The Role of Ubiquitination and the E3 Ligase Nedd4 in Regulating Corneal Epithelial Wound Healing.

Purpose: Ubiquitination serves as a fundamental post-translational modification in numerous cellular events. Yet, its role in regulating corneal epithelial wound healing (CEWH) remains elusive. This study endeavored to determine the function and mechanism of ubiquitination in CEWH. Methods: Western blot and immunoprecipitation were used to discern ubiquitination alterations during CEWH in mice. Interventions, including neuronally expressed developmentally downregulated 4 (Nedd4) siRNA and proteasome/lysosome inhibitor, assessed their impact on CEWH. In vitro analyses, such as the scratch wound assay, MTS assay, and EdU staining, were conducted to gauge cell migration and proliferation in human corneal epithelial cells (HCECs). Moreover, transfection of miR-30/200 coupled with a luciferase activity assay ascertained their regulatory mechanism on Nedd4. Results: Global ubiquitination levels were markedly increased during the mouse CEWH. Importantly, the application of either proteasomal or lysosomal inhibitors notably impeded the healing process both in vivo and in vitro. Furthermore, Nedd4 was identified as an essential E3 ligase for CEWH. Nedd4 expression was significantly upregulated during CEWH. In vivo studies revealed that downregulation of Nedd4 substantially delayed CEWH, whereas further investigations underscored its role in regulating cell proliferation and migration, through the Stat3 pathway by targeting phosphatase and tensin homolog (PTEN). Notably, our findings pinpointed miR-30/200 family members as direct regulators of Nedd4. Conclusions: Ubiquitination holds pivotal significance in orchestrating CEWH. The critical E3 ligase Nedd4, under the regulatory purview of miR-30 and miR-200, facilitates CEWH through PTEN-mediated Stat3 signaling. This revelation sheds light on a prospective therapeutic target within the realm of CEWH.

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.

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.

NEDD4 and NEDD4L: Ubiquitin Ligases Closely Related to Digestive Diseases.

Protein ubiquitination is an enzymatic cascade reaction and serves as an important protein post-translational modification (PTM) that is involved in the vast majority of cellular life activities. The key enzyme in the ubiquitination process is E3 ubiquitin ligase (E3), which catalyzes the binding of ubiquitin (Ub) to the protein substrate and influences substrate specificity. In recent years, the relationship between the subfamily of neuron-expressed developmental downregulation 4 (NEDD4), which belongs to the E3 ligase system, and digestive diseases has drawn widespread attention. Numerous studies have shown that NEDD4 and NEDD4L of the NEDD4 family can regulate the digestive function, as well as a series of related physiological and pathological processes, by controlling the subsequent degradation of proteins such as PTEN, c-Myc, and P21, along with substrate ubiquitination. In this article, we reviewed the appropriate functions of NEDD4 and NEDD4L in digestive diseases including cell proliferation, invasion, metastasis, chemotherapeutic drug resistance, and multiple signaling pathways, based on the currently available research evidence for the purpose of providing new ideas for the prevention and treatment of digestive diseases.

Involvement of E3 ubiquitin ligase NEDD4-mediated YY1 ubiquitination in alleviating idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and lethal lung disease characterized by progressive lung scarring. This study aims to elucidate the role of the E3 ubiquitin ligase NEDD4 in the ubiquitination of YY1 and its subsequent impact on TAB1 transcription, revealing a possible molecular mechanism in the development of IPF. Through bioinformatics analysis and both in vitro and in vivo experiments, we observed differential expression levels of NEDD4 and YY1 between normal and IPF samples, identifying NEDD4 as an upstream E3 ubiquitin ligase of YY1. Furthermore, binding sites for the transcription factor YY1 on the promoter region of TAB1 were discovered, indicating a direct interaction. In vitro experiments using HEPF cells showed that NEDD4 mediates the ubiquitination and degradation of YY1, leading to suppressed TAB1 transcription, thereby inhibiting cell proliferation and fibrogenesis. These findings were corroborated by in vivo experiments in an IPF mouse model, where the ubiquitination pathway facilitated by NEDD4 attenuated IPF progression through the downregulation of YY1 and TAB1 transcription. These results suggest that NEDD4 plays a crucial role in the development of IPF by modulating YY1 ubiquitination and TAB1 transcription, providing new insights into potential therapeutic targets for treating IPF.

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.

TMED3 stabilizes SMAD2 by counteracting NEDD4-mediated ubiquitination to promote ovarian cancer.

Ovarian cancer is a major cause of death among cancer patients. Recent research has shown that the transmembrane emp24 domain (TMED) protein family plays a role in the progression of various types of cancer. In this study, we investigated the expression of TMED3 in ovarian cancer tumors compared to nontumor tissues using immunohistochemical staining. We found that TMED3 was overexpressed in ovarian cancer tumors, and its high expression was associated with poor disease-free and overall survival. To understand the functional implications of TMED3 overexpression in ovarian cancer, we conducted experiments to knockdown TMED3 using short hairpin RNA (shRNA). We observed that TMED3 knockdown resulted in reduced cell viability and migration, as well as increased cell apoptosis. Additionally, in subcutaneous xenograft models in BALB-c nude mice, TMED3 knockdown inhibited tumor growth. Further investigation revealed that SMAD family member 2 (SMAD2) was a downstream target of TMED3, driving ovarian cancer progression. TMED3 stabilized SMAD2 by inhibiting the E3 ligase NEDD4-mediated ubiquitination of SMAD2. To confirm the importance of SMAD2 in TMED3-mediated ovarian cancer, we performed functional rescue experiments and found that SMAD2 played a critical role in this process. Moreover, we discovered that the PI3K-AKT pathway was involved in the promoting effects of TMED3 overexpression on ovarian cancer cells. Overall, our study identifies TMED3 as a prognostic indicator and tumor promoter in ovarian cancer. Its function is likely mediated through the regulation of the SMAD2 and PI3K-AKT signaling pathway. These findings contribute to our understanding of the molecular mechanisms underlying ovarian cancer progression and provide potential targets for therapeutic intervention.

E3 ubiquitin ligase NEDD4L inhibits epithelial-mesenchymal transition by suppressing the ß-catenin/HIF-1α positive feedback loop in chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyp (CRSwNP) is a refractory inflammatory disease with epithelial-mesenchymal transition (EMT) as one of the key features. Since ubiquitin modification has been shown to regulate the EMT process in other diseases, targeting ubiquitin ligases may be a potential strategy for the treatment of CRSwNP. In this study we investigated whether certain E3 ubiquitin ligases could regulate the EMT process in CRSwNP, and whether these regulations could be the potential drug targets as well as the underlying mechanisms. After screening the potential drug target by bioinformatic analyses, the expression levels of three potential E3 ubiquitin ligases were compared among the control, eosinophilic nasal polyp (ENP) and non-eosinophilic nasal polyp (NENP) group in clinical samples, and the significant decrement of the expression level of NEDD4L was found. Then, IP-MS, bioinformatics and immunohistochemistry studies suggested that low NEDD4L expression may be associated with the EMT process. In human nasal epithelial cells (hNECs) and human nasal epithelial cell line RPMI 2650, knockdown of NEDD4L promoted EMT, while upregulating NEDD4L reversed this effect, suggesting that NEDD4L inhibited EMT in nasal epithelial cells. IP-MS and Co-IP studies revealed that NEDD4L mediated the degradation of DDR1. We demonstrated that NEDD4L inhibited the ß-catenin/HIF-1α positive feedback loop either directly (degrading ß-catenin and HIF-1α) or indirectly (mediating DDR1 degradation). These results were confirmed in a murine NP model in vivo. This study for the first time reveals the regulatory role of ubiquitin in the EMT process of nasal epithelial cells, and identifies a novel drug target NEDD4L, which has promising efficacy against both ENP and NENP by suppressing ß-catenin/HIF-1α positive feedback loop.

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.

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.

E3 ligase Nedd4L promotes macrophage M1 polarization and exacerbates brain damage by TRAF3/TBK1 signaling pathway after ICH in mice.

BACKGROUND: Intracerebral hemorrhage (ICH) is a serious medical problem, and promising strategy is limited. Macrophage initiated brain inflammatory injury following ICH, but the molecular mechanism had not been well identified. E3 ligase Nedd4L is implicated in the pathogenesis of the inflammatory immune response. METHODS: In the present study, we detected the levels of Nedd4L in macrophages following ICH. Furthermore, Macrophage M1 polarization, pro-inflammatory cytokine production, BBB disruption, brain water content and neurological function were examined in ICH mice. RESULTS: Here, we demonstrated that E3 ligase Nedd4L levels of macrophage increased following ICH, promoted M1 polarization inflammation by TRAF3. Nedd4L promoted BBB disruption, as well as neurological deficits. Inhibition of Nedd4L significantly attenuated M1 polarization in vivo. Inhibition of Nedd4L decreased TRAF3 and TBK1 levels, and subsequent phosphorylation of p38 and NF-κB p65 subunit following ICH. CONCLUSIONS: Our data demonstrated that Nedd4L was involved in the pathogenesis of ICH, which promoted inflammatory responses and exacerbated brain damage by TRAF3 following ICH.

Nedd4-1 regulates human sodium-dependent vitamin C transporter-2 functional expression in neuronal and epithelial cells.

The ubiquitin-proteasomal pathway regulates the functional expression of many membrane transporters in a variety of cellular systems. Nothing is currently known about the role of ubiquitin E3 ligase, neural precursor cell-expressed developmentally down-regulated gene 4 (Nedd4-1) and the proteasomal degradation pathway in regulating human vitamin C transporter-2 (hSVCT2) in neuronal cells. hSVCT2 mediates the uptake of ascorbic acid (AA) and is the predominantly expressed vitamin C transporter isoform in neuronal systems. Therefore, we addressed this knowledge gap in our study. Analysis of mRNA revealed markedly higher expression of Nedd4-1 in neuronal samples than that of Nedd4-2. Interestingly, Nedd4-1 expression in the hippocampus was higher in patients with Alzheimer's disease (AD) and age-dependently increased in the J20 mouse model of AD. The interaction of Nedd4-1 and hSVCT2 was confirmed by coimmunoprecipitation and colocalization. While the coexpression of Nedd4-1 with hSVCT2 displayed a significant decrease in AA uptake, siRNA-mediated knockdown of Nedd4-1 expression up-regulated the AA uptake. Further, we mutated a classical Nedd4 protein interacting motif ("PPXY") within the hSVCT2 polypeptide and observed markedly decreased AA uptake due to the intracellular localization of the mutated hSVCT2. Also, we determined the role of the proteasomal degradation pathway in hSVCT2 functional expression in SH-SY5Y cells and the results indicated that the proteasomal inhibitor (MG132) significantly up-regulated the AA uptake and hSVCT2 protein expression level. Taken together, our findings show that the regulation of hSVCT2 functional expression is at least partly mediated by the Nedd4-1 dependent ubiquitination and proteasomal pathways.

E3 ubiquitin ligase neural precursor cell-expressed developmentally downregulated gene 4 motivates FOXA1 ubiquitination and restrains proliferation of diffuse large B-cell lymphoma cells via the Wnt/ß-Catenin pathway.

Neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) is an E3 ubiquitin ligase that recognizes substrates via protein-protein interactions and takes part in tumor development. This study aims to clarify NEDD4's functions in diffuse large B-cell lymphoma (DLBCL) and its downstream mechanisms. Collection of 53 DLBCL tissues and adjacent normal lymphoid tissues, and detection of NEDD4 and Forkhead box protein A1 (FOXA1) in the tissues were conducted. The selection of DLBCL cells was for FARAGE, and test of cells' advancement was after transfection. Analysis of NEDD4 and FOXA1's link, and test of Wnt/ß-catenin pathway were implemented. In vivo tumor xenograft experiments were put into effect. Detection of the pathological conditions of tumor tissues and the positive Ki67 in the family was implemented. It came out NEDD4 was reduced in DLBCL tissues and cell lines, and FOXA1 was elevated; Enhancing NEDD4 or repressing FOXA1 refrained DLBCL cells' advancement; NEDD4 could combine with FOXA1 and trigger its ubiquitination and degradation; NEDD4 inactivates the Wnt/ß-catenin pathway by motivating FOXA1 ubiquitination; NEDD4 enhancement refrained DLBCL growth in vivo. In conclusion, the E3 ubiquitin ligase NEDD4 accelerates FOXA1 ubiquitination but refrains DLBCL cell proliferation via the Wnt/ß-Catenin pathway.

NEDD4 E3 ubiquitin ligases: Promising biomarkers and therapeutic targets for cancer.

Accumulating evidence has demonstrated that NEDD4 E3 ubiquitin ligase family plays a pivotal oncogenic role in a variety of malignancies via mediating ubiquitin dependent degradation processes. Moreover, aberrant expression of NEDD4 E3 ubiquitin ligases is often indicative of cancer progression and correlated with poor prognosis. In this review, we are going to address association of expression of NEDD4 E3 ubiquitin ligases with cancers, the signaling pathways and the molecular mechanisms by which the NEDD4 E3 ubiquitin ligases regulate oncogenesis and progression, and the therapies targeting the NEDD4 E3 ubiquitin ligases. This review provides the systematic and comprehensive summary of the latest research status of E3 ubiquitin ligases in the NEDD4 subfamily, and proposes that NEDD4 family E3 ubiquitin ligases are promising anti-cancer drug targets, aiming to provide research direction for clinical targeting of NEDD4 E3 ubiquitin ligase therapy.

Expression and regulatory network of E3 ubiquitin ligase NEDD4 family in cancers.

NEDD4 family represent an important group of E3 ligases, which regulate various cellular pathways of cell proliferation, cell junction and inflammation. Emerging evidence suggested that NEDD4 family members participate in the initiation and development of tumor. In this study, we systematically investigated the molecular alterations as well as the clinical relevance regarding NEDD4 family genes in 33 cancer types. Finally, we found that NEDD4 members showed increased expression in pancreas cancer and decreased expression in thyroid cancer. NEDD4 E3 ligase family genes had an average mutation frequency in the range of 0-32.1%, of which HECW1 and HECW2 demonstrated relatively high mutation rate. Breast cancer harbors large amount of NEDD4 copy number amplification. NEDD4 family members interacted proteins were enriched in various pathways including p53, Akt, apoptosis and autophagy, which were confirmed by further western blot and flow cytometric analysis in A549 and H1299 lung cancer cells. In addition, expression of NEDD4 family genes were associated with survival of cancer patients. Our findings provide novel insight into the effect of NEDD4 E3 ligase genes on cancer progression and treatment in the future.

Divergent regulation of α-arrestin ARRDC3 function by ubiquitination.

The α-arrestin ARRDC3 is a recently discovered tumor suppressor in invasive breast cancer that functions as a multifaceted adaptor protein to control protein trafficking and cellular signaling. However, the molecular mechanisms that control ARRDC3 function are unknown. Other arrestins are known to be regulated by posttranslational modifications, suggesting that ARRDC3 may be subject to similar regulatory mechanisms. Here we report that ubiquitination is a key regulator of ARRDC3 function and is mediated primarily by two proline-rich PPXY motifs in the ARRDC3 C-tail domain. Ubiquitination and the PPXY motifs are essential for ARRDC3 function in regulating GPCR trafficking and signaling. Additionally, ubiquitination and the PPXY motifs mediate ARRDC3 protein degradation, dictate ARRDC3 subcellular localization, and are required for interaction with the NEDD4-family E3 ubiquitin ligase WWP2. These studies demonstrate a role for ubiquitination in regulating ARRDC3 function and reveal a mechanism by which ARRDC3 divergent functions are controlled.

Identification of Ndfip1 as a novel negative regulator for spatial memory formation associated with increased ubiquitination of Beclin 1 and PTEN.

Long-term memory formation requires de novo RNA and protein synthesis. By using the differential display-polymerase chain reaction strategy, we have presently identified the Nedd4 family interacting protein 1 (Ndfip1) cDNA fragment that is differentially expressed between the slow learners and the fast learners from the water maze learning task in rats. Further, the fast learners show decreased Ndfip1 mRNA and protein expression levels than the slow learners. Spatial training similarly decreases the Ndfip1 mRNA and protein expression levels. Conversely, the Ndfip1 conditional heterozygous (cHet) mice show enhanced spatial memory performance compared to the Ndfip1flox/WT control mice. Result from co-immunoprecipitation experiment indicates that spatial training decreases the association between Ndfip1 and the E3 ubiquitin ligase Nedd4 (Nedd4-1), and we have shown that both Beclin 1 and PTEN are endogenous ubiquitination targets of Nedd4 in the hippocampus. Further, spatial training decreases endogenous Beclin 1 and PTEN ubiquitination, and increases Beclin 1 and PTEN expression in the hippocampus. On the other hand, the Becn1 conditional knockout (cKO) mice and the Pten cKO mice both show impaired spatial learning and memory performance. Moreover, the expression level of Beclin 1 and PTEN is higher in the Ndfip1 cHet mice compared with the Ndfip1flox/WT control mice. Here, we have identified Ndfip1 as a candidate novel negative regulation for spatial memory formation and this is associated with increased ubiquitination of Beclin 1 and PTEN in the hippocampus.

Semisynthetic Approach to the Analysis of Tumor Suppressor PTEN Ubiquitination.

Phosphatase and tensin homologue (PTEN) tumor suppressor protein is a PIP3 lipid phosphatase that is subject to multifaceted post-translational modifications. One such modification is the monoubiquitination of Lys13 that may alter its cellular localization but is also positioned in a manner that could influence several of its cellular functions. To explore the regulatory influence of ubiquitin on PTEN's biochemical properties and its interaction with ubiquitin ligases and a deubiquitinase, the generation of a site-specifically and stoichiometrically ubiquitinated protein could be beneficial. Here, we describe a semisynthetic method that relies upon sequential expressed protein ligation steps to install ubiquitin at a Lys13 mimic in near full-length PTEN. This approach permits the concurrent installation of C-terminal modifications in PTEN, thereby facilitating an analysis of the interplay between N-terminal ubiquitination and C-terminal phosphorylation. We find that the N-terminal ubiquitination of PTEN inhibits its enzymatic function, reduces its binding to lipid vesicles, modulates its processing by NEDD4-1 E3 ligase, and is efficiently cleaved by the deubiquitinase, USP7. Our ligation approach should motivate related efforts for uncovering the effects of ubiquitination of complex proteins.

The role of NEDD4 related HECT-type E3 ubiquitin ligases in defective autophagy in cancer cells: molecular mechanisms and therapeutic perspectives.

The homologous to the E6-AP carboxyl terminus (HECT)-type E3 ubiquitin ligases are the selective executers in the protein ubiquitination, playing a vital role in modulation of the protein function and stability. Evidence shows the regulatory role of HECT-type E3 ligases in various steps of the autophagic process. Autophagy is an intracellular digestive and recycling process that controls the cellular hemostasis. Defective autophagy is involved in tumorigenesis and has been detected in various types of cancer cells. A growing body of findings indicates that HECT-type E3 ligases, in particular members of the neural precursor cell expressed developmentally downregulated protein 4 (NEDD4) including NEDD4-1, NEDD4-L, SMURFs, WWPs, and ITCH, play critical roles in dysregulation or dysfunction of autophagy in cancer cells. The present review focuses on NEDD4 E3 ligases involved in defective autophagy in cancer cells and discusses their autophagic function in different cancer cells as well as substrates and the signaling pathways in which they participate, conferring a basis for the cancer treatment through the modulating of these E3 ligases.

Emerging roles of the HECT E3 ubiquitin ligases in gastric cancer.

Gastric cancer (GC) is one of the most pernicious gastrointestinal tumors with extraordinarily high incidence and mortality. Ubiquitination modification of cellular signaling proteins has been shown to play important roles in GC tumorigenesis, progression, and prognosis. The E3 ubiquitin ligase is the crucial enzyme in the ubiquitination reaction and determines the specificity of ubiquitination substrates, and thus, the cellular effects. The HECT E3 ligases are the second largest E3 ubiquitin ligase family characterized by containing a HECT domain that has E3 ubiquitin ligase activity. The HECT E3 ubiquitin ligases have been found to engage in GC progression. However, whether HECT E3 ligases function as tumor promoters or tumor suppressors in GC remains controversial. In this review, we will focus on recent discoveries about the role of the HECT E3 ubiquitin ligases, especially members of the NEDD4 and other HECT E3 ligase subfamilies, in GC.