Nedd4 Family Research Articles

Distinctive regulation of Cav3.1 and Cav3.2 low-voltage-activated calcium channels by NEDD4 family E3 ubiquitin ligases

CaV3.1 and CaV3.2 calcium channels serve critical roles in the cardiovascular and nervous systems. CaV3 dysregulation underlies diverse diseases, including chronic pain. Interventions that tune the functional expression and gating of CaV3 channels represent powerful mechanisms to regulate physiology, and opportunities for developing new therapeutics. Ubiquitination is a prevalent posttranslational modification that regulates different aspects of protein lifecycles, including subcellular localization and stability.

Downregulation of NEDD4L by EGFR signaling promotes the development of lung adenocarcinoma

Cumulative evidence indicates that the abnormal regulation of the NEDD4 family of E3-ubiquitin ligases participates in the tumorigenesis and development of cancer. However, their role in lung adenocarcinoma (LUAD) remains unclear. This study comprehensively analyzed the NEDD4 family in LUAD data sets from public databases and found only NEDD4L was associated with the overall survival of LUAD patients. Gene set enrichment analysis (GSEA) indicated that NEDD4L might be involved in the regulation of mTORC1 pathway. Both cytological and clinical assays showed that NEDD4L inhibited the activity of the mTOR signaling pathway. In vivo and in vitro experiments showed that NEDD4L could significantly inhibit the proliferation of LUAD cells. In addition, this study also found that the expression of NEDD4L was regulated by EGFR signaling. These findings firstly revealed that NEDD4L mediates an interplay between EGFR and mTOR pathways in LUAD, and suggest that NEDD4L held great potential as a novel biomarker and therapeutic target for LUAD.

FGFR3 Destabilizes PD-L1 via NEDD4 to Control T-cell-Mediated Bladder Cancer Immune Surveillance.

Fibroblast growth factor receptor 3 (FGFR3) is frequently activated by mutation or overexpression, and it is a validated therapeutic target in urothelial carcinoma (UC) of the bladder. However, the role and detailed molecular mechanism of FGFR3 in the immune microenvironment of bladder cancer remain largely unknown. Here, we demonstrate that inhibition of FGFR3 in FGFR3-activated bladder cancer elevates PD-L1 protein levels by affecting its ubiquitination, thereby inhibiting the antitumor activity of CD8+ T cells. Tissue microarray analysis in human UC showed an inverse correlation between FGFR3 and PD-L1. Furthermore, NEDD4, an E3 ubiquitin ligase of the NEDD4 family of proteins, was phosphorylated by FGFR3 activation and served as a regulator of PD-L1 ubiquitination. Mechanistically, NEDD4 interacted with PD-L1 and catalyzed Lys48 (K48)-linked polyubiquitination of PD-L1. In mice bearing NEDD4 knockout bladder cancer, CD8+ T-cell infiltration and antitumor activity were significantly inhibited due to PD-L1 upregulation in bladder cancer cells. Furthermore, multiple FGFR3-activated tumor-bearing mouse models suggested that attenuated CD8+ T-cell-mediated antitumor efficacy following FGFR3-targeted therapy could be rescued by a combination with anti-PD-1 immunotherapy, which leads to effective tumor suppression. This study establishes a key molecular link between targeted therapy and immune surveillance and identifies NEDD4 as a crucial E3 ubiquitin ligase that targets PD-L1 for degradation in FGFR3-activated bladder cancer. These findings may potentially be exploited for combination therapies in UC of the bladder and possibly other malignancies with activated FGFR3. SIGNIFICANCE: NEDD4 links two important molecules associated with targeted therapy and immune surveillance, providing mechanistic rationale and preclinical support for immuno-targeted combination therapy for FGFR3-activated bladder cancer.

Predicting PY motif-mediated protein-protein interactions in the Nedd4 family of ubiquitin ligases.

The Nedd4 family contains several structurally related but functionally distinct HECT-type ubiquitin ligases. The members of the Nedd4 family are known to recognize substrates through their multiple WW domains, which recognize PY motifs (PPxY, LPxY) or phospho-threonine or phospho-serine residues. To better understand protein interactor recognition mechanisms across the Nedd4 family, we report the development and implementation of a python-based tool, PxYFinder, to identify PY motifs in the primary sequences of previously identified interactors of Nedd4 and related ligases. Using PxYFinder, we find that, on average, half of Nedd4 family interactions are likely PY-motif mediated. Further, we find that PPxY motifs are more prevalent than LPxY motifs and are more likely to occur in proline-rich regions and that PPxY regions are more disordered on average relative to LPxY-containing regions. Informed by consensus sequences for PY motifs across the Nedd4 interactome, we rationally designed a focused peptide library and employed a computational screen, revealing sequence- and biomolecular interaction-dependent determinants of WW-domain/PY-motif interactions. Cumulatively, our efforts provide a new bioinformatic tool and expand our understanding of sequence and structural factors that contribute to PY-motif mediated interactor recognition across the Nedd4 family.

Human cytomegalovirus UL42 protein inhibits the degradation of glycoprotein B through inhibition of Nedd4 family ubiquitin E3 ligases.

Human cytomegalovirus (HCMV) is a globally ubiquitous pathogen and causes congenital infection as well as opportunistic infection in immunocompromised patients. The HCMV UL42 gene encodes a membrane protein that regulates the function of Nedd4 family ubiquitin E3 ligases through its PPxY motif. As HCMV envelope glycoprotein B (gB) also has a PPxY motif at its C-terminal cytoplasmic domain, we examined whether there was any relationship between UL42 protein and gB. Among the Nedd4 family proteins, Nedd4, Nedd4L, and Itch induced the degradation of gB in transiently expressing cells. The degradation of gB by Nedd4 was inhibited by proteasome inhibitor MG132, lysosome inhibitor chloroquine, and the co-expression of UL42 proteins. Among those Nedd4 family proteins, Itch was relocalized by the co-expression of gB to the perinuclear region of the cytoplasm. A co-immunoprecipitation assay demonstrated an interaction between gB and Itch through its PPxY motif. The 150 kDa gB precursor was aberrantly ubiquitinated, and the total amount of gB was quickly decreased in the absence of UL42. Our results indicate that UL42 prevents the degradation of gB by the inhibition of Nedd4 family proteins.

Integrated bioinformatics analysis of the NEDD4 family reveals a prognostic value of NEDD4L in clear-cell renal cell cancer.

The members of the Nedd4-like E3 family participate in various biological processes. However, their role in clear cell renal cell carcinoma (ccRCC) is not clear. This study systematically analyzed the Nedd4-like E3 family members in ccRCC data sets from multiple publicly available databases. NEDD4L was identified as the only NEDD4 family member differentially expressed in ccRCC compared with normal samples. Bioinformatics tools were used to characterize the function of NEDD4L in ccRCC. It indicated that NEDD4L might regulate cellular energy metabolism by co-expression analysis, and subsequent gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. A prognostic model developed by the LASSO Cox regression method showed a relatively good predictive value in training and testing data sets. The result revealed that NEDD4L was associated with biosynthesis and metabolism of ccRCC. Since NEDD4L is downregulated and dysregulation of metabolism is involved in tumor progression, NEDD4L might be a potential therapeutic target in ccRCC.

CD97 stabilises the immunological synapse between dendritic cells and T cells and is targeted for degradation by the Salmonella effector SteD.

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII β chain. This requires the Nedd4 family HECT E3 ubiquitin ligase Wwp2 and a tumor-suppressing transmembrane protein adaptor Tmem127. Here, through a proteomic screen of dendritic cells, we found that SteD targets the plasma membrane protein CD97 for degradation by a similar mechanism. SteD enhanced ubiquitination of CD97 on K555 and mutation of this residue eliminated the effect of SteD on CD97 surface levels. We showed that CD97 localises to and stabilises the immunological synapse between dendritic cells and T cells. Removal of CD97 by SteD inhibited dendritic cell-T cell interactions and reduced T cell activation, independently of its effect on MHCII. Therefore, SteD suppresses T cell immunity by two distinct processes.

Molecular Interactions between Two LMP2A PY Motifs of EBV and WW Domains of E3 Ubiquitin Ligase AIP4

Interactions involving Epstein–Barr virus (EBV) LMP2A and Nedd4 family E3 ubiquitin–protein ligases promote the ubiquitination of LMP2A-associated proteins, which results in the perturbation of normal B-cell signaling. Here, we solved the solution structure of the WW2 domain of hAIP4 and investigated the binding mode involving the N-terminal domain of LMP2A and the WW2 domain. The WW2 domain presented a conserved WW domain scaffold with a three-stranded anti-parallel β-sheet and bound two PY motifs via different binding mechanisms. Our NMR titration and ITC data demonstrated that the PY motifs of LMP2A can recognize and interact weakly with the XP groove of the WW2 domain (residues located around the third β-strand), and then residues between two PY motifs optimize the binding by interacting with the loop 1 region of the WW2 domain. In particular, the residue Val15 in the hairpin loop region between β1 and β2 of the WW2 domain exhibited unique changes depending on the terminal residues of the PY motif. This result suggested that the hairpin loop is responsible for additional interactions outside the XP groove, and this hypothesis was confirmed in a deuterium exchange experiment. These weak but wide interactions can stabilize the complex formed between the PY and WW domains.

PMEPA1 and NEDD4 control the proton production of osteoclasts by regulating vesicular trafficking.

Osteoclast bone resorption activity is critically regulated to maintain bone homeostasis. Osteoclasts resorb bone by producing protons and acid hydrolase via lysosomal secretion, however, a detailed mechanism remains elusive. PMEPA1 is a vesicular membrane protein, which binds to the NEDD4 family member of ubiquitin ligases. We have previously reported that Pmepa1 is highly expressed in bone resorbing osteoclasts, and regulates bone resorption. Here, we investigated the mechanism of bone resorption regulated by PMEPA1. Mutant mice lacking NEDD4-binding domains of PMEPA1 displayed enhanced bone volume, and reduced bone resorption activity in comparison with those of WT mice. Analysis with pH-sensitive fluorescence probe revealed that proton secretion from osteoclasts significantly decreased in Pmepa1 mutant osteoclasts. Immunofluorescence analysis revealed that PMEPA1 was colocalized with NEDD4, V0A3, and V0D2 subunits of vacuolar ATPase, which regulate the proton production of osteoclasts. In addition, Nedd4 knockdown reduced bone resorption and proton secretion of osteoclasts. Furthermore, Pmepa1 mutation and Nedd4 knockdown altered the cytoplasmic distribution of components of V-ATPase and expression of autophagy-related proteins, suggesting that lysosomal secretion is affected. Collectively, these findings indicate that PMEPA1 controls proton secretion from osteoclasts via NEDD4 by regulating vesicular trafficking, and NEDD4 is an important regulator of bone resorption.

Ndfip1 Prevents Rotenone-Induced Neurotoxicity and Upregulation of α-Synuclein in SH-SY5Y Cells.

Nedd4 family interacting protein 1 (Ndfip1) is an adaptor of Nedd4-family ubiquitin ligases. Experimental results showed that Ndfip1 had a potential neuroprotective effect in neurology diseases. However, the neuroprotective effect and the underlying mechanisms of Ndfip1 in Parkinson's disease (PD) have not yet been fully elucidated. Therefore, in this study, we explored the neuroprotective effect of Ndfip1 against mitochondrial complex I inhibitor rotenone in a human dopaminergic neuroblastoma SH-SY5Y cell line and further elucidated its possible underlying mechanisms. Our results showed that rotenone could induce the up-regulation of α-synuclein (α-syn) in both mRNA and protein levels. The expression of Ndfip1 decreased at 24 h after rotenone treatment. Further study showed that high expression of Ndfip1 could protect SH-SY5Y cells against rotenone-induced neurotoxicity and antagonize the rotenone-induced increase in α-syn protein levels. In addition, high expression of Ndfip1 inhibited rotenone-induced increase in the protein levels of caspase-3 and decrease in tyrosine hydroxylase (TH). Further study showed that Ndfip1 did not affect the protein expression of iron regulatory protein 1 (IRP1), transferrin receptor 1 (TfR1), while antagonized the increase in protein levels of P62 and ferritin L caused by rotenone. Our findings provide specific identification of Ndfip1 proteins to inhibit the increase of α-syn in rotenone-induced SH-SY5Y cells. Ndfip1 might be a new theoretical drug target for the prevention and treatment of PD.

The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination.

ALG-2 is a penta-EF-hand Ca2+-binding protein and interacts with a variety of proteins in mammalian cells. In order to find new ALG-2-binding partners, we searched a human protein database and retrieved sequences containing the previously identified ALG-2-binding motif type 2 (ABM-2). After selecting 12 high-scored sequences, we expressed partial or full-length GFP-fused proteins in HEK293 cells and performed a semi-quantitative in vitro binding assay. SARAF, a negative regulator of store-operated Ca2+ entry (SOCE), showed the strongest binding activity. Biochemical analysis of Strep-tagged and GFP-fused SARAF proteins revealed ubiquitination that proceeded during pulldown assays under certain buffer conditions. Overexpression of ALG-2 interfered with ubiquitination of wild-type SARAF but not ubiquitination of the F228S mutant that had impaired ALG-2-binding activity. The SARAF cytosolic domain (CytD) contains two PPXY motifs targeted by the WW domains of NEDD4 family E3 ubiquitin ligases. The PPXY motif proximal to the ABM-2 sequence was found to be more important for both in-cell ubiquitination and post-cell lysis ubiquitination. A ubiquitination-defective mutant of SARAF with Lys-to-Arg substitutions in the CytD showed a slower degradation rate by half-life analysis. ALG-2 promoted Ca2+-dependent CytD-to-CytD interactions of SARAF. The ALG-2 dimer may modulate the stability of SARAF by sterically blocking ubiquitination and by bridging SARAF molecules at the CytDs.

Deletion of ubiquitin ligase Nedd4l exacerbates ischemic brain damage

Ubiquitination by Nedd4 (neuronally expressed developmentally downregulated 4) family of HECT type E3 ligases plays a key role in degrading misfolded and damaged proteins, and its disruption leads to neurodegeneration. Parkinson's disease-causing protein α-Synuclein (α-Syn) is ubiquitinated by the Nedd4 family and degraded by endosomes. Nedd4l is the only Nedd4 homolog that showed upregulation in post-stroke surviving cortical neurons where it correlated with neuroprotection. We tested the role of Nedd4l after stroke by subjecting the Nedd4l−/− mice to transient middle cerebral artery occlusion. Focal ischemia significantly increased Nedd4l expression and poly-ubiquitinated α-Syn levels, and knockout of Nedd4l reduced post-ischemic poly-ubiquitinated α-Syn that is majorly located in the peri-infarct neurons. Co-immunoprecipitation further shows that focal ischemia enhances the α-Syn-Nedd4l interaction resulting in increased ubiquitination of α-Syn. Nedd4l knockout mice (n = 7 mice/group) showed exacerbated post-ischemic motor dysfunction manifested by decreased time on the rotarod and increased number of foot faults, and significantly increased ischemic brain damage. This suggests that Nedd4l might be a potential therapeutic target to minimize α-Syn-mediated toxicity after cerebral ischemia.

The Tumour Suppressor TMEM127 Is a Nedd4-Family E3 Ligase Adaptor Required by Salmonella SteD to Ubiquitinate and Degrade MHC Class II Molecules

SummaryThe Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII β chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4+ T cell activation. We found that TMEM127 contains a canonical PPxY motif, which was required for binding to WWP2. SteD bound to TMEM127 and enabled TMEM127 to interact with and induce ubiquitination of mature MHCII. Furthermore, SteD also underwent TMEM127- and WWP2-dependent ubiquitination, which both contributed to its degradation and augmented its activity on mMHCII.

Identification of novel interacting partners of the NEDD4 ubiquitin ligase in mouse testis

Posttranslational modification by ubiquitination targets proteins for degradation, recycling, stabilization or altered trafficking, and as such can alter cellular signaling pathways. The substrate specificity of this multistep process is controlled by ubiquitin ligases, including those of the HECT domain-containing NEDD4 family. In the testis, ubiquitination of many proteins contributes to organ development and maturation of spermatozoa and NEDD4 is known to be important in the control of spermatogonial stem cell homeostasis. However, a comprehensive understanding of NEDD4 substrates in testis development is lacking. Here we demonstrate high expression of Nedd4 in somatic cells of the mouse testis and in the murine Leydig cell-like cell line TM3. Immunoprecipitation of NEDD4 tagged with GFP at either the amino or carboxyl terminus was subjected to proteomic analysis for interacting proteins. We identified a substantial list of potential interaction partners, including known NEDD4 substrates, proteins involved in ubiquitination and proteins important for testis development and spermatogenesis. We confirmed the interaction of NEDD4 with a subset of these putative interacting proteins, validating the integrity of the dataset. These potential interactors may be further explored to reveal important roles of NEDD4-mediated ubiquitination in the testis. SignificanceUbiquitination is important for testis development and function, and NEDD4 is known to ubiquitinate various proteins to affect cellular signaling and development, including those implicated in spermatogenesis. However, substrates of NEDD4 that are important during testis development remain to be identified. Here we report NEDD4 expression in the developing testis and TM3 testicular cell line. This study identifies a substantial list of NEDD4 interacting proteins in the TM3 testicular cell line, with validation of some of these interactions. Hence, this provides novel NEDD4 targets that may contribute to testis development and function that may be further explored.

Calcineurin-dependent regulation of endocytosis by a plasma membrane ubiquitin ligase adaptor, Rcr1.

Rsp5, the Nedd4 family member in yeast, is an E3 ubiquitin ligase involved in numerous cellular processes, many of which require Rsp5 to interact with PY-motif containing adaptor proteins. Here, we show that two paralogous transmembrane Rsp5 adaptors, Rcr1 and Rcr2, are sorted to distinct cellular locations: Rcr1 is a plasma membrane (PM) protein, whereas Rcr2 is sorted to the vacuole. Rcr2 is delivered to the vacuole using ubiquitin as a sorting signal. Rcr1 is delivered to the PM by the exomer complex using a newly uncovered PM sorting motif. Further, we show that Rcr1, but not Rcr2, is up-regulated via the calcineurin/Crz1 signaling pathway. Upon exogenous calcium treatment, Rcr1 ubiquitinates and down-regulates the chitin synthase Chs3. We propose that the PM-anchored Rsp5/Rcr1 ubiquitin ligase-adaptor complex can provide an acute response to degrade unwanted proteins under stress conditions, thereby maintaining cell integrity.

Nicotine promotes tumor progression and epithelial-mesenchymal transition by regulating the miR-155-5p/NDFIP1 axis in pancreatic ductal adenocarcinoma

BackgroundNicotine, the major component of cigarette smoke, has been reported to promote pancreatic ductal adenocarcinoma (PDAC) growth and invasion. Deregulation of microRNA (miRNA) expression is found in many cancers, including PDAC. The effects of nicotine on miRNAs change in PDAC progression remain unknown. MethodsThe effects of cigarette smoking/nicotine exposure on PDAC cell lines and tissues were evaluated. Quantitative real-time PCR and in situ hybridization assays were used to determine miR-155-5p expression in human PDAC tissue and cell lines upon cigarette smoking/nicotine exposure. Bioinformatics, loss-of-function experiments, luciferase reporter assay were performed to validate Nedd4 family interacting protein 1 (NDFIP1) as a direct target of miR-155-5p. The potentials of systemic miR-155-5p inhibitor-based therapy in overcoming nicotine exposure were evaluated in tumor xenograft model. ResultsNicotine promoted PDAC cells proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in a dose-response manner. MiR-155-5p was found to be highly expressed in PDAC cell lines and tissues upon cigarette smoking/nicotine exposure. Functional studies showed that miR-155-5p knockdown could override the enhancement of oncogenic activity due to nicotine exposure in vitro and in vivo by directly interacting with the 3′ untranslated regions (UTRs) of NDFIP1. ConclusionsThese data demonstrate that nicotine-regulated miR-155-5p/NDFIP1 promotes tumor progression and EMT of PDAC.

ITCH as a potential therapeutic target in human cancers.

The ITCH/AIP4 ubiquitin E3 ligase was discovered independently by two groups searching for atrophin-1 interacting proteins and studying the genetics of mouse coat color alteration, respectively. ITCH is classified as a NEDD4 family E3 ligase featured with the C-terminal HECT domain for E3 ligase function and WW domains for substrate recruiting. ITCH deficiency in the mouse causes severe multi-organ autoimmune disease. Its roles in maintaining a balanced immune response have been extensively characterized over the past two and a half decades. A wealth of reports demonstrate a multifaceted role of ITCH in human cancers. Given the versatility of ITCH in catalyzing both proteolytic and non-proteolytic ubiquitination of its over fifty substrates, ITCH's role in malignancies is believed to be context-dependent. In this review, we summarize the downstream substrates of ITCH, the functions of ITCH in both tumor cells and the immune system, as well as the implications of such functions in human cancers. Moreover, we describe the upstream regulatory mechanisms of ITCH and the efforts have been made to target ITCH using small molecule inhibitors.

Molecular Analysis of Membrane Targeting by the C2 Domain of the E3 Ubiquitin Ligase Smurf1.

SMAD ubiquitination regulatory factor 1 (Smurf1) is a Nedd4 family E3 ubiquitin ligase that regulates cell motility, polarity and TGFβ signaling. Smurf1 contains an N-terminal protein kinase C conserved 2 (C2) domain that targets cell membranes and is required for interactions with membrane-localized substrates such as RhoA. Here, we investigated the lipid-binding mechanism of Smurf1 C2, revealing a general affinity for anionic membranes in addition to a selective affinity for phosphoinositides (PIPs). We found that Smurf1 C2 localizes not only to the plasma membrane but also to negatively charged intracellular sites, acting as an anionic charge sensor and selective PIP-binding domain. Site-directed mutagenesis combined with docking/molecular dynamics simulations revealed that the Smurf1 C2 domain loop region primarily interacts with PIPs and cell membranes, as opposed to the β-surface cationic patch employed by other C2 domains. By depleting PIPs from the inner leaflet of the plasma membrane, we found that PIP binding is necessary for plasma membrane localization. Finally, we used a Smurf1 cellular ubiquitination assay to show that the amount of ubiquitin at the plasma membrane interface depends on the lipid-binding properties of Smurf1. This study shows the mechanism by which Smurf1 C2 targets membrane-based substrates and reveals a novel interaction for non-calcium-dependent C2 domains and membrane lipids.

A Cycle of Ubiquitination Regulates Adaptor Function of the Nedd4-Family Ubiquitin Ligase Rsp5.

In yeast, the main ubiquitin ligase responsible for the sorting of proteins to the lysosomal vacuole is Rsp5, a member of the Nedd4 family of ligases whose distinguishing features are a catalytic homologous to E6AP C terminus (HECT) domain and 3 central WW domains that bind PY motifs in target proteins. Many substrates do not bind Rsp5 directly and instead rely on PY-containing adaptor proteins that interact with Rsp5. Recent studies indicate that the activities of these adaptors are elevated when they undergo ubiquitination, yet the mechanism whereby ubiquitination activates the adaptors and how this process is regulated remain unclear. Here, we report on a mechanism that explains how ubiquitination stimulates adaptor function and how this process can be regulated by the Rsp5-associated deubiquitinase, Ubp2. Our overexpression experiments revealed that several adaptors compete for Rsp5 invivo. We found that the ability of the adaptors to compete effectively was enhanced by their ubiquitination and diminished by a block of their ubiquitination. Ubiquitination-dependent adaptor activation required a ubiquitin-binding surface within the Rsp5 catalytic HECT domain. Finally, like constitutively ubiquitinated adaptors, a Ubp2 deficiency increased both the adaptor activity and the ability to compete for Rsp5. Our data support a model whereby ubiquitinated Rsp5 adaptors are more active when "locked" onto Rsp5 via its N-lobe ubiquitin-binding surface and less active when they are "unlocked" by Ubp2-mediated deubiquitination.

E3 Ubiquitin ligase NEDD4 family‑regulatory network in cardiovascular disease.

Protein ubiquitination represents a critical modification occurring after translation. E3 ligase catalyzes the covalent binding of ubiquitin to the protein substrate, which could be degraded. Ubiquitination as an important protein post-translational modification is closely related to cardiovascular disease. The NEDD4 family, belonging to HECT class of E3 ubiquitin ligases can recognize different substrate proteins, including PTEN, ENaC, Nav1.5, SMAD2, PARP1, Septin4, ALK1, SERCA2a, TGFβR3 and so on, via the WW domain to catalyze ubiquitination, thus participating in multiple cardiovascular-related disease such as hypertension, arrhythmia, myocardial infarction, heart failure, cardiotoxicity, cardiac hypertrophy, myocardial fibrosis, cardiac remodeling, atherosclerosis, pulmonary hypertension and heart valve disease. However, there is currently no review comprehensively clarifying the important role of NEDD4 family proteins in the cardiovascular system. Therefore, the present review summarized recent studies about NEDD4 family members in cardiovascular disease, providing novel insights into the prevention and treatment of cardiovascular disease. In addition, assessing transgenic animals and performing gene silencing would further identify the ubiquitination targets of NEDD4. NEDD4 quantification in clinical samples would also constitute an important method for determining NEDD4 significance in cardiovascular disease.