a reference for the study of fatty liver in other animals.

Rupatadine-inhibited OTUD3 promotes DLBCL progression and immune evasion through deubiquitinating MYL12A and PD-L1.

The obstacle to effectively treating Diffuse Large B-cell Lymphoma (DLBCL) lies in the resistance observed toward standard therapies. Identifying therapeutic targets that prove effective for relapsed or refractory patients poses a significant challenge. OTUD3, a deubiquitinase enzyme, is overexpressed in DLBCL tissues. However, its role in DLBCL has not been investigated. Our study has brought to light the multifaceted impact of OTUD3 in DLBCL. Not only does it enhance cell survival through the deubiquitination of MYL12A, but it also induces CD8+ T cell exhaustion within the local environment by deubiquitinating PD-L1. Our findings indicate that the OTUD3 inhibitor, Rupatadine, exerts its influence through competitive binding with OTUD3. This operation diminishes the deubiquitination of both MYL12A and PD-L1 by OTUD3. This research unveils the central and oncogenic role of OTUD3 in DLBCL and highlights the potential clinical application value of the OTUD3 inhibitor, Rupatadine. These findings contribute valuable insights into addressing the challenges of resistant DLBCL cases and offer a promising avenue for further clinical exploration.

HDAC6 inhibition disrupts HDAC6-P300 interaction reshaping the cancer chromatin landscape.

BACKGROUND: Histone deacetylases (HDACs) are crucial regulators of gene expression, DNA synthesis, and cellular processes, making them essential targets in cancer research. HDAC6, specifically, influences protein stability and chromatin dynamics. Despite HDAC6's potential therapeutic value, its exact role in gene regulation and chromatin remodeling needs further clarification. This study examines how HDAC6 inactivation influences lysine acetyltransferase P300 stabilization and subsequent effects on chromatin structure and function in cancer cells. METHODS AND RESULTS: We employed the HDAC6 inhibitor ITF3756, siRNA, or CRISPR/Cas9 gene editing to inactivate HDAC6 in different epigenomic backgrounds. Constantly, this inactivation led to significant changes in chromatin accessibility, particularly increased acetylation of histone H3 lysines 9, 14, and 27 (ATAC-seq and H3K27Ac ChIP-seq analysis). Transcriptomics, proteomics, and gene ontology analysis revealed gene changes in cell proliferation, adhesion, migration, and apoptosis. Significantly, HDAC6 inactivation altered P300 ubiquitination, stabilizing P300 and leading to downregulating genes critical for cancer cell survival. CONCLUSIONS: Our study highlights the substantial impact of HDAC6 inactivation on the chromatin landscape of cancer cells and suggests a role for P300 in contributing to the anticancer effects. The stabilization of P300 with HDAC6 inhibition proposes a potential shift in therapeutic focus from HDAC6 itself to its interaction with P300. This finding opens new avenues for developing targeted cancer therapies, improving our understanding of epigenetic mechanisms in cancer cells.

Alantolactone facilitates ferroptosis in non-small cell lung cancer through promoting FTH1 ubiquitination and degradation.

Alantolactone (ALT), a natural sesquiterpene lactone from Inula helenium L., demonstrates potent antitumor activity in various human cancers, notably non-small cell lung cancer (NSCLC). Despite its recognized efficacy, the precise mechanisms of action remain elusive. Our study aimed to elucidate ALT's impact on NSCLC. Our findings suggested that ALT triggered apoptosis both in vitro and in vivo, underscoring its anticancer potential. Interestingly, the ferroptosis inhibitor (Fer-1), rather than necrostatin-1 (Nec-1) or Z-VAD-FMK, rescued ALT-induced cell death, implicating ferroptosis as pivotal. Subsequent analyses revealed ferroptosis as the primary mechanism underlying ALT-induced NSCLC cell death, supported by markers including ROS accumulation, MDA elevation, GSH depletion, Fe2+ generation, and GPX4 reduction. Through DARTS/MS proteomics, we identified FTH1 as the target of ALT-induced ferroptosis. Immunoblotting confirmed ALT's inhibition of FTH1 protein expression and accelerated its degradation in NSCLC cells. Immunoprecipitation assays demonstrated increased FTH1 ubiquitination induced by ALT. Additionally, ALT induced ferroptosis and facilitated Fe2+ accumulation via FTH1 ubiquitination. Importantly, ALT displayed potent antitumor effects in a subcutaneous xenograft model in BALB/c-nu/nu nude mice by enhancing ferroptosis. In summary, ALT induced ferroptosis by promoting intracellular Fe2+ accumulation through accelerated FTH1 degradation, highlighting its potential as an antitumor agent targeting ferroptosis.

Endogenously produced itaconate negatively regulates innate-driven cytokine production and drives global ubiquitination in human macrophages.

A wide variety of electrophilic derivatives of itaconate, the Kreb's cycle-derived metabolite, are immunomodulatory, yet these derivatives have overlapping and sometimes contradictory activities. Therefore, we generated a genetic system to interrogate the immunomodulatory functions of endogenously produced itaconate in human macrophages. Endogenous itaconate is driven by multiple innate signals restraining inflammatory cytokine production. Endogenous itaconate directly targets cysteine 13 in IRAK4 (disrupting IRAK4 autophosphorylation and activation), drives the degradation of nuclear factor κB, and modulates global ubiquitination patterns. As a result, cells unable to make itaconate overproduce inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and IL-1ß in response to these innate activators. In contrast, the production of interferon (IFN)ß, downstream of LPS, requires the production of itaconate. These data demonstrate that itaconate is a critical arbiter of inflammatory cytokine production downstream of multiple innate signaling pathways, laying the groundwork for the development of itaconate mimetics for the treatment of autoimmunity.

Triptolide induced spermatogenesis dysfunction via ferroptosis activation by promoting K63-linked GPX4 polyubiquitination in spermatocytes.

Triptolide (TP) is a major bioactive compound derived from Tripterygium wilfordii Hook. F. (TwHF) known for its medicinal properties, but it also exhibits potential toxic effects. It has been demonstrated to induce severe male reproductive toxicity, yet the precise mechanism behind this remains unclear, which limits its broad clinical application. This study aimed to investigate the mechanisms underlying testicular damage and spermatogenesis dysfunction induced by TP in mice, using both mouse models and the spermatocyte-derived cell line GC-2spd. In the present study, it was found that TP displayed significant testicular microstructure damaged and spermatogenesis defects including lower concentration and abnormal morphology by promoting ROS formation, MDA production and restraining GSH level, glutathione peroxidase 4 (GPX4) expression in vivo. Furthermore, Ferrostatin-1 (FER-1), a ferroptosis inhibitor, was found to significantly reduce the accumulation of lipid peroxidation, alleviate testicular microstructural damage, and enhance spermatogenic function in mice. Besides, notably decreased cell viability, collapsed mitochondrial membrane potential, and elevated DNA damage were observed in vitro. The above-mentioned phenomenon could be reversed by pre-treatment of FER-1, indicating that ferroptosis participated in the TP-mediated spermatogenesis dysfunction. Mechanistically, TP could enhance GPX4 ubiquitin degradation via triggering K63-linked polyubiquitination of GPX4, thereby stimulating ferroptosis in spermatocytes. Functionally, GPX4 deletion intensified ferroptosis and exacerbated DNA damage in GC-2 cells, while GPX4 overexpression mitigated ferroptosis induced by TP. Overall, these findings for the first time indicated a vital role of ferroptosis in TP induced-testicular injury and spermatogenic dysfunction through promoting GPX4 K63-linked polyubiquitination, which hopefully offers a potential therapeutic avenue for TP-related male reproductive damage. In addition, this study also provides a theoretical foundation for the improved clinical application of TP or TwHF in the future.

Lens autophagy protein ATG16L1: a potential target for cataract treatment.

Rationale: Cataract is the leading cause of blindness and low vision worldwide, yet its pathological mechanism is not fully understood. Although macroautophagy/autophagy is recognized as essential for lens homeostasis and has shown potential in alleviating cataracts, its precise mechanism remains unclear. Uncovering the molecular details of autophagy in the lens could provide targeted therapeutic interventions alongside surgery. Methods: We monitored autophagic activities in the lens and identified the key autophagy protein ATG16L1 by immunofluorescence staining, Western blotting, and transmission electron microscopy. The regulatory mechanism of ATG16L1 ubiquitination was analyzed by co-immunoprecipitation and Western blotting. We used the crystal structure of E3 ligase gigaxonin and conducted the docking screening of a chemical library. The effect of the identified compound riboflavin was tested in vitro in cells and in vivo animal models. Results: We used HLE cells and connexin 50 (cx50)-deficient cataract zebrafish model and confirmed that ATG16L1 was crucial for lens autophagy. Stabilizing ATG16L1 by attenuating its ubiquitination-dependent degradation could promote autophagy activity and relieve cataract phenotype in cx50-deficient zebrafish. Mechanistically, the interaction between E3 ligase gigaxonin and ATG16L1 was weakened during this process. Leveraging these mechanisms, we identified riboflavin, an E3 ubiquitin ligase-targeting drug, which suppressed ATG16L1 ubiquitination, promoted autophagy, and ultimately alleviated the cataract phenotype in autophagy-related models. Conclusions: Our study identified an unrecognized mechanism of cataractogenesis involving ATG16L1 ubiquitination in autophagy regulation, offering new insights for treating cataracts.

Deubiquitylase OTUD3 regulates integrated stress response to suppress progression and sorafenib resistance of liver cancer.

The integrated stress response (ISR) is activated in response to intrinsic and extrinsic stimuli, playing a role in tumor progression and drug resistance. The regulatory role and mechanism of ISR in liver cancer, however, remain largely unexplored. Here, we demonstrate that OTU domain-containing protein 3 (OTUD3) is a deubiquitylase of eukaryotic initiation factor 2α (eIF2α), antagonizing ISR and suppressing liver cancer. OTUD3 decreases interactions between eIF2α and the kinase EIF2ΑK3 by removing K27-linked polyubiquitylation on eIF2α. OTUD3 deficiency in mice leads to enhanced ISR and accelerated progression of N-nitrosodiethylamine-induced hepatocellular carcinoma. Additionally, decreased OTUD3 expression associated with elevated eIF2α phosphorylation correlates with the progression of human liver cancer. Moreover, ISR activation due to decreased OTUD3 expression renders liver cancer cells resistant to sorafenib, while the combined use of the ISR inhibitor ISRIB significantly improves their sensitivity to sorafenib. Collectively, these findings illuminate the regulatory mechanism of ISR in liver cancer and provide a potential strategy to counteract sorafenib resistance.

Kaempferol Alleviates Injury in Human Retinal Pigment Epithelial Cells via STAT1 Ubiquitination-Mediated Degradation of IRF7.

BACKGROUND: Retinal pigment epithelial (RPE) cells have a pivotal function in preserving the equilibrium of the retina and moderating the immunological interaction between the choroid and the retina. This study primarily focuses on delineating the protective effect offered by Kaempferol (Kae) against RPE cell damage. METHODS: Bioinformatics analysis was performed on the GSE30719 dataset to identify hub genes associated with RPE. Subsequently, we analyzed the impact of Kae on RPE apoptosis, cell viability, and inflammatory response through cell experiments, and explored the interaction between hub genes and Kae. RESULTS: Based on the GSE30719 dataset, nine hub genes (ISG15, IFIT1, IFIT3, STAT1, OASL, RSAD2, IRF7, MX2, and MX1) were identified, all of which were highly expressed in the GSE30719 case group. Kae could boost the proliferative activity of RPE cells caused by lipopolysaccharide (LPS), as well as reduce apoptosis and the generation of inflammatory factors (tumor necrosis factor receptor (TNFR), interleukin-1beta (IL-1ß)) and cytokines (IL-1, IL-6, IL-12). STAT1 was shown to inhibit cell proliferation, promote apoptosis, and secrete IL-1/IL-6/IL-12 in LPS-induced RPE cells. Moreover, IRF7 was found to interact with STAT1 in LPS-induced RPE cells, and STAT1 could maintain IRF7 levels through deubiquitination. In addition, we also found that the protective effect of Kae on LPS-induced RPE cell injury was mediated through STAT1/IRF7 axis. CONCLUSION: This study provided evidence that Kae protects RPE cells via regulating the STAT1/IRF7 signaling pathways, indicating its potential therapeutic relevance in the diagnosis and management of retinal disorders linked with RPE cell damage.

Astragaloside IV relieves passive heymann nephritis and podocyte injury by suppressing the TRAF6/NF-κb axis.

The pathogenesis of membranous nephropathy (MN) involves podocyte injury that is attributed to inflammatory responses induced by local immune deposits. Astragaloside IV (AS-IV) is known for its robust anti-inflammatory properties. Here, we investigated the effects of AS-IV on passive Heymann nephritis (PHN) rats and TNF-α-induced podocytes to determine the underlying molecular mechanisms of MN. Serum biochemical parameters, 24-h urine protein excretion and renal histopathology were evaluated in PHN and control rats. The expression of tumor necrosis factor receptor associated factor 6 (TRAF6), the phosphorylation of nuclear factor kappa B (p-NF-κB), the expression of associated proinflammatory cytokines (TNF-α, IL-6 and IL-1ß) and the ubiquitination of TRAF6 were measured in PHN rats and TNF-α-induced podocytes. We detected a marked increase in mRNA expression of TNF-α, IL-6 and IL-1ß and in the protein abundance of p-NF-κB and TRAF6 within the renal tissues of PHN rats and TNF-α-induced podocytes. Conversely, there was a reduction in the K48-linked ubiquitination of TRAF6. Additionally, AS-IV was effective in ameliorating serum creatinine, proteinuria, and renal histopathology in PHN rats. This effect was concomitant with the suppression of NF-κB pathway activation and decreased expression of TNF-α, IL-6, IL-1ß and TRAF6. AS-IV decreased TRAF6 levels by promoting K48-linked ubiquitin conjugation to TRAF6, which triggered ubiquitin-mediated degradation. In summary, AS-IV averted renal impairment in PHN rats and TNF-α-induced podocytes, likely by modulating the inflammatory response through the TRAF6/NF-κB axis. Targeting TRAF6 holds therapeutic promise for managing MN.

USP19 Stabilizes TAK1 to Regulate High Glucose/Free Fatty Acid-induced Dysfunction in HK-2 Cells.

OBJECTIVE: Obesity-induced kidney injury contributes to the development of diabetic nephropathy (DN). Here, we identified the functions of ubiquitin-specific peptidase 19 (USP19) in HK-2 cells exposed to a combination of high glucose (HG) and free fatty acid (FFA) and determined its association with TGF-beta-activated kinase 1 (TAK1). METHODS: HK-2 cells were exposed to a combination of HG and FFA. USP19 mRNA expression was detected by quantitative RT-PCR (qRT-PCR), and protein analysis was performed by immunoblotting (IB). Cell growth was assessed by Cell Counting Kit-8 (CCK-8) viability and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assays. Cell cycle distribution and apoptosis were detected by flow cytometry. The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation (Co-IP) assays and IB. RESULTS: In HG+FFA-challenged HK-2 cells, USP19 was highly expressed. USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells. Moreover, USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1 (PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species (ROS) generation in HK-2 cells. Mechanistically, USP19 stabilized the TAK1 protein through deubiquitination. Importantly, increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells. CONCLUSION: The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1, providing a potential therapeutic strategy for combating DN.