Directed RNase H Cleavage of Nascent Transcripts Causes Transcription Termination.

An attractive approach to reduce gene expression is via the use of antisense oligonucleotides (ASOs) that harness the RNase H1 mechanism. Here we show that RNase H ASOs targeted to introns or exons robustly reduce the level of spliced RNA associated with chromatin. Surprisingly, intron-targeted ASOs reduce the level of pre-mRNA associated with chromatin to a greater extent than exon-targeted ASOs. This indicates that exon-targeted ASOs achieve full activity after the pre-mRNA has undergone splicing, but before the mRNA is released from chromatin. Even though RNase H ASOs can reduce the level of RNA associated with chromatin, the effect of ASO-directed RNA degradation on transcription has never been documented. Here we show that intron-targeted ASOs and, to a lesser extent, exon-targeted ASOs cause RNA polymerase II (Pol II) transcription termination in cultured cells and mice. Furthermore, ASO-directed transcription termination is mediated by the nuclear exonuclease XRN2.

Dismissal of RNA Polymerase II Underlies a Large Ligand-Induced Enhancer Decommissioning Program.

Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers.

NEDD4L intramolecular interactions regulate its auto and substrate NaV1.5 ubiquitination.

NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, NaV1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For NaV1.5, this process is critical as alterations in Na+ current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of NaV1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of NaV1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na+ current reduction using the NEDD4L HECT domain as cargo of a NaV1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding NaV1.5 ubiquitination.

Ubiquitin ligase NEDD4 promotes the proliferation of hepatocellular carcinoma cells through targeting PCDH17 protein for ubiquitination and degradation.

Neural precursor cell expressed developmentally downregulated 4 (NEDD4), an E3 ubiquitin ligase, is commonly upregulated in human hepatocellular carcinoma (HCC) and functions as an oncogenic factor in the progression of HCC, but the molecular mechanism needs be further explored. In this study, we found that NEDD4 could facilitate the proliferation of HCC cells, which was associated with regulating the ERK signaling. Further investigation showed that protocadherin 17 (PCDH17) was a potential substrate of NEDD4, and restoration of PCDH17 could block the facilitation of ERK signaling and HCC cells proliferation induced by NEDD4 overexpression. Whereafter, we confirmed that NEDD4 interacted with PCDH17 and promoted the Lys33-linked polyubiquitination and degradation of it via the proteasome pathway. Finally, NEDD4 protein level was found to be inversely correlated with that of PCDH17 in human HCC tissues. In conclusion, these results suggest that NEDD4 acts as an E3 ubiquitin ligase for PCDH17 ubiquitination and degradation thereby promoting the proliferation of HCC cells through regulating the ERK signaling, which may provide novel evidence for NEDD4 to be a promising therapeutic target for HCC.

Physiological Functions of the Ubiquitin Ligases Nedd4-1 and Nedd4-2.

The Nedd4 family of E3 ubiquitin ligases, consisting of a C2-WW(n)-HECT domain architecture, includes the closely related Nedd4/Nedd4-1 and Nedd4L/Nedd4-2, which play critical roles in human physiology and pathophysiology.This review focuses on the regulation of enzymatic activity of these Nedd4 proteins, as well as on their roles in regulating stability and function of membrane and other signaling proteins, such as ion channels, ion transporters, and growth factor receptors. The diseases caused by impairment of such regulation are discussed, as well as opportunities and challenges for targeting these enzymes for therapy.

Development and validation of AI/ML derived splice-switching oligonucleotides.

Splice-switching oligonucleotides (SSOs) are antisense compounds that act directly on pre-mRNA to modulate alternative splicing (AS). This study demonstrates the value that artificial intelligence/machine learning (AI/ML) provides for the identification of functional, verifiable, and therapeutic SSOs. We trained XGboost tree models using splicing factor (SF) pre-mRNA binding profiles and spliceosome assembly information to identify modulatory SSO binding sites on pre-mRNA. Using Shapley and out-of-bag analyses we also predicted the identity of specific SFs whose binding to pre-mRNA is blocked by SSOs. This step adds considerable transparency to AI/ML-driven drug discovery and informs biological insights useful in further validation steps. We applied this approach to previously established functional SSOs to retrospectively identify the SFs likely to regulate those events. We then took a prospective validation approach using a novel target in triple negative breast cancer (TNBC), NEDD4L exon 13 (NEDD4Le13). Targeting NEDD4Le13 with an AI/ML-designed SSO decreased the proliferative and migratory behavior of TNBC cells via downregulation of the TGFß pathway. Overall, this study illustrates the ability of AI/ML to extract actionable insights from RNA-seq data.

Elevated intracellular Na+ and osmolarity stimulate catalytic activity of the ubiquitin ligase Nedd4-2.

Regulation of catalytic activity of E3 ubiquitin ligases is critical for their cellular functions. We identified an unexpected mode of regulation of E3 catalytic activity by ions and osmolarity; enzymatic activity of the HECT family E3 Nedd4-2/Nedd4L is enhanced by increased intracellular Na+ ([Na+]i) and by hyperosmolarity. This stimulated activity is mediated by activation of p38-MAPK and is inhibited by WNKs. Moreover, protease (Furin)-mediated activation of the epithelial Na+ channel ENaC (a bona fide Nedd4-2 substrate), which leads to increased [Na+]i and osmolarity, results in enhanced Nedd4-2 catalytic activity. This enhancement is inhibited by a Furin inhibitor, by a protease-resistant ENaC mutant, or by treatment with the ENaC inhibitor amiloride. Moreover, WNK inhibition, which stimulates catalytic activity of Nedd4-2, leads to reduced levels of cell-surface ENaC and reduced channel activity. ENaC activity does not affect Nedd4-2:ENaC binding. Therefore, these results demonstrate activation of a ubiquitin ligase by Na+ and osmotic changes. Importantly, they reveal a negative feedback loop in which active ENaC leads to stimulation of catalytic activity of its own suppressor, Nedd4-2, to protect cells from excessive Na+ loading and hyperosmotic stress and to protect the animal from hypertension.

Targeted ubiquitination of sensory neuron calcium channels reduces the development of neuropathic pain.

Neuropathic pain caused by lesions to somatosensory neurons due to injury or disease is a widespread public health problem that is inadequately managed by small-molecule therapeutics due to incomplete pain relief and devastating side effects. Genetically encoded molecules capable of interrupting nociception have the potential to confer long-lasting analgesia with minimal off-target effects. Here, we utilize a targeted ubiquitination approach to achieve a unique posttranslational functional knockdown of high-voltage-activated calcium channels (HVACCs) that are obligatory for neurotransmission in dorsal root ganglion (DRG) neurons. CaV-aßlator comprises a nanobody targeted to CaV channel cytosolic auxiliary ß subunits fused to the catalytic HECT domain of the Nedd4-2 E3 ubiquitin ligase. Subcutaneous injection of adeno-associated virus serotype 9 encoding CaV-aßlator in the hind paw of mice resulted in the expression of the protein in a subset of DRG neurons that displayed a concomitant ablation of CaV currents and also led to an increase in the frequency of spontaneous inhibitory postsynaptic currents in the dorsal horn of the spinal cord. Mice subjected to spare nerve injury displayed a characteristic long-lasting mechanical, thermal, and cold hyperalgesia underlain by a dramatic increase in coordinated phasic firing of DRG neurons as reported by in vivo Ca2+ spike recordings. CaV-aßlator significantly dampened the integrated Ca2+ spike activity and the hyperalgesia in response to nerve injury. The results advance the principle of targeting HVACCs as a gene therapy for neuropathic pain and demonstrate the therapeutic potential of posttranslational functional knockdown of ion channels achieved by exploiting the ubiquitin-proteasome system.

NEDD4L affects KLF5 stability through ubiquitination to control ferroptosis and radiotherapy resistance in oesophageal squamous cell carcinoma.

Oesophageal squamous cell carcinoma (ESCC) contributes to high mortality. Modulating ferroptosis may reverse resistance to radiotherapy. This article was to explore the ubiquitination modification of KLF5 and its effect on ferroptosis in ESCC. KLF5 was under-expressed by shRNA plasmids in the cells and ROS levels were analysed by flow cytometry, ferroptotic gene expression was detected by qRT-PCR, MDA and GSH levels were determined by ELISA, cell morphology was observed by transmission electron microscopy, and Fe ion levels were analysed by immunofluorescence. Cells were treated with Ferrostatin-1 and NAC and analysed for cell proliferation by colony formation assay, cell migration and invasion by Transwell assays, and apoptosis by flow cytometry. DNA damage in cells was also analysed using comet assay, EdU doping assay, γH2AX fluorescence, DNA-PKcs and PCR. NEDD4L and KLF5 binding was analysed by immunoprecipitation. Changes in ferroptosis, DNA damage and resistance were analysed in cells with both silencing NEDD4L and KLF5. Changes in tumour resistance to radiation were analysed in mice underexpressing NEDD4L and KLF5. Low expression of KLF5 significantly promotes cellular lipid peroxidation levels, with decreased expression of SOD and GPX4, and increased expression of ACSL4. Concurrently, MDA levels deplete GSH, and cells exhibit typical ferroptotic morphology with increased Fe2+ content. KLF5 inhibition results in enhanced cellular clonogenicity, migration and invasion activities, reduced apoptosis, increased tail DNA, nuclear EdU incorporation, nuclear γH2AX foci and elevated expression of DNA-PKcs, LIG4, RAD9B and BMI1. Ferrostatin-1 and NAC reverse these effects. NEDD4L ubiquitination modifies and degrades KLF5, with NEDD4L/KLF5 inhibition mitigating cellular ferroptosis and DNA damage, thereby promoting radiosensitivity both in vitro and in vivo. NEDD4L increases radiosensitivity by accelerating cellular ferroptosis via ubiquitination modification of KLF5.

ECHDC2 inhibits the proliferation of gastric cancer cells by binding with NEDD4 to degrade MCCC2 and reduce aerobic glycolysis.

BACKGROUND: The Enoyl-CoA hydratase/isomerase family plays a crucial role in the metabolism of tumors, being crucial for maintaining the energy balance and biosynthetic needs of cancer cells. However, the enzymes within this family that are pivotal in gastric cancer (GC) remain unclear. METHODS: We employed bioinformatics techniques to identify key Enoyl-CoA hydratase/isomerase in GC. The expression of ECHDC2 and its clinical significance were validated through tissue microarray analysis. The role of ECHDC2 in GC was further assessed using colony formation assays, CCK8 assay, EDU assay, Glucose and lactic acid assay, and subcutaneous tumor experiments in nude mice. The mechanism of action of ECHDC2 was validated through Western blotting, Co-immunoprecipitation, and immunofluorescence experiments. RESULTS: Our analysis of multiple datasets indicates that low expression of ECHDC2 in GC is significantly associated with poor prognosis. Overexpression of ECHDC2 notably inhibits aerobic glycolysis and proliferation of GC cells both in vivo and in vitro. Further experiments revealed that overexpression of ECHDC2 suppresses the P38 MAPK pathway by inhibiting the protein level of MCCC2, thereby restraining glycolysis and proliferation in GC cells. Ultimately, it was discovered that ECHDC2 promotes the ubiquitination and subsequent degradation of MCCC2 protein by binding with NEDD4. CONCLUSIONS: These findings underscore the pivotal role of the ECHDC2 in regulating aerobic glycolysis and proliferation in GC cells, suggesting ECHDC2 as a potential therapeutic target in GC.

Aberrant expression of NEDD4L disrupts mitochondrial homeostasis by downregulating CaMKKß in diabetic kidney disease.

Disturbance in mitochondrial homeostasis within proximal tubules is a critical characteristic associated with diabetic kidney disease (DKD). CaMKKß/AMPK signaling plays an important role in regulating mitochondrial homeostasis. Despite the downregulation of CaMKKß in DKD pathology, the underlying mechanism remains elusive. The expression of NEDD4L, which is primarily localized to renal proximal tubules, is significantly upregulated in the renal tubules of mice with DKD. Coimmunoprecipitation (Co-IP) assays revealed a physical interaction between NEDD4L and CaMKKß. Moreover, deletion of NEDD4L under high glucose conditions prevented rapid CaMKKß protein degradation. In vitro studies revealed that the aberrant expression of NEDD4L negatively influences the protein stability of CaMKKß. This study also explored the role of NEDD4L in DKD by using AAV-shNedd4L in db/db mice. These findings confirmed that NEDD4L inhibition leads to a decrease in urine protein excretion, tubulointerstitial fibrosis, and oxidative stress, and mitochondrial dysfunction. Further in vitro studies demonstrated that si-Nedd4L suppressed mitochondrial fission and reactive oxygen species (ROS) production, effects antagonized by si-CaMKKß. In summary, the findings provided herein provide strong evidence that dysregulated NEDD4L disturbs mitochondrial homeostasis by negatively modulating CaMKKß in the context of DKD. This evidence underscores the potential of therapeutic interventions targeting NEDD4L and CaMKKß to safeguard renal tubular function in the management of DKD.

Ubiquitination of angiotensin-converting enzyme 2 contributes to the development of pulmonary arterial hypertension mediated by neural precursor cell-expressed developmentally down-regulated gene 4-Like.

OBJECTIVES: In this study, we investigated whether neural precursor cell-expressed developmentally down-regulated gene 4-like (NEDD4L) is the E3 enzyme of angiotensin-converting enzyme 2 (ACE2) and whether NEDD4L degrades ACE2 via ubiquitination, leading to the progression of pulmonary arterial hypertension (PAH). METHODS: Bioinformatic analyses were used to explore the E3 ligase that ubiquitinates ACE2. Cultured pulmonary arterial smooth muscle cells (PASMCs) and specimens from patients with PAH were used to investigate the crosstalk between NEDD4L and ACE2 and its ubiquitination in the context of PAH. RESULTS: The inhibition of ubiquitination attenuated hypoxia-induced proliferation of PASMCs. The levels of NEDD4L were increased, and those of ACE2 were decreased in lung tissues from patients with PAH and in PASMCs. NEDD4L, the E3 ligase of ACE2, inhibited the expression of ACE2 in PASMCs, possibly through ubiquitination-mediated degradation. PAH was associated with upregulation of NEDD4L expression and downregulation of ACE2 expression. CONCLUSIONS: NEDD4L, the E3 ubiquitination enzyme of ACE2, promotes the proliferation of PASMCs, ultimately leading to PAH.

Gstp1 negatively regulates blood pressure in hypertensive rat via promoting APLNR ubiquitination degradation mediated by Nedd4.

Hypertension is a leading risk factor for disease burden worldwide. Vascular contraction and remodeling contribute to the development of hypertension. Glutathione S-transferase P1 (Gstp1) plays several critical roles in both normal and neoplastic cells. In this study, we investigated the effect of Gstp1 on hypertension as well as on vascular smooth muscle cell (VSMC) contraction and phenotypic switching. We identified the higher level of Gstp1 in arteries and VSMCs from hypertensive rats compared with normotensive rats for the first time. We then developed Adeno-associated virus 9 (AAV9) mediated Gstp1 down-regulation and overexpression in rats and measured rat blood pressure by using the tail-cuff and the carotid catheter method. We found that the blood pressure of spontaneously hypertensive rats (SHR) rose significantly with Gstp1 down-regulation and reduced apparently after Gstp1 overexpression. Similar results were obtained from the observations of 2-kidney-1-clip renovascular (2K1C) hypertensive rats. Gstp1 did not influence blood pressure of normotensive Wistar-Kyoto (WKY) rats and Sprague-Dawley (SD) rats. Further in vitro study indicated that Gstp1 knockdown in SHR-VSMCs promoted cell proliferation, migration, dedifferentiation and contraction, while Gstp1 overexpression showed opposite effects. Results from bioinformatic analysis showed that the Apelin/APLNR system was involved in the effect of Gstp1 on SHR-VSMCs. The rise in blood pressure of SHR induced by Gstp1 knockdown could be reversed by APLNR antagonist F13A. We further found that Gstp1 enhanced the association between APLNR and Nedd4 E3 ubiquitin ligases to induce APLNR ubiquitination degradation. Thus, in the present study, we discovered a novel anti-hypertensive role of Gstp1 in hypertensive rats and provided the experimental basis for designing an effective anti-hypertensive therapeutic strategy.

Tudor-SN exacerbates pathological vascular remodeling by promoting the polyubiquitination of PTEN via NEDD4-1.

BACKGROUND: Dysregulation of vascular homeostasis can induce cardiovascular diseases and increase global mortality rates. Although lineage tracing studies have confirmed the pivotal role of modulated vascular smooth muscle cells (VSMCs) in the progression of pathological vascular remodeling, the underlying mechanisms are still unclear. METHODS: The expression of Tudor-SN was determined in VSMCs of artery stenosis, PDGF-BB-treated VSMCs and atherosclerotic plaque. Loss- and gain-of-function approaches were used to explore the role of Tudor-SN in the modulation of VSMCs phenotype both in vivo and in vitro. RESULTS: In this study, we demonstrate that Tudor-SN expression is significantly elevated in injury-induced arteries, atherosclerotic plaques, and PDGF-BB-stimulated VSMCs. Tudor-SN deficiency attenuates, but overexpression aggravates the synthetic phenotypic switching of VSMCs and pathological vascular remodeling. Loss of Tudor-SN also reduces atherosclerotic plaque formation and increases plaque stability. Mechanistically, PTEN, the major regulator of the MAPK and PI3K-AKT signaling pathways, plays a vital role in Tudor-SN-mediated regulation on proliferation and migration of VSMCs. Tudor-SN facilitates the polyubiquitination and degradation of PTEN via NEDD4-1, thus exacerbating vascular remodeling under pathological conditions. BpV (HOpic), a specific inhibitor of PTEN, not only counteracts the protective effect of Tudor-SN deficiency on proliferation and migration of VSMCs, but also abrogates the negative effect of carotid artery injury-induced vascular remodeling in mice. CONCLUSIONS: Our findings reveal that Tudor-SN deficiency significantly ameliorated pathological vascular remodeling by reducing NEDD4-1-dependent PTEN polyubiquitination, suggesting that Tudor-SN may be a novel target for preventing vascular diseases.

NEDD4L mediates ITGB4 ubiquitination and degradation to suppress esophageal carcinoma progression.

The ubiquitination-mediated protein degradation exerts a vital role in the progression of multiple tumors. NEDD4L, which belongs to the E3 ubiquitin ligase NEDD4 family, is related to tumor genesis, metastasis and drug resistance. However, the anti-tumor role of NEDD4L in esophageal carcinoma, and the potential specific recognition substrate remain unclear. Based on public esophageal carcinoma database and clinical sample data, it was discovered in this study that the expression of NEDD4L in esophageal carcinoma was apparently lower than that in atypical hyperplastic esophageal tissue and esophageal squamous epithelium. Besides, patients with high expression of NEDD4L in esophageal carcinoma tissue had longer progression-free survival than those with low expression. Experiments in vivo and in vitro also verified that NEDD4L suppressed the growth and metastasis of esophageal carcinoma. Based on co-immunoprecipitation and proteome analysis, the NEDD4L ubiquitination-degraded protein ITGB4 was obtained. In terms of the mechanism, the HECT domain of NEDD4L specifically bound to the Galx-ß domain of ITGB4, which modified the K915 site of ITGB4 in an ubiquitination manner, and promoted the ubiquitination degradation of ITGB4, thus suppressing the malignant phenotype of esophageal carcinoma.

Activity-based protein profiling and global proteome analysis reveal MASTL as a potential therapeutic target in gastric cancer.

BACKGROUND: Gastric cancer (GC) is a prevalent malignancy with limited therapeutic options for advanced stages. This study aimed to identify novel therapeutic targets for GC by profiling HSP90 client kinases. METHODS: We used mass spectrometry-based activity-based protein profiling (ABPP) with a desthiobiotin-ATP probe, combined with sensitivity analysis of HSP90 inhibitors, to profile kinases in a panel of GC cell lines. We identified kinases regulated by HSP90 in inhibitor-sensitive cells and investigated the impact of MASTL knockdown on GC cell behavior. Global proteomic analysis following MASTL knockdown was performed, and bioinformatics tools were used to analyze the resulting data. RESULTS: Four kinases-MASTL, STK11, CHEK1, and MET-were identified as HSP90-regulated in HSP90 inhibitor-sensitive cells. Among these, microtubule-associated serine/threonine kinase-like (MASTL) was upregulated in GC and associated with poor prognosis. MASTL knockdown decreased migration, invasion, and proliferation of GC cells. Global proteomic profiling following MASTL knockdown revealed NEDD4-1 as a potential downstream mediator of MASTL in GC progression. NEDD4-1 was also upregulated in GC and associated with poor prognosis. Similar to MASTL inhibition, NEDD4-1 knockdown suppressed migration, invasion, and proliferation of GC cells. CONCLUSIONS: Our multi-proteomic analyses suggest that targeting MASTL could be a promising therapy for advanced gastric cancer, potentially through the reduction of tumor-promoting proteins including NEDD4-1. This study enhances our understanding of kinase signaling pathways in GC and provides new insights for potential treatment strategies.

Deoxyribonuclease 1-like 3 inhibits colorectal malignancy through antagonizing NEDD4-triggered CDKN1A ubiquitination.

Deoxyribonuclease 1-like 3 (DNASE1L3) has been shown to play nonnegligible roles in several types of carcinomas. Nevertheless, the biological function, clinical relevance, and influence of DNASE1L3 in colorectal cancer (CRC) remain obscure. Immunohistochemistry was adopted to examine DNASE1L3 and CDKN1A expression in CRC tissue, and the clinical significance of DNASE1L3 was assessed. Cell counting kit-8, colony formation, and transwell assays were employed for assessing tumor proliferation and migration. The mechanisms underlying the impact of DNASE1L3 were explored via western blot analysis, co-immunoprecipitation, and ubiquitination assay. It was observed that DNASE1L3 was downregulated in CRC tissues and was tightly associated with patient prognosis. DNASE1L3 impaired CRC cell proliferation and migration through elevating CDKN1A via suppressing CDKN1A ubiquitination. Meanwhile, DNASE1L3 was positively related to CDKN1A. In mechanism, DNASE1L3 and CDKN1A interacted with the E3 ubiquitin ligase NEDD4. Moreover, DNASE1L3 was competitively bound to NEDD4, thus repressing NEDD4-mediated CDKN1A ubiquitination and degradation. These discoveries implied the potential mechanisms of DNASE1L3 during tumorigenesis, suggesting that DNASE1L3 may serve as a new potential therapeutic agent for CRC.

Tril dampens Nodal signaling through Pellino2- and Traf6-mediated activation of Nedd4l.

Toll-like receptor 4 (Tlr) interactor with leucine-rich repeats (Tril) functions as a Tlr coreceptor to mediate innate immunity in adults. In Xenopus embryos, Tril triggers degradation of the transforming growth factor ß (Tgf-ß) family inhibitor, Smad7. This enhances bone morphogenetic protein (Bmp) signaling to enable ventral mesoderm to commit to a blood fate. Here, we show that Tril simultaneously dampens Nodal signaling by catalytically activating the ubiquitin ligase NEDD4 Like (Nedd4l). Nedd4l then targets Nodal receptors for degradation. How Tril signals are transduced in a nonimmune context is unknown. We identify the ubiquitin ligase Pellino2 as a protein that binds to the cytoplasmic tail of Tril and subsequently forms a complex with Nedd4l and another E3 ligase, TNF-receptor associated factor 6 (Traf6). Pellino2 and Traf6 are essential for catalytic activation of Nedd4l, both in Xenopus and in mammalian cells. Traf6 ubiquitinates Nedd4l, which is then recruited to membrane compartments where activation occurs. Collectively, our findings reveal that Tril initiates a noncanonical Tlr-like signaling cascade to activate Nedd4l, thereby coordinately regulating the Bmp and Nodal arms of the Tgf-ß superfamily during vertebrate development.

PUM2 promoted osteoarthritis progression through PTEN-mediated chondrocyte ferroptosis by facilitating NEDD4 mRNA degradation.

Osteoarthritis (OA) is a prevalent degenerative joint disease with a lack of effective therapeutic. Chondrocyte ferroptosis contributes to the progression of OA. PUM2 is shown to exacerbate ischemia-reperfusion-induced neuroinflammation by promoting ferroptosis, but its role in OA remains unexplored. Here, primary mouse chondrocytes were stimulated with IL-1ß to mimic OA chondrocyte injury in vitro. And PUM2 was upregulated in OA cartilage tissues and IL-1ß-induced chondrocytes. Silencing PUM2 alleviated IL-1ß-induced chondrocyte inflammation and ECM degradation. Mechanistically, PUM2 facilitated the degradation of NEDD4 mRNA by binding to the 3'UTR of NEDD4 mRNA, which in turn inhibited NEDD4 induced PTEN ubiquitination and degradation. Consistently, NEDD4 silencing reversed the ameliorative effect of PUM2 knockdown on chondrocyte injury, and overexpression of PTEN abolished the improved role of NEDD4 in chondrocyte injury. Moreover, PTEN aggravated IL-1ß-induced ferroptosis in chondrocytes through the Nrf2/HO-1 pathway by increasing the levels of Fe2+, ROS, MDA, and ACSL4 protein, decreasing the activity of SOD and the levels of GSH and GPX4 protein, and aggravating mitochondrial damage. Additionally, destabilized medial meniscus (DMM) were conducted to establish the OA mouse model, and adenovirus-mediated PUM2 shRNA was administered intra-articularly. Silencing PUM2 attenuated OA-induced cartilage damage in vivo. In conclusion, PUM2 promoted OA progression through PTEN-mediated chondrocyte ferroptosis by facilitating NEDD4 mRNA degradation.

The Ubiquitin Ligase Adaptor NDFIP1 Interacts with TRESK and Negatively Regulates the Background K+ Current.

The TRESK (K2P18.1, KCNK18) background potassium channel is expressed in primary sensory neurons and has been reported to contribute to the regulation of pain sensations. In the present study, we examined the interaction of TRESK with NDFIP1 (Nedd4 family-interacting protein 1) in the Xenopus oocyte expression system by two-electrode voltage clamp and biochemical methods. We showed that the coexpression of NDFIP1 abolished the TRESK current under the condition where the other K+ channels were not affected. Mutations in the three PPxY motifs of NDFIP1, which are responsible for the interaction with the Nedd4 ubiquitin ligase, prevented a reduction in the TRESK current. Furthermore, the overexpression of a dominant-negative Nedd4 construct in the oocytes coexpressing TRESK with NDFIP1 partially reversed the down-modulating effect of the adaptor protein on the K+ current. The biochemical data were also consistent with the functional results. An interaction between epitope-tagged versions of TRESK and NDFIP1 was verified by co-immunoprecipitation experiments. The coexpression of NDFIP1 with TRESK induced the ubiquitination of the channel protein. Altogether, the results suggest that TRESK is directly controlled by and highly sensitive to the activation of the NDFIP1-Nedd4 system. The NDFIP1-mediated reduction in the TRESK component may induce depolarization, increase excitability, and attenuate the calcium dependence of the membrane potential by reducing the calcineurin-activated fraction in the ensemble background K+ current.