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.

circNEIL3 inhibits tumor metastasis through recruiting the E3 ubiquitin ligase Nedd4L to degrade YBX1.

Distant metastasis is a major contributor to cancer-related mortality. However, the role of circRNAs in this process remains unclear. Herein, we profiled the circRNA expression in a cohort of 68 colorectal carcinoma (CRC) primary tumors and their paired liver metastatic lesions. By overlapping with the TGFß-responsive circRNAs, circNEIL3 (hsa_circ_0001460) was identified as a TGFß-repressive and metastasis-related circRNA. Functionally, circNEIL3 effectively inhibited tumor metastasis in both and in vivo and in vivo models of various cancer types. Mechanistically, circNEIL3 exerts its metastasis-repressive function through its direct interaction with oncogenic protein, Y-box-binding protein 1 (YBX1), which consequently promotes the Nedd4L-mediated proteasomal degradation of YBX1. Importantly, circNEIL3 expression was negatively correlated to YBX1 protein level and metastatic tendency in CRC patient samples. Collectively, our findings indicate the YBX1-dependent antimetastatic function of circNEIL3 and highlight the potential of circNEIL3 as a biomarker and therapeutic option in cancer treatment.

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.

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.

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.

Activation of nicotinic acetylcholine receptor α7 subunit limits Zika viral infection via promoting autophagy and ferroptosis.

Vagus nerve regulates viral infection and inflammation via the alpha 7 nicotinic acetylcholine receptor (α7 nAChR); however, the role of α7 nAChR in ZIKA virus (ZIKV) infection, which can cause severe neurological diseases such as microcephaly and Guillain-Barré syndrome, remains unknown. Here, we first examined the role of α7 nAChR in ZIKV infection in vitro. A broad effect of α7 nAChR activation was identified in limiting ZIKV infection in multiple cell lines. Combined with transcriptomics analysis, we further demonstrated that α7 nAChR activation promoted autophagy and ferroptosis pathways to limit cellular ZIKV viral loads. Additionally, activation of α7 nAChR prevented ZIKV-induced p62 nucleus accumulation, which mediated an enhanced autophagy pathway. By regulating proteasome complex and an E3 ligase NEDD4, activation of α7 nAChR resulted in increased amount of cellular p62, which further enhanced the ferroptosis pathway to reduce ZIKV infection. Moreover, utilizing in vivo neonatal mouse models, we showed that α7 nAChR is essential in controlling the disease severity of ZIKV infection. Taken together, our findings identify an α7 nAChR-mediated effect that critically contributes to limiting ZIKV infection, and α7 nAChR activation offers a novel strategy for combating ZIKV infection and its complications.

The E3 ubiquitin ligase ITCH negatively regulates intercellular communication via gap junctions by targeting connexin43 for lysosomal degradation.

Intercellular communication via gap junctions has a fundamental role in regulating cell growth and tissue homeostasis, and its dysregulation may be involved in cancer development and radio- and chemotherapy resistance. Connexin43 (Cx43) is the most ubiquitously expressed gap junction channel protein in human tissues. Emerging evidence indicates that dysregulation of the sorting of Cx43 to lysosomes is important in mediating the loss of Cx43-based gap junctions in cancer cells. However, the molecular basis underlying this process is currently poorly understood. Here, we identified the E3 ubiquitin ligase ITCH as a novel regulator of intercellular communication via gap junctions. We demonstrate that ITCH promotes loss of gap junctions in cervical cancer cells, which is associated with increased degradation of Cx43 in lysosomes. The data further indicate that ITCH interacts with and regulates Cx43 ubiquitination and that the ITCH-induced loss of Cx43-based gap junctions requires its catalytic HECT (homologous to E6-AP C-terminus) domain. The data also suggest that the ability of ITCH to efficiently promote loss of Cx43-based gap junctions and degradation of Cx43 depends on a functional PY (PPXY) motif in the C-terminal tail of Cx43. Together, these data provide new insights into the molecular basis underlying the degradation of Cx43 and have implications for the understanding of how intercellular communication via gap junctions is lost during cancer development.

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.

NEDD4L is a promoter for angiogenesis and cell proliferation in human umbilical vein endothelial cells.

Dysregulated angiogenesis leads to neovascularization, which can promote or exacerbate various diseases. Previous studies have proved that NEDD4L plays an important role in hypertension and atherosclerosis. Hence, we hypothesized that NEDD4L may be a critical regulator of endothelial cell (EC) function. This study aimed to define the role of NEDD4L in regulating EC angiogenesis and elucidate their underlying mechanisms. Loss- and gain-of-function of NEDD4L detected the angiogenesis and mobility role in human umbilical vein endothelial cells (HUVECs) using Matrigel tube formation assay, cell proliferation and migration. Pharmacological pathway inhibitors and western blot were used to determine the underlying mechanism of NEDD4L-regulated endothelial functions. Knockdown of NEDD4L suppressed tube formation, cell proliferation and cell migration in HUVECs, whereas NEDD4L overexpression promoted these functions. Moreover, NEDD4L-regulated angiogenesis and cell progression are associated with the phosphorylation of Akt, Erk1/2 and eNOS and the expression of VEGFR2 and cyclin D1 and D3. Mechanically, further evidence was confirmed by using Akt blocker MK-2206, Erk1/2 blocker U0126 and eNOS blocker L-NAME. Overexpression NEDD4L-promoted angiogenesis, cell migration and cell proliferation were restrained by these inhibitors. In addition, overexpression NEDD4L-promoted cell cycle-related proteins cyclin D1 and D3 were also suppressed by Akt blocker MK-2206, Erk1/2 blocker U0126 and eNOS blocker L-NAME. Our results demonstrated a novel finding that NEDD4L promotes angiogenesis and cell progression by regulating the Akt/Erk/eNOS pathways.

Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

Acute myeloid leukemia (AML) is challenging to treat due to its heterogeneity, prompting a deep understanding of its pathogenesis mechanisms, diagnosis, and treatment. Here, we found reduced expression and acetylation levels of WISP2 in bone marrow mononuclear cells from AML patients and that AML patients with lower WISP2 expression tended to have reduced survival. At the functional level, overexpression of WISP2 in leukemia cells (HL-60 and Kasumi-1) suppressed cell proliferation, induced cell apoptosis, and exerted antileukemic effects in an in vivo model of AML. Our mechanistic investigation demonstrated that WISP2 deacetylation was regulated by the deacetylase histone deacetylase (HDAC)3. In addition, we determined that crosstalk between acetylation and ubiquitination was involved in the modulation of WISP2 expression in AML. Deacetylation of WISP2 decreased the stability of the WISP2 protein by boosting its ubiquitination mediated by NEDD4 and proteasomal degradation. Moreover, pan-HDAC inhibitors (valproic acid and trichostatin A) and an HDAC3-specific inhibitor (RGFP966) induced WISP2 acetylation at lysine K6 and prevented WISP2 degradation. This regulation led to inhibition of proliferation and induction of apoptosis in AML cells. In summary, our study revealed that WISP2 contributes to tumor suppression in AML, which provided an experimental framework for WISP2 as a candidate for gene therapy of AML.

HECT domain interaction with ubiquitin binding sites on Tsg101-UEV controls HIV-1 egress, maturation, and infectivity.

The HECT domain of HECT E3 ligases consists of flexibly linked N- and C-terminal lobes, with a ubiquitin (Ub) donor site on the C-lobe that is directly involved in substrate modification. HECT ligases also possess a secondary Ub binding site in the N-lobe, which is thought to play a role in processivity, specificity, or regulation. Here, we report the use of paramagnetic solution NMR to characterize a complex formed between the isolated HECT domain of neural precursor cell-expressed developmentally downregulated 4-1 and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). Both proteins are involved in endosomal trafficking, a process driven by Ub signaling, and are hijacked by viral pathogens for particle assembly; however, a direct interaction between them has not been described, and the mechanism by which the HECT E3 ligase contributes to pathogen formation has not been elucidated. We provide evidence for their association, consisting of multiple sites on the neural precursor cell-expressed developmentally downregulated 4-1 HECT domain and elements of the Tsg101 UEV domain involved in noncovalent ubiquitin binding. Furthermore, we show using an established reporter assay that HECT residues perturbed by UEV proximity define determinants of viral maturation and infectivity. These results suggest the UEV interaction is a determinant of HECT activity in Ub signaling. As the endosomal trafficking pathway is hijacked by several human pathogens for egress, the HECT-UEV interaction could represent a potential novel target for therapeutic intervention.

Chronic social defeat stress induces the down-regulation of the Nedd4L-GLT-1 ubiquitination pathway in the prefrontal cortex of mice.

Stressful life events contribute to the onset of major depressive disorder (MDD). We recently demonstrated abnormalities in ubiquitination in the pathophysiology of MDD. However, the underlying molecular mechanisms remain unclear. We investigated the involvement of the ubiquitination system-mediated glutamatergic dysfunction in social impairment induced by chronic social defeat stress (CSDS). Adult C57BL/6J mice were exposed to aggressor ICR male mice for 10 consecutive days. Social impairment was induced by CSDS in the social interaction test 1 days after the last stress exposure. In terms of brain microdialysis, CSDS reduced depolarization-evoked glutamate release in the prefrontal cortex (PFC), which was reversed by a glutamate transporter 1 (GLT-1) inhibitor. Interestingly, the expression of ubiquitinated, but not total GLT-1, was decreased in the PFC of mice exposed to CSDS. The expression of neural precursor cells expressing developmentally downregulated gene 4-like (Nedd4L: E3 ligase for GLT-1), and ubiquitin-conjugating enzyme E2D2 (Ube2d2: E2 ubiquitin-conjugating enzyme for Nedd4L) was also reduced in CSDS mice. Furthermore, the downregulation of the Nedd4L-GLT-1 ubiquitination pathway decreased SIT ratio, but up-regulation increased it even in non-CSDS mice. Taken together, the decrease in GLT-1 ubiquitination may reduce the release of extracellular glutamate induced by high-potassium stimulation, which may lead to social impairment, while we could not find differences in GLT-1 ubiquitination between susceptible and resistant CSDS mice. In conclusion, GLT-1 ubiquitination could play a crucial role in the pathophysiology of MDD and is an attractive target for the development of novel antidepressants.

NEDD4 and NEDD4L regulate Wnt signalling and intestinal stem cell priming by degrading LGR5 receptor.

The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis, regeneration and cancer, little is known about the post-translational regulation of LGR5. Here, we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation, increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically, we find that both NEDD4 and NEDD4L negatively regulate Wnt/ß-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers, which subsequently enhances tumour predisposition and progression.

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.

Vagal-α7 nicotinic acetylcholine receptor signaling exacerbates influenza severity by promoting lung epithelial cell infection.

The vagus nerve circuit, operating through the alpha-7 nicotinic acetylcholine receptor (α7 nAChR), regulates the inflammatory response by influencing immune cells. However, the role of vagal-α7 nAChR signaling in influenza virus infection is unclear. In particular, does vagal-α7 nAChR signaling impact the infection of alveolar epithelial cells (AECs), the primary target cells of influenza virus? Here, we demonstrated a distinct role of α7 nAChR in type II AECs compared to its role in immune cells during influenza infection. We found that deletion of Chrna7 (encoding gene of α7 nAChR) in type II AECs or disruption of vagal circuits reduced lung influenza infection and protected mice from influenza-induced lung injury. We further unveiled that activation of α7 nAChR enhanced influenza infection through PTP1B-NEDD4L-ASK1-p38MAPK pathway. Mechanistically, activation of α7 nAChR signaling decreased p38MAPK phosphorylation during infection, facilitating the nuclear export of influenza viral ribonucleoproteins and thereby promoting infection. Taken together, our findings reveal a mechanism mediated by vagal-α7 nAChR signaling that promotes influenza viral infection and exacerbates disease severity. Targeting vagal-α7 nAChR signaling may offer novel strategies for combating influenza virus infections.

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 and 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 downregulation 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) and 2-kidney-1-clip (2K1C) renovascular hypertensive rats rose significantly with Gstp1 downregulation and reduced apparently after Gstp1 overexpression. 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. 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.

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.

E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2.

Aberrant activation of inflammation signaling triggered by tumor necrosis factor α (TNF-α), interleukin-1 (IL-1), and interleukin-17 (IL-17) is associated with immunopathology. Here, we identify neural precursor cells expressed developmentally down-regulated gene 4-like (NEDD4L), a HECT type E3 ligase, as a common negative regulator of signaling induced by TNF-α, IL-1, and IL-17. NEDD4L modulates the degradation of mitogen-activated protein kinase kinase kinase 2 (MEKK2) via constitutively and directly binding to MEKK2 and promotes its poly-ubiquitination. In interleukin-17 receptor (IL-17R) signaling, Nedd4l knockdown or deficiency enhances IL-17-induced p38 and NF-κB activation and the production of proinflammatory cytokines and chemokines in a MEKK2-dependent manner. We further show that IL-17-induced MEKK2 Ser520 phosphorylation is required not only for downstream p38 and NF-κB activation but also for NEDD4L-mediated MEKK2 degradation and the subsequent shutdown of IL-17R signaling. Importantly, Nedd4l-deficient mice show increased susceptibility to IL-17-induced inflammation and aggravated symptoms of experimental autoimmune encephalomyelitis (EAE) in IL-17R signaling-dependent manner. These data suggest that NEDD4L acts as an inhibitor of IL-17R signaling, which ameliorates the pathogenesis of IL-17-mediated autoimmune diseases.

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.