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.

Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2-4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4-1) and Nedd4L (Nedd4-2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4-1 and Nedd4-2, and rat-Nedd4-1, using protein microarrays spotted with approximately 8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4-1 and Nedd4-2, others were specific to only one, with several Tyr kinases preferred by Nedd4-1 and some ion channels by Nedd4-2; this was subsequently validated in vivo. Accordingly, Nedd4-1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4-1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases.

To define the links between paramyxovirus budding and cellular ESCRT machinery, we previously identified angiomotin-like 1 (AMOTL1) in a screen for host factors that bind to the matrix (M) protein of parainfluenza virus 5 (PIV5). This protein harbors three L/PPXY sequences, allowing it to interact with WW domain containing proteins including NEDD4 family members. We hypothesize that paramyxoviruses use AMOTL1 as a linker to indirectly recruit the same NEDD4 ubiquitin ligases for budding that other enveloped viruses recruit directly through their PPXY late domains. In support of this hypothesis, we found that AMOTL1 could link together M proteins and NEDD4 family proteins in three-way co-IP experiments. Both PIV5 and mumps virus M proteins could be linked to the NEDD4 family proteins NEDD4-1, NEDD4L, and NEDL1, provided that AMOTL1 was co-expressed as a bridging protein. AMOT and AMOTL2 could not substitute for AMOTL1, as they lacked the ability to bind with paramyxovirus M proteins. Attachment of a PPXY late domain sequence to PIV5 M protein obviated the need for AMOTL1 as a linker between M and NEDD4 proteins. Together, these results suggest a novel host factor recruitment strategy for paramyxoviruses to achieve particle release.

Nedd4 is a family of ubiquitin E3 ligases that regulate numerous cellular processes. In this report, we showed that alpha- and beta-herpesviruses have membrane proteins that regulate the function of the Nedd4 family members. Although the homology search score was quite low, UL56 of herpes simplex virus type 1 and 2, ORF0 of varicella-zoster virus, UL42 of human cytomegalovirus, and U24 of human herpesvirus 6A, 6B, and 7 all possess at least one PPxY (PY) motif in their cytoplasmic domain, and are able to bind with Itch, a member of the Nedd4 family. These viral proteins altered the localization of Itch and decreased Itch expression in co-expressing cells. In addition, these viral proteins reduced the production of retrovirus vectors through the regulation of the Nedd4 family of proteins. U24, but not the other proteins, effectively reduced CD3ε expression on the T cell surface. These viral molecules are thought to contribute to the specific function of each virus through the regulation of Nedd4 family activity.

The Drosophila Discs large (Dlg) scaffolding protein acts as a tumor suppressor regulating basolateral epithelial polarity and proliferation. In mammals, four Dlg homologs have been identified; however, their functions in cell polarity remain poorly understood. Here, we demonstrate that the X-linked mental retardation gene product Dlg3 contributes to apical-basal polarity and epithelial junction formation in mouse organizer tissues, as well as to planar cell polarity in the inner ear. We purified complexes associated with Dlg3 in polarized epithelial cells, including proteins regulating directed trafficking and tight junction formation. Remarkably, of the four Dlg family members, Dlg3 exerts a distinct function by recruiting the ubiquitin ligases Nedd4 and Nedd4-2 through its PPxY motifs. We found that these interactions are required for Dlg3 monoubiquitination, apical membrane recruitment, and tight junction consolidation. Our findings reveal an unexpected evolutionary diversification of the vertebrate Dlg family in basolateral epithelium formation.

Ubiquitin-modification of proteins by E3 ubiquitin ligases is an important post-translational mechanism implicated in neuronal survival and injury following cerebral ischemia. However, of the 500 or so E3s thought to be present in mammalian cells, very few specific E3s have been identified and associated with brain ischemia. Here, we demonstrate endogenous induction of HECT-type E3 ligases of the Nedd4 family and their adaptor Nedd4-family interacting protein 1 (Ndfip1) following transient focal cerebral ischemia in rats. Ndfip1 is upregulated in surviving cortical neurons and its neuroprotective activity is correlated with Nedd4-2 upregulation, but not two other Nedd4 family members examined (Nedd4-1 and Itch). Immunoprecipitation assays confirmed biochemical binding of Ndfip1 with Nedd4-2 in the brain, with or without ischemic stroke, indicating their endogenous interaction. While Ndfip1 and Itch have been previously shown to interact outside of the nervous system, ischemic induction of Itch in the present study was associated with cellular survival independent of Ndfip1. Together, these findings demonstrate specific and differential regulation of Nedd4 family E3 ligases under ischemic conditions, and identify two E3 ligases and their adaptor that potentially regulate ubiquitination in ischemic stroke to provide neuroprotection.

Alpha-synuclein can form beta-sheet filaments, the accumulation of which plays a key role in the development of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. It has previously been shown that alpha-synuclein is a substrate for the HECT domain-containing ubiquitin ligase Nedd4, and is subject to ubiquitin-mediated endosomal degradation. We show here that alpha-synuclein filaments are much better substrates for ubiquitination in vitro than monomeric alpha-synuclein, and that this increased susceptibility cannot be mimicked by the mere clustering of monomers. Recognition by Nedd4 family enzymes is not through the conventional binding of PPxY-containing sequences to WW domains of the ligase, but it also involves C2 and HECT domains. The disease-causing alpha-synuclein mutant A53T is a much less efficient substrate for Nedd4 ligases than the wild-type protein. We suggest that preferential recognition, ubiquitination and degradation of beta-sheet-containing filaments may help to limit toxicity, and that A53T alpha-synuclein may be more toxic, at least in part because it avoids this fate.

Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.

Retroviruses engage the ESCRT pathway through late assembly (L) domains in Gag to promote virus release. HIV-1 uses a PTAP motif as its primary L domain, which interacts with the ESCRT-I component Tsg101. In contrast, certain other retroviruses primarily use PPxY-type L domains, which constitute ligands for NEDD4-type ubiquitin ligases. Surprisingly, although HIV-1 Gag lacks PPxY motifs, the release of HIV-1 L domain mutants is potently enhanced by ectopic NEDD4-2s, a native isoform with a naturally truncated C2 domain that appears to account for the residual titer of L domain-defective HIV-1. The reason for the unique potency of the NEDD4-2s isoform has remained unclear. We now show that the naturally truncated C2 domain of NEDD4-2s functions as an autonomous Gag-targeting module that can be functionally replaced by the unrelated Gag-binding protein cyclophilin A (CypA). The residual C2 domain of NEDD4-2s was sufficient to transfer the ability to stimulate HIV-1 budding to other NEDD4 family members, including the yeast homologue Rsp5, and even to isolated catalytic HECT domains. The isolated catalytic domain of NEDD4-2s also efficiently promoted HIV-1 budding when targeted to Gag via CypA. We conclude that the regions typically required for substrate recognition by HECT ubiquitin ligases are all dispensable to stimulate HIV-1 release, implying that the relevant target for ubiquitination is Gag itself or can be recognized by divergent isolated HECT domains. However, the mere ability to ubiquitinate Gag was not sufficient to stimulate HIV-1 budding. Rather, our results indicate that the synthesis of K63-linked ubiquitin chains is critical for ubiquitin ligase-mediated virus release.

While the role of Polycomb group protein-mediated "cell memory" is well established in developmental contexts, little is known about their role in adult tissues and in particular in post-mitotic cells. Emerging evidence assigns a pivotal role in cell plasticity and adaptation. PRC2-Ezh1α/β signaling pathway from cytoplasm to chromatin protects skeletal muscle cells from oxidative stress. However, detailed mechanisms controlling degradation of cytoplasmic Ezh1β and assembly of canonical PRC2-Ezh1α repressive complex remain to be clarified.

Viral late domains are used by many viruses to recruit the cellular endosomal sorting complex required for transport (ESCRT) to mediate membrane scission during viral budding. Unlike the P(S/T)AP and YPX(1-3)L late domains, which interact directly with the ESCRT proteins Tsg101 and ALIX, the molecular linkage connecting the PPXY late domain to ESCRT proteins is unclear. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) matrix protein, Z, contains only one late domain, PPXY. We previously found that this domain in LCMV Z, as well as the ESCRT pathway, are required for the release of defective interfering (DI) particles but not infectious virus. To better understand the molecular mechanism of ESCRT recruitment by the PPXY late domain, affinity purification-mass spectrometry was used to identify host proteins that interact with the Z proteins of the Old World mammarenaviruses LCMV and Lassa virus. Several Nedd4 family E3 ubiquitin ligases interact with these matrix proteins and in the case of LCMV Z, the interaction was PPXY-dependent. We demonstrated that these ligases directly ubiquitinate LCMV Z and mapped the specific lysine residues modified. A recombinant LCMV containing a Z that cannot be ubiquitinated maintained its ability to produce both infectious virus and DI particles, suggesting that direct ubiquitination of LCMV Z alone is insufficient for recruiting ESCRT proteins to mediate virus release. However, Nedd4 ligases appear to be important for DI particle release suggesting that ubiquitination of targets other than the Z protein itself is required for efficient viral ESCRT recruitment.

Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network.

Downregulation of ubiquitin (Ub) ligase activity prevents premature ubiquitination and is critical for cellular homeostasis. Nedd4 Ub ligases share a common domain architecture and yet are regulated in distinct ways through interactions of the catalytic HECT domain with the N-terminal C2 domain or the central WW domain region. Smurf1 and Smurf2 are two highly related Nedd4 ligases with ~70% overall sequence identity. Here, we show that the Smurf1 C2 domain interacts with the HECT domain and inhibits ligase activity in trans. However, in contrast to Smurf2, we find that full-length Smurf1 is a highly active Ub ligase, and we can attribute this striking difference in regulation to the lack of one WW domain (WW1) in Smurf1. Using NMR spectroscopy and biochemical assays, we identified the WW1 region as an additional inhibitory element in Smurf2 that cooperates with the C2 domain to enhance HECT domain binding and Smurf2 inhibition. Our work provides important insights into Smurf regulation and highlights that the activities of highly related proteins can be controlled in distinct ways.

Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY motif containing adaptor proteins. Several arrestin-related trafficking adaptors (ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63-linked di-ubiquitination by Rsp5. This modification enhances the plasma membrane recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the pombe orthologs of Rsp5 and Art1, Pub1, and Any1. Furthermore, we discover that the homologous to E6AP C-terminus (HECT) domain exosite protects the K63-linked di-ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment, and activity of Rsp5 for the turnover of membrane proteins.

The functional repertoire of surface ion channels is sustained by dynamic processes of trafficking, sorting, and degradation. Dysregulation of these processes underlies diverse ion channelopathies including cardiac arrhythmias and cystic fibrosis. Ubiquitination powerfully regulates multiple steps in the channel lifecycle, yet basic mechanistic understanding is confounded by promiscuity among E3 ligase/substrate interactions and ubiquitin code complexity. Here we targeted the catalytic domain of E3 ligase, CHIP, to YFP-tagged KCNQ1 ± KCNE1 subunits with a GFP-nanobody to selectively manipulate this channel complex in heterologous cells and adult rat cardiomyocytes. Engineered CHIP enhanced KCNQ1 ubiquitination, eliminated KCNQ1 surface-density, and abolished reconstituted K+ currents without affecting protein expression. A chemo-genetic variation enabling chemical control of ubiquitination revealed KCNQ1 surface-density declined with a ~ 3.5 hr t1/2 by impaired forward trafficking. The results illustrate utility of engineered E3 ligases to elucidate mechanisms underlying ubiquitin regulation of membrane proteins, and to achieve effective post-translational functional knockdown of ion channels.

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.

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. SIGNIFICANCE: Ubiquitination 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.

Skeletal muscle atrophy is a consequence of muscle inactivity resulting from denervation, unloading and immobility. It accompanies many chronic disease states and also occurs as a pathophysiologic consequence of normal aging. In all these conditions, ubiquitin-dependent proteolysis is a key regulator of the loss of muscle mass, and ubiquitin ligases confer specificity to this process by interacting with, and linking ubiquitin moieties to target substrates through protein:protein interaction domains. Our previous work suggested that the ubiquitin-protein ligase Nedd4-1 is a potential mediator of skeletal muscle atrophy associated with inactivity (denervation, unloading and immobility). Here we generated a novel tool, the Nedd4-1 skeletal muscle-specific knockout mouse (myo(Cre);Nedd4-1(flox/flox)) and subjected it to a well validated model of denervation induced skeletal muscle atrophy. The absence of Nedd4-1 resulted in increased weights and cross-sectional area of type II fast twitch fibres of denervated gastrocnemius muscle compared with wild type littermates controls, at seven and fourteen days following tibial nerve transection. These effects are not mediated by the Nedd4-1 substrates MTMR4, FGFR1 and Notch-1. These results demonstrate that Nedd4-1 plays an important role in mediating denervation-induced skeletal muscle atrophy in vivo.

The ubiquitin-proteasome pathway (UPP) is a hallmark of the eukaryotic cell. In jawed vertebrates, it has been co-opted by the adaptive immune system, where proteasomal degradation produces endogenous peptides for major histocompatibility complex class I antigen presentation. However, proteolytic products are also necessary for the phylogenetically widespread innate immune system, as they often play a role as host defense peptides (HDPs), pivotal effectors against pathogens. Here, we report the identification of the arachnid HDP oligoventin, which shares homology to a core member of the UPP, E3 ubiquitin ligases. Oligoventin has broad antimicrobial activity and shows strong synergy with lysozymes. Using computational and phylogenetic approaches, we show high conservation of the oligoventin signature in HECT E3s. In silico simulation of HECT E3s self-proteolysis provides evidence that HDPs can be generated by fine-tuned 26S proteasomal degradation, and therefore are consistent with the hypothesis that oligoventin is a cryptic peptide released by the proteolytic processing of an Nedd4 E3 precursor protein. Finally, we compare the production of HDPs and endogenous antigens from orthologous HECT E3s by proteasomal degradation as a means of analyzing the UPP coupling to metazoan immunity. Our results highlight the functional plasticity of the UPP in innate and adaptive immune systems as a possibly recurrent mechanism to generate functionally diverse peptides.

Several ATP-Binding Cassette (ABC) transporters, including ABCG1 and the related ABCG4, are essential regulators of cellular lipid homeostasis. ABCG1 is expressed ubiquitously and is functional in the context of atherosclerosis. However, ABCG4 is expressed almost exclusively in brain and has been linked to Alzheimer's disease (AD). These transporters are highly regulated post-translationally by E3 ubiquitin ligases, with the ligase NEDD4-1 (Neural precursor cell-expressed developmentally downregulated gene 4) implicated in their protein stability. In this study, we investigated interacting partners of ABCG1 using peptide-mass spectrometry and identified the potential adaptor protein, Alix (apoptosis-linked gene 2-interacting protein X). In this paper, we hypothesized and investigated whether Alix could facilitate the interaction between NEDD4-1 and the ABC transporters. We showed that Alix and NEDD4-1 proteins were co-expressed in several commonly used cell lines. Knockdown of Alix in cells overexpressing ABCG1 or ABCG4 increased transporter protein expression while co-immunoprecipitation experiments showed interaction between NEDD4-1, Alix, and ABC transporters. In summary, we provide evidence that Alix serves as a co-factor for the interaction between the E3-ubiquitin ligase NEDD4-1 and the ABC transporter targets, ABCG1 and ABCG4.