Journal articles on the topic 'Ubiquitin ligase'
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1
Yoshida, Yukiko, Yasushi Saeki, Arisa Murakami, Junko Kawawaki, Hikaru Tsuchiya, Hidehito Yoshihara, Mayumi Shindo, and Keiji Tanaka. "A comprehensive method for detecting ubiquitinated substrates using TR-TUBE." Proceedings of the National Academy of Sciences 112, no. 15 (March 31, 2015): 4630–35. http://dx.doi.org/10.1073/pnas.1422313112.
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The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE–expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.
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Lee, Jaeseok, Youngjun Lee, Young Mee Jung, Ju Hyun Park, Hyuk Sang Yoo, and Jongmin Park. "Discovery of E3 Ligase Ligands for Target Protein Degradation." Molecules 27, no. 19 (October 2, 2022): 6515. http://dx.doi.org/10.3390/molecules27196515.
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Target protein degradation has emerged as a promising strategy for the discovery of novel therapeutics during the last decade. Proteolysis-targeting chimera (PROTAC) harnesses a cellular ubiquitin-dependent proteolysis system for the efficient degradation of a protein of interest. PROTAC consists of a target protein ligand and an E3 ligase ligand so that it enables the target protein degradation owing to the induced proximity with ubiquitin ligases. Although a great number of PROTACs has been developed so far using previously reported ligands of proteins for their degradation, E3 ligase ligands have been mostly limited to either CRBN or VHL ligands. Those PROTACs showed their limitation due to the cell type specific expression of E3 ligases and recently reported resistance toward PROTACs with CRBN ligands or VHL ligands. To overcome these hurdles, the discovery of various E3 ligase ligands has been spotlighted to improve the current PROTAC technology. This review focuses on currently reported E3 ligase ligands and their application in the development of PROTACs.
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Kilroy, Gail, Heather Kirk-Ballard, Lauren E. Carter, and Z. Elizabeth Floyd. "The Ubiquitin Ligase Siah2 Regulates PPARγ Activity in Adipocytes." Endocrinology 153, no. 3 (March 1, 2012): 1206–18. http://dx.doi.org/10.1210/en.2011-1725.
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Moderate reductions in peroxisome proliferator-activated receptor (PPAR)γ levels control insulin sensitivity as effectively as activation of PPARγ in adipocytes by the thiazolidinediones. That observation suggests that PPARγ activity can be regulated by modulating the amount of PPARγ protein in adipocytes. Activation of PPARγ in adipocytes is linked to changes in PPARγ protein levels via increased degradation of PPARγ proteins by the ubiquitin proteasome system. Identification of the ubiquitin ligase or ligases that recognize ligand bound PPARγ is an essential step in determining the physiological significance of the relationship between activation and ubiquitin-dependent degradation of PPARγ. Using an RNA interference-based screen, we identified five RING (really interesting new gene)-type ubiquitin ligases that alter PPARγ protein levels in adipocytes. Here, we demonstrate that Drosophila seven-in-absentia homolog 2 (Siah2), a mammalian homolog of Drosophila seven-in-absentia, regulates PPARγ ubiquitylation and ligand-dependent activation of PPARγ in adipocytes. We also demonstrate that Siah2 expression is up-regulated during adipogenesis and that PPARγ interacts with Siah2 during adipogenesis. In addition, Siah2 is required for adipogenesis. These data suggest that modulation of PPARγ protein levels by the ubiquitin ligase Siah2 is essential in determining the physiological effects of PPARγ activation in adipocytes.
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Fang, Nancy N., and Thibault Mayor. "Hul5 ubiquitin ligase." Prion 6, no. 3 (July 2012): 240–44. http://dx.doi.org/10.4161/pri.19929.
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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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Ibarra, Rebeca, Heather R. Borror, Bryce Hart, Richard G. Gardner, and Gary Kleiger. "The San1 Ubiquitin Ligase Avidly Recognizes Misfolded Proteins through Multiple Substrate Binding Sites." Biomolecules 11, no. 11 (November 2, 2021): 1619. http://dx.doi.org/10.3390/biom11111619.
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Cellular homeostasis depends on robust protein quality control (PQC) pathways that discern misfolded proteins from functional ones in the cell. One major branch of PQC involves the controlled degradation of misfolded proteins by the ubiquitin-proteasome system. Here ubiquitin ligases must recognize and bind to misfolded proteins with sufficient energy to form a complex and with an adequate half-life to achieve poly-ubiquitin chain formation, the signal for protein degradation, prior to its dissociation from the ligase. It is not well understood how PQC ubiquitin ligases accomplish these tasks. Employing a fully reconstituted enzyme and substrate system to perform quantitative biochemical experiments, we demonstrate that the yeast PQC ubiquitin ligase San1 contains multiple substrate binding sites along its polypeptide chain that appear to display specificity for unique misfolded proteins. The results are consistent with a model where these substrate binding sites enable San1 to bind to misfolded substrates avidly, resulting in high affinity ubiquitin ligase-substrate complexes.
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Kelsall, Ian R., Jiazhen Zhang, Axel Knebel, J. Simon C. Arthur, and Philip Cohen. "The E3 ligase HOIL-1 catalyses ester bond formation between ubiquitin and components of the Myddosome in mammalian cells." Proceedings of the National Academy of Sciences 116, no. 27 (June 17, 2019): 13293–98. http://dx.doi.org/10.1073/pnas.1905873116.
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The linear ubiquitin assembly complex (LUBAC) comprises 3 components: HOIP, HOIL-1, and Sharpin, of which HOIP and HOIL-1 are both members of the RBR subfamily of E3 ubiquitin ligases. HOIP catalyses the formation of Met1-linked ubiquitin oligomers (also called linear ubiquitin), but the function of the E3 ligase activity of HOIL-1 is unknown. Here, we report that HOIL-1 is an atypical E3 ligase that forms oxyester bonds between the C terminus of ubiquitin and serine and threonine residues in its substrates. Exploiting the sensitivity of HOIL-1–generated oxyester bonds to cleavage by hydroxylamine, and macrophages from knock-in mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant, we identify IRAK1, IRAK2, and MyD88 as physiological substrates of the HOIL-1 E3 ligase during Toll-like receptor signaling. HOIL-1 is a monoubiquitylating E3 ubiquitin ligase that initiates the de novo synthesis of polyubiquitin chains that are attached to these proteins in macrophages. HOIL-1 also catalyses its own monoubiquitylation in cells and most probably the monoubiquitylation of Sharpin, in which ubiquitin is also attached by an oxyester bond. Our study establishes that oxyester-linked ubiquitylation is used as an intracellular signaling mechanism.
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Marblestone, Jeffrey G., K. G. Suresh Kumar, Michael J. Eddins, Craig A. Leach, David E. Sterner, Michael R. Mattern, and Benjamin Nicholson. "Novel Approach for Characterizing Ubiquitin E3 Ligase Function." Journal of Biomolecular Screening 15, no. 10 (September 23, 2010): 1220–28. http://dx.doi.org/10.1177/1087057110380456.
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The ubiquitin-proteasome system is central to the regulation of numerous cellular events, and dysregulation may lead to disease pathogenesis. E3 ubiquitin ligases typically function in concert with E1 and E2 enzymes to recruit specific substrates, thereby coordinating their ubiquitylation and subsequent proteasomal degradation or cellular activity. E3 ligases have been implicated in a wide range of pathologies, and monitoring their activity in a rapid and cost-effective manner would be advantageous in drug discovery. The relative lack of high-throughput screening (HTS)–compliant E3 ligase assays has significantly hindered the discovery of E3 inhibitors. Herein, the authors describe a novel HTS-compliant E3 ligase assay platform that takes advantage of a ubiquitin binding domain’s inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase-dependent manner. This assay has been used successfully with members of both the RING and HECT families, demonstrating the platform’s broad utility for analyzing a wide range of E3 ligases. The utility of the assay platform is demonstrated by the identification of inhibitors of the E3 ligase CARP2. As the number of E3 ligases associated with various disease states increases, the ability to quantitate the activity of these enzymes in an expeditious manner becomes imperative in drug discovery.
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Toma-Fukai, Sachiko, and Toshiyuki Shimizu. "Structural Diversity of Ubiquitin E3 Ligase." Molecules 26, no. 21 (November 4, 2021): 6682. http://dx.doi.org/10.3390/molecules26216682.
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The post-translational modification of proteins regulates many biological processes. Their dysfunction relates to diseases. Ubiquitination is one of the post-translational modifications that target lysine residue and regulate many cellular processes. Three enzymes are required for achieving the ubiquitination reaction: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). E3s play a pivotal role in selecting substrates. Many structural studies have been conducted to reveal the molecular mechanism of the ubiquitination reaction. Recently, the structure of PCAF_N, a newly categorized E3 ligase, was reported. We present a review of the recent progress toward the structural understanding of E3 ligases.
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Kelley, Dior R. "E3 Ubiquitin Ligases: Key Regulators of Hormone Signaling in Plants." Molecular & Cellular Proteomics 17, no. 6 (March 7, 2018): 1047–54. http://dx.doi.org/10.1074/mcp.mr117.000476.
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Ubiquitin-mediated control of protein stability is central to most aspects of plant hormone signaling. Attachment of ubiquitin to target proteins occurs via an enzymatic cascade with the final step being catalyzed by a family of enzymes known as E3 ubiquitin ligases, which have been classified based on their protein domains and structures. Although E3 ubiquitin ligases are conserved among eukaryotes, in plants they are well-known to fulfill unique roles as central regulators of phytohormone signaling, including hormone perception and regulation of hormone biosynthesis. This review will highlight up-to-date findings that have refined well-known E3 ligase-substrate interactions and defined novel E3 ligase substrates that mediate numerous hormone signaling pathways. Additionally, examples of how particular E3 ligases may mediate hormone crosstalk will be discussed as an emerging theme. Looking forward, promising experimental approaches and methods that will provide deeper mechanistic insight into the roles of E3 ubiquitin ligases in plants will be considered.
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Kolesar, Peter, Karel Stejskal, David Potesil, Johanne M. Murray, and Jan J. Palecek. "Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase." Cells 11, no. 1 (January 4, 2022): 165. http://dx.doi.org/10.3390/cells11010165.
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Structural Maintenance of Chromosomes (SMC) complexes are important for many aspects of the chromosomal organization. Unlike cohesin and condensin, the SMC5/6 complex contains a variant RING domain carried by its Nse1 subunit. RING domains are characteristic for ubiquitin ligases, and human NSE1 has been shown to possess ubiquitin-ligase activity in vitro. However, other studies were unable to show such activity. Here, we confirm Nse1 ubiquitin-ligase activity using purified Schizosaccharomyces pombe proteins. We demonstrate that the Nse1 ligase activity is stimulated by Nse3 and Nse4. We show that Nse1 specifically utilizes Ubc13/Mms2 E2 enzyme and interacts directly with ubiquitin. We identify the Nse1 mutation (R188E) that specifically disrupts its E3 activity and demonstrate that the Nse1-dependent ubiquitination is particularly important under replication stress. Moreover, we determine Nse4 (lysine K181) as the first known SMC5/6-associated Nse1 substrate. Interestingly, abolition of Nse4 modification at K181 leads to suppression of DNA-damage sensitivity of other SMC5/6 mutants. Altogether, this study brings new evidence for Nse1 ubiquitin ligase activity, significantly advancing our understanding of this enigmatic SMC5/6 function.
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Martin-Serrano, Juan, Scott W. Eastman, Wayne Chung, and Paul D. Bieniasz. "HECT ubiquitin ligases link viral and cellular PPXY motifs to the vacuolar protein-sorting pathway." Journal of Cell Biology 168, no. 1 (December 28, 2004): 89–101. http://dx.doi.org/10.1083/jcb.200408155.
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Many enveloped viruses exploit the class E vacuolar protein-sorting (VPS) pathway to bud from cells, and use peptide motifs to recruit specific class E VPS factors. Homologous to E6AP COOH terminus (HECT) ubiquitin ligases have been implicated as cofactors for PPXY motif–dependent budding, but precisely which members of this family are responsible, and how they access the VPS pathway is unclear. Here, we show that PPXY-dependent viral budding is unusually sensitive to inhibitory fragments derived from specific HECT ubiquitin ligases, namely WWP1 and WWP2. We also show that WWP1, WWP2, or Itch ubiquitin ligase recruitment promotes PPXY-dependent virion release, and that this function requires that the HECT ubiquitin ligase domain be catalytically active. Finally, we show that several mammalian HECT ubiquitin ligases, including WWP1, WWP2, and Itch are recruited to class E compartments induced by dominant negative forms of the class E VPS ATPase, VPS4. These data indicate that specific HECT ubiquitin ligases can link PPXY motifs to the VPS pathway to induce viral budding.
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Yuan, Yuan, Xiaogang Chen, and Enying Huang. "Upregulation of Circular RNA Itchy E3 Ubiquitin Protein Ligase Inhibits Cell Proliferation and Promotes Cell Apoptosis Through Targeting MiR-197 in Prostate Cancer." Technology in Cancer Research & Treatment 18 (January 1, 2019): 153303381988686. http://dx.doi.org/10.1177/1533033819886867.
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Objective: This study aimed to investigate the effect of circular RNA itchy E3 ubiquitin protein ligase on cell proliferation and apoptosis and to explore its target micro-RNAs in prostate cancer cells. Methods: Circular RNA itchy E3 ubiquitin protein ligase expression in human prostate cancer cells and normal prostate epithelial cells was determined by real time-quantitative polymerase chain reaction assay. Circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids (circular RNA itchy E3 ubiquitin protein ligase(+) group and control overexpression plasmids group were transfected with PC-3 cells. Rescue experiment was performed by transfection of circular RNA itchy E3 ubiquitin protein ligase overexpression and micro-197 overexpression plasmids (circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids/micro RNA (+) group) into PC-3 cells. Cell Counting Kit-8 and annexin V/propidium iodide assays were conducted to evaluate cell proliferation and apoptosis, respectively. Western blot was performed to determine the expressions of apoptotic-related markers. Results: Circular RNA itchy E3 ubiquitin protein ligase expression was decreased in DU 145, 22RV1, VCaP, and PC-3 cells compared to RWPE cells. In PC-3 cells, cell proliferation rate was reduced in circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids group compared to control overexpression plasmids group at 48 hours and 72 hours. Cell apoptosis rate was elevated in circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids group compared to control overexpression plasmids group at 48 hours, and Western blot showed the similar results. Micro RNA-197 but not micro RNA-31 or micro RNA-432 was the target micro-RNA of circular RNA itchy E3 ubiquitin protein ligase. In rescue experiments, cell proliferation rate was elevated, but apoptosis rate was reduced in circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids/micro RNA (+) group compared to circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids group, indicating that circular RNA itchy E3 ubiquitin protein ligase upregulation inhibited cell proliferation but promoted apoptosis through downregulating micro RNA-197. Conclusion: Circular RNA itchy E3 ubiquitin protein ligase upregulation suppresses cell proliferation but promotes apoptosis through targeting micro RNA-197 in prostate cancer. Our study may provide a new insight for the treatment of prostate cancer.
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Rittinger, Katrin. "Ubiquitin-dependent regulation of immune and inflammatory signaling pathways." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C241. http://dx.doi.org/10.1107/s2053273314097587.
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Modification of proteins with ubiquitin is a key mechanism for the regulation of a wide range of cellular functions. The outcome of the modification is determined by the way ubiquitin molecules are linked to each other. Linear (M1-linked) ubiquitin chains play an important role in the regulation of immune and inflammatory signaling pathways and contribute to the activation of NF-κB. They are synthesized by the E3 ubiquitin ligase LUBAC (linear ubiquitin chain assembly complex) that is composed of at least three subunits named HOIL-1L, HOIP and SHARPIN. LUBAC belongs to the RBR (RING-inbetween-RING) family of E3 ligases that combine the properties of RING and HECT ligases and act as RING/HECT hybrids. Indeed, we have recently shown that linear ubiquitin chain synthesis proceeds via ubiquitin thioester intermediate formed by the HOIP subunit before subsequent transfer onto the target. I will present a combination of structural and biochemical data that provide a molecular explanation how this unusual E3 ligase complex promotes the synthesis of linear ubiquitin chains with high specificity, regardless of the E2 conjugating enzyme it works with.
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Williams, Erin R. "Lipidating a ubiquitin ligase." Science Signaling 12, no. 588 (July 2, 2019): eaay5593. http://dx.doi.org/10.1126/scisignal.aay5593.
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Jackson, Peter K., and Adam G. Eldridge. "The SCF Ubiquitin Ligase." Molecular Cell 9, no. 5 (May 2002): 923–25. http://dx.doi.org/10.1016/s1097-2765(02)00538-5.
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Vriend, Jerry, and Mark W. Nachtigal. "Ubiquitin Proteasome Pathway Transcriptome in Epithelial Ovarian Cancer." Cancers 13, no. 11 (May 28, 2021): 2659. http://dx.doi.org/10.3390/cancers13112659.
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In this article, we reviewed the transcription of genes coding for components of the ubiquitin proteasome pathway in publicly available datasets of epithelial ovarian cancer (EOC). KEGG analysis was used to identify the major pathways distinguishing EOC of low malignant potential (LMP) from invasive high-grade serous ovarian carcinomas (HGSOC), and to identify the components of the ubiquitin proteasome system that contributed to these pathways. We identified elevated transcription of several genes encoding ubiquitin conjugases associated with HGSOC. Fifty-eight genes coding for ubiquitin ligases and more than 100 genes encoding ubiquitin ligase adaptors that were differentially expressed between LMP and HGSOC were also identified. Many differentially expressed genes encoding E3 ligase adaptors were Cullin Ring Ligase (CRL) adaptors, and 64 of them belonged to the Cullin 4 DCX/DWD family of CRLs. The data suggest that CRLs play a role in HGSOC and that some of these proteins may be novel therapeutic targets. Differential expression of genes encoding deubiquitinases and proteasome subunits was also noted.
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Wimuttisuk, Wananit, and Jeffrey D. Singer. "The Cullin3 Ubiquitin Ligase Functions as a Nedd8-bound Heterodimer." Molecular Biology of the Cell 18, no. 3 (March 2007): 899–909. http://dx.doi.org/10.1091/mbc.e06-06-0542.
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Cullins are members of a family of scaffold proteins that assemble multisubunit ubiquitin ligase complexes to confer substrate specificity for the ubiquitination pathway. Cullin3 (Cul3) forms a catalytically inactive BTB-Cul3-Rbx1 (BCR) ubiquitin ligase, which becomes functional upon covalent attachment of the ubiquitin homologue neural-precursor-cell-expressed and developmentally down regulated 8 (Nedd8) near the C terminus of Cul3. Current models suggest that Nedd8 activates cullin complexes by providing a recognition site for a ubiquitin-conjugating enzyme. Based on the following evidence, we propose that Nedd8 activates the BCR ubiquitin ligase by mediating the dimerization of Cul3. First, Cul3 is found as a neddylated heterodimer bound to a BTB domain-containing protein in vivo. Second, the formation of a Cul3 heterodimer is mediated by a Nedd8 molecule, which covalently attaches itself to one Cul3 molecule and binds to the winged-helix B domain at the C terminus of the second Cul3 molecule. Third, complementation experiments revealed that coexpression of two distinct nonfunctional Cul3 mutants can rescue the ubiquitin ligase function of the BCR complex. Likewise, a substrate of the BCR complex binds heterodimeric Cul3, suggesting that the Cul3 complex is active as a dimer. These findings not only provide insight into the architecture of the active BCR complex but also suggest assembly as a regulatory mechanism for activation of all cullin-based ubiquitin ligases.
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Kühnle, Simone, Ulrike Kogel, Sandra Glockzin, Andreas Marquardt, Aaron Ciechanover, Konstantin Matentzoglu, and Martin Scheffner. "Physical and Functional Interaction of the HECT Ubiquitin-protein Ligases E6AP and HERC2." Journal of Biological Chemistry 286, no. 22 (April 14, 2011): 19410–16. http://dx.doi.org/10.1074/jbc.m110.205211.
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Deregulation of the ubiquitin-protein ligase E6AP contributes to the development of the Angelman syndrome and to cervical carcinogenesis suggesting that the activity of E6AP needs to be under tight control. However, how E6AP activity is regulated at the post-translational level under non-pathologic conditions is poorly understood. In this study, we report that the giant protein HERC2, which is like E6AP a member of the HECT family of ubiquitin-protein ligases, binds to E6AP. The interaction is mediated by the RCC1-like domain 2 of HERC2 and a region spanning amino acid residues 150–200 of E6AP. Furthermore, we provide evidence that HERC2 stimulates the ubiquitin-protein ligase activity of E6AP in vitro and within cells and that this stimulatory effect does not depend on the ubiquitin-protein ligase activity of HERC2. Thus, the data obtained indicate that HERC2 acts as a regulator of E6AP.
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Takedachi, Arato, Masafumi Saijo, and Kiyoji Tanaka. "DDB2 Complex-Mediated Ubiquitylation around DNA Damage Is Oppositely Regulated by XPC and Ku and Contributes to the Recruitment of XPA." Molecular and Cellular Biology 30, no. 11 (April 5, 2010): 2708–23. http://dx.doi.org/10.1128/mcb.01460-09.
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ABSTRACT UV-damaged-DNA-binding protein (UV-DDB) is a heterodimer comprised of DDB1 and DDB2 and integrated in a complex that includes a ubiquitin ligase component, cullin 4A, and Roc1. Here we show that the ubiquitin ligase activity of the DDB2 complex is required for efficient global genome nucleotide excision repair (GG-NER) in chromatin. Mutant DDB2 proteins derived from xeroderma pigmentosum group E patients are not able to mediate ubiquitylation around damaged sites in chromatin. We also found that CSN, a negative regulator of cullin-based ubiquitin ligases, dissociates from the DDB2 complex when the complex binds to damaged DNA and that XPC and Ku oppositely regulate the ubiquitin ligase activity, especially around damaged sites. Furthermore, the DDB2 complex-mediated ubiquitylation plays a role in recruiting XPA to damaged sites. These findings shed some light on the early stages of GG-NER.
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Seenivasan, Ramkumar, Thomas Hermanns, Tamara Blyszcz, Michael Lammers, Gerrit J. K. Praefcke, and Kay Hofmann. "Mechanism and chain specificity of RNF216/TRIAD3, the ubiquitin ligase mutated in Gordon Holmes syndrome." Human Molecular Genetics 28, no. 17 (April 24, 2019): 2862–73. http://dx.doi.org/10.1093/hmg/ddz098.
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AbstractGordon Holmes syndrome (GDHS) is an adult-onset neurodegenerative disorder characterized by ataxia and hypogonadotropic hypogonadism. GDHS is caused by mutations in the gene encoding the RING-between-RING (RBR)-type ubiquitin ligase RNF216, also known as TRIAD3. The molecular pathology of GDHS is not understood, although RNF216 has been reported to modify several substrates with K48-linked ubiquitin chains, thereby targeting them for proteasomal degradation. We identified RNF216 in a bioinformatical screen for putative SUMO-targeted ubiquitin ligases and confirmed that a cluster of predicted SUMO-interaction motifs (SIMs) indeed recognizes SUMO2 chains without targeting them for ubiquitination. Surprisingly, purified RNF216 turned out to be a highly active ubiquitin ligase that exclusively forms K63-linked ubiquitin chains, suggesting that the previously reported increase of K48-linked chains after RNF216 overexpression is an indirect effect. The linkage-determining region of RNF216 was mapped to a narrow window encompassing the last two Zn-fingers of the RBR triad, including a short C-terminal extension. Neither the SIMs nor a newly discovered ubiquitin-binding domain in the central portion of RNF216 contributes to chain specificity. Both missense mutations reported in GDHS patients completely abrogate the ubiquitin ligase activity. For the R660C mutation, ligase activity could be restored by using a chemical ubiquitin loading protocol that circumvents the requirement for ubiquitin-conjugating (E2) enzymes. This result suggests Arg-660 to be required for the ubiquitin transfer from the E2 to the catalytic cysteine. Our findings necessitate a re-evaluation of the previously assumed degradative role of RNF216 and rather argue for a non-degradative K63 ubiquitination, potentially acting on SUMOylated substrates.
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Bhaduri, Utsa, and Giuseppe Merla. "Ubiquitination, Biotech Startups, and the Future of TRIM Family Proteins: A TRIM-Endous Opportunity." Cells 10, no. 5 (April 25, 2021): 1015. http://dx.doi.org/10.3390/cells10051015.
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Ubiquitination is a post-translational modification that has pivotal roles in protein degradation and diversified cellular processes, and for more than two decades it has been a subject of interest in the biotech or biopharmaceutical industry. Tripartite motif (TRIM) family proteins are known to have proven E3 ubiquitin ligase activities and are involved in a multitude of cellular and physiological events and pathophysiological conditions ranging from cancers to rare genetic disorders. Although in recent years many kinds of E3 ubiquitin ligases have emerged as the preferred choices of big pharma and biotech startups in the context of protein degradation and disease biology, from a surface overview it appears that TRIM E3 ubiquitin ligases are not very well recognized yet in the realm of drug discovery. This article will review some of the blockbuster scientific discoveries and technological innovations from the world of ubiquitination and E3 ubiquitin ligases that have impacted the biopharma community, from biotech colossuses to startups, and will attempt to evaluate the future of TRIM family proteins in the province of E3 ubiquitin ligase-based drug discovery.
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Hong, Jeongkwan, Minho Won, and Hyunju Ro. "The Molecular and Pathophysiological Functions of Members of the LNX/PDZRN E3 Ubiquitin Ligase Family." Molecules 25, no. 24 (December 15, 2020): 5938. http://dx.doi.org/10.3390/molecules25245938.
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The ligand of Numb protein-X (LNX) family, also known as the PDZRN family, is composed of four discrete RING-type E3 ubiquitin ligases (LNX1, LNX2, LNX3, and LNX4), and LNX5 which may not act as an E3 ubiquitin ligase owing to the lack of the RING domain. As the name implies, LNX1 and LNX2 were initially studied for exerting E3 ubiquitin ligase activity on their substrate Numb protein, whose stability was negatively regulated by LNX1 and LNX2 via the ubiquitin-proteasome pathway. LNX proteins may have versatile molecular, cellular, and developmental functions, considering the fact that besides these proteins, none of the E3 ubiquitin ligases have multiple PDZ (PSD95, DLGA, ZO-1) domains, which are regarded as important protein-interacting modules. Thus far, various proteins have been isolated as LNX-interacting proteins. Evidence from studies performed over the last two decades have suggested that members of the LNX family play various pathophysiological roles primarily by modulating the function of substrate proteins involved in several different intracellular or intercellular signaling cascades. As the binding partners of RING-type E3s, a large number of substrates of LNX proteins undergo degradation through ubiquitin-proteasome system (UPS) dependent or lysosomal pathways, potentially altering key signaling pathways. In this review, we highlight recent and relevant findings on the molecular and cellular functions of the members of the LNX family and discuss the role of the erroneous regulation of these proteins in disease progression.
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Kim, Jong Hum, Seok Keun Cho, Tae Rin Oh, Moon Young Ryu, Seong Wook Yang, and Woo Taek Kim. "MPSR1 is a cytoplasmic PQC E3 ligase for eliminating emergent misfolded proteins in Arabidopsis thaliana." Proceedings of the National Academy of Sciences 114, no. 46 (October 30, 2017): E10009—E10017. http://dx.doi.org/10.1073/pnas.1713574114.
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Ubiquitin E3 ligases are crucial for eliminating misfolded proteins before they form cytotoxic aggregates that threaten cell fitness and survival. However, it remains unclear how emerging misfolded proteins in the cytoplasm can be selectively recognized and eliminated by E3 ligases in plants. We found that Misfolded Protein Sensing RING E3 ligase 1 (MPSR1) is an indispensable E3 ligase required for plant survival after protein-damaging stress. Under no stress, MPSR1 is prone to rapid degradation by the 26S proteasome, concealing its protein quality control (PQC) E3 ligase activity. Upon proteotoxic stress, MPSR1 directly senses incipient misfolded proteins and tethers ubiquitins for subsequent degradation. Furthermore, MPSR1 sustains the structural integrity of the proteasome complex at the initial stage of proteotoxic stress. Here, we suggest that the MPSR1 pathway is a constitutive mechanism for proteostasis under protein-damaging stress, as a front-line surveillance system in the cytoplasm.
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Fuseya, Yasuhiro, and Kazuhiro Iwai. "Biochemistry, Pathophysiology, and Regulation of Linear Ubiquitination: Intricate Regulation by Coordinated Functions of the Associated Ligase and Deubiquitinase." Cells 10, no. 10 (October 9, 2021): 2706. http://dx.doi.org/10.3390/cells10102706.
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The ubiquitin system modulates protein functions by decorating target proteins with ubiquitin chains in most cases. Several types of ubiquitin chains exist, and chain type determines the mode of regulation of conjugated proteins. LUBAC is a ubiquitin ligase complex that specifically generates N-terminally Met1-linked linear ubiquitin chains. Although linear ubiquitin chains are much less abundant than other types of ubiquitin chains, they play pivotal roles in cell survival, proliferation, the immune response, and elimination of bacteria by selective autophagy. Because linear ubiquitin chains regulate inflammatory responses by controlling the proinflammatory transcription factor NF-κB and programmed cell death (including apoptosis and necroptosis), abnormal generation of linear chains can result in pathogenesis. LUBAC consists of HOIP, HOIL-1L, and SHARPIN; HOIP is the catalytic center for linear ubiquitination. LUBAC is unique in that it contains two different ubiquitin ligases, HOIP and HOIL-1L, in the same ligase complex. Furthermore, LUBAC constitutively interacts with the deubiquitinating enzymes (DUBs) OTULIN and CYLD, which cleave linear ubiquitin chains generated by LUBAC. In this review, we summarize the current status of linear ubiquitination research, and we discuss the intricate regulation of LUBAC-mediated linear ubiquitination by coordinate function of the HOIP and HOIL-1L ligases and OTULIN. Furthermore, we discuss therapeutic approaches to targeting LUBAC-mediated linear ubiquitin chains.
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Bashir, Tarig, N. Valerio Dorrello, Virginia Amador, Daniele Guardavaccaro, and Michele Pagano. "Control of the SCFSkp2–Cks1 ubiquitin ligase by the APC/CCdh1 ubiquitin ligase." Nature 428, no. 6979 (March 2004): 190–93. http://dx.doi.org/10.1038/nature02330.
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Chen, An, Beixue Gao, Jingping Zhang, Tamara McEwen, Shui Q. Ye, Donna Zhang, and Deyu Fang. "The HECT-Type E3 Ubiquitin Ligase AIP2 Inhibits Activation-Induced T-Cell Death by Catalyzing EGR2 Ubiquitination." Molecular and Cellular Biology 29, no. 19 (August 3, 2009): 5348–56. http://dx.doi.org/10.1128/mcb.00407-09.
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ABSTRACT E3 ubiquitin ligases, which target specific molecules for proteolytic destruction, have emerged as key regulators of immune functions. Several E3 ubiquitin ligases, including c-Cbl, Cbl-b, GRAIL, Itch, and Nedd4, have been shown to negatively regulate T-cell activation. Here, we report that the HECT-type E3 ligase AIP2 positively regulates T-cell activation. Ectopic expression of AIP2 in mouse primary T cells enhances their proliferation and interleukin-2 production by suppressing the apoptosis of T cells. AIP2 interacts with and promotes ubiquitin-mediated degradation of EGR2, a zinc finger transcription factor that has been found to regulate Fas ligand (FasL) expression during activation-induced T-cell death. Suppression of AIP2 expression by small RNA interference upregulates EGR2, inhibits EGR2 ubiquitination and FasL expression, and enhances the apoptosis of T cells. Therefore, AIP2 regulates activation-induced T-cell death by suppressing EGR2-mediated FasL expression via the ubiquitin pathway.
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Lin, You-Sheng, Yung-Chi Chang, Ting-Yu Lai, Chih-Yuan Lee, Tsung-Hsien Chuang, and Li-Chung Hsu. "The role of novel E3 ubiquitin ligase in the regulation of TLR3 signaling pathway." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 226.26. http://dx.doi.org/10.4049/jimmunol.204.supp.226.26.
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Abstract Toll-like receptors (TLRs) are critical players in the host’s defense against infection by recognizing pathogen-associated molecular patterns (PAMPs) derived from microbes, and subsequently induce inflammatory responses, which eventually eliminate pathogens and repair damage tissues. However, excessive inflammation is detrimental to the host, and has been associated with the pathogenesis of various inflammatory and autoimmune diseases. Ubiquitination is a crucial strategy to alter protein function, expression, or cellular localization at the post-translational level, and has been shown to participate in the regulation of innate immune responses. We previously found that a novel E3 ubiquitin ligase was induced upon endosomal TLRs (TLR3/7/9) activation. In this project, we demonstrate that this E3 ubiquitin ligase negatively regulates TLR3-driven immune responses, which is dependent on its ubiquitin ligase activity. This novel E3 ubiquitin ligase controls TLR3 trafficking from the endosomes to the lysosomes for degradation. In addition, this E3 ubiquitin ligase interacts with TLR3 and promotes the ubiquitination of TLR3. We also found that lysosomal activity and intracellular pH value were altered in macrophages lacking this E3 ubiquitin ligase after poly(I:C) stimulation. Furthermore, we showed that mice with this E3 ubiquitin ligase deficiency were resistant to encephalomyocarditis virus (EMCV) infection due to enhanced type I interferon production. Our study together reveals this novel E3 ubiquitin ligase as a negative regulator of TLR3-induced inflammatory responses and proposes a novel mechanism for the termination of TLR3 signaling and immune response.
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van der Reijden, Bert A., Laurens van der Meer, Jurgen Marteijn, Theo de Witte, and Joop H. Jansen. "Triad1 Regulates Myelopoiesis through Different Ubiquitin Ligase Activities." Blood 110, no. 11 (November 16, 2007): 3292. http://dx.doi.org/10.1182/blood.v110.11.3292.3292.
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Abstract The modification of cellular proteins with poly-ubiquitin chains plays an essential role in hematopoiesis. Different types of ubiquitin chains may have opposite effects on the marked proteins. Chains linked through lysine 48 of ubiquitin are recognized by the proteasome resulting in progressive degradation of the ubiquitylated proteins. Ubiquitin chains linked through lysine 63 are not recognized by the proteasome. Instead, these chains can bind proteins that regulate signal transduction and gene transcription. Although it is known that ubiquitylation is essential for hematopoiesis and that alterations in ubiquitylation have been implicated in malignant hematopoiesis the ubiquitin ligases that catalyze protein ubiquitylation remain largely unknown. Triad1 is an ubiquitin ligase that inhibits the proliferation of myeloid progenitor cells through its ligase activity. Triad1 belongs to a unique class of ligases that harbor two RING finger protein domains. This domain specifically binds ubiquitin conjugating enzymes (Ubcs). Together with the Ubcs the ligases determine which type of ubiquitin chain is catalyzed. To understand how Triad1 regulates myelopoiesis we screened a panel of Ubcs for Triad1 interaction and found that Triad1 binds UbcH7 through its N-terminal RING domain and Ubc13 through its C-terminal RING domain. UbcH7 catalyzes the formation of ubiquitin chains linked through lysine 48 that are recognized by the proteasome. Importantly, ubiquitin chains catalyzed by Ubc13 are linked through lysine 63 and are not recognized by the proteasome. In agreement with these interactions, in vitro ubiquitylation assays using different ubiquitin mutants containing only one lysine residue showed that Triad1 can catalyze the formation of both types of ubiquitin chains. The relevance of these findings in myelopoiesis was studied by generating a panel of Triad1 deletion mutants that lack the coiled coil, DRIL or RING domains. Next, the growth inhibitory effect of these mutants was tested in clonogenic assays by retroviral transduction of U937 cells. Like in primary cells, wild type Triad1 inhibited U937 colony formation by over 60% compared to empty vector transduced cells. Three Triad1 mutants lacking the DRIL domain or either one or two of the coiled coil domains inhibited clonogenic growth at a comparable rate as wild type Triad1. In contrast, deletion of either the N- or C-terminal RING finger completely abrogated the inhibitory effect of Triad1 in clonogenic growth. Thus, loss of either the UbcH7 or Ubc13 binding domain of Triad1 affects its inhibitory function in myeloid cell proliferation. Recently, we reported that Triad1 binds the transcription factor Gfi1. Gfi1 plays an important role during many hematopoietic developmental stages and is essential for neutrophilic differentiation. Remarkably, Triad1 inhibited Gfi1 ubiquitylation and proteasomal degradation. The finding here that Triad1 can catalyze the formation of different ubiquitin chains might suggest that Triad1 may modify Gfi1 with ubiquitin in a functional way, rather than marking it for proteasomal degradation. Indeed, in vivo ubiquitylation experiments showed that Gfi1 can be modified with ubiquitin chains not linked through lysine 48. The relevance of this finding and the role of Triad1 in this process is currently studied. Together, these data indicate that the dual ubiquitin ligase activity that results in the formation of different poly-ubiquitin chains is crucial to the central role of Triad1 in myelopoiesis.
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Sung, George. "Similar but Different: RBR E3 Ligases and their Domains that are Crucial for Function." McGill Science Undergraduate Research Journal 12, no. 1 (April 9, 2017): 50–53. http://dx.doi.org/10.26443/msurj.v12i1.45.
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Background: The E3 ubiquitin ligases can be subdivided into four distinct types (RING, HECT, U-box, and RBR type) based on their domain architecture and ubiquitin transfer mechanism. Recent structures of different RBR E3 ligases have been solved showing enzymes in their autoinhibited state. The only exception is HOIP/ HOIL-1L which was recently solved in its “active” conformation. This review discusses the structural and functional characteristics of three different members of the RBR E3 ubiquitin ligase family: Parkin, HOIP/HOIL-1L, and HHARI. Methods: Searches were performed using PubMed. Search term includes “RBR E3 Ligase”, “Parkin”, “HOIP/ HOIL-1L”, “HHARI”, “UbcH7”, and “E2”. In the end, 25 journal articles were selected as the foundation of this review. The structural coordinates of Parkin, HOIP, and HHARI were accessed from the PDB (www.rcsb.org) with the PDB IDs 4ZYN, 5EDV, and 4KBL, respectively. Summary: Currently, most solved RBR E3 ligase structures are only in their inactive forms, except for HOIP/ HOIL-1L, and these inactive forms provide valuable information on how these proteins are regulated in vivo. All the RBR E3 ligases have common domains, but their structures and functions are heavily dependent on their accessory domains, which serve as regulators that orchestrate certain ubiquitin chain syntheses and play a role in the autoinhibition of RBR E3 ligases. Although these domains are structurally different, they use distinct molecular interactions to achieve the same goal. While the regulation of most RBR E3 ligases has been extensively studied, more structural studies are required to further characterize the mechanism that these enzymes use to build different ubiquitin chains. Understanding the mechanisms underlying the formation of each type of ubiquitin chain could help elucidate their functions and related pathways.
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Horn-Ghetko, Daniel, David T. Krist, J. Rajan Prabu, Kheewoong Baek, Monique P. C. Mulder, Maren Klügel, Daniel C. Scott, Huib Ovaa, Gary Kleiger, and Brenda A. Schulman. "Ubiquitin ligation to F-box protein targets by SCF–RBR E3–E3 super-assembly." Nature 590, no. 7847 (February 3, 2021): 671–76. http://dx.doi.org/10.1038/s41586-021-03197-9.
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AbstractE3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates1,2. However, rather than functioning individually, many neddylated cullin–RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family—which together account for nearly half of all ubiquitin ligases in humans—form E3–E3 super-assemblies3–7. Here, by studying CRLs in the SKP1–CUL1–F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3–E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3–E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3–E3 super-assembly may therefore underlie widespread ubiquitylation.
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Lukashchuk, Natalia, and Karen H. Vousden. "Ubiquitination and Degradation of Mutant p53." Molecular and Cellular Biology 27, no. 23 (October 1, 2007): 8284–95. http://dx.doi.org/10.1128/mcb.00050-07.
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ABSTRACT While wild-type p53 is normally a rapidly degraded protein, mutant forms of p53 are stabilized and accumulate to high levels in tumor cells. In this study, we show that mutant and wild-type p53 proteins are ubiquitinated and degraded through overlapping but distinct pathways. While Mdm2 can drive the degradation of both mutant and wild-type p53, our data suggest that the ability of Mdm2 to function as a ubiquitin ligase is less important in the degradation of mutant p53, which is heavily ubiquitinated in an Mdm2-independent manner. Our initial attempts to identify ubiquitin ligases that are responsible for the ubiquitination of mutant p53 have suggested a role for the chaperone-associated ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein), although other unidentified ubiquitin ligases also appear to contribute. The contribution of Mdm2 to the degradation of mutant p53 may reflect the ability of Mdm2 to deliver the ubiquitinated mutant p53 to the proteasome.
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Saravanan, Konda Mani, Muthu Kannan, Prabhakar Meera, Nagaraj Bharathkumar, and Thirunavukarasou Anand. "E3 ligases: a potential multi-drug target for different types of cancers and neurological disorders." Future Medicinal Chemistry 14, no. 3 (January 2022): 187–201. http://dx.doi.org/10.4155/fmc-2021-0157.
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Ubiquitylation is a posttranslational modification of proteins that is necessary for a variety of cellular processes. E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase are all involved in transferring ubiquitin to the target substrate to regulate cellular function. The objective of this review is to provide an overview of different aspects of E3 ubiquitin ligases that can lead to major biological system failure in several deadly diseases. The first part of this review covers the important characteristics of E3 ubiquitin ligases and their classification based on structural domains. Further, the authors provide some online resources that help researchers explore the data relevant to the enzyme. The following section delves into the involvement of E3 ubiquitin ligases in various diseases and biological processes, including different types of cancer and neurological disorders.
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Fang, Deyu, An Chen, and Sang-Myeong Lee. "Inhibition of activation-induced T cell death by AIP2-mediated ubiquitination of EGR2 (35.20)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 35.20. http://dx.doi.org/10.4049/jimmunol.182.supp.35.20.
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Abstract E3 ubiquitin ligases, which target specific molecules for proteolytic destruction, have emerged as key regulators of immune functions. Several E3 ubiquitin ligases, including c-Cbl, Cbl-b, GRAIL, Itch, and Nedd4, have been shown to negatively regulate T-cell activation. Here we report that the HECT-type E3 ligase, AIP2, positively regulates T-cell activation. Ectopic expression of AIP2 in mouse primary T cells enhances their proliferation and IL-2 production by suppressing apoptosis of T cells. AIP2 interacts with and promotes ubiquitin-mediated degradation of EGR2, a zinc finger transcription factor that has been found to regulate Fas ligand (FasL) expression during activation-induced T cell death. Suppression of AIP2 expression by small RNA interference upregulates EGR2 and FasL expression and enhances the apoptosis of T cells. Therefore, AIP2 regulates activation-induced T cell death by suppressing EGR2-mediated FasL expression via the ubiquitin pathway.
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Leverson, Joel D., Claudio A. P. Joazeiro, Andrew M. Page, Han-kuei Huang, Philip Hieter, and Tony Hunter. "The APC11 RING-H2 Finger Mediates E2-Dependent Ubiquitination." Molecular Biology of the Cell 11, no. 7 (July 2000): 2315–25. http://dx.doi.org/10.1091/mbc.11.7.2315.
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Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that theSaccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.
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Trempe, Jean-François, Jonathan Krett, Véronique Sauvé, Marjan Seirafi, Karl Grenier, Matthew Tang, kalle Gehring, and Edward Fon. "Catalysis of ubiquitin transfer by the RBR ubiquitin ligase parkin." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C845. http://dx.doi.org/10.1107/s2053273314091542.
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Mutations in the Parkin gene are responsible for an autosomal recessive form of Parkinson's disease. The parkin protein is a RING1-In-Between-RING2 (RBR) E3 ubiquitin ligase, which functions through a two-step mechanism involving a parkin~ubiquitin thioester intermediate [1]. However, compared to other ubiquitin ligases, parkin exhibits low basal activity and requires activation both in vitro and in cells. As parkin is neuroprotective in various models of Parkinson's disease, understanding how it catalyses ubiquitin transfer will be critical. We previously reported the crystal structure of full-length parkin [2]. The structure shows parkin in an auto-inhibited state and provides insight into how it is activated. The RING0 domain occludes the ubiquitin acceptor site Cys431 in RING2 whereas a novel Repressor Element of Parkin (REP) binds RING1 and blocks its E2-binding site. Remarkably, mutations that disrupt these inhibitory interactions activate parkin both in vitro and in cells. The structure also reveals that His433 and Glu444 form a catalytic dyad adjacent to Cys431. Here, we show that His433 catalyses the acyl transfer of ubiquitin carboxy terminus from Cys431 to a target lysine side-chain amino group. Mutation of His433 does not affect UbcH7~ubiquitin discharging or thioester intermediate formation, but prevents formation of polyubiquitin chains on parkin. However, mutation of His433 does not affect significantly parkin's mitochondrial recruitment and substrate ubiquitination, suggesting that other factors might be at play in vivo. We also investigate the catalytic role of other residues located around the Cys431, such as Trp462. The work provides insight into the mechanism of ubiquitination by RBR E3 ligases with important implications for Parkinson's disease.
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Antoniou, Nikolaos, Nefeli Lagopati, Dimitrios Ilias Balourdas, Michail Nikolaou, Alexandros Papalampros, Panagiotis V. S. Vasileiou, Vassilios Myrianthopoulos, et al. "The Role of E3, E4 Ubiquitin Ligase (UBE4B) in Human Pathologies." Cancers 12, no. 1 (December 24, 2019): 62. http://dx.doi.org/10.3390/cancers12010062.
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The genome is exposed daily to many deleterious factors. Ubiquitination is a mechanism that regulates several crucial cellular functions, allowing cells to react upon various stimuli in order to preserve their homeostasis. Ubiquitin ligases act as specific regulators and actively participate among others in the DNA damage response (DDR) network. UBE4B is a newly identified member of E3 ubiquitin ligases that appears to be overexpressed in several human neoplasms. The aim of this review is to provide insights into the role of UBE4B ubiquitin ligase in DDR and its association with p53 expression, shedding light particularly on the molecular mechanisms of carcinogenesis.
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Moore, D. J. "Parkin: a multifaceted ubiquitin ligase." Biochemical Society Transactions 34, no. 5 (October 1, 2006): 749–53. http://dx.doi.org/10.1042/bst0340749.
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Mutations in the parkin gene are a common cause of autosomal recessive early-onset parkinsonism. Parkin functions as an E3 ubiquitin ligase where it can polyubiquitinate a number of its protein substrates, thus targeting them for degradation by the 26 S proteasomal complex. Recent studies have demonstrated that alternative modes of parkin-mediated ubiquitination may serve other non-degradative regulatory roles. In addition, parkin appears to function as a multipurpose neuroprotectant in a number of toxic paradigms. Coupled with these observations, parkin may integrate other gene products associated with parkinsonism, including α-synuclein, LRRK2 (leucine-rich repeat kinase 2), DJ-1 and PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1], into a common biochemical pathway of potential relevance to disease pathogenesis. Parkin therefore represents a unique multifaceted ubiquitin ligase consistent with an important housekeeping role in maintaining the integrity or survival of dopaminergic neurons.
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Skaar, Jeffrey R., Julia K. Pagan, and Michele Pagano. "SCF ubiquitin ligase-targeted therapies." Nature Reviews Drug Discovery 13, no. 12 (November 14, 2014): 889–903. http://dx.doi.org/10.1038/nrd4432.
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Gabelli, Sandra B., Zan Chen, Hanjie Jiang, Daniel Dempsey, L. Mario Amzel, Cynthia Wolberger, Peter Devreotes, and Phil Cole. "Regulation of WWP2 ubiquitin ligase." Acta Crystallographica Section A Foundations and Advances 73, a2 (December 1, 2017): C833. http://dx.doi.org/10.1107/s2053273317087411.
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Hochstrasser, Mark. "Ubiquitin Ligation without a Ligase." Developmental Cell 13, no. 1 (July 2007): 4–6. http://dx.doi.org/10.1016/j.devcel.2007.06.003.
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Kane, Lesley A., Michael Lazarou, Adam I. Fogel, Yan Li, Koji Yamano, Shireen A. Sarraf, Soojay Banerjee, and Richard J. Youle. "PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity." Journal of Cell Biology 205, no. 2 (April 21, 2014): 143–53. http://dx.doi.org/10.1083/jcb.201402104.
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PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin’s ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.
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Bruce, M. Christine, Voula Kanelis, Fatemeh Fouladkou, Anne Debonneville, Olivier Staub, and Daniela Rotin. "Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain." Biochemical Journal 415, no. 1 (September 12, 2008): 155–63. http://dx.doi.org/10.1042/bj20071708.
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Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein–protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse–chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain–HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.
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Zhang, Ting, Yue Xu, Yanfen Liu, and Yihong Ye. "gp78 functions downstream of Hrd1 to promote degradation of misfolded proteins of the endoplasmic reticulum." Molecular Biology of the Cell 26, no. 24 (December 2015): 4438–50. http://dx.doi.org/10.1091/mbc.e15-06-0354.
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Eukaryotic cells eliminate misfolded proteins from the endoplasmic reticulum (ER) via a conserved process termed ER-associated degradation (ERAD). Central regulators of the ERAD system are membrane-bound ubiquitin ligases, which are thought to channel misfolded proteins through the ER membrane during retrotranslocation. Hrd1 and gp78 are mammalian ubiquitin ligases homologous to Hrd1p, an ubiquitin ligase essential for ERAD in Saccharomyces cerevisiae. However, the functional relevance of these proteins to Hrd1p is unclear. In this paper, we characterize the gp78-containing ubiquitin ligase complex and define its functional interplay with Hrd1 using biochemical and recently developed CRISPR-based genetic tools. Our data show that transient inactivation of the gp78 complex by short hairpin RNA–mediated gene silencing causes significant stabilization of both luminal and membrane ERAD substrates, but unlike Hrd1, which plays an essential role in retrotranslocation and ubiquitination of these ERAD substrates, knockdown of gp78 does not affect either of these processes. Instead, gp78 appears to act downstream of Hrd1 to promote ERAD via cooperation with the BAG6 chaperone complex. We conclude that the Hrd1 complex forms an essential retrotranslocation module that is evolutionarily conserved, but the mammalian ERAD system uses additional ubiquitin ligases to assist Hrd1 during retrotranslocation.
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Boutell, Chris, Seth Sadis, and Roger D. Everett. "Herpes Simplex Virus Type 1 Immediate-Early Protein ICP0 and Its Isolated RING Finger Domain Act as Ubiquitin E3 Ligases In Vitro." Journal of Virology 76, no. 2 (January 15, 2002): 841–50. http://dx.doi.org/10.1128/jvi.76.2.841-850.2002.
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ABSTRACT Proteasome-dependent degradation of ubiquitinated proteins plays a key role in many important cellular processes. Ubiquitination requires the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and frequently a substrate-specific ubiquitin protein ligase (E3). One class of E3 ubiquitin ligases has been shown to contain a common zinc-binding RING finger motif. We have previously shown that herpes simplex virus type 1 ICP0, itself a RING finger protein, induces the proteasome-dependent degradation of several cellular proteins and induces the accumulation of colocalizing conjugated ubiquitin in vivo. We now report that both full-length ICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains in vitro in the presence of E1 and the E2 enzymes UbcH5a and UbcH6. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in ICP0 activity in other assays. We conclude that ICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and to target specific cellular proteins for destruction by the 26S proteasome.
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Jones, Jessica M., Carrie Simkus, and Anamika Bhattacharyya. "KPNA1 is a putative substrate of the RAG1 ubiquitin ligase (138.11)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 138.11. http://dx.doi.org/10.4049/jimmunol.182.supp.138.11.
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Abstract The RAG1 V(D)J recombinase encompasses DNA binding/cleavage and ubiquitin ligase activities. The nuclear transport protein karyopherin alpha 1 (KPNA1) binds to RAG1 upstream of its ubiquitin ligase domain, but this interaction is not required for nuclear localization of RAG1. We found that the isolated ubiquitin ligase domain of RAG1 (amino acids 218-389) promoted ubiquitylation of purified KPNA1 in a reaction supported by the ubiquitin conjugating (E2) enzyme UbcH2/Rad6 or UbcH5a. KPNA1 is the first putative substrate identified for the RAG1 ubiquitin ligase. Ubiquitylation of KPNA1 required the lysine/arginine-rich region spanning RAG1 amino acids 218-263 upstream of the RAG1 ubiquitin ligase domain, but RAG1 was still able to undergo auto-ubiquitylation in this region even in the presence of KPNA1. RAG1 did not promote rapid ubiquitin chain extension following mono-ubiquitylation of substrate, regardless of the E2 used. Substitutions of amino acids surrounding the third, non-canonical Zn coordination site of the RAG1 RING domain abrogated functional interaction with E2 enzymes, and this was significantly correlated with reduction in the ability of full length RAG1 to support recombination of extra-chromosomal substrates. These data suggest that RAG1-dependent mono-ubiquitylation of a substrate, possibly KPNA1, is required for optimal levels of V(D)J recombination.
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Park, Hyeong Cheol, Sung Cheol Koo, Hun Kim, Wonkyun Choi, and Dae-Jin Yun. "Characterization of small ubiquitin-like modifier E3 ligase, OsSIZ1, mutant in rice." Journal of Plant Biotechnology 39, no. 4 (December 31, 2012): 235–41. http://dx.doi.org/10.5010/jpb.2012.39.4.235.
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Stanley, Bradford J., Elana S. Ehrlich, Leslie Short, Yunkai Yu, Zuoxiang Xiao, Xiao-Fang Yu, and Yong Xiong. "Structural Insight into the Human Immunodeficiency Virus Vif SOCS Box and Its Role in Human E3 Ubiquitin Ligase Assembly." Journal of Virology 82, no. 17 (June 18, 2008): 8656–63. http://dx.doi.org/10.1128/jvi.00767-08.
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ABSTRACT Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.
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Kozlov, Guennadi, Long Nguyen, Tong Lin, Gregory De Crescenzo, Morag Park, and Kalle Gehring. "Structural Basis of Ubiquitin Recognition by the Ubiquitin-associated (UBA) Domain of the Ubiquitin Ligase EDD." Journal of Biological Chemistry 282, no. 49 (September 25, 2007): 35787–95. http://dx.doi.org/10.1074/jbc.m705655200.
Full textAbstract:
EDD (or HYD) is an E3 ubiquitin ligase in the family of HECT (homologous to E6-AP C terminus) ligases. EDD contains an N-terminal ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Here, we use isothermal titration calorimetry (ITC), NMR titrations, and pull-down assays to show that the EDD UBA domain binds ubiquitin. The 1.85Å crystal structure of the complex with ubiquitin reveals the structural basis of ubiquitin recognition by UBA helices α1 and α3. The structure shows a larger number of intermolecular hydrogen bonds than observed in previous UBA/ubiquitin complexes. Two of these involve ordered water molecules. The functional importance of residues at the UBA/ubiquitin interface was confirmed using site-directed mutagenesis. Surface plasmon resonance (SPR) measurements show that the EDD UBA domain does not have a strong preference for polyubiquitin chains over monoubiquitin. This suggests that EDD binds to monoubiquitinated proteins, which is consistent with its involvement in DNA damage repair pathways.
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Spratt, Donald E., Helen Walden, and Gary S. Shaw. "RBR E3 ubiquitin ligases: new structures, new insights, new questions." Biochemical Journal 458, no. 3 (February 28, 2014): 421–37. http://dx.doi.org/10.1042/bj20140006.
Full textAbstract:
The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology.