Journal articles: 'Nedd4 Family E3 Ligases'

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Jiang, Hanjie, Stefani N. Thomas, Zan Chen, Claire Y. Chiang, and Philip A. Cole. "Comparative analysis of the catalytic regulation of NEDD4-1 and WWP2 ubiquitin ligases." Journal of Biological Chemistry 294, no. 46 (October 2, 2019): 17421–36. http://dx.doi.org/10.1074/jbc.ra119.009211.

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NEDD4-1 E3 ubiquitin protein ligase (NEDD4-1) and WW domain-containing E3 ubiquitin ligase (WWP2) are HECT family ubiquitin E3 ligases. They catalyze Lys ubiquitination of themselves and other proteins and are important in cell growth and differentiation. Regulation of NEDD4-1 and WWP2 catalytic activities is important for controlling cellular protein homeostasis, and their dysregulation may lead to cancer and other diseases. Previous work has implicated noncatalytic regions, including the C2 domain and/or WW domain linkers in NEDD4-1 and WWP2, in contributing to autoinhibition of the catalytic HECT domains by intramolecular interactions. Here, we explored the molecular mechanisms of these NEDD4-1 and WWP2 regulatory regions and their interplay with allosteric binding proteins such as Nedd4 family-interacting protein (NDFIP1), engineered ubiquitin variants, and linker phosphomimics. We found that in addition to influencing catalytic activities, the WW domain linker regions in NEDD4-1 and WWP2 can impact product distribution, including the degree of polyubiquitination and Lys-48 versus Lys-63 linkages. We show that allosteric activation by NDFIP1 or engineered ubiquitin variants is largely mediated by relief of WW domain linker autoinhibition. WWP2-mediated ubiquitination of WW domain-binding protein 2 (WBP2), phosphatase and tensin homolog (PTEN), and p62 proteins by WWP2 suggests that substrate ubiquitination can also be influenced by WW linker autoinhibition, although to differing extents. Overall, our results provide a deeper understanding of the intricate and multifaceted set of regulatory mechanisms in the control of NEDD4-1–related ubiquitin ligases.

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Qian, Hao, Ying Zhang, Boquan Wu, Shaojun Wu, Shilong You, Naijin Zhang, and Yingxian Sun. "Structure and function of HECT E3 ubiquitin ligases and their role in oxidative stress." Journal of Translational Internal Medicine 8, no. 2 (June 30, 2020): 71–79. http://dx.doi.org/10.2478/jtim-2020-0012.

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AbstractUbiquitination is a modification after protein transcription that plays a vital role in maintaining the homeostasis of the cellular environment. The Homologous to E6AP C-terminus (HECT) family E3 ubiquitin ligases are a kind of E3 ubiquitin ligases with a C-terminal HECT domain that mediates the binding of ubiquitin to substrate proteins and a variable-length N-terminal extension. HECT-ubiquitinated ligases can be divided into three categories: NEDD4 superfamily, HERC superfamily, and other HECT superfamilies. HECT ubiquitin ligase plays an essential role in the development of many human diseases. In this review, we focus on the physiological and pathological processes involved in oxidative stress and the role of E3 ubiquitin ligase of the HECT family.

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An, Heeseon, David T. Krist, and Alexander V. Statsyuk. "Crosstalk between kinases and Nedd4 family ubiquitin ligases." Mol. BioSyst. 10, no. 7 (2014): 1643–57. http://dx.doi.org/10.1039/c3mb70572b.

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Understanding the interplay between kinase and E3 ligase signaling pathways will allow better understanding of therapeutically relevant pathways and the design of small molecule therapeutics targeting these pathways.

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Ikeda, Akiko, Robert G. Caldwell, Richard Longnecker, and Masato Ikeda. "Itchy, a Nedd4 Ubiquitin Ligase, Downregulates Latent Membrane Protein 2A Activity in B-Cell Signaling." Journal of Virology 77, no. 9 (May 1, 2003): 5529–34. http://dx.doi.org/10.1128/jvi.77.9.5529-5534.2003.

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ABSTRACT Nedd4 family ubiquitin protein ligases (E3s) specifically associate with latent membrane protein 2A (LMP2A) of Epstein-Barr virus. Our previous studies analyzing LMP2A function in vitro have suggested that Nedd4 family E3s regulate LMP2A function. To determine the role of Nedd4 family E3s in LMP2A B-cell signaling, LMP2A transgenic (LMP2A+) mice were crossed with mice with the Itch-deficient (Itch−/−) background. Itchy, a mouse homologue of human AIP4, is a Nedd4 family E3 and is also the most abundant Nedd4 family E3 found in LMP2A affinity precipitates from B cells. There were significantly fewer B-cell receptor-positive B cells in spleen and bone marrow B cells in LMP2A+ Itch−/− mice than in LMP2A+ mice. In addition, LMP2A+ Itch−/− bone marrow B cells formed larger colonies in cultures treated with interleukin-7 (IL-7) than control bone marrow B cells did. Finally, there was a dramatic increase in tyrosine phosphorylation of LMP2A and Syk in IL-7-cultured LMP2A+ Itch−/− B cells. These results indicate that Nedd4 family E3s, in particular Itchy, downmodulate LMP2A activity in B-cell signaling.

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Chen, Ceshi, and Lydia E. Matesic. "The Nedd4-like family of E3 ubiquitin ligases and cancer." Cancer and Metastasis Reviews 26, no. 3-4 (August 29, 2007): 587–604. http://dx.doi.org/10.1007/s10555-007-9091-x.

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Winberg, Gösta, Liudmila Matskova, Fu Chen, Pamela Plant, Daniela Rotin, Gerald Gish, Robert Ingham, Ingemar Ernberg, and Tony Pawson. "Latent Membrane Protein 2A of Epstein-Barr Virus Binds WW Domain E3 Protein-Ubiquitin Ligases That Ubiquitinate B-Cell Tyrosine Kinases." Molecular and Cellular Biology 20, no. 22 (November 15, 2000): 8526–35. http://dx.doi.org/10.1128/mcb.20.22.8526-8535.2000.

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ABSTRACT The latent membrane protein (LMP) 2A of Epstein-Barr virus (EBV) is implicated in the maintenance of viral latency and appears to function in part by inhibiting B-cell receptor (BCR) signaling. The N-terminal cytoplasmic region of LMP2A has multiple tyrosine residues that upon phosphorylation bind the SH2 domains of the Syk tyrosine kinase and the Src family kinase Lyn. The LMP2A N-terminal region also has two conserved PPPPY motifs. Here we show that the PPPPY motifs of LMP2A bind multiple WW domains of E3 protein-ubiquitin ligases of the Nedd4 family, including AIP4 and KIAA0439, and demonstrate that AIP4 and KIAA0439 form physiological complexes with LMP2A in EBV-positive B cells. In addition to a C2 domain and four WW domains, these proteins have a C-terminal Hect catalytic domain implicated in the ubiquitination of target proteins. LMP2A enhances Lyn and Syk ubiquitination in vivo in a fashion that depends on the activity of Nedd4 family members and correlates with destabilization of the Lyn tyrosine kinase. These results suggest that LMP2A serves as a molecular scaffold to recruit both B-cell tyrosine kinases and C2/WW/Hect domain E3 protein-ubiquitin ligases. This may promote Lyn and Syk ubiquitination in a fashion that contributes to a block in B-cell signaling. LMP2A may potentiate a normal mechanism by which Nedd4 family E3 enzymes regulate B-cell signaling.

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Brigui, Amira, Line Hofmann, Camilla Argüelles, Matthieu Sanial, Robert A. Holmgren, and Anne Plessis. "Control of the dynamics and homeostasis of the Drosophila Hedgehog receptor Patched by two C2-WW-HECT-E3 Ubiquitin ligases." Open Biology 5, no. 10 (October 2015): 150112. http://dx.doi.org/10.1098/rsob.150112.

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The conserved Hedgehog (HH) signals control animal development, adult stem cell maintenance and oncogenesis. In Drosophila , the HH co-receptor Patched (PTC) controls both HH gradient formation and signalling. PTC is post-translationally downregulated by HH, which promotes its endocytosis and destabilization, but the mechanisms of PTC trafficking and its importance in the control of PTC remain to be understood. PTC interacts with E3 Ubiquitin (UB)-ligases of the C2-WW-HECT family; two of them—SMURF and NEDD4—are known to regulate its levels. We demonstrate that mutation of the PTC PY motif, which mediates binding of C2-WW-HECT family members, inhibits its internalization but not its autonomous and non-autonomous signalling activities. In addition, we show that the two related UB-C2-WW-HECT ligases NEDD4 and SU(DX) regulate PTC trafficking and finely tune its accumulation through partially redundant but distinct functions. While both NEDD4 and SU(DX) promote PTC endocytosis, only SU(DX) is able to induce its lysosomal targeting and degradation. In conclusion, PTC trafficking and homeostasis are tightly regulated by a family of UB-ligases.

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Yao, Weiyi, Zelin Shan, Aihong Gu, Minjie Fu, Zhifeng Shi, and Wenyu Wen. "WW domain–mediated regulation and activation of E3 ubiquitin ligase Suppressor of Deltex." Journal of Biological Chemistry 293, no. 43 (September 13, 2018): 16697–708. http://dx.doi.org/10.1074/jbc.ra118.003781.

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The Nedd4 family E3 ligases Itch and WWP1/2 play crucial roles in the regulation of cell cycle progression and apoptosis and are closely correlated with cancer development and metastasis. It has been recently shown that the ligase activities of Itch and WWP1/2 are tightly regulated, with the HECT domain sequestered intramolecularly by a linker region connecting WW2 and WW3. Here, we show that a similar autoinhibitory mechanism is utilized by the Drosophila ortholog of Itch and WWP1/2, Suppressor of Deltex (Su(dx)). We show that Su(dx) adopts an inactive steady state with the WW domain region interacting with the HECT domain. We demonstrate that both the linker and preceding WW2 are required for the efficient binding and regulation of Su(dx) HECT. Recruiting the multiple-PY motif–containing adaptor dNdfip via WW domains relieves the inhibitory state of Su(dx) and leads to substrate (e.g. Notch) ubiquitination. Our study demonstrates an evolutionarily conservative mechanism governing the regulation and activation of some Nedd4 family E3 ligases. Our results also suggest a dual regulatory mechanism for specific Notch down-regulation via dNdfip–Su(dx)–mediated Notch ubiquitination.

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Kotorashvili, Adam, Scott J. Russo, Surafel Mulugeta, Susan Guttentag, and Michael F. Beers. "Anterograde Transport of Surfactant Protein C Proprotein to Distal Processing Compartments Requires PPDY-mediated Association with Nedd4 Ubiquitin Ligases." Journal of Biological Chemistry 284, no. 24 (April 14, 2009): 16667–78. http://dx.doi.org/10.1074/jbc.m109.002816.

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Biosynthesis of surfactant protein C (SP-C) by alveolar type 2 cells requires proteolytic processing of a 21-kDa propeptide (proSP-C21) in post-Golgi compartments to yield a 3.7-kDa mature form. Scanning alanine mutagenesis, binding assays, and co-immunoprecipitation were used to characterize the proSP-C targeting domain. Delivery of proSP-C21 to distal processing organelles is dependent upon the NH2-terminal cytoplasmic SP-C propeptide, which contains a conserved PPDY motif. In A549 cells, transfection of EGFP/proSP-C21 constructs containing polyalanine substitution for Glu11–Thr18, 13PPDY16, or 14P,16Y produced endoplasmic reticulum retention of the fusion proteins. Protein-protein interactions of proSP-C with known WW domains were screened using a solid-phase array that revealed binding of the proSP-C NH2 terminus to several WW domains found in the Nedd4 family of E3 ligases. Specificity of the interaction was confirmed by co-immunoprecipitation of proSP-C and Nedd4 or Nedd4-2 in epithelial cell lines. By Western blotting and reverse transcription-PCR, both forms were detected in primary human type 2 cells. Knockdown of Nedd4-2 by small interference RNA transfection of cultured human type 2 cells blocked processing of 35S-labeled proSP-C21. Mutagenesis of potential acceptor sites for ubiquitination in the cytosolic domain of proSP-C (Lys6, Lys34, or both) failed to inhibit trafficking of EGFP/proSP-C21. These results indicate that PPDY-mediated interaction with Nedd4 E3-ligases is required for trafficking of proSP-C. We speculate that the Nedd4/proSP-C tandem is part of a larger protein complex containing a ubiquitinated component that further directs its transport.

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Ingham, Robert J., Gerald Gish, and Tony Pawson. "The Nedd4 family of E3 ubiquitin ligases: functional diversity within a common modular architecture." Oncogene 23, no. 11 (March 2004): 1972–84. http://dx.doi.org/10.1038/sj.onc.1207436.

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Darko-Boateng, Arden, and Henry M. Colecraft. "Regulation of Low Voltage-Activated Calcium Channels by NEDD4 Family of E3 Ubiquitin Ligases." Biophysical Journal 120, no. 3 (February 2021): 159a. http://dx.doi.org/10.1016/j.bpj.2020.11.1143.

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Rougier, Jean-Sébastien, Miguel X. van Bemmelen, M. Christine Bruce, Thomas Jespersen, Bruno Gavillet, Florine Apothéloz, Sophie Cordonier, Olivier Staub, Daniela Rotin, and Hugues Abriel. "Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins." American Journal of Physiology-Cell Physiology 288, no. 3 (March 2005): C692—C701. http://dx.doi.org/10.1152/ajpcell.00460.2004.

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The voltage-gated Na+ channels (Nav) form a family composed of 10 genes. The COOH termini of Nav contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Nav1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Nav proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Nav1.2 and Nav1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Nav1.2, Nav1.3, and Nav1.5 indicated that mouse brain Nedd4-2 binds to the Nav PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Nav1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a Kd of ∼55 μM. We tested the binding properties and the ability to ubiquitinate and downregulate Nav1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Nav1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Nav1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Nav1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Nav channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane.

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Bhandari, Deepali, Seth L. Robia, and Adriano Marchese. "The E3 Ubiquitin Ligase Atrophin Interacting Protein 4 Binds Directly To The Chemokine Receptor CXCR4 Via a Novel WW Domain-mediated Interaction." Molecular Biology of the Cell 20, no. 5 (March 2009): 1324–39. http://dx.doi.org/10.1091/mbc.e08-03-0308.

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The E3 ubiquitin ligase atrophin interacting protein 4 (AIP4) mediates ubiquitination and down-regulation of the chemokine receptor CXCR4. AIP4 belongs to the Nedd4-like homologous to E6-AP carboxy terminus domain family of E3 ubiquitin ligases, which typically bind proline-rich motifs within target proteins via the WW domains. The intracellular domains of CXCR4 lack canonical WW domain binding motifs; thus, whether AIP4 is targeted to CXCR4 directly or indirectly via an adaptor protein remains unknown. Here, we show that AIP4 can interact directly with CXCR4 via a novel noncanonical WW domain-mediated interaction involving serine residues 324 and 325 within the carboxy-terminal tail of CXCR4. These serine residues are critical for mediating agonist-promoted binding of AIP4 and subsequent ubiquitination and degradation of CXCR4. These residues are phosphorylated upon agonist activation and phosphomimetic mutants show enhanced binding to AIP4, suggesting a mechanism whereby phosphorylation mediates the interaction between CXCR4 and AIP4. Our data reveal a novel noncanonical WW domain-mediated interaction involving phosphorylated serine residues in the absence of any proline residues and suggest a novel mechanism whereby an E3 ubiquitin ligase is targeted directly to an activated G protein-coupled receptor.

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Kang, Yudi, Jun Guo, Tonghua Yang, Wentao Li, and Shetuan Zhang. "Regulation of the human ether-a-go-go-related gene (hERG) potassium channel by Nedd4 family interacting proteins (Ndfips)." Biochemical Journal 472, no. 1 (October 30, 2015): 71–82. http://dx.doi.org/10.1042/bj20141282.

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The human ether-a-go-go-related gene (hERG)-encoded K+ channel is critical for cardiac repolarization. In the present study, we demonstrate that the E3 ubiquitin (Ub) ligase neural precursor cell expressed developmentally down-regulated protein 4-2 (Nedd4-2) is directed to specific cellular compartments by Nedd4 family-interacting proteins (Ndfips) to selectively target the mature hERG channels for degradation.

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Scott, Jordan L., and Robert V. Stahelin. "Spatial and Temporal Regulation of the Nedd4 Family of E3 Ubiquitin Ligases through Phospholipid Binding." Biophysical Journal 104, no. 2 (January 2013): 595a. http://dx.doi.org/10.1016/j.bpj.2012.11.3304.

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Zhang, Ying, Hao Qian, Boquan Wu, Shilong You, Shaojun Wu, Saien Lu, Pingyuan Wang, Liu Cao, Naijin Zhang, and Yingxian Sun. "E3 Ubiquitin ligase NEDD4 family‑regulatory network in cardiovascular disease." International Journal of Biological Sciences 16, no. 14 (2020): 2727–40. http://dx.doi.org/10.7150/ijbs.48437.

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Vana, Marcy L., Yi Tang, Aiping Chen, Gisselle Medina, Carol Carter, and Jonathan Leis. "Role of Nedd4 and Ubiquitination of Rous Sarcoma Virus Gag in Budding of Virus-Like Particles from Cells." Journal of Virology 78, no. 24 (December 15, 2004): 13943–53. http://dx.doi.org/10.1128/jvi.78.24.13943-13953.2004.

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ABSTRACT Rous sarcoma virus (RSV) budding requires an interaction of the L domain within the p2b region of Gag with cellular Nedd4-family E3 ubiquitin protein ligases. Members of our laboratories previously demonstrated that overexpression of a fragment of the chicken Nedd4-like protein (LDI-1 WW) inhibits Gag release in a dominant-negative manner (A. Kikonyogo, F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis, Proc. Natl. Acad. Sci. USA 98:11199-11204, 2001). We have now identified the complete 3′ end of LDI-1 and determined that it has a C-terminal ubiquitin ligase HECT domain, similar to other Nedd4 family members. While overexpression of the full-length LDI-1 clone (LDI-1 FL) had little effect on Gag budding, an LDI-1 FL mutant with a substitution in the HECT domain catalytic site blocked Gag release, similar to LDI-1 WW. The coexpression of Gag and hemagglutinin-tagged ubiquitin (HA-Ub) resulted in the detection of mono- and polyubiquitinated forms of Gag in cells and mostly monoubiquitinated Gag in virus-like particles (VLPs). When the Nedd4-binding site (L domain) was deleted, ubiquitinated Gag was not detected. Interestingly, the release of Gag with ubiquitin covalently linked to the C terminus (Gag-Ub) was still blocked by LDI-1 WW. To understand the mechanism of this inhibition, we examined cells expressing Gag and LDI-1 WW by electron microscopy. In the presence of LDI-1 WW, VLPs were found in electron-dense inclusion bodies in the cytoplasm of transfected cells. In contrast, when cells that coexpressed Gag-Ub and LDI-1 WW were examined, inclusion bodies were detected but did not contain VLPs. These results indicate that the ubiquitination of Gag is dependent upon Nedd4 binding to the L domain and suggest that Nedd4 has additional functions during RSV release besides the ubiquitination of Gag.

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Mund, Thomas, Michael Graeb, Juliusz Mieszczanek, Melissa Gammons, Hugh R. B. Pelham, and Mariann Bienz. "Disinhibition of the HECT E3 ubiquitin ligase WWP2 by polymerized Dishevelled." Open Biology 5, no. 12 (December 2015): 150185. http://dx.doi.org/10.1098/rsob.150185.

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Dishevelled is a pivot in Wnt signal transduction, controlling both β-catenin-dependent transcription to specify proliferative cell fates, and cell polarity and other non-nuclear events in post-mitotic cells. In response to Wnt signals, or when present at high levels, Dishevelled forms signalosomes by dynamic polymerization. Its levels are controlled by ubiquitylation, mediated by various ubiquitin ligases, including NEDD4 family members that bind to a conserved PPxY motif in Dishevelled (mammalian Dvl1–3). Here, we show that Dvl2 binds to the ubiquitin ligase WWP2 and unlocks its ligase activity from autoinhibition. This disinhibition of WWP2 depends on several features of Dvl2 including its PPxY motif and to a lesser extent its DEP domain, but crucially on the ability of Dvl2 to polymerize, indicating that WWP2 is activated in Wnt signalosomes. We show that Notch intracellular domains are substrates for Dvl-activated WWP2 and their transcriptional activity is consequently reduced, providing a molecular mechanism for cross-talk between Wnt and Notch signalling. These regulatory interactions are conserved in Drosophila whose WWP2 orthologue, Suppressor-of-deltex, downregulates Notch signalling upon activation by Dishevelled in developing wing tissue. Attentuation of Notch signalling by Dishevelled signalosomes could be important during the transition of cells from the proliferative to the post-mitotic state.

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Vecchione, Andrea, Adriano Marchese, Pauline Henry, Daniela Rotin, and Andrea Morrione. "The Grb10/Nedd4 Complex Regulates Ligand-Induced Ubiquitination and Stability of the Insulin-Like Growth Factor I Receptor." Molecular and Cellular Biology 23, no. 9 (May 1, 2003): 3363–72. http://dx.doi.org/10.1128/mcb.23.9.3363-3372.2003.

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ABSTRACT The adapter protein Grb10 belongs to a superfamily of related proteins, including Grb7, -10, and -14 and Caenorhabditis elegans Mig10. Grb10 is an interacting partner of the insulin-like growth factor I receptor (IGF-IR) and the insulin receptor (IR). Previous work showed an inhibitory effect of mouse Grb10 (mGrb10α) on IGF-I-mediated mitogenesis (A. Morrione et al., J. Biol. Chem. 272:26382-26387, 1997). With mGrb10α as bait in a yeast two-hybrid screen, mouse Nedd4 (mNedd4-1), a ubiquitin protein ligase, was previously isolated as an interacting protein of Grb10 (A. Morrione et al., J. Biol. Chem. 274:24094-24099, 1999). However, Grb10 is not ubiquitinated by Nedd4 in cells. Here we show that in mouse embryo fibroblasts overexpressing Grb10 and the IGF-IR (p6/Grb10), there is a strong ligand-dependent increase in ubiquitination of the IGF-IR compared with that in parental cells (p6). This increased ubiquitination is associated with a shorter half-life and increased internalization of the IGF-IR. The IGF-IR is stabilized following treatment with both MG132 and chloroquine, indicating that both the proteasome and lysosomal pathways mediate degradation of the receptor. Ubiquitination of the IGF-IR likely occurs at the plasma membrane, prior to the formation of endocytic vesicles, as it is insensitive to dansylcadaverine, an inhibitor of early endosome formation in IGF-IR endocytosis. Grb10 coimmunoprecipitates with the IGF-IR and endogenous Nedd4 in p6/Grb10 cells, suggesting the presence of a Grb10/Nedd4/IGF-IR complex. Ubiquitination of the IGF-IR in p6/Grb10 cells is severely impaired by overexpression of a catalytically inactive Nedd4 mutant (Nedd4-CS), which also stabilizes the receptor. Likewise, overexpression of a Grb10 mutant lacking the Src homology 2 (SH2) domain impaired ubiquitination of the IGF-IR in parental p6 and p6/Grb10 cells, indicating that Grb10 binding to Nedd4 is critical for ubiquitination of the receptor. These results suggest a role for the Grb10/Nedd4 complex in regulating ubiquitination and stability of the IGF-IR, and they suggest that Grb10 serves as an adapter to form a bridge between Nedd4 and the IGF-IR. This is the first demonstration of regulation of stability of a tyrosine kinase receptor by the Nedd4 (HECT) family of E3 ligases.

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Novoselova, Tatiana V., Kiran Zahira, Ruth-Sarah Rose, and James A. Sullivan. "Bul Proteins, a Nonredundant, Antagonistic Family of Ubiquitin Ligase Regulatory Proteins." Eukaryotic Cell 11, no. 4 (February 3, 2012): 463–70. http://dx.doi.org/10.1128/ec.00009-12.

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ABSTRACT Like other Nedd4 ligases, Saccharomyces cerevisiae E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of BUL gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using in vitro ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination.

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Liang, Changxiang, Guoyan Liang, Xiaoqing Zheng, Yongxiong Huang, Shuaihao Huang, and Dong Yin. "RSP5 Positively Regulates the Osteogenic Differentiation of Mesenchymal Stem Cells by Activating the K63-Linked Ubiquitination of Akt." Stem Cells International 2020 (April 6, 2020): 1–13. http://dx.doi.org/10.1155/2020/7073805.

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Mesenchymal stem cells (MSCs) are multipotent stem cells that have a strong osteogenic differentiation capacity. However, the molecular mechanism underlying the osteogenic differentiation of MSCs remains largely unknown and thus hinders further development of MSC-based cell therapies for bone repair in the clinic. RSP5, also called NEDD4L (NEDD4-like E3 ubiquitin protein ligase), belongs to the HECT (homologous to E6-AP carboxyl terminus) domain-containing E3 ligase family. Nevertheless, although many studies have been conducted to elucidate the role of RSP5 in various biological processes, its effect on osteogenesis remains elusive. In this study, we demonstrated that the expression of RSP5 was elevated during the osteogenesis of MSCs and positively regulated the osteogenic capacity of MSCs by inducing K63-linked polyubiquitination and activation of the Akt pathway. Taken together, our findings suggest that RSP5 may be a promising target to improve therapeutic efficiency by using MSCs for bone regeneration and repair.

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Howitt, Jason, Jenny Lackovic, Ley-Hian Low, Adam Naguib, Alison Macintyre, Choo-Peng Goh, Jennifer K. Callaway, et al. "Ndfip1 regulates nuclear Pten import in vivo to promote neuronal survival following cerebral ischemia." Journal of Cell Biology 196, no. 1 (January 2, 2012): 29–36. http://dx.doi.org/10.1083/jcb.201105009.

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PTEN (phosphatase and tensin homologue deleted on chromosome TEN) is the major negative regulator of phosphatidylinositol 3-kinase signaling and has cell-specific functions including tumor suppression. Nuclear localization of PTEN is vital for tumor suppression; however, outside of cancer, the molecular and physiological events driving PTEN nuclear entry are unknown. In this paper, we demonstrate that cytoplasmic Pten was translocated into the nuclei of neurons after cerebral ischemia in mice. Critically, this transport event was dependent on a surge in the Nedd4 family–interacting protein 1 (Ndfip1), as neurons in Ndfip1-deficient mice failed to import Pten. Ndfip1 binds to Pten, resulting in enhanced ubiquitination by Nedd4 E3 ubiquitin ligases. In vitro, Ndfip1 overexpression increased the rate of Pten nuclear import detected by photobleaching experiments, whereas Ndfip1−/− fibroblasts showed negligible transport rates. In vivo, Ndfip1 mutant mice suffered larger infarct sizes associated with suppressed phosphorylated Akt activation. Our findings provide the first physiological example of when and why transient shuttling of nuclear Pten occurs and how this process is critical for neuron survival.

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Keuss, Matthew J., Yann Thomas, Robin Mcarthur, Nicola T. Wood, Axel Knebel, and Thimo Kurz. "Characterization of the mammalian family of DCN-type NEDD8 E3 ligases." Journal of Cell Science 129, no. 7 (February 18, 2016): 1441–54. http://dx.doi.org/10.1242/jcs.181784.

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Zhu, Kang, Zelin Shan, Xing Chen, Yuqun Cai, Lei Cui, Weiyi Yao, Zhen Wang, et al. "Allosteric auto‐inhibition and activation of the Nedd4 family E3 ligase Itch." EMBO reports 18, no. 9 (July 26, 2017): 1618–30. http://dx.doi.org/10.15252/embr.201744454.

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Kim, Hyung-Seok, Yangsook Song Green, Yuanyuan Xie, and Jan L. Christian. "Tril dampens Nodal signaling through Pellino2- and Traf6-mediated activation of Nedd4l." Proceedings of the National Academy of Sciences 118, no. 36 (September 2, 2021): e2104661118. http://dx.doi.org/10.1073/pnas.2104661118.

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

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Baillet, Nicolas, Sophie Krieger, Xavier Carnec, Mathieu Mateo, Alexandra Journeaux, Othmann Merabet, Valérie Caro, Frédéric Tangy, Pierre-Olivier Vidalain, and Sylvain Baize. "E3 Ligase ITCH Interacts with the Z Matrix Protein of Lassa and Mopeia Viruses and Is Required for the Release of Infectious Particles." Viruses 12, no. 1 (December 31, 2019): 49. http://dx.doi.org/10.3390/v12010049.

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Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related, rodent-born mammarenaviruses. LASV is the causative agent of Lassa fever, a deadly hemorrhagic fever endemic in West Africa, whereas MOPV is non-pathogenic in humans. The Z matrix protein of arenaviruses is essential to virus assembly and budding by recruiting host factors, a mechanism that remains partially defined. To better characterize the interactions involved, a yeast two-hybrid screen was conducted using the Z proteins from LASV and MOPV as a bait. The cellular proteins ITCH and WWP1, two members of the Nedd4 family of HECT E3 ubiquitin ligases, were found to bind the Z proteins of LASV, MOPV and other arenaviruses. The PPxY late-domain motif of the Z proteins is required for the interaction with ITCH, although the E3 ubiquitin-ligase activity of ITCH is not involved in Z ubiquitination. The silencing of ITCH was shown to affect the replication of the old-world mammarenaviruses LASV, MOPV, Lymphocytic choriomeningitis virus (LCMV) and to a lesser extent Lujo virus (LUJV). More precisely, ITCH was involved in the egress of virus-like particles and the release of infectious progeny viruses. Thus, ITCH constitutes a novel interactor of LASV and MOPV Z proteins that is involved in virus assembly and release.

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Dores, Michael R., Huilan Lin, Neil J. Grimsey, Francisco Mendez, and JoAnn Trejo. "The α-arrestin ARRDC3 mediates ALIX ubiquitination and G protein–coupled receptor lysosomal sorting." Molecular Biology of the Cell 26, no. 25 (December 15, 2015): 4660–73. http://dx.doi.org/10.1091/mbc.e15-05-0284.

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The sorting of G protein–coupled receptors (GPCRs) to lysosomes is critical for proper signaling and cellular responses. We previously showed that the adaptor protein ALIX regulates lysosomal degradation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, independent of ubiquitin-binding ESCRTs and receptor ubiquitination. However, the mechanisms that regulate ALIX function during PAR1 lysosomal sorting are not known. Here we show that the mammalian α-arrestin arrestin domain–containing protein-3 (ARRDC3) regulates ALIX function in GPCR sorting via ubiquitination. ARRDC3 colocalizes with ALIX and is required for PAR1 sorting at late endosomes and degradation. Depletion of ARRDC3 by small interfering RNA disrupts ALIX interaction with activated PAR1 and the CHMP4B ESCRT-III subunit, suggesting that ARRDC3 regulates ALIX activity. We found that ARRDC3 is required for ALIX ubiquitination induced by activation of PAR1. A screen of nine mammalian NEDD4-family E3 ubiquitin ligases revealed a critical role for WWP2. WWP2 interacts with ARRDC3 and not ALIX. Depletion of WWP2 inhibited ALIX ubiquitination and blocked ALIX interaction with activated PAR1 and CHMP4B. These findings demonstrate a new role for the α-arrestin ARRDC3 and the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs.

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Shea, Fortune F., Jennie L. Rowell, Yechaowei Li, Tien-Hsien Chang, and Carlos E. Alvarez. "Mammalian Alpha Arrestins Link Activated Seven Transmembrane Receptors to Nedd4 Family E3 Ubiquitin Ligases and Interact with Beta Arrestins." PLoS ONE 7, no. 12 (December 7, 2012): e50557. http://dx.doi.org/10.1371/journal.pone.0050557.

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Kamada, Shinji. "Inhibitor of apoptosis proteins as E3 ligases for ubiquitin and NEDD8." BioMolecular Concepts 4, no. 2 (April 1, 2013): 161–71. http://dx.doi.org/10.1515/bmc-2012-0036.

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AbstractThe inhibitors of apoptosis proteins (IAPs) are endogenous inhibitors for apoptosis. Apoptosis is carried out by caspases, which are the family of cystein proteases. IAPs regulate caspases through two conserved regions, the baculovirus IAP repeats (BIRs) and the really interesting new gene (RING) domains. Although the BIRs are responsible for binding to caspases, the RING domain can act as a ubiquitin-E3 ligase, leading to ubiquitylation of IAPs themselves and their pro-apoptotic IAP counterparts such as caspases. Recently, it is reported that another ubiquitin-like protein, neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8), is also involved in the regulation of apoptosis through neddylation of caspases mediated by IAPs. On the contrary, the results against the function of IAPs as a NEDD8-E3 ligase are also suggested. This review presents the summary of IAPs, caspases, and the ubiquitin-proteasome system and how their interactions influence the regulation of apoptosis.

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Huang, Xi, Enfan Zhang, Xing Guo, Jing Chen, Xuanru Lin, Qingxiao Chen, Haimeng Yan, et al. "NEDD4-1 Modulates the Resistance of Multiple Myeloma to Bortezomib." Blood 128, no. 22 (December 2, 2016): 2068. http://dx.doi.org/10.1182/blood.v128.22.2068.2068.

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Abstract Background: Multiple myeloma (MM) is among the most common hematologic malignancies. Proteasome inhibitor bortezomib (Bor) is one of the most effective drugs for treatment of MM. However, during long-term Bor treatment, MM cells may eventually develop acquired-resistance to Bor which results in recurrence and a poor prognosis of MM. Several researches show that E3 ubiquitin ligases (E3s) primarily determine the substrate specificity of ubiquitin proteasome system and play an essential role in Bor resistance of MM. NEDD4-1 E3s, a founding member of the Neural precursor cell-Expressed Developmentally Downregulated gene 4 (NEDD4) family, was proved to involve in the proliferation, migration, invasion of cancer cells and the sensitivity of anticancer therapies. Our current study aims to explore the role and underlying mechanism of NEDD4-1 in acquired resistance of Bor in MM. Methods: The mRNA and protein levels of NEDD4-1 and its substrates in MM cell lines (H929, LP-1, RPMI8226, OPM-2 and ARP-1) and MM patients were detected by Quantitative Realtime PCR and Western Blotting. Lentiviral plasmids containing shRNA against NEDD4-1 were transfected into MM cells. Cell viability, proliferation and apoptosis of MM cells were measured by Cell Counting kit8 (CCK8) and flow cytometry. Gene array was used to compare the gene expression profiles of a panel of Bor treated MM cells vs vehicle-treated MM cells. Results: Gene array showed NEDD4-1 was significantly increased in MM cells treated with Bor. MM cells (CD138+ plasma cells of the bone marrow) from refractory/recurrence patients expressed lower NEDD4-1 than primary patient myeloma cells. Also, MM cell lines H929, ARP-1, LP-1 highly expressed NEDD4-1 at mRNA and protein levels. RPMI8226 and OPM-2 were relatively low expressed. Cell growth assay displayed no significant difference in proliferation between the NEDD4-1 knockdown (KD) and the control group (P>0.05). After suppression of NEDD4-1 using shRNAs, the killing effect of Bor in MM was significantly weaker than the control group (P<0.05). We also found that PTEN was decreased in the NEDD4-1 KD H929 cell line. Otherwise, phospho-STAT3 (ser727) and oncoprotein c-Myc and Bcl-2 were upregulated. Conclusion: Collectively, our study reveals that inhibition of NEDD4-1 can reduce MM sensitivity to Bor via regulating PTEN, c-Myc and Bcl-2, may be related to JAK/STAT signaling pathway, which suggests that NEDD4-1 probably acts as a novel drug target and therapeutic paradigm in the battle against multiple myeloma. Disclosures No relevant conflicts of interest to declare.

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Singh, Sunil, and J. Sivaraman. "Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity." Biochemical Journal 477, no. 5 (March 4, 2020): 905–23. http://dx.doi.org/10.1042/bcj20200027.

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The HECT family of E3 ubiquitin ligase is divided into three subfamilies: the NEDD4, the HERC, and the ‘other’. Previous studies have mostly targeted members of the NEDD4 subfamily for structural and functional analysis. The UBE3C E3 ligase is a member of the ‘other’ subfamily HECT and influences several crucial cellular processes, including innate immunity, proteasome processivity, and cancer metastasis. Here, we report the crystal structure of the HECT domain of UBE3C (amino acids (aa) 744–1083) with an additional fifty N-terminal amino acids (aa 693–743) at 2.7 Å, along with multiple in vitro ubiquitination assays to understand its enzymatic activity. The UBE3C HECT domain forms an open, L-shaped, bilobed conformation, having a large N-lobe and a small C-lobe. We show that the N-terminal region (aa 693–743) preceding the UBE3C HECT domain as well as a loop region (aa 758–762) in the N-lobe of the HECT domain affect the stability and activity of UBE3C HECT domain. Moreover, we identified Lys903 in the UBE3C HECT domain as a major site of autoubiquitination. The deletion of the last three amino acids at the C-terminal completely abrogated UBE3C activity while mutations of Gln961 and Ser1049 residues in the HECT domain substantially decreased its autoubiquitination activity. We demonstrate that these region/residues are involved in the E2–E3 transthiolation process and affect the UBE3C mediated autoubiquitination. Collectively, our study identified key residues crucial for UBE3C enzymatic activity, and it may assist in the development of suitable inhibitors to regulate its activity in multiple cancers.

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Seo, Min-Duk, Seung-Hyeon Seok, Ji-Hun Kim, Ji Woong Choi, Sung Jean Park, and Bong-Jin Lee. "Molecular Interactions between Two LMP2A PY Motifs of EBV and WW Domains of E3 Ubiquitin Ligase AIP4." Life 11, no. 5 (April 22, 2021): 379. http://dx.doi.org/10.3390/life11050379.

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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.

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Belgareh-Touzé, Naima, Sébastien Léon, Zoi Erpapazoglou, Marta Stawiecka-Mirota, Danièle Urban-Grimal, and Rosine Haguenauer-Tsapis. "Versatile role of the yeast ubiquitin ligase Rsp5p in intracellular trafficking." Biochemical Society Transactions 36, no. 5 (September 19, 2008): 791–96. http://dx.doi.org/10.1042/bst0360791.

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The ubiquitin ligase (E3) Rsp5p is the only member of the Nedd (neural-precursor-cell-expressed, developmentally down-regulated) 4 family of E3s present in yeast. Rsp5p has several proteasome-independent functions in membrane protein trafficking, including a role in the ubiquitination of most plasma membrane proteins, leading to their endocytosis. Rsp5p is also required for the ubiquitination of endosomal proteins, leading to their sorting to the internal vesicles of MVBs (multivesicular bodies). Rsp5p catalyses the attachment of non-conventional ubiquitin chains, linked through ubiquitin Lys-63, to some endocytic and MVB cargoes. This modification appears to be required for efficient sorting, possibly because these chains have a greater affinity for the ubiquitin-binding domains present within endocytic or MVB sorting complexes. The mechanisms involved in the recognition of plasma membrane and MVB substrates by Rsp5p remain unclear. A subset of Rsp5/Nedd4 substrates have a ‘PY motif’ and are recognized directly by the WW (Trp-Trp) domains of Rsp5p. Most Rsp5p substrates do not carry PY motifs, but some may depend on PY-containing proteins for their ubiquitination by Rsp5p, consistent with the latter's acting as specificity factors or adaptors. As in other ubiquitin-conjugating systems, these adaptors are also Rsp5p substrates and undergo ubiquitin-dependent trafficking. In the present review, we discuss recent examples illustrating the role of Rsp5p in membrane protein trafficking and providing new insights into the regulation of this E3 by adaptor proteins.

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Choo, Yin Yin, Boon Kim Boh, Jessica Jie Wei Lou, Jolane Eng, Yee Chin Leck, Benjamin Anders, Peter G. Smith, and Thilo Hagen. "Characterization of the role of COP9 signalosome in regulating cullin E3 ubiquitin ligase activity." Molecular Biology of the Cell 22, no. 24 (December 15, 2011): 4706–15. http://dx.doi.org/10.1091/mbc.e11-03-0251.

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Cullin RING ligases (CRLs) are the largest family of cellular E3 ubiquitin ligases and mediate polyubiquitination of a number of cellular substrates. CRLs are activated via the covalent modification of the cullin protein with the ubiquitin-like protein Nedd8. This results in a conformational change in the cullin carboxy terminus that facilitates the ubiquitin transfer onto the substrate. COP9 signalosome (CSN)-mediated cullin deneddylation is essential for CRL activity in vivo. However, the mechanism through which CSN promotes CRL activity in vivo is currently unclear. In this paper, we provide evidence that cullin deneddylation is not intrinsically coupled to substrate polyubiquitination as part of the CRL activation cycle. Furthermore, inhibiting substrate-receptor autoubiquitination is unlikely to account for the major mechanism through which CSN regulates CRL activity. CSN also did not affect recruitment of the substrate-receptor SPOP to Cul3, suggesting it may not function to facilitate the exchange of Cul3 substrate receptors. Our results indicate that CSN binds preferentially to CRLs in the neddylation-induced, active conformation. Binding of the CSN complex to active CRLs may recruit CSN-associated proteins important for CRL regulation. The deneddylating activity of CSN would subsequently promote its own dissociation to allow progression through the CRL activation cycle.

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Cerny, Ondrej, Camilla Godlee, Romina Tocci, Nancy E. Cross, Haoran Shi, James C. Williamson, Eric Alix, Paul J. Lehner, and David W. Holden. "CD97 stabilises the immunological synapse between dendritic cells and T cells and is targeted for degradation by the Salmonella effector SteD." PLOS Pathogens 17, no. 7 (July 27, 2021): e1009771. http://dx.doi.org/10.1371/journal.ppat.1009771.

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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.

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Barajas, Daniel, Zhenghe Li, and Peter D. Nagy. "The Nedd4-Type Rsp5p Ubiquitin Ligase Inhibits Tombusvirus Replication by Regulating Degradation of the p92 Replication Protein and Decreasing the Activity of the Tombusvirus Replicase." Journal of Virology 83, no. 22 (September 16, 2009): 11751–64. http://dx.doi.org/10.1128/jvi.00789-09.

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ABSTRACT Recent in vitro proteomics screens revealed that many host proteins could interact with the replication proteins of Tomato bushy stunt virus (TBSV), which is a small, plus-stranded RNA virus (Z. Li, D. Barajas, T. Panavas, D. A. Herbst, and P. D. Nagy, J. Virol. 82:6911-6926, 2008). To further our understanding of the roles of host factors in TBSV replication, we have tested the effect of Rsp5p, which is a member of the Nedd4 family of E3 ubiquitin ligases. The full-length Rsp5p, via its WW domain, is shown to interact with p33 and the central portion of p92pol replication proteins. We find that overexpression of Rsp5p inhibits TBSV replication in Saccharomyces cerevisiae yeast, while downregulation of Rsp5p leads to increased TBSV accumulation. The inhibition is caused by Rsp5p-guided degradation of p92pol, while the negative effect on the p33 level is less pronounced. Interestingly, recombinant Rsp5p also inhibits TBSV RNA replication in a cell-free replication assay, likely due to its ability to bind to p33 and p92pol. We show that the WW domain of Rsp5p, which is involved in protein interactions, is responsible for inhibition of TBSV replication, whereas the HECT domain, involved in protein ubiquitination, is not necessary for Rsp5p-mediated inhibition of viral replication. Overall, our data suggest that direct binding between Rsp5p and p92pol reduces the stability of p92pol, with consequent inhibition of TBSV replicase activity.

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Zhang, Wei, Ayaka Muramatsu, Rina Matsuo, Naoki Teranishi, Yui Kahara, Terunao Takahara, Hideki Shibata, and Masatoshi Maki. "The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination." International Journal of Molecular Sciences 21, no. 17 (August 31, 2020): 6315. http://dx.doi.org/10.3390/ijms21176315.

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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.

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Santonico, Elena. "Old and New Concepts in Ubiquitin and NEDD8 Recognition." Biomolecules 10, no. 4 (April 7, 2020): 566. http://dx.doi.org/10.3390/biom10040566.

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Post-translational modifications by ubiquitin and ubiquitin-like proteins (Ubls) have known roles in a myriad of cellular processes. Ubiquitin- and Ubl-binding domains transmit the information conferred by these post-translational modifications by recognizing functional surfaces and, when present, different chain structures. Numerous domains binding to ubiquitin have been characterized and their structures solved. Analogously, motifs selectively interacting with SUMO (small ubiquitin-like modifier) have been identified in several proteins and their role in SUMO-dependent processes investigated. On the other hand, proteins that specifically recognize other Ubl modifications are known only in a few cases. The high sequence identity between NEDD8 and ubiquitin has made the identification of specific NEDD8-binding domains further complicated due to the promiscuity in the recognition by several ubiquitin-binding domains. Two evolutionarily related domains, called CUBAN (cullin-binding domain associating with NEDD8) and CoCUN (cousin of CUBAN), have been recently described. The CUBAN binds monomeric NEDD8 and neddylated cullins, but it also interacts with di-ubiquitin chains. Conversely, the CoCUN domain only binds ubiquitin. CUBAN and CoCUN provide an intriguing example of how nature solved the issue of promiscuity versus selectivity in the recognition of these two highly related molecules. The structural information available to date suggests that the ancestor of CUBAN and CoCUN was a three-helix bundle domain that diversified in KHNYN (KH and NYN domain-containing) and N4BP1 (NEDD4-binding protein-1) by acquiring different features. Indeed, these domains diverged towards two recognition modes, that recall respectively the electrostatic interaction utilized by the E3-ligase RBX1/2 in the interaction with NEDD8, and the hydrophobic features described in the recognition of ubiquitin by CUE (coupling ubiquitin conjugation to ER degradation) domains. Intriguingly, CUBAN and CoCUN domains are only found in KHNYN and N4BP1, respectively, both proteins belonging to the PRORP family whose members are characterized by the combination of protein modules involved in RNA metabolism with domains mediating ubiquitin/NEDD8 recognition. This review recapitulates the current knowledge and recent findings of CUBAN and CoCUN domains and the proteins containing them.

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Feng, Shu-Mang, Rebecca S. Muraoka-Cook, Debra Hunter, Melissa A. Sandahl, Laura S. Caskey, Keiji Miyazawa, Azeddine Atfi, and H. Shelton Earp. "The E3 Ubiquitin Ligase WWP1 Selectively Targets HER4 and Its Proteolytically Derived Signaling Isoforms for Degradation." Molecular and Cellular Biology 29, no. 3 (December 1, 2008): 892–906. http://dx.doi.org/10.1128/mcb.00595-08.

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ABSTRACT In general, epidermal growth factor receptor family members stimulate cell proliferation. In contrast, at least one HER4 isoform, JM-a/Cyt1, inhibits cell growth after undergoing a two-step proteolytic cleavage that first produces a membrane-anchored 80-kDa fragment (m80HER4) and subsequently liberates a soluble 80-kDa fragment, s80HER4. Here we report that s80HER4 Cyt1 action increased the expression of WWP1 (for WW domain-containing protein 1), an E3 ubiquitin ligase, but not other members of the Nedd4 E3 ligase family. The HER4 Cyt1 isoform contains three proline-rich tyrosine (PY) WW binding motifs, while Cyt2 has only two. WWP1 binds to all three Cyt1 PY motifs; the interaction with PY2 found exclusively in Cyt1 was strongest. WWP1 ubiquitinated and caused the degradation of HER4 but not of EGFR, HER2, or HER3. The HER4-WWP1 interaction also accelerated WWP1 degradation. Membrane HER4 (full length and m80HER4, the product of the first proteolytic cleavage) were the preferred targets of WWP1, correlating with the membrane localization of WWP1. Conversely s80HER4, a poorer WWP1 substrate, was found in the cell nucleus, while WWP1 was not. Deletion of the C2 membrane association domain of WWP1 allowed more efficient s80HER4 degradation, suggesting that WWP1 is normally part of a membrane complex that regulates HER4 membrane species levels, with a predilection for the growth-inhibitory Cyt1 isoform. Finally, WWP1 expression diminished HER4 biologic activity in MCF-7 cells. We previously showed that nuclear s80HER4 is ubiquitinated and degraded by the anaphase-promoting complex, suggesting that HER4 ubiquitination within specific cellular compartments helps regulate the unique HER4 signaling capabilities.

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Scott, Jordan L., Cary T. Frick, Kristen A. Johnson, Haining Liu, Sylvia S. Yong, Allyson G. Varney, Olaf Wiest, and Robert V. Stahelin. "Molecular Analysis of Membrane Targeting by the C2 Domain of the E3 Ubiquitin Ligase Smurf1." Biomolecules 10, no. 2 (February 4, 2020): 229. http://dx.doi.org/10.3390/biom10020229.

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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.

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Wawrzycka, Donata, and Katarzyna Mizio. "Ubiquitination, quality control and degradation of membrane proteins – chance for therapies?" Postępy Higieny i Medycyny Doświadczalnej 72 (June 7, 2018): 512–25. http://dx.doi.org/10.5604/01.3001.0012.0823.

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Plasma membrane integrity maintenance is crucial for cell survival. Plasma membrane proteins are under tight regulation and under certain conditions are actively removed from the membrane allowing cells to adapt to changing environment. Proteins blocked in the cell membrane may interact with other molecules and form toxic aggregates. Ubiquitin is one of the most important modifiers targeting proteins for degradation and/or regulating protein functions. Several quality control mechanisms have been identified in eukaryotic cells: chaperone- dependent system that recognizes and ubiquitinates misfolded or redundant membrane proteins; protein-intrinsic LID-degron system, based on recognition of degron and ARTs-Rsp5 network that controls quality of membrane transporters. Rsp5, a Nedd4-family E3 ubiquitin ligase, is crucial for plasma membrane proteins ubiquitination. Rsp5-dependent ubiquitin action acts as a sorting signal for internalization of a membrane protein via endocytosis, recognition by the ESCRT system and vacuolar degradation. Rsp5 builds poliUb-chains on K63 and recognizes substrates through various adaptor proteins. Most of the identified Rsp5 adaptors belongs to the α-arrestin family. Plasma membrane protein ubiquitination and degradation disorders may cause neurodegenerative and cardiovascular diseases. The yeast Saccharomyces cerevisiae is one of the best models for studying trafficking pathways of membrane proteins and ubiquitination systems.

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Alix, Eric, Camilla Godlee, Ondrej Cerny, Samkeliso Blundell, Romina Tocci, Sophie Matthews, Mei Liu, et al. "The Tumour Suppressor TMEM127 Is a Nedd4-Family E3 Ligase Adaptor Required by Salmonella SteD to Ubiquitinate and Degrade MHC Class II Molecules." Cell Host & Microbe 28, no. 1 (July 2020): 54–68. http://dx.doi.org/10.1016/j.chom.2020.04.024.

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Luke-Glaser, Sarah, Marcia Roy, Brett Larsen, Thierry Le Bihan, Pavel Metalnikov, Mike Tyers, Matthias Peter, and Lionel Pintard. "CIF-1, a Shared Subunit of the COP9/Signalosome and Eukaryotic Initiation Factor 3 Complexes, Regulates MEL-26 Levels in the Caenorhabditis elegans Embryo." Molecular and Cellular Biology 27, no. 12 (April 2, 2007): 4526–40. http://dx.doi.org/10.1128/mcb.01724-06.

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ABSTRACT The COP9/signalosome (CSN) is an evolutionarily conserved macromolecular complex that regulates the cullin-RING ligase (CRL) class of E3 ubiquitin ligases, primarily by removing the ubiquitin-like protein Nedd8 from the cullin subunit. In the Caenorhabditis elegans embryo, the CSN controls the degradation of the microtubule-severing protein MEI-1 through CUL-3 deneddylation. However, the molecular mechanisms of CSN function and its subunit composition remain to be elucidated. Here, using a proteomic approach, we have characterized the CSN and CUL-3 complexes from C. elegans embryos. We show that the CSN physically interacts with the CUL-3-based CRL and regulates its activity by counteracting the autocatalytic instability of the substrate-specific adaptor MEL-26. Importantly, we identified the uncharacterized protein K08F11.3/CIF-1 (for CSN-eukaryotic initiation factor 3 [eIF3]) as a stoichiometric and functionally important subunit of the CSN complex. CIF-1 appears to be the only ortholog of Csn7 encoded by the C. elegans genome, but it also exhibits extensive sequence similarity to eIF3m family members, which are required for the initiation of protein translation. Indeed, CIF-1 binds eIF-3.F and inactivation of cif-1 impairs translation in vivo. Taken together, our results indicate that CIF-1 is a shared subunit of the CSN and eIF3 complexes and may therefore link protein translation and degradation.

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Pericole, Fernando V., Joao Machado-Neto, Fernando Ferreira Costa, and Sara T. Olalla Saad. "Abnormal Expression of Ndfip2 and Cbl in Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients: Role of Ubiquitin Proteasome System in Myeloid Neoplasms and Normal Hematopoiesis." Blood 118, no. 21 (November 18, 2011): 2567. http://dx.doi.org/10.1182/blood.v118.21.2567.2567.

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Abstract Abstract 2567 Introduction: Nedd4-like E3 ligases regulate a wide variety of cellular processes, including ubiquitin-mediated trafficking, lysosomal or proteasomal degradation and nuclear translocation of proteins. Ndfip1 and Ndfip2 are endosomal PY-motif containing proteins and are activators of Nedd4 family members. Ndfip proteins are also regulators of PTEN/Akt/mTOR and MAP kinase and are induced upon T-cell activation. Cbl (or c-Cbl) is a highly conserved RING finger ubiquitin ligase (E3) and regulates signaling by multiple tyrosine kinases (TKs) through interaction with their SH2 and SH3 domains. Cbl proteins negatively regulate TK receptors, mediating the ubiquitination and degradation of activated TKs. In addition to its E3-dependent negative role, Cbl mediates positive pro-oncogenic effect when E3 activity is lost. Hematopoietic stem cells (HSC) of Cbl −/− mice exhibit augmented pool size, hyperproliferation and enhanced long-term repopulation capacity. HSC of Cbl −/− mice are also hyper responsive for thrombopoietin and display elevated levels of STAT5 phosphorylation. Aim: The aim was to determine NDFIP1, NDFIP2 and CBL expression in acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) and myelodysplastic syndrome (MDS) patients and to correlate gene expression with clinical features. Furthermore, we analyzed the expression during erythroid, granulocytic and megakaryocytic differentiation, using cell line models. Methods: NDFIP1, NDFIP2 and CBL expression was verified, by q-PCR, in bone marrow aspirates from 15 normal donors, 38 MDS (24 low-risk and 14 high-risk MDS), 28 AML and 5 APL patients, at the time of diagnosis. NB4, a promyelocytic leukemia cell line, was treated with 10−6 M of All-trans retinoic acid (ATRA) for granulocytic differentiation. KU812 was treated with 100μM Hydroxyurea (HU) and 50μM Hemin (HE) for erythroid differentiation. K562 was stimulated with 20nM of Phorbol-13 myristate-12 acetate (PMA) for megakaryocytic differentiation. Results: NDFIP2 gene expression was significantly decreased in AML (0.34 [1.7–0.03] vs. 0.59 [1.98–0.01], P=0.04) and was increased in APL, compared with normal donors (3.33 [4.73–0.32] vs. 0.59 [1.98–0.01], P=0.018) and was also different between each other (P=0.002). CBL was significantly lower in MDS, AML and APL patients, compared with control group (0.36 [3.13–0.05], 0.22 [0.68–0.002], 0.10 [0.18–0.01] vs 0.55 [1.52–0], respectively, with all P<0.01). NDFIP1 expression was not different between all subgroups (P>0.05). In AML, NDFIP1 expression was negatively correlated with peripheral lymphocyte counts (r= −0.39 and P=0.044) and blast counts (r=-0.44 and P=0.02). NDFIP2 expression in AML showed a negative correlation with peripheral lymphocyte counts (r=-0.41, P=0.03) and CBL expression was positively correlated with platelet counts in the same population (r=0.54, P=0.003). In MDS patients, NDFIP1 and NDFIP2 expression was not different from the control group. However, in high-risk MDS, NDFIP1 expression was negatively correlated with bone marrow and peripheral neutrophil counts (r= −0.63, P=0.01; r=−0.60, P=0.002, respectively). During differentiation assay for granulocytic (NB4), erythroid (KU812) and megakaryocytic (K562) lineages, there was an increased expression of NDFIP1 (eight-fold, two-fold and three-fold increase, respectively), NDFIP2 (three-fold, two-fold and four-fold increase, respectively) and CBL (ten-fold, two-fold and three-fold increase, respectively). Conclusions: Ubiquitin-proteasome system (UPS) plays an essential role in the homeostasis of cellular protein traffic and degradation, regulating cell fate, together with autophagy and apoptosis. Disruption of UPS is essential for leukemogenesis and has potential therapeutic implications. NDFIP2 and CBL are important elements of UPS. Our results show that CBL and NDFIP2 are less expressed in AML (except APL) and correlate with platelet, peripheral blasts and lymphocyte counts. The upregulation of these genes during normal hematopoiesis sheds light on their importance in the myeloid differentiation process. The higher expression of NDFIP2 in APL may be related to a better differentiation potential and autophagy activation. Moreover, the correlation of higher levels of NDFIP1 with lower neutrophil counts in high risk MDS suggests that this disease may still have functional response to T-cell activation. Disclosures: No relevant conflicts of interest to declare.

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Zhou, Lihong, and Felicity Z. Watts. "Nep1, a Schizosaccharomyces pombe deneddylating enzyme." Biochemical Journal 389, no. 2 (July 5, 2005): 307–14. http://dx.doi.org/10.1042/bj20041991.

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Nedd8 is a ubiquitin-like modifier that is attached to the cullin components of E3 ubiquitin ligases. More recently, p53 has also been shown to be Nedd8-modified. Nedd8 attachment occurs in a manner similar to that observed for other ubiquitin-like modifiers. In the present study, we report on the characterization of Nep1, a deneddylating enzyme in fission yeast (Schizosaccharomyces pombe). Unlike loss of ned8, deletion of the nep1 gene is not lethal, although nep1.d cells are heterogeneous in length, suggesting a defect in cell-cycle progression. Viability of nep1.d cells is dependent on a functional spindle checkpoint but not on the DNA integrity checkpoint. Deletion of a related gene (nep2), either alone or in combination with nep1.d, also has little effect on cell viability. We show that Nep1 can deneddylate the Pcu1, Pcu3 and Pcu4 cullins in vitro and that its activity is sensitive to N-ethylmaleimide, consistent with the idea that it is a member of the cysteine protease family. nep1.d cells accumulate Nedd8-modified proteins, although these do not correspond to modified forms of the cullins, suggesting that, although Nep1 can deneddylate cullins in vitro, this is not its main function in vivo. Nep1 can be co-precipitated with the signalosome subunit Csn5. Nep1 itself is present in a high-molecular-mass complex, but the presence of this complex is not dependent on the production of intact signalosomes. Our results suggest that, in vivo, Nep1 may be responsible for deneddylating proteins other than cullins.

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Wang, Chenji, Jian An, Pingzhao Zhang, Chen Xu, Kun Gao, Di Wu, Dejie Wang, Hongxiu Yu, Jun O. Liu, and Long Yu. "The Nedd4-like ubiquitin E3 ligases target angiomotin/p130 to ubiquitin-dependent degradation." Biochemical Journal 444, no. 2 (May 11, 2012): 279–89. http://dx.doi.org/10.1042/bj20111983.

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AMOT (angiomotin) is a membrane-associated protein that is expressed in ECs (endothelial cells) and controls migration, TJ (tight junction) formation, cell polarity and angiogenesis. Recent studies have revealed that AMOT and two AMOT-like proteins, AMOTL1 and AMOTL2, play critical roles in the Hippo pathway by regulating the subcellular localization of the co-activators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif). However, it has been unclear how AMOT is regulated. In the present study, we report that AMOT undergoes proteasomal degradation. We identify three members of Nedd4 (neural-precursor-cell-expressed developmentally down-regulated)-like ubiquitin E3 ligases, Nedd4, Nedd4-2 and Itch, as the ubiquitin E3 ligases for the long isoform of AMOT, AMOT/p130. We demonstrate that Nedd4, Nedd4-2 and Itch mediate poly-ubiquitination of AMOT/p130 in vivo. Overexpression of Nedd4, Nedd4-2 or Itch leads to AMOT/p130 proteasomal degradation. Knockdown of Nedd4, Nedd4-2 and Itch causes an accumulation of steady-state level of AMOT/p130. We also show that three L/P-PXY motifs of AMOT/p130 and the WW domains of Nedd4 mediate their interaction. Furthermore, Nedd4-like ubiquitin E3 ligases might compete with YAP for the binding to AMOT/p130, and subsequently targeting AMOT/p130 for ubiquitin-dependent degradation. Together, these observations reveal a novel post-translational regulatory mechanism of AMOT/p130.

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Sheedlo, Michael J., Jiazhang Qiu, Yunhao Tan, Lake N. Paul, Zhao-Qing Luo, and Chittaranjan Das. "Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination." Proceedings of the National Academy of Sciences 112, no. 49 (November 23, 2015): 15090–95. http://dx.doi.org/10.1073/pnas.1514568112.

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Manipulation of the host’s ubiquitin network is emerging as an important strategy for counteracting and repurposing the posttranslational modification machineries of the host by pathogens. Ubiquitin E3 ligases encoded by infectious agents are well known, as are a variety of viral deubiquitinases (DUBs). Bacterial DUBs have been discovered, but little is known about the structure and mechanism underlying their ubiquitin recognition. In this report, we found that members of theLegionella pneumophilaSidE effector family harbor a DUB module important for ubiquitin dynamics on the bacterial phagosome. Structural analysis of this domain alone and in complex with ubiquitin vinyl methyl ester (Ub-VME) reveals unique molecular contacts used in ubiquitin recognition. Instead of relying on the Ile44 patch of ubiquitin, as commonly used in eukaryotic counterparts, the SdeADubmodule engages Gln40 of ubiquitin. The architecture of the active-site cleft presents an open arrangement with conformational plasticity, permitting deubiquitination of three of the most abundant polyubiquitin chains, with a distinct preference for Lys63 linkages. We have shown that this preference enables efficient removal of Lys63 linkages from the phagosomal surface. Remarkably, the structure reveals by far the most parsimonious use of molecular contacts to achieve deubiquitination, with less than 1,000 Å2of accessible surface area buried upon complex formation with ubiquitin. This type of molecular recognition appears to enable dual specificity toward ubiquitin and the ubiquitin-like modifier NEDD8.

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Medvar, Barbara, Viswanathan Raghuram, Trairak Pisitkun, Abhijit Sarkar, and Mark A. Knepper. "Comprehensive database of human E3 ubiquitin ligases: application to aquaporin-2 regulation." Physiological Genomics 48, no. 7 (July 1, 2016): 502–12. http://dx.doi.org/10.1152/physiolgenomics.00031.2016.

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Aquaporin-2 (AQP2) is regulated in part via vasopressin-mediated changes in protein half-life that are in turn dependent on AQP2 ubiquitination. Here we addressed the question, “What E3 ubiquitin ligase is most likely to be responsible for AQP2 ubiquitination?” using large-scale data integration based on Bayes' rule. The first step was to bioinformatically identify all E3 ligase genes coded by the human genome. The 377 E3 ubiquitin ligases identified in the human genome, consisting predominant of HECT, RING, and U-box proteins, have been used to create a publically accessible and downloadable online database ( https://hpcwebapps.cit.nih.gov/ESBL/Database/E3-ligases/ ). We also curated a second database of E3 ligase accessory proteins that included BTB domain proteins, cullins, SOCS-box proteins, and F-box proteins. Using Bayes' theorem to integrate information from multiple large-scale proteomic and transcriptomic datasets, we ranked these 377 E3 ligases with respect to their probability of interaction with AQP2. Application of Bayes' rule identified the E3 ligases most likely to interact with AQP2 as (in order of probability): NEDD4 and NEDD4L (tied for first), AMFR, STUB1, ITCH, ZFPL1. Significantly, the two E3 ligases tied for top rank have also been studied extensively in the reductionist literature as regulatory proteins in renal tubule epithelia. The concordance of conclusions from reductionist and systems-level data provides strong motivation for further studies of the roles of NEDD4 and NEDD4L in the regulation of AQP2 protein turnover.

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Zou, Xiao, Gal Levy-Cohen, and Michael Blank. "Molecular functions of NEDD4 E3 ubiquitin ligases in cancer." Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1856, no. 1 (August 2015): 91–106. http://dx.doi.org/10.1016/j.bbcan.2015.06.005.

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KURATOMI, Go, Akiyoshi KOMURO, Kouichiro GOTO, Masahiko SHINOZAKI, Keiji MIYAZAWA, Kohei MIYAZONO, and Takeshi IMAMURA. "NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) negatively regulates TGF-β (transforming growth factor-β) signalling by inducing ubiquitin-mediated degradation of Smad2 and TGF-β type I receptor." Biochemical Journal 386, no. 3 (March 8, 2005): 461–70. http://dx.doi.org/10.1042/bj20040738.

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Inhibitory Smad, Smad7, is a potent inhibitor of TGF-β (transforming growth factor-β) superfamily signalling. By binding to activated type I receptors, it prevents the activation of R-Smads (receptor-regulated Smads). To identify new components of the Smad pathway, we performed yeast two-hybrid screening using Smad7 as bait, and identified NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) as a direct binding partner of Smad7. NEDD4-2 is structurally similar to Smurfs (Smad ubiquitin regulatory factors) 1 and 2, which were identified previously as E3 ubiquitin ligases for R-Smads and TGF-β superfamily receptors. NEDD4-2 functions like Smurfs 1 and 2 in that it associates with TGF-β type I receptor via Smad7, and induces its ubiquitin-dependent degradation. Moreover, NEDD4-2 bound to TGF-β-specific R-Smads, Smads 2 and 3, in a ligand-dependent manner, and induced degradation of Smad2, but not Smad3. However, in contrast with Smurf2, NEDD4-2 failed to induce ubiquitination of SnoN (Ski-related novel protein N), although NEDD4-2 bound to SnoN via Smad2 more strongly than Smurf2. We showed further that overexpressed NEDD4-2 prevents transcriptional activity induced by TGF-β and BMP, whereas silencing of the NEDD4-2 gene by siRNA (small interfering RNA) resulted in enhancement of the responsiveness to TGF-β superfamily cytokines. These data suggest that NEDD4-2 is a member of the Smurf-like C2-WW-HECT (WW is Trp-Trp and HECT is homologous to the E6-accessory protein) type E3 ubiquitin ligases, which negatively regulate TGF-β superfamily signalling through similar, but not identical, mechanisms to those used by Smurfs.

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Journal articles on the topic 'Nedd4 Family E3 Ligases'

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