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Sample records for 1-ring ubiquitin ligase

  1. Structure of a BMI-1-Ring1B Polycomb Group Ubiquitin Ligase Complex

    SciTech Connect

    Li,Z.; Cao, R.; Wang, M.; Myers, M.; Zhang, Y.; Xu, R.

    2006-01-01

    Polycomb group (PcG) proteins Bmi-1 and Ring1B are core subunits of the PRC1 complex which plays important roles in the regulation of Hox gene expression, X-chromosome inactivation, tumorigenesis and stem cell self-renewal. The RING finger protein Ring1B is an E3 ligase that participates in the ubiquitination of lysine 119 of histone H2A, and the binding of Bmi-1 stimulates the E3 ligase activity. We have mapped the regions of Bmi-1 and Ring1B required for efficient ubiquitin transfer and determined a 2.5 Angstroms structure of the Bmi-1-Ring1B core domain complex. The structure reveals that Ring1B 'hugs' Bmi-1 through extensive RING domain contacts and its N-terminal tail wraps around Bmi-1. The two regions of interaction have a synergistic effect on the E3 ligase activity. Our analyses suggest a model where the Bmi-1-Ring1B complex stabilizes the interaction between the E2 enzyme and the nucleosomal substrate to allow efficient ubiquitin transfer.

  2. Types of Ubiquitin Ligases.

    PubMed

    Morreale, Francesca Ester; Walden, Helen

    2016-03-24

    Ubiquitination is a post-translational modification of proteins involved in a variety of cellular processes. Ubiquitination requires the sequential action of three enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin ligases). This SnapShot highlights the main types of E3 ubiquitin ligases, which can be classified in three families depending on the presence of characteristic domains and on the mechanism of ubiquitin transfer to the substrate protein.

  3. SCF ubiquitin ligase targeted therapies

    PubMed Central

    Skaar, Jeffrey R.; Pagan, Julia K.; Pagano, Michele

    2015-01-01

    Summary The recent clinical successes of inhibitors of the proteasome for the treatment of cancer have highlighted the therapeutic potential of this protein degradation system. Proteasome inhibitors prevent the degradation of numerous proteins, so increased specificity could be achieved by inhibiting the components of the ubiquitin-proteasome system that target specific subsets of proteins for degradation. F-box proteins are the substrate-targeting subunits of SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complexes. Through the degradation of a plethora of diverse substrates, SCF ubiquitin ligases control a large number of processes at the cellular and organismal levels, and their misregulation is implicated in many pathologies. SCF ligases are characterized by a high specificity for their substrates, so they represent promising drug targets. However, the potential for therapeutic manipulation of SCF complexes remains an underdeveloped area. This review will explore and discuss potential strategies to target SCF-mediated biology to treat human diseases. PMID:25394868

  4. Origin and diversification of TRIM ubiquitin ligases.

    PubMed

    Marín, Ignacio

    2012-01-01

    Most proteins of the TRIM family (also known as RBCC family) are ubiquitin ligases that share a peculiar protein structure, characterized by including an N-terminal RING finger domain closely followed by one or two B-boxes. Additional protein domains found at their C termini have been used to classify TRIM proteins into classes. TRIMs are involved in multiple cellular processes and many of them are essential components of the innate immunity system of animal species. In humans, it has been shown that mutations in several TRIM-encoding genes lead to diverse genetic diseases and contribute to several types of cancer. They had been hitherto detected only in animals. In this work, by comprehensively analyzing the available diversity of TRIM and TRIM-like protein sequences and evaluating their evolutionary patterns, an improved classification of the TRIM family is obtained. Members of one of the TRIM subfamilies defined, called Subfamily A, turn to be present not only in animals, but also in many other eukaryotes, such as fungi, apusozoans, alveolates, excavates and plants. The rest of subfamilies are animal-specific and several of them originated only recently. Subfamily A proteins are characterized by containing a MATH domain, suggesting a potential evolutionary connection between TRIM proteins and a different type of ubiquitin ligases, known as TRAFs, which contain quite similar MATH domains. These results indicate that the TRIM family emerged much earlier than so far thought and contribute to our understanding of its origin and diversification. The structural and evolutionary links with the TRAF family of ubiquitin ligases can be experimentally explored to determine whether functional connections also exist.

  5. Dysregulation of ubiquitin ligases in cancer

    PubMed Central

    Ronai, Ze’ev A.

    2015-01-01

    Ubiquitin ligases are critical components of the ubiquitin proteasome system (UPS), which governs fundamental processes regulating normal cellular homeostasis, metabolism, and cell cycle in response to external stress signals and DNA damage. Among multiple steps of the UPS system required to regulate protein ubiquitination and stability, UBLs define specificity, as they recognize and interact with substrates in a temporally- and spatially-regulated manner. Such interactions are required for substrate modification by ubiquitin chains, which marks proteins for recognition and degradation by the proteasome, or alters their subcellular localization or assembly into functional complexes. UBLs are often deregulated in cancer, altering substrate availability or activity in a manner that can promote cellular transformation. Such deregulation can occur at the epigenetic, genomic, or post-translational levels. Alterations in UBL can be used to predict their contributions, affecting tumor suppressors or oncogenes in select tumors. Better understanding of mechanisms underlying UBL expression and activities is expected to drive the development of next generation modulators that can serve as novel therapeutic modalities. This review summarizes our current understanding of UBL deregulation in cancer and highlights novel opportunities for therapeutic interventions. PMID:26690337

  6. Signaling-mediated control of ubiquitin ligases in endocytosis.

    PubMed

    Polo, Simona

    2012-03-15

    Ubiquitin-dependent regulation of endocytosis plays an important part in the control of signal transduction, and a critical issue in the understanding of signal transduction therefore relates to regulation of ubiquitination in the endocytic pathway. We discuss here what is known of the mechanisms by which signaling controls the activity of the ubiquitin ligases that specifically recognize the targets of ubiquitination on the endocytic pathway, and suggest alternative mechanisms that deserve experimental investigation.

  7. A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function

    PubMed Central

    Liu, Yaobin; Huang, Xiangao; He, Xian; Zhou, Yanqing; Jiang, Xiaogang; Chen-Kiang, Selina; Jaffrey, Samie R.; Xu, Guoqiang

    2015-01-01

    The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 μM) and its structural analog lenalidomide (10 μM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.—Liu, Y., Huang, X., He, X., Zhou, Y., Jiang, X., Chen-Kiang, S., Jaffrey, S. R., Xu, G. A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function. PMID:26231201

  8. Regulation of Parkin E3 ubiquitin ligase activity.

    PubMed

    Walden, Helen; Martinez-Torres, R Julio

    2012-09-01

    Parkin is an E3 ubiquitin ligase mutated in autosomal recessive juvenile Parkinson's disease. In addition, it is a putative tumour suppressor, and has roles outside its enzymatic activity. It is critical for mitochondrial clearance through mitophagy, and is an essential protein in most eukaryotes. As such, it is a tightly controlled protein, regulated through an array of external interactions with multiple proteins, posttranslational modifications including phosphorylation and S-nitrosylation, and self-regulation through internal associations. In this review, we highlight some of the recent studies into Parkin regulation and discuss future challenges for gaining a full molecular understanding of the regulation of Parkin E3 ligase activity.

  9. TRIM proteins as RING finger E3 ubiquitin ligases.

    PubMed

    Ikeda, Kazuhiro; Inoue, Satoshi

    2012-01-01

    The tripartite motif(TRIM) proteins harboring the RING finger, B-box and coiled-coil (RBCC) domain motifs form a large protein family. The members of this family are involved in various biological processes, including growth, differentiation, apoptosis and transcription and also in diseases and oncogenesis. Recent studies have revealed that TRIM proteins play key roles in innate antiviral immunity. An accumulating body of evidence has demonstrated that some TRIM proteins function as E3 ubiquitin ligases in specific ubiquitin-mediated protein degradation pathways; however, the precise mechanisms underlying this function have not been fully elucidated. In this chapter, we focus on the TRIM family of proteins specially with regard to E3 ligase.

  10. Bacteria-host relationship: ubiquitin ligases as weapons of invasion

    PubMed Central

    Maculins, Timurs; Fiskin, Evgenij; Bhogaraju, Sagar; Dikic, Ivan

    2016-01-01

    Eukaryotic cells utilize the ubiquitin (Ub) system for maintaining a balanced functioning of cellular pathways. Although the Ub system is exclusive to eukaryotes, prokaryotic bacteria have developed an armory of Ub ligase enzymes that are capable of employing the Ub systems of various hosts, ranging from plant to animal cells. These enzymes have been acquired through the evolution and can be classified into three main classes, RING (really interesting new gene), HECT (homologous to the E6-AP carboxyl terminus) and NEL (novel E3 ligases). In this review we describe the roles played by different classes of bacterial Ub ligases in infection and pathogenicity. We also provide an overview of the different mechanisms by which bacteria mimic specific components of the host Ub system and outline the gaps in our current understanding of their functions. Additionally, we discuss approaches and experimental tools for validating this class of enzymes as potential novel antibacterial therapy targets. PMID:26964724

  11. SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.

    PubMed Central

    Willems, A R; Goh, T; Taylor, L; Chernushevich, I; Shevchenko, A; Tyers, M

    1999-01-01

    Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis. PMID:10582239

  12. SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.

    PubMed

    Willems, A R; Goh, T; Taylor, L; Chernushevich, I; Shevchenko, A; Tyers, M

    1999-09-29

    Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.

  13. Screening for E3-Ubiquitin ligase inhibitors: challenges and opportunities

    PubMed Central

    Landré, Vivien; Rotblat, Barak; Melino, Sonia; Bernassola, Francesca; Melino, Gerry

    2014-01-01

    The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, and its deregulation has been implicated in a wide range of human pathologies that include cancer, neurodegenerative and immunological disorders and viral infections. Targeting the UPS by small molecular regulators thus provides an opportunity for the development of therapeutics for the treatment of several diseases. The proteasome inhibitor Bortezomib was approved for treatment of hematologic malignancies by the FDA in 2003, becoming the first drug targeting the ubiquitin proteasome system in the clinic. Development of drugs targeting specific components of the ubiquitin proteasome system, however, has lagged behind, mainly due to the complexity of the ubiquitination reaction and its outcomes. However, significant advances have been made in recent years in understanding the molecular nature of the ubiquitination system and the vast variety of cellular signals that it produces. Additionally, improvement of screening methods, both in vitro and in silico, have led to the discovery of a number of compounds targeting components of the ubiquitin proteasome system, and some of these have now entered clinical trials. Here, we discuss the current state of drug discovery targeting E3 ligases and the opportunities and challenges that it provides. PMID:25237759

  14. Substrates of IAP ubiquitin ligases identified with a designed orthogonal E3 ligase, the NEDDylator

    PubMed Central

    Zhuang, Min; Guan, Shenheng; Wang, Haopeng; Burlingame, Alma L.; Wells, James A.

    2012-01-01

    SUMMARY Inhibitors of Apoptosis Proteins (IAPs) are guardian ubiquitin ligases that keep classic pro-apoptotic proteins in check. Systematic identification of additional IAP substrates is challenged by the heterogeneity and sheer number of ubiquitinated proteins (>5000). Here we report a powerful catalytic tagging tool, the NEDDylator, which fuses a NEDD8 E2 conjugating enzyme, Ubc12, to the ubiquitin ligase, XIAP or cIAP1. This permits transfer of the rare ubiquitin homolog NEDD8 to the ubiquitin E3 substrates allowing them to be efficiently purified for LC/MS/MS identification. We have identified >50 potential IAP substrates of both cytosolic and mitochondrial origin that bear hallmark N-terminal IAP binding motifs. These substrates include the recently discovered protein phosphatase, PGAM5, which we show is proteolytically processed, accumulates in cytosol during apoptosis, and sensitizes cells to death. These studies reveal mechanisms and antagonistic partners for specific IAPs, and provide a powerful technology for labeling binding partners in transient protein-protein complexes. PMID:23201124

  15. UHRF2, another E3 ubiquitin ligase for p53

    SciTech Connect

    Bai, Lu; Wang, Xiaohui; Jin, Fangmin; Yang, Yan; Qian, Guanhua; Duan, Changzhu

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer UHRF2 associates with p53 in vivo and in vitro. Black-Right-Pointing-Pointer UHRF2 interacts with p53 through its SRA/YDG domain. Black-Right-Pointing-Pointer UHRF2 ubiquitinates p53 in vivo and in vitro. -- Abstract: UHRF2, ubiquitin-like with PHD and ring finger domains 2, is a nuclear E3 ubiquitin ligase, which is involved in cell cycle and epigenetic regulation. UHRF2 interacts with multiple cell cycle proteins, including cyclins (A2, B1, D1, and E1), CDK2, and pRb; moreover, UHRF2 could ubiquitinate cyclin D1 and cyclin E1. Also, UHRF2 has been shown to be implicated in epigenetic regulation by associating with DNMTs, G9a, HDAC1, H3K9me2/3 and hemi-methylated DNA. We found that UHRF2 associates with tumor suppressor protein p53, and p53 is ubiquitinated by UHRF2 in vivo and in vitro. Given that both UHRF2 and p53 are involved in cell cycle regulation, this study may suggest a novel signaling pathway on cell proliferation.

  16. Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

    NASA Technical Reports Server (NTRS)

    Lecker, Stewart H.; Goldberg, A. L. (Principal Investigator)

    2003-01-01

    PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

  17. A novel ubiquitin ligase is deficient in Fanconi anemia.

    PubMed

    Meetei, Amom Ruhikanta; de Winter, Johan P; Medhurst, Annette L; Wallisch, Michael; Waisfisz, Quinten; van de Vrugt, Henri J; Oostra, Anneke B; Yan, Zhijiang; Ling, Chen; Bishop, Colin E; Hoatlin, Maureen E; Joenje, Hans; Wang, Weidong

    2003-10-01

    Fanconi anemia is a recessively inherited disease characterized by congenital defects, bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Fanconi anemia proteins function in a DNA damage response pathway involving breast cancer susceptibility gene products, BRCA1 and BRCA2 (refs. 1,2). A key step in this pathway is monoubiquitination of FANCD2, resulting in the redistribution of FANCD2 to nuclear foci containing BRCA1 (ref. 3). The underlying mechanism is unclear because the five Fanconi anemia proteins known to be required for this ubiquitination have no recognizable ubiquitin ligase motifs. Here we report a new component of a Fanconi anemia protein complex, called PHF9, which possesses E3 ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitination in vivo. Because PHF9 is defective in a cell line derived from an individual with Fanconi anemia, we conclude that PHF9 (also called FANCL) represents a novel Fanconi anemia complementation group (FA-L). Our data suggest that PHF9 has a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for monoubiquitination of FANCD2.

  18. A design principle underlying the paradoxical roles of E3 ubiquitin ligases

    PubMed Central

    Lee, Daewon; Kim, Minjin; Cho, Kwang-Hyun

    2014-01-01

    E3 ubiquitin ligases are important cellular components that determine the specificity of proteolysis in the ubiquitin-proteasome system. However, an increasing number of studies have indicated that E3 ubiquitin ligases also participate in transcription. Intrigued by the apparently paradoxical functions of E3 ubiquitin ligases in both proteolysis and transcriptional activation, we investigated the underlying design principles using mathematical modeling. We found that the antagonistic functions integrated in E3 ubiquitin ligases can prevent any undesirable sustained activation of downstream genes when E3 ubiquitin ligases are destabilized by unexpected perturbations. Interestingly, this design principle of the system is similar to the operational principle of a safety interlock device in engineering systems, which prevents a system from abnormal operation unless stability is guaranteed. PMID:24994517

  19. Selective multifaceted E3 ubiquitin ligases barricade extreme defense: Potential therapeutic targets for neurodegeneration and ageing.

    PubMed

    Upadhyay, Arun; Amanullah, Ayeman; Chhangani, Deepak; Mishra, Ribhav; Mishra, Amit

    2015-11-01

    Efficient and regular performance of Ubiquitin Proteasome System and Autophagy continuously eliminate deleterious accumulation of nonnative protiens. In cellular quality control system, E3 ubiquitin ligases are significant employees for defense mechanism against abnormal toxic proteins. Few findings indicate that lack of functions of E3 ubiquitin ligases can be a causative factor of neurodevelopmental disorders, neurodegeneration, cancer and ageing. However, the detailed molecular pathomechanism implying E3 ubiquitin ligases in cellular functions in multifactorial disease conditions are not well understood. This article systematically represents the unique characteristics, molecular nature, and recent developments in the knowledge of neurobiological functions of few crucial E3 ubiquitin ligases. Here, we review recent literature on the roles of E6-AP, HRD1 and ITCH E3 ubiquitin ligases in the neuro-pathobiological mechanisms, with precise focus on the processes of neurodegeneration, and thereby propose new lines of potential targets for therapeutic interventions.

  20. A design principle underlying the paradoxical roles of E3 ubiquitin ligases

    NASA Astrophysics Data System (ADS)

    Lee, Daewon; Kim, Minjin; Cho, Kwang-Hyun

    2014-07-01

    E3 ubiquitin ligases are important cellular components that determine the specificity of proteolysis in the ubiquitin-proteasome system. However, an increasing number of studies have indicated that E3 ubiquitin ligases also participate in transcription. Intrigued by the apparently paradoxical functions of E3 ubiquitin ligases in both proteolysis and transcriptional activation, we investigated the underlying design principles using mathematical modeling. We found that the antagonistic functions integrated in E3 ubiquitin ligases can prevent any undesirable sustained activation of downstream genes when E3 ubiquitin ligases are destabilized by unexpected perturbations. Interestingly, this design principle of the system is similar to the operational principle of a safety interlock device in engineering systems, which prevents a system from abnormal operation unless stability is guaranteed.

  1. A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function.

    PubMed

    Liu, Yaobin; Huang, Xiangao; He, Xian; Zhou, Yanqing; Jiang, Xiaogang; Chen-Kiang, Selina; Jaffrey, Samie R; Xu, Guoqiang

    2015-12-01

    The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 μM) and its structural analog lenalidomide (10 μM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.

  2. The Ubiquitin Ligase Hul5 Promotes Proteasomal Processivity▿

    PubMed Central

    Aviram, Sharon; Kornitzer, Daniel

    2010-01-01

    The 26S proteasome is a large cytoplasmic protease that degrades polyubiquitinated proteins to short peptides in a processive manner. The proteasome 19S regulatory subcomplex tethers the target protein via its polyubiquitin adduct and unfolds the target polypeptide, which is then threaded into the proteolytic site-containing 20S subcomplex. Hul5 is a 19S subcomplex-associated ubiquitin ligase that elongates ubiquitin chains on proteasome-bound substrates. We isolated hul5Δ as a mutation with which fusions of an unstable cyclin to stable reporter proteins accumulate as partially processed products. These products appear transiently in the wild type but are strongly stabilized in 19S ATPase mutants and in the hul5Δ mutant, supporting a role for the ATPase subunits in the unfolding of proteasome substrates before insertion into the catalytic cavity and suggesting a role for Hul5 in the processive degradation of proteins that are stalled on the proteasome. PMID:20008553

  3. Suramin inhibits cullin-RING E3 ubiquitin ligases

    PubMed Central

    Wu, Kenneth; Chong, Robert A.; Yu, Qing; Bai, Jin; Spratt, Donald E.; Ching, Kevin; Lee, Chan; Miao, Haibin; Tappin, Inger; Hurwitz, Jerard; Zheng, Ning; Shaw, Gary S.; Sun, Yi; Felsenfeld, Dan P.; Sanchez, Roberto; Zheng, Jun-nian; Pan, Zhen-Qiang

    2016-01-01

    Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3′s cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1’s conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2–E3 interface through small-molecule modulators. PMID:27001857

  4. Ubiquitination-dependent degradation of p73 by the mitochondrial E3 ubiquitin ligase Hades.

    PubMed

    Min, Bumki; Ryu, Jiwon; Chi, Seung-Wook; Yi, Gwan-Su

    2015-11-13

    p73 is a member of the p53 family of transcription factors which plays an essential role in tumor suppression. p73 is associated with the sensitivity of cancer cells to chemotherapy and the prognosis of many cancers. In this study, we showed the ubiquitination-dependent degradation of p73 by the mitochondrial E3 ubiquitin ligase Hades. First, the binding between p73 and Hades was identified by co-immunoprecipitation experiments, and it was found that the Hades RING-finger domain mediates the interaction with p73. Immunofluorescence analysis showed that p73 moves to the mitochondria and colocalizes with Hades during etoposide-induced apoptosis. By performing in vivo and in vitro ubiquitination assays, we observed that the Hades RING-finger domain promotes ubiquitination of p73. Finally, it was shown that SiRNA-mediated depletion of Hades stabilizes p73. Taken together, our results showed that Hades mediates the ubiquitination-dependent degradation of mitochondrial p73 under apoptotic conditions. These findings suggest that Hades-mediated p73 ubiquitination is a novel regulatory mechanism for the exonuclear function of p73.

  5. THE ROLE OF E3 LIGASES IN THE UBIQUITIN-DEPENDENT REGULATION OF SPERMATOGENESIS*

    PubMed Central

    Richburg, John H.; Myers, Jessica L.; Bratton, Shawn B.

    2014-01-01

    The ubiquitination of proteins is a post-translational modification that was first described as a means to target misfolded or unwanted proteins for degradation by the proteasome. It is now appreciated that the ubiquitination of proteins also serves as a mechanism to modify protein function and cellular functions such as protein trafficking, cell signaling, DNA repair, chromatin modifications, cell-cycle progression and cell death. The ubiquitination of proteins occurs through the hierarchal transfer of ubiquitin from an E1 ubiquitin-activating enzyme to an E2 ubiquitin-conjugating enzyme and finally to an E3 ubiquitin ligase that transfers the ubiquitin to its target protein. It is the final E3 ubiquitin ligase that confers the substrate specificity for ubiquitination and is the focus of this review. Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells undergo mitotic proliferation and expansion of the diploid spermatogonial population, differentiate into spermatocytes and progress through two meiotic divisions to produce haploid spermatids that proceed through a final morphogenesis to generate mature spermatozoa. The ubiquitination of proteins in the cells of the testis occurs in many of the processes required for the progression of mature spermatozoa. Since it is the E3 ubiquitin ligase that recognizes the target protein and provides the specificity and selectivity for ubiquitination, this review highlights known examples of E3 ligases in the testis and the differing roles that they play in maintaining functional spermatogenesis. PMID:24632385

  6. Essential Roles of E3 Ubiquitin Ligases in p53 Regulation

    PubMed Central

    Sane, Sanam; Rezvani, Khosrow

    2017-01-01

    The ubiquitination pathway and proteasomal degradation machinery dominantly regulate p53 tumor suppressor protein stability, localization, and functions in both normal and cancerous cells. Selective E3 ubiquitin ligases dominantly regulate protein levels and activities of p53 in a large range of physiological conditions and in response to cellular changes induced by exogenous and endogenous stresses. The regulation of p53’s functions by E3 ubiquitin ligases is a complex process that can lead to positive or negative regulation of p53 protein in a context- and cell type-dependent manner. Accessory proteins bind and modulate E3 ubiquitin ligases, adding yet another layer of regulatory control for p53 and its downstream functions. This review provides a comprehensive understanding of p53 regulation by selective E3 ubiquitin ligases and their potential to be considered as a new class of biomarkers and therapeutic targets in diverse types of cancers. PMID:28218667

  7. Building and remodelling Cullin–RING E3 ubiquitin ligases

    PubMed Central

    Lydeard, John R; Schulman, Brenda A; Harper, J Wade

    2013-01-01

    Cullin–RING E3 ubiquitin ligases (CRLs) control a plethora of biological pathways through targeted ubiquitylation of signalling proteins. These modular assemblies use substrate receptor modules to recruit specific targets. Recent efforts have focused on understanding the mechanisms that control the activity state of CRLs through dynamic alterations in CRL architecture. Central to these processes are cycles of cullin neddylation and deneddylation, as well as exchange of substrate receptor modules to re-sculpt the CRL landscape, thereby responding to the cellular requirements to turn over distinct proteins in different contexts. This review is focused on how CRLs are dynamically controlled with an emphasis on how cullin neddylation cycles are integrated with receptor exchange. PMID:24232186

  8. Antagonistic roles of ubiquitin ligase HEI10 and SUMO ligase RNF212 regulate meiotic recombination.

    PubMed

    Qiao, Huanyu; Prasada Rao, H B D; Yang, Ye; Fong, Jared H; Cloutier, Jeffrey M; Deacon, Dekker C; Nagel, Kathryn E; Swartz, Rebecca K; Strong, Edward; Holloway, J Kim; Cohen, Paula E; Schimenti, John; Ward, Jeremy; Hunter, Neil

    2014-02-01

    Crossover recombination facilitates the accurate segregation of homologous chromosomes during meiosis. In mammals, poorly characterized regulatory processes ensure that every pair of chromosomes obtains at least one crossover, even though most recombination sites yield non-crossovers. Designation of crossovers involves selective localization of the SUMO ligase RNF212 to a minority of recombination sites, where it stabilizes pertinent factors such as MutSγ (ref. 4). Here we show that the ubiquitin ligase HEI10 (also called CCNB1IP1) is essential for this crossover/non-crossover differentiation process. In HEI10-deficient mice, RNF212 localizes to most recombination sites, and dissociation of both RNF212 and MutSγ from chromosomes is blocked. Consequently, recombination is impeded, and crossing over fails. In wild-type mice, HEI10 accumulates at designated crossover sites, suggesting that it also has a late role in implementing crossing over. As with RNF212, dosage sensitivity for HEI10 indicates that it is a limiting factor for crossing over. We suggest that SUMO and ubiquitin have antagonistic roles during meiotic recombination that are balanced to effect differential stabilization of recombination factors at crossover and non-crossover sites.

  9. Role of a ribosome-associated E3 ubiquitin ligase in protein quality control.

    PubMed

    Bengtson, Mario H; Joazeiro, Claudio A P

    2010-09-23

    Messenger RNA lacking stop codons ('non-stop mRNA') can arise from errors in gene expression, and encode aberrant proteins whose accumulation could be deleterious to cellular function. In bacteria, these 'non-stop proteins' become co-translationally tagged with a peptide encoded by ssrA/tmRNA (transfer-messenger RNA), which signals their degradation by energy-dependent proteases. How eukaryotic cells eliminate non-stop proteins has remained unknown. Here we show that the Saccharomyces cerevisiae Ltn1 RING-domain-type E3 ubiquitin ligase acts in the quality control of non-stop proteins, in a process that is mechanistically distinct but conceptually analogous to that performed by ssrA: Ltn1 is predominantly associated with ribosomes, and it marks nascent non-stop proteins with ubiquitin to signal their proteasomal degradation. Ltn1-mediated ubiquitylation of non-stop proteins seems to be triggered by their stalling in ribosomes on translation through the poly(A) tail. The biological relevance of this process is underscored by the finding that loss of Ltn1 function confers sensitivity to stress caused by increased non-stop protein production. We speculate that defective protein quality control may underlie the neurodegenerative phenotype that results from mutation of the mouse Ltn1 homologue Listerin.

  10. ATLs and BTLs, plant-specific and general eukaryotic structurally-related E3 ubiquitin ligases.

    PubMed

    Guzmán, Plinio

    2014-02-01

    Major components of the ubiquitin proteasome system are the enzymes that operate on the transfer of ubiquitin to selected target substrate, known as ubiquitin ligases. The RING finger is a domain that is present in key classes of ubiquitin ligases. This domain coordinates the interaction with a suitable E2 conjugase and the transfer of ubiquitin from the E2 to protein targets. Additional domains coupled to the same polypeptide are important for modulating the function of these ubiquitin ligases. Plants contain several types of E3 ubiquitin ligases that in many cases have expanded as multigene families. Some families are specific to the plant lineage, whereas others may have a common ancestor among plants and other eukaryotic lineages. Arabidopsis Tóxicos en Levadura (ATLs) and BCA2 zinc finger ATLs (BTLs) are two families of ubiquitin ligases that share some common structural features. These are intronless genes that encode a highly related RING finger domain, and yet during evolutionary history, their mode of gene expansion and function is rather different. In each of these two families, the co-occurrence of transmembrane helices or C2/C2 (BZF finger) domains with a selected variation on the RING finger has been subjected to strong selection pressure in order to preserve their unique domain architectures during evolution.

  11. Mechanism of auxin perception by the TIR1 ubiquitin ligase.

    PubMed

    Tan, Xu; Calderon-Villalobos, Luz Irina A; Sharon, Michal; Zheng, Changxue; Robinson, Carol V; Estelle, Mark; Zheng, Ning

    2007-04-05

    Auxin is a pivotal plant hormone that controls many aspects of plant growth and development. Perceived by a small family of F-box proteins including transport inhibitor response 1 (TIR1), auxin regulates gene expression by promoting SCF ubiquitin-ligase-catalysed degradation of the Aux/IAA transcription repressors, but how the TIR1 F-box protein senses and becomes activated by auxin remains unclear. Here we present the crystal structures of the Arabidopsis TIR1-ASK1 complex, free and in complexes with three different auxin compounds and an Aux/IAA substrate peptide. These structures show that the leucine-rich repeat domain of TIR1 contains an unexpected inositol hexakisphosphate co-factor and recognizes auxin and the Aux/IAA polypeptide substrate through a single surface pocket. Anchored to the base of the TIR1 pocket, auxin binds to a partially promiscuous site, which can also accommodate various auxin analogues. Docked on top of auxin, the Aux/IAA substrate peptide occupies the rest of the TIR1 pocket and completely encloses the hormone-binding site. By filling in a hydrophobic cavity at the protein interface, auxin enhances the TIR1-substrate interactions by acting as a 'molecular glue'. Our results establish the first structural model of a plant hormone receptor.

  12. A conformational switch regulates the ubiquitin ligase HUWE1

    PubMed Central

    Sander, Bodo; Xu, Wenshan; Eilers, Martin; Popov, Nikita; Lorenz, Sonja

    2017-01-01

    The human ubiquitin ligase HUWE1 has key roles in tumorigenesis, yet it is unkown how its activity is regulated. We present the crystal structure of a C-terminal part of HUWE1, including the catalytic domain, and reveal an asymmetric auto-inhibited dimer. We show that HUWE1 dimerizes in solution and self-associates in cells, and that both occurs through the crystallographic dimer interface. We demonstrate that HUWE1 is inhibited in cells and that it can be activated by disruption of the dimer interface. We identify a conserved segment in HUWE1 that counteracts dimer formation by associating with the dimerization region intramolecularly. Our studies reveal, intriguingly, that the tumor suppressor p14ARF binds to this segment and may thus shift the conformational equilibrium of HUWE1 toward the inactive state. We propose a model, in which the activity of HUWE1 underlies conformational control in response to physiological cues—a mechanism that may be exploited for cancer therapy. DOI: http://dx.doi.org/10.7554/eLife.21036.001 PMID:28193319

  13. Signal-induced disassembly of the SCF ubiquitin ligase complex by Cdc48/p97

    PubMed Central

    Yen, James L.; Flick, Karin; Papagiannis, Christie V.; Mathur, Radhika; Tyrrell, An; Ouni, Ikram; Kaake, Robyn M.; Huang, Lan; Kaiser, Peter

    2012-01-01

    Summary A large group of E3 ubiquitin ligases is formed by the multisubunit SCF complex, whose core complex (Rbx1/Cul1-Cdc53/Skp1) binds one of many substrate recruiting F-box proteins to form an array of SCF ligases with diverse substrate specificities. It has long been thought that ubiquitylation by SCF ligases is regulated at the level of substrate binding. Here we describe an alternative mechanism of SCF regulation by active dissociation of the F-box subunit. We show that cadmium stress induces selective recruitment of the AAA+ ATPase Cdc48/p97 to catalyze dissociation of the F-box subunit from the yeast SCFMet30 ligase to block substrate ubiquitylation and trigger downstream events. Our results not only provide an additional layer of ubiquitin ligase regulation but also suggest that targeted, signal-dependent dissociation of multisubunit enzyme complexes is an important mechanism in control of enzyme function. PMID:23000173

  14. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation

    PubMed Central

    Lechtenberg, Bernhard C.; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K.; Ware, Carl F.; Mace, Peter D.; Riedl, Stefan J.

    2015-01-01

    Ubiquitination is a central process affecting all facets of cellular signaling and function1. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate2,3. The RBR family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases4–6. The RBR family includes Parkin4 and HOIP, the central catalytic factor of the linear ubiquitin chain assembly complex (LUBAC)7. While structural insights into the RBR E3 ligases Parkin and HHARI in their overall autoinhibited forms are available8–13, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely enigmatic. Here we present the first structure of the fully active HOIP-RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP-RBR adopts a conformation markedly different from that of autoinhibited RBRs. HOIP-RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centers ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, surprisingly, three distinct helix–IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~Ub conjugate as well as an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP-RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

  15. Activation of the E3 ubiquitin ligase Parkin.

    PubMed

    Caulfield, Thomas R; Fiesel, Fabienne C; Springer, Wolfdieter

    2015-04-01

    The PINK1 (phosphatase and tensin homologue-induced putative kinase 1)/Parkin-dependent mitochondrial quality control pathway mediates the clearance of damaged organelles, but appears to be disrupted in Parkinson's disease (PD) [Springer and Kahle (2011) Autophagy 7, 266-278]. Upon mitochondrial stress, PINK1 activates the E3 ubiquitin (Ub) ligase Parkin through phosphorylation of the Ub-like (UBL) domain of Parkin and of the small modifier Ub itself at a conserved residue [Sauvé and Gehring (2014) Cell Res. 24, 1025-1026]. Recently resolved partial crystal structures of Parkin showed a 'closed', auto-inhibited conformation, consistent with its notoriously weak enzymatic activity at steady state [Wauer and Komander (2013) EMBO J. 32, 2099-2112; Riley et al. (2013) Nat. Commun. 4, 1982; Trempe et al. (2013) Science 340, 1451-1455; Spratt et al. (2013) Nat. Commun. 4, 1983]. It has thus become clear that Parkin must undergo major structural rearrangements in order to unleash its catalytic functions. Recent published findings derived from X-ray structures and molecular modelling present a complete structural model of human Parkin at an all-atom resolution [Caulfield et al. (2014) PLoS Comput. Biol. 10, e1003935]. The results of the combined in silico simulations-based and experimental assay-based study indicates that PINK1-dependent Ser65 phosphorylation of Parkin is required for its activation and triggering of 'opening' conformations. Indeed, the obtained structures showed a sequential release of Parkin's intertwined domains and allowed docking of an Ub-charged E2 coenzyme, which could enable its enzymatic activity. In addition, using cell-based screening, select E2 enzymes that redundantly, cooperatively or antagonistically regulate Parkin's activation and/or enzymatic functions at different stages of the mitochondrial autophagy (mitophagy) process were identified [Fiesel et al. (2014) J. Cell Sci. 127, 3488-3504]. Other work that aims to pin-point the particular

  16. RING-type E3 ligases: Master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination

    PubMed Central

    Metzger, Meredith B.; Pruneda, Jonathan N.; Klevit, Rachel E.; Weissman, Allan M.

    2013-01-01

    RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. PMID:23747565

  17. Assembly of the Elongin A Ubiquitin Ligase Is Regulated by Genotoxic and Other Stresses*

    PubMed Central

    Weems, Juston C.; Slaughter, Brian D.; Unruh, Jay R.; Hall, Shawn M.; McLaird, Merry B.; Gilmore, Joshua M.; Washburn, Michael P.; Florens, Laurence; Yasukawa, Takashi; Aso, Teijiro; Conaway, Joan W.; Conaway, Ronald C.

    2015-01-01

    Elongin A performs dual functions in cells as a component of RNA polymerase II (Pol II) transcription elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that has been shown to target Pol II stalled at sites of DNA damage. Here we investigate the mechanism(s) governing conversion of the Elongin complex from its elongation factor to its ubiquitin ligase form. We report the discovery that assembly of the Elongin A ubiquitin ligase is a tightly regulated process. In unstressed cells, Elongin A is predominately present as part of Pol II elongation factor Elongin. Assembly of Elongin A into the ubiquitin ligase is strongly induced by genotoxic stress; by transcriptional stresses that lead to accumulation of stalled Pol II; and by other stimuli, including endoplasmic reticulum and nutrient stress and retinoic acid signaling, that activate Elongin A-dependent transcription. Taken together, our findings shed new light on mechanisms that control the Elongin A ubiquitin ligase and suggest that it may play a role in Elongin A-dependent transcription. PMID:25878247

  18. RBR E3 ubiquitin ligases: new structures, new insights, new questions

    PubMed Central

    Spratt, Donald E.; Walden, Helen; Shaw, Gary S.

    2014-01-01

    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. PMID:24576094

  19. USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase

    PubMed Central

    Harada, Kumi; Kato, Masako; Nakamura, Nobuhiro

    2016-01-01

    In the endoplasmic reticulum (ER), misfolded and unfolded proteins are eliminated by a process called ER-associated protein degradation (ERAD) in order to maintain cell homeostasis. In the ERAD pathway, several ER-localized E3 ubiquitin ligases target ERAD substrate proteins for ubiquitination and subsequent proteasomal degradation. However, little is known about how the functions of the ERAD ubiquitin ligases are regulated. Recently, USP19, an ER-anchored deubiquitinating enzyme (DUB), has been suggested to be involved in the regulation of ERAD. In this study, HRD1, an ERAD ubiquitin ligase, is shown to be a novel substrate for USP19. We demonstrate that USP19 rescues HRD1 from proteasomal degradation by deubiquitination of K48-linked ubiquitin chains. In addition, the altered expression of USP19 affects the steady-state levels of HRD1. These results suggest that USP19 regulates the stability of HRD1 and provide insight into the regulatory mechanism of the ERAD ubiquitin ligases. PMID:27827840

  20. Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4

    PubMed Central

    Xu, Yingqi; Plechanovová, Anna; Simpson, Peter; Marchant, Jan; Leidecker, Orsolya; Kraatz, Sebastian; Hay, Ronald T.; Matthews, Steve J.

    2014-01-01

    The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer. PMID:24969970

  1. Role of Ubiquitin Ligases and the Proteasome in Oncogenesis: Novel Targets for Anticancer Therapies

    PubMed Central

    Micel, Lindsey N.; Tentler, John J.; Smith, Peter G.; Eckhardt, Gail S.

    2013-01-01

    The ubiquitin proteasome system (UPS) regulates the ubiquitination, and thus degradation and turnover, of many proteins vital to cellular regulation and function. The UPS comprises a sequential series of enzymatic processes using four key enzyme families: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-carrier proteins), E3 (ubiquitin-protein ligases), and E4 (ubiquitin chain assembly factors). Because the UPS is a crucial regulator of the cell cycle, and abnormal cell-cycle control can lead to oncogenesis, aberrancies within the UPS pathway can result in a malignant cellular phenotype and thus has become an attractive target for novel anticancer agents. This article will provide an overall review of the mechanics of the UPS, describe aberrancies leading to cancer, and give an overview of current drug therapies selectively targeting the UPS. PMID:23358974

  2. Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

    SciTech Connect

    Gorelik, Maryna; Orlicky, Stephen; Sartori, Maria A.; Tang, Xiaojing; Marcon, Edyta; Kurinov, Igor; Greenblatt, Jack F.; Tyers, Mike; Moffat, Jason; Sicheri, Frank; Sidhu, Sachdev S.

    2016-03-14

    The ubiquitin proteasome components are often misregulated in numerous diseases, encouraging the search for drug targets and inhibitors. E3 ligases that specify ubiquitination targets are of particular interest. Multimeric Skp1–Cul1–F-box (SCF) E3 ligases constitute one of the largest E3 families connected to every cellular process and multiple diseases; however, their characterization as therapeutic targets is impeded by functional diversity and poor characterization of its members. Herein we describe a strategy to inhibit SCF E3 ligases using engineered ubiquitin-based binders. We identify a previously uncharacterized inhibitory site and design ubiquitin-based libraries targeting this site. Our strategy to target SCF E3 ligases with small-molecule–like agents will have broad applications for basic research and drug development relating to SCF E3 ligase function.

  3. RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

    SciTech Connect

    Sheren, Jamie E.; Kassenbrock, C. Kenneth

    2013-11-01

    Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

  4. The Ubiquitin Ligase Itch and Ubiquitination Regulate BFRF1-Mediated Nuclear Envelope Modification for Epstein-Barr Virus Maturation

    PubMed Central

    Lee, Chung-Pei; Liu, Guan-Ting; Kung, Hsiu-Ni; Liu, Po-Ting; Liao, Yen-Tzu; Chow, Lu-Ping; Chang, Ling-Shih; Chang, Yu-Hsin; Chang, Chou-Wei; Shu, Wen-Chi; Angers, Annie; Farina, Antonella; Tsai, Ching-Hwa; Bouamr, Fadila

    2016-01-01

    ABSTRACT The cellular endosomal sorting complex required for transport (ESCRT) was recently found to mediate important morphogenesis processes at the nuclear envelope (NE). We previously showed that the Epstein-Barr virus (EBV) BFRF1 protein recruits the ESCRT-associated protein Alix to modulate NE structure and promote EBV nuclear egress. Here, we uncover new cellular factors and mechanisms involved in this process. BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. BFRF1 is ubiquitinated, and elimination of possible ubiquitination by either lysine mutations or fusion of a deubiquitinase hampers NE-derived vesicle formation and virus maturation. While it interacts with multiple Nedd4-like ubiquitin ligases, BFRF1 preferentially binds Itch ligase. We show that Itch associates with Alix and BFRF1 and is required for BFRF1-induced NE vesicle formation. Our data demonstrate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE and EBV maturation, uncovering novel regulatory mechanisms of nuclear egress of viral nucleocapsids. IMPORTANCE The nuclear envelope (NE) of eukaryotic cells not only serves as a transverse scaffold for cellular processes, but also as a natural barrier for most DNA viruses that assemble their nucleocapsids in the nucleus. Previously, we showed that the cellular endosomal sorting complex required for transport (ESCRT) machinery is required for the nuclear egress of EBV. Here, we further report the molecular interplay among viral BFRF1, the ESCRT adaptor Alix, and the ubiquitin ligase Itch. We found that BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. The lysine residues and the ubiquitination of BFRF1 regulate the formation of BFRF1-induced NE-derived vesicles and EBV maturation. During the process, a ubiquitin ligase, Itch, preferably associates with BFRF1 and is required for BFRF1-induced NE vesicle formation. Therefore, our data indicate that Itch

  5. A network of ubiquitin ligases is important for the dynamics of misfolded protein aggregates in yeast.

    PubMed

    Theodoraki, Maria A; Nillegoda, Nadinath B; Saini, Jagdeep; Caplan, Avrom J

    2012-07-06

    Quality control ubiquitin ligases promote degradation of misfolded proteins by the proteasome. If the capacity of the ubiquitin/proteasome system is exceeded, then misfolded proteins accumulate in aggregates that are cleared by the autophagic system. To identify components of the ubiquitin/proteasome system that protect against aggregation, we analyzed a GFP-tagged protein kinase, Ste11ΔN(K444R)-GFP, in yeast strains deleted for 14 different ubiquitin ligases. We show that deletion of almost all of these ligases affected the proteostatic balance in untreated cells such that Ste11ΔN(K444R)-GFP aggregation was changed significantly compared with the levels found in wild type cells. By contrast, aggregation was increased significantly in only six E3 deletion strains when Ste11ΔN(K444R)-GFP folding was impaired due to inhibition of the molecular chaperone Hsp90 with geldanamycin. The increase in aggregation of Ste11ΔN(K444R)-GFP due to deletion of UBR1 and UFD4 was partially suppressed by deletion of UBR2 due to up-regulation of Rpn4, which controls proteasome activity. Deletion of UBR1 in combination with LTN1, UFD4, or DOA10 led to a marked hypersensitivity to azetidine 2-carboxylic acid, suggesting some redundancy in the networks of quality control ubiquitin ligases. Finally, we show that Ubr1 promotes clearance of protein aggregates when the autophagic system is inactivated. These results provide insight into the mechanics by which ubiquitin ligases cooperate and provide feedback regulation in the clearance of misfolded proteins.

  6. A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase.

    PubMed

    Low, Teck Yew; Peng, Mao; Magliozzi, Roberto; Mohammed, Shabaz; Guardavaccaro, Daniele; Heck, Albert J R

    2014-12-16

    Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases. Identification of substrates of ubiquitin ligases is crucial not only to unravel the molecular mechanisms by which the UPS controls protein degradation but also for drug discovery purposes because many established UPS substrates are implicated in disease. We developed a combined bioinformatics and affinity purification-mass spectrometry (AP-MS) workflow for the system-wide identification of substrates of SCF(βTrCP), a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are characterized by a multisubunit architecture typically consisting of the invariable subunits Rbx1, Cul1, and Skp1 and one of 69 F-box proteins. The F-box protein of this member of the family is βTrCP. SCF(βTrCP) binds, through the WD40 repeats of βTrCP, to the DpSGXX(X)pS diphosphorylated motif in its substrates. We recovered 27 previously reported SCF(βTrCP) substrates, of which 22 were verified by two independent statistical protocols, thereby confirming the reliability of this approach. In addition to known substrates, we identified 221 proteins that contained the DpSGXX(X)pS motif and also interacted specifically with the WD40 repeats of βTrCP. Thus, with SCF(βTrCP), as the example, we showed that integration of structural information, AP-MS, and degron motif mining constitutes an effective method to screen for substrates of ubiquitin ligases.

  7. Control of cell growth by the SCF and APC/C ubiquitin ligases

    PubMed Central

    Skaar, Jeffrey R.; Pagano, Michele

    2009-01-01

    The ubiquitin-proteasome system plays key roles in the control of cell growth. The cell cycle in particular is highly regulated by the functions of the SCF and APC/C ubiquitin ligases, and perturbation of their function can result in tumorigenesis. Although the SCF and APC/C complexes are well-established in growth control pathways, many aspects of their function remain unknown. Recent studies have shed light on the mechanism of SCF-mediated ubiquitination and new functions for the SCF complex and APC/C. Our expanding understanding of the roles of the SCF and APC/C complexes highlight the potential for targeted molecular therapies. PMID:19775879

  8. Composition, Roles, and Regulation of Cullin-Based Ubiquitin E3 Ligases

    PubMed Central

    Choi, Christina M.; Gray, William M.; Mooney, Sutton; Hellmann, Hanjo

    2014-01-01

    Due to their sessile nature, plants depend on flexible regulatory systems that allow them to adequately regulate developmental and physiological processes in context with environmental cues. The ubiquitin proteasome pathway, which targets a great number of proteins for degradation, is cellular tool that provides the necessary flexibility to accomplish this task. Ubiquitin E3 ligases provide the needed specificity to the pathway by selectively binding to particular substrates and facilitating their ubiquitylation. The largest group of E3 ligases known in plants is represented by CULLIN-REALLY INTERESTING NEW GENE (RING) E3 ligases (CRLs). In recent years, a great amount of knowledge has been generated to reveal the critical roles of these enzymes across all aspects of plant life. This review provides an overview of the different classes of CRLs in plants, their specific complex compositions, the variety of biological processes they control, and the regulatory steps that can affect their activities. PMID:25505853

  9. E3 ubiquitin ligases and abscisic acid signaling

    PubMed Central

    Liu, Hongxia

    2011-01-01

    The ubiquitin proteasome system is involved in the regulation of nearly every aspect of plant growth and development. Protein ubiquitination involves the covalent attachment of ubiquitin to target proteins through a cascade catalyzed by three enzymes known as E1, E2 and E3. E3s are of particular interest as they confer substrate specificity during ubiquitination through their diverse substrate recognition domains. Recently, a number of E3s have been identified that actively participate in abscisic acid hormone biology, including regulation of biosynthesis, de-repression or activation of abscisic acid response and degradation of signaling components. In this review, we summarize recent exciting studies of the different types of E3s that target specific mediators of abscisic acid signaling or affect the plants response to the hormone. PMID:21364320

  10. The E3 ubiquitin ligase RNF185 facilitates the cGAS-mediated innate immune response

    PubMed Central

    Lv, Zhongshi; Mao, Zhaomin; Tang, Yijun; Kong, Xiufang; Li, Senlin; Cui, Ye; Liu, Heng; Zhang, Lele; Zhang, Xiaojie; Jiang, Lindi; Zhou, Qin

    2017-01-01

    The cyclic GMP-AMP synthase (cGAS), upon cytosolic DNA stimulation, catalyzes the formation of the second messenger 2′3′-cGAMP, which then binds to stimulator of interferon genes (STING) and activates downstream signaling. It remains to be elucidated how the cGAS enzymatic activity is modulated dynamically. Here, we reported that the ER ubiquitin ligase RNF185 interacted with cGAS during HSV-1 infection. Ectopic-expression or knockdown of RNF185 respectively enhanced or impaired the IRF3-responsive gene expression. Mechanistically, RNF185 specifically catalyzed the K27-linked poly-ubiquitination of cGAS, which promoted its enzymatic activity. Additionally, Systemic Lupus Erythematosus (SLE) patients displayed elevated expression of RNF185 mRNA. Collectively, this study uncovers RNF185 as the first E3 ubiquitin ligase of cGAS, shedding light on the regulation of cGAS activity in innate immune responses. PMID:28273161

  11. Identification of TRIM22 as a RING finger E3 ubiquitin ligase

    SciTech Connect

    Duan Zhijian; Gao Bo; Xu Wei; Xiong Sidong

    2008-09-26

    TRIM22, a member of the TRIM family proteins which contain RING finger, B-box, and coiled-coil domains, has been reported as a transcriptional regulator and involved in various cellular processes. In this study, the E3 ubiquitin ligase activity, a novel property of TRIM22, was demonstrated. It was found that TRIM22 underwent self-ubiquitylation in vitro in combination with the E2 enzyme UbcH5B and the ubiquitylation was dependent on its RING finger domain. Further evidences showed that TRIM22 could also be self-ubiquitylated in vivo. Importantly, TRIM22 was conjugated with poly-ubiquitin chains and stabilized by the proteasome inhibitor in 293T cells, suggesting that TRIM22 targeted itself for proteasomal degradation through the poly-ubiquitylation. We also found that TRIM22 was located in the nucleus, indicating that TRIM22 might function as a nuclear E3 ubiquitin ligase.

  12. Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation.

    PubMed

    Riling, Christopher; Kamadurai, Hari; Kumar, Suresh; O'Leary, Claire E; Wu, Kuen-Phon; Manion, Erica E; Ying, Mingjie; Schulman, Brenda A; Oliver, Paula M

    2015-09-25

    Nedd4-family E3 ubiquitin ligases regulate an array of biologic processes. Autoinhibition maintains these catalytic ligases in an inactive state through several mechanisms. However, although some Nedd4 family members are activated by binding to Nedd4 family-interacting proteins (Ndfips), how binding activates E3 function remains unclear. Our data reveal how these two regulatory processes are linked functionally. In the absence of Ndfip1, the Nedd4 family member Itch can bind an E2 but cannot accept ubiquitin onto its catalytic cysteine. This is because Itch is autoinhibited by an intramolecular interaction between its HECT (homologous to the E6-AP carboxy terminus domain) and two central WW domains. Ndfip1 binds these WW domains to release the HECT, allowing trans-thiolation and Itch catalytic activity. This molecular switch also regulates the closely related family member WWP2. Importantly, multiple PY motifs are required for Ndfip1 to activate Itch, functionally distinguishing Ndfips from single PY-containing substrates. These data establish a novel mechanism for control of the function of a subfamily of Nedd4 E3 ligases at the level of E2-E3 trans-thiolation.

  13. Smurf1 ubiquitin ligase targets Kruppel-like factor KLF2 for ubiquitination and degradation in human lung cancer H1299 cells.

    PubMed

    Xie, Ping; Tang, Ying; Shen, Shan; Wang, Yunyan; Xing, Guichun; Yin, Yuxin; He, Fuchu; Zhang, Lingqiang

    2011-04-01

    Krüppel-like factor 2 (KLF2) has been demonstrated to be essential for normal lung development, erythroid differentiation, T-cell differentiation, migration and homing. However, the mechanisms underlying the regulation of KLF2, in particular its responsible E3 ligase is still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets KLF2 for poly-ubiquitination and proteasomal degradation specifically in lung cancer H1299 cells. The catalytic ligase activity of Smurf1 is required for it to regulate KLF2. Consequently, Smurf1 represses the transcriptional factor activity of KLF2 and regulates the expression its downstream genes such as CD62L and Wee1. This study provided the first evidence that Smurf1 functions as an E3 ligase to promote the ubiquitination and proteasomal degradation of KLF2.

  14. The pineal gland: A model for adrenergic modulation of ubiquitin ligases

    PubMed Central

    Liu, Wenjun; Reiter, Russel J.

    2017-01-01

    Introduction A recent study of the pineal gland of the rat found that the expression of more than 3000 genes showed significant day/night variations (The Hartley dataset). The investigators of this report made available a supplemental table in which they tabulated the expression of many genes that they did not discuss, including those coding for components of the ubiquitin proteasome system. Herein we identify the genes of the ubiquitin proteasome system whose expression were significantly influenced by environmental lighting in the Hartley dataset, those that were stimulated by DBcAMP in pineal glands in culture, and those that were stimulated by norepinephrine. Purpose Using the Ubiquitin and Ubiquitin-like Conjugation Database (UUCA) we identified ubiquitin ligases and conjugases, and deubiquitinases in the Hartley dataset for the purpose of determining whether expression of genes of the ubiquitin proteasome pathway were significantly influenced by day/night variations and if these variations were regulated by autonomic innervation of the pineal gland from the superior cervical ganglia. Methods In the Hartley experiments pineal glands groups of rats sacrificed during the day and groups sacrificed during the night were examined for gene expression. Additional groups of rats had their superior cervical ganglia removed surgically or surgically decentralized and the pineal glands likewise examined for gene expression. Results The genes with at least a 2-fold day/night significant difference in expression included genes for 5 ubiquitin conjugating enzymes, genes for 58 ubiquitin E3 ligases and genes for 6 deubiquitinases. A 35-fold day/night difference was noted in the expression of the gene Sik1, which codes for a protein containing both an ubiquitin binding domain (UBD) and an ubiquitin-associated (UBA) domain. Most of the significant differences in these genes were prevented by surgical removal, or disconnection, of the superior cervical ganglia, and most were

  15. Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

    PubMed Central

    Lutz, Lindy M.; Pace, Chandler R.

    2016-01-01

    ABSTRACT The rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or β-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or β-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity. IMPORTANCE The ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin

  16. Control of Amino Acid Homeostasis by a Ubiquitin Ligase-Coactivator Protein Complex.

    PubMed

    Guerra, Damian; Chapiro, Sonia M; Pratelli, Réjane; Yu, Shi; Jia, Weitao; Leary, Julie; Pilot, Guillaume; Callis, Judy

    2017-03-03

    Intercellular amino acid transport is essential for the growth of all multicellular organisms, and its dysregulation is implicated in developmental disorders. By an unknown mechanism, amino acid efflux is stimulated in plants by overexpression of a membrane-localized protein (GLUTAMINE DUMPER 1 (GDU1)) that requires a ubiquitin ligase (LOSS OF GDU 2 (LOG2). Here we further explore the physiological consequences of the interaction between these two proteins. LOG2 ubiquitin ligase activity is necessary for GDU1-dependent tolerance to exogenous amino acids, and LOG2 self-ubiquitination was markedly stimulated by the GDU1 cytosolic domain, suggesting that GDU1 functions as an adaptor or coactivator of amino acid exporter(s). However, other consequences more typical of a ligase-substrate relationship are observed: disruption of the LOG2 gene increased the in vivo half-life of GDU1, mass spectrometry confirmed that LOG2 ubiquitinates GDU1 at cytosolic lysines, and GDU1 protein levels decreased upon co-expression with active, but not enzymatically inactive LOG2. Altogether these data indicate LOG2 negatively regulates GDU1 protein accumulation by a mechanism dependent upon cytosolic GDU1 lysines. Although GDU1-lysine substituted protein exhibited diminished in vivo ubiquitination, overexpression of GDU1 lysine mutants still conferred amino acid tolerance in a LOG2-dependent manner, consistent with GDU1 being both a substrate and facilitator of LOG2 function. From these data, we offer a model in which GDU1 activates LOG2 to stimulate amino acid export, a process that could be negatively regulated by GDU1 ubiquitination and LOG2 self-ubiquitination.

  17. A Ubiquitin Ligase Complex Regulates Caspase Activation During Sperm Differentiation in Drosophila

    PubMed Central

    Arama, Eli; Bader, Maya; Rieckhof, Gabrielle E; Steller, Hermann

    2007-01-01

    In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, we screened a large collection of sterile male flies for mutants that block effector caspase activation at the onset of spermatid individualization. Here, we describe the identification and characterization of a testis-specific, Cullin-3–dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3Testis), the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC) domain of Cul3Testis that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis–like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation. PMID:17880263

  18. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide.

    PubMed

    Fischer, Eric S; Böhm, Kerstin; Lydeard, John R; Yang, Haidi; Stadler, Michael B; Cavadini, Simone; Nagel, Jane; Serluca, Fabrizio; Acker, Vincent; Lingaraju, Gondichatnahalli M; Tichkule, Ritesh B; Schebesta, Michael; Forrester, William C; Schirle, Markus; Hassiepen, Ulrich; Ottl, Johannes; Hild, Marc; Beckwith, Rohan E J; Harper, J Wade; Jenkins, Jeremy L; Thomä, Nicolas H

    2014-08-07

    In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4(CRBN). Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4(CRBN) and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4(CRBN). Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4(CRBN) while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

  19. Ubiquitin protein ligase Nedd4 binds to connexin43 by a phosphorylation-modulated process.

    PubMed

    Leykauf, Kerstin; Salek, Mojibrahman; Bomke, Jörg; Frech, Matthias; Lehmann, Wolf-Dieter; Dürst, Matthias; Alonso, Angel

    2006-09-01

    Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization.

  20. A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes.

    PubMed

    Araki, Kazuaki; Kawamura, Meiko; Suzuki, Toshiaki; Matsuda, Noriyuki; Kanbe, Daiji; Ishii, Kyoko; Ichikawa, Tomio; Kumanishi, Toshiro; Chiba, Tomoki; Tanaka, Keiji; Nawa, Hiroyuki

    2003-08-01

    Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).

  1. Ubiquitination of ERMES components by the E3 ligase Rsp5 is involved in mitophagy

    PubMed Central

    Belgareh-Touzé, Naïma; Cavellini, Laetitia; Cohen, Mickael M.

    2017-01-01

    ABSTRACT Mitochondria are dynamic organelles that undergo permanent fission and fusion events. These processes play an essential role in maintaining normal cellular function. In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum-mitochondrial encounter structure (ERMES) is a marker of sites of mitochondrial division, but it is also involved in a plethora of other mitochondrial functions. However, it remains unclear how these different functions are regulated. We show here that Mdm34 and Mdm12, 2 components of ERMES, are ubiquitinated by the E3 ligase Rsp5. This ubiquitination is not involved in mitochondrial dynamics or in the distribution and turnover of ERMES. Nevertheless, the ubiquitination of Mdm34 and Mdm12 was required for efficient mitophagy. We thus report here the first identification of ubiquitinated substrates participating in yeast mitophagy. PMID:27846375

  2. Senescence-inducing stress promotes proteolysis of phosphoglycerate mutase via ubiquitin ligase Mdm2

    PubMed Central

    Mikawa, Takumi; Maruyama, Takeshi; Okamoto, Koji; Nakagama, Hitoshi; Lleonart, Matilde E.; Tsusaka, Takeshi; Hori, Kousuke; Murakami, Itsuo; Izumi, Taisuke; Takaori-Kondo, Akifumi; Yokode, Masayuki; Peters, Gordon; Beach, David

    2014-01-01

    Despite the well-documented clinical significance of the Warburg effect, it remains unclear how the aggressive glycolytic rates of tumor cells might contribute to other hallmarks of cancer, such as bypass of senescence. Here, we report that, during oncogene- or DNA damage–induced senescence, Pak1-mediated phosphorylation of phosphoglycerate mutase (PGAM) predisposes the glycolytic enzyme to ubiquitin-mediated degradation. We identify Mdm2 as a direct binding partner and ubiquitin ligase for PGAM in cultured cells and in vitro. Mutations in PGAM and Mdm2 that abrogate ubiquitination of PGAM restored the proliferative potential of primary cells under stress conditions and promoted neoplastic transformation. We propose that Mdm2, a downstream effector of p53, attenuates the Warburg effect via ubiquitination and degradation of PGAM. PMID:24567357

  3. Senescence-inducing stress promotes proteolysis of phosphoglycerate mutase via ubiquitin ligase Mdm2.

    PubMed

    Mikawa, Takumi; Maruyama, Takeshi; Okamoto, Koji; Nakagama, Hitoshi; Lleonart, Matilde E; Tsusaka, Takeshi; Hori, Kousuke; Murakami, Itsuo; Izumi, Taisuke; Takaori-Kondo, Akifumi; Yokode, Masayuki; Peters, Gordon; Beach, David; Kondoh, Hiroshi

    2014-03-03

    Despite the well-documented clinical significance of the Warburg effect, it remains unclear how the aggressive glycolytic rates of tumor cells might contribute to other hallmarks of cancer, such as bypass of senescence. Here, we report that, during oncogene- or DNA damage-induced senescence, Pak1-mediated phosphorylation of phosphoglycerate mutase (PGAM) predisposes the glycolytic enzyme to ubiquitin-mediated degradation. We identify Mdm2 as a direct binding partner and ubiquitin ligase for PGAM in cultured cells and in vitro. Mutations in PGAM and Mdm2 that abrogate ubiquitination of PGAM restored the proliferative potential of primary cells under stress conditions and promoted neoplastic transformation. We propose that Mdm2, a downstream effector of p53, attenuates the Warburg effect via ubiquitination and degradation of PGAM.

  4. Ubiquitin Ligase Substrate Identification through Quantitative Proteomics at Both the Protein and Peptide Levels

    PubMed Central

    Lee, Kimberly A.; Hammerle, Lisa P.; Andrews, Paul S.; Stokes, Matthew P.; Mustelin, Tomas; Silva, Jeffrey C.; Black, Roy A.; Doedens, John R.

    2011-01-01

    Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His6-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology. PMID:21987572

  5. Parkin is a component of an SCF-like ubiquitin ligase complex and protects postmitotic neurons from kainate excitotoxicity.

    PubMed

    Staropoli, John F; McDermott, Caroline; Martinat, Cécile; Schulman, Brenda; Demireva, Elena; Abeliovich, Asa

    2003-03-06

    Mutations in parkin, which encodes a RING domain protein associated with ubiquitin ligase activity, lead to autosomal recessive Parkinson's disease characterized by midbrain dopamine neuron loss. Here we show that parkin functions in a multiprotein ubiquitin ligase complex that includes the F-box/WD repeat protein hSel-10 and Cullin-1. HSel-10 serves to target the parkin ubiquitin ligase activity to cyclin E, an hSel-10-interacting protein previously implicated in the regulation of neuronal apoptosis. Consistent with the notion that cyclin E is a substrate of the parkin ubiquitin ligase complex, parkin deficiency potentiates the accumulation of cyclin E in cultured postmitotic neurons exposed to the glutamatergic excitotoxin kainate and promotes their apoptosis. Furthermore, parkin overexpression attenuates the accumulation of cyclin E in toxin-treated primary neurons, including midbrain dopamine neurons, and protects them from apoptosis.

  6. PEX2 is the E3 ubiquitin ligase required for pexophagy during starvation

    PubMed Central

    Sargent, Graeme; van Zutphen, Tim; Shatseva, Tatiana; Zhang, Ling; Di Giovanni, Valeria

    2016-01-01

    Peroxisomes are metabolic organelles necessary for anabolic and catabolic lipid reactions whose numbers are highly dynamic based on the metabolic need of the cells. One mechanism to regulate peroxisome numbers is through an autophagic process called pexophagy. In mammalian cells, ubiquitination of peroxisomal membrane proteins signals pexophagy; however, the E3 ligase responsible for mediating ubiquitination is not known. Here, we report that the peroxisomal E3 ubiquitin ligase peroxin 2 (PEX2) is the causative agent for mammalian pexophagy. Expression of PEX2 leads to gross ubiquitination of peroxisomes and degradation of peroxisomes in an NBR1-dependent autophagic process. We identify PEX5 and PMP70 as substrates of PEX2 that are ubiquitinated during amino acid starvation. We also find that PEX2 expression is up-regulated during both amino acid starvation and rapamycin treatment, suggesting that the mTORC1 pathway regulates pexophagy by regulating PEX2 expression levels. Finally, we validate our findings in vivo using an animal model. PMID:27597759

  7. Ubiquitin-Activated Interaction Traps (UBAITs) identify E3 ligase binding partners.

    PubMed

    O'Connor, Hazel F; Lyon, Nancy; Leung, Justin W; Agarwal, Poonam; Swaim, Caleb D; Miller, Kyle M; Huibregtse, Jon M

    2015-12-01

    We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ubiquitin-Activated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ--a histone protein involved in DNA repair--as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.

  8. Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase.

    PubMed Central

    Lahav-Baratz, S; Sudakin, V; Ruderman, J V; Hershko, A

    1995-01-01

    Cyclin B/cdc2 is responsible both for driving cells into mitosis and for activating the ubiquitin-dependent degradation of mitotic cyclins near the end of mitosis, an event required for the completion of mitosis and entry into interphase of the next cell cycle. Previous work with cell-free extracts of rapidly dividing clam embryos has identified two specific components required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase that purifies as part of a approximately 1500-kDa complex, termed the cyclosome, and which is active only near the end of mitosis. Here, we have separated the cyclosome from its ultimate upstream activator, cdc2. The mitotic, active form of the cyclosome can be inactivated by incubation with a partially purified, endogenous okadaic acid-sensitive phosphatase; addition of cdc2 restores activity to the cyclosome after a lag that reproduces that seen previously in intact cells and in crude extracts. These results demonstrate that activity of cyclin-ubiquitin ligase is controlled by reversible phosphorylation of the cyclosome complex. Images Fig. 3 PMID:7568122

  9. Deficiency for the ubiquitin ligase UBE3B in a blepharophimosis-ptosis-intellectual-disability syndrome.

    PubMed

    Basel-Vanagaite, Lina; Dallapiccola, Bruno; Ramirez-Solis, Ramiro; Segref, Alexandra; Thiele, Holger; Edwards, Andrew; Arends, Mark J; Miró, Xavier; White, Jacqueline K; Désir, Julie; Abramowicz, Marc; Dentici, Maria Lisa; Lepri, Francesca; Hofmann, Kay; Har-Zahav, Adi; Ryder, Edward; Karp, Natasha A; Estabel, Jeanne; Gerdin, Anna-Karin B; Podrini, Christine; Ingham, Neil J; Altmüller, Janine; Nürnberg, Gudrun; Frommolt, Peter; Abdelhak, Sonia; Pasmanik-Chor, Metsada; Konen, Osnat; Kelley, Richard I; Shohat, Mordechai; Nürnberg, Peter; Flint, Jonathan; Steel, Karen P; Hoppe, Thorsten; Kubisch, Christian; Adams, David J; Borck, Guntram

    2012-12-07

    Ubiquitination plays a crucial role in neurodevelopment as exemplified by Angelman syndrome, which is caused by genetic alterations of the ubiquitin ligase-encoding UBE3A gene. Although the function of UBE3A has been widely studied, little is known about its paralog UBE3B. By using exome and capillary sequencing, we here identify biallelic UBE3B mutations in four patients from three unrelated families presenting an autosomal-recessive blepharophimosis-ptosis-intellectual-disability syndrome characterized by developmental delay, growth retardation with a small head circumference, facial dysmorphisms, and low cholesterol levels. UBE3B encodes an uncharacterized E3 ubiquitin ligase. The identified UBE3B variants include one frameshift and two splice-site mutations as well as a missense substitution affecting the highly conserved HECT domain. Disruption of mouse Ube3b leads to reduced viability and recapitulates key aspects of the human disorder, such as reduced weight and brain size and a downregulation of cholesterol synthesis. We establish that the probable Caenorhabditis elegans ortholog of UBE3B, oxi-1, functions in the ubiquitin/proteasome system in vivo and is especially required under oxidative stress conditions. Our data reveal the pleiotropic effects of UBE3B deficiency and reinforce the physiological importance of ubiquitination in neuronal development and function in mammals.

  10. Viral modulators of cullin RING ubiquitin ligases: culling the host defense.

    PubMed

    Barry, Michele; Früh, Klaus

    2006-05-16

    Cullin RING ubiquitin ligases (CRULs) are found in all eukaryotes and play an essential role in targeting proteins for ubiquitin-mediated destruction, thus regulating a plethora of cellular processes. Viruses manipulate CRULs by redirecting this destruction machinery to eliminate unwanted host cell proteins, thus allowing viruses to slip past host immune barriers. Depending on the host organism, virus-modified CRULs can perform an amazing range of tasks, including the elimination of crucial signal transduction molecules in the human interferon pathway and suppression of virus-induced gene silencing in plants. This Perspective summarizes recent advances in our understanding of how viral proteins manipulate the function of CRULs.

  11. TRIMmunity: The roles of the TRIM E3-ubiquitin ligase family in innate antiviral immunity

    PubMed Central

    Rajsbaum, Ricardo; García-Sastre, Adolfo; Versteeg, Gijs A.

    2014-01-01

    Tripartite motif (TRIM) proteins have been implicated in multiple cellular functions, including antiviral activity. Research efforts so far indicate that the antiviral activity of TRIMs relies, for the most part, on their function as E3-ubiquitin ligases. A substantial number of the TRIM-family members have been demonstrated to mediate innate immune cell signal transduction and subsequent cytokine induction. In addition, a subset of TRIMs has been shown to restrict viral replication by directly targeting viral proteins. Although the body of work on the cellular roles of TRIM E3 ubiquitin ligases has rapidly grown over the last years, many aspects of their molecular workings and multi-functionality remain unclear. The antiviral function of many TRIMs seems to be conferred by specific isoforms, sub-cellular localization, and in cell-type specific contexts. Here we review recent findings on TRIM antiviral functions, current limitations and an outlook for future research. PMID:24333484

  12. Neuromuscular regulation in zebrafish by a large AAA+ ATPase/ubiquitin ligase, mysterin/RNF213

    PubMed Central

    Kotani, Yuri; Morito, Daisuke; Yamazaki, Satoru; Ogino, Kazutoyo; Kawakami, Koichi; Takashima, Seiji; Hirata, Hiromi; Nagata, Kazuhiro

    2015-01-01

    Mysterin (also known as RNF213) is a huge intracellular protein with two AAA+ ATPase modules and a RING finger ubiquitin ligase domain. Mysterin was originally isolated as a significant risk factor for the cryptogenic cerebrovascular disorder moyamoya disease, and was found to be involved in physiological angiogenesis in zebrafish. However, the function and the physiological significance of mysterin in other than blood vessels remain largely unknown, although mysterin is ubiquitously expressed in animal tissues. In this study, we performed antisense-mediated suppression of a mysterin orthologue in zebrafish larvae and revealed that mysterin-deficient larvae showed significant reduction in fast myofibrils and immature projection of primary motoneurons, leading to severe motor deficits. Fast muscle-specific restoration of mysterin expression cancelled these phenotypes, and interestingly both AAA+ ATPase and ubiquitin ligase activities of mysterin were indispensable for proper fast muscle formation, demonstrating an essential role of mysterin and its enzymatic activities in the neuromuscular regulation in zebrafish. PMID:26530008

  13. E3 ubiquitin ligase Hades negatively regulates the exonuclear function of p53

    PubMed Central

    Jung, J H; Bae, S; Lee, J Y; Woo, S R; Cha, H J; Yoon, Y; Suh, K-S; Lee, S-J; Park, I-C; Jin, Y-W; Lee, K-H; An, S; Lee, J H

    2011-01-01

    Following DNA damage, p53 translocates to the cytoplasm and mitochondria, where it triggers transcription-independent apoptosis by binding to Bcl-2 family proteins. However, little is known about how this exonuclear function of p53 is regulated. Here, we identify and characterize a p53-interacting protein called Hades, an E3 ligase that interacts with p53 in the mitochondria. Hades reduces p53 stability via a mechanism that requires its RING-finger domain with ubiquitin ligase activity. Hades polyubiquitinates p53 in vitro independent of Mdm2 and targets a critical lysine residue in p53 (lysine 24) distinct from those targeted by Mdm2. Hades inhibits a p53-dependent mitochondrial cell death pathway by inhibiting p53 and Bcl-2 interactions. These findings show that Hades-mediated p53 ubiquitination is a novel mechanism for negatively regulating the exonuclear function of p53. PMID:21597459

  14. E3 ubiquitin ligase Hades negatively regulates the exonuclear function of p53.

    PubMed

    Jung, J H; Bae, S; Lee, J Y; Woo, S R; Cha, H J; Yoon, Y; Suh, K-S; Lee, S-J; Park, I-C; Jin, Y-W; Lee, K-H; An, S; Lee, J H

    2011-12-01

    Following DNA damage, p53 translocates to the cytoplasm and mitochondria, where it triggers transcription-independent apoptosis by binding to Bcl-2 family proteins. However, little is known about how this exonuclear function of p53 is regulated. Here, we identify and characterize a p53-interacting protein called Hades, an E3 ligase that interacts with p53 in the mitochondria. Hades reduces p53 stability via a mechanism that requires its RING-finger domain with ubiquitin ligase activity. Hades polyubiquitinates p53 in vitro independent of Mdm2 and targets a critical lysine residue in p53 (lysine 24) distinct from those targeted by Mdm2. Hades inhibits a p53-dependent mitochondrial cell death pathway by inhibiting p53 and Bcl-2 interactions. These findings show that Hades-mediated p53 ubiquitination is a novel mechanism for negatively regulating the exonuclear function of p53.

  15. TRIMmunity: the roles of the TRIM E3-ubiquitin ligase family in innate antiviral immunity.

    PubMed

    Rajsbaum, Ricardo; García-Sastre, Adolfo; Versteeg, Gijs A

    2014-03-20

    Tripartite motif (TRIM) proteins have been implicated in multiple cellular functions, including antiviral activity. Research efforts so far indicate that the antiviral activity of TRIMs relies, for the most part, on their function as E3-ubiquitin ligases. A substantial number of the TRIM family members have been demonstrated to mediate innate immune cell signal transduction and subsequent cytokine induction. In addition, a subset of TRIMs has been shown to restrict viral replication by directly targeting viral proteins. Although the body of work on the cellular roles of TRIM E3-ubiquitin ligases has rapidly grown over the last years, many aspects of their molecular workings and multi-functionality remain unclear. The antiviral function of many TRIMs seems to be conferred by specific isoforms, by sub-cellular localization and in cell-type-specific contexts. Here we review recent findings on TRIM antiviral functions, current limitations and an outlook for future research.

  16. The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy.

    PubMed

    Sun, Aiqin; Wei, Jing; Childress, Chandra; Shaw Iv, John H; Peng, Ke; Shao, Genbao; Yang, Wannian; Lin, Qiong

    2017-01-13

    The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.

  17. Substrate Trapping Proteomics Reveals Targets of the βTrCP2/FBXW11 Ubiquitin Ligase

    PubMed Central

    Kim, Tai Young; Siesser, Priscila F.; Rossman, Kent L.; Goldfarb, Dennis; Mackinnon, Kathryn; Yan, Feng; Yi, XianHua; MacCoss, Michael J.; Moon, Randall T.; Der, Channing J.

    2014-01-01

    Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study βTrCP2/FBXW11, a substrate adaptor for the SKP1–CUL1–F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to “trap” ubiquitylated substrates on the SCFFBXW11 E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCFFBXW11 bound, polyubiquitylated, and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and, consequently, multinucleation. Surprisingly, this occurred independently of the guanine nucleotide exchange factor (GEF) catalytic activity and of the presence of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this report supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062. PMID:25332235

  18. ITCH E3 Ubiquitin Ligase Interacts with Ebola Virus VP40 To Regulate Budding

    PubMed Central

    Han, Ziying; Sagum, Cari A.; Bedford, Mark T.; Sidhu, Sachdev S.; Sudol, Marius

    2016-01-01

    ABSTRACT Ebola virus (EBOV) and Marburg virus (MARV) belong to the Filoviridae family and can cause outbreaks of severe hemorrhagic fever, with high mortality rates in humans. The EBOV VP40 (eVP40) and MARV VP40 (mVP40) matrix proteins play a central role in virion assembly and egress, such that independent expression of VP40 leads to the production and egress of virus-like particles (VLPs) that accurately mimic the budding of infectious virus. Late (L) budding domains of eVP40 recruit host proteins (e.g., Tsg101, Nedd4, and Alix) that are important for efficient virus egress and spread. For example, the PPxY-type L domain of eVP40 and mVP40 recruits the host Nedd4 E3 ubiquitin ligase via its WW domains to facilitate budding. Here we sought to identify additional WW domain host interactors and demonstrate that the PPxY L domain motif of eVP40 interacts specifically with the WW domain of the host E3 ubiquitin ligase ITCH. ITCH, like Nedd4, is a member of the HECT class of E3 ubiquitin ligases, and the resultant physical and functional interaction with eVP40 facilitates VLP and virus budding. Identification of this novel eVP40 interactor highlights the functional interplay between cellular E3 ligases, ubiquitination, and regulation of VP40-mediated egress. IMPORTANCE The unprecedented magnitude and scope of the recent 2014-2015 EBOV outbreak in West Africa and its emergence here in the United States and other countries underscore the critical need for a better understanding of the biology and pathogenesis of this emerging pathogen. We have identified a novel and functional EBOV VP40 interactor, ITCH, that regulates VP40-mediated egress. This virus-host interaction may represent a new target for our previously identified small-molecule inhibitors of virus egress. PMID:27489272

  19. The Role of beta-TrCP Ubiquitin Ligase Receptor in the Development of Breast Cancer

    DTIC Science & Technology

    2007-06-01

    Receptor in the Development of Breast Cancer PRINCIPAL INVESTIGATOR: Vladimir Spiegelman, M.D., Ph.D. CONTRACTING ORGANIZATION...CONTRACT NUMBER The Role of beta-TrCP Ubiquitin Ligase Receptor in the Development of Breast Cancer 5b. GRANT NUMBER W81XWH-05-1-0415 5c...apoptosis of cancer cells induced by various pro-apoptotic stimuli. These and other data indicate that NF-kappaB inhibiting agents could become useful

  20. The EEL-1 ubiquitin ligase promotes DNA damage-induced germ cell apoptosis in C. elegans

    PubMed Central

    Ross, A J; Li, M; Yu, B; Gao, M X; Derry, W B

    2011-01-01

    E3 ubiquitin ligases target a growing number of pro- and anti-apoptotic proteins, including tumour suppressor p53, caspases, and the Bcl-2 family. The core apoptosis pathway is well conserved between mammals and Caenorhabditis elegans, but the extent to which ubiquitin ligases regulate apoptotic cell death is not known. To investigate the role of E3 ligases in apoptosis, we inhibited 108 of the 165 predicted E3 ubiquitin ligase genes by RNA interference and quantified apoptosis in the C. elegans germline after genotoxic stress. From this screen, we identified the homologous to E6-associated protein C terminus-domain E3 ligase EEL-1 as a positive regulator of apoptosis. Intriguingly, the human homologue of EEL-1, Huwe1/ARF-BP1/Mule/HectH9, has been reported to possess both pro- and anti-apoptotic functions through its ability to stimulate Mcl-1 and p53 degradation, respectively. Here, we demonstrate that eel-1 is required to promote DNA damage-induced germ cell apoptosis, but does not have a role in physiological germ cell apoptosis or developmental apoptosis in somatic tissue. Furthermore, eel-1 acts in parallel to the p53-like gene cep-1 and intersects the core apoptosis pathway upstream of the Bcl-2/Mcl-1 orthologue ced-9. Although ee1-1 mutants exhibit hypersensitivity to genotoxic stress they do not appear to be defective in DNA repair, suggesting a distinct role for EEL-1 in promoting damage-induced apoptosis in the germline. PMID:21233842

  1. The EEL-1 ubiquitin ligase promotes DNA damage-induced germ cell apoptosis in C. elegans.

    PubMed

    Ross, A J; Li, M; Yu, B; Gao, M X; Derry, W B

    2011-07-01

    E3 ubiquitin ligases target a growing number of pro- and anti-apoptotic proteins, including tumour suppressor p53, caspases, and the Bcl-2 family. The core apoptosis pathway is well conserved between mammals and Caenorhabditis elegans, but the extent to which ubiquitin ligases regulate apoptotic cell death is not known. To investigate the role of E3 ligases in apoptosis, we inhibited 108 of the 165 predicted E3 ubiquitin ligase genes by RNA interference and quantified apoptosis in the C. elegans germline after genotoxic stress. From this screen, we identified the homologous to E6-associated protein C terminus-domain E3 ligase EEL-1 as a positive regulator of apoptosis. Intriguingly, the human homologue of EEL-1, Huwe1/ARF-BP1/Mule/HectH9, has been reported to possess both pro- and anti-apoptotic functions through its ability to stimulate Mcl-1 and p53 degradation, respectively. Here, we demonstrate that eel-1 is required to promote DNA damage-induced germ cell apoptosis, but does not have a role in physiological germ cell apoptosis or developmental apoptosis in somatic tissue. Furthermore, eel-1 acts in parallel to the p53-like gene cep-1 and intersects the core apoptosis pathway upstream of the Bcl-2/Mcl-1 orthologue ced-9. Although ee1-1 mutants exhibit hypersensitivity to genotoxic stress they do not appear to be defective in DNA repair, suggesting a distinct role for EEL-1 in promoting damage-induced apoptosis in the germline.

  2. Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins.

    PubMed

    Crowder, Justin J; Geigges, Marco; Gibson, Ryan T; Fults, Eric S; Buchanan, Bryce W; Sachs, Nadine; Schink, Andrea; Kreft, Stefan G; Rubenstein, Eric M

    2015-07-24

    Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3'-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane.

  3. Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins*

    PubMed Central

    Crowder, Justin J.; Geigges, Marco; Gibson, Ryan T.; Fults, Eric S.; Buchanan, Bryce W.; Sachs, Nadine; Schink, Andrea; Kreft, Stefan G.; Rubenstein, Eric M.

    2015-01-01

    Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3′-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane. PMID:26055716

  4. The SCF Slimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication.

    PubMed

    Rogers, Gregory C; Rusan, Nasser M; Roberts, David M; Peifer, Mark; Rogers, Stephen L

    2009-01-26

    Restricting centriole duplication to once per cell cycle is critical for chromosome segregation and genomic stability, but the mechanisms underlying this block to reduplication are unclear. Genetic analyses have suggested an involvement for Skp/Cullin/F box (SCF)-class ubiquitin ligases in this process. In this study, we describe a mechanism to prevent centriole reduplication in Drosophila melanogaster whereby the SCF E3 ubiquitin ligase in complex with the F-box protein Slimb mediates proteolytic degradation of the centrosomal regulatory kinase Plk4. We identified SCF(Slimb) as a regulator of centriole duplication via an RNA interference (RNAi) screen of Cullin-based ubiquitin ligases. We found that Plk4 binds to Slimb and is an SCF(Slimb) target. Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase. Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

  5. Stabilization of the E3 Ubiquitin Ligase Nrdp1 by the Deubiquitinating Enzyme USP8

    PubMed Central

    Wu, Xiuli; Yen, Lily; Irwin, Lisa; Sweeney, Colleen; Carraway, Kermit L.

    2004-01-01

    Nrdp1 is a RING finger-containing E3 ubiquitin ligase that physically interacts with and regulates steady-state cellular levels of the ErbB3 and ErbB4 receptor tyrosine kinases and has been implicated in the degradation of the inhibitor-of-apoptosis protein BRUCE. Here we demonstrate that the Nrdp1 protein undergoes efficient proteasome-dependent degradation and that mutations in its RING finger domain that disrupt ubiquitin ligase activity enhance stability. These observations suggest that Nrdp1 self-ubiquitination and stability could play an important role in regulating the activity of this protein. Using affinity chromatography, we identified the deubiquitinating enzyme USP8 (also called Ubpy) as a protein that physically interacts with Nrdp1. Nrdp1 and USP8 could be coimmunoprecipitated, and in transfected cells USP8 specifically bound to Nrdp1 but not cbl, a RING finger E3 ligase involved in ligand-stimulated epidermal growth factor receptor down-regulation. The USP8 rhodanese and catalytic domains mediated Nrdp1 binding. USP8 markedly enhanced the stability of Nrdp1, and a point mutant that disrupts USP8 catalytic activity destabilized endogenous Nrdp1. Our results indicate that Nrdp1 is a specific target for the USP8 deubiquitinating enzyme and are consistent with a model where USP8 augments Nrdp1 activity by mediating its stabilization. PMID:15314180

  6. Stabilization of the E3 ubiquitin ligase Nrdp1 by the deubiquitinating enzyme USP8.

    PubMed

    Wu, Xiuli; Yen, Lily; Irwin, Lisa; Sweeney, Colleen; Carraway, Kermit L

    2004-09-01

    Nrdp1 is a RING finger-containing E3 ubiquitin ligase that physically interacts with and regulates steady-state cellular levels of the ErbB3 and ErbB4 receptor tyrosine kinases and has been implicated in the degradation of the inhibitor-of-apoptosis protein BRUCE. Here we demonstrate that the Nrdp1 protein undergoes efficient proteasome-dependent degradation and that mutations in its RING finger domain that disrupt ubiquitin ligase activity enhance stability. These observations suggest that Nrdp1 self-ubiquitination and stability could play an important role in regulating the activity of this protein. Using affinity chromatography, we identified the deubiquitinating enzyme USP8 (also called Ubpy) as a protein that physically interacts with Nrdp1. Nrdp1 and USP8 could be coimmunoprecipitated, and in transfected cells USP8 specifically bound to Nrdp1 but not cbl, a RING finger E3 ligase involved in ligand-stimulated epidermal growth factor receptor down-regulation. The USP8 rhodanese and catalytic domains mediated Nrdp1 binding. USP8 markedly enhanced the stability of Nrdp1, and a point mutant that disrupts USP8 catalytic activity destabilized endogenous Nrdp1. Our results indicate that Nrdp1 is a specific target for the USP8 deubiquitinating enzyme and are consistent with a model where USP8 augments Nrdp1 activity by mediating its stabilization.

  7. E3 Ubiquitin Ligase RLIM Negatively Regulates c-Myc Transcriptional Activity and Restrains Cell Proliferation

    PubMed Central

    Wang, Lan; Cai, Hao; Zhu, Jingjing; Yu, Long

    2016-01-01

    RNF12/RLIM is a RING domain-containing E3 ubiquitin ligase whose function has only begun to be elucidated recently. Although RLIM was reported to play important roles in some biological processes such as imprinted X-chromosome inactivation and regulation of TGF-β pathway etc., other functions of RLIM are largely unknown. Here, we identified RLIM as a novel E3 ubiquitin ligase for c-Myc, one of the most frequently deregulated oncoproteins in human cancers. RLIM associates with c-Myc in vivo and in vitro independently of the E3 ligase activity of RLIM. Moreover, RLIM promotes the polyubiquitination of c-Myc protein independently of Ser62 and Thr58 phosphorylation of c-Myc. However, RLIM-mediated ubiquitination does not affect c-Myc stability. Instead, RLIM inhibits the transcriptional activity of c-Myc through which RLIM restrains cell proliferation. Our results suggest that RLIM may function as a tumor suppressor by controlling the activity of c-Myc oncoprotein. PMID:27684546

  8. The TRC8 E3 ligase ubiquitinates MHC class I molecules before dislocation from the ER

    PubMed Central

    Stagg, Helen R.; Thomas, Mair; van den Boomen, Dick; Wiertz, Emmanuel J.H.J.; Drabkin, Harry A.; Gemmill, Robert M.

    2009-01-01

    The US2 and US11 gene products of human cytomegalovirus promote viral evasion by hijacking the endoplasmic reticulum (ER)–associated degradation (ERAD) pathway. US2 and US11 initiate dislocation of newly translocated major histocompatibility complex class I (MHC I) from the ER to the cytosol for proteasome-mediated degradation, thereby decreasing cell surface MHC I. Despite being instrumental in elucidating the mammalian ERAD pathway, the responsible E3 ligase or ligases remain unknown. Using a functional small interfering RNA library screen, we now identify TRC8 (translocation in renal carcinoma, chromosome 8 gene), an ER-resident E3 ligase previously implicated as a hereditary kidney cancer gene, as required for US2-mediated MHC I ubiquitination. Depletion of TRC8 prevents MHC I ubiquitination and dislocation by US2 and restores cell surface MHC I. TRC8 forms an integral part of a novel multiprotein ER complex that contains MHC I, US2, and signal peptide peptidase. Our data show that the TRC8 E3 ligase is required for MHC I dislocation from the ER and identify a new complex associated with mammalian ERAD. PMID:19720873

  9. The prolific ATL family of RING-H2 ubiquitin ligases.

    PubMed

    Guzmán, Plinio

    2012-08-01

    An abundant class of E3 ubiquitin ligases encodes the RING-finger domain. The RING finger binds to the E2 ubiquitin-conjugating enzyme and brings together both the E2 and substrate. It is predicted that 477 RING finger E3 ligases exist in Arabidopsis thaliana. A particular family among them, named Arabidopsis Tóxicos en Levadura (ATL), consists of 91 members that contain the RING-H2 variation and a hydrophobic domain located at the N-terminal end. Transmembrane E3 ligases are important in several biological processes. For instance, some transmembrane RING finger E3 ligases are main participants in the endoplasmic reticulum-associated degradation pathway that targets misfolded proteins. Functional analysis of a number of ATLs has shown that some of them regulate distinct pathways in plants. Several ATLs have been shown to participate in defense responses, while others play a role in the regulation of the carbon/nitrogen response during post-germinative seedling growth transition, in the regulation of cell death during root development, in endosperm development, or in the transition to flowering under short day conditions. The ATL family has also been instrumental in evolution studies for showing how gene families are expanded in plant genomes.

  10. The prolific ATL family of RING-H2 ubiquitin ligases

    PubMed Central

    Guzmán, Plinio

    2012-01-01

    An abundant class of E3 ubiquitin ligases encodes the RING-finger domain. The RING finger binds to the E2 ubiquitin-conjugating enzyme and brings together both the E2 and substrate. It is predicted that 477 RING finger E3 ligases exist in Arabidopsis thaliana. A particular family among them, named Arabidopsis Tóxicos en Levadura (ATL), consists of 91 members that contain the RING-H2 variation and a hydrophobic domain located at the N-terminal end. Transmembrane E3 ligases are important in several biological processes. For instance, some transmembrane RING finger E3 ligases are main participants in the endoplasmic reticulum-associated degradation pathway that targets misfolded proteins. Functional analysis of a number of ATLs has shown that some of them regulate distinct pathways in plants. Several ATLs have been shown to participate in defense responses, while others play a role in the regulation of the carbon/nitrogen response during post-germinative seedling growth transition, in the regulation of cell death during root development, in endosperm development, or in the transition to flowering under short day conditions. The ATL family has also been instrumental in evolution studies for showing how gene families are expanded in plant genomes. PMID:22827943

  11. Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage

    PubMed Central

    Kang, Ho Chul; Lee, Yun-Il; Shin, Joo-Ho; Andrabi, Shaida A.; Chi, Zhikai; Gagné, Jean-Philippe; Lee, Yunjong; Ko, Han Seok; Lee, Byoung Dae; Poirier, Guy G.; Dawson, Valina L.; Dawson, Ted M.

    2011-01-01

    Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3 ligase. Iduna’s E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate Iduna’s PAR binding and Iduna’s E3 ligase activity. Iduna’s E3 ligase activity also requires an intact really interesting new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86, XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna’s PAR-dependent ubiquitination of PARP1 targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA repair. PMID:21825151

  12. Characterization of a novel RING-type ubiquitin E3 ligase GhRING2 differentially expressed in cotton fiber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome proteolysis pathway is responsible for the degradation of abnormal and short-lived proteins to regulate many important biochemical activities in eukaryotes. By employing affymetrix microarray analysis, we have identified a novel ubiquitin ligase E3 gene GhRING2 that is diffe...

  13. New Insights into the RNA-Binding and E3 Ubiquitin Ligase Activities of Roquins

    PubMed Central

    Zhang, Qi; Fan, Lixin; Hou, Feng; Dong, Aiping; Wang, Yun-Xing; Tong, Yufeng

    2015-01-01

    Roquins are a family of highly conserved RNA-binding proteins that also contain a RING-type E3 ubiquitin ligase domain. They repress constitutive decay elements containing mRNAs and play a critical role in RNA homeostasis and immunological self-tolerance. Here we present the crystal structures of the RNA-binding region of Roquin paralog RC3H2 in both apo- and RNA-bound forms. The RNA-binding region has a bipartite architecture composed of ROQ and HEPN domains, and can bind to stem-loop and double-stranded RNAs simultaneously. The two domains undergo a large orientation change to accommodate RNA duplex binding. We profiled E2 ubiquitin-conjugating enzymes that pair with Roquins and found that RC3H1 and RC3H2 interact with two sets of overlapping but not identical E2 enzymes to drive the assembly of polyubiquitin chains of different linkages. Crystal structures, small-angle X-ray scattering, and E2 profiling revealed that while the two paralogs are highly homologous, RC3H2 and RC3H1 are different in their structures and functions. We also demonstrated that RNA duplex binding to RC3H2 cross-talks with its E3 ubiquitin ligase function using an in vitro auto-ubiquitination assay. PMID:26489670

  14. Deficiency in ubiquitin ligase TRIM2 causes accumulation of neurofilament light chain and neurodegeneration

    PubMed Central

    Balastik, Martin; Ferraguti, Francesco; Pires-da Silva, André; Lee, Tae Ho; Alvarez-Bolado, Gonzalo; Lu, Kun Ping; Gruss, Peter

    2008-01-01

    TRIM RING finger proteins have been shown to play an important role in cancerogenesis, in the pathogenesis of some human hereditary disorders, and in the defense against viral infection, but the function of the majority of TRIM proteins remains unknown. Here, we show that TRIM RING finger protein TRIM2, highly expressed in the nervous system, is an UbcH5a-dependent ubiquitin ligase. We further demonstrate that TRIM2 binds to neurofilament light subunit (NF-L) and regulates NF-L ubiquitination. Additionally, we show that mice deficient in TRIM2 have increased NF-L level in axons and NF-L-filled axonal swellings in cerebellum, retina, spinal cord, and cerebral cortex. The axonopathy is followed by progressive neurodegeneration accompanied by juvenile-onset tremor and ataxia. Our results demonstrate that TRIM2 is an ubiquitin ligase and point to a mechanism regulating NF-L metabolism through an ubiquitination pathway that, if deregulated, triggers neurodegeneration. PMID:18687884

  15. Genomic and Phenomic Screens for Flower Related RING Type Ubiquitin E3 Ligases in Arabidopsis

    PubMed Central

    Pavicic, Mirko; Mouhu, Katriina; Wang, Feng; Bilicka, Marcelina; Chovanček, Erik; Himanen, Kristiina

    2017-01-01

    Flowering time control integrates endogenous as well as environmental signals to promote flower development. The pathways and molecular networks involved are complex and integrate many modes of signal transduction. In plants ubiquitin mediated protein degradation pathway has been proposed to be as important mode of signaling as phosphorylation and transcription. To systematically study the role of ubiquitin signaling in the molecular regulation of flowering we have taken a genomic approach to identify flower related Ubiquitin Proteasome System components. As a large and versatile gene family the RING type ubiquitin E3 ligases were chosen as targets of the genomic screen. The complete list of Arabidopsis RING E3 ligases were retrieved and verified in the Arabidopsis genome v11 and their differential expression was used for their categorization into flower organs or developmental stages. Known regulators of flowering time or floral organ development were identified in these categories through literature search and representative mutants for each category were purchased for functional characterization by growth and morphological phenotyping. To this end, a workflow was developed for high throughput phenotypic screening of growth, morphology and flowering of nearly a thousand Arabidopsis plants in one experimental round.

  16. Ubiquitination and regulation of AURKA identifies a hypoxia-independent E3 ligase activity of VHL.

    PubMed

    Hasanov, E; Chen, G; Chowdhury, P; Weldon, J; Ding, Z; Jonasch, E; Sen, S; Walker, C L; Dere, R

    2017-01-23

    The hypoxia-regulated tumor-suppressor von Hippel-Lindau (VHL) is an E3 ligase that recognizes its substrates as part of an oxygen-dependent prolyl hydroxylase (PHD) reaction, with hypoxia-inducible factor α (HIFα) being its most notable substrate. Here we report that VHL has an equally important function distinct from its hypoxia-regulated activity. We find that Aurora kinase A (AURKA) is a novel, hypoxia-independent target for VHL ubiquitination. In contrast to its hypoxia-regulated activity, VHL mono-, rather than poly-ubiquitinates AURKA, in a PHD-independent reaction targeting AURKA for degradation in quiescent cells, where degradation of AURKA is required to maintain the primary cilium. Tumor-associated variants of VHL differentiate between these two functions, as a pathogenic VHL mutant that retains intrinsic ability to ubiquitinate HIFα is unable to ubiquitinate AURKA. Together, these data identify VHL as an E3 ligase with important cellular functions under both normoxic and hypoxic conditions.Oncogene advance online publication, 23 January 2017; doi:10.1038/onc.2016.495.

  17. E3 ubiquitin ligase SP1 regulates peroxisome biogenesis in Arabidopsis

    DOE PAGES

    Pan, Ronghui; Satkovich, John; Hu, Jianping

    2016-10-31

    Peroxisomes are ubiquitous eukaryotic organelles that play pivotal roles in a suite of metabolic processes and often act coordinately with other organelles, such as chloroplasts and mitochondria. Peroxisomes import proteins to the peroxisome matrix by peroxins (PEX proteins), but how the function of the PEX proteins is regulated is poorly understood. In this study, we identified the Arabidopsis RING (really interesting new gene) type E3 ubiquitin ligase SP1 [suppressor of plastid protein import locus 1 (ppi1) 1] as a peroxisome membrane protein with a regulatory role in peroxisome protein import. SP1 interacts physically with the two components of the peroxisomemore » protein docking complex PEX13–PEX14 and the (RING)-finger peroxin PEX2. Loss of SP1 function suppresses defects of the pex14-2 and pex13-1 mutants, and SP1 is involved in the degradation of PEX13 and possibly PEX14 and all three RING peroxins. An in vivo ubiquitination assay showed that SP1 has the ability to promote PEX13 ubiquitination. Our study has revealed that, in addition to its previously reported function in chloroplast biogenesis, SP1 plays a role in peroxisome biogenesis. The same E3 ubiquitin ligase promotes the destabilization of components of two distinct protein-import machineries, indicating that degradation of organelle biogenesis factors by the ubiquitin–proteasome system may constitute an important regulatory mechanism in coordinating the biogenesis of metabolically linked organelles in eukaryotes.« less

  18. Cullin-RING ubiquitin ligases in salicylic acid-mediated plant immune signaling

    PubMed Central

    Furniss, James J.; Spoel, Steven H.

    2015-01-01

    Plant immune responses against biotrophic pathogens are regulated by the signaling hormone salicylic acid (SA). SA establishes immunity by regulating a variety of cellular processes, including programmed cell death (PCD) to isolate and kill invading pathogens, and development of systemic acquired resistance (SAR) which provides long-lasting, broad-spectrum resistance throughout the plant. Central to these processes is post-translational modification of SA-regulated signaling proteins by ubiquitination, i.e., the covalent addition of small ubiquitin proteins. Emerging evidence indicates SA-induced protein ubiquitination is largely orchestrated by Cullin-RING ligases (CRLs), which recruit specific substrates for ubiquitination using interchangeable adaptors. Ligation of ubiquitin chains interlinked at lysine 48 leads to substrate degradation by the 26S proteasome. Here we discuss how CRL-mediated degradation of both nucleotide-binding/leucine-rich repeat domain containing immune receptors and SA-induced transcription regulators are critical for functional PCD and SAR responses, respectively. By placing these recent findings in context of knowledge gained in other eukaryotic model species, we highlight potential alternative roles for processive ubiquitination in regulating the activity of SA-mediated immune responses. PMID:25821454

  19. Identification of the endocytic sorting signal recognized by the Art1-Rsp5 ubiquitin ligase complex

    PubMed Central

    Guiney, Evan L.; Klecker, Till; Emr, Scott D.

    2016-01-01

    Targeted endocytosis of plasma membrane (PM) proteins allows cells to adjust their complement of membrane proteins to changing extracellular conditions. For a wide variety of PM proteins, initiation of endocytosis is triggered by ubiquitination. In yeast, arrestin-related trafficking adaptors (ARTs) enable a single ubiquitin ligase, Rsp5, to specifically and selectively target a wide range of PM proteins for ubiquitination and endocytosis. However, the mechanisms that allow ARTs to specifically recognize their appropriate substrates are unknown. We present the molecular features in the methionine permease Mup1 that are required for Art1-Rsp5–mediated ubiquitination and endocytosis. A combination of genetics, fluorescence microscopy, and biochemistry reveals three critical features that comprise an ART sorting signal in the Mup1 N-terminal cytosolic tail: 1) an extended acidic patch, 2) in close proximity to the first Mup1 transmembrane domain, and 3) close to the ubiquitinated lysines. We show that a functionally similar ART sorting signal is also required for the endocytosis of a second Art1-dependent cargo, Can1, suggesting a common mechanism for recognition of Art1 substrates. We isolate two separate suppressor mutations in the Art1 C-terminal domain that allele-specifically restore endocytosis of two Mup1 acidic patch mutants, consistent with an interaction between the Art1 C-terminus and the Mup1 acidic patch. We propose that this interaction is required for recruitment of the Art1-Rsp5 ubiquitination complex. PMID:27798240

  20. TRIM37 defective in mulibrey nanism is a novel RING finger ubiquitin E3 ligase

    SciTech Connect

    Kallijaervi, Jukka; Lahtinen, Ulla; Haemaelaeinen, Riikka; Lipsanen-Nyman, Marita; Palvimo, Jorma J.; Lehesjoki, Anna-Elina . E-mail: anna-elina.lehesjoki@helsinki.fi

    2005-08-01

    Mulibrey nanism is an autosomal recessive prenatal-onset growth disorder characterized by dysmorphic features, cardiomyopathy, and hepatomegaly. Mutations in TRIM37 encoding a tripartite motif (TRIM, RING-B-box-coiled-coil)-family protein underlie mulibrey nanism. We investigated the ubiquitin ligase activity predicted for the RING domain of TRIM37 by analyzing its autoubiquitination. Full-length TRIM37 and its TRIM domain were highly polyubiquitinated when co-expressed with ubiquitin. Polyubiquitination was decreased in a mutant protein with disrupted RING domain (Cys35Ser;Cys36Ser) and in the Leu76Pro mutant protein, a disease-associated missense mutation affecting the TRIM domain of TRIM37. Bacterially produced GST-TRIM domain fusion protein, but not its Cys35Ser;Cys36Ser or Leu76Pro mutants, were polyubiquitinated in cell-free conditions, implying RING-dependent modification. Ubiquitin was also identified as an interaction partner for TRIM37 in a yeast two-hybrid screen. Ectopically expressed TRIM37 rapidly formed aggregates that were ubiquitin-, proteasome subunit-, and chaperone-positive in immunofluorescence analysis, defining them as aggresomes. The Cys35Ser;Cys36Ser mutant and the Leu76Pro and Gly322Val patient mutant proteins were markedly less prone to aggregation, implying that aggresomal targeting reflects a physiological function of TRIM37. These findings suggest that TRIM37 acts as a TRIM domain-dependent E3 ubiquitin ligase and imply defective ubiquitin-dependent degradation of an as-yet-unidentified target protein in the pathogenesis of mulibrey nanism.

  1. Identification of HECT E3 ubiquitin ligase family genes involved in stem cell regulation and regeneration in planarians.

    PubMed

    Henderson, Jordana M; Nisperos, Sean V; Weeks, Joi; Ghulam, Mahjoobah; Marín, Ignacio; Zayas, Ricardo M

    2015-08-15

    E3 ubiquitin ligases constitute a large family of enzymes that modify specific proteins by covalently attaching ubiquitin polypeptides. This post-translational modification can serve to regulate protein function or longevity. In spite of their importance in cell physiology, the biological roles of most ubiquitin ligases remain poorly understood. Here, we analyzed the function of the HECT domain family of E3 ubiquitin ligases in stem cell biology and tissue regeneration in planarians. Using bioinformatic searches, we identified 17 HECT E3 genes that are expressed in the Schmidtea mediterranea genome. Whole-mount in situ hybridization experiments showed that HECT genes were expressed in diverse tissues and most were expressed in the stem cell population (neoblasts) or in their progeny. To investigate the function of all HECT E3 ligases, we inhibited their expression using RNA interference (RNAi) and determined that orthologs of huwe1, wwp1, and trip12 had roles in tissue regeneration. We show that huwe1 RNAi knockdown led to a significant expansion of the neoblast population and death by lysis. Further, our experiments showed that wwp1 was necessary for both neoblast and intestinal tissue homeostasis as well as uncovered an unexpected role of trip12 in posterior tissue specification. Taken together, our data provide insights into the roles of HECT E3 ligases in tissue regeneration and demonstrate that planarians will be a useful model to evaluate the functions of E3 ubiquitin ligases in stem cell regulation.

  2. Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

    PubMed Central

    Xie, Chuan-Ming; Wei, Wenyi; Sun, Yi

    2013-01-01

    Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainly controlled by the ubiquitin—proteasome system (UPS), which consists of two distinct steps: (1) ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and (2) subsequent degradation by the 26S proteasome. Among all E3 ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such as cancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore, altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis. PMID:23522382

  3. Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

    PubMed

    Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

    2015-01-01

    Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

  4. E2 conjugating enzyme selectivity and requirements for function of the E3 ubiquitin ligase CHIP.

    PubMed

    Soss, Sarah E; Yue, Yuanyuan; Dhe-Paganon, Sirano; Chazin, Walter J

    2011-06-17

    The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2∼Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2∼Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2∼Ub conjugate is derived from other molecular determinants.

  5. TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

    PubMed Central

    Hoffmann, Saskia; Smedegaard, Stine; Nakamura, Kyosuke; Mortuza, Gulnahar B.; Räschle, Markus; Ibañez de Opakua, Alain; Oka, Yasuyoshi; Feng, Yunpeng; Blanco, Francisco J.; Mann, Matthias; Montoya, Guillermo; Groth, Anja; Bekker-Jensen, Simon

    2016-01-01

    Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication. PMID:26711499

  6. Disinhibition of the HECT E3 ubiquitin ligase WWP2 by polymerized Dishevelled

    PubMed Central

    Mund, Thomas; Graeb, Michael; Mieszczanek, Juliusz; Gammons, Melissa; Pelham, Hugh R. B.; Bienz, Mariann

    2015-01-01

    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. PMID:26701932

  7. MDM2 E3 ligase-mediated ubiquitination and degradation of HDAC1 in vascular calcification

    PubMed Central

    Kwon, Duk-Hwa; Eom, Gwang Hyeon; Ko, Jeong Hyeon; Shin, Sera; Joung, Hosouk; Choe, Nakwon; Nam, Yoon Seok; Min, Hyun-Ki; Kook, Taewon; Yoon, Somy; Kang, Wanseok; Kim, Yong Sook; Kim, Hyung Seok; Choi, Hyuck; Koh, Jeong-Tae; Kim, Nacksung; Ahn, Youngkeun; Cho, Hyun-Jai; Lee, In-Kyu; Park, Dong Ho; Suk, Kyoungho; Seo, Sang Beom; Wissing, Erin R.; Mendrysa, Susan M.; Nam, Kwang-Il; Kook, Hyun

    2016-01-01

    Vascular calcification (VC) is often associated with cardiovascular and metabolic diseases. However, the molecular mechanisms linking VC to these diseases have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity via either chemical inhibitor or genetic ablation enhances VC. HDAC1 protein, but not mRNA, is reduced in cell and animal calcification models and in human calcified coronary artery. Under calcification-inducing conditions, proteasomal degradation of HDAC1 precedes VC and it is mediated by MDM2 E3 ubiquitin ligase that initiates HDAC1 K74 ubiquitination. Overexpression of MDM2 enhances VC, whereas loss of MDM2 blunts it. Decoy peptide spanning HDAC1 K74 and RG 7112, an MDM2 inhibitor, prevent VC in vivo and in vitro. These results uncover a previously unappreciated ubiquitination pathway and suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC. PMID:26832969

  8. Deubiquitinase FAM/USP9X Interacts with the E3 Ubiquitin Ligase SMURF1 Protein and Protects It from Ligase Activity-dependent Self-degradation

    PubMed Central

    Xie, Yang; Avello, Monika; Schirle, Markus; McWhinnie, Elizabeth; Feng, Yan; Bric-Furlong, Eva; Wilson, Christopher; Nathans, Robin; Zhang, Jing; Kirschner, Marc W.; Huang, Shih-Min A.; Cong, Feng

    2013-01-01

    Ubiquitination is an essential post-translational modification that mediates diverse cellular functions. SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) belongs to the Nedd4 family of HECT ubiquitin ligases that directly catalyzes ubiquitin conjugation onto diverse substrates. As a result, SMURF1 regulates a great variety of cellular physiologies including bone morphogenetic protein (BMP) signaling, cell migration, and planar cell polarity. Structurally, SMURF1 consists of a C2 domain, two WW domain repeats, and a catalytic HECT domain essential for its E3 ubiquitin ligase activity. This modular architecture allows for interactions with other proteins, which are either substrates or adaptors of SMURF1. Despite the increasing number of SMURF1 substrates identified, current knowledge regarding regulatory proteins and their modes of action on controlling SMURF1 activity is still limited. In this study, we employed quantitative mass spectrometry to analyze SMURF1-associated cellular complexes, and identified the deubiquitinase FAM/USP9X as a novel interacting protein for SMURF1. Through domain mapping study, we found the second WW domain of SMURF1 and the carboxyl terminus of USP9X critical for this interaction. SMURF1 is autoubiquitinated through its intrinsic HECT E3 ligase activity, and is degraded by the proteasome. USP9X association antagonizes this activity, resulting in deubiquitination and stabilization of SMURF1. In MDA-MB-231 breast cancer cells, SMURF1 expression is elevated and is required for cellular motility. USP9X stabilizes endogenous SMURF1 in MDA-MB-231 cells. Depletion of USP9X led to down-regulation of SMURF1 and significantly impaired cellular migration. Taken together, our data reveal USP9X as an important regulatory protein of SMURF1 and suggest that the association between deubiquitinase and E3 ligase may serve as a common strategy to control the cellular protein dynamics through modulating E3 ligase stability. PMID:23184937

  9. Lenalidomide Stabilizes the Erythropoietin Receptor by Inhibiting the E3 Ubiquitin Ligase RNF41.

    PubMed

    Basiorka, Ashley A; McGraw, Kathy L; De Ceuninck, Leentje; Griner, Lori N; Zhang, Ling; Clark, Justine A; Caceres, Gisela; Sokol, Lubomir; Komrokji, Rami S; Reuther, Gary W; Wei, Sheng; Tavernier, Jan; List, Alan F

    2016-06-15

    In a subset of patients with non-del(5q) myelodysplastic syndrome (MDS), lenalidomide promotes erythroid lineage competence and effective erythropoiesis. To determine the mechanism by which lenalidomide promotes erythropoiesis, we investigated its action on erythropoietin receptor (EpoR) cellular dynamics. Lenalidomide upregulated expression and stability of JAK2-associated EpoR in UT7 erythroid cells and primary CD71+ erythroid progenitors. The effects of lenalidomide on receptor turnover were Type I cytokine receptor specific, as evidenced by coregulation of the IL3-Rα receptor but not c-Kit. To elucidate this mechanism, we investigated the effects of lenalidomide on the E3 ubiquitin ligase RNF41. Lenalidomide promoted EpoR/RNF41 association and inhibited RNF41 auto-ubiquitination, accompanied by a reduction in EpoR ubiquitination. To confirm that RNF41 is the principal target responsible for EpoR stabilization, HEK293T cells were transfected with EpoR and/or RNF41 gene expression vectors. Steady-state EpoR expression was reduced in EpoR/RNF41 cells, whereas EpoR upregulation by lenalidomide was abrogated, indicating that cellular RNF41 is a critical determinant of drug-induced receptor modulation. Notably, shRNA suppression of CRBN gene expression failed to alter EpoR upregulation, indicating that drug-induced receptor modulation is independent of cereblon. Immunohistochemical staining showed that RNF41 expression decreased in primary erythroid cells of lenalidomide-responding patients, suggesting that cellular RNF41 expression merits investigation as a biomarker for lenalidomide response. Our findings indicate that lenalidomide has E3 ubiquitin ligase inhibitory effects that extend to RNF41 and that inhibition of RNF41 auto-ubiquitination promotes membrane accumulation of signaling competent JAK2/EpoR complexes that augment Epo responsiveness. Cancer Res; 76(12); 3531-40. ©2016 AACR.

  10. The bacterial effector Cif interferes with SCF ubiquitin ligase function by inhibiting deneddylation of Cullin1.

    PubMed

    Morikawa, Hanako; Kim, Minsoo; Mimuro, Hitomi; Punginelli, Claire; Koyama, Tomohiro; Nagai, Shinya; Miyawaki, Atsushi; Iwai, Kazuhiro; Sasakawa, Chihiro

    2010-10-15

    Cycle inhibiting factor (Cif) is one of the effectors delivered into epithelial cells by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) via the type III secretion system (TTSS). Cif family proteins, which inhibit host cell-cycle progression via mechanisms not yet precisely understood, are highly conserved among EPEC, EHEC, Yersinia pseudotuberculosis, Photorhabdus luminescens and Burkholderia pseudomallei. Levels of several proteins relevant to cell-cycle progression are modulated by Cullin-RING ligases (CRLs), which in turn are activated by conjugation and deconjugation of NEDD8 to Cullins. Here we show that Cif interacts with NEDD8 and interferes with SCF (Skp1-Cullin1-F-box protein) complex ubiquitin ligase function. We found that neddylated Cullin family proteins accumulated and ubiquitination of p27 decreased in cells infected with EPEC. Consequently, Cif stabilized SCF substrates such as CyclinD1, Cdt1, and p27, and caused G1 cell-cycle arrest. Using time-lapse-imaging of fluorescent ubiquitination-based cell-cycle indicator (Fucci)-expressing cells, we were able to monitor cell-cycle progression during EPEC infection and confirmed the arrest of infected cells at G1. Our in vitro and in vivo data show that Cif-NEDD8 interaction inhibits deneddylation of Cullins, suppresses CRL activity and induces G1 arrest. We thus conclude that the bacterial effector Cif interferes with neddylation-mediated cell-cycle control.

  11. RING finger ubiquitin-protein isopeptide ligase Nrdp1/FLRF regulates parkin stability and activity.

    PubMed

    Zhong, Ling; Tan, Ying; Zhou, An; Yu, Qingming; Zhou, Jianhua

    2005-03-11

    Parkin is a ubiquitin-protein isopeptide ligase. It has been suggested that loss of function in parkin causes accumulation and aggregation of its substrates, leading to death of dopaminergic neurons in Parkinson disease. Using the yeast two-hybrid screen, we isolated a RING finger protein that interacted with the N terminus of parkin in a Drosophila cDNA library. Interaction between human parkin and the mammalian RING finger protein homologue Nrdp1/FLRF, a ubiquitin-protein isopeptide ligase that ubiquitinates ErbB3 and ErbB4, was validated by in vitro binding assay, co-immunoprecipitation, and immunofluorescence co-localization. Significantly, pulse-chase experiments showed that cotransfection of Nrdp1 and parkin reduced the half-life of parkin from 5 to 2.5 h. Consistent with these findings, we further observed that degradation of CDCrel-1, a parkin substrate, was facilitated by overexpression of parkin protein. However, co-transfection of Nrdp1 with parkin reversed the effects of parkin on CDCrel-1 degradation. We conclude that Nrdp1 is a parkin modifier that accelerates degradation of parkin, resulting in a reduction of parkin activity.

  12. Protein-protein interactions involved in the recognition of p27 by E3 ubiquitin ligase.

    PubMed Central

    Xu, Kui; Belunis, Charles; Chu, Wei; Weber, David; Podlaski, Frank; Huang, Kuo-Sen; Reed, Steven I; Vassilev, Lyubomir T

    2003-01-01

    The p27(Kip1) protein is a potent cyclin-dependent kinase inhibitor, the level of which is decreased in many common human cancers as a result of enhanced ubiquitin-dependent degradation. The multiprotein complex SCF(Skp2) has been identified as the ubiquitin ligase that targets p27, but the functional interactions within this complex are not well understood. One component, the F-box protein Skp2, binds p27 when the latter is phosphorylated on Thr(187), thus providing substrate specificity for the ligase. Recently, we and others have shown that the small cell cycle regulatory protein Cks1 plays a critical role in p27 ubiquitination by increasing the binding affinity of Skp2 for p27. Here we report the development of a homogeneous time-resolved fluorescence assay that allows the quantification of the molecular interactions between human recombinant Skp2, Cks1 and a p27-derived peptide phosphorylated on Thr(187). Using this assay, we have determined the dissociation constant of the Skp2-Cks1 complex (K(d) 140 +/- 14 nM) and have shown that Skp2 binds phosphorylated p27 peptide with high affinity only in the presence of Cks1 (K(d) 37 +/- 2 nM). Cks1 does not bind directly to the p27 phosphopeptide or to Skp1, which confirms its suggested role as an allosteric effector of Skp2. PMID:12529174

  13. Ubiquitin ligase gene neurl3 plays a role in spermatogenesis of half-smooth tongue sole (Cynoglossus semilaevis) by regulating testis protein ubiquitination.

    PubMed

    Xu, Wenteng; Li, Hailong; Dong, Zhongdian; Cui, Zhongkai; Zhang, Ning; Meng, Liang; Zhu, Ying; Liu, Yang; Li, Yangzhen; Guo, Hua; Ma, Jialu; Wei, Zhanfei; Zhang, Nianwei; Yang, Yingming; Chen, Songlin

    2016-10-30

    E3 ubiquitin ligases are a large gene family that plays a diversity of roles in spermatogenesis. In this study, the functional characterization of a neuralized E3 ubiquitin protein ligase 3 (neurl3) revealed its potential participation in spermatogenesis. Firstly, we found that neurl3 exhibited male-biased transcription and that its translation was predominant in testis germ cells. The knockdown of neurl3 by RNA interference caused increased transcription of spermatogenesis-related genes. These results corroborate previous studies indicating a role for neurl3 in spermatogenesis. Moreover, the levels of neurl3 transcription and testis protein ubiquitination were closely correlated. Based on these findings, we speculate that neurl3 modulates testis protein ubiquitination in a dosage-dependent manner and that this influences spermatogenesis.

  14. The HERC2 ubiquitin ligase is essential for embryonic development and regulates motor coordination.

    PubMed

    Cubillos-Rojas, Monica; Schneider, Taiane; Hadjebi, Ouadah; Pedrazza, Leonardo; de Oliveira, Jarbas Rodrigues; Langa, Francina; Guénet, Jean-Louis; Duran, Joan; de Anta, Josep Maria; Alcántara, Soledad; Ruiz, Rocio; Pérez-Villegas, Eva María; Aguilar-Montilla, Francisco J; Carrión, Ángel M; Armengol, Jose Angel; Baple, Emma; Crosby, Andrew H; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis

    2016-08-30

    A mutation in the HERC2 gene has been linked to a severe neurodevelopmental disorder with similarities to the Angelman syndrome. This gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p53 and is involved in important cellular processes such as DNA repair, cell cycle, cancer, and iron metabolism. Despite the critical role of HERC2 in these physiological and pathological processes, little is known about its relevance in vivo. Here, we described a mouse with targeted inactivation of the Herc2 gene. Homozygous mice were not viable. Distinct from other ubiquitin ligases that interact with p53, such as MDM2 or MDM4, p53 depletion did not rescue the lethality of homozygous mice. The HERC2 protein levels were reduced by approximately one-half in heterozygous mice. Consequently, HERC2 activities, including ubiquitin ligase and stimulation of p53 activity, were lower in heterozygous mice. A decrease in HERC2 activities was also observed in human skin fibroblasts from individuals with an Angelman-like syndrome that express an unstable mutant protein of HERC2. Behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. This effect was not observed in p53 knockout mice, indicating that a mechanism independent of p53 activity is involved. Morphological analysis showed the presence of HERC2 in Purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. In these animals, an increase of autophagosomes and lysosomes was observed. Our findings establish a crucial role of HERC2 in embryonic development and motor coordination.

  15. The HERC2 ubiquitin ligase is essential for embryonic development and regulates motor coordination

    PubMed Central

    Cubillos-Rojas, Monica; Schneider, Taiane; Hadjebi, Ouadah; Pedrazza, Leonardo; de Oliveira, Jarbas Rodrigues; Langa, Francina; Guénet, Jean-Louis; Duran, Joan; de Anta, Josep Maria; Alcántara, Soledad; Ruiz, Rocio; Pérez-Villegas, Eva María; Aguilar, Francisco J.; Carrión, Ángel M.; Armengol, Jose Angel; Baple, Emma; Crosby, Andrew H.; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis

    2016-01-01

    A mutation in the HERC2 gene has been linked to a severe neurodevelopmental disorder with similarities to the Angelman syndrome. This gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p53 and is involved in important cellular processes such as DNA repair, cell cycle, cancer, and iron metabolism. Despite the critical role of HERC2 in these physiological and pathological processes, little is known about its relevance in vivo. Here, we described a mouse with targeted inactivation of the Herc2 gene. Homozygous mice were not viable. Distinct from other ubiquitin ligases that interact with p53, such as MDM2 or MDM4, p53 depletion did not rescue the lethality of homozygous mice. The HERC2 protein levels were reduced by approximately one-half in heterozygous mice. Consequently, HERC2 activities, including ubiquitin ligase and stimulation of p53 activity, were lower in heterozygous mice. A decrease in HERC2 activities was also observed in human skin fibroblasts from individuals with an Angelman-like syndrome that express an unstable mutant protein of HERC2. Behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. This effect was not observed in p53 knockout mice, indicating that a mechanism independent of p53 activity is involved. Morphological analysis showed the presence of HERC2 in Purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. In these animals, an increase of autophagosomes and lysosomes was observed. Our findings establish a crucial role of HERC2 in embryonic development and motor coordination. PMID:27528230

  16. Pathogenic Role of the CRL4 Ubiquitin Ligase in Human Disease

    PubMed Central

    Lee, Jennifer; Zhou, Pengbo

    2012-01-01

    The cullin 4-RING ubiquitin ligase (CRL4) family employs multiple DDB1–CUL4 associated factors substrate receptors to direct the degradation of proteins involved in a wide spectrum of cellular functions. Aberrant expression of the cullin 4A (CUL4A) gene is found in many tumor types, while mutations of the cullin 4B (CUL4B) gene are causally associated with human X-linked mental retardation. This focused review will summarize our current knowledge of the two CUL4 family members in the pathogenesis of human malignancy and neuronal disease, and discuss their potential as new targets for cancer prevention and therapeutic intervention. PMID:22649780

  17. Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex.

    PubMed

    Peng, Zhimin; Liao, Zhongping; Matsumoto, Yoshihiro; Yang, Austin; Tomkinson, Alan E

    2016-10-14

    The synthesis, processing, and joining of Okazaki fragments during DNA replication is complex, requiring the sequential action of a large number of proteins. Proliferating cell nuclear antigen, a DNA sliding clamp, interacts with and coordinates the activity of several DNA replication proteins, including the enzymes flap endonuclease 1 (FEN-1) and DNA ligase I that complete the processing and joining of Okazaki fragments, respectively. Although it is evident that maintaining the appropriate relative stoichiometry of FEN-1 and DNA ligase I, which compete for binding to proliferating cell nuclear antigen, is critical to prevent genomic instability, little is known about how the steady state levels of DNA replication proteins are regulated, in particular the proteolytic mechanisms involved in their turnover. Because DNA ligase I has been reported to be ubiquitylated, we used a proteomic approach to map ubiquitylation sites and screen for DNA ligase I-associated E3 ubiquitin ligases. We identified three ubiquitylated lysine residues and showed that DNA ligase I interacts with and is targeted for ubiquitylation by DCAF7, a specificity factor for the Cul4-DDB1 complex. Notably, knockdown of DCAF7 reduced the degradation of DNA ligase I in response to inhibition of proliferation and replacement of ubiquitylated lysine residues reduced the in vitro ubiquitylation of DNA ligase I by Cul4-DDB1 and DCAF7. In contrast, a different E3 ubiquitin ligase regulates FEN-1 turnover. Thus, although the expression of many of the genes encoding DNA replication proteins is coordinately regulated, our studies reveal that different mechanisms are involved in the turnover of these proteins.

  18. Bovine Papillomavirus Replicative Helicase E1 Is a Target of the Ubiquitin Ligase APC

    PubMed Central

    Mechali, Francisca; Hsu, Chiung-Yueh; Castro, Anna; Lorca, Thierry; Bonne-Andrea, Catherine

    2004-01-01

    The papillomavirus E1 replicative helicase is essential for replication and maintenance of extrachromosomal viral genomes in infected cells. We previously found that the bovine papillomavirus E1 protein is a substrate of the ubiquitin-dependent proteolytic pathway. Here we show that E1 is targeted for degradation by the anaphase-promoting complex (APC). Inhibition of APC activity by the specific inhibitor Emi1 or point mutations in the D-box and KEN-box motifs of E1 stabilize the protein and increase viral DNA replication in both a cell-free system and in living cells. These findings involve APC as the ubiquitin ligase that controls E1 levels to maintain a constant low copy number of the viral genome during latent infection. PMID:14963168

  19. The role and mechanism of CRL4 E3 ubiquitin ligase in cancer and its potential therapy implications

    PubMed Central

    Sang, Youzhou; Yan, Fan; Ren, Xiubao

    2015-01-01

    CRLs (Cullin-RING E3 ubiquitin ligases) are the largest E3 ligase family in eukaryotes, which ubiquitinate a wide range of substrates involved in cell cycle regulation, signal transduction, transcriptional regulation, DNA damage response, genomic integrity, tumor suppression and embryonic development. CRL4 E3 ubiquitin ligase, as one member of CRLs family, consists of a RING finger domain protein, cullin4 (CUL4) scaffold protein and DDB1–CUL4 associated substrate receptors. The CUL4 subfamily includes two members, CUL4A and CUL4B, which share extensively sequence identity and functional redundancy. Aberrant expression of CUL4 has been found in a majority of tumors. Given the significance of CUL4 in cancer, understanding its detailed aspects of pathogenesis of human malignancy would have significant value for the treatment of cancer. Here, the work provides an overview to address the role of CRL4 E3 ubiquitin ligase in cancer development and progression, and discuss the possible mechanisms of CRL4 ligase involving in many cellular processes associated with tumor. Finally, we discuss its potential value in cancer therapy. PMID:26460955

  20. Overexpression of a Soybean Ariadne-Like Ubiquitin Ligase Gene GmARI1 Enhances Aluminum Tolerance in Arabidopsis

    PubMed Central

    Zhang, Xiaolian; Wang, Ning; Chen, Pei; Gao, Mengmeng; Liu, Juge; Wang, Yufeng; Zhao, Tuanjie; Li, Yan; Gai, Junyi

    2014-01-01

    Ariadne (ARI) subfamily of RBR (Ring Between Ring fingers) proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene) finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L.) Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2–4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress. PMID:25364908

  1. The ubiquitin conjugating enzyme UbcH10 competes with UbcH3 for binding to the SCF complex, a ubiquitin ligase involved in cell cycle progression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ubiquitylation, which regulates most biological pathways, occurs through an enzymatic cascade involving a ubiquitin (ub) activating enzyme (E1), a ub conjugating enzyme (E2) and a ub ligase (E3). UbcH3 is the E2 that interacts with SCF (Skp1/Cul1/F-box protein) complex and ubiquitylates many protein...

  2. The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates a defense-related NAC transcription factor and is involved in defense signaling.

    PubMed

    Miao, Min; Niu, Xiangli; Kud, Joanna; Du, Xinran; Avila, Julian; Devarenne, Timothy P; Kuhl, Joseph C; Liu, Yongsheng; Xiao, Fangming

    2016-07-01

    We recently identified a defense-related tomato (Solanum lycopersicum) NAC (NAM, ATAF1,2, CUC2) transcription factor, NAC1, that is subjected to ubiquitin-proteasome system-dependent degradation in plant cells. In this study, we identified a tomato ubiquitin ligase (termed SEVEN IN ABSENTIA3; SINA3) that ubiquitinates NAC1, promoting its degradation. We conducted coimmunoprecipitation and bimolecular fluorescence complementation to determine that SINA3 specifically interacts with the NAC1 transcription factor in the nucleus. Moreover, we found that SINA3 ubiquitinates NAC1 in vitro and promotes NAC1 degradation via polyubiquitination in vivo, indicating that SINA3 is a ubiquitin ligase that ubiquitinates NAC1, promoting its degradation. Our real-time PCR analysis indicated that, in contrast to our previous finding that NAC1 mRNA abundance increases upon Pseudomonas infection, the SINA3 mRNA abundance decreases in response to Pseudomonas infection. Moreover, using Agrobacterium-mediated transient expression, we found that overexpression of SINA3 interferes with the hypersensitive response cell death triggered by multiple plant resistance proteins. These results suggest that SINA3 ubiquitinates a defense-related NAC transcription factor for degradation and plays a negative role in defense signaling.

  3. Chlorovirus Skp1-Binding Ankyrin Repeat Protein Interplay and Mimicry of Cellular Ubiquitin Ligase Machinery

    PubMed Central

    Noel, Eric A.; Kang, Ming; Adamec, Jiri; Oyler, George A.

    2014-01-01

    ABSTRACT The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins. IMPORTANCE Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their

  4. Early Origin and Evolution of the Angelman Syndrome Ubiquitin Ligase Gene Ube3a

    PubMed Central

    Sato, Masaaki

    2017-01-01

    The human Ube3a gene encodes an E3 ubiquitin ligase and exhibits brain-specific genomic imprinting. Genetic abnormalities that affect the maternal copy of this gene cause the neurodevelopmental disorder Angelman syndrome (AS), which is characterized by severe mental retardation, speech impairment, seizure, ataxia and some unique behavioral phenotypes. In this review article, I highlight the evolution of the Ube3a gene and its imprinting to provide evolutionary insights into AS. Recent comparative genomic studies have revealed that Ube3a is most phylogenetically similar to HECTD2 among the human HECT (homologous to the E6AP carboxyl terminus) family of E3 ubiquitin ligases, and its distant evolutionary origin can be traced to common ancestors of fungi and animals. Moreover, a gene more similar to Ube3a than HECTD2 is found in a range of eukaryotes from amoebozoans to basal metazoans, but is lost in later lineages. Unlike in mice and humans, Ube3a expression is biallelic in birds, monotremes, marsupials and insects. The imprinting domain that governs maternal expression of Ube3a was formed from non-imprinted elements following multiple chromosomal rearrangements after diversification of marsupials and placental mammals. Hence, the evolutionary origins of Ube3a date from long before the emergence of the nervous system, although its imprinted expression was acquired relatively recently. These observations suggest that exogenous expression and functional analyses of ancient Ube3a orthologs in mammalian neurons will facilitate the evolutionary understanding of AS. PMID:28326016

  5. Huwe1 ubiquitin ligase is essential to synchronize neuronal and glial differentiation in the developing cerebellum

    PubMed Central

    D’Arca, Domenico; Zhao, Xudong; Xu, Wenming; Ramirez-Martinez, Nadya C.; Iavarone, Antonio; Lasorella, Anna

    2010-01-01

    We have generated a knockout mouse strain in which the gene coding for the ubiquitin ligase Huwe1 has been inactivated in cerebellar granule neuron precursors (CGNPs) and radial glia. These mice have a high rate of postnatal lethality and profound cerebellar abnormalities. The external granule layer of the cerebellum, which contains CGNPs, is expanded and displays aberrant proliferation and impaired differentiation of the progenitor cell population. The uncontrolled proliferation of the CGNPs is associated with accumulation of the N-Myc oncoprotein, a substrate of Huwe1, and con-sequent activation of the signaling events downstream to N-Myc. Furthermore, loss of Huwe1 in Bergmann glia leads to extensive disorganization of this cell population with layering aberrations, severe granule neuron migration defects, and persistence of ectopic clusters of granule neurons in the external granule layer. Our findings uncover an unexpected role for Huwe1 in regulating Berg-mann glia differentiation and indicate that this ubiquitin ligase orchestrates the programming of the neural progenitors that give rise to neurons and glia in the cerebellum. PMID:20231446

  6. Huwe1 ubiquitin ligase is essential to synchronize neuronal and glial differentiation in the developing cerebellum.

    PubMed

    D'Arca, Domenico; Zhao, Xudong; Xu, Wenming; Ramirez-Martinez, Nadya C; Iavarone, Antonio; Lasorella, Anna

    2010-03-30

    We have generated a knockout mouse strain in which the gene coding for the ubiquitin ligase Huwe1 has been inactivated in cerebellar granule neuron precursors (CGNPs) and radial glia. These mice have a high rate of postnatal lethality and profound cerebellar abnormalities. The external granule layer of the cerebellum, which contains CGNPs, is expanded and displays aberrant proliferation and impaired differentiation of the progenitor cell population. The uncontrolled proliferation of the CGNPs is associated with accumulation of the N-Myc oncoprotein, a substrate of Huwe1, and con-sequent activation of the signaling events downstream to N-Myc. Furthermore, loss of Huwe1 in Bergmann glia leads to extensive disorganization of this cell population with layering aberrations, severe granule neuron migration defects, and persistence of ectopic clusters of granule neurons in the external granule layer. Our findings uncover an unexpected role for Huwe1 in regulating Berg-mann glia differentiation and indicate that this ubiquitin ligase orchestrates the programming of the neural progenitors that give rise to neurons and glia in the cerebellum.

  7. The Ubiquitin Ligase Siah2 Regulates Obesity-induced Adipose Tissue Inflammation

    PubMed Central

    Kilroy, Gail; Carter, Lauren E.; Newman, Susan; Burk, David H.; Manuel, Justin; Möller, Andreas; Bowtell, David D.; Mynatt, Randall L.; Ghosh, Sujoy; Floyd, Z. Elizabeth

    2015-01-01

    Objective Chronic, low-grade adipose tissue inflammation associated with adipocyte hypertrophy is an important link in the relationship between obesity and insulin resistance. Although ubiquitin ligases regulate inflammatory processes, the role of these enzymes in metabolically driven adipose tissue inflammation is relatively unexplored. Herein, we examined the effect of the ubiquitin ligase Siah2 on obesity-related adipose tissue inflammation. Methods Wild-type and Siah2KO mice were fed a low or high fat diet for 16 weeks. Indirect calorimetry, body composition, glucose and insulin tolerance were assayed along with glucose and insulin levels. Gene and protein expression, immunohistochemistry, adipocyte size distribution and lipolysis were also analyzed. Results Enlarged adipocytes in obese Siah2KO mice are not associated with obesity-induced insulin resistance. Proinflammatory gene expression, stress kinase signaling, fibrosis and crown-like structures are reduced in the Siah2KO adipose tissue and Siah2KO adipocytes are more responsive to insulin-dependent inhibition of lipolysis. Loss of Siah2 increases expression of PPARγ target genes involved in lipid metabolism and decreases expression of proinflammatory adipokines regulated by PPARγ. Conclusions Siah2 links adipocyte hypertrophy with adipocyte dysfunction and recruitment of proinflammatory immune cells to adipose tissue. Selective regulation of PPARγ activity is a Siah2-mediated mechanism contributing to obesity-induced adipose tissue inflammation. PMID:26380945

  8. The Ubiquitin Ligase CBLC Maintains the Network Organization of the Golgi Apparatus.

    PubMed

    Lee, Wan Yin; Goh, Germaine; Chia, Joanne; Boey, Adrian; Gunko, Natalia V; Bard, Frederic

    2015-01-01

    The Golgi apparatus plays a pivotal role in the sorting and post-translational modifications of secreted and membrane proteins. In mammalian cells, the Golgi is organized in stacks of cisternae linked together to form a network with a ribbon shape. Regulation of Golgi ribbon formation is poorly understood. Here we find in an image-based RNAi screen that depletion of the ubiquitin-ligase CBLC induces Golgi fragmentation. Depletions of the close homologues CBL and CBLB do not induce any visible defects. In CBLC-depleted cells, Golgi stacks appear relatively unperturbed at both the light and electron microscopy levels, suggesting that CBLC controls mostly network organization. CBLC partially localizes on Golgi membranes and this localization is enhanced after activation of the SRC kinase. Inhibition of SRC reverts CBLC depletion effects, suggesting interplay between the two. CBLC's regulation of Golgi network requires its ubiquitin ligase activity. However, SRC levels are not significantly affected by CBLC, and CBLC knockdown does not phenocopy SRC activation, suggesting that CBLC's action at the Golgi is not direct downregulation of SRC. Altogether, our results demonstrate a role of CBLC in regulating Golgi ribbon by antagonizing the SRC tyrosine kinase.

  9. Targeting the mTOR-DEPTOR Pathway by CRL E3 Ubiquitin Ligases: Therapeutic Application1

    PubMed Central

    Zhao, Yongchao; Sun, Yi

    2012-01-01

    The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine/threonine protein kinase, integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth, proliferation, survival, and autophagy. The mTOR pathway is frequently activated in many human cancers, mainly resulting from alterations in the upstream regulators, such as phosphoinositide 3-kinase (PI3K)/AKT activation, PTEN loss or dysregulation of mTOR-negative regulators (e.g., TSC1/2), leading to uncontrolled proliferation. Thus, inhibiting the PI3K/AKT/mTOR pathways is widely considered as an effective approach for targeted cancer therapy. Recently, we and others found that DEPTOR, a naturally occurring inhibitor of both mTORC1 and mTORC2, was degraded by SCF (Skp1-Cullin-F box proteins) E3 ubiquitin ligase, the founding member of cullin-RING-ligases (CRLs), resulting in mTOR activation and cell proliferation. In addition to DEPTOR, previous studies have demonstrated that several other negative regulators of mTOR pathway are also substrates of CRL/SCF E3s. Thus, targeting CRL/SCF E3s is expected to cause the accumulation of these mTOR signal inhibitors to effectively block the mTOR pathway. In this review, we will discuss mTOR signaling pathway, how DEPTOR regulates mTOR/AKT axis, thus acting as a tumor suppressor or oncogene in some cases, how DEPTOR is ubiquitinated and degraded by SCFβ-TrCP E3, and how MLN4924, a small-molecule indirect inhibitor of CRL/SCF E3 ligases through blocking cullin neddylation, might be useful as a novel approach of mTOR pathway targeting for cancer therapy. PMID:22745582

  10. The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein-coupled receptor CXCR4.

    PubMed

    Holleman, Justine; Marchese, Adriano

    2014-06-15

    G protein-coupled receptor (GPCR) sorting into the degradative pathway is important for limiting the duration and magnitude of signaling. Agonist activation of the GPCR CXCR4 induces its rapid ubiquitination and sorting to lysosomes via the endosomal sorting complex required for transport (ESCRT) pathway. We recently reported that ESCRT-0 ubiquitination is linked to the efficiency with which CXCR4 is sorted for lysosomal degradation; however mechanistic insight is lacking. Here we define a novel role for the really interesting new gene-domain E3 ubiquitin ligase deltex-3-like (DTX3L) in regulating CXCR4 sorting from endosomes to lysosomes. We show that DTX3L localizes to early endosomes upon CXCR4 activation and interacts directly with and inhibits the activity of the E3 ubiquitin ligase atrophin-1 interacting protein 4. This serves to limit the extent to which ESCRT-0 is ubiquitinated and is able to sort CXCR4 for lysosomal degradation. Therefore we define a novel role for DTX3L in GPCR endosomal sorting and reveal an unprecedented link between two distinct E3 ubiquitin ligases to control the activity of the ESCRT machinery.

  11. The Anaphase-Promoting Complex (APC) ubiquitin ligase affects chemosensory behavior in C. elegans

    PubMed Central

    Wang, Julia; Jennings, Alexandra K.

    2016-01-01

    The regulation of fundamental aspects of neurobiological function has been linked to the ubiquitin signaling system (USS), which regulates the degradation and activity of proteins and is catalyzed by E1, E2, and E3 enzymes. The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that controls diverse developmental and signaling processes in post-mitotic neurons; however, potential roles for the APC in sensory function have yet to be explored. In this study, we examined the effect of the APC ubiquitin ligase on chemosensation in Caenorhabditis elegans by testing chemotaxis to the volatile odorants, diacetyl, pyrazine, and isoamyl alcohol, to which wild-type worms are attracted. Animals with loss of function mutations in either of two alleles (g48 and ye143) of the gene encoding the APC subunit EMB-27 APC6 showed increased chemotaxis towards diacetyl and pyrazine, odorants sensed by AWA neurons, but exhibited normal chemotaxis to isoamyl alcohol, which is sensed by AWC neurons. The statistically significant increase in chemotaxis in the emb-27 APC6 mutants suggests that the APC inhibits AWA-mediated chemosensation in C. elegans. Increased chemotaxis to pyrazine was also seen with mutants lacking another essential APC subunit, MAT-2 APC1; however, mat-2 APC1 mutants exhibited wild type responses to diacetyl. The difference in responsiveness of these two APC subunit mutants may be due to differential strength of these hypomorphic alleles or may indicate the presence of functional sub-complexes of the APC at work in this process. These findings are the first evidence for APC-mediated regulation of chemosensation and lay the groundwork for further studies aimed at identifying the expression levels, function, and targets of the APC in specific sensory neurons. Because of the similarity between human and C. elegans nervous systems, the role of the APC in sensory neurons may also advance our understanding of human sensory function and disease. PMID

  12. The E3 Ubiquitin Ligase TMEM129 Is a Tri-Spanning Transmembrane Protein

    PubMed Central

    van de Weijer, Michael L.; van Muijlwijk, Guus H.; Visser, Linda J.; Costa, Ana I.; Wiertz, Emmanuel J. H. J.; Lebbink, Robert Jan

    2016-01-01

    Misfolded proteins from the endoplasmic reticulum (ER) are transported back into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 hijacks this ER-associated protein degradation (ERAD) pathway to downregulate human leukocyte antigen (HLA) class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. Recently, we identified the E3 ubiquitin ligase transmembrane protein 129 (TMEM129) as a key player in this process, where interference with TMEM129 activity in human cells completely abrogates US11-mediated class I degradation. Here, we set out to further characterize TMEM129. We show that TMEM129 is a non-glycosylated protein containing a non-cleaved signal anchor sequence. By glycosylation scanning mutagenesis, we show that TMEM129 is a tri-spanning ER-membrane protein that adopts an Nexo–Ccyto orientation. This insertion in the ER membrane positions the C-terminal really interesting new gene (RING) domain of TMEM129 in the cytosol, making it available to catalyze ubiquitination reactions that are required for cytosolic degradation of secretory proteins. PMID:27854284

  13. Regulation of EGFR protein stability by the HECT-type ubiquitin ligase SMURF2.

    PubMed

    Ray, Dipankar; Ahsan, Aarif; Helman, Abigail; Chen, Guoan; Hegde, Ashok; Gurjar, Susmita Ramanand; Zhao, Lili; Kiyokawa, Hiroaki; Beer, David G; Lawrence, Theodore S; Nyati, Mukesh K

    2011-07-01

    Epidermal growth factor receptor (EGFR) is overexpressed in a variety of epithelial tumors and is considered to be an important therapeutic target. Although gene amplification is responsible for EGFR overexpression in certain human malignancies including lung and head and neck cancers, additional molecular mechanisms are likely. Here, we report a novel interaction of EGFR with an HECT-type ubiquitin ligase SMURF2, which can ubiquitinate, but stabilize EGFR by protecting it from c-Cbl-mediated degradation. Conversely, small interfering RNA (siRNA)-mediated knockdown of SMURF2 destabilized EGFR, induced an autophagic response and reduced the clonogenic survival of EGFR-expressing cancer cell lines, with minimal effects on EGFR-negative cancer cells, normal fibroblasts, and normal epithelial cells. UMSCC74B head and neck squamous cancer cells, which form aggressive tumors in nude mice, significantly lost in vivo tumor-forming ability on siRNA-mediated SMURF2 knockdown. Gene expression microarray data from 443 lung adenocarcinoma patients, and tissue microarray data from 67 such patients, showed a strong correlation of expression between EGFR and SMURF2 at the messenger RNA and protein levels, respectively. Our findings suggest that SMURF2-mediated protective ubiquitination of EGFR may be responsible for EGFR overexpression in certain tumors and support targeting SMURF2-EGFR interaction as a novel therapeutic approach in treating EGFR-addicted tumors.

  14. The Ubiquitin Ligase Praja1 Reduces NRAGE Expression and Inhibits Neuronal Differentiation of PC12 Cells

    PubMed Central

    Teuber, Jan; Mueller, Bettina; Fukabori, Ryoji; Lang, Daniel; Albrecht, Anne; Stork, Oliver

    2013-01-01

    Evidence suggests that regulated ubiquitination of proteins plays a critical role in the development and plasticity of the central nervous system. We have previously identified the ubiquitin ligase Praja1 as a gene product induced during fear memory consolidation. However, the neuronal function of this enzyme still needs to be clarified. Here, we investigate its involvement in the nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma (PC12) cells. Praja1 co-localizes with cytoskeleton components and the neurotrophin receptor interacting MAGE homologue (NRAGE). We observed an enhanced expression of Praja1 after 3 days of NGF treatment and a suppression of neurite formation upon Praja1 overexpression in stably transfected PC12 cell lines, which was associated with a proteasome-dependent reduction of NRAGE levels. Our data suggest that Praja1, through ubiquitination and degradation of NRAGE, inhibits neuronal differentiation. The two murine isoforms, Praja1.1 and Praja1.2, appear to be functionally homologous in this respect. PMID:23717400

  15. The ubiquitin ligase TRIM25 targets ERG for degradation in prostate cancer

    PubMed Central

    Wang, Shan; Kollipara, Rahul K.; Humphries, Caroline G.; Ma, Shi-Hong; Hutchinson, Ryan; Li, Rui; Siddiqui, Javed; Tomlins, Scott A.; Raj, Ganesh V.; Kittler, Ralf

    2016-01-01

    Ets related gene (ERG) is a transcription factor that is overexpressed in 40% of prostate tumors due to a gene fusion between ERG and TMPRSS2. Because ERG functions as a driver of prostate carcinogenesis, understanding the mechanisms that influence its turnover may provide new molecular handles to target the protein. Previously, we found that ERG undergoes ubiquitination and then is deubiquitinated by USP9X in prostate cancer cells to prevent its proteasomal degradation. Here, we identify Tripartite motif-containing protein 25 (TRIM25) as the E3 ubiquitin ligase that ubiquitinates the protein prior to its degradation. TRIM25 binds full-length ERG, and it also binds the N-terminally truncated variants of ERG that are expressed in tumors with TMPRSS2-ERG fusions. We demonstrate that TRIM25 polyubiquitinates ERG in vitro and that inactivation of TRIM25 resulted in reduced polyubiquitination and stabilization of ERG. TRIM25 mRNA and protein expression was increased in ERG rearrangement-positive prostate cancer specimens, and we provide evidence that ERG upregulates TRIM25 expression. Thus, overexpression of ERG in prostate cancer may cause an increase in TRIM25 activity, which is mitigated by the expression of the deubiquitinase USP9X, which is required to stabilize ERG. PMID:27626314

  16. The G1 phase E3 ubiquitin ligase TRUSS that gets deregulated in human cancers is a novel substrate of the S-phase E3 ubiquitin ligase Skp2.

    PubMed

    Jamal, Azfar; Swarnalatha, Manickavinayaham; Sultana, Sarwat; Joshi, Prashant; Panda, Subrat K; Kumar, Vijay

    2015-01-01

    E3 ubiquitin ligases have been implicated in the ubiquitination and proteasome-mediated degradation of several key regulators of cell cycle. Owing to their pleotropic behavior, E3 ubiquitin ligases are tightly regulated both at transcriptional and post-translational levels. The E3 ubiquitin ligase TRUSS (tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein) which negatively regulates c-Myc, are found down-regulated in most human cancer cell lines. However, the mechanism of regulation of intracellular levels of TRUSS remains elusive. Here we show that TRUSS is expressed majorly during the G1 phase of cell cycle and its level starts to decline with the expression of S-phase specific E3 ligase Skp2. Enforced expression of Skp2 led to a marked increase in the ubiquitination of TRUSS after its phosphorylation by GSK3β and followed by rapid proteolytic degradation. Our co-immunoprecipitation studies suggested a direct interaction between Skp2 and TRUSS through the LRR motif of Skp2. Interestingly, the human tumor samples that exhibited elevated expression of Skp2, showed relatively poor expression of TRUSS. Further, enforced expression of HBx, the oncoprotein of Hepatitis B virus which is known to stabilize c-Myc and enhance its oncogenic potential, led to the intracellular accumulation of TRUSS as well as c-Myc. Apparently, HBx also interacted with TRUSS which negatively impacted the TRUSS-c-Myc and TRUSS-Skp2 interactions leading to stabilization of TRUSS. Thus, the present study suggests that TRUSS is a novel substrate of E3 ligase Skp2 and that disruption of TRUSS-Skp2 interaction by viral oncoproteins could lead to pathophysiological sequelae.

  17. Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface.

    PubMed

    Gorelik, Maryna; Orlicky, Stephen; Sartori, Maria A; Tang, Xiaojing; Marcon, Edyta; Kurinov, Igor; Greenblatt, Jack F; Tyers, Mike; Moffat, Jason; Sicheri, Frank; Sidhu, Sachdev S

    2016-03-29

    Skp1-Cul1-F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

  18. Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

    DOE PAGES

    Gorelik, Maryna; Orlicky, Stephen; Sartori, Maria A.; ...

    2016-03-14

    Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface.more » Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.« less

  19. Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

    SciTech Connect

    Gorelik, Maryna; Orlicky, Stephen; Sartori, Maria A.; Tang, Xiaojing; Marcon, Edyta; Kurinov, Igor; Greenblatt, Jack F.; Tyers, Mike; Moffat, Jason; Sicheri, Frank; Sidhu, Sachdev S.

    2016-03-14

    Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

  20. Pulling a Ligase out of a “HAT”: pCAF Mediates Ubiquitination of the Class II Transactivator

    PubMed Central

    2017-01-01

    The Class II Transactivator (CIITA) is essential to the regulation of Major Histocompatibility Class II (MHC II) genes transcription. As the “master regulator” of MHC II transcription, CIITA regulation is imperative and requires various posttranslational modifications (PTMs) in order to facilitate its role. Previously we identified various ubiquitination events on CIITA. Monoubiquitination is important for CIITA transactivity, while K63 linked ubiquitination is involved in crosstalk with ERK1/2 phosphorylation, where together they mediate cellular movement from the cytoplasm to nuclear region. Further, CIITA is also modified by degradative K48 polyubiquitination. However, the E3 ligase responsible for these modifications was unknown. We show CIITA ubiquitination and transactivity are enhanced with the histone acetyltransferase (HAT), p300/CBP associated factor (pCAF), and the E3 ligase region within pCAF is necessary for both. Additionally, pCAF mediated ubiquitination is independent of pCAF's HAT domain, and acetylation deficient CIITA is K48 polyubiquitinated and degraded in the presence of pCAF. Lastly, we identify the histone acetyltransferase, pCAF, as the E3 ligase responsible for CIITA's ubiquitination. PMID:28286521

  1. GCN5 is a required cofactor for a ubiquitin ligase that targets NF-κB/RelA

    PubMed Central

    Mao, Xicheng; Gluck, Nathan; Li, Duo; Maine, Gabriel N.; Li, Haiying; Zaidi, Iram W.; Repaka, Aparna; Mayo, Marty W.; Burstein, Ezra

    2009-01-01

    The transcription factor NF-κB is a critical regulator of inflammatory and cell survival signals. Proteasomal degradation of NF-κB subunits plays an important role in the termination of NF-κB activity, and at least one of the identified ubiquitin ligases is a multimeric complex containing Copper Metabolism Murr1 Domain 1 (COMMD1) and Cul2. We report here that GCN5, a histone acetyltransferase, associates with COMMD1 and other components of the ligase, promotes RelA ubiquitination, and represses κB-dependent transcription. In this role, the acetyltransferase activity of GCN5 is not required. Interestingly, GCN5 binds more avidly to RelA after phosphorylation on Ser 468, an event that is dependent on IKK activity. Consistent with this, we find that both GCN5 and the IκB Kinase (IKK) complex promote RelA degradation. Collectively, the data indicate that GCN5 participates in the ubiquitination process as an accessory factor for a ubiquitin ligase, where it provides a novel link between phosphorylation and ubiquitination. PMID:19339690

  2. A conserved RAD6-MDM2 ubiquitin ligase machinery targets histone chaperone ASF1A in tumorigenesis.

    PubMed

    Wang, Chen; Chang, Jian-Feng; Yan, Hongli; Wang, Da-Liang; Liu, Yan; Jing, Yuanya; Zhang, Meng; Men, Yu-Long; Lu, Dongdong; Yang, Xiao-Mei; Chen, Su; Sun, Fang-Lin

    2015-10-06

    Chromatin is a highly organized and dynamic structure in eukaryotic cells. The change of chromatin structure is essential in many cellular processes, such as gene transcription, DNA damage repair and others. Anti-silencing function 1 (ASF1) is a histone chaperone that participates in chromatin higher-order organization and is required for appropriate chromatin assembly. In this study, we identified the E2 ubiquitin-conjugating enzyme RAD6 as an evolutionary conserved interacting protein of ASF1 in D. melanogaster and H. sapiens that promotes the turnover of ASF1A by cooperating with a well-known E3 ligase, MDM2, via ubiquitin-proteasome pathway in H. sapiens. Further functional analyses indicated that the interplay between RAD6 and ASF1A associates with tumorigenesis. Together, these data suggest that the RAD6-MDM2 ubiquitin ligase machinery is critical for the degradation of chromatin-related proteins.

  3. The ubiquitin ligase HUWE1 regulates hematopoietic stem cell maintenance and lymphoid commitment

    PubMed Central

    King, Bryan; Boccalatte, Francesco; Moran-Crusio, Kelly; Wolf, Elmar; Wang, Jingjing; Kayembe, Clarisse; Lazaris, Charalampos; Yu, Xiaofeng; Aranda-Orgilles, Beatriz; Lasorella, Anna; Aifantis, Iannis

    2016-01-01

    Hematopoietic stem cells (HSCs) are dormant in the bone marrow and can be activated in response to diverse stresses to replenish all blood cell types. Here we identify the ubiquitin ligase Huwe1 as a crucial regulator of HSC functions via its post-translational control of N-myc. We found Huwe1 to be essential for HSC self-renewal, quiescence and lymphoid fate specification. Using a novel fluorescent fusion allele (MycnM), we observed that N-myc expression was restricted to the most immature, multipotent stem and progenitor populations. N-myc was upregulated in response to stress or upon loss of Huwe1, leading to increased proliferation and stem cell exhaustion. Mycn depletion reversed most of these phenotypes in vivo, suggesting that the attenuation of N-myc by Huwe1 is essential to reestablish homeostasis following stress. PMID:27668798

  4. A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration.

    PubMed

    Chu, Jessie; Hong, Nancy A; Masuda, Claudio A; Jenkins, Brian V; Nelms, Keats A; Goodnow, Christopher C; Glynne, Richard J; Wu, Hua; Masliah, Eliezer; Joazeiro, Claudio A P; Kay, Steve A

    2009-02-17

    A mouse neurological mutant, lister, was identified through a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Homozygous lister mice exhibit profound early-onset and progressive neurological and motor dysfunction. lister encodes a RING finger protein, LISTERIN, which functions as an E3 ubiquitin ligase in vitro. Although lister is widely expressed in all tissues, motor and sensory neurons and neuronal processes in the brainstem and spinal cord are primarily affected in the mutant. Pathological signs include gliosis, dystrophic neurites, vacuolated mitochondria, and accumulation of soluble hyperphosphorylated tau. Analysis with a different lister allele generated through targeted gene trap insertion reveals LISTERIN is required for embryonic development and confirms that direct perturbation of a LISTERIN-regulated process causes neurodegeneration. The lister mouse uncovers a pathway involved in neurodegeneration and may serves as a model for understanding the molecular mechanisms underlying human neurodegenerative disorders.

  5. A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration

    PubMed Central

    Chu, Jessie; Hong, Nancy A.; Masuda, Claudio A.; Jenkins, Brian V.; Nelms, Keats A.; Goodnow, Christopher C.; Glynne, Richard J.; Wu, Hua; Masliah, Eliezer; Joazeiro, Claudio A. P.; Kay, Steve A.

    2009-01-01

    A mouse neurological mutant, lister, was identified through a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Homozygous lister mice exhibit profound early-onset and progressive neurological and motor dysfunction. lister encodes a RING finger protein, LISTERIN, which functions as an E3 ubiquitin ligase in vitro. Although lister is widely expressed in all tissues, motor and sensory neurons and neuronal processes in the brainstem and spinal cord are primarily affected in the mutant. Pathological signs include gliosis, dystrophic neurites, vacuolated mitochondria, and accumulation of soluble hyperphosphorylated tau. Analysis with a different lister allele generated through targeted gene trap insertion reveals LISTERIN is required for embryonic development and confirms that direct perturbation of a LISTERIN-regulated process causes neurodegeneration. The lister mouse uncovers a pathway involved in neurodegeneration and may serves as a model for understanding the molecular mechanisms underlying human neurodegenerative disorders. PMID:19196968

  6. Merlin's tumor suppression linked to inhibition of the E3 ubiquitin ligase CRL4DCAF1

    PubMed Central

    Li, Wei

    2010-01-01

    The mechanism by which the FERM domain protein Merlin, encoded by the tumor suppressor NF2, restrains cell proliferation is poorly understood. Prior studies have suggested that Merlin exerts its antimitogenic effect by interacting with multiple signaling proteins located at or near the plasma membrane. We have recently observed that Merlin translocates into the nucleus and binds to and inhibits the E3 ubiquitin ligase CRL4DCAF1. Genetic evidence indicates that inactivation of Merlin induces oncogenic gene expression, hyperproliferation, and tumorigenicity by unleashing the activity of CRL4DCAF1. In addition to providing a potential explanation for the diverse effects that loss of Merlin exerts in multiple cell types, these findings suggest that compounds inhibiting CRL4DCAF1 may display therapeutic efficacy in Neurofibromatosis type 2 and other cancers driven by Merlin inactivation. PMID:21084862

  7. The Ubiquitin E3 Ligase NOSIP Modulates Protein Phosphatase 2A Activity in Craniofacial Development

    PubMed Central

    Hoffmeister, Meike; Prelle, Carola; Küchler, Philipp; Kovacevic, Igor; Moser, Markus; Müller-Esterl, Werner; Oess, Stefanie

    2014-01-01

    Holoprosencephaly is a common developmental disorder in humans characterised by incomplete brain hemisphere separation and midface anomalies. The etiology of holoprosencephaly is heterogeneous with environmental and genetic causes, but for a majority of holoprosencephaly cases the genes associated with the pathogenesis could not be identified so far. Here we report the generation of knockout mice for the ubiquitin E3 ligase NOSIP. The loss of NOSIP in mice causes holoprosencephaly and facial anomalies including cleft lip/palate, cyclopia and facial midline clefting. By a mass spectrometry based protein interaction screen we identified NOSIP as a novel interaction partner of protein phosphatase PP2A. NOSIP mediates the monoubiquitination of the PP2A catalytic subunit and the loss of NOSIP results in an increase in PP2A activity in craniofacial tissue in NOSIP knockout mice. We conclude, that NOSIP is a critical modulator of brain and craniofacial development in mice and a candidate gene for holoprosencephaly in humans. PMID:25546391

  8. Fbxw7β, E3 ubiquitin ligase, negative regulation of primary myoblast differentiation, proliferation and migration.

    PubMed

    Shin, Kyungshin; Hwang, Sang-Gu; Choi, Ik Joon; Ko, Young-Gyu; Jeong, Jaemin; Kwon, Heechung

    2017-04-01

    Satellite cells attached to skeletal muscle fibers play a crucial role in skeletal muscle regeneration. During regeneration, the satellite cells proliferate, migrate to the damaged region, and fuse to each other. Although it is important to determine the cellular mechanisms controlling myoblast behavior, their regulators are not well understood. In this study, we evaluated the roles of Fbxw7 in primary myoblasts and determined its potential as a therapeutic target for muscle disease. We originally found that Fbxw7β, one of the E3 ubiquitin ligase Fbxw7 subtypes, negatively regulates differentiation, proliferation and migration of myoblasts and satellite cells on muscle fiber. However, these phenomena were not observed in myoblasts expressing a dominant-negative, F-box deleted Fbxw7β, mutant. Our results suggest that myoblast differentiation potential and muscle regeneration can be regulated by Fbxw7β.

  9. E3 ubiquitin ligase Cbl-b suppresses human ORMDL3 expression through STAT6 mediation.

    PubMed

    Yang, Wei-Xia; Jin, Rui; Jiang, Chun-Ming; Wang, Xiao-Hua; Shu, Jin; Li, Ling; Zhu, Liang-Hua; Zhuang, Li-Li; Gao, Chao; Zhou, Guo-Ping

    2015-07-08

    Orosomucoid 1-Like Protein 3 (ORMDL3) is an asthma candidate gene and Casitas B lineage lymphoma b (Cbl-b), an E3 ubiquitin ligase, is a critical factor in maintaining airway immune tolerance. However, the association of Cbl-b with ORMDL3 for asthma is unclear. Here, we show that expression of ORMDL3 is significantly increased and shows a strong linear correlation with decreased Cbl-b in the peripheral blood of recurrent wheeze patients. To elucidate the molecular mechanisms underlying this correlation, we identified that Cbl-b suppressed the transcriptional activity and mRNA expression of ORMDL3 in vivo. Further investigation showed that phosphorylation of signal transducer and activator of transcription 6 (STAT6) was induced by interleukin 4 bound to the ORMDL3 promoter, while Cbl-b reduced the phosphorylation of STAT6. Our results show that Cbl-b suppresses human ORMDL3 expression through STAT6.

  10. Cortical dynamics during cell motility are regulated by CRL3KLHL21 E3 ubiquitin ligase

    PubMed Central

    Courtheoux, Thibault; Enchev, Radoslav I.; Lampert, Fabienne; Gerez, Juan; Beck, Jochen; Picotti, Paola; Sumara, Izabela; Peter, Matthias

    2016-01-01

    Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3KLHL21 E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3KLHL21 activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions. PMID:27641145

  11. Nrdp1/FLRF is a ubiquitin ligase promoting ubiquitination and degradation of the epidermal growth factor receptor family member, ErbB3

    PubMed Central

    Qiu, Xiao-Bo; Goldberg, Alfred L.

    2002-01-01

    The epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases plays fundamental roles in the regulation of cell survival, proliferation, and differentiation. Here, we present evidence that ErbB3 is degraded by proteasomes, and that Nrdp1 (referred to as FLRF in mice) associates with ErbB3 and stimulates its ubiquitination and degradation by proteasomes. Nrdp1 mRNAs are expressed in a variety of human tissues. The N-terminal half of Nrdp1 possesses an atypical RING finger domain, which is required for enhancing ErbB3 degradation. Its C-terminal half by itself associates with ErbB3 and raises ErbB3 levels in cells, probably by acting as a dominant–negative form of Nrdp1. In cell-free systems, Nrdp1 has ubiquitin ligase (E3) activity and ubiquitinates ErbB3, as well as itself, in the presence of the ubiquitin-carrier protein (E2), UbcH5. These data indicate that Nrdp1 is a RING finger-type of ubiquitin ligase, which promotes degradation of ErbB3 by proteasomes and, thus, may be an important factor influencing cell growth. PMID:12411582

  12. Nrdp1/FLRF is a ubiquitin ligase promoting ubiquitination and degradation of the epidermal growth factor receptor family member, ErbB3.

    PubMed

    Qiu, Xiao-Bo; Goldberg, Alfred L

    2002-11-12

    The epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases plays fundamental roles in the regulation of cell survival, proliferation, and differentiation. Here, we present evidence that ErbB3 is degraded by proteasomes, and that Nrdp1 (referred to as FLRF in mice) associates with ErbB3 and stimulates its ubiquitination and degradation by proteasomes. Nrdp1 mRNAs are expressed in a variety of human tissues. The N-terminal half of Nrdp1 possesses an atypical RING finger domain, which is required for enhancing ErbB3 degradation. Its C-terminal half by itself associates with ErbB3 and raises ErbB3 levels in cells, probably by acting as a dominant-negative form of Nrdp1. In cell-free systems, Nrdp1 has ubiquitin ligase (E3) activity and ubiquitinates ErbB3, as well as itself, in the presence of the ubiquitin-carrier protein (E2), UbcH5. These data indicate that Nrdp1 is a RING finger-type of ubiquitin ligase, which promotes degradation of ErbB3 by proteasomes and, thus, may be an important factor influencing cell growth.

  13. Mining and characterization of ubiquitin E3 ligases expressed in the mouse testis

    PubMed Central

    2012-01-01

    Background Ubiquitin-mediated protein modification and degradation are believed to play important roles in mammalian spermatogenesis. The catalogues of ubiquitin activating enzymes, conjugating enzymes, and ligases (E3s) have been known for mammals such as mice and humans. However, a systematic characterization of E3s expressed during spermatogenesis has not been carried out. Results In present study, we set out to mine E3s from the mouse genome and to characterize their expression pattern, subcellular localization, and enzymatic activities based on microarray data and biochemical assays. We identified 398 putative E3s belonging to the RING, U-box, and HECT subfamilies and found that most genes were conserved between mice and humans. We discovered that 73 of them were highly or specifically expressed in the testes based on the microarray expression data. We selected 10 putative E3 genes to examine their mRNA expression pattern, and several genes to study their subcellular localization and E3 ligase activity. RT-PCR results showed that all the selected genes were predominately expressed in the testis. Some putative E3s were localized in the cytoplasm while others were in both the cytoplasm and the nucleus. Moreover, all the selected proteins were enzymatically active as demonstrated by in vitro and in vivo assays. Conclusions We have identified a large number of putative E3s that are expressed during mouse spermatogenesis. Among these, a significant portion is highly or specifically expressed in the testis. Subcellular localization and enzymatic activity assays suggested that these E3s might execute diverse functions in mammalian spermatogenesis. Our results may serve as an initial guide to the field for further functional analysis. PMID:22992278

  14. Probes of Ubiquitin E3 ligases distinguish different stages of Parkin activation

    PubMed Central

    Pao, Kuan-Chuan; Stanley, Mathew; Han, Cong; Lai, Yu-Chiang; Murphy, Paul; Balk, Kristin; Wood, Nicola T.; Corti, Olga; Corvol, Jean-Christophe; Muqit, Miratul M.K.; Virdee, Satpal

    2016-01-01

    E3 ligases represent an important class of enzymes, yet there are currently no chemical probes to profile their activity. We develop a new class of activity-based probe by reengineering of a ubiquitin-charged E2 conjugating enzyme and demonstrate their utility by profiling the transthiolation activity of the RING-in-between-RING (RBR) E3 ligase Parkin in vitro and in cellular extracts. Our study provides valuable insight into the roles, and cellular hierarchy, of distinct phosphorylation events in Parkin activation. We also profile Parkin patient disease-associated mutations and strikingly demonstrate that they largely mediate their effect by altering transthiolation activity. Furthermore, our probes enable direct and quantitative measurement of endogenous Parkin activity revealing that endogenous Parkin is activated in neuronal cell lines (≥75 %) in response to mitochondrial depolarization. This new technology also holds promise as a novel biomarker of PINK1-Parkin signalling as demonstrated by compatibility with Parkinson’s disease patient-derived samples. PMID:26928937

  15. Defective in Mitotic Arrest 1 (Dma1) Ubiquitin Ligase Controls G1 Cyclin Degradation*

    PubMed Central

    Hernández-Ortega, Sara; Bru, Samuel; Ricco, Natalia; Ramírez, Sara; Casals, Núria; Jiménez, Javier; Isasa, Marta; Crosas, Bernat; Clotet, Josep

    2013-01-01

    Progression through the G1 phase of the cell cycle is controlled by diverse cyclin-dependent kinases (CDKs) that might be associated to numerous cyclin isoforms. Given such complexity, regulation of cyclin degradation should be crucial for coordinating progression through the cell cycle. In Saccharomyces cerevisiae, SCF is the only E3 ligase known to date to be involved in G1 cyclin degradation. Here, we report the design of a genetic screening that uncovered Dma1 as another E3 ligase that targets G1 cyclins in yeast. We show that the cyclin Pcl1 is ubiquitinated in vitro and in vivo by Dma1, and accordingly, is stabilized in dma1 mutants. We demonstrate that Pcl1 must be phosphorylated by its own CDK to efficiently interact with Dma1 and undergo degradation. A nonphosphorylatable version of Pcl1 accumulates throughout the cell cycle, demonstrating the physiological relevance of the proposed mechanism. Finally, we present evidence that the levels of Pcl1 and Cln2 are independently controlled in response to nutrient availability. This new previously unknown mechanism for G1 cyclin degradation that we report here could help elucidate the specific roles of the redundant CDK-cyclin complexes in G1. PMID:23264631

  16. SVIP regulates Z variant alpha-1 antitrypsin retro-translocation by inhibiting ubiquitin ligase gp78

    PubMed Central

    Khodayari, Nazli; Wang, Rejean liqun; Marek, George; Krotova, Karina; Kirst, Mariana; Liu, Chen; Rouhani, Farshid; Brantly, Mark

    2017-01-01

    Alpha-1 antitrypsin deficiency (AATD) is an inherited disorder characterized by early-onset emphysema and liver disease. The most common disease-causing mutation is a single amino acid substitution (Glu/Lys) at amino acid 342 of the mature protein, resulting in disruption of the 290–342 salt bridge (an electrophoretic abnormality defining the mutation [Z allele, or ZAAT]), protein misfolding, polymerization, and accumulation in the endoplasmic reticulum of hepatocytes and monocytes. The Z allele causes a toxic gain of function, and the E3 ubiquitin ligase gp78 promotes degradation and increased solubility of endogenous ZAAT. We hypothesized that the accumulation of ZAAT is influenced by modulation of gp78 E3 ligase and SVIP (small VCP-interacting protein) interaction with p97/VCP in ZAAT-expressing hepatocytes. We showed that the SVIP inhibitory effect on ERAD due to overexpression causes the accumulation of ZAAT in a human Z hepatocyte–like cell line (AT01). Overexpression of gp78, as well as SVIP suppression, induces gp78-VCP/p97 interaction in AT01 cells. This interaction leads to retro-translocation of ZAAT and reduction of the SVIP inhibitory role in ERAD. In this context, overexpression of gp78 or SVIP suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD. PMID:28301499

  17. The von Hippel-Lindau tumor suppressor protein is a component of an E3 ubiquitin-protein ligase activity.

    PubMed

    Lisztwan, J; Imbert, G; Wirbelauer, C; Gstaiger, M; Krek, W

    1999-07-15

    pVHL, the product of the VHL tumor suppressor gene, plays an important role in the regulation of cell growth and differentiation of human kidney cells, and inactivation of the VHL gene is the most frequent genetic event in human kidney cancer. The biochemical function of pVHL is unknown. Here we report that pVHL exists in vivo in a complex that displays ubiquitination-promoting activity in conjunction with the universally required components E1, E2, and ubiquitin. pVHL-associated ubiquitination activity requires, at a minimum, pVHL to bind elongin C and Cul-2, relatives of core components of SCF (Skp1-Cdc53/Cul-1-F-box protein) E3 ligase complexes. Notably, certain tumor-derived mutants of pVHL demonstrate loss of associated ubiquitination promoting activity. These results identify pVHL as a component of a potential SCF-like E3 ubiquitin-protein ligase complex and suggest a direct link between pVHL tumor suppressor and the process of ubiquitination.

  18. Genome-wide identification and gene expression profiling of ubiquitin ligases for endoplasmic reticulum protein degradation

    PubMed Central

    Kaneko, Masayuki; Iwase, Ikuko; Yamasaki, Yuki; Takai, Tomoko; Wu, Yan; Kanemoto, Soshi; Matsuhisa, Koji; Asada, Rie; Okuma, Yasunobu; Watanabe, Takeshi; Imaizumi, Kazunori; Nomura, Yausyuki

    2016-01-01

    Endoplasmic reticulum (ER)-associated degradation (ERAD) is a mechanism by which unfolded proteins that accumulate in the ER are transported to the cytosol for ubiquitin–proteasome-mediated degradation. Ubiquitin ligases (E3s) are a group of enzymes responsible for substrate selectivity and ubiquitin chain formation. The purpose of this study was to identify novel E3s involved in ERAD. Thirty-seven candidate genes were selected by searches for proteins with RING-finger motifs and transmembrane regions, which are the major features of ERAD E3s. We performed gene expression profiling for the identified E3s in human and mouse tissues. Several genes were specifically or selectively expressed in both tissues; the expression of four genes (RNFT1, RNF185, CGRRF1 and RNF19B) was significantly upregulated by ER stress. To determine the involvement of the ER stress-responsive genes in ERAD, we investigated their ER localisation, in vitro autoubiquitination activity and ER stress resistance. All were partially localised to the ER, whereas CGRRF1 did not possess E3 activity. RNFT1 and RNF185, but not CGRRF1 and RNF19B, exhibited significant resistance to ER stressor in an E3 activity-dependent manner. Thus, these genes are possible candidates for ERAD E3s. PMID:27485036

  19. CRL4A(CRBN) E3 ubiquitin ligase restricts BK channel activity and prevents epileptogenesis.

    PubMed

    Liu, Jiye; Ye, Jia; Zou, Xiaolong; Xu, Zhenghao; Feng, Yan; Zou, Xianxian; Chen, Zhong; Li, Yuezhou; Cang, Yong

    2014-05-21

    Ion channels regulate membrane excitation, and mutations of ion channels often cause serious neurological disorders including epilepsy. Compared with extensive analyses of channel protein structure and function, much less is known about the fine tuning of channel activity by post-translational modification. Here we report that the large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels are targeted by the E3 ubiquitin ligase CRL4A(CRBN) for polyubiquitination and retained in the endoplasmic reticulum (ER). Inactivation of CRL4A(CRBN) releases deubiquitinated BK channels from the ER to the plasma membrane, leading to markedly enhanced channel activity. Mice with CRL4A(CRBN) mutation in the brain or treated with a CRL4A(CRBN) inhibitor are very sensitive to seizure induction, which can be attenuated by blocking BK channels. Finally, the mutant mice develop spontaneous epilepsy when aged. Therefore, ubiquitination of BK channels before their cell surface expression is an important step to prevent systemic neuronal excitability and epileptogenesis.

  20. A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase.

    PubMed

    Matyskiela, Mary E; Lu, Gang; Ito, Takumi; Pagarigan, Barbra; Lu, Chin-Chun; Miller, Karen; Fang, Wei; Wang, Nai-Yu; Nguyen, Derek; Houston, Jack; Carmel, Gilles; Tran, Tam; Riley, Mariko; Nosaka, Lyn'Al; Lander, Gabriel C; Gaidarova, Svetlana; Xu, Shuichan; Ruchelman, Alexander L; Handa, Hiroshi; Carmichael, James; Daniel, Thomas O; Cathers, Brian E; Lopez-Girona, Antonia; Chamberlain, Philip P

    2016-07-14

    Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4(CRBN) E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumour activity. The anti-tumour activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degradation of the translation termination factor GSPT1. Patient-derived acute myeloid leukaemia tumour cells exhibit high sensitivity to CC-885, indicating the clinical potential of this mechanism. Crystallographic studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn containing a glycine residue at a key position, interacting with both CC-885 and a 'hotspot' on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homology to previously known cereblon substrates, mutational analysis and modelling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon 'neosubstrate' selectivity, and identify an anti-tumour target rendered druggable by cereblon modulation.

  1. Evidence of an Antimicrobial Peptide Signature Encrypted in HECT E3 Ubiquitin Ligases

    PubMed Central

    Candido-Ferreira, Ivan Lavander; Kronenberger, Thales; Sayegh, Raphael Santa Rosa; Batista, Isabel de Fátima Correia; da Silva Junior, Pedro Ismael

    2017-01-01

    The ubiquitin-proteasome pathway (UPP) is a hallmark of the eukaryotic cell. In jawed vertebrates, it has been co-opted by the adaptive immune system, where proteasomal degradation produces endogenous peptides for major histocompatibility complex class I antigen presentation. However, proteolytic products are also necessary for the phylogenetically widespread innate immune system, as they often play a role as host defense peptides (HDPs), pivotal effectors against pathogens. Here, we report the identification of the arachnid HDP oligoventin, which shares homology to a core member of the UPP, E3 ubiquitin ligases. Oligoventin has broad antimicrobial activity and shows strong synergy with lysozymes. Using computational and phylogenetic approaches, we show high conservation of the oligoventin signature in HECT E3s. In silico simulation of HECT E3s self-proteolysis provides evidence that HDPs can be generated by fine-tuned 26S proteasomal degradation, and therefore are consistent with the hypothesis that oligoventin is a cryptic peptide released by the proteolytic processing of an Nedd4 E3 precursor protein. Finally, we compare the production of HDPs and endogenous antigens from orthologous HECT E3s by proteasomal degradation as a means of analyzing the UPP coupling to metazoan immunity. Our results highlight the functional plasticity of the UPP in innate and adaptive immune systems as a possibly recurrent mechanism to generate functionally diverse peptides. PMID:28119686

  2. The HECTD3 E3 ubiquitin ligase suppresses cisplatin-induced apoptosis via stabilizing MALT1.

    PubMed

    Li, Yi; Chen, Xi; Wang, Zehua; Zhao, Dong; Chen, Hui; Chen, Wenlin; Zhou, Zhongmei; Zhang, Junran; Zhang, Jing; Li, Hongmin; Chen, Ceshi

    2013-01-01

    Homologous to the E6-associated protein carboxyl terminus domain containing 3 (HECTD3) is an E3 ubiquitin ligase with unknown functions. Here, we show that HECTD3 confers cancer cell resistance to cisplatin. To understand the molecular mechanisms, we performed a yeast two-hybrid analysis and identified mucosa-associated lymphoid tissue 1 (MALT1) as an HECTD3-interacting protein. HECTD3 promotes MALT1 ubiquitination with nondegradative polyubiquitin chains by direct interacting with the MALT1 through its N-terminal destruction of cyclin domain. HECTD3 does not target MALT1 for degradation but stabilize it. HECTD3 depletion dramatically decreases the levels of MALT1 in MCF7 and HeLa cells treated with cisplatin, which is correlated to an increase in apoptosis. Knockdown of MALT1 likewise increases cisplatin-induced apoptosis in these cancer cells. However, HECTD3 over-expression leads to a decreased cisplatin-induced apoptosis, whereas overexpression of MALT1 partially rescues HECTD3 depletion-induced apoptosis. These findings suggest that HECTD3 promotes cell survival through stabilizing MALT1. Our data have important implications in cancer therapy by providing novel molecular targets.

  3. Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2.

    PubMed

    Heyne, Kristina; Förster, Juliane; Schüle, Roland; Roemer, Klaus

    2014-04-01

    NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator.

  4. Ubiquitination of HLA-DO by MARCH family E3 ligases

    PubMed Central

    Jahnke, Martin; Trowsdale, John; Kelly, Adrian P

    2013-01-01

    HLA-DO (DO) is a nonclassical MHC class II (MHCII) molecule that negatively regulates the ability of HLA-DM to catalyse the removal of invariant chain-derived CLIP peptides from classical MHCII molecules. Here, we show that DO is posttranslationally modified by ubiquitination. The location of the modified lysine residue is shared with all classical MHCII beta chains, suggesting a conserved function. Three membrane-associated RING-CH (MARCH1, 8 and 9) family E3 ligases that polyubiquitinate MHCII induce similar profiles of polyubiquitination on DOβ. All three MARCH proteins also influenced trafficking of DO indirectly by a mechanism that required the DOβ encoded di-leucine and tyrosine-based endocytosis motifs. This may be the result of MARCH-induced ubiquitination of components of the endocytic machinery. MARCH9 was by far the most efficient at inducing intracellular redistribution of DO but did not target molecules for lysosomal degradation. The specificity of MARCH9 for HLA-DQ and HLA-DO suggests a need for common regulation of these two MHC-encoded molecules. PMID:23400868

  5. Acquired platinum resistance involves epithelial to mesenchymal transition through ubiquitin ligase FBXO32 dysregulation

    PubMed Central

    Tanaka, Nobuyuki; Miyazaki, Yasumasa; Mikami, Shuji; Niwa, Naoya; Otsuka, Yutaro; Mizuno, Ryuichi; Kikuchi, Eiji; Miyajima, Akira; Sabe, Hisataka; Okada, Yasunori; Suematsu, Makoto; Oya, Mototsugu

    2016-01-01

    To identify the molecules involved in epithelial to mesenchymal transition (EMT) in urothelial carcinoma (UC) after acquisition of platinum resistance, here we examined the changes in global gene expression before and after platinum treatment. Four invasive UC cell lines, T24, 5637, and their corresponding sublines T24PR and 5637PR with acquired platinum resistance, were assessed by microarray, and the ubiquitin E3 ligase FBXO32 was newly identified as a negative regulator of EMT in UC tumors after acquisition of platinum resistance. In vitro and in vivo studies showed an intimate relationship between FBXO32 expression and EMT, demonstrating that FBXO32 dysregulation in T24PR cells results in elevated expression of the mesenchymal molecules SNAIL and vimentin and decreased expression of the epithelial molecule E-cadherin. The association between FBXO32 expression and EMT was further validated using clinical samples. Knockdown of MyoD expression, a specific target of FBXO32 polyubiquitination, revealed upregulation of E-cadherin expression and downregulation of SNAIL and vimentin expression in T24PR cells. Comparative genomic hybridization array analysis demonstrated loss of heterozygosity at 8q24.13 in T24PR cells, which harbors FBXO32. Our findings suggest the importance of the association between EMT and ubiquitin-proteasome regulation when tumors develop acquired platinum resistance. PMID:27812537

  6. Acquired platinum resistance involves epithelial to mesenchymal transition through ubiquitin ligase FBXO32 dysregulation.

    PubMed

    Tanaka, Nobuyuki; Kosaka, Takeo; Miyazaki, Yasumasa; Mikami, Shuji; Niwa, Naoya; Otsuka, Yutaro; Minamishima, Yoji Andrew; Mizuno, Ryuichi; Kikuchi, Eiji; Miyajima, Akira; Sabe, Hisataka; Okada, Yasunori; Uhlén, Per; Suematsu, Makoto; Oya, Mototsugu

    2016-11-03

    To identify the molecules involved in epithelial to mesenchymal transition (EMT) in urothelial carcinoma (UC) after acquisition of platinum resistance, here we examined the changes in global gene expression before and after platinum treatment. Four invasive UC cell lines, T24, 5637, and their corresponding sublines T24PR and 5637PR with acquired platinum resistance, were assessed by microarray, and the ubiquitin E3 ligase FBXO32 was newly identified as a negative regulator of EMT in UC tumors after acquisition of platinum resistance. In vitro and in vivo studies showed an intimate relationship between FBXO32 expression and EMT, demonstrating that FBXO32 dysregulation in T24PR cells results in elevated expression of the mesenchymal molecules SNAIL and vimentin and decreased expression of the epithelial molecule E-cadherin. The association between FBXO32 expression and EMT was further validated using clinical samples. Knockdown of MyoD expression, a specific target of FBXO32 polyubiquitination, revealed upregulation of E-cadherin expression and downregulation of SNAIL and vimentin expression in T24PR cells. Comparative genomic hybridization array analysis demonstrated loss of heterozygosity at 8q24.13 in T24PR cells, which harbors FBXO32. Our findings suggest the importance of the association between EMT and ubiquitin-proteasome regulation when tumors develop acquired platinum resistance.

  7. Rines E3 ubiquitin ligase regulates MAO-A levels and emotional responses.

    PubMed

    Kabayama, Miyuki; Sakoori, Kazuto; Yamada, Kazuyuki; Ornthanalai, Veravej G; Ota, Maya; Morimura, Naoko; Katayama, Kei-ichi; Murphy, Niall P; Aruga, Jun

    2013-08-07

    Monoamine oxidase A (MAO-A), the catabolic enzyme of norepinephrine and serotonin, plays a critical role in emotional and social behavior. However, the control and impact of endogenous MAO-A levels in the brain remains unknown. Here we show that the RING finger-type E3 ubiquitin ligase Rines/RNF180 regulates brain MAO-A subset, monoamine levels, and emotional behavior. Rines interacted with MAO-A and promoted its ubiquitination and degradation. Rines knock-out mice displayed impaired stress responses, enhanced anxiety, and affiliative behavior. Norepinephrine and serotonin levels were altered in the locus ceruleus, prefrontal cortex, and amygdala in either stressed or resting conditions, and MAO-A enzymatic activity was enhanced in the locus ceruleus in Rines knock-out mice. Treatment of Rines knock-out mice with MAO inhibitors showed genotype-specific effects on some of the abnormal affective behaviors. These results indicated that the control of emotional behavior by Rines is partly due to the regulation of MAO-A levels. These findings verify that Rines is a critical regulator of the monoaminergic system and emotional behavior and identify a promising candidate drug target for treating diseases associated with emotion.

  8. An allosteric inhibitor of substrate recognition by the SCF[superscript Cdc4] ubiquitin ligase

    SciTech Connect

    Orlicky, Stephen; Tang, Xiaojing; Neduva, Victor; Elowe, Nadine; Brown, Eric D.; Sicheri, Frank; Tyers, Mike

    2010-09-17

    The specificity of SCF ubiquitin ligase-mediated protein degradation is determined by F-box proteins. We identified a biplanar dicarboxylic acid compound, called SCF-I2, as an inhibitor of substrate recognition by the yeast F-box protein Cdc4 using a fluorescence polarization screen to monitor the displacement of a fluorescein-labeled phosphodegron peptide. SCF-I2 inhibits the binding and ubiquitination of full-length phosphorylated substrates by SCF{sup Cdc4}. A co-crystal structure reveals that SCF-I2 inserts itself between the {beta}-strands of blades 5 and 6 of the WD40 propeller domain of Cdc4 at a site that is 25 {angstrom} away from the substrate binding site. Long-range transmission of SCF-I2 interactions distorts the substrate binding pocket and impedes recognition of key determinants in the Cdc4 phosphodegron. Mutation of the SCF-I2 binding site abrogates its inhibitory effect and explains specificity in the allosteric inhibition mechanism. Mammalian WD40 domain proteins may exhibit similar allosteric responsiveness and hence represent an extensive class of druggable target.

  9. Yeast Rsp5 ubiquitin ligase affects the actin cytoskeleton in vivo and in vitro.

    PubMed

    Kaminska, Joanna; Spiess, Matthias; Stawiecka-Mirota, Marta; Monkaityte, Rasa; Haguenauer-Tsapis, Rosine; Urban-Grimal, Daniele; Winsor, Barbara; Zoladek, Teresa

    2011-12-01

    Yeast Rsp5 ubiquitin ligase is involved in several cellular processes, including endocytosis. Actin patches are sites of endocytosis, a process involving actin assembly and disassembly. Here we show Rsp5 localization in cortical patches and demonstrate its involvement in actin cytoskeleton organization and dynamics. We found that the Rsp5-F1-GFP2 N-terminal fragment and full length GFP-Rsp5 were recruited to peripheral patches that temporarily co-localized with Abp1-mCherry, a marker of actin patches. Actin cytoskeleton organization was defective in a strain lacking RSP5 or overexpressing RSP5, and this phenotype was accompanied by morphological abnormalities. Overexpression of RSP5 caused hypersensitivity of cells to Latrunculin A, an actin-depolymerizing drug and was toxic to cells lacking Las17, an activator of actin nucleation. Moreover, Rsp5 was required for efficient actin polymerization in a whole cell extract based in vitro system. Rsp5 interacted with Las17 and Las17-binding proteins, Lsb1 and Lsb2, in a GST-Rsp5-WW2/3 pull down assay. Rsp5 ubiquitinated Lsb1-HA and Lsb2-HA without directing them for degradation. Overexpression of RSP5 increased the cellular level of HA-Las17 in wild type and in lsb1Δ lsb2Δ strains in which the basal level of Las17 was already elevated. This increase was prevented in a strain devoid of Las17-binding protein Sla1 which is also a target of Rsp5 ubiquitination. Thus, Rsp5 together with Lsb1, Lsb2 and Sla1 regulate the level of Las17, an important activator of actin polymerization.

  10. The ubiquitin ligase RNF126 regulates the retrograde sorting of the cation-independent mannose 6-phosphate receptor.

    PubMed

    Smith, Christopher J; McGlade, C Jane

    2014-01-15

    The ubiquitin proteasome system is central to the regulation of a number of intracellular sorting pathways in mammalian cells including quality control at the endoplasmic reticulum and the internalization and endosomal sorting of cell surface receptors. Here we describe that RNF126, an E3 ubiquitin ligase, is involved in the sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR). In cells transiently depleted of RNF126, the CI-MPR is dispersed into Rab4 positive endosomes and the efficiency of retrograde sorting is delayed. Furthermore, the stable knockdown of RNF126 leads to the lysosomal degradation of CI-MPR and missorting of cathepsin D. RNF126 specifically regulates the sorting of the CI-MPR as other cargo that follow the retrograde sorting route including the cholera toxin, furin and TGN38 are unaffected in the absence of RNF126. Lastly we show that the RING finger domain of RNF126 is required to rescue the decrease in CI-MPR levels, suggesting that the ubiquitin ligase activity of RNF126 is required for CI-MPR sorting. Together, our data indicate that the ubiquitin ligase RNF126 has a role in the retrograde sorting of the CI-MPR.

  11. A Family of Salmonella Virulence Factors Functions as a Distinct Class of Autoregulated E3 Ubiquitin Ligases

    SciTech Connect

    Quezada, C.; Hicks, S; Galan, J; Stebbins, C

    2009-01-01

    Processes as diverse as receptor binding and signaling, cytoskeletal dynamics, and programmed cell death are manipulated by mimics of host proteins encoded by pathogenic bacteria. We show here that the Salmonella virulence factor SspH2 belongs to a growing class of bacterial effector proteins that harness and subvert the eukaryotic ubiquitination pathway. This virulence protein possesses ubiquitination activity that depends on a conserved cysteine residue. A crystal structure of SspH2 reveals a canonical leucine-rich repeat (LRR) domain that interacts with a unique E{sub 3} ligase [which we have termed NEL for Novel E{sub 3} Ligase] C-terminal fold unrelated to previously observed HECT or RING-finger E{sub 3} ligases. Moreover, the LRR domain sequesters the catalytic cysteine residue contained in the NEL domain, and we suggest a mechanism for activation of the ligase requiring a substantial conformational change to release the catalytic domain for function. We also show that the N-terminal domain targets SspH2 to the apical plasma membrane of polarized epithelial cells and propose a model whereby binding of the LRR to proteins at the target site releases the ligase domain for site-specific function.

  12. Regulation of neddylation and deneddylation of cullin1 in SCFSkp2 ubiquitin ligase by F-box protein and substrate

    PubMed Central

    Bornstein, Gil; Ganoth, Dvora; Hershko, Avram

    2006-01-01

    The activity of cullin-containing ubiquitin protein ligase complexes is stimulated by linkage to cullin of the ubiquitin-like protein Nedd8 (“neddylation”). Neddylation is inhibited by the tight binding of cullins to CAND1 (cullin-associated and neddylation-dissociated 1) protein, and Nedd8 is removed from cullins by specific isopeptidase activity of the COP9/signalosome (CSN) complex. The mechanisms that regulate neddylation and deneddylation of cullins were unknown. We examined this problem for the case of SCFSkp2, a cullin1 (Cul1)-containing ubiquitin ligase complex that contains the S phase-associated protein Skp2 as the substrate-binding F-box protein subunit. SCFSkp2 targets for degradation the cyclin-dependent kinase (cdk) inhibitor p27 in the G1-to-S phase transition, a process that requires its phosphorylation and binding to cdk2-cyclin E. Because levels of Skp2, cyclin E, and the accessory protein Cks1 (cyclin kinase subunit 1) all rise at the end of G1 phase, it seemed possible that the neddylation of Cul1 in SCFSkp2 is regulated by the availability of the F-box protein and/or the substrate. We found that the supplementation of Skp2–Skp1 and substrate (along with further components necessary for substrate presentation to the ubiquitin ligase) to extracts of HeLa cells synergistically increased levels of neddylated Cul1. Skp2–Skp1 abrogates the inhibitory influence of CAND1 on the neddylation of Cul1 by promoting the dissociation of the cullin–CAND1 complex, whereas substrate, together with substrate-presenting components, prevents the action of CSN to deneddylate cullin. We propose a sequence of events in which the increased availability of Skp2 and substrate in the transition of cells to S phase promotes the neddylation and assembly of the SCFSkp2 ubiquitin ligase complex. PMID:16861300

  13. Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo.

    PubMed

    Pires-Oliveira, Marcelo; Maragno, Ana Leticia G C; Parreiras-e-Silva, Lucas T; Chiavegatti, Tiago; Gomes, Marcelo D; Godinho, Rosely O

    2010-02-01

    Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. In the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. In addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.

  14. The multidrug resistance pump ABCB1 is a substrate for the ubiquitin ligase NEDD4-1

    PubMed Central

    Akkaya, Begum G.; Zolnerciks, Joseph K.; Ritchie, Tasha K.; Bauer, Bjoern; Hartz, Anika M.S.; Sullivan, James A.; Linton, Kenneth J.

    2016-01-01

    The ATP Binding Cassette transporter ABCB1 can export the neurotoxic peptide β-amyloid from endothelial cells that line the blood-brain barrier (BBB). This has the potential to lower cerebral levels of β-amyloid, but ABCB1 expression in the BBB appears to be progressively reduced in patients with Alzheimer's disease. The surface density of many membrane proteins is regulated by ubiquitination catalysed by ubiquitin E3 ligases. In brain capillaries of mice challenged with β-amyloid ex vivo, we show that the level of the ubiquitin ligase Nedd4 increases concomitant with reduction in Abcb1. In vitro we show that human ABCB1 is a substrate for human NEDD4-1 ligase. Recombinant ABCB1 was purified from Sf21 insect cells and incubated with recombinant NEDD4-1 purified from E. coli. The treated ABCB1 had reduced mobility on SDS-PAGE, and mass spectrometry identified eight lysine residues, K271, K272, K575, K685, K877, K885, K887 and K1062 that were ubiquitinated by NEDD4-1. Molecular modelling showed that all of the residues are exposed on the surface of the intracellular domains of ABCB1. K877, K885 and K887 in particular, are located in the intracellular loop of transmembrane helix 10 (TMH10) in close proximity, in the tertiary fold, to a putative NEDD4-1 binding site in the intracellular helix extending from TMH12 (PxY motif, residues 996-998). Transient expression of NEDD4-1 in HEK293 Flp-In cells stably expressing ABCB1 was shown to reduce the surface density of the transporter. Together, the data identify this ubiquitin ligase as a potential target for intervention in the pathophysiology of Alzheimer's disease. PMID:26006083

  15. Inactivation of the putative ubiquitin-E3 ligase PDLIM2 in classical Hodgkin and anaplastic large cell lymphoma

    PubMed Central

    Wurster, K D; Hummel, F; Richter, J; Giefing, M; Hartmann, S; Hansmann, M-L; Kreher, S; Köchert, K; Krappmann, D; Klapper, W; Hummel, M; Wenzel, S-S; Lenz, G; Janz, M; Dörken, B; Siebert, R; Mathas, S

    2017-01-01

    Apart from its unique histopathological appearance with rare tumor cells embedded in an inflammatory background of bystander cells, classical Hodgkin lymphoma (cHL) is characterized by an unusual activation of a broad range of signaling pathways involved in cellular activation. This includes constitutive high-level activity of nuclear factor-κB (NF-κB), Janus kinase/signal transducer and activator of transcription (JAK/STAT), activator protein-1 (AP-1) and interferon regulatory factor (IRF) transcription factors (TFs) that are physiologically only transiently activated. Here, we demonstrate that inactivation of the putative ubiquitin E3-ligase PDLIM2 contributes to this TF activation. PDLIM2 expression is lost at the mRNA and protein levels in the majority of cHL cell lines and Hodgkin and Reed–Sternberg (HRS) cells of nearly all cHL primary samples. This loss is associated with PDLIM2 genomic alterations, promoter methylation and altered splicing. Reconstitution of PDLIM2 in HRS cell lines inhibits proliferation, blocks NF-κB transcriptional activity and contributes to cHL-specific gene expression. In non-Hodgkin B-cell lines, small interfering RNA-mediated PDLIM2 knockdown results in superactivation of TFs NF-κB and AP-1 following phorbol 12-myristate 13-acetate (PMA) stimulation. Furthermore, expression of PDLIM2 is lost in anaplastic large cell lymphoma (ALCL) that shares key biological aspects with cHL. We conclude that inactivation of PDLIM2 is a recurrent finding in cHL and ALCL, promotes activation of inflammatory signaling pathways and thereby contributes to their pathogenesis. PMID:27538486

  16. Gsk3β and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson's disease

    PubMed Central

    Teixeira, Felipe Roberti; Randle, Suzanne J.; Patel, Shachi P.; Mevissen, Tycho E.T.; Zenkeviciute, Grasilda; Koide, Tie; Komander, David; Laman, Heike

    2016-01-01

    Fbxo7 is a clinically relevant F-box protein, associated with both cancer and Parkinson's disease (PD). Additionally, SNPs within FBXO7 are correlated with alterations in red blood cell parameters. Point mutations within FBXO7 map within specific functional domains, including near its F-box domain and its substrate recruiting domains, suggesting that deficiencies in SCFFbxo7/PARK15 ubiquitin ligase activity are mechanistically linked to early-onset PD. To date, relatively few substrates of the ligase have been identified. These include HURP (hepatoma up-regulated protein), whose ubiquitination results in proteasome-mediated degradation, and c-IAP1 (inhibitor of apoptosis protein 1), TNF receptor-associated factor 2 (TRAF2), and NRAGE, which are not destabilized as a result of ubiquitination. None of these substrates have been linked directly to PD, nor has it been determined whether they would directly engage neuronal cell death pathways. To discover ubiquitinated substrates of SCFFbxo7 implicated more directly in PD aetiology, we conducted a high-throughput screen using protein arrays to identify new candidates. A total of 338 new targets were identified and from these we validated glycogen synthase kinase 3β (Gsk3β), which can phosphorylate α-synuclein, and translocase of outer mitochondrial membrane 20 (Tomm20), a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCFFbxo7 substrates both in vitro and in vivo. Ubiquitin chain restriction analyses revealed that Fbxo7 modified Gsk3β using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3β activity, rather than its levels or localization. In addition, Fbxo7 ubiquitinated Tomm20, and its levels correlated with Fbxo7 expression, indicating a stabilizing effect. None of the PD-associated mutations in Fbxo7 impaired Tomm20 ubiquitination. Our findings demonstrate that SCFFbxo7 has an impact directly on two proteins implicated in pathological processes leading to PD

  17. Mitochondrial E3 Ubiquitin Protein Ligase 1 Mediates Cigarette Smoke-Induced Endothelial Cell Death and Dysfunction.

    PubMed

    Kim, Sun-Yong; Kim, Hyo Jeong; Park, Mi Kyeong; Huh, Jin Won; Park, Hye Yun; Ha, Sang Yun; Shin, Joo-Ho; Lee, Yun-Song

    2016-02-01

    By virtue of the critical roles of Akt in vascular endothelial cell (EC) survival and function, cigarette smoke-induced Akt reduction may contribute to EC death and dysfunction in smokers' lungs. One of the negative Akt regulatory mechanisms is K48-linked Akt ubiquitination and subsequent proteasomal degradation. Here, we assessed the involvement of mitochondrial E3 ubiquitin protein ligase 1 (MUL1), recently revealed as a novel Akt ubiquitin E3 ligase, in cigarette smoke-induced Akt ubiquitination and its contribution to pulmonary EC death and dysfunction. In human lung microvascular ECs (HLMVECs), cigarette smoke extract (CSE) noticeably elevated MUL1 expression and K48-linked Akt ubiquitination, whereas Akt, p-Akt, eNOS, and p-eNOS levels were decreased. MUL1 knockdown suppressed CSE-induced Akt ubiquitination/degradation and cytoplasmic reductions of Akt and p-Akt. Furthermore, MUL1 knockdown attenuated reductions of eNOS and p-eNOS and alleviated EC survival, migration, and tube formation in the presence of CSE exposure. In addition, overexpression of K284R Akt, a mutant for a MUL1-ubiquitination site, produced similar effects. In HLMVECs exposed to CSE, Akt-MUL1 interaction was increased in coimmunoprecipitation and in situ proximity ligation assays. Similarly, the proximity ligation assay signals were elevated in rat lungs exposed to cigarette smoke for 3 months, during which Mul1 levels were noticeably increased. Finally, we found that CSE-mediated MUL1 induction in HLMVECs is mediated by retinoic acid receptor-related orphan receptor α. Taken together, these data suggest that cigarette smoke-induced MUL1 elevation mediates Akt ubiquitination/degradation, potentially leading to pulmonary EC death and functional impairment.

  18. The Ubiquitin Receptor DA1 Interacts with the E3 Ubiquitin Ligase DA2 to Regulate Seed and Organ Size in Arabidopsis[C][W

    PubMed Central

    Xia, Tian; Li, Na; Dumenil, Jack; Li, Jie; Kamenski, Andrei; Bevan, Michael W.; Gao, Fan; Li, Yunhai

    2013-01-01

    Seed size in higher plants is determined by the coordinated growth of the embryo, endosperm, and maternal tissue. Several factors that act maternally to regulate seed size have been identified, such as AUXIN RESPONSE FACTOR2, APETALA2, KLUH, and DA1, but the genetic and molecular mechanisms of these factors in seed size control are almost totally unknown. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligase ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to regulate the final size of seeds in Arabidopsis thaliana. Here, we describe another RING-type protein with E3 ubiquitin ligase activity, encoded by DA2, which regulates seed size by restricting cell proliferation in the maternal integuments of developing seeds. The da2-1 mutant forms large seeds, while overexpression of DA2 decreases seed size of wild-type plants. Overexpression of rice (Oryza sativa) GRAIN WIDTH AND WEIGHT2, a homolog of DA2, restricts seed growth in Arabidopsis. Genetic analyses show that DA2 functions synergistically with DA1 to regulate seed size, but does so independently of EOD1. Further results reveal that DA2 interacts physically with DA1 in vitro and in vivo. Therefore, our findings define the genetic and molecular mechanisms of three ubiquitin-related proteins DA1, DA2, and EOD1 in seed size control and indicate that they are promising targets for crop improvement. PMID:24045020

  19. Protein Kinase C (PKC)-promoted Endocytosis of Glutamate Transporter GLT-1 Requires Ubiquitin Ligase Nedd4-2-dependent Ubiquitination but Not Phosphorylation*

    PubMed Central

    García-Tardón, Noemí; González-González, Inmaculada M.; Martínez-Villarreal, Jaime; Fernández-Sánchez, Enrique; Giménez, Cecilio; Zafra, Francisco

    2012-01-01

    Glutamate transporter-1 (GLT-1) is the main glutamate transporter in the central nervous system, and its concentration severely decreases in neurodegenerative diseases. The number of transporters in the plasma membrane reflects the balance between their insertion and removal, and it has been reported that the regulated endocytosis of GLT-1 depends on its ubiquitination triggered by protein kinase C (PKC) activation. Here, we identified serine 520 of GLT-1 as the primary target for PKC-dependent phosphorylation, although elimination of this serine did not impair either GLT-1 ubiquitination or endocytosis in response to phorbol esters. In fact, we present evidence indicating that the ubiquitin ligase Nedd4-2 mediates the PKC-dependent ubiquitination and down-regulation of GLT-1. Overexpression of Nedd4-2 increased the ubiquitination of the transporter and promoted its degradation. Moreover, phorbol myristate acetate enhanced Nedd4-2 phosphorylation and the formation of GLT-1·Nedd4-2 complexes, whereas siRNA knockdown of Nedd4-2 prevented ubiquitination, endocytosis, and the concomitant decrease in GLT-1 activity triggered by PKC activation. These results indicate that GLT-1 endocytosis is independent of its phosphorylation and that Nedd4-2 mediates PKC-dependent down-regulation of the transporter. PMID:22505712

  20. The ubiquitin ligase MuRF1 regulates PPARα activity in the heart by enhancing nuclear export via monoubiquitination

    PubMed Central

    Rodríguez, Jessica E.; Liao, Jie-Ying; He, Jun; Schisler, Jonathan C.; Newgard, Christopher B.; Drujan, Doreen; Glass, David L.; Frederick, C.Brandon; Yoder, Bryan C.; Lalush, David S.; Patterson, Cam; Willis, Monte S.

    2015-01-01

    The transcriptional regulation of peroxisome proliferator-activated receptor (PPAR) α by post-translational modification, such as ubiquitin, has not been described. We report here for the first time an ubiquitin ligase (muscle ring finger-1/MuRF1) that inhibits fatty acid oxidation by inhibiting PPARα, but not PPARβ/δ or PPARγ in cardiomyocytes in vitro. Similarly, MuRF1 Tg+ hearts showed significant decreases in nuclear PPARα activity and acyl-carnitine intermediates, while MuRF1−/− hearts exhibited increased PPARα activity and acyl-carnitine intermediates. MuRF1 directly interacts with PPARα, mono-ubiquitinates it, and targets it for nuclear export to inhibit fatty acid oxidation in a proteasome independent manner. We then identified a previously undescribed nuclear export sequence in PPARα, along with three specific lysines (292, 310, 388) required for MuRF1s targeting of nuclear export. These studies identify the role of ubiquitination in regulating cardiac PPARα, including the ubiquitin ligase that may be responsible for this critical regulation of cardiac metabolism in heart failure. PMID:26116825

  1. The Ubiquitin Ligase Smurf1 Functions in Selective Autophagy of Mycobacterium tuberculosis and Anti-tuberculous Host Defense.

    PubMed

    Franco, Luis H; Nair, Vidhya R; Scharn, Caitlyn R; Xavier, Ramnik J; Torrealba, Jose R; Shiloh, Michael U; Levine, Beth

    2017-01-11

    During antibacterial autophagy, ubiquitination of intracellular bacteria recruits proteins that mediate bacterial delivery to the lysosome for degradation. Smurf1 is an E3 ubiquitin ligase whose role in selective bacterial autophagy is unknown. We show that Smurf1 facilitates selective autophagy of the human pathogen Mycobacterium tuberculosis (Mtb). Smurf1(-/-) macrophages are defective in recruiting polyubiquitin, the proteasome, the ubiquitin-binding autophagy adaptor NBR1, the autophagy protein LC3, and the lysosomal marker LAMP1 to Mtb-associated structures and are more permissive for Mtb growth. This function of Smurf1 requires both its ubiquitin-ligase and C2 phospholipid-binding domains, and involves K48- rather than K63-linked ubiquitination. Chronically infected Smurf1(-/-) mice have increased bacterial load, increased lung inflammation, and accelerated mortality. SMURF1 controls Mtb replication in human macrophages and associates with bacteria in lungs of patients with pulmonary tuberculosis. Thus, Smurf1 is required for selective autophagy of Mtb and host defense against tuberculosis infection.

  2. A SPOPL/Cullin-3 ubiquitin ligase complex regulates endocytic trafficking by targeting EPS15 at endosomes

    PubMed Central

    Gschweitl, Michaela; Ulbricht, Anna; Barnes, Christopher A; Enchev, Radoslav I; Stoffel-Studer, Ingrid; Meyer-Schaller, Nathalie; Huotari, Jatta; Yamauchi, Yohei; Greber, Urs F; Helenius, Ari; Peter, Matthias

    2016-01-01

    Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets. DOI: http://dx.doi.org/10.7554/eLife.13841.001 PMID:27008177

  3. Oligomerization of the Nrdp1 E3 Ubiquitin Ligase Is Necessary for Efficient Autoubiquitination but Not ErbB3 Ubiquitination*

    PubMed Central

    Printsev, Ignat; Yen, Lily; Sweeney, Colleen; Carraway, Kermit L.

    2014-01-01

    Overexpression of the ErbB3 receptor tyrosine kinase protein in breast and other cancers contributes to tumor malignancy and therapeutic resistance. The RBCC/TRIM family RING finger E3 ubiquitin ligase Nrdp1 mediates the ubiquitination of ErbB3 in normal mammary epithelial cells to facilitate receptor degradation and suppress steady-state receptor levels. Post-transcriptional loss of Nrdp1 in patient breast tumors allows ErbB3 overexpression and receptor contribution to tumor progression, and elevated lability through autoubiquitination contributes to the observed loss of Nrdp1 in tumors relative to normal tissue. To begin to understand the mechanisms underlying Nrdp1 protein self-regulation through lability, we investigated the structural determinants required for efficient autoubiquitination and ErbB3 ubiquitination. Using mutagenesis, chemical cross-linking, size exclusion chromatography, and native polyacrylamide gel electrophoresis, we demonstrate that Nrdp1 self-associates into a stable oligomeric complex in cells. Deletion of its coiled-coil domain abrogates oligomerization but does not affect Nrdp1-mediated ErbB3 ubiquitination or degradation. On the other hand, the presence of the coiled-coil domain is necessary for efficient Nrdp1 autoubiquitination via a trans mechanism, indicating that Nrdp1 ubiquitination of its various targets is functionally separable. Finally, a GFP fusion of the coiled-coil domain stabilizes Nrdp1 and potentiates ErbB3 ubiquitination and degradation. These observations point to a model whereby the coiled-coil domain plays a key role in regulating Nrdp1 lability by promoting its assembly into an oligomeric complex, and raise the possibility that inhibition of ligase oligomerization via its coiled-coil domain could be of therapeutic benefit to breast cancer patients by restoring Nrdp1 protein. PMID:24519943

  4. Oligomerization of the Nrdp1 E3 ubiquitin ligase is necessary for efficient autoubiquitination but not ErbB3 ubiquitination.

    PubMed

    Printsev, Ignat; Yen, Lily; Sweeney, Colleen; Carraway, Kermit L

    2014-03-21

    Overexpression of the ErbB3 receptor tyrosine kinase protein in breast and other cancers contributes to tumor malignancy and therapeutic resistance. The RBCC/TRIM family RING finger E3 ubiquitin ligase Nrdp1 mediates the ubiquitination of ErbB3 in normal mammary epithelial cells to facilitate receptor degradation and suppress steady-state receptor levels. Post-transcriptional loss of Nrdp1 in patient breast tumors allows ErbB3 overexpression and receptor contribution to tumor progression, and elevated lability through autoubiquitination contributes to the observed loss of Nrdp1 in tumors relative to normal tissue. To begin to understand the mechanisms underlying Nrdp1 protein self-regulation through lability, we investigated the structural determinants required for efficient autoubiquitination and ErbB3 ubiquitination. Using mutagenesis, chemical cross-linking, size exclusion chromatography, and native polyacrylamide gel electrophoresis, we demonstrate that Nrdp1 self-associates into a stable oligomeric complex in cells. Deletion of its coiled-coil domain abrogates oligomerization but does not affect Nrdp1-mediated ErbB3 ubiquitination or degradation. On the other hand, the presence of the coiled-coil domain is necessary for efficient Nrdp1 autoubiquitination via a trans mechanism, indicating that Nrdp1 ubiquitination of its various targets is functionally separable. Finally, a GFP fusion of the coiled-coil domain stabilizes Nrdp1 and potentiates ErbB3 ubiquitination and degradation. These observations point to a model whereby the coiled-coil domain plays a key role in regulating Nrdp1 lability by promoting its assembly into an oligomeric complex, and raise the possibility that inhibition of ligase oligomerization via its coiled-coil domain could be of therapeutic benefit to breast cancer patients by restoring Nrdp1 protein.

  5. Direct recognition of the C-terminal polylysine residues of nonstop protein by Ltn1, an E3 ubiquitin ligase.

    PubMed

    Sung, Kwang Hoon; Song, Hyun Kyu

    2014-10-24

    When mRNAs lack stop codons, errors in gene expression and coding of aberrant proteins that are harmful in cells can result. In Saccharomyces cerevisiae, a 180-kDa E3-ubiquitin ligase, Ltn1 has been known to associate with ribosomes and marks translationally-arrested aberrant nascent polypeptides for proteasomal degradation. Here, we demonstrate the Ltn1 E3-ubiquitin ligase directly binds to the nonstop proteins and efficiently ubiquitylates them. The middle domain of Ltn1 is responsible for recognizing the polylysine residues of the nonstop protein with an affinity of 2-3μM. This biochemical characterization of Ltn1 expands our knowledge regarding the fundamental process that removes aberrant nascent polypeptides in eukaryotes.

  6. Expansion and diversification of BTL ring-H2 ubiquitin ligases in angiosperms: putative Rabring7/BCA2 orthologs.

    PubMed

    Aguilar-Hernández, Victor; Medina, Juliana; Aguilar-Henonin, Laura; Guzmán, Plinio

    2013-01-01

    RING finger E3 ligases are components of the ubiquitin proteasome system (UPS) that mediate the transfer of ubiquitin to substrates. Single-subunit RING finger E3s binds the E2 ubiquitin-conjugating enzyme and contains recognition sequences for the substrate within the same polypeptide. Here we describe the characterization of a class of RING finger E3 ligases that is conserved among eukaryotes. This class encodes a RING-H2 domain related in sequence to the ATL RING-H2 domain, another class of E3 ligases, and a C2/C2 zing finger at the amino-terminus, formerly described as BZF. In viridiplantae (green algae and land plants), we designed this family as BTL for BZF ATLs. BTLs are putative orthologs of the mammalian Rabring7/BCA2 RING-H2 E3s that have expanded in angiosperms. They are found in numbers ranging from three to thirty-one, which is in contrast to the one to three members normally found in animals, fungi, and protists. Furthermore, the number of sequence LOGOs generated in angiosperms is four times greater than that in other eukaryotes. In contrast to ATLs, which show expansion by tandem duplication, tandemly duplicated BTLs are scarce. The mode of action of Rabring7/BCA2 and BTLs may be similar since both the Rabring7/BCA2 BZF and the ath|BTL4 BZF are likely to mediate the binding of ubiquitin. This study introduces valuable information on the evolution and domain structure of the Rabring7/BCA2/BTL class of E3 ligases which may be important for core eukaryotic genes.

  7. Expansion and Diversification of BTL Ring-H2 Ubiquitin Ligases in Angiosperms: Putative Rabring7/BCA2 Orthologs

    PubMed Central

    Aguilar-Henonin, Laura; Guzmán, Plinio

    2013-01-01

    RING finger E3 ligases are components of the ubiquitin proteasome system (UPS) that mediate the transfer of ubiquitin to substrates. Single-subunit RING finger E3s binds the E2 ubiquitin-conjugating enzyme and contains recognition sequences for the substrate within the same polypeptide. Here we describe the characterization of a class of RING finger E3 ligases that is conserved among eukaryotes. This class encodes a RING-H2 domain related in sequence to the ATL RING-H2 domain, another class of E3 ligases, and a C2/C2 zing finger at the amino-terminus, formerly described as BZF. In viridiplantae (green algae and land plants), we designed this family as BTL for BZF ATLs. BTLs are putative orthologs of the mammalian Rabring7/BCA2 RING-H2 E3s that have expanded in angiosperms. They are found in numbers ranging from three to thirty-one, which is in contrast to the one to three members normally found in animals, fungi, and protists. Furthermore, the number of sequence LOGOs generated in angiosperms is four times greater than that in other eukaryotes. In contrast to ATLs, which show expansion by tandem duplication, tandemly duplicated BTLs are scarce. The mode of action of Rabring7/BCA2 and BTLs may be similar since both the Rabring7/BCA2 BZF and the ath|BTL4 BZF are likely to mediate the binding of ubiquitin. This study introduces valuable information on the evolution and domain structure of the Rabring7/BCA2/BTL class of E3 ligases which may be important for core eukaryotic genes. PMID:23951330

  8. Human SHPRH is a ubiquitin ligase for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen.

    PubMed

    Unk, Ildiko; Hajdú, Ildikó; Fátyol, Károly; Szakál, Barnabás; Blastyák, András; Bermudez, Vladimir; Hurwitz, Jerard; Prakash, Louise; Prakash, Satya; Haracska, Lajos

    2006-11-28

    Human SHPRH gene is located at the 6q24 chromosomal region, and loss of heterozygosity in this region is seen in a wide variety of cancers. SHPRH is a member of the SWI/SNF family of ATPases/helicases, and it possesses a C(3)HC(4) RING motif characteristic of ubiquitin ligase proteins. In both of these features, SHPRH resembles the yeast Rad5 protein, which, together with Mms2-Ubc13, promotes replication through DNA lesions via an error-free postreplicational repair pathway. Genetic evidence in yeast has indicated a role for Rad5 as a ubiquitin ligase in mediating the Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Here we show that SHPRH is a functional homolog of Rad5. Similar to Rad5, SHPRH physically interacts with the Rad6-Rad18 and Mms2-Ubc13 complexes, and we show that SHPRH protein is a ubiquitin ligase indispensable for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Based on these observations, we predict a role for SHPRH in promoting error-free replication through DNA lesions. Such a role for SHPRH is consistent with the observation that this gene is mutated in a number of cancer cell lines, including those from melanomas and ovarian cancers, which raises the strong possibility that SHPRH function is an important deterrent to mutagenesis and carcinogenesis in humans.

  9. Ataxia and hypogonadism caused by the loss of ubiquitin ligase activity of the U box protein CHIP

    PubMed Central

    Shi, Chang-He; Schisler, Jonathan C.; Rubel, Carrie E.; Tan, Song; Song, Bo; McDonough, Holly; Xu, Lei; Portbury, Andrea L.; Mao, Cheng-Yuan; True, Cadence; Wang, Rui-Hao; Wang, Qing-Zhi; Sun, Shi-Lei; Seminara, Stephanie B.; Patterson, Cam; Xu, Yu-Ming

    2014-01-01

    Gordon Holmes syndrome (GHS) is a rare Mendelian neurodegenerative disorder characterized by ataxia and hypogonadism. Recently, it was suggested that disordered ubiquitination underlies GHS though the discovery of exome mutations in the E3 ligase RNF216 and deubiquitinase OTUD4. We performed exome sequencing in a family with two of three siblings afflicted with ataxia and hypogonadism and identified a homozygous mutation in STUB1 (NM_005861) c.737C→T, p.Thr246Met, a gene that encodes the protein CHIP (C-terminus of HSC70-interacting protein). CHIP plays a central role in regulating protein quality control, in part through its ability to function as an E3 ligase. Loss of CHIP function has long been associated with protein misfolding and aggregation in several genetic mouse models of neurodegenerative disorders; however, a role for CHIP in human neurological disease has yet to be identified. Introduction of the Thr246Met mutation into CHIP results in a loss of ubiquitin ligase activity measured directly using recombinant proteins as well as in cell culture models. Loss of CHIP function in mice resulted in behavioral and reproductive impairments that mimic human ataxia and hypogonadism. We conclude that GHS can be caused by a loss-of-function mutation in CHIP. Our findings further highlight the role of disordered ubiquitination and protein quality control in the pathogenesis of neurodegenerative disease and demonstrate the utility of combining whole-exome sequencing with molecular analyses and animal models to define causal disease polymorphisms. PMID:24113144

  10. RPM-1 Uses Both Ubiquitin Ligase and Phosphatase-Based Mechanisms to Regulate DLK-1 during Neuronal Development

    PubMed Central

    Baker, Scott T.; Opperman, Karla J.; Tulgren, Erik D.; Turgeon, Shane M.; Bienvenut, Willy; Grill, Brock

    2014-01-01

    The Pam/Highwire/RPM-1 (PHR) proteins are key regulators of neuronal development that function in axon extension and guidance, termination of axon outgrowth, and synapse formation. Outside of development, the PHR proteins also regulate axon regeneration and Wallerian degeneration. The PHR proteins function in part by acting as ubiquitin ligases that degrade the Dual Leucine zipper-bearing Kinase (DLK). Here, we show that the Caenorhabditis elegans PHR protein, Regulator of Presynaptic Morphology 1 (RPM-1), also utilizes a phosphatase-based mechanism to regulate DLK-1. Using mass spectrometry, we identified Protein Phosphatase Magnesium/Manganese dependent 2 (PPM-2) as a novel RPM-1 binding protein. Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PPM-2 acts on S874 of DLK-1, a residue implicated in regulation of DLK-1 binding to a short, inhibitory isoform of DLK-1 (DLK-1S). Our study demonstrates that PHR proteins function through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK. Thus, PHR proteins are potentially more accurate and sensitive regulators of DLK than originally thought. Our results also highlight an important and expanding role for the PP2C phosphatase family in neuronal development. PMID:24810406

  11. Allosteric Interactions by p53 mRNA Govern HDM2 E3 Ubiquitin Ligase Specificity under Different Conditions

    PubMed Central

    Medina-Medina, Ixaura; García-Beltrán, Paola; de la Mora-de la Mora, Ignacio; Oria-Hernández, Jesús; Millot, Guy; Fahraeus, Robin; Reyes-Vivas, Horacio; Sampedro, José G.

    2016-01-01

    HDM2 and HDMX are key negative regulatory factors of the p53 tumor suppressor under normal conditions by promoting its degradation or preventing its trans activity, respectively. It has more recently been shown that both proteins can also act as positive regulators of p53 after DNA damage. This involves phosphorylation by ATM on serine residues HDM2(S395) and HDMX(S403), promoting their respective interaction with the p53 mRNA. However, the underlying molecular mechanisms of how these phosphorylation events switch HDM2 and HDMX from negative to positive regulators of p53 is not known. Our results show that these phosphorylation events reside within intrinsically disordered domains and change the conformation of the proteins. The modifications promote the exposition of N-terminal interfaces that support the formation of a new HDMX-HDM2 heterodimer independent of the C-terminal RING-RING interaction. The E3 ubiquitin ligase activity of this complex toward p53 is prevented by the p53 mRNA ligand but, interestingly, does not affect the capacity to ubiquitinate HDMX and HDM2. These results show how ATM-mediated modifications of HDMX and HDM2 switch HDM2 E3 ubiquitin ligase activity away from p53 but toward HDMX and itself and illustrate how the substrate specificity of HDM2 E3 ligase activity is regulated. PMID:27215386

  12. TRIM16 acts as an E3 ubiquitin ligase and can heterodimerize with other TRIM family members.

    PubMed

    Bell, Jessica L; Malyukova, Alena; Holien, Jessica K; Koach, Jessica; Parker, Michael W; Kavallaris, Maria; Marshall, Glenn M; Cheung, Belamy B

    2012-01-01

    The TRIM family of proteins is distinguished by its tripartite motif (TRIM). Typically, TRIM proteins contain a RING finger domain, one or two B-box domains, a coiled-coil domain and the more variable C-terminal domains. TRIM16 does not have a RING domain but does harbour two B-box domains. Here we showed that TRIM16 homodimerized through its coiled-coil domain and heterodimerized with other TRIM family members; TRIM24, Promyelocytic leukaemia (PML) protein and Midline-1 (MID1). Although, TRIM16 has no classic RING domain, three-dimensional modelling of TRIM16 suggested that its B-box domains adopts RING-like folds leading to the hypothesis that TRIM16 acts as an ubiquitin ligase. Consistent with this hypothesis, we demonstrated that TRIM16, devoid of a classical RING domain had auto-polyubiquitination activity and acted as an E3 ubiquitin ligase in vivo and in vitro assays. Thus via its unique structure, TRIM16 possesses both heterodimerization function with other TRIM proteins and also has E3 ubiquitin ligase activity.

  13. Allosteric Interactions by p53 mRNA Govern HDM2 E3 Ubiquitin Ligase Specificity under Different Conditions.

    PubMed

    Medina-Medina, Ixaura; García-Beltrán, Paola; de la Mora-de la Mora, Ignacio; Oria-Hernández, Jesús; Millot, Guy; Fahraeus, Robin; Reyes-Vivas, Horacio; Sampedro, José G; Olivares-Illana, Vanesa

    2016-08-15

    HDM2 and HDMX are key negative regulatory factors of the p53 tumor suppressor under normal conditions by promoting its degradation or preventing its trans activity, respectively. It has more recently been shown that both proteins can also act as positive regulators of p53 after DNA damage. This involves phosphorylation by ATM on serine residues HDM2(S395) and HDMX(S403), promoting their respective interaction with the p53 mRNA. However, the underlying molecular mechanisms of how these phosphorylation events switch HDM2 and HDMX from negative to positive regulators of p53 is not known. Our results show that these phosphorylation events reside within intrinsically disordered domains and change the conformation of the proteins. The modifications promote the exposition of N-terminal interfaces that support the formation of a new HDMX-HDM2 heterodimer independent of the C-terminal RING-RING interaction. The E3 ubiquitin ligase activity of this complex toward p53 is prevented by the p53 mRNA ligand but, interestingly, does not affect the capacity to ubiquitinate HDMX and HDM2. These results show how ATM-mediated modifications of HDMX and HDM2 switch HDM2 E3 ubiquitin ligase activity away from p53 but toward HDMX and itself and illustrate how the substrate specificity of HDM2 E3 ligase activity is regulated.

  14. The Blue Light-Dependent Polyubiquitination and Degradation of Arabidopsis Cryptochrome2 Requires Multiple E3 Ubiquitin Ligases.

    PubMed

    Liu, Qing; Wang, Qin; Liu, Bin; Wang, Wei; Wang, Xu; Park, Joon; Yang, Zhenming; Du, Xinglin; Bian, Mingdi; Lin, Chentao

    2016-10-01

    Cryptochromes are blue light receptors regulated by light-dependent ubiquitination and degradation in both plant and animal lineages. The Arabidopsis genome encodes two cryptochromes, CRY1 and CRY2, of which CRY2 undergoes blue light-dependent ubiquitination and 26S proteasome-dependent degradation. The molecular mechanism regulating blue light-dependent proteolysis of CRY2 is still not fully understood. We found that the F-box proteins ZEITLUPE (ZTL) and Lov Kelch Protein2 (LKP2), which mediate blue light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1-SPA1 protein complex in blue light-dependent CRY2 degradation is more likely to be attributable to its cullin 4 (CUL4)-based E3 ubiquitin ligase activity than its activity as the cryptochrome signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4 mutant, the cop1-5 null mutant and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele (axr6-3), especially under the non-permissive temperature. Based on these and other results presented, we propose that photoexcited CRY2 undergoes Lys48-linked polyubiquitination catalyzed by the CUL4- and CUL1-based E3 ubiquitin ligases.

  15. E6-AP/UBE3A protein acts as a ubiquitin ligase toward SOX9 protein.

    PubMed

    Hattori, Takako; Kishino, Tetsuya; Stephen, Shelley; Eberspaecher, Heidi; Maki, Sayumi; Takigawa, Masaharu; de Crombrugghe, Benoit; Yasuda, Hideyo

    2013-12-06

    SOX9 is a transcription factor that acts as a key regulator at various stages of cartilage differentiation. There is ample evidence that intracellular SOX9 protein levels are tightly regulated both by sumoylation and by degradation through the ubiquitin-proteasome pathway. Using a proteomics approach, here we report the identification of a SOX9-binding protein, E6-AP/UBE3A, that may act as a ubiquitin ligase toward Sox9. E6-AP bound SOX9 through the region consisting mostly of its high mobility group domain in vitro. In nuclear lysates, FLAG-tagged E6-AP coprecipitated with Sox9 and its high mobility group domain. This finding was estimated using nuclear lysates from a chondrocytic cell line that endogenously expresses E6-AP and SOX9. Accordingly, ectopically expressed E6-AP and SOX9 colocalized in the nucleus. We show that E6-AP ubiquitinates SOX9 in vitro and in vivo and that SOX9 levels are enhanced after addition of the proteasome inhibitor bortezomib. Similar, siRNA knockdown of E6-AP and the E2 ligase Ubc9 increased cellular SOX9 amounts, supporting the notion that SOX9 may be ubiquitinated in hypertrophic chondrocytes by E6-AP and degraded by proteasomes. This is in accordance with the distribution of SOX9 levels, which are high in proliferating and prehypertrophic chondrocytes but low in hypertrophic chondrocytes, whereas E6-AP levels are high in hypertrophic chondrocytes and low in prehypertrophic chondrocytes. Furthermore, E6-AP-deficient mice showed SOX9 accumulation in chondrocytes and the brain. These findings support the concept that E6-AP regulates SOX9 levels in developing cartilage by acting as a ubiquitin ligase.

  16. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks

    PubMed Central

    Dantuma, Nico P.; Pfeiffer, Annika

    2016-01-01

    Ubiquitin and the ubiquitin-like modifier SUMO are intimately connected with the cellular response to various types of DNA damage. A striking feature is the local accumulation of these proteinaceous post-translational modifications in the direct vicinity to DNA double-strand breaks, which plays a critical role in the formation of ionizing radiation-induced foci. The functional significance of these modifications is the coordinated recruitment and removal of proteins involved in DNA damage signaling and repair in a timely manner. The central orchestrators of these processes are the ubiquitin and SUMO ligases that are responsible for accurately tagging a broad array of chromatin and chromatin-associated proteins thereby changing their behavior or destination. Despite many differences in the mode of action of these enzymes, they share some striking features that are of direct relevance for their function in the DNA damage response. In this review, we outline the molecular mechanisms that are responsible for the recruitment of ubiquitin and SUMO ligases and discuss the importance of chromatin proximity in this process. PMID:27148355

  17. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks.

    PubMed

    Dantuma, Nico P; Pfeiffer, Annika

    2016-01-01

    Ubiquitin and the ubiquitin-like modifier SUMO are intimately connected with the cellular response to various types of DNA damage. A striking feature is the local accumulation of these proteinaceous post-translational modifications in the direct vicinity to DNA double-strand breaks, which plays a critical role in the formation of ionizing radiation-induced foci. The functional significance of these modifications is the coordinated recruitment and removal of proteins involved in DNA damage signaling and repair in a timely manner. The central orchestrators of these processes are the ubiquitin and SUMO ligases that are responsible for accurately tagging a broad array of chromatin and chromatin-associated proteins thereby changing their behavior or destination. Despite many differences in the mode of action of these enzymes, they share some striking features that are of direct relevance for their function in the DNA damage response. In this review, we outline the molecular mechanisms that are responsible for the recruitment of ubiquitin and SUMO ligases and discuss the importance of chromatin proximity in this process.

  18. The E3 ubiquitin ligase Itch controls the protein stability of p63.

    PubMed

    Rossi, Mario; Aqeilan, Rami I; Neale, Michael; Candi, Eleonora; Salomoni, Paolo; Knight, Richard A; Croce, Carlo M; Melino, Gerry

    2006-08-22

    p63, a member of the p53 family of transcription factors, plays an important role in epithelial development, regulating both cell cycle and apoptosis. Even though p63 activity is regulated mainly at the posttranslational level, the control of p63 protein stability is far from being fully understood. Here, we show that the Hect (homologous to the E6-associated protein C terminus)-containing Nedd4-like ubiquitin protein ligase Itch binds, ubiquitylates, and promotes the degradation of p63. The physical interaction occurs at the border between the PY and the SAM (sterile alpha motif) domains; a single Y504F mutation significantly affects p63 degradation. Itch and p63 are coexpressed in the epidermis and in primary keratinocytes where Itch controls the p63 protein steady-state level. Accordingly, p63 protein levels are significantly increased in Itch knockout keratinocytes. These data suggest that Itch has a fundamental role in the mechanism that controls endogenous p63 protein levels and therefore contributes to regulation of p63 in physiological conditions.

  19. Viral ubiquitin ligase WSSV222 is required for efficient white spot syndrome virus replication in shrimp.

    PubMed

    He, Fang; Syed, Syed Musthaq; Hameed, A S Sahul; Kwang, Jimmy

    2009-06-01

    The E3 ligase WSSV222 of white spot syndrome virus (WSSV) is involved in anti-apoptosis regulation by ubiquitin-mediated degradation of tumour suppressor-like protein (TSL), a shrimp tumour suppressor. In the present study, WSSV222 gene expression was silenced by using specific small interfering RNA (siRNA) in Sf9 and BHK cells. Based on the results of the in vitro silencing, WSSV-challenged shrimp were treated with anti-WSSV222 siRNA to knock down WSSV222 protein expression. The survival rate of shrimp and the efficiency of WSSV replication were assessed to evaluate the efficacy of anti-WSSV222 siRNA in regulating WSSV infection in shrimp. The anti-WSSV222 siRNA reduced the cumulative mortality in shrimp challenged with 10(3) copies of WSSV and delayed the mean time to death in shrimp challenged with the higher dose of 10(6) copies. The results of real-time quantitative PCR showed that virus replication was delayed and reduced in WSSV-challenged shrimp treated with anti-WSSV222 siRNA in comparison with challenged shrimp treated with random-control siRNA. Co-immunoprecipitation assays revealed that WSSV222 silencing inhibited the degradation of TSL in WSSV-challenged shrimp, indicating the requirement for WSSV222 for efficient replication of WSSV in shrimp.

  20. Regulating the Regulators: Recent Revelations in the Control of E3 Ubiquitin Ligases*

    PubMed Central

    Vittal, Vinayak; Stewart, Mikaela D.; Brzovic, Peter S.; Klevit, Rachel E.

    2015-01-01

    Since its discovery as a post-translational signal for protein degradation, our understanding of ubiquitin (Ub) has vastly evolved. Today, we recognize that the role of Ub signaling is expansive and encompasses diverse processes including cell division, the DNA damage response, cellular immune signaling, and even organismal development. With such a wide range of functions comes a wide range of regulatory mechanisms that control the activity of the ubiquitylation machinery. Ub attachment to substrates occurs through the sequential action of three classes of enzymes, E1s, E2s, and E3s. In humans, there are 2 E1s, ∼35 E2s, and hundreds of E3s that work to attach Ub to thousands of cellular substrates. Regulation of ubiquitylation can occur at each stage of the stepwise Ub transfer process, and substrates can also impact their own modification. Recent studies have revealed elegant mechanisms that have evolved to control the activity of the enzymes involved. In this minireview, we highlight recent discoveries that define some of the various mechanisms by which the activities of E3-Ub ligases are regulated. PMID:26187467

  1. The ubiquitin ligase FbxL7 regulates the Dachsous-Fat-Dachs system in Drosophila

    PubMed Central

    Rodrigues-Campos, Mariana; Thompson, Barry J.

    2014-01-01

    The atypical cadherins Dachsous (Ds) and Fat (Ft) are required to control the size and shape of tissues and organs in animals. In Drosophila, a key effector of Ds and Ft is the atypical myosin Dachs, which becomes planar polarised along the proximal-distal axis in developing epithelia to regulate tissue size via the Hippo pathway and tissue shape via modulating tension at junctions. How Ds and Ft control Dachs polarisation remains unclear. Here, we identify a ubiquitin ligase, FbxL7, as a novel component of the Ds-Ft-Dachs system that is required to control the level and localisation of Dachs. Loss of FbxL7 results in accumulation of Dachs, similar to loss of Ft. Overexpression of FbxL7 causes downregulation of Dachs, similar to overexpression of the Ft intracellular domain. In addition to regulating Dachs, FbxL7 also influences Ds in a similar manner. GFP-tagged FbxL7 localises to the plasma membrane in a Ft-dependent manner and is planar polarised. We propose that Ft recruits FbxL7 to the proximal side of the cell to help restrict Ds and Dachs to the distal side of the cell. PMID:25256343

  2. Central role of E3 ubiquitin ligase MG53 in insulin resistance and metabolic disorders.

    PubMed

    Song, Ruisheng; Peng, Wei; Zhang, Yan; Lv, Fengxiang; Wu, Hong-Kun; Guo, Jiaojiao; Cao, Yongxing; Pi, Yanbin; Zhang, Xin; Jin, Li; Zhang, Mao; Jiang, Peng; Liu, Fenghua; Meng, Shaoshuai; Zhang, Xiuqin; Jiang, Ping; Cao, Chun-Mei; Xiao, Rui-Ping

    2013-02-21

    Insulin resistance is a fundamental pathogenic factor present in various metabolic disorders including obesity and type 2 diabetes. Although skeletal muscle accounts for 70-90% of insulin-stimulated glucose disposal, the mechanism underlying muscle insulin resistance is poorly understood. Here we show in mice that muscle-specific mitsugumin 53 (MG53; also called TRIM72) mediates the degradation of the insulin receptor and insulin receptor substrate 1 (IRS1), and when upregulated, causes metabolic syndrome featuring insulin resistance, obesity, hypertension and dyslipidaemia. MG53 expression is markedly elevated in models of insulin resistance, and MG53 overexpression suffices to trigger muscle insulin resistance and metabolic syndrome sequentially. Conversely, ablation of MG53 prevents diet-induced metabolic syndrome by preserving the insulin receptor, IRS1 and insulin signalling integrity. Mechanistically, MG53 acts as an E3 ligase targeting the insulin receptor and IRS1 for ubiquitin-dependent degradation, comprising a central mechanism controlling insulin signal strength in skeletal muscle. These findings define MG53 as a novel therapeutic target for treating metabolic disorders and associated cardiovascular complications.

  3. Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4

    PubMed Central

    Lu, Jing; Qian, Yimin; Altieri, Martha; Dong, Hanqing; Wang, Jing; Raina, Kanak; Hines, John; Winkler, James D.; Crew, Andrew P.; Coleman, Kevin; Crews, Craig M.

    2015-01-01

    Summary BRD4, a bromodomain and extraterminal domain (BET) family member, is an attractive target in multiple pathological settings, particularly cancer. While BRD4 inhibitors have shown some promise in MYC-driven malignancies such as Burkitt’s Lymphoma (BL), we show that BRD4 inhibitors lead to robust BRD4 protein accumulation, which may account for their limited suppression of MYC expression, modest anti-proliferative activity and lack of apoptotic induction. To address these limitations, we designed ARV-825, a heterobifunctional PROTAC (Proteolysis Targeting Chimera) that recruits BRD4 to the E3 ubiquitin ligase cereblon leading to fast, efficient, and prolonged degradation of BRD4 in all BL cell lines tested. Consequently, ARV-825 more effectively suppresses c-MYC levels and downstream signaling than small molecule BRD4 inhibitors resulting in more effective cell proliferation inhibition and apoptosis induction in BL. Our findings provide strong evidence that cereblon-based PROTACs provide a better and more efficient strategy in targeting BRD4 than traditional small molecule inhibitors. PMID:26051217

  4. The ubiquitin ligase Ubr4 controls stability of podocin/MEC-2 supercomplexes

    PubMed Central

    Rinschen, Markus M.; Bharill, Puneet; Wu, Xiongwu; Kohli, Priyanka; Reinert, Matthäus J.; Kretz, Oliver; Saez, Isabel; Schermer, Bernhard; Höhne, Martin; Bartram, Malte P.; Aravamudhan, Sriram; Brooks, Bernard R.; Vilchez, David; Huber, Tobias B.; Müller, Roman-Ulrich; Krüger, Marcus; Benzing, Thomas

    2016-01-01

    The PHB-domain protein podocin maintains the renal filtration barrier and its mutation is an important cause of hereditary nephrotic syndrome. Podocin and its Caenorhabditis elegans orthologue MEC-2 have emerged as key components of mechanosensitive membrane protein signalling complexes. Whereas podocin resides at a specialized cell junction at the podocyte slit diaphragm, MEC-2 is found in neurons required for touch sensitivity. Here, we show that the ubiquitin ligase Ubr4 is a key component of the podocin interactome purified both from cultured podocytes and native glomeruli. It colocalizes with podocin and regulates its stability. In C. elegans, this process is conserved. Here, Ubr4 is responsible for the degradation of mislocalized MEC-2 multimers. Ubiquitylomic analysis of mouse glomeruli revealed that podocin is ubiquitylated at two lysine residues. These sites were Ubr4-dependent and were conserved across species. Molecular dynamics simulations revealed that ubiquitylation of one site, K301, do not only target podocin/MEC-2 for proteasomal degradation, but may also affect stability and disassembly of the multimeric complex. We suggest that Ubr4 is a key regulator of podocyte foot process proteostasis. PMID:26792178

  5. An allosteric conduit facilitates dynamic multisite substrate recognition by the SCFCdc4 ubiquitin ligase

    NASA Astrophysics Data System (ADS)

    Csizmok, Veronika; Orlicky, Stephen; Cheng, Jing; Song, Jianhui; Bah, Alaji; Delgoshaie, Neda; Lin, Hong; Mittag, Tanja; Sicheri, Frank; Chan, Hue Sun; Tyers, Mike; Forman-Kay, Julie D.

    2017-01-01

    The ubiquitin ligase SCFCdc4 mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4WD40 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4WD40 domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation.

  6. The SCF ubiquitin ligase protein slimb regulates centrosome duplication in Drosophila.

    PubMed

    Wojcik, E J; Glover, D M; Hays, T S

    2000-09-21

    The duplication of the centrosome is a key event in the cell-division cycle. Although defects in centrosome duplication are thought to contribute to genomic instability [1-3] and are a hallmark of certain transformed cells and human cancer [4-6], the mechanism responsible for centrosome duplication is not understood. Recent experiments have established that centrosome duplication requires the activity of cyclin-dependent kinase 2 (Cdk2) and cyclins E and A [7-9]. The stability of cyclin E is regulated by the ubiquitin ligase SCF, which is a protein complex composed of Skp1, Cdc53 (Cullin) and F-box proteins [10-12]. The Skp1 and Cullin components have been detected on mammalian centrosomes, and shown to be essential for centrosome duplication and separation in Xenopus [13]. Here, we report that Slimb, an F-box protein that targets proteins to the SCFcomplex [14,15], plays a role in limiting centrosome replication. We found that, in the fruit fly Drosophila, the hypomorphic mutation slimb(crd) causes the appearance of additional centrosomes and mitotic defects in mutant larval neuroblasts.

  7. The mitochondrial ubiquitin ligase MARCH5 resolves MAVS aggregates during antiviral signalling

    PubMed Central

    Yoo, Young-Suk; Park, Yong-Yea; Kim, Jae-Hoon; Cho, Hyeseon; Kim, Song-Hee; Lee, Ho-Soo; Kim, Tae-Hwan; Sun Kim, You; Lee, Youngsoo; Kim, Chul-Joong; Jung, Jae U; Lee, Jong-Soo; Cho, Hyeseong

    2015-01-01

    Mitochondria serve as platforms for innate immunity. The mitochondrial antiviral signalling (MAVS) protein forms aggregates that elicit robust type-I interferon induction on viral infection, but persistent MAVS signalling leads to host immunopathology; it remains unknown how these signalling aggregates are resolved. Here we identify the mitochondria-resident E3 ligase, MARCH5, as a negative regulator of MAVS aggregates. March5+/− mice and MARCH5-deficient immune cells exhibit low viral replication and elevated type-I interferon responses to RNA viruses. MARCH5 binds MAVS only during viral stimulation when MAVS forms aggregates, and these interactions require the RING domain of MARCH5 and the CARD domain of MAVS. MARCH5, but not its RING mutant (MARCH5H43W), reduces the level of MAVS aggregates. MARCH5 transfers ubiquitin to Lys7 and Lys500 of MAVS and promotes its proteasome-mediated degradation. Our results indicate that MARCH5 modulates MAVS-mediated antiviral signalling, preventing excessive immune reactions. PMID:26246171

  8. Levels of the Mahogunin Ring Finger 1 E3 Ubiquitin Ligase Do Not Influence Prion Disease

    PubMed Central

    Silvius, Derek; Pitstick, Rose; Ahn, Misol; Meishery, Delisha; Oehler, Abby; Barsh, Gregory S.; DeArmond, Stephen J.; Carlson, George A.; Gunn, Teresa M.

    2013-01-01

    Prion diseases are rare but invariably fatal neurodegenerative disorders. They are associated with spongiform encephalopathy, a histopathology characterized by the presence of large, membrane-bound vacuolar structures in the neuropil of the brain. While the primary cause is recognized as conversion of the normal form of prion protein (PrPC) to a conformationally distinct, pathogenic form (PrPSc), the cellular pathways and mechanisms that lead to spongiform change, neuronal dysfunction and death are not known. Mice lacking the Mahogunin Ring Finger 1 (MGRN1) E3 ubiquitin ligase develop spongiform encephalopathy by 9 months of age but do not become ill. In cell culture, PrP aberrantly present in the cytosol was reported to interact with and sequester MGRN1. This caused endo-lysosomal trafficking defects similar to those observed when Mgrn1 expression is knocked down, implicating disrupted MGRN1-dependent trafficking in the pathogenesis of prion disease. As these defects were rescued by over-expression of MGRN1, we investigated whether reduced or elevated Mgrn1 expression influences the onset, progression or pathology of disease in mice inoculated with PrPSc. No differences were observed, indicating that disruption of MGRN1-dependent pathways does not play a significant role in the pathogenesis of transmissible spongiform encephalopathy. PMID:23383230

  9. An essential role of CBL and CBL-B ubiquitin ligases in mammary stem cell maintenance.

    PubMed

    Mohapatra, Bhopal; Zutshi, Neha; An, Wei; Goetz, Benjamin; Arya, Priyanka; Bielecki, Timothy A; Mustaq, Insha; Storck, Matthew D; Meza, Jane L; Band, Vimla; Band, Hamid

    2017-03-15

    The ubiquitin ligases CBL and CBL-B are negative regulators of tyrosine kinase signaling with established roles in the immune system. However, their physiological roles in epithelial tissues are unknown. Here, we used MMTV-Cre-mediated Cbl gene deletion on a Cbl-b null background, as well as a tamoxifen-inducible mammary stem cell (MaSC)-specific Cbl and Cbl-b double knockout (Cbl/Cbl-b DKO) using Lgr5-EGFP-IRES-CreERT2, to demonstrate a mammary epithelial cell-autonomous requirement of CBL and CBL-B in the maintenance of MaSCs. Using a newly engineered tamoxifen-inducible Cbl and Cbl-b deletion model with a dual fluorescent reporter (Cbl(flox/flox); Cbl-b(flox/flox); Rosa26-CreERT; mT/mG), we show that Cbl/Cbl-b DKO in mammary organoids leads to hyperactivation of AKT-mTOR signaling with depletion of MaSCs. Chemical inhibition of AKT or mTOR rescued MaSCs from Cbl/Cbl-b DKO-induced depletion. Our studies reveal a novel, cell-autonomous requirement of CBL and CBL-B in epithelial stem cell maintenance during organ development and remodeling through modulation of mTOR signaling.

  10. Aberrant substrate engagement of the ER translocon triggers degradation by the Hrd1 ubiquitin ligase

    PubMed Central

    Rubenstein, Eric M.; Kreft, Stefan G.; Greenblatt, Wesley; Swanson, Robert

    2012-01-01

    Little is known about quality control of proteins that aberrantly or persistently engage the endoplasmic reticulum (ER)-localized translocon en route to membrane localization or the secretory pathway. Hrd1 and Doa10, the primary ubiquitin ligases that function in ER-associated degradation (ERAD) in yeast, target distinct subsets of misfolded or otherwise abnormal proteins based primarily on degradation signal (degron) location. We report the surprising observation that fusing Deg1, a cytoplasmic degron normally recognized by Doa10, to the Sec62 membrane protein rendered the protein a Hrd1 substrate. Hrd1-dependent degradation occurred when Deg1-Sec62 aberrantly engaged the Sec61 translocon channel and underwent topological rearrangement. Mutations that prevent translocon engagement caused a reversion to Doa10-dependent degradation. Similarly, a variant of apolipoprotein B, a protein known to be cotranslocationally targeted for proteasomal degradation, was also a Hrd1 substrate. Hrd1 therefore likely plays a general role in targeting proteins that persistently associate with and potentially obstruct the translocon. PMID:22689655

  11. An allosteric conduit facilitates dynamic multisite substrate recognition by the SCFCdc4 ubiquitin ligase

    PubMed Central

    Csizmok, Veronika; Orlicky, Stephen; Cheng, Jing; Song, Jianhui; Bah, Alaji; Delgoshaie, Neda; Lin, Hong; Mittag, Tanja; Sicheri, Frank; Chan, Hue Sun; Tyers, Mike; Forman-Kay, Julie D.

    2017-01-01

    The ubiquitin ligase SCFCdc4 mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4WD40 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4WD40 domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation. PMID:28045046

  12. The SCFFBW7 ubiquitin ligase complex as a tumor suppressor in T cell leukemia.

    PubMed

    Thompson, Benjamin J; Buonamici, Silvia; Sulis, Maria Luisa; Palomero, Teresa; Vilimas, Tomas; Basso, Giuseppe; Ferrando, Adolfo; Aifantis, Iannis

    2007-08-06

    Recent studies have shown that activating mutations of NOTCH1 are responsible for the majority of T cell acute lymphoblastic leukemia (T-ALL) cases. Most of these mutations truncate its C-terminal domain, a region that is important for the NOTCH1 proteasome-mediated degradation. We report that the E3 ligase FBW7 targets NOTCH1 for ubiquitination and degradation. Our studies map in detail the amino acid degron sequence required for NOTCH1-FBW7 interaction. Furthermore, we identify inactivating FBW7 mutations in a large fraction of human T-ALL lines and primary leukemias. These mutations abrogate the binding of FBW7 not only to NOTCH1 but also to the two other characterized targets, c-Myc and cyclin E. The majority of the FBW7 mutations were present during relapse, and they were associated with NOTCH1 HD mutations. Interestingly, most of the T-ALL lines harboring FBW7 mutations were resistant to gamma-secretase inhibitor treatment and this resistance appeared to be related to the stabilization of the c-Myc protein. Our data suggest that FBW7 is a novel tumor suppressor in T cell leukemia, and implicate the loss of FBW7 function as a potential mechanism of drug resistance in T-ALL.

  13. Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4

    PubMed Central

    Hewawasam, Geetha; Shivaraju, Manjunatha; Mattingly, Mark; Venkatesh, Swaminathan; Martin-Brown, Skylar; Florens, Laurence; Workman, Jerry L.; Gerton, Jennifer L.

    2010-01-01

    Cse4 is a variant of histone H3 that is incorporated into a single nucleosome at each centromere in budding yeast. We have discovered an E3 ubiquitin ligase, called Psh1, which controls the cellular level of Cse4 via ubiquitylation and proteolysis. The activity of Psh1 is dependent on both its RING and Zinc finger domains. We demonstrate the specificity of the ubiquitylation activity of Psh1 toward Cse4 in vitro and map the sites of ubiquitylation. Mutation of key lysines prevents ubiquitylation of Cse4 by Psh1 in vitro and stabilizes Cse4 in vivo. While deletion of Psh1 stabilizes Cse4, elimination of the Cse4-specific chaperone Scm3 destabilizes Cse4 and the addition of Scm3 to the Psh1-Cse4 ubiquitylation reaction prevents Cse4 ubiquitylation, together suggesting Scm3 may protect Cse4 from ubiquitylation. Without Psh1, Cse4 overexpression is toxic and Cse4 is found at ectopic locations. Our results suggest Psh1 functions to prevent the mislocalization of Cse4. PMID:21070970

  14. The Highwire Ubiquitin Ligase Promotes Axonal Degeneration by Tuning Levels of Nmnat Protein

    PubMed Central

    Xiong, Xin; Hao, Yan; Sun, Kan; Li, Jiaxing; Li, Xia; Mishra, Bibhudatta; Soppina, Pushpanjali; Wu, Chunlai; Hume, Richard I.; Collins, Catherine A.

    2012-01-01

    Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury. PMID:23226106

  15. RANKL coordinates multiple osteoclastogenic pathways by regulating expression of ubiquitin ligase RNF146.

    PubMed

    Matsumoto, Yoshinori; Larose, Jose; Kent, Oliver A; Lim, Melissa; Changoor, Adele; Zhang, Lucia; Storozhuk, Yaryna; Mao, Xiaohong; Grynpas, Marc D; Cong, Feng; Rottapel, Robert

    2017-04-03

    Bone undergoes continuous remodeling due to balanced bone formation and resorption mediated by osteoblasts and osteoclasts, respectively. Osteoclasts arise from the macrophage lineage, and their differentiation is dependent on RANKL, a member of the TNF family of cytokines. Here, we have provided evidence that RANKL controls the expression of 3BP2, an adapter protein that is required for activation of SRC tyrosine kinase and simultaneously coordinates the attenuation of β-catenin, both of which are required to execute the osteoclast developmental program. We found that RANKL represses the transcription of the E3 ubiquitin ligase RNF146 through an NF-κB-related inhibitory element in the RNF146 promoter. RANKL-mediated suppression of RNF146 results in the stabilization of its substrates, 3BP2 and AXIN1, which consequently triggers the activation of SRC and attenuates the expression of β-catenin, respectively. Depletion of RNF146 caused hypersensitivity to LPS-induced TNF-α production in vivo. RNF146 thus acts as an inhibitory switch to control osteoclastogenesis and cytokine production and may be a control point underlying the pathogenesis of chronic inflammatory diseases.

  16. Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels

    PubMed Central

    Schreiber, Joerg; Végh, Marlene J.; Dawitz, Julia; Kroon, Tim; Loos, Maarten; Labonté, Dorthe; Li, Ka Wan; Van Nierop, Pim; Van Diepen, Michiel T.; De Zeeuw, Chris I.; Kneussel, Matthias; Meredith, Rhiannon M.; Smit, August B.

    2015-01-01

    Synaptic plasticity requires remodeling of the actin cytoskeleton. Although two actin isoforms, β- and γ-actin, are expressed in dendritic spines, the specific contribution of γ-actin in the expression of synaptic plasticity is unknown. We show that synaptic γ-actin levels are regulated by the E3 ubiquitin ligase TRIM3. TRIM3 protein and Actg1 transcript are colocalized in messenger ribonucleoprotein granules responsible for the dendritic targeting of messenger RNAs. TRIM3 polyubiquitylates γ-actin, most likely cotranslationally at synaptic sites. Trim3−/− mice consequently have increased levels of γ-actin at hippocampal synapses, resulting in higher spine densities, increased long-term potentiation, and enhanced short-term contextual fear memory consolidation. Interestingly, hippocampal deletion of Actg1 caused an increase in long-term fear memory. Collectively, our findings suggest that temporal control of γ-actin levels by TRIM3 is required to regulate the timing of hippocampal plasticity. We propose a model in which TRIM3 regulates synaptic γ-actin turnover and actin filament stability and thus forms a transient inhibitory constraint on the expression of hippocampal synaptic plasticity. PMID:26527743

  17. Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4.

    PubMed

    Lu, Jing; Qian, Yimin; Altieri, Martha; Dong, Hanqing; Wang, Jing; Raina, Kanak; Hines, John; Winkler, James D; Crew, Andrew P; Coleman, Kevin; Crews, Craig M

    2015-06-18

    BRD4, a bromodomain and extraterminal domain (BET) family member, is an attractive target in multiple pathological settings, particularly cancer. While BRD4 inhibitors have shown some promise in MYC-driven malignancies such as Burkitt's lymphoma (BL), we show that BRD4 inhibitors lead to robust BRD4 protein accumulation, which may account for their limited suppression of MYC expression, modest antiproliferative activity, and lack of apoptotic induction. To address these limitations we designed ARV-825, a hetero-bifunctional PROTAC (Proteolysis Targeting Chimera) that recruits BRD4 to the E3 ubiquitin ligase cereblon, leading to fast, efficient, and prolonged degradation of BRD4 in all BL cell lines tested. Consequently, ARV-825 more effectively suppresses c-MYC levels and downstream signaling than small-molecule BRD4 inhibitors, resulting in more effective cell proliferation inhibition and apoptosis induction in BL. Our findings provide strong evidence that cereblon-based PROTACs provide a better and more efficient strategy in targeting BRD4 than traditional small-molecule inhibitors.

  18. K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression

    PubMed Central

    Hao, Zhenyue; Sheng, Yi; Duncan, Gordon S.; Li, Wanda Y.; Dominguez, Carmen; Sylvester, Jennifer; Su, Yu-Wen; Lin, Gloria H.Y.; Snow, Bryan E.; Brenner, Dirk; You-Ten, Annick; Haight, Jillian; Inoue, Satoshi; Wakeham, Andrew; Elford, Alisha; Hamilton, Sara; Liang, Yi; Zúñiga-Pflücker, Juan C.; He, Housheng Hansen; Ohashi, Pamela S.; Mak, Tak W.

    2017-01-01

    T-cell proliferation is regulated by ubiquitination but the underlying molecular mechanism remains obscure. Here we report that Lys-48-linked ubiquitination of the transcription factor KLF4 mediated by the E3 ligase Mule promotes T-cell entry into S phase. Mule is elevated in T cells upon TCR engagement, and Mule deficiency in T cells blocks proliferation because KLF4 accumulates and drives upregulation of its transcriptional targets E2F2 and the cyclin-dependent kinase inhibitors p21 and p27. T-cell-specific Mule knockout (TMKO) mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), show impaired generation of antigen-specific CD8+ T cells with reduced cytokine production, and fail to clear LCMV infections. Thus, Mule-mediated ubiquitination of the novel substrate KLF4 regulates T-cell proliferation, autoimmunity and antiviral immune responses in vivo. PMID:28084302

  19. Drosophila Nedd4, a ubiquitin ligase, is recruited by Commissureless to control cell surface levels of the roundabout receptor.

    PubMed

    Myat, Anna; Henry, Pauline; McCabe, Veronica; Flintoft, Louisa; Rotin, Daniela; Tear, Guy

    2002-08-01

    Crossing the midline produces changes in axons such that they are no longer attracted to the midline. In Drosophila, Roundabout reaches high levels on axons once they have crossed the midline, and this prohibits recrossing. Roundabout protein levels are regulated by Commissureless. We show that Commissureless binds to and is regulated by the ubiquitin ligase DNedd4. We further show that the ability of Commissureless to regulate Roundabout protein levels requires an intact DNedd4 binding site and ubiquitin acceptor sites within the Commissureless protein. The ability of Commissureless to regulate Robo in the embryo also requires a Commissureless/DNedd4 interaction. Our results show that changes in axonal sensitivity to external cues during pathfinding across the midline makes use of ubiquitin-dependent mechanisms to regulate transmembrane protein levels.

  20. Cbl-c Ubiquitin Ligase Activity Is Increased via the Interaction of Its RING Finger Domain with a LIM Domain of the Paxillin Homolog, Hic 5

    PubMed Central

    Ryan, Philip E.; Kales, Stephen C.; Yadavalli, Rajgopal; Nau, Marion M.; Zhang, Han; Lipkowitz, Stanley

    2012-01-01

    Cbl proteins (Cbl, Cbl-b and Cbl-c) are ubiquitin ligases that are critical regulators of tyrosine kinase signaling. In this study we identify a new Cbl-c interacting protein, Hydrogen peroxide Induced Construct 5 (Hic-5). The two proteins interact through a novel interaction mediated by the RING finger of Cbl-c and the LIM2 domain of Hic-5. Further, this interaction is mediated and dependent on specific zinc coordinating complexes within the RING finger and LIM domain. Binding of Hic-5 to Cbl-c leads to an increase in the ubiquitin ligase activity of Cbl-c once Cbl-c has been activated by Src phosphorylation or through an activating phosphomimetic mutation. In addition, co-transfection of Hic-5 with Cbl-c leads to an increase in Cbl-c mediated ubiquitination of the EGFR. These data suggest that Hic-5 enhances Cbl-c ubiquitin ligase activity once Cbl-c has been phosphorylated and activated. Interactions between heterologous RING fingers have been shown to activate E3s. This is the first demonstration of enhancement of ubiquitin ligase activity of a RING finger ubiquitin ligase by the direct interaction of a LIM zinc coordinating domain. PMID:23145173

  1. Rad7 E3 Ubiquitin Ligase Attenuates Polyubiquitylation of Rpn10 and Dsk2 Following DNA Damage in Saccharomyces cerevisiae

    PubMed Central

    Benoun, Joseph M.; Lalimar-Cortez, Danielle; Valencia, Analila; Granda, Adriana; Moore, Destaye M.; Kelson, Eric P.

    2016-01-01

    During Nucleotide Excision Repair (NER) in the yeast S. cerevisiae, ubiquitylation of Rad4 is carried out by the E3 ubiquitin ligase that includes Rad7-Elc1-Cul3 and is critical to optimal NER. Rad7 E3 activity targets Rad4 for degradation by the proteaseome but, in principle, could also trigger other DNA damage responses. We observed increased nuclear ubiquitin foci (Ub-RFP) formation in S. cerevisiae containing a Rad7 E3 ligase mutant (rad7SOCS) in response to DNA damage by benzo[a]pyrenediolepoxide (BPDE) in dividing cells. Immunoblots reveal that ubiquitin conjugates of Rpn10 and Dsk2 accumulate in greater abundance in rad7SOCS compared to RAD7 in dividing cells in response to BPDE which makes Rpn10 and Dsk2 candidates for being the ubiquitylated species observed in our microscopy experiments. Microscopy analysis with strains containing Dsk2-GFP shows that Dsk2-GFP and Dsk2-GFP/Ub-RFP colocalized in nuclear foci form to an increased extent in a rad7SOCS mutant background in dividing cells than in a RAD7 wild-type strain. Further, Dsk2-GFP in the rad7SOCS strain formed more foci at the plasma membrane following BPDE treatment in dividing cells relative to strains containing RAD7 or a rad7Δ deletion mutant. In response to a different agent, UV irradiation, levels of ubiquitylated proteins were increased in rad7SOCS relative to RAD7, and the proteasomal deubiquitylase subunit, Rpn11 was even monoubiquitylated in the absence of damaging agents. Together these data show that Rad7 E3 activity attenuates ubiquitylation of proteins regulating the shuttling of polyubiquitylated proteins to the proteasome (Dsk2 and Rpn10) and removal of ubiquitin chains just prior to degradation (Rpn11). Since Rad7 E3 ligase activity has been shown to increase ubiquitylation of its target proteins, yet our results show increased ubiquitylation in the absence of Rad7 E3, we suggest that Rad7 E3 action regulates ubiquitin ligase and deubiquitylase (DUB) activities that act on Rpn10, Dsk2

  2. Functional characterization of SAG/RBX2/ROC2/RNF7, an antioxidant protein and an E3 ubiquitin ligase.

    PubMed

    Sun, Yi; Li, Hua

    2013-02-01

    SAG (Sensitive to Apoptosis Gene), also known as RBX2 (RING box protein 2), ROC2 (Regulator of Cullins 2), or RNF7 (RING Finger Protein 7), was originally cloned in our laboratory as a redox inducible antioxidant protein and later characterized as the second member of the RBX/ROC RING component of the SCF (SKP1-CUL-F-box Proteins) E3 ubiquitin ligase. When acting alone, SAG scavenges oxygen radicals by forming inter- and intra-molecular disulfide bonds, whereas by forming a complex with other components of the SCF E3 ligase, SAG promotes ubiquitination and degradation of a number of protein substrates, including c-JUN, DEPTOR, HIF-1α, IκBα, NF1, NOXA, p27, and procaspase-3, thus regulating various signaling pathways and biological processes. Specifically, SAG protects cells from apoptosis, confers radioresistance, and plays an essential and non-redundant role in mouse embryogenesis and vasculogenesis. Furthermore, stress-inducible SAG is overexpressed in a number of human cancers and SAG overexpression correlates with poor patient prognosis. Finally, SAG transgenic expression in epidermis causes an early stage inhibition, but later stage promotion, of skin tumorigenesis triggered by DMBA/TPA. Given its major role in promoting targeted degradation of tumor suppressive proteins, leading to apoptosis suppression and accelerated tumorigenesis, SAG E3 ligase appears to be an attractive anticancer target.

  3. The E3 ubiquitin ligase Nrdp1 'preferentially' promotes TLR-mediated production of type I interferon.

    PubMed

    Wang, Chen; Chen, Taoyong; Zhang, Jia; Yang, Mingjin; Li, Nan; Xu, Xiongfei; Cao, Xuetao

    2009-07-01

    E3 ubiquitin ligases are important in both innate and adaptive immunity. Here we report that Nrdp1, an E3 ubiquitin ligase, inhibited the production of proinflammatory cytokines but increased interferon-beta production in Toll-like receptor-triggered macrophages by suppressing adaptor MyD88-dependent activation of transcription factors NF-kappaB and AP-1 while promoting activation of the kinase TBK1 and transcription factor IRF3. Nrdp1 directly bound and polyubiquitinated MyD88 and TBK1, which led to degradation of MyD88 and activation of TBK1. Knockdown of Nrdp1 inhibited the degradation of MyD88 and the activation of TBK1 and IRF3. Nrdp1-transgenic mice showed resistance to lipopolysaccharide-induced endotoxin shock and to infection with vesicular stomatitis virus. Our data suggest that Nrdp1 functions as both an adaptor protein and an E3 unbiquitin ligase to regulate TLR responses in different ways.

  4. ROC1/RBX1 E3 ubiquitin ligase silencing suppresses tumor cell growth via sequential induction of G2/M arrest, apoptosis, and senescence

    PubMed Central

    Jia, Lijun; Soengas, Maria S.; Sun, Yi

    2009-01-01

    ROC1 (Regulator of Cullins-1) or RBX1 (Ring Box Protein-1) is a RING component of SCF (Skp-1, cullins, F-box proteins) E3 ubiquitin ligases, which regulate diverse cellular processes by targeting a variety of substrates for degradation. However, little is known about the role of ROC1 in human cancer. Here we reported that ROC1 is ubiquitously over-expressed in primary human tumor tissues and human cancer cell lines. ROC1 silencing by siRNA significantly inhibited the growth of multiple human cancer cells via induction of senescence and apoptosis as well as G2/M arrest. Senescence induction is coupled with DNA damage in p53/p21 and p16/pRB-independent manners. Apoptosis is associated with accumulation of Puma and reduction of Bcl-2, Mcl-1, and survivin; and G2/M arrest is associated with accumulation of 14-3-3σ and elimination of cyclin B1 and Cdc2. In U87 glioblastoma cells, these phenotypic changes occur sequentially upon ROC1 silencing, starting with G2/M arrest, followed by apoptosis and senescence. Thus, ROC1 silencing triggers multiple death and growth arrest pathways to effectively suppress tumor cell growth, suggesting that ROC1 may serve as a potential anti-cancer target. PMID:19509229

  5. The nucleoprotein of influenza A virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase RNF43

    PubMed Central

    Nailwal, H; Sharma, S; Mayank, A K; Lal, S K

    2015-01-01

    The interplay between influenza virus and host factors to support the viral life cycle is well documented. Influenza A virus (IAV) proteins interact with an array of cellular proteins and hijack host pathways which are at the helm of cellular responses to facilitate virus invasion. The multifaceted nature of the ubiquitination pathway for protein regulation makes it a vulnerable target of many viruses including IAV. To this end we conducted a yeast two-hybrid screen to search for cellular ubiquitin ligases important for influenza virus replication. We identified host protein, RING finger protein 43 (RNF43), a RING-type E3 ubiquitin ligase, as a novel interactor of nucleoprotein (NP) of IAV and an essential partner to induce NP-driven p53-mediated apoptosis in IAV-infected cells. In this study, we demonstrate that IAV leads to attenuation of RNF43 transcripts and hence its respective protein levels in the cellular milieu whereas in RNF43 depleted cells, viral replication was escalated several folds. Moreover, RNF43 polyubiquitinates p53 which further leads to its destabilization resulting in a decrease in induction of the p53 apoptotic pathway, a hitherto unknown process targeted by NP for p53 stabilization and accumulation. Collectively, these results conclude that NP targets RNF43 to modulate p53 ubiquitination levels and hence causes p53 stabilization which is conducive to an enhanced apoptosis level in the host cells. In conclusion, our study unravels a novel strategy adopted by IAV for utilizing the much conserved ubiquitin proteasomal pathway. PMID:25996295

  6. Targeting Cullin–RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation

    PubMed Central

    Bulatov, Emil; Ciulli, Alessio

    2015-01-01

    In the last decade, the ubiquitin–proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin–RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs. PMID:25886174

  7. E3 ubiquitin ligase NKLAM positively regulates macrophage inducible nitric oxide synthase expression.

    PubMed

    Lawrence, Donald W; Gullickson, Gail; Kornbluth, Jacki

    2015-01-01

    Stimulated macrophages generate potent anti-microbial reactive oxygen and nitrogen species within their phagosomes. Previous studies have shown that the E3 ubiquitin ligase natural killer lytic-associated molecule (NKLAM) is a macrophage phagosomal protein that plays a role in macrophage anti-bacterial activity. In vivo, NKLAM-knockout (KO) mice produce less nitric oxide (NO) upon exposure to lipopolysaccharide (LPS) than wild type (WT) mice. In vitro, we found that NO production and inducible nitric oxide synthase (iNOS) protein were diminished in LPS-stimulated NKLAM-KO bone marrow-derived and splenic macrophages. Additionally, LPS-stimulated NKLAM-KO macrophages displayed defects in STAT1 tyrosine phosphorylation and production of interferon beta (IFNβ). The JAK/STAT pathway is critical for the production of IFNβ, which augments iNOS protein expression in mice. iNOS protein expression is also regulated by the transcription factor NFκB, thus we investigated whether NKLAM influences NFκB function. LPS-stimulated NKLAM-KO macrophages showed evidence of delayed nuclear translocation of the NFκB subunit p65. This was associated with a reduction in p65/DNA colocalization. The defect in p65 translocation was independent of IKBα degradation. NKLAM-KO macrophages also expressed less p65 and showed evidence of defective p65 phosphorylation at serine 536. Importantly, LPS-stimulated NKLAM-KO macrophages have diminished NFκB transcriptional activity as assessed by transfection of a luciferase reporter plasmid. Collectively, our data implicate NKLAM as a novel modulator of macrophage iNOS expression.

  8. Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas.

    PubMed

    Won, Minho; Ro, Hyunju; Dawid, Igor B

    2015-10-06

    The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.

  9. The E3 ubiquitin ligase skp2 regulates neural differentiation independent from the cell cycle

    PubMed Central

    Boix-Perales, Hector; Horan, Ian; Wise, Helen; Lin, Horng-Ru; Chuang, Li-Chiou; Yew, P Renee; Philpott, Anna

    2007-01-01

    Background The SCFskp2 complex is an E3 ubiquitin ligase that is known to target a number of cell cycle regulators, including cyclin-dependent kinase inhibitors, for proteolysis. While its role in regulation of cell division has been well documented, additional functions in differentiation, including in the nervous system, have not been investigated. Results Using Xenopus as a model system, here we demonstrate that skp2 has an additional role in regulation of differentiation of primary neurons, the first neurons to differentiate in the neural plate. Xenopus skp2 shows a dynamic expression pattern in early embryonic neural tissue and depletion of skp2 results in generation of extra primary neurons. In contrast, over-expression of skp2 inhibits neurogenesis in a manner dependent on its ability to act as part of the SCFskp2 complex. Moreover, inhibition of neurogenesis by skp2 occurs upstream of the proneural gene encoding NeuroD and prior to cell cycle exit. We have previously demonstrated that the Xenopus cyclin dependent kinase inhibitor Xic1 is essential for primary neurogenesis at an early stage, and before these cells exit the cell cycle. We show that SCFskp2 degrades Xic1 in embryos and this contributes to the ability of skp2 to regulate neurogenesis. Conclusion We conclude that the SCFskp2 complex has functions in the control of neuronal differentiation additional to its role in cell cycle regulation. Thus, it is well placed to be a co-ordinating factor regulating both cell proliferation and cell differentiation directly. PMID:18081928

  10. The expression of the ubiquitin ligase subunit Cks1 in human breast cancer

    PubMed Central

    Slotky, Merav; Shapira, Ma'anit; Ben-Izhak, Ofer; Linn, Shai; Futerman, Boris; Tsalic, Medy; Hershko, Dan D

    2005-01-01

    Introduction Loss of the cell-cycle inhibitory protein p27Kip1 is associated with a poor prognosis in breast cancer. The decrease in the levels of this protein is the result of increased proteasome-dependent degradation, mediated and rate-limited by its specific ubiquitin ligase subunits S-phase kinase protein 2 (Skp2) and cyclin-dependent kinase subunit 1 (Cks1). Skp2 was recently found to be overexpressed in breast cancers, but the role of Cks1 in these cancers is unknown. The present study was undertaken to examine the role of Cks1 expression in breast cancer and its relation to p27Kip1 and Skp2 expression and to tumor aggressiveness. Methods The expressions of Cks1, Skp2, and p27Kip1 were examined immunohistochemically on formalin-fixed, paraffin-wax-embedded tissue sections from 50 patients with breast cancer and by immunoblot analysis on breast cancer cell lines. The relation between Cks1 levels and patients' clinical and histological parameters were examined by Cox regression and the Kaplan–Meier method. Results The expression of Cks1 was strongly associated with Skp2 expression (r = 0.477; P = 0.001) and inversely with p27Kip1 (r = -0.726; P < 0.0001). Overexpression of Cks1 was associated with loss of tumor differentiation, young age, lack of expression of estrogen receptors and of progesterone receptors, and decreased disease-free (P = 0.0007) and overall (P = 0.041) survival. In addition, Cks1 and Skp2 expression were increased by estradiol in estrogen-dependent cell lines but were down-regulated by tamoxifen. Conclusion These results suggest that Cks1 is involved in p27Kip1 down-regulation and may have an important role in the development of aggressive tumor behavior in breast cancer. PMID:16168119

  11. Copy number variation of E3 ubiquitin ligase genes in peripheral blood leukocyte and colorectal cancer

    PubMed Central

    Bi, Haoran; Tian, Tian; Zhu, Lin; Zhou, Haibo; Hu, Hanqing; Liu, Yanhong; Li, Xia; Hu, Fulan; Zhao, Yashuang; Wang, Guiyu

    2016-01-01

    Given that E3 ubiquitin ligases (E3) regulate specific protein degradation in many cancer-related biological processes. E3 copy number variation (CNV) may affect the development and prognosis of colorectal cancer (CRC). Therefore, we detected CNVs of five E3 genes in 518 CRC patients and 518 age, gender and residence matched controls in China, and estimated the association between E3 gene CNVs and CRC risk and prognosis. We also estimated their interactions with environmental factors and CRC risk. We find a significant association between the CNVs of MDM2 and CRC risk (amp v.s. wt: odds ratio = 14.37, 95% confidence interval: 1.27, 163.74, P = 0.032), while SKP2 CNVs may significantly decrease CRC risk (del v.s. wt: odds ratio = 0.32, 95% confidence interval: 0.10, 1.00, P = 0.050). However, we find no significant association between the CNVs of other genes and CRC risk. The only significant gene-environment interaction effects are between SKP2 CNVs and consumption of fish and/or fruit (P = 0.014 and P = 0.035) and between FBXW7 CNVs and pork intake (P = 0.040). Finally, we find marginally significant association between β-TRCP CNVs and CRC prognosis (amp v.s. wt, hazard ratio = 0.42, 95% confidence interval: 0.19, 0.97, P = 0.050). PMID:27417709

  12. E3 ubiquitin ligase UBR5 drives the growth and metastasis of triple negative breast cancer.

    PubMed

    Liao, Liqiu; Song, Mei; Li, Xin; Tang, Lili; Zhang, Tuo; Zhang, Lixing; Pan, Yihang; Chouchane, Lotfi; Ma, Xiaojing

    2017-03-22

    Patients with triple negative breast cancers (TNBC) are at high risk for recurrence and metastasis at an early time despite standard treatment, underscoring the need for novel therapeutic modalities. Here we report for the first time a distinctive and profound role of the E3 ubiquitin ligase UBR5 in the growth and metastasis of TNBC. An analysis of primary TNBC specimen by whole exon sequencing revealed strong gene amplifications of UBR5 associated with the disease. UBR5 overexpression in TNBC tissues was confirmed at mRNA and protein levels. CRISPR/Cas9-mediated deletion of ubr5 in an experimental murine mammary carcinoma model of TNBC dramatically abrogated tumor growth and metastasis in vivo, which could be reversed completely via reconstitution with wild type UBR5 but not a catalytically inactive mutant. Loss of UBR5 caused an impairment in angiogenesis within the tumor, associated with increased apoptosis, necrosis, and growth arrest. Absence of UBR5 in the tumor triggered aberrant epithelial to mesenchymal transition (EMT), principally via abrogated expression of E-cadherin, which resulted in severely reduced tumor metastasis to secondary organs. Use of NOD/SCID mice revealed that tumor-derived UBR5 facilitated tumor growth in a manner completely dependent upon immune cells in the microenvironment, whereas it promoted metastasis in a tumor cell-autonomous fashion. Our findings unveil UBR5 as a novel and critical regulator of tumor growth, metastasis, and immune response, and highlight the potential for UBR5 as an effective therapeutic target for the treatment of highly aggressive breast and ovarian cancers that fail conventional therapy.

  13. Role of β-TrCP ubiquitin ligase receptor in UVB mediated responses in skin

    PubMed Central

    Bhatia, Neehar; Demmer, Tara A.; Sharma, Alok K.; Elcheva, Irina; Spiegelman, Vladimir S.

    2011-01-01

    Skin cancers are the most common cancers in the United States. Exposure to UVB radiation is a major risk factor for skin cancer induction. SCFβ-TrCP E3 ubiquitin ligase has been found to be involved in cell cycle, cell proliferation and transformation. Aberrant up-regulation of beta-transducin repeats-containing proteins (β-TrCP) is often found in cancer cell lines and primary tumors. We have previously demonstrated that β-TrCP2 is over-expressed in chemically induced mouse skin tumors [1]. Various cellular stress stimuli, including UVB, induce an increase in β-TrCP1 mRNA and protein levels in human cells [2]. We have previously shown that inhibition of β-TrCP function, by induction of dominant negative β-TrCP2 (β-TrCP2ΔF), in vitro in hTERT immortalized normal keratinocytes, results in increase in UVB induced apoptosis [3]. We have generated transgenic mice with inducible, selective expression of dominant negative β-TrCP2 in epidermis with the Keratin 5 promoter (K5-rTA × TRE-HA-β-TrCPΔF). Here we report that inhibition of β-TrCP function in mouse epidermis results in decrease in UVB-induced edema, hyperplasia, and inflammatory response and increment in UVB-induced apoptosis in skin. Our results suggest that β-TrCP may be an essential player in UVB induced responses in skin and can be a potential therapeutic target for skin cancer. PMID:21187057

  14. High throughput screening for inhibitors of the HECT ubiquitin E3 ligase ITCH identifies antidepressant drugs as regulators of autophagy.

    PubMed

    Rossi, M; Rotblat, B; Ansell, K; Amelio, I; Caraglia, M; Misso, G; Bernassola, F; Cavasotto, C N; Knight, R A; Ciechanover, A; Melino, G

    2014-05-01

    Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 ligase that promotes the ubiquitylation and degradation of several proteins, including p73, p63, c-Jun, JunB, Notch and c-FLIP, thus affecting cell fate. Accordingly, ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening of ITCH auto-ubiquitylation, we identified several putative ITCH inhibitors, one of which is clomipramine--a clinically useful antidepressant drug. Previously, we have shown that clomipramine inhibits autophagy by blocking autophagolysosomal fluxes and thus could potentiate chemotherapy in vitro. Here, we found that clomipramine specifically blocks ITCH auto-ubiquitylation, as well as p73 ubiquitylation. By screening structural homologs of clomipramine, we identified several ITCH inhibitors and putative molecular moieties that are essential for ITCH inhibition. Treating a panel of breast, prostate and bladder cancer cell lines with clomipramine, or its homologs, we found that they reduce cancer cell growth, and synergize with gemcitabine or mitomycin in killing cancer cells by blocking autophagy. We also discuss a potential mechanism of inhibition. Together, our study (i) demonstrates the feasibility of using high throughput screening to identify E3 ligase inhibitors and (ii) provides insight into how clomipramine and its structural homologs might interfere with ITCH and other HECT E3 ligase catalytic activity in (iii) potentiating chemotherapy by regulating autophagic fluxes. These results may have direct clinical applications.

  15. Ubiquitin Ligase NEDD4 Regulates PPARγ Stability and Adipocyte Differentiation in 3T3-L1 Cells

    PubMed Central

    Li, Jing Jing; Wang, Ruishan; Lama, Rati; Wang, Xinjiang; Floyd, Z. Elizabeth; Park, Edwards A.; Liao, Francesca-Fang

    2016-01-01

    Peroxisome proliferator–activated receptor-γ (PPARγ) is a ligand-activated nuclear receptor which controls lipid and glucose metabolism. It is also the master regulator of adipogenesis. In adipocytes, ligand-dependent PPARγ activation is associated with proteasomal degradation; therefore, regulation of PPARγ degradation may modulate its transcriptional activity. Here, we show that neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4), an E3 ubiquitin ligase, interacts with the hinge and ligand binding domains of PPARγ and is a bona fide E3 ligase for PPARγ. NEDD4 increases PPARγ stability through the inhibition of its proteasomal degradation. Knockdown of NEDD4 in 3T3-L1 adipocytes reduces PPARγ protein levels and suppresses adipocyte conversion. PPARγ correlates positively with NEDD4 in obese adipose tissue. Together, these findings support NEDD4 as a novel regulator of adipogenesis by modulating the stability of PPARγ. PMID:27917940

  16. Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy

    PubMed Central

    Li, Jie; Qi, Wei; Chen, Guo; Feng, Du; Liu, Jinhua; Ma, Biao; Zhou, Changqian; Mu, Chenglong; Zhang, Weilin; Chen, Quan; Zhu, Yushan

    2015-01-01

    Mitochondria serve as membrane sources and signaling platforms for regulating autophagy. Accumulating evidence has also shown that damaged mitochondria are removed through both selective mitophagy and general autophagy in response to mitochondrial and oxidative stresses. Protein ubiquitination through mitochondrial E3 ligases plays an integrative role in mitochondrial outer membrane protein degradation, mitochondrial dynamics, and mitophagy. Here we showed that MUL1, a mitochondria-localized E3 ligase, regulates selenite-induced mitophagy in an ATG5 and ULK1-dependent manner. ULK1 partially translocated to mitochondria after selenite treatment and interacted with MUL1. We also demonstrated that ULK1 is a novel substrate of MUL1. These results suggest the association of mitochondria with autophagy regulation and provide a new mechanism for the beneficial effects of selenium as a chemopreventive agent. PMID:26018823

  17. Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition.

    PubMed

    Palle, Komaraiah; Vaziri, Cyrus

    2011-05-15

    Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA Topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.

  18. Identification of essential sequences for cellular localization in the muscle-specific ubiquitin E3 ligase MAFbx/Atrogin 1.

    PubMed

    Julie, Lagirand-Cantaloube; Sabrina, Batonnet-Pichon; Marie-Pierre, Leibovitch; Leibovitch, Serge A

    2012-02-17

    In skeletal muscle atrophy, upregulation and nuclear accumulation of the Ubiquitin E3 ligase MAFbx is essential for accelerated muscle protein loss, but the nuclear/cytoplasmic shuttling of MAFbx is undefined. Here we found that MAFbx contains two functional nuclear localization signals (NLS). Mutation or deletion of only one NLS induced cytoplasmic localization of MAFbx. We identified a non-classical NES located in the leucine charged domain (LCD) of MAFbx, which is leptomycin B insensitive. We demonstrated that mutation (L169Q) in LLXXL motif of LCD suppressed cytoplasmic retention of MAFbx. Nucleocytoplasmic shuttling of MAFbx represents a novel mechanism for targeting its substrates and its cytosolic partners in muscle atrophy.

  19. Balancing protein stability and activity in cancer: a new approach for identifying driver mutations affecting CBL ubiquitin ligase activation

    PubMed Central

    Li, Minghui; Kales, Stephen C.; Ma, Ke; Shoemaker, Benjamin A.; Crespo-Barreto, Juan; Cangelosi, Andrew L.; Lipkowitz, Stanley; Panchenko, Anna R.

    2015-01-01

    Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved depicting the protein at different stages of its activation cycle and thus provide mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than did random non-cancer mutations. We further tested the ability of these computational models assessing the changes in CBL stability and its binding to ubiquitin conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two-thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL. PMID:26676746

  20. Reconstruction of an active SOCS3-based E3 ubiquitin ligase complex in vitro: identification of the active components and JAK2 and gp130 as substrates.

    PubMed

    Kershaw, Nadia J; Laktyushin, Artem; Nicola, Nicos A; Babon, Jeffrey J

    2014-02-01

    SOCS3 (suppressor of cytokine signaling 3) inhibits the intracellular signaling cascade initiated by exposure of cells to cytokines. SOCS3 regulates signaling via two distinct mechanisms: directly inhibiting the catalytic activity of Janus kinases (JAKs) that initiate the intracellular signaling cascade and catalysing the ubiquitination of signaling components by recruiting components of an E3 ubiquitin ligase complex. Here we investigate the latter mode-of-action biochemically by reconstructing a SOCS3-based E3 ubiquitin ligase complex in vitro using fully purified, recombinant components and examining its ability to promote the ubiquitination of molecules involved in the cytokine signaling cascade. We show that SOCS3 is an active substrate recruitment module for a Cullin5-based E3 ligase and have defined the core protein components required for ubiquitination. SOCS3-induced polyubiquitination was rapid and could proceed through a number of different ubiquitin lysines. SOCS3 catalyzed the ubiquitination of both the IL-6 receptor common chain (gp130) and JAK2.

  1. The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.

    PubMed Central

    Sudakin, V; Ganoth, D; Dahan, A; Heller, H; Hershko, J; Luca, F C; Ruderman, J V; Hershko, A

    1995-01-01

    The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles. Images PMID:7787245

  2. MARCH1 E3 Ubiquitin Ligase Dampens the Innate Inflammatory Response by Modulating Monocyte Functions in Mice.

    PubMed

    Galbas, Tristan; Raymond, Maxime; Sabourin, Antoine; Bourgeois-Daigneault, Marie-Claude; Guimont-Desrochers, Fanny; Yun, Tae Jin; Cailhier, Jean-François; Ishido, Satoshi; Lesage, Sylvie; Cheong, Cheolho; Thibodeau, Jacques

    2017-01-15

    Ubiquitination was recently identified as a central process in the pathogenesis and development of numerous inflammatory diseases, such as obesity, atherosclerosis, and asthma. Treatment with proteasomal inhibitors led to severe side effects because ubiquitination is heavily involved in a plethora of cellular functions. Thus, new players regulating ubiquitination processes must be identified to improve therapies for inflammatory diseases. In addition to their role in adaptive immunity, endosomal MHC class II (MHCII) molecules were shown to modulate innate immune responses by fine tuning the TLR4 signaling pathway. However, the role of MHCII ubiquitination by membrane associated ring-CH-type finger 1 (MARCH1) E3 ubiquitin ligase in this process remains to be assessed. In this article, we demonstrate that MARCH1 is a key inhibitor of innate inflammation in response to bacterial endotoxins. The higher mortality of March1(-/-) mice challenged with a lethal dose of LPS was associated with significantly stronger systemic production of proinflammatory cytokines and splenic NK cell activation; however, we did not find evidence that MARCH1 modulates LPS or IL-10 signaling pathways. Instead, the mechanism by which MARCH1 protects against endotoxic shock rests on its capacity to promote the transition of monocytes from Ly6C(Hi) to Ly6C(+/-) Moreover, in competitive bone marrow chimeras, March1(-/-) monocytes and polymorphonuclear neutrophils outcompeted wild-type cells with regard to bone marrow egress and homing to peripheral organs. We conclude that MARCH1 exerts MHCII-independent effects that regulate the innate arm of immunity. Thus, MARCH1 might represent a potential new target for emerging therapies based on ubiquitination reactions in inflammatory diseases.

  3. E3 ubiquitin ligase Cbl-b negatively regulates C-type lectin receptor–mediated antifungal innate immunity

    PubMed Central

    Zhu, Le-Le; Xu, Xia; Zhao, Xue-Qiang; Wang, Ting-Ting; Tang, Bing; Jiang, Yuan-Ying

    2016-01-01

    Activation of various C-type lectin receptors (CLRs) initiates potent proinflammatory responses against various microbial infections. However, how activated CLRs are negatively regulated remains unknown. In this study, we report that activation of CLRs Dectin-2 and Dectin-3 by fungi infections triggers them for ubiquitination and degradation in a Syk-dependent manner. Furthermore, we found that E3 ubiquitin ligase Casitas B–lineage lymphoma protein b (Cbl-b) mediates the ubiquitination of these activated CLRs through associating with each other via adapter protein FcR-γ and tyrosine kinase Syk, and then the ubiquitinated CLRs are sorted into lysosomes for degradation by an endosomal sorting complex required for transport (ESCRT) system. Therefore, the deficiency of either Cbl-b or ESCRT subunits significantly decreases the degradation of activated CLRs, thereby resulting in the higher expression of proinflammatory cytokines and inflammation. Consistently, Cbl-b–deficient mice are more resistant to fungi infections compared with wild-type controls. Together, our study indicates that Cbl-b negatively regulates CLR-mediated antifungal innate immunity, which provides molecular insight for designing antifungal therapeutic agents. PMID:27432944

  4. Ectromelia Virus BTB/kelch Proteins, EVM150 and EVM167, Interact with Cullin-3 Based Ubiquitin Ligases

    PubMed Central

    Wilton, Brianne A.; Campbell, Stephanie; Van Buuren, Nicholas; Garneau, Robyn; Furukawa, Manabu; Xiong, Yue; Barry., Michele

    2008-01-01

    Cellular proteins containing BTB and kelch domains have been shown to function as adapters for the recruitment of substrates to cullin-3-based ubiquitin ligases. Poxviruses are the only family of viruses known to encode multiple BTB/kelch proteins, suggesting that poxviruses may modulate the ubiquitin pathway through interaction with cullin-3. Ectromelia virus encodes four BTB/kelch proteins and one BTB-only protein. Here we demonstrate that two of the ectromelia virus encoded BTB/kelch proteins, EVM150 and EVM167, interacted with cullin-3. Similar to cellular BTB proteins, the BTB domain of EVM150 and EVM167 was necessary and sufficient for cullin-3 interaction. During infection, EVM150 and EVM167 localized to discrete cytoplasmic regions, which co-localized with cullin-3. Furthermore, EVM150 and EVM167 co-localized and interacted with conjugated ubiquitin, as demonstrated by confocal microscopy and co-immunoprecipitation. Our findings suggest that the ectromelia virus encoded BTB/kelch proteins, EVM150 and EVM167, interact with cullin-3 potentially functioning to recruit unidentified substrates for ubiquitination. PMID:18221766

  5. Ectromelia virus BTB/kelch proteins, EVM150 and EVM167, interact with cullin-3-based ubiquitin ligases.

    PubMed

    Wilton, Brianne A; Campbell, Stephanie; Van Buuren, Nicholas; Garneau, Robyn; Furukawa, Manabu; Xiong, Yue; Barry, Michele

    2008-04-25

    Cellular proteins containing BTB and kelch domains have been shown to function as adapters for the recruitment of substrates to cullin-3-based ubiquitin ligases. Poxviruses are the only family of viruses known to encode multiple BTB/kelch proteins, suggesting that poxviruses may modulate the ubiquitin pathway through interaction with cullin-3. Ectromelia virus encodes four BTB/kelch proteins and one BTB-only protein. Here we demonstrate that two of the ectromelia virus-encoded BTB/kelch proteins, EVM150 and EVM167, interacted with cullin-3. Similar to cellular BTB proteins, the BTB domain of EVM150 and EVM167 was necessary and sufficient for cullin-3 interaction. During infection, EVM150 and EVM167 localized to discrete cytoplasmic regions, which co-localized with cullin-3. Furthermore, EVM150 and EVM167 co-localized and interacted with conjugated ubiquitin, as demonstrated by confocal microscopy and co-immunoprecipitation. Our findings suggest that the ectromelia virus-encoded BTB/kelch proteins, EVM150 and EVM167, interact with cullin-3 potentially functioning to recruit unidentified substrates for ubiquitination.

  6. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex.

    PubMed

    Zheng, Ning; Schulman, Brenda A; Song, Langzhou; Miller, Julie J; Jeffrey, Philip D; Wang, Ping; Chu, Claire; Koepp, Deanna M; Elledge, Stephen J; Pagano, Michele; Conaway, Ronald C; Conaway, Joan W; Harper, J Wade; Pavletich, Nikola P

    2002-04-18

    SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 A apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold.

  7. Using the E4orf6-Based E3 Ubiquitin Ligase as a Tool To Analyze the Evolution of Adenoviruses

    PubMed Central

    Gilson, Timra; Ballmann, Mónika Z.; Papp, Tibor; Pénzes, Judit J.; Benkő, Mária; Harrach, Balázs; Branton, Philip E.

    2016-01-01

    ABSTRACT E4orf6 proteins from all human adenoviruses form Cullin-based ubiquitin ligase complexes that, in association with E1B55K, target cellular proteins for degradation. While most are assembled with Cul5, a few utilize Cul2. BC-box motifs enable all these E4orf6 proteins to assemble ligase complexes with Elongins B and C. We also identified a Cul2-box motif used for Cul2 selection in all Cul2-based complexes. With this information, we set out to determine if other adenoviruses also possess the ability to form the ligase complex and, if so, to predict their Cullin usage. Here we report that all adenoviruses known to encode an E4orf6-like protein (mastadenoviruses and atadenoviruses) maintain the potential to form the ligase complex. We could accurately predict Cullin usage for E4orf6 products of mastadenoviruses and all but one atadenovirus. Interestingly, in nonhuman primate adenoviruses, we found a clear segregation of Cullin binding, with Cul5 utilized by viruses infecting great apes and Cul2 by Old/New World monkey viruses, suggesting that a switch from Cul2 to Cul5 binding occurred during the period when great apes diverged from monkeys. Based on the analysis of Cullin selection, we also suggest that the majority of human adenoviruses, which exhibit a broader tropism for the eye and the respiratory tract, exhibit Cul5 specificity and resemble viruses infecting great apes, whereas those that infect the gastrointestinal tract may have originated from monkey viruses that share Cul2 specificity. Finally, aviadenoviruses also appear to contain E4orf6 genes that encode proteins with a conserved XCXC motif followed by, in most cases, a BC-box motif. IMPORTANCE Two early adenoviral proteins, E4orf6 and E1B55K, form a ubiquitin ligase complex with cellular proteins to ubiquitinate specific substrates, leading to their degradation by the proteasome. In studies with representatives of each human adenovirus species, we (and others) previously discovered that some

  8. Investigation of the intermolecular recognition mechanism between the E3 ubiquitin ligase Keap1 and substrate based on multiple substrates analysis

    NASA Astrophysics Data System (ADS)

    Jiang, Zheng-Yu; Xu, Li-Li; Lu, Meng-Chen; Pan, Yang; Huang, Hao-Ze; Zhang, Xiao-Jin; Sun, Hao-Peng; You, Qi-Dong

    2014-12-01

    E3 ubiquitin ligases are attractive drug targets due to their specificity to the ubiquitin machinery. However, the development of E3 ligase inhibitors has proven challenging for the fact that they must disrupt protein-protein interactions (PPIs). The E3 ligase involved in interactome provide new hope for the discovery of the E3 ligase inhibitors. These currently known natural binding partners of the E3 ligase can benefit the discovery of other unknown substrates and also the E3 ligase inhibitors. Herein, we present a novel strategy that using multiple substrates to elucidate the molecular recognition mechanism of E3 ubiquitin ligase. Molecular dynamics simulation, molecular mechanics-generalized born surface area (MM-GBSA) binding energy calculation and energy decomposition scheme were incorporated to evaluate the quantitative contributions of sub-pocket and per-residue to binding. In this case, Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor component of the Cullin-RING ubiquitin ligases complex, is applied for the investigation of how it recognize its substrates, especially Nrf2, a master regulator of the antioxidant response. By analyzing multiple substrates binding determinants, we found that both the polar sub-pockets (P1 and P2) and the nonpolar sub-pockets (P4 and P5) of Keap1 can make remarkable contributions to intermolecular interactions. This finding stresses the requirement for substrates to interact with the polar and nonpolar sub-pockets simultaneously. The results discussed in this paper not only show the binding determinants of the Keap1 substrates but also provide valuable implications for both Keap1 substrate discovery and PPI inhibitor design.

  9. CUL4-DDB1-CDT2 E3 Ligase Regulates the Molecular Clock Activity by Promoting Ubiquitination-Dependent Degradation of the Mammalian CRY1

    PubMed Central

    Tong, Xin; Zhang, Deqiang; Guha, Anirvan; Arthurs, Blake; Cazares, Victor; Gupta, Neil; Yin, Lei

    2015-01-01

    The CUL4-DDB1 E3 ligase complex serves as a critical regulator in various cellular processes, including cell proliferation, DNA damage repair, and cell cycle progression. However, whether this E3 ligase complex regulates clock protein turnover and the molecular clock activity in mammalian cells is unknown. Here we show that CUL4-DDB1-CDT2 E3 ligase ubiquitinates CRY1 and promotes its degradation both in vitro and in vivo. Depletion of the major components of this E3 ligase complex, including Ddb1, Cdt2, and Cdt2-cofactor Pcna, leads to CRY1 stabilization in cultured cells or in the mouse liver. CUL4A-DDB1-CDT2 E3 ligase targets lysine 585 within the C-terminal region of CRY1 protein, shown by the CRY1 585KA mutant’s resistance to ubiquitination and degradation mediated by the CUL4A-DDB1 complex. Surprisingly, both depletion of Ddb1 and over-expression of Cry1-585KA mutant enhance the oscillatory amplitude of the Bmal1 promoter activity without altering its period length, suggesting that CUL4A-DDB1-CDT2 E3 targets CRY1 for degradation and reduces the circadian amplitude. All together, we uncovered a novel biological role for CUL4A-DDB1-CDT2 E3 ligase that regulates molecular circadian behaviors via promoting ubiquitination-dependent degradation of CRY1. PMID:26431207

  10. A HECT Ubiquitin-Protein Ligase as a Novel Candidate Gene for Altered Quinine and Quinidine Responses in Plasmodium falciparum

    PubMed Central

    Sanchez, Cecilia P.; Cyrklaff, Marek; Mu, Jianbing; Ferdig, Michael T.; Stein, Wilfred D.; Lanzer, Michael

    2014-01-01

    The emerging resistance to quinine jeopardizes the efficacy of a drug that has been used in the treatment of malaria for several centuries. To identify factors contributing to differential quinine responses in the human malaria parasite Plasmodium falciparum, we have conducted comparative quantitative trait locus analyses on the susceptibility to quinine and also its stereoisomer quinidine, and on the initial and steady-state intracellular drug accumulation levels in the F1 progeny of a genetic cross. These data, together with genetic screens of field isolates and laboratory strains associated differential quinine and quinidine responses with mutated pfcrt, a segment on chromosome 13, and a novel candidate gene, termed MAL7P1.19 (encoding a HECT ubiquitin ligase). Despite a strong likelihood of association, episomal transfections demonstrated a role for the HECT ubiquitin-protein ligase in quinine and quinidine sensitivity in only a subset of genetic backgrounds, and here the changes in IC50 values were moderate (approximately 2-fold). These data show that quinine responsiveness is a complex genetic trait with multiple alleles playing a role and that more experiments are needed to unravel the role of the contributing factors. PMID:24830312

  11. MM-1 facilitates degradation of c-Myc by recruiting proteasome and a novel ubiquitin E3 ligase.

    PubMed

    Kimura, Yumiko; Nagao, Arisa; Fujioka, Yuko; Satou, Akiko; Taira, Takahiro; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2007-10-01

    We have reported that a novel c-Myc-binding protein, MM-1, repressed the E-box-dependent transcription activity of c-Myc by recruiting the HDAC1 complex via TIF1beta/KAP1, a transcriptional corepressor. We have also reported that a mutation of A157R in MM-1, which is often observed in patients with leukemia or lymphoma, abrogated all of the repressive activities of MM-1 toward c-Myc, indicating that MM-1 is a novel tumor suppressor. In this study, we found that MM-1 was bound to a component of proteasome and stimulated degradation of c-Myc in human cells. Knockdown of endogenous MM-1 in human HeLa cells by introduction of siRNA against MM-1 stabilized the endogenous c-Myc. To identify proteins that participate in c-Myc degradation by MM-1, in vivo and in vitro binding assays were carried out. The results showed that MM-1 directly bound to Rpt3, a subunit of 26S proteasome, and that c-Myc directly bound to Skp2, which recruited ElonginC, ElonginB and Cullin2, thereby forming a novel ubiquitin E3 ligase. Knockdown of endogenous Cullin2 stabilized the endogenous c-Myc. Thus, MM-1 is a factor that connects c-Myc to the ubiquitin E3 ligase and the proteasome.

  12. E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors

    PubMed Central

    Zhang, Yan; Yokoyama, Shigetoshi; Herriges, John C.; Zhang, Zhen; Young, Randee E.; Verheyden, Jamie M.; Sun, Xin

    2016-01-01

    The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis. PMID:27335464

  13. Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1

    PubMed Central

    Heck, Jarrod W.; Cheung, Samantha K.; Hampton, Randolph Y.

    2009-01-01

    Eukaryotic cells maintain proteostasis by quality control (QC) degradation. These pathways can specifically target a wide variety of distinct misfolded proteins, and so are important for management of cellular stress. Although a number of conserved QC pathways have been described in yeast, the E3 ligases responsible for cytoplasmic QC are unknown. We now show that Ubr1 and San1 mediate chaperone-dependent ubiquitination of numerous misfolded cytoplasmic proteins. This action of Ubr1 is distinct from its role in the “N-end rule.” In this capacity, Ubr1 functions to protect cells from proteotoxic stresses. Our phenotypic and biochemical studies of Ubr1 and San1 indicate that two strategies are employed for cytoplasmic QC: chaperone-assisted ubiquitination by Ubr1 and chaperone-dependent delivery to nuclear San1. The broad conservation of Ubr ligases and the relevant chaperones indicates that these mechanisms will be important in understanding both basic and biomedical aspects of cellular proteostasis. PMID:20080635

  14. Identification of a novel motif that affects the conformation and activity of the MARCH1 E3 ubiquitin ligase.

    PubMed

    Bourgeois-Daigneault, Marie-Claude; Thibodeau, Jacques

    2013-02-15

    MARCH1, a member of the membrane-associated RING-CH family of E3 ubiquitin ligases, regulates antigen presentation by downregulating the cell surface expression of Major Histocompatibility Complex class II and CD86 molecules. MARCH1 is a transmembrane protein that exposes both its N- and C-terminus to the cytoplasm. We have conducted a structure-function analysis of its two cytoplasmic tails to gain insights into the trafficking of MARCH1 in the endocytic pathway. Fusion of the N-terminal portion of MARCH1 to a type II transmembrane reporter molecule revealed that this cytoplasmic tail contains endosomal sorting motifs. The C-terminal domain also appears to contain intracellular sorting signals because it reduced surface expression of a type I transmembrane reporter molecule. Mutation of the two putative C-terminal tyrosine-based sorting signals did not affect the activity of human MARCH1; however, it did reduce its incorporation into exosomes. Moreover, site-directed mutagenesis pointed to a functional C-terminal 221VQNC224 sequence that affects the spatial organization of the two cytoplasmic regions. This motif is also found in other RING-type E3 ubiquitin ligases, such as parkin. Altogether, these findings highlight the complex regulation of MARCH1 trafficking in the endocytic pathway as well as the intricate interactions between its cytoplasmic tails.

  15. Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-beta signaling.

    PubMed

    Gao, Sheng; Alarcón, Claudio; Sapkota, Gopal; Rahman, Sadia; Chen, Pan-Yu; Goerner, Nina; Macias, Maria J; Erdjument-Bromage, Hediye; Tempst, Paul; Massagué, Joan

    2009-11-13

    TGF-beta induces phosphorylation of the transcription factors Smad2 and Smad3 at the C terminus as well as at an interdomain linker region. TGF-beta-induced linker phosphorylation marks the activated Smad proteins for proteasome-mediated destruction. Here, we identify Nedd4L as the ubiquitin ligase responsible for this step. Through its WW domain, Nedd4L specifically recognizes a TGF-beta-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 polyubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGF-beta-activated Smads and restricts the amplitude and duration of TGF-beta gene responses, and in mouse embryonic stem cells, it limits the induction of mesoendodermal fates by Smad2/3-activating factors. Hierarchical regulation is provided by SGK1, which phosphorylates Nedd4L to prevent binding of Smad2/3. Previously identified as a regulator of renal sodium channels, Nedd4L is shown here to play a broader role as a general modulator of Smad turnover during TGF-beta signal transduction.

  16. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1

    PubMed Central

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-01-01

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1–3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4–9 did not influence the cell cycle–regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4–9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1. PMID:27226481

  17. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1.

    PubMed

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-07-15

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1-3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4-9 did not influence the cell cycle-regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4-9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.

  18. Curcumin-induced degradation of ErbB2: A role for the E3 ubiquitin ligase CHIP and the Michael reaction acceptor activity of curcumin.

    PubMed

    Jung, Yunjin; Xu, Wanping; Kim, Heejung; Ha, Namchul; Neckers, Len

    2007-03-01

    We investigated the molecular mechanism underlying curcumin depletion of ErbB2 protein. Curcumin induced ErbB2 ubiquitination but pretreatment with proteasome inhibitors neither prevented curcumin depletion of ErbB2 protein nor further accumulated ubiquitinated ErbB2. Curcumin increased association of endogenous and ectopically expressed CHIP, a chaperone-dependent ubiquitin ligase, with ErbB2. In COS7 cells cotransfected with ErbB2 and various CHIP plasmids followed by curcumin treatment, CHIP-H260Q (a mutant lacking ubiquitin ligase activity) promoted less curcumin-induced ErbB2 ubiquitination than did wild type CHIP, and CHIP-K30A (a mutant incapable of binding Hsp90 and Hsp70) neither associated with ErbB2 nor promoted its ubiquitination. ErbB2 mutants lacking the kinase domain failed to associate with CHIP and were completely resistant to ubiquitination and depletion induced by curcumin. Finally, curcumin's Michael reaction acceptor functionality was required for both covalent association of curcumin with ErbB2 and curcumin-mediated ErbB2 depletion. These data suggest (1) that CHIP-dependent ErbB2 ubiquitination is implicated in curcumin-stimulated ErbB2 depletion, and (2) that covalent modification of ErbB2 by curcumin is the proximal signal which initiates this process.

  19. Lysine 63-Linked TANK-Binding Kinase 1 Ubiquitination by Mindbomb E3 Ubiquitin Protein Ligase 2 Is Mediated by the Mitochondrial Antiviral Signaling Protein

    PubMed Central

    Ye, Jung Sook; Kim, Nari; Lee, Kyoung Jin; Nam, Young Ran; Lee, Uk

    2014-01-01

    ABSTRACT Beta interferon (IFN-β) is involved in a wide range of cellular functions, and its secretion must be tightly controlled to inhibit viral spreading while minimizing cellular damage. Intracellular viral replication triggers cellular signaling cascades leading to the activation of the transcription factors NF-κB and interferon regulatory factor 3 (IRF3) and IRF7 (IRF3/7), which synergistically bind to the IFN-β gene promoter to induce its expression. The mitochondrial antiviral signaling protein (MAVS) is a governing adaptor protein that mediates signaling communications between virus-sensing proteins and transcription factors. The activity of MAVS in the regulation of IFN-β secretion is affected by many cellular factors. However, the mechanism of MAVS-mediated IRF3/7 activation is not completely understood. Here, we identified a highly conserved DLAIS motif at amino acid positions 438 to 442 of MAVS that is indispensable for IRF3/7 activation. Specifically, the L439S and A440R mutations suppress IRF3/7 activation. Pulldown experiments using wild-type and mutant MAVS showed that mindbomb E3 ubiquitin protein ligase 2 (MIB2) binds to the DLAIS motif. Furthermore, the DLAIS motif was found to be critical for MIB2 binding, the ligation of K63-linked ubiquitin to TANK-binding kinase 1, and phosphorylation-mediated IRF3/7 activation. Our results suggest that MIB2 plays a putative role in MAVS-mediated interferon signaling. IMPORTANCE Mitochondrial antiviral signaling protein (MAVS) mediates signaling from virus-sensing proteins to transcription factors for the induction of beta interferon. However, the mechanism underlying activation of MAVS-mediated interferon regulatory factors 3 and 7 (IRF3/7) is not completely understood. We found a highly conserved DLAIS motif in MAVS that is indispensable for IRF3/7 activation through TANK-binding kinase 1 (TBK1) and identified it as the binding site for mindbomb E3 ubiquitin protein ligase 2 (MIB2). The mutations that

  20. Direct role for proliferating cell nuclear antigen in substrate recognition by the E3 ubiquitin ligase CRL4Cdt2.

    PubMed

    Havens, Courtney G; Shobnam, Nadia; Guarino, Estrella; Centore, Richard C; Zou, Lee; Kearsey, Stephen E; Walter, Johannes C

    2012-03-30

    The E3 ubiquitin ligase Cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) is emerging as an important cell cycle regulator that targets numerous proteins for destruction in S phase and after DNA damage, including Cdt1, p21, and Set8. CRL4(Cdt2) substrates contain a "PIP degron," which consists of a canonical proliferating cell nuclear antigen (PCNA) interaction motif (PIP box) and an adjacent basic amino acid. Substrates use their PIP box to form a binary complex with PCNA on chromatin and the basic residue to recruit CRL4(Cdt2) for substrate ubiquitylation. Using Xenopus egg extracts, we identify an acidic residue in PCNA that is essential to support destruction of all CRL4(Cdt2) substrates. This PCNA residue, which adjoins the basic amino acid of the bound PIP degron, is dispensable for substrate binding to PCNA but essential for CRL4(Cdt2) recruitment to chromatin. Our data show that the interaction of CRL4(Cdt2) with substrates requires molecular determinants not only in the substrate degron but also on PCNA. The results illustrate a potentially general mechanism by which E3 ligases can couple ubiquitylation to the formation of protein-protein interactions.

  1. The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress

    PubMed Central

    Melnik, Andre; Wilson-Zbinden, Caroline; Schellhaas, René; Kastner, Lisa; Piwko, Wojciech; Dees, Martina; Picotti, Paola; Maric, Marija; Labib, Karim; Luke, Brian; Peter, Matthias

    2016-01-01

    Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1’s replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks. PMID:26849847

  2. Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene

    PubMed Central

    Cook, Emma C. L.; Nelson, Jessica K.; Sorrentino, Vincenzo; Koenis, Duco; Moeton, Martina; Scheij, Saskia; Ottenhoff, Roelof; Bleijlevens, Boris; Loregger, Anke

    2017-01-01

    Cellular cholesterol metabolism is subject to tight regulation to maintain adequate levels of this central lipid molecule. Herein, the sterol-responsive Liver X Receptors (LXRs) play an important role owing to their ability to reduce cellular cholesterol load. In this context, identifying the full set of LXR-regulated genes will contribute to our understanding of their role in cholesterol metabolism. Using global transcriptional analysis we report here the identification of RNF145 as an LXR-regulated target gene. We demonstrate that RNF145 is regulated by LXRs in both human and mouse primary cells and cell lines, and in vivo in mice. Regulation of RNF145 by LXR depends on a functional LXR-element in its proximal promotor. Consistent with LXR-dependent regulation of Rnf145 we show that regulation is lost in macrophages and fibroblasts from Lxrαβ(-/-) mice, and also in vivo in livers of Lxrα(-/-) mice treated with the LXR synthetic ligand T0901317. RNF145 is closely related to RNF139/TRC8, an E3 ligase implicated in control of SREBP processing. However, silencing of RNF145 in HepG2 or HeLa cells does not impair SREBP1/2 processing and sterol-responsive gene expression in these cells. Similar to TRC8, we demonstrate that RNF145 is localized to the ER and that it possesses intrinsic E3 ubiquitin ligase activity. In summary, we report the identification of RNF145 as an ER-resident E3 ubiquitin ligase that is transcriptionally controlled by LXR. PMID:28231341

  3. Structural basis of lenalidomide-induced CK1α degradation by the CRL4(CRBN) ubiquitin ligase.

    PubMed

    Petzold, Georg; Fischer, Eric S; Thomä, Nicolas H

    2016-04-07

    Thalidomide and its derivatives, lenalidomide and pomalidomide, are immune modulatory drugs (IMiDs) used in the treatment of haematologic malignancies. IMiDs bind CRBN, the substrate receptor of the CUL4-RBX1-DDB1-CRBN (also known as CRL4(CRBN)) E3 ubiquitin ligase, and inhibit ubiquitination of endogenous CRL4(CRBN) substrates. Unexpectedly, IMiDs also repurpose the ligase to target new proteins for degradation. Lenalidomide induces degradation of the lymphoid transcription factors Ikaros and Aiolos (also known as IKZF1 and IKZF3), and casein kinase 1α (CK1α), which contributes to its clinical efficacy in the treatment of multiple myeloma and 5q-deletion associated myelodysplastic syndrome (del(5q) MDS), respectively. How lenalidomide alters the specificity of the ligase to degrade these proteins remains elusive. Here we present the 2.45 Å crystal structure of DDB1-CRBN bound to lenalidomide and CK1α. CRBN and lenalidomide jointly provide the binding interface for a CK1α β-hairpin-loop located in the kinase N-lobe. We show that CK1α binding to CRL4(CRBN) is strictly dependent on the presence of an IMiD. Binding of IKZF1 to CRBN similarly requires the compound and both, IKZF1 and CK1α, use a related binding mode. Our study provides a mechanistic explanation for the selective efficacy of lenalidomide in del(5q) MDS therapy. We anticipate that high-affinity protein-protein interactions induced by small molecules will provide opportunities for drug development, particularly for targeted protein degradation.

  4. A Novel Retinoblastoma Protein (RB) E3 Ubiquitin Ligase (NRBE3) Promotes RB Degradation and Is Transcriptionally Regulated by E2F1 Transcription Factor.

    PubMed

    Wang, Yingshuang; Zheng, Zongfang; Zhang, Jingyi; Wang, You; Kong, Ruirui; Liu, Jiangying; Zhang, Ying; Deng, Hongkui; Du, Xiaojuan; Ke, Yang

    2015-11-20

    Retinoblastoma protein (RB) plays critical roles in tumor suppression and is degraded through the proteasomal pathway. However, E3 ubiquitin ligases responsible for proteasome-mediated degradation of RB are largely unknown. Here we characterize a novel RB E3 ubiquitin ligase (NRBE3) that binds RB and promotes RB degradation. NRBE3 contains an LXCXE motif and bound RB in vitro. NRBE3 interacted with RB in cells when proteasome activity was inhibited. NRBE3 promoted RB ubiquitination and degradation via the ubiquitin-proteasome pathway. Importantly, purified NRBE3 ubiquitinated recombinant RB in vitro, and a U-box was identified as essential for its E3 activity. Surprisingly, NRBE3 was transcriptionally activated by E2F1/DP1. Consequently, NRBE3 affected the cell cycle by promoting G1/S transition. Moreover, NRBE3 was up-regulated in breast cancer tissues. Taken together, we identified NRBE3 as a novel ubiquitin E3 ligase for RB that might play a role as a potential oncoprotein in human cancers.

  5. Down-regulation of intestinal apical calcium entry channel TRPV6 by ubiquitin E3 ligase Nedd4-2.

    PubMed

    Zhang, Wei; Na, Tao; Wu, Guojin; Jing, Haiyan; Peng, Ji-Bin

    2010-11-19

    Nedd4-2 is an archetypal HECT ubiquitin E3 ligase that disposes target proteins for degradation. Because of the proven roles of Nedd4-2 in degradation of membrane proteins, such as epithelial Na(+) channel, we examined the effect of Nedd4-2 on the apical Ca(2+) channel TRPV6, which is involved in transcellular Ca(2+) transport in the intestine using the Xenopus laevis oocyte system. We demonstrated that a significant amount of Nedd4-2 protein was distributed to the absorptive epithelial cells in ileum, cecum, and colon along with TRPV6. When co-expressed in oocytes, Nedd4-2 and, to a lesser extent, Nedd4 down-regulated the protein abundance and Ca(2+) influx of TRPV6 and TRPV5, respectively. TRPV6 ubiquitination was increased, and its stability was decreased by Nedd4-2. The Nedd4-2 inhibitory effects on TRPV6 were partially blocked by proteasome inhibitor MG132 but not by the lysosome inhibitor chloroquine. The rate of TRPV6 internalization was not significantly altered by Nedd4-2. The HECT domain was essential to the inhibitory effect of Nedd4-2 on TRPV6 and to their association. The WW1 and WW2 domains interacted with TRPV6 terminal regions, and a disruption of the interactions by D204H and D376H mutations in the WW1 and WW2 domains increased TRPV6 ubiquitination and degradation. Thus, WW1 and WW2 may serve as a molecular switch to limit the ubiquitination of TRPV6 by the HECT domain. In conclusion, Nedd4-2 may regulate TRPV6 protein abundance in intestinal epithelia by controlling TRPV6 ubiquitination.

  6. Structure of a Fbw7-Skp1-Cyclin E Complex: Multisite-Phosphorylated Substrate Recognition by SCF Ubiquitin Ligases

    SciTech Connect

    Hao,B.; Oehlmann, S.; Sowa, M.; Harper, J.; Pavletich, N.

    2007-01-01

    The ubiquitin-mediated proteolysis of cyclin E plays a central role in cell-cycle progression, and cyclin E accumulation is a common event in cancer. Cyclin E degradation is triggered by multisite phosphorylation, which induces binding to the SCFFbw7 ubiquitin ligase complex. Structures of the Skp1-Fbw7 complex bound to cyclin E peptides identify a doubly phosphorylated pThr380/pSer384 cyclin E motif as an optimal, high-affinity degron and a singly phosphorylated pThr62 motif as a low-affinity one. Biochemical data indicate that the closely related yeast SCFCdc4 complex recognizes the multisite phosphorylated Sic1 substrate similarly and identify three doubly phosphorylated Sic1 degrons, each capable of high-affinity interactions with two Cdc4 phosphate binding sites. A model that explains the role of multiple cyclin E/Sic1 degrons is provided by the findings that Fbw7 and Cdc4 dimerize, that Fbw7 dimerization enhances the turnover of a weakly associated cyclin E in vivo, and that Cdc4 dimerization increases the rate and processivity of Sic1 ubiquitination in vitro.

  7. High throughput screening for inhibitors of the HECT ubiquitin E3 ligase ITCH identifies antidepressant drugs as regulators of autophagy

    PubMed Central

    Rossi, M; Rotblat, B; Ansell, K; Amelio, I; Caraglia, M; Misso, G; Bernassola, F; Cavasotto, C N; Knight, R A; Ciechanover, A; Melino, G

    2014-01-01

    Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 ligase that promotes the ubiquitylation and degradation of several proteins, including p73, p63, c-Jun, JunB, Notch and c-FLIP, thus affecting cell fate. Accordingly, ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening of ITCH auto-ubiquitylation, we identified several putative ITCH inhibitors, one of which is clomipramine—a clinically useful antidepressant drug. Previously, we have shown that clomipramine inhibits autophagy by blocking autophagolysosomal fluxes and thus could potentiate chemotherapy in vitro. Here, we found that clomipramine specifically blocks ITCH auto-ubiquitylation, as well as p73 ubiquitylation. By screening structural homologs of clomipramine, we identified several ITCH inhibitors and putative molecular moieties that are essential for ITCH inhibition. Treating a panel of breast, prostate and bladder cancer cell lines with clomipramine, or its homologs, we found that they reduce cancer cell growth, and synergize with gemcitabine or mitomycin in killing cancer cells by blocking autophagy. We also discuss a potential mechanism of inhibition. Together, our study (i) demonstrates the feasibility of using high throughput screening to identify E3 ligase inhibitors and (ii) provides insight into how clomipramine and its structural homologs might interfere with ITCH and other HECT E3 ligase catalytic activity in (iii) potentiating chemotherapy by regulating autophagic fluxes. These results may have direct clinical applications. PMID:24787015

  8. Inactivation of Sag/Rbx2/Roc2 e3 ubiquitin ligase triggers senescence and inhibits kras-induced immortalization.

    PubMed

    Tan, Mingjia; Li, Hua; Sun, Yi

    2015-01-01

    Our recent study showed that SAG/RBX2 E3 ubiquitin ligase regulates apoptosis and vasculogenesis by promoting degradation of NOXA and NF1, and co-operates with Kras to promote lung tumorigenesis by activating NFκB and mTOR pathways via targeted degradation of tumor suppressive substrates including IκB, DEPTOR, p21 and p27. Here we investigated the role of Sag/Rbx2 E3 ligase in cellular senescence and immortalization of mouse embryonic fibroblasts (MEFs) and report that Sag is required for proper cell proliferation and Kras(G12D)-induced immortalization. Sag inactivation by genetic deletion remarkably suppresses cell proliferation by inducing senescence, which is associated with accumulation of p16, but not p53. Mechanistically, Sag deletion caused accumulation of Jun-B, a substrate of Sag-Fbxw7 E3 ligase and a transcription factor that drives p16 transcription. Importantly, senescence triggered by Sag deletion can be largely rescued by simultaneous deletion of Cdkn2a, the p16 encoding gene, indicating its causal role. Furthermore, Kras(G12D)-induced immortalization can also be abrogated by Sag deletion via senescence induction, which is again rescued by simultaneous deletion of Cdkn2a. Finally, we found that Sag deletion inactivates Kras(G12D) activity and block the MAPK signaling pathway, together with accumulated p16, to induce senescence. Taken together, our results demonstrated that Sag is a Kras(G12D)-cooperating oncogene required for Kras(G12D)-induced immortalization and transformation, and targeting SAG-SCF E3 ligase may, therefore, have therapeutic value for senescence-based cancer treatment.

  9. Inactivation of Sag/Rbx2/Roc2 E3 Ubiquitin Ligase Triggers Senescence and Inhibits Kras-Induced Immortalization

    PubMed Central

    Tan, Mingjia; Li, Hua; Sun, Yi

    2015-01-01

    Our recent study showed that SAG/RBX2 E3 ubiquitin ligase regulates apoptosis and vasculogenesis by promoting degradation of NOXA and NF1, and co-operates with Kras to promote lung tumorigenesis by activating NFκB and mTOR pathways via targeted degradation of tumor suppressive substrates including IκB, DEPTOR, p21 and p27. Here we investigated the role of Sag/Rbx2 E3 ligase in cellular senescence and immortalization of mouse embryonic fibroblasts (MEFs) and report that Sag is required for proper cell proliferation and KrasG12D-induced immortalization. Sag inactivation by genetic deletion remarkably suppresses cell proliferation by inducing senescence, which is associated with accumulation of p16, but not p53. Mechanistically, Sag deletion caused accumulation of Jun-B, a substrate of Sag-Fbxw7 E3 ligase and a transcription factor that drives p16 transcription. Importantly, senescence triggered by Sag deletion can be largely rescued by simultaneous deletion of Cdkn2a, the p16 encoding gene, indicating its causal role. Furthermore, KrasG12D-induced immortalization can also be abrogated by Sag deletion via senescence induction, which is again rescued by simultaneous deletion of Cdkn2a. Finally, we found that Sag deletion inactivates KrasG12D activity and block the MAPK signaling pathway, together with accumulated p16, to induce senescence. Taken together, our results demonstrated that Sag is a KrasG12D-cooperating oncogene required for KrasG12D-induced immortalization and transformation, and targeting SAG-SCF E3 ligase may, therefore, have therapeutic value for senescence-based cancer treatment. PMID:25622904

  10. Deficiency of UBE2T, the E2 Ubiquitin Ligase Necessary for FANCD2 and FANCI Ubiquitination, Causes FA-T Subtype of Fanconi Anemia.

    PubMed

    Rickman, Kimberly A; Lach, Francis P; Abhyankar, Avinash; Donovan, Frank X; Sanborn, Erica M; Kennedy, Jennifer A; Sougnez, Carrie; Gabriel, Stacey B; Elemento, Olivier; Chandrasekharappa, Settara C; Schindler, Detlev; Auerbach, Arleen D; Smogorzewska, Agata

    2015-07-07

    Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT.

  11. Multiple interactions drive adaptor-mediated recruitment of the ubiquitin ligase rsp5 to membrane proteins in vivo and in vitro.

    PubMed

    Sullivan, James A; Lewis, Michael J; Nikko, Elina; Pelham, Hugh R B

    2007-07-01

    Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain "PY" motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY-WW interactions is required for the ubiquitination of Smf1.

  12. Ube3a, the E3 ubiquitin ligase causing Angelman syndrome and linked to autism, regulates protein homeostasis through the proteasomal shuttle Rpn10.

    PubMed

    Lee, So Young; Ramirez, Juanma; Franco, Maribel; Lectez, Benoît; Gonzalez, Monika; Barrio, Rosa; Mayor, Ugo

    2014-07-01

    Ubiquitination, the covalent attachment of ubiquitin to a target protein, regulates most cellular processes and is involved in several neurological disorders. In particular, Angelman syndrome and one of the most common genomic forms of autism, dup15q, are caused respectively by lack of or excess of UBE3A, a ubiquitin E3 ligase. Its Drosophila orthologue, Ube3a, is also active during brain development. We have now devised a protocol to screen for substrates of this particular ubiquitin ligase. In a neuronal cell system, we find direct ubiquitination by Ube3a of three proteasome-related proteins Rpn10, Uch-L5, and CG8209, as well as of the ribosomal protein Rps10b. Only one of these, Rpn10, is targeted for degradation upon ubiquitination by Ube3a, indicating that degradation might not be the only effect of Ube3a on its substrates. Furthermore, we report the genetic interaction in vivo between Ube3a and the C-terminal part of Rpn10. Overexpression of these proteins leads to an enhanced accumulation of ubiquitinated proteins, further supporting the biochemical evidence of interaction obtained in neuronal cells.

  13. The ubiquitin ligase UBE4A inhibits prostate cancer progression by targeting interleukin-like EMT inducer (ILEI).

    PubMed

    Sun, Yanan; Jia, Xiaopeng; Gao, Qiang; Liu, Xing; Hou, Lianguo

    2017-01-01

    Epithelial to mesenchymal transition (EMT) is an important prerequisite for metastasis to secondary organs. Interleukin-like EMT inducer (ILEI) protein has been shown to translationally upregulated during EMT and metastatic progression as a consequence of aberrant TGF-β signaling. Our initial evaluation of FAM3C (encoding ILEI) and ILEI expression in normal prostate (PCS-440-010) and prostate cancer cell lines (DU145, LNCaP, and PC3) revealed detectable protein expression in only LNCaP cell line even though all cell lines tested had comparable FAM3C expression. Given that PC3 and DU145 cell lines did not have detectable ILEI expression hinted at additional level of regulation of ILEI expression. Treatment with MG-132 resulted in robust detection of ILEI in the PCS-440-010, PC3 and DU145 cell lines, suggesting that at least in these cell lines, ILEI is actively degraded by the proteasome. Mass spectrometric analysis of FLAG immunoprecipitates of untreated and MG-132 treated FLAG-ILEI transfected cells indicated that UBE4A and UBE3C ubiquitin ligases were interacting with ILEI. Ectopic overexpression of UBE4A, but not UBE3C, resulted in destabilization of ILEI in LNCaP cells, whereas RNAi-mediated silencing of UBE4A in PCS-440-010, PC3 and DU145 cell lines resulted in robust accumulation of ILEI, indicating UBE4A as the cognate ubiquitin ligase for ILEI. Co-immunoprecipitation experiments established direct interaction of endogenous ILEI and UBE4A. Furthermore, co-immunoprecipitation of FLAG-tagged ILEI in cells co-transfected with either HA-UBE4A or HA-UBE3C revealed robust polyubiquitinated smear of ILEI in cells transfected with UBE4A, but not UBE3C, thus confirming UBE4A as the ubiquitin ligase for ILEI degradation. Ectopic overexpression of UBE4A, but not UBE3C, in cells was downregulated in vitro migration and invasion in these cells. Cumulatively, our data reveals a novel post-translational regulatory mechanism of regulating ILEI1 expression, a protein

  14. PDLIM7 is a novel target of the ubiquitin ligase Nedd4-1 in skeletal muscle.

    PubMed

    D'Cruz, Robert; Plant, Pamela J; Pablo, Lesley A; Lin, Shouzhe; Chackowicz, Joshua; Correa, Judy; Bain, James; Batt, Jane

    2016-02-01

    Skeletal muscle atrophy remains a complication occurring both as a natural response to muscle disuse and as a pathophysiological response to illness such as diabetes mellitus and nerve injury, such as traumatic muscle denervation. The ubiquitin-proteasome system (UPS) is the predominant proteolytic machinery responsible for atrophy of skeletal muscle, and Nedd4-1 (neural precursor cell-expressed developmentally down-regulated 4-1) is one of a series of E3 ubiquitin ligases identified to mediate inactivity-induced muscle wasting. Targets of Nedd4-1 mediated ubiquitination in skeletal muscle remain poorly understood. In the present study, we identified PDLIM7 (PDZ and LIM domain 7, Enigma), a member of the PDZ-LIM family of proteins, as a novel target of Nedd4-1 in skeletal muscle. The PDZ-LIM family of proteins is known to regulate muscle development and function. We show that Nedd4-1 expression in muscle atrophied by denervation is co-incident with a decrease in PDLIM7 and that PDLIM7 protein levels are stabilized in denervated muscle of Nedd4-1 skeletal muscle-specific knockout mice (SMS-KO). Exogenous PDLIM7 and Nedd4-1 transfected into human embryonic kidney (HEK)293 cells co-immunoprecipitate through binding between the PY motif of PDLIM7 and the second and third WW domains of Nedd4-1 and endogenous PDLIM7 and Nedd4-1 interact in the cytoplasm of differentiated C2C12 myotubes, leading to PDLIM7 ubiquitination. These results identify PDLIM7 as a bona fide skeletal muscle substrate of Nedd4-1 and suggest that this interaction may underlie the progression of skeletal muscle atrophy. This offers a novel therapeutic target that could be potentially used to attenuate muscle atrophy.

  15. The ubiquitin-protein ligase Nedd4 targets Notch1 in skeletal muscle and distinguishes the subset of atrophies caused by reduced muscle tension.

    PubMed

    Koncarevic, Alan; Jackman, Robert W; Kandarian, Susan C

    2007-02-01

    Ubiquitination-dependent proteolysis is a fundamental process underlying skeletal muscle atrophy. Thus, the role of ubiquitin ligases is of great interest. There are no focused studies in muscle on the ubiquitin ligase Nedd4. We first confirmed increased mRNA expression in rat soleus muscles due to 1-14 days of hind limb unloading. Nedd4 protein localized to the sarcolemmal region of muscle fibers. Hind limb unloading, sciatic nerve denervation, starvation, and diabetes led to atrophy of soleus, plantaris, and gastrocnemius muscles, but only unloaded and denervated muscles showed a marked increase in Nedd4 protein expression. This increase was strongly correlated with decreased Notch1 expression, a known target of Nedd4 in other cell types. Overexpression of dominant negative Nedd4 in soleus muscles completely reversed the unloading-induced decrease of Notch1 expression, indicating that Nedd4 is required for Notch1 inactivation. Overexpression of wild-type Nedd4 in soleus muscles of weight bearing rats caused a decrease in Notch1 protein, indicating that Nedd4 is sufficient for Notch1 down-regulation. To further show that Notch1 is a Nedd4 substrate in muscle, conditional overexpression of Nedd4 in C2C12 myotubes induced ubiquitination of Notch1. This is the first finding of a Nedd4 substrate in muscle and of an ubiquitin ligase, the activity of which distinguishes disuse from cachexia atrophy.

  16. Genetically engineered mouse models for functional studies of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases

    PubMed Central

    Zhou, Weihua; Wei, Wenyi; Sun, Yi

    2013-01-01

    The SCF (SKP1 (S-phase-kinase-associated protein 1), Cullin-1, F-box protein) E3 ubiquitin ligases, the founding member of Cullin-RING ligases (CRLs), are the largest family of E3 ubiquitin ligases in mammals. Each individual SCF E3 ligase consists of one adaptor protein SKP1, one scaffold protein cullin-1 (the first family member of the eight cullins), one F-box protein out of 69 family members, and one out of two RING (Really Interesting New Gene) family proteins RBX1/ROC1 or RBX2/ROC2/SAG/RNF7. Various combinations of these four components construct a large number of SCF E3s that promote the degradation of many key regulatory proteins in cell-context, temporally, and spatially dependent manners, thus controlling precisely numerous important cellular processes, including cell cycle progression, apoptosis, gene transcription, signal transduction, DNA replication, maintenance of genome integrity, and tumorigenesis. To understand how the SCF E3 ligases regulate these cellular processes and embryonic development under in vivo physiological conditions, a number of mouse models with transgenic (Tg) expression or targeted deletion of components of SCF have been established and characterized. In this review, we will provide a brief introduction to the ubiquitin-proteasome system (UPS) and the SCF E3 ubiquitin ligases, followed by a comprehensive overview on the existing Tg and knockout (KO) mouse models of the SCF E3s, and discuss the role of each component in mouse embryogenesis, cell proliferation, apoptosis, carcinogenesis, as well as other pathogenic processes associated with human diseases. We will end with a brief discussion on the future directions of this research area and the potential applications of the knowledge gained to more effective therapeutic interventions of human diseases. PMID:23528706

  17. The E3 Ubiquitin Ligase Siah2 Contributes to Castration-Resistant Prostate Cancer by Regulation of Androgen Receptor Transcriptional Activity

    PubMed Central

    Qi, Jianfei; Tripathi, Manisha; Mishra, Rajeev; Sahgal, Natasha; Fazil, Ladan; Ettinger, Susan; Placzek, William J.; Claps, Giuseppina; Chung, Leland W.K.; Bowtell, David; Gleave, Martin; Bhowmick, Neil; Ronai, Ze'ev A.

    2013-01-01

    SUMMARY Understanding the mechanism underlying the regulation of the androgen receptor (AR), a central player in the development of castration-resistant prostate cancer (CRPC), holds promise for overcoming the challenge of treating CRPC. We demonstrate that the ubiquitin ligase Siah2 targets a select pool of NCOR1-bound, transcriptionally-inactive AR for ubiquitin-dependent degradation, thereby promoting expression of select AR target genes implicated in lipid metabolism, cell motility, and proliferation. Siah2 is required for prostate cancer cell growth under androgen-deprivation conditions in vitro and in vivo, and Siah2 inhibition promotes prostate cancer regression upon castration. Notably, Siah2 expression is markedly increased in human CRPCs. Collectively, we find that selective regulation of AR transcriptional activity by the ubiquitin ligase Siah2 is important for CRPC development. PMID:23518348

  18. The E3 ubiquitin ligase Siah2 contributes to castration-resistant prostate cancer by regulation of androgen receptor transcriptional activity.

    PubMed

    Qi, Jianfei; Tripathi, Manisha; Mishra, Rajeev; Sahgal, Natasha; Fazli, Ladan; Fazil, Ladan; Ettinger, Susan; Placzek, William J; Claps, Giuseppina; Chung, Leland W K; Bowtell, David; Gleave, Martin; Bhowmick, Neil; Ronai, Ze'ev A

    2013-03-18

    Understanding the mechanism underlying the regulation of the androgen receptor (AR), a central player in the development of castration-resistant prostate cancer (CRPC), holds promise for overcoming the challenge of treating CRPC. We demonstrate that the ubiquitin ligase Siah2 targets a select pool of NCOR1-bound, transcriptionally-inactive AR for ubiquitin-dependent degradation, thereby promoting expression of select AR target genes implicated in lipid metabolism, cell motility, and proliferation. Siah2 is required for prostate cancer cell growth under androgen-deprivation conditions in vitro and in vivo, and Siah2 inhibition promotes prostate cancer regression upon castration. Notably, Siah2 expression is markedly increased in human CRPCs. Collectively, we find that selective regulation of AR transcriptional activity by the ubiquitin ligase Siah2 is important for CRPC development.

  19. Investigation of the molecular mechanism of δ-catenin ubiquitination: Implication of β-TrCP-1 as a potential E3 ligase.

    PubMed

    Shrestha, Hridaya; Yuan, Tingting; He, Yongfeng; Moon, Pyong-Gon; Shrestha, Nensi; Ryu, Taeyong; Park, So-Yeon; Cho, Young-Chang; Lee, Chan-Hyeong; Baek, Moon-Chang; Cho, Sayeon; Simkhada, Shishli; Kim, Hangun; Kim, Kwonseop

    2016-09-01

    Ubiquitination, a post-translational modification, involves the covalent attachment of ubiquitin to the target protein. The ubiquitin-proteasome pathway and the endosome-lysosome pathway control the degradation of the majority of eukaryotic proteins. Our previous study illustrated that δ-catenin ubiquitination occurs in a glycogen synthase kinase-3 (GSK-3) phosphorylation-dependent manner. However, the molecular mechanism of δ-catenin ubiquitination is still unknown. Here, we show that the lysine residues required for ubiquitination are located mainly in the C-terminal portion of δ-catenin. In addition, we provide evidence that β-TrCP-1 interacts with δ-catenin and functions as an E3 ligase, mediating δ-catenin ubiquitin-proteasome degradation. Furthermore, we prove that both the ubiquitin-proteasome pathway and the lysosome degradation pathway are involved in δ-catenin degradation. Our novel findings on the mechanism of δ-catenin ubiquitination will add a new perspective to δ-catenin degradation and the effects of δ-catenin on E-cadherin involved in epithelial cell-cell adhesion, which is implicated in prostate cancer progression.

  20. The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ.

    PubMed

    Watanabe, Masashi; Takahashi, Hidehisa; Saeki, Yasushi; Ozaki, Takashi; Itoh, Shihori; Suzuki, Masanobu; Mizushima, Wataru; Tanaka, Keiji; Hatakeyama, Shigetsugu

    2015-04-23

    Adipocyte differentiation is a strictly controlled process regulated by a series of transcriptional activators. Adipogenic signals activate early adipogenic activators and facilitate the transient formation of early enhanceosomes at target genes. These enhancer regions are subsequently inherited by late enhanceosomes. PPARγ is one of the late adipogenic activators and is known as a master regulator of adipogenesis. However, the factors that regulate PPARγ expression remain to be elucidated. Here, we show that a novel ubiquitin E3 ligase, tripartite motif protein 23 (TRIM23), stabilizes PPARγ protein and mediates atypical polyubiquitin conjugation. TRIM23 knockdown caused a marked decrease in PPARγ protein abundance during preadipocyte differentiation, resulting in a severe defect in late adipogenic differentiation, whereas it did not affect the formation of early enhanceosomes. Our results suggest that TRIM23 plays a critical role in the switching from early to late adipogenic enhanceosomes by stabilizing PPARγ protein possibly via atypical polyubiquitin conjugation.

  1. Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1.

    PubMed

    Bodine, Sue C; Baehr, Leslie M

    2014-09-15

    Muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1 were identified more than 10 years ago as two muscle-specific E3 ubiquitin ligases that are increased transcriptionally in skeletal muscle under atrophy-inducing conditions, making them excellent markers of muscle atrophy. In the past 10 years much has been published about MuRF1 and MAFbx with respect to their mRNA expression patterns under atrophy-inducing conditions, their transcriptional regulation, and their putative substrates. However, much remains to be learned about the physiological role of both genes in the regulation of mass and other cellular functions in striated muscle. Although both MuRF1 and MAFbx are enriched in skeletal, cardiac, and smooth muscle, this review will focus on the current understanding of MuRF1 and MAFbx in skeletal muscle, highlighting the critical questions that remain to be answered.

  2. NIRF, a Novel Ubiquitin Ligase, Inhibits Hepatitis B Virus Replication Through Effect on HBV Core Protein and H3 Histones.

    PubMed

    Qian, Guanhua; Hu, Bin; Zhou, Danlin; Xuan, Yanyan; Bai, Lu; Duan, Changzhu

    2015-05-01

    Np95/ICBP90-like RING finger protein (NIRF), a novel E3 ubiquitin ligase, has been shown to interact with HBc and promote its degradation. This study investigated the effects of NIRF on replication of hepatitis B virus (HBV) and the mechanisms. We have shown that NIRF inhibits replication of HBV DNA and secretion of HBsAg and HBeAg in HepG2 cells transfected with pAAV-HBV1.3. NIRF also inhibits the replication and secretion of HBV in a mouse model that expressed HBV. NIRF reduces acetylation of HBV cccDNA-bound H3 histones. These results showed that NIRF is involved in the HBV replication cycle not only through direct interaction with HBc but also reduces acetylation of HBV cccDNA-bound H3 histones.

  3. The E3 ubiquitin ligase HOS1 is involved in ethylene regulation of leaf expansion in Arabidopsis.

    PubMed

    Lee, Kyounghee; Seo, Pil Joon

    2015-01-01

    Ethylene regulates a variety of physiological processes, such as flowering, senescence, abscission, and fruit ripening. In particular, leaf expansion is also controlled by ethylene in Arabidopsis. Exogenous treatment with ethylene inhibits leaf expansion, and consistently, ethylene insensitive mutants show increased leaf area. Here, we report that the RING finger-containing E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) regulates leaf expansion in an ethylene signaling pathway. The HOS1-deficient mutant showed reduced leaf area and was insensitive to ethylene perception inhibitor, silver thiosulfate (STS). Accordingly, genes encoding ethylene signaling components were significantly up-regulated in hos1-3. This study demonstrates that the HOS1 protein is involved in ethylene signal transduction for the proper regulation of leaf expansion possibly under environmentally stressful conditions.

  4. Identification of a Protein Network Interacting with TdRF1, a Wheat RING Ubiquitin Ligase with a Protective Role against Cellular Dehydration1[C][W

    PubMed Central

    Guerra, Davide; Mastrangelo, Anna Maria; Lopez-Torrejon, Gema; Marzin, Stephan; Schweizer, Patrick; Stanca, Antonio Michele; del Pozo, Juan Carlos; Cattivelli, Luigi; Mazzucotelli, Elisabetta

    2012-01-01

    Plants exploit ubiquitination to modulate the proteome with the final aim to ensure environmental adaptation and developmental plasticity. Ubiquitination targets are specifically driven to degradation through the action of E3 ubiquitin ligases. Genetic analyses have indicated wide functions of ubiquitination in plant life; nevertheless, despite the large number of predicted E3s, only a few of them have been characterized so far, and only a few ubiquitination targets are known. In this work, we characterized durum wheat (Triticum durum) RING Finger1 (TdRF1) as a durum wheat nuclear ubiquitin ligase. Moreover, its barley (Hordeum vulgare) homolog was shown to protect cells from dehydration stress. A protein network interacting with TdRF1 has been defined. The transcription factor WHEAT BEL1-TYPE HOMEODOMAIN1 (WBLH1) was degraded in a TdRF1-dependent manner through the 26S proteasome in vivo, the mitogen-activated protein kinase TdWNK5 [for Triticum durum WITH NO LYSINE (K)5] was able to phosphorylate TdRF1 in vitro, and the RING-finger protein WHEAT VIVIPAROUS-INTERACTING PROTEIN2 (WVIP2) was shown to have a strong E3 ligase activity. The genes coding for the TdRF1 interactors were all responsive to cold and/or dehydration stress, and a negative regulative function in dehydration tolerance was observed for the barley homolog of WVIP2. A role in the control of plant development was previously known, or predictable based on homology, for wheat BEL1-type homeodomain1(WBLH1). Thus, TdRF1 E3 ligase might act regulating the response to abiotic stress and remodeling plant development in response to environmental constraints. PMID:22167118

  5. Shigella IpaH7.8 E3 ubiquitin ligase targets glomulin and activates inflammasomes to demolish macrophages

    PubMed Central

    Suzuki, Shiho; Mimuro, Hitomi; Kim, Minsoo; Ogawa, Michinaga; Ashida, Hiroshi; Toyotome, Takahito; Franchi, Luigi; Suzuki, Masato; Sanada, Takahito; Suzuki, Toshihiko; Tsutsui, Hiroko; Núñez, Gabriel; Sasakawa, Chihiro

    2014-01-01

    When nucleotide-binding oligomerization domain–like receptors (NLRs) sense cytosolic-invading bacteria, they induce the formation of inflammasomes and initiate an innate immune response. In quiescent cells, inflammasome activity is tightly regulated to prevent excess inflammation and cell death. Many bacterial pathogens provoke inflammasome activity and induce inflammatory responses, including cell death, by delivering type III secreted effectors, the rod component flagellin, and toxins. Recent studies indicated that Shigella deploy multiple mechanisms to stimulate NLR inflammasomes through type III secretion during infection. Here, we show that Shigella induces rapid macrophage cell death by delivering the invasion plasmid antigen H7.8 (IpaH7.8) enzyme 3 (E3) ubiquitin ligase effector via the type III secretion system, thereby activating the NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasomes and caspase-1 and leading to macrophage cell death in an IpaH7.8 E3 ligase-dependent manner. Mice infected with Shigella possessing IpaH7.8, but not with Shigella possessing an IpaH7.8 E3 ligase-null mutant, exhibited enhanced bacterial multiplication. We defined glomulin/flagellar-associated protein 68 (GLMN) as an IpaH7.8 target involved in IpaH7.8 E3 ligase-dependent inflammasome activation. This protein originally was identified through its association with glomuvenous malformations and more recently was described as a member of a Cullin ring ligase inhibitor. Modifying GLMN levels through overexpression or knockdown led to reduced or augmented inflammasome activation, respectively. Macrophages stimulated with lipopolysaccharide/ATP induced GLMN puncta that localized with the active form of caspase-1. Macrophages from GLMN+/− mice were more responsive to inflammasome activation than those from GLMN+/+ mice. Together, these results highlight a unique bacterial adaptation that hijacks inflammasome activation via

  6. Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin

    PubMed Central

    Moussaud-Lamodière, Elisabeth L.; Dourado, Daniel F. A. R.; Flores, Samuel C.; Springer, Wolfdieter

    2014-01-01

    Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through

  7. HDAC7 Ubiquitination by the E3 Ligase CBX4 Is Involved in Contextual Fear Conditioning Memory Formation.

    PubMed

    Jing, Xu; Sui, Wen-Hai; Wang, Shuai; Xu, Xu-Feng; Yuan, Rong-Rong; Chen, Xiao-Rong; Ma, Hui-Xian; Zhu, Ying-Xiao; Sun, Jin-Kai; Yi, Fan; Chen, Zhe-Yu; Wang, Yue

    2017-04-05

    Histone acetylation, an epigenetic modification, plays an important role in long-term memory formation. Recently, histone deacetylase (HDAC) inhibitors were demonstrated to promote memory formation, which raises the intriguing possibility that they may be used to rescue memory deficits. However, additional research is necessary to clarify the roles of individual HDACs in memory. In this study, we demonstrated that HDAC7, within the dorsal hippocampus of C57BL6J mice, had a late and persistent decrease after contextual fear conditioning (CFC) training (4-24 h), which was involved in long-term CFC memory formation. We also showed that HDAC7 decreased via ubiquitin-dependent degradation. CBX4 was one of the HDAC7 E3 ligases involved in this process. Nur77, as one of the target genes of HDAC7, increased 6-24 h after CFC training and, accordingly, modulated the formation of CFC memory. Finally, HDAC7 was involved in the formation of other hippocampal-dependent memories, including the Morris water maze and object location test. The current findings facilitate an understanding of the molecular and cellular mechanisms of HDAC7 in the regulation of hippocampal-dependent memory.SIGNIFICANCE STATEMENT The current findings demonstrated the effects of histone deacetylase 7 (HDAC7) on hippocampal-dependent memories. Moreover, we determined the mechanism of decreased HDAC7 in contextual fear conditioning (CFC) through ubiquitin-dependent protein degradation. We also verified that CBX4 was one of the HDAC7 E3 ligases. Finally, we demonstrated that Nur77, as one of the important targets for HDAC7, was involved in CFC memory formation. All of these proteins, including HDAC7, CBX4, and Nur77, could be potential therapeutic targets for preventing memory deficits in aging and neurological diseases.

  8. A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A*

    PubMed Central

    Fishman-Jacob, Tali; Reznichenko, Lydia; Youdim, Moussa B. H.; Mandel, Silvia A.

    2009-01-01

    The aim of this study was to develop a new model of sporadic Parkinson disease (PD) based on silencing of the SKP1A gene, a component of the ubiquitin-proteasome/E3 ligase complex, Skp1, Cullin 1, F-box protein, which was found to be highly decreased in the substantia nigra of sporadic PD patients. Initially, an embryonic mouse substantia nigra-derived cell line (SN4741 cells) was infected with short hairpin RNA lentiviruses encoding the murine transcript of the SKP1A gene or with scrambled vector. SKP1A silencing resulted in increased susceptibility to neuronal damages induced by the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium ion and serum starvation, in parallel with a decline in the expression of the dopaminergic markers, dopamine transporter and vesicular monoamine transporter-2. SKP1A-deficient cells presented a delay in completion of the cell cycle and the inability to arrest at the G0/G1 phase when induced to differentiate. Instead, the cells progressed through S phase, developing rounded aggregates with characteristics of aggresomes including immunoreactivity for γ-tubulin, α-synuclein, ubiquitin, tyrosine hydroxylase, Hsc-70 (70-kDa heat shock cognate protein), and proteasome subunit, and culminating in a lethal phenotype. Conversely, stably enforced expression of wild type SKP1A duplicated the survival index of naïve SN4741 cells under proteasomal inhibition injury, suggesting a new structural role of SKP1 in dopaminergic neuronal function, besides its E3 ligase activity. These results link, for the first time, SKP1 to dopamine neuronal function and survival, suggesting an essential role in sporadic PD. In summary, this new model has reproduced to a significant extent the molecular alterations described in sporadic PD at the cellular level, implicating Skp1 as a potential modifier in sporadic PD neurodegeneration. PMID:19748892

  9. A sporadic Parkinson disease model via silencing of the ubiquitin-proteasome/E3 ligase component SKP1A.

    PubMed

    Fishman-Jacob, Tali; Reznichenko, Lydia; Youdim, Moussa B H; Mandel, Silvia A

    2009-11-20

    The aim of this study was to develop a new model of sporadic Parkinson disease (PD) based on silencing of the SKP1A gene, a component of the ubiquitin-proteasome/E3 ligase complex, Skp1, Cullin 1, F-box protein, which was found to be highly decreased in the substantia nigra of sporadic PD patients. Initially, an embryonic mouse substantia nigra-derived cell line (SN4741 cells) was infected with short hairpin RNA lentiviruses encoding the murine transcript of the SKP1A gene or with scrambled vector. SKP1A silencing resulted in increased susceptibility to neuronal damages induced by the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium ion and serum starvation, in parallel with a decline in the expression of the dopaminergic markers, dopamine transporter and vesicular monoamine transporter-2. SKP1A-deficient cells presented a delay in completion of the cell cycle and the inability to arrest at the G(0)/G(1) phase when induced to differentiate. Instead, the cells progressed through S phase, developing rounded aggregates with characteristics of aggresomes including immunoreactivity for gamma-tubulin, alpha-synuclein, ubiquitin, tyrosine hydroxylase, Hsc-70 (70-kDa heat shock cognate protein), and proteasome subunit, and culminating in a lethal phenotype. Conversely, stably enforced expression of wild type SKP1A duplicated the survival index of naïve SN4741 cells under proteasomal inhibition injury, suggesting a new structural role of SKP1 in dopaminergic neuronal function, besides its E3 ligase activity. These results link, for the first time, SKP1 to dopamine neuronal function and survival, suggesting an essential role in sporadic PD. In summary, this new model has reproduced to a significant extent the molecular alterations described in sporadic PD at the cellular level, implicating Skp1 as a potential modifier in sporadic PD neurodegeneration.

  10. Ubiquitin ligase EL5 maintains the viability of root meristems by influencing cytokinin-mediated nitrogen effects in rice

    PubMed Central

    Nishizawa, Yoko

    2014-01-01

    Root formation is dependent on meristematic activity and is influenced by nitrogen supply. We have previously shown that ubiquitin ligase, EL5, in rice (Oryza sativa) is involved in the maintenance of root meristematic viability. When mutant EL5 protein is overexpressed to dominantly inhibit the endogenous EL5 function in rice, primordial and meristematic necrosis ia observed. Here, we analysed the cause of root cell death in transgenic rice plants (mEL5) overexpressing EL5V162A, which encodes a partly inactive ubiquitin ligase. The mEL5 mutants showed increased sensitivity to nitrogen that was reflected in the inhibition of root formation. Treatment of mEL5 with nitrate or nitrite caused meristematic cell death accompanied by browning. Transcriptome profiling of whole roots exhibited overlaps between nitrite-responsive genes in non-transgenic (NT) rice plants and genes with altered basal expression levels in mEL5. Phytohormone profiling of whole roots revealed that nitrite treatment increased cytokinin levels, but mEL5 constitutively contained more cytokinin than NT plants and showed increased sensitivity to exogenous cytokinin. More superoxide was detected in mEL5 roots after treatment with nitrite or cytokinin, and treatment with an inhibitor of superoxide production prevented mEL5 roots from both nitrite- and cytokinin-induced meristematic cell death. These results indicate a nitrogen-triggered pathway that leads to changes in root formation through the production of cytokinin and superoxide, on which EL5 acts to prevent meristematic cell death. PMID:24663342

  11. Endothelial deletion of Sag/Rbx2/Roc2 E3 ubiquitin ligase causes embryonic lethality and blocks tumor angiogenesis.

    PubMed

    Tan, M; Li, H; Sun, Y

    2014-10-30

    SAG (Sensitive to Apoptosis Gene), also known as RBX2 or ROC2, is a RING protein required for the activity of Cullin-RING ligase (CRL). Our recent study showed that Sag total knockout caused embryonic lethality at E11.5-12.5 days with associated defects in vasculogenesis. Whether Sag is required for de novo vasculogenesis in embryos and angiogenesis in tumors is totally unknown. Here, we report that Sag endothelial deletion also causes embryonic lethality at E15.5 with poor vasculogenesis. Sag deletion in primary endothelial cells (ECs) or knockdown in MS-1 ECs inhibits migration, proliferation and tube formation, with p27 accumulation being responsible for the suppression of migration and proliferation. Furthermore, Sag deletion significantly inhibits angiogenesis in an in vivo Matrigel plug assay, and tumor angiogenesis and tumorigenesis in a B16F10 melanoma model. Finally, MLN4924, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE) that inhibits CRL, suppresses in vitro migration, proliferation and tube formation, as well as in vivo angiogenesis and tumorigenesis. Taken together, our study, using both genetic and pharmaceutical approaches, demonstrates that Sag is essential for embryonic vasculogenesis and tumor angiogenesis, and provides the proof-of-concept evidence that targeting Sag E3 ubiquitin ligase may have clinical value for anti-angiogenesis therapy of human cancer.

  12. Insights into Cullin-RING E3 ubiquitin ligase recruitment: Structure of the VHL–EloBC–Cul2 complex

    PubMed Central

    Nguyen, Henry C.; Yang, Haitao; Fribourgh, Jennifer L.; Wolfe, Leslie S.; Xiong, Yong

    2015-01-01

    Summary The von Hippel-Lindau tumor suppressor protein (VHL) recruits a Cullin 2 (Cul2) E3 ubiquitin ligase to downregulate HIF-1α, an essential transcription factor for the hypoxia response. Mutations in VHL lead to VHL disease and renal cell carcinomas. Inhibition of this pathway to upregulate erythropoietin production is a promising new therapy to treat ischemia and chronic anemia. Here we report the crystal structure of VHL bound to a Cul2 N-terminal domain, Elongin B (EloB), and Elongin C (EloC). Cul2 interacts with both the VHL BC box and cullin box and a novel EloC site. Comparison to other cullin E3 ligase structures shows that there is a conserved, yet flexible, cullin recognition module and that cullin selectivity is influenced by distinct electrostatic interactions. Our structure provides a structural basis for the study of the pathogenesis of VHL disease and the rationale design of novel compounds that may modulate cullin–substrate receptor interactions. PMID:25661653

  13. Insights into Cullin-RING E3 ubiquitin ligase recruitment: structure of the VHL-EloBC-Cul2 complex.

    PubMed

    Nguyen, Henry C; Yang, Haitao; Fribourgh, Jennifer L; Wolfe, Leslie S; Xiong, Yong

    2015-03-03

    The von Hippel-Lindau tumor suppressor protein (VHL) recruits a Cullin 2 (Cul2) E3 ubiquitin ligase to downregulate HIF-1α, an essential transcription factor for the hypoxia response. Mutations in VHL lead to VHL disease and renal cell carcinomas. Inhibition of this pathway to upregulate erythropoietin production is a promising new therapy to treat ischemia and chronic anemia. Here, we report the crystal structure of VHL bound to a Cul2 N-terminal domain, Elongin B, and Elongin C (EloC). Cul2 interacts with both the VHL BC box and cullin box and a novel EloC site. Comparison with other cullin E3 ligase structures shows that there is a conserved, yet flexible, cullin recognition module and that cullin selectivity is influenced by distinct electrostatic interactions. Our structure provides a structural basis for the study of the pathogenesis of VHL disease and rationale for the design of novel compounds that may modulate cullin-substrate receptor interactions.

  14. Endothelial deletion of Sag/Rbx2/Roc2 E3 ubiquitin ligase causes embryonic lethality and blocks tumor angiogenesis

    PubMed Central

    Tan, Mingjia; Li, Hua; Sun, Yi

    2014-01-01

    SAG (Sensitive to Apoptosis Gene), also known as RBX2 or ROC2, is a RING protein required for the activity of Cullin-RING ligase (CRL). Our recent study showed that Sag total knockout caused embryonic lethality at E11.5–12.5 days with associated defects in vasculogenesis. Whether Sag is required for de novo vasculogenesis in embryos and angiogenesis in tumors is totally unknown. Here, we report that Sag endothelial deletion also causes embryonic lethality at E15.5 with poor vasculogenesis. Sag deletion in primary endothelial cells or knockdown in MS-1 endothelial cells inhibits migration, proliferation and tube formation with p27 accumulation being responsible for the suppression of migration and proliferation. Furthermore, Sag deletion significantly inhibits angiogenesis in an in vivo Matrigel plug assay, and tumor angiogenesis and tumorigenesis in a B16F10 melanoma model. Finally, MLN4924, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE) that inhibits CRL, suppresses in vitro migration, proliferation, and tube formation, as well as in vivo angiogenesis and tumorigenesis. Taken together, our study, using both genetic and pharmaceutical approaches, demonstrates that Sag is essential for embryonic vasculogenesis and tumor angiogenesis, and provides the proof-of-concept evidence that targeting Sag E3 ubiquitin ligase may have clinical value for anti-angiogenesis therapy of human cancer. PMID:24213570

  15. Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase.

    PubMed

    Aghajan, Mariam; Jonai, Nao; Flick, Karin; Fu, Fei; Luo, Manlin; Cai, Xiaolu; Ouni, Ikram; Pierce, Nathan; Tang, Xiaobo; Lomenick, Brett; Damoiseaux, Robert; Hao, Rui; Del Moral, Pierre M; Verma, Rati; Li, Ying; Li, Cheng; Houk, Kendall N; Jung, Michael E; Zheng, Ning; Huang, Lan; Deshaies, Raymond J; Kaiser, Peter; Huang, Jing

    2010-07-01

    The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)(Met30) ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCF(Met30) in vivo and in vitro, but not the closely related SCF(Cdc4). Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

  16. Distinct Functional Domains Contribute to Degradation of the Low Density Lipoprotein Receptor (LDLR) by the E3 Ubiquitin Ligase Inducible Degrader of the LDLR (IDOL)

    PubMed Central

    Sorrentino, Vincenzo; Scheer, Lilith; Santos, Ana; Reits, Eric; Bleijlevens, Boris; Zelcer, Noam

    2011-01-01

    We recently identified the liver X receptor-regulated E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) as a modulator of lipoprotein metabolism. Acting as an E3 ubiquitin ligase, IDOL triggers ubiquitination and subsequent degradation of the low density lipoprotein receptor (LDLR). We demonstrate here that this outcome requires the conserved FERM and RING domains present in IDOL. The RING domain promotes ubiquitination in vitro and Lys-63-specific ubiquitination of the LDLR in vivo in response to IDOL or liver X receptor activation. We further identify RING residues that differentially influence ubiquitination of the LDLR or stability of IDOL. The FERM domain interacts with the LDLR and in living cells co-localizes with the receptor at the plasma membrane. Homology modeling revealed a phosphotyrosine-binding element embedded in the FERM domain. Mutating residues within this region or residues in the LDLR preceding the NPVY endocytosis motif abrogate LDLR degradation by IDOL. Collectively, our results indicate that both the FERM and RING domains are required for promoting lysosomal degradation of the LDLR by IDOL. Our findings may facilitate development of structure-based IDOL inhibitors aimed at increasing LDLR abundance in therapeutic strategies to treat cardiovascular disease. PMID:21734303

  17. Inactivation of SAG/RBX2 E3 ubiquitin ligase suppresses KrasG12D-driven lung tumorigenesis.

    PubMed

    Li, Hua; Tan, Mingjia; Jia, Lijun; Wei, Dongping; Zhao, Yongchao; Chen, Guoan; Xu, Jie; Zhao, Lili; Thomas, Dafydd; Beer, David G; Sun, Yi

    2014-02-01

    Cullin-RING ligases (CRLs) are a family of E3 ubiquitin ligase complexes that rely on either RING-box 1 (RBX1) or sensitive to apoptosis gene (SAG), also known as RBX2, for activity. RBX1 and SAG are both overexpressed in human lung cancer; however, their contribution to patient survival and lung tumorigenesis is unknown. Here, we report that overexpression of SAG, but not RBX1, correlates with poor patient prognosis and more advanced disease. We found that SAG is overexpressed in murine KrasG12D-driven lung tumors and that Sag deletion suppressed lung tumorigenesis and extended murine life span. Using cultured lung cancer cells, we showed that SAG knockdown suppressed growth and survival, inactivated both NF-κB and mTOR pathways, and resulted in accumulation of tumor suppressor substrates, including p21, p27, NOXA, and BIM. Importantly, growth suppression by SAG knockdown was partially rescued by simultaneous knockdown of p21 or the mTOR inhibitor DEPTOR. Treatment with MLN4924, a small molecule inhibitor of CRL E3s, also inhibited the formation of KrasG12D-induced lung tumors through a similar mechanism involving inactivation of NF-κB and mTOR and accumulation of tumor suppressor substrates. Together, our results demonstrate that Sag is a Kras-cooperating oncogene that promotes lung tumorigenesis and suggest that targeting SAG-CRL E3 ligases may be an effective therapeutic approach for Kras-driven lung cancers.

  18. Inactivation of SAG E3 ubiquitin ligase blocks embryonic stem cell differentiation and sensitizes leukemia cells to retinoid acid.

    PubMed

    Tan, Mingjia; Li, Yun; Yang, Ruiguo; Xi, Ning; Sun, Yi

    2011-01-01

    Sensitive to Apoptosis Gene (SAG), also known as RBX2 (RING box protein-2), is the RING component of SCF (SKP1, Cullin, and F-box protein) E3 ubiquitin ligase. Our previous studies have demonstrated that SAG is an anti-apoptotic protein and an attractive anti-cancer target. We also found recently that Sag knockout sensitized mouse embryonic stem cells (mES) to radiation and blocked mES cells to undergo endothelial differentiation. Here, we reported that compared to wild-type mES cells, the Sag(-/-) mES cells were much more sensitive to all-trans retinoic acid (RA)-induced suppression of cell proliferation and survival. While wild-type mES cells underwent differentiation upon exposure to RA, Sag(-/-) mES cells were induced to death via apoptosis instead. The cell fate change, reflected by cellular stiffness, can be detected as early as 12 hrs post RA exposure by AFM (Atomic Force Microscopy). We then extended this novel finding to RA differentiation therapy of leukemia, in which the resistance often develops, by testing our hypothesis that SAG inhibition would sensitize leukemia to RA. Indeed, we found a direct correlation between SAG overexpression and RA resistance in multiple leukemia lines. By using MLN4924, a small molecule inhibitor of NEDD8-Activating Enzyme (NAE), that inactivates SAG-SCF E3 ligase by blocking cullin neddylation, we were able to sensitize two otherwise resistant leukemia cell lines, HL-60 and KG-1 to RA. Mechanistically, RA sensitization by MLN4924 was mediated via enhanced apoptosis, likely through accumulation of pro-apoptotic proteins NOXA and c-JUN, two well-known substrates of SAG-SCF E3 ligase. Taken together, our study provides the proof-of-concept evidence for effective treatment of leukemia patients by RA-MLN4924 combination.

  19. A mechanism for transcriptional repression dependent on the BRCA1 E3 ubiquitin ligase.

    PubMed

    Horwitz, Andrew A; Affar, El Bachir; Heine, George F; Shi, Yang; Parvin, Jeffrey D

    2007-04-17

    Loss of function of the tumor suppressor protein BRCA1 is responsible for a high percentage of familial and also sporadic breast cancers. Early work identified a stimulatory transcriptional coactivator function for the BRCA1 protein, and more recently, BRCA1 has been implicated in transcriptional repression, although few examples of repressed genes have been characterized. We recently used an in vitro transcription assay to identify a biochemical mechanism that explained the BRCA1 stimulatory activity. In this study, we identified an ubiquitin-dependent mechanism by which BRCA1 inhibits transcription. BRCA1 ubiquitinates the transcriptional preinitiation complex, preventing stable association of TFIIE and TFIIH, and thus blocks the initiation of mRNA synthesis. What is striking about this mechanism of regulation by BRCA1 is that the ubiquitination of the preinitiation complex is not targeting proteins for degradation by the proteasome, nor are ubiquitin receptors modifying the activity, but rather the ubiquitin moiety itself interferes with the assembly of basal transcription factors at the promoter. Using RNAi to knockdown expression of the endogenous BRCA1 protein, we assessed the level of repression dependent on BRCA1 in the cell, and we found that BRCA1 is at least as significant a transcriptional repressor as it is an activator. These results define a biochemical mechanism by which the BRCA1 enzymatic activity regulates a key cellular process.

  20. The ubiquitin ligase PDZRN3 is required for vascular morphogenesis through Wnt/planar cell polarity signalling.

    PubMed

    Sewduth, Raj N; Jaspard-Vinassa, Béatrice; Peghaire, Claire; Guillabert, Aude; Franzl, Nathalie; Larrieu-Lahargue, Frederic; Moreau, Catherine; Fruttiger, Marcus; Dufourcq, Pascale; Couffinhal, Thierry; Duplàa, Cécile

    2014-09-08

    Development and stabilization of a vascular plexus requires the coordination of multiple signalling processes. Wnt planar cell polarity (PCP) signalling is critical in vertebrates for diverse morphogenesis events, which coordinate cell orientation within a tissue-specific plane. However, its functional role in vascular morphogenesis is not well understood. Here we identify PDZRN3, an ubiquitin ligase, and report that Pdzrn3 deficiency impairs embryonic angiogenic remodelling and postnatal retinal vascular patterning, with a loss of two-dimensional polarized orientation of the intermediate retinal plexus. Using in vitro and ex vivo Pdzrn3 loss-of-function and gain-of-function experiments, we demonstrate a key role of PDZRN3 in endothelial cell directional and coordinated extension. PDZRN3 ubiquitinates Dishevelled 3 (Dvl3), to promote endocytosis of the Frizzled/Dvl3 complex, for PCP signal transduction. These results highlight the role of PDZRN3 to direct Wnt PCP signalling, and broadly implicate this pathway in the planar orientation and highly branched organization of vascular plexuses.

  1. Negative regulation of the Hippo pathway by E3 ubiquitin ligase ITCH is sufficient to promote tumorigenicity.

    PubMed

    Salah, Zaidoun; Melino, Gerry; Aqeilan, Rami I

    2011-03-01

    The Hippo tumor suppressor pathway, originally defined in fruit flies, regulates cellular proliferation and survival and exerts profound effects on normal mammalian cell fate and tumorigenesis. The present understanding of Hippo pathway components and mechanisms remains incomplete in cancer. WW domain-containing proteins regulate diverse biological processes through interaction with proline-tyrosine (PPxY)-containing targets. In this study, we report that the E3 ubiquitin ligase ITCH regulates stability of LATS1, a serine/threonine kinase in the Hippo pathway, through protein-protein interaction of the PPxY motifs of LATS1 with the WW domains of ITCH. Ubiquitination of LATS1 catalyzed by ITCH stimulated the proteasomal degradation of LATS1. Furthermore, ITCH-mediated degradation of LATS1 was associated with enhanced cell growth, induction of epithelial-mesenchymal transition, and increased tumorigenicity. Conversely, ITCH depletion increased LATS1 levels, enhancing FAS-induced apoptosis and reducing proliferation, survival, and migration. These phenotypes were rescued when both ITCH and LATS1 were depleted. Together, our results reveal a novel functional link between ITCH and the Hippo pathway, deepening their critical roles in tumorigenesis.

  2. The Steroidogenic Enzyme AKR1C3 Regulates Stability of the Ubiquitin Ligase Siah2 in Prostate Cancer Cells*

    PubMed Central

    Fan, Lingling; Peng, Guihong; Hussain, Arif; Fazli, Ladan; Guns, Emma; Gleave, Martin; Qi, Jianfei

    2015-01-01

    Re-activation of androgen receptor (AR) activity is the main driver for development of castration-resistant prostate cancer. We previously reported that the ubiquitin ligase Siah2 enhanced AR transcriptional activity and prostate cancer cell growth. Among the genes we found to be regulated by Siah2 was AKR1C3, which encodes a key androgen biosynthetic enzyme implicated in castration-resistant prostate cancer development. Here, we found that Siah2 inhibition in CWR22Rv1 prostate cancer cells decreased AKR1C3 expression as well as intracellular androgen levels, concomitant with inhibition of cell growth in vitro and in orthotopic prostate tumors. Re-expression of either wild-type or catalytically inactive forms of AKR1C3 partially rescued AR activity and growth defects in Siah2 knockdown cells, suggesting a nonenzymatic role for AKR1C3 in these outcomes. Unexpectedly, AKR1C3 re-expression in Siah2 knockdown cells elevated Siah2 protein levels, whereas AKR1C3 knockdown had the opposite effect. We further found that AKR1C3 can bind Siah2 and inhibit its self-ubiquitination and degradation, thereby increasing Siah2 protein levels. We observed parallel expression of Siah2 and AKR1C3 in human prostate cancer tissues. Collectively, our findings identify a new role for AKR1C3 in regulating Siah2 stability and thus enhancing Siah2-dependent regulation of AR activity in prostate cancer cells. PMID:26160177

  3. The role of E3 ubiquitin-ligases MuRF-1 and MAFbx in loss of skeletal muscle mass.

    PubMed

    Rom, Oren; Reznick, Abraham Z

    2016-09-01

    The ubiquitin-proteasome system (UPS) is the main regulatory mechanism of protein degradation in skeletal muscle. The ubiquitin-ligase enzymes (E3s) have a central role in determining the selectivity and specificity of the UPS. Since their identification in 2001, the muscle specific E3s, muscle RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx), have been shown to be implicated in the regulation of skeletal muscle atrophy in various pathological and physiological conditions. This review aims to explore the involvement of MuRF-1 and MAFbx in catabolism of skeletal muscle during various pathologies, such as cancer cachexia, sarcopenia of aging, chronic kidney disease (CKD), diabetes, and chronic obstructive pulmonary disease (COPD). In addition, the effects of various lifestyle and modifiable factors (e.g. nutrition, exercise, cigarette smoking, and alcohol) on MuRF-1 and MAFbx regulation will be discussed. Finally, evidence of potential strategies to protect against skeletal muscle wasting through inhibition of MuRF-1 and MAFbx expression will be explored.

  4. Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection.

    PubMed

    Ferretti, Lorenza P; Himmels, Sarah-Felicitas; Trenner, Anika; Walker, Christina; von Aesch, Christine; Eggenschwiler, Aline; Murina, Olga; Enchev, Radoslav I; Peter, Matthias; Freire, Raimundo; Porro, Antonio; Sartori, Alessandro A

    2016-08-26

    Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein-protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity.

  5. The Steroidogenic Enzyme AKR1C3 Regulates Stability of the Ubiquitin Ligase Siah2 in Prostate Cancer Cells.

    PubMed

    Fan, Lingling; Peng, Guihong; Hussain, Arif; Fazli, Ladan; Guns, Emma; Gleave, Martin; Qi, Jianfei

    2015-08-21

    Re-activation of androgen receptor (AR) activity is the main driver for development of castration-resistant prostate cancer. We previously reported that the ubiquitin ligase Siah2 enhanced AR transcriptional activity and prostate cancer cell growth. Among the genes we found to be regulated by Siah2 was AKR1C3, which encodes a key androgen biosynthetic enzyme implicated in castration-resistant prostate cancer development. Here, we found that Siah2 inhibition in CWR22Rv1 prostate cancer cells decreased AKR1C3 expression as well as intracellular androgen levels, concomitant with inhibition of cell growth in vitro and in orthotopic prostate tumors. Re-expression of either wild-type or catalytically inactive forms of AKR1C3 partially rescued AR activity and growth defects in Siah2 knockdown cells, suggesting a nonenzymatic role for AKR1C3 in these outcomes. Unexpectedly, AKR1C3 re-expression in Siah2 knockdown cells elevated Siah2 protein levels, whereas AKR1C3 knockdown had the opposite effect. We further found that AKR1C3 can bind Siah2 and inhibit its self-ubiquitination and degradation, thereby increasing Siah2 protein levels. We observed parallel expression of Siah2 and AKR1C3 in human prostate cancer tissues. Collectively, our findings identify a new role for AKR1C3 in regulating Siah2 stability and thus enhancing Siah2-dependent regulation of AR activity in prostate cancer cells.

  6. The ETS protein MEF is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCFSkp2.

    PubMed

    Liu, Yan; Hedvat, Cyrus V; Mao, Shifeng; Zhu, Xin-Hua; Yao, Jinjuan; Nguyen, Hoang; Koff, Andrew; Nimer, Stephen D

    2006-04-01

    MEF is an ETS-related transcription factor with strong transcriptional activating activity that affects hematopoietic stem cell behavior and is required for normal NK cell and NK T-cell development. The MEF (also known as ELF4) gene is repressed by several leukemia-associated fusion transcription factor proteins (PML-retinoic acid receptor alpha and AML1-ETO), but it is also activated by retroviral insertion in several cancer models. We have previously shown that cyclin A-dependent phosphorylation of MEF largely restricts its activity to the G(1) phase of the cell cycle; we now show that MEF is a short-lived protein whose expression level also peaks during late G(1) phase. Mutagenesis studies show that the rapid turnover of MEF in S phase is dependent on the specific phosphorylation of threonine 643 and serine 648 at the C terminus of MEF by cdk2 and on the Skp1/Cul1/F-box (SCF) E3 ubiquitin ligase complex SCF(Skp2), which targets MEF for ubiquitination and proteolysis. Overexpression of MEF drives cells through the G(1)/S transition, thereby promoting cell proliferation. The tight regulation of MEF levels during the cell cycle contributes to its effects on regulating cell cycle entry and cell proliferation.

  7. Complementary genetic screens identify the E3 ubiquitin ligase CBLC, as a modifier of PARP inhibitor sensitivity

    PubMed Central

    Brough, Rachel; Hodny, Zdenek; Ashworth, Alan; Bartek, Jiri; Lord, Christopher J.

    2015-01-01

    Based on a series of basic, preclinical and clinical studies, the Poly (ADP-ribose) Polymerase 1 (PARP1) inhibitor, olaparib, has recently been approved for use in ovarian cancer patients with BRCA1 or BRCA2 mutations. By identifying novel predictive biomarkers of tumour cell sensitivity to olaparib, it is possible that the utility of PARP inhibitors could be extended beyond this patient subgroup. Many of the known genetic determinants of PARP inhibitor response have key roles in DNA damage response (DDR) pathways. Although protein ubiquitylation is known to play an important role in regulating the DDR, the exact mechanisms by which this occurs are not fully understood. Using two parallel RNA interference-based screening approaches, we identified the E3 ubiquitin ligase, CBLC, as a candidate biomarker of response to olaparib. We validated this observation by demonstrating that silencing of CBLC causes increased sensitivity to olaparib in breast cancer cell line models and that defective homologous recombination (HR) DNA repair is the likely cause. This data provides an example of how defects in the ubiquitin machinery have the potential to influence the response of tumour cells to PARP inhibitors. PMID:25883215

  8. Structural and biochemical basis for ubiquitin ligase recruitment by arrestin-related domain-containing protein-3 (ARRDC3).

    PubMed

    Qi, Shiqian; O'Hayre, Morgan; Gutkind, J Silvio; Hurley, James H

    2014-02-21

    After protracted stimulation, the β2-adrenergic receptor and many other G-protein-coupled receptors are ubiquitinated and down-regulated. Arrestin-related domain-containing protein-3 (ARRDC3) has been proposed to recruit the ubiquitin ligase Nedd4 to the β2-adrenergic receptor. ARRDC3 contains two PPXY motifs that could potentially interact with any of the four WW domains of Nedd4. Here we dissect the interaction determinants. ARRDC3 PPXY-Nedd4 WW dissociation constants vary from unmeasurable to Kd = 3 μM for the third WW domain of Nedd4 binding to the first PPXY motif of ARRDC3. Structures of the uncomplexed and PPXY1-bound WW3 domain were determined at 1.1 and 1.7 Å resolution. The structures revealed conformational changes upon binding and the hydrogen bonding network in exquisite detail. Tight packing of ARRDC3 Val-352', part of a 310 helix at the C terminus of PPXY1, is important for high affinity binding to WW3. Although no single WW domain is strictly essential for the binding of Nedd4 and ARRDC3 expressed in HEK293 cells, high affinity binding of full-length ARRDC3 and Nedd4 is driven by the avid interaction of both PPXY motifs with either the WW2-WW3 or WW3-WW4 combinations, with Kd values as low as 300 nM.

  9. Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection

    PubMed Central

    Ferretti, Lorenza P.; Himmels, Sarah-Felicitas; Trenner, Anika; Walker, Christina; von Aesch, Christine; Eggenschwiler, Aline; Murina, Olga; Enchev, Radoslav I.; Peter, Matthias; Freire, Raimundo; Porro, Antonio; Sartori, Alessandro A.

    2016-01-01

    Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein–protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity. PMID:27561354

  10. Ubiquitin Ligase, MuRF-1 regulates myosin heavy chain type IIa transcripts during muscle atrophy under microgravity conditions

    NASA Astrophysics Data System (ADS)

    Kagawa, Sachiko

    Skeletal muscles are vulnerable to marked atrophy under microgravity conditions. We previously reported that gastrocnemius muscle atrophy by spaceflight was specifically sensitive to the ubiquitin-proteasome proteolytic pathway. We also screened more over 26,000 skeletal muscle genes in rats exposed to real weightlessness and found that the expression of Ubiquitin Ligase, Muscle specific Ring Finger-1 (MuRF-1) upregulated under microgravity. In the present study, we examined the role of MuRF-1 in microgravity-induced muscle atrophy. The amounts of MuRF-1 transcripts significantly increased in skeletal muscle after denervation, an in vivo model of microgravity-induced unloading. MuRF-1 deficient (MuRF-1-/-) mice significantly inhibited reduction of muscle weight for muscle atrophy, compared with wild type mice. Interestingly, MuRF-1-/- mice significantly inhibited upregulation of myosin heavy chain (MyHC) type IIa transcrips, while wild type mice significantly increased expression of MyHC type IIa transcripts in denervated skeletal muscle. Our present results suggest that MuRF-1 may play an important role in regulation of MyHC type IIa during muscle atrophy under microgravity conditions.

  11. Host Cell-catalyzed S-Palmitoylation Mediates Golgi Targeting of the Legionella Ubiquitin Ligase GobX*

    PubMed Central

    Lin, Yi-Han; Doms, Alexandra G.; Cheng, Eric; Kim, Byoungkwan; Evans, Timothy R.; Machner, Matthias P.

    2015-01-01

    The facultative intracellular pathogen Legionella pneumophila, the causative agent of Legionnaires disease, infects and replicates within human alveolar macrophages. L. pneumophila delivers almost 300 effector proteins into the besieged host cell that alter signaling cascades and create conditions that favor intracellular bacterial survival. In order for the effectors to accomplish their intracellular mission, their activity needs to be specifically directed toward the correct host cell protein or target organelle. Here, we show that the L. pneumophila effector GobX possesses E3 ubiquitin ligase activity that is mediated by a central region homologous to mammalian U-box domains. Furthermore, we demonstrate that GobX exploits host cell S-palmitoylation to specifically localize to Golgi membranes. The hydrophobic palmitate moiety is covalently attached to a cysteine residue at position 175, which is part of an amphipathic α-helix within the C-terminal region of GobX. Site-directed mutagenesis of cysteine 175 or residues on the hydrophobic face of the amphipathic helix strongly attenuated palmitoylation and Golgi localization of GobX. Together, our study provides evidence that the L. pneumophila effector GobX exploits two post-translational modification pathways of host cells, ubiquitination and S-palmitoylation. PMID:26316537

  12. CRL4-DCAF1 ubiquitin E3 ligase directs protein phosphatase 2A degradation to control oocyte meiotic maturation.

    PubMed

    Yu, Chao; Ji, Shu-Yan; Sha, Qian-Qian; Sun, Qing-Yuan; Fan, Heng-Yu

    2015-08-18

    Oocyte meiosis is a specialized cell cycle that gives rise to fertilizable haploid gametes and is precisely controlled in various dimensions. We recently found that E3 ubiquitin ligase CRL4 is required for female fertility by regulating DNA hydroxymethylation to maintain oocyte survival and to promote zygotic genome reprogramming. However, not all phenotypes of CRL4-deleted oocytes could be explained by this mechanism. Here we show that CRL4 controls oocyte meiotic maturation by proteasomal degradation of protein phosphatase 2A scaffold subunit, PP2A-A. Oocyte-specific deletion of DDB1 or DCAF1 (also called VPRBP) results in delayed meiotic resumption and failure to complete meiosis I along with PP2A-A accumulation. DCAF1 directly binds to and results in the poly-ubiquitination of PP2A-A. Moreover, combined deletion of Ppp2r1a rescues the meiotic defects caused by DDB1/DCAF1 deficiency. These results provide in vivo evidence that CRL4-directed PP2A-A degradation is physiologically essential for regulating oocyte meiosis and female fertility.

  13. A genomic survey of HECT ubiquitin ligases in eukaryotes reveals independent expansions of the HECT system in several lineages.

    PubMed

    Grau-Bové, Xavier; Sebé-Pedrós, Arnau; Ruiz-Trillo, Iñaki

    2013-01-01

    The posttranslational modification of proteins by the ubiquitination pathway is an important regulatory mechanism in eukaryotes. To date, however, studies on the evolutionary history of the proteins involved in this pathway have been restricted to E1 and E2 enzymes, whereas E3 studies have been focused mainly in metazoans and plants. To have a wider perspective, here we perform a genomic survey of the HECT family of E3 ubiquitin-protein ligases, an important part of this posttranslational pathway, in genomes from representatives of all major eukaryotic lineages. We classify eukaryotic HECTs and reconstruct, by phylogenetic analysis, the putative repertoire of these proteins in the last eukaryotic common ancestor (LECA). Furthermore, we analyze the diversity and complexity of protein domain architectures of HECTs along the different extant eukaryotic lineages. Our data show that LECA had six different HECTs and that protein expansion and N-terminal domain diversification shaped HECT evolution. Our data reveal that the genomes of animals and unicellular holozoans considerably increased the molecular and functional diversity of their HECT system compared with other eukaryotes. Other eukaryotes, such as the Apusozoa Thecanomas trahens or the Heterokonta Phytophthora infestans, independently expanded their HECT repertoire. In contrast, plant, excavate, rhodophyte, chlorophyte, and fungal genomes have a more limited enzymatic repertoire. Our genomic survey and phylogenetic analysis clarifies the origin and evolution of different HECT families among eukaryotes and provides a useful phylogenetic framework for future evolutionary studies of this regulatory pathway.

  14. A CUL-2 ubiquitin ligase containing three FEM proteins degrades TRA-1 to regulate C. elegans sex determination

    PubMed Central

    Starostina, Natalia G.; Lim, Jae-min; Schvarzstein, Mara; Wells, Lance; Spence, Andrew M.; Kipreos, Edward T.

    2007-01-01

    Summary In Caenorhabditis elegans, the Gli-family transcription factor TRA-1 is the terminal effector of the sex determination pathway. TRA-1 activity inhibits male development and allows female fates. Genetic studies have indicated that TRA-1 is negatively regulated by the fem-1, fem-2, and fem-3 genes. However, the mechanism of this regulation has not been understood. Here, we present data that TRA-1 is regulated by degradation mediated by a CUL-2-based ubiquitin ligase complex that contains FEM-1 as the substrate-recognition subunit, and FEM-2 and FEM-3 as cofactors. CUL-2 physically associates with both FEM-1 and TRA-1 in vivo, and cul-2 mutant males share feminization phenotypes with fem mutants. CUL-2 and the FEM proteins negatively regulate TRA-1 protein levels in C. elegans. When expressed in human cells, the FEM proteins interact with human CUL2 and induce the proteasome-dependent degradation of TRA-1. This work demonstrates that the terminal step in C. elegans sex determination is controlled by ubiquitin-mediated proteolysis. PMID:17609115

  15. Heterologous expression of the gourd E3 ubiquitin ligase gene LsRZF1 compromises the drought stress tolerance in Arabidopsis thaliana.

    PubMed

    Min, Ji-Hee; Ju, Hyun-Woo; Yang, Kwang-Yeol; Chung, Jung-Sung; Cho, Baik-Ho; Kim, Cheol Soo

    2014-04-01

    Protein ubiquitination is one of the major regulatory processes used by eukaryotic cells. The ubiquitin E3 ligase acts as a main determinant of substrate specificity. However, the precise roles of E3 ligase in plants to drought stress are poorly understood. In this study, a gourd family (Lagenaria siceraria) ortholog of Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1) gene, designated LsRZF1, was identified and characterized. LsRZF1 was reduced by abscisic acid (ABA), osmotic stress, and drought conditions. Compared to wild type, transgenic Arabidopsis plants ectopic expressing LsRZF1 were hypersensitive to ABA and osmotic stress during early seedling development, indicating that LsRZF1 negatively regulates drought-mediated control of early seedling development. Moreover, the ectopic expression of the LsRZF1 gene was very influential in drought sensitive parameters including proline content, water loss, and the expression of dehydration stress-related genes. Furthermore, ubiquitin E3 ligase activity and genetic data indicate that AtRZF1 and LsRZF1 function in similar pathway to control proline metabolism in Arabidopsis under drought condition. Together, these results suggest that the E3 ligase LsRZF1 is an important regulator of water deficit stress during early seedling development.

  16. Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth*

    PubMed Central

    Mertz, Joseph; Tan, Haiyan; Pagala, Vishwajeeth; Bai, Bing; Chen, Ping-Chung; Li, Yuxin; Cho, Ji-Hoon; Shaw, Timothy; Wang, Xusheng; Peng, Junmin

    2015-01-01

    The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 down-regulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interactomes; and Mib1 analysis provides a comprehensive interactome for investigating its role in signaling networks and neuronal development. PMID:25931508

  17. Cbl-family ubiquitin ligases and their recruitment of CIN85 are largely dispensable for epidermal growth factor receptor endocytosis

    PubMed Central

    Ahmad, Gulzar; Mohapatra, Bhopal; Schulte, Nancy A.; Nadeau, Scott; Luan, Haitao; Zutshi, Neha; Tom, Eric; Ortega-Cava, Cesar; Tu, Chun; Sanada, Masashi; Ogawa, Seishi; Toews, Myron L.; Band, Vimla; Band, Hamid

    2014-01-01

    Members of the Casitas B-Lineage Lymphoma (Cbl) family (Cbl, Cbl-b and Cbl-c) of ubiquitin ligases serve as negative regulators of receptor tyrosine kinases (RTKs). An essential role of Cbl-family protein-dependent ubiquitination for efficient ligand-induced lysosomal targeting and degradation is now well-accepted. However, a more proximal role of Cbl and Cbl-b as adapters for CIN85-endophilin recruitment to mediate ligand-induced initial internalization of RTKs is supported by some studies but refuted by others. Overexpression and/or incomplete depletion of Cbl proteins in these studies is likely to have contributed to this dichotomy. To address the role of endogenous Cbl and Cbl-b in the internalization step of RTK endocytic traffic, we established Cbl/Cbl-b double-knockout (DKO) mouse embryonic fibroblasts (MEFs) and demonstrated that these cells lack the expression of both Cbl-family members as well as endophilin A, while they express CIN85. We show that ligand-induced ubiquitination of EGFR, as a prototype RTK, was abolished in DKO MEFs, and EGFR degradation was delayed. These traits were reversed by ectopic human Cbl expression. EGFR endocytosis, assessed using the internalization of 125I-labeled or fluorescent EGF, or of EGFR itself, was largely retained in Cbl/Cbl-b DKO compared to wild type MEFs. EGFR internalization was also largely intact in Cbl/Cbl-b depleted MCF-10A human mammary epithelial cell line. Inducible shRNA-mediated knockdown of CIN85 in wild type or Cbl/Cbl-b DKO MEFs had no impact on EGFR internalization. Our findings, establish that, at physiological expression levels, Cbl, Cbl-b and CIN85 are largely dispensable for EGFR internalization. Our results support the model that Cbl-CIN85-endophilin complex is not required for efficient internalization of EGFR, a prototype RTK. PMID:25449262

  18. Ubiquitin ligase gp78 increases solubility and facilitates degradation of the Z variant of {alpha}-1-antitrypsin

    SciTech Connect

    Shen Yuxian; Ballar, Petek; Fang, Shengyun . E-mail: fangs@umbi.umd.edu

    2006-11-03

    Deficiency of circulating {alpha}-1-antitrypsin (AAT) is the most widely recognized abnormality of a proteinase inhibitor that causes lung disease. AAT-deficiency is caused by mutations of the AAT gene that lead to AAT protein retention in the endoplasmic reticulum (ER). Moreover, the mutant AAT accumulated in the ER predisposes the homozygote to severe liver injuries, such as neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Despite the fact that mutant AAT protein is subject to ER-associated degradation (ERAD), yeast genetic studies have determined that the ubiquitination machinery, Hrd1/Der3p-cue1p-Ubc7/6p, which plays a prominent role in ERAD, is not involved in degradation of mutant AAT. Here we report that gp78, a ubiquitin ligase (E3) pairing with mammalian Ubc7 for ERAD, ubiquitinates and facilitates degradation of ATZ, the classic deficiency variant of AAT having a Z mutation (Glu 342 Lys). Unexpectedly, gp78 over-expression also significantly increases ATZ solubility. p97/VCP, an AAA ATPase essential for retrotranslocation of misfolded proteins from the ER during ERAD, is involved in gp78-mediated degradation of ATZ. Surprisingly, unlike other ERAD substrates that cause ER stress leading to apoptosis when accumulated in the ER, ATZ, in fact, increases cell proliferation when over-expressed in cells. This effect can be partially inhibited by gp78 over-expression. These data indicate that gp78 assumes multiple unique quality control roles over ATZ, including the facilitation of degradation and inhibition of aggregation of ATZ.

  19. Effects of ageing on expression of the muscle-specific E3 ubiquitin ligases and Akt-dependent regulation of Foxo transcription factors in skeletal muscle.

    PubMed

    Wagatsuma, Akira; Shiozuka, Masataka; Takayama, Yuzo; Hoshino, Takayuki; Mabuchi, Kunihiko; Matsuda, Ryoichi

    2016-01-01

    Controversy exists as to whether the muscle-specific E3 ubiquitin ligases MAFbx and MuRF1 are transcriptionally upregulated in the process of sarcopenia. In the present study, we investigated the effects of ageing on mRNA/protein expression of muscle-specific E3 ubiquitin ligases and Akt/Foxo signalling in gastrocnemius muscles of female mice. Old mice exhibited a typical sarcopenic phenotype, characterized by loss of muscle mass and strength, decreased amount of myofibrillar proteins, incidence of aberrant muscle fibres, and genetic signature to sarcopenia. Activation levels of Akt were lower in adult and old mice than in young mice. Consequently, Akt-mediated phosphorylation levels of Foxo1 and Foxo3 proteins were decreased. Nuclear levels of Foxo1 and Foxo3 proteins showed an overall increasing trend in old mice. MAFbx mRNA expression was decreased in old mice relative to adult mice, whereas MuRF1 mRNA expression was less affected by ageing. At the protein level, MAFbx was less affected by ageing, whereas MuRF1 was increased in old mice relative to adult mice, with ubiquitin-protein conjugates being increased with ageing. In conclusion, we provided evidence for no mRNA upregulation of muscle-specific E3 ubiquitin ligases and disconnection between their expression and Akt/Foxo signalling in sarcopenic mice. Their different responsiveness to ageing may reflect different roles in sarcopenia.

  20. E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells

    PubMed Central

    Samant, Rahul S.; Clarke, Paul A.; Workman, Paul

    2014-01-01

    The molecular chaperone heat shock protein 90 (HSP90) is required for the activity and stability of its client proteins. Pharmacologic inhibition of HSP90 leads to the ubiquitin-mediated degradation of clients, particularly activated or mutant oncogenic protein kinases. Client ubiquitination occurs via the action of one or more E3 ubiquitin ligases. We sought to identify the role of Cullin-RING family E3 ubiquitin ligases in the cellular response to HSP90 inhibition. Through a focused siRNA screen of 28 Cullin-RING ligase family members, we found that CUL5 and RBX2 were required for degradation of several HSP90 clients upon treatment of human cancer cells with the clinical HSP90 inhibitor 17-AAG. Surprisingly, silencing Cullin-5 (CUL5) also delayed the earlier loss of HSP90 client protein activity at the same time as delaying cochaperone dissociation from inhibited HSP90–client complexes. Expression of a dominant-negative CUL5 showed that NEDD8 conjugation of CUL5 is required for client degradation but not for loss of client activity or recruitment of clients and HSP90 to CUL5. Silencing CUL5 reduced cellular sensitivity to three distinct HSP90 inhibitors, across four cancer types driven by different protein kinases. Our results reveal the importance of CUL5 in multiple aspects of the cellular response to HSP90 inhibition. PMID:24760825

  1. Inhibition of the ubiquitin ligase activity improves the production of biologically active fusion protein HSA-HGF in Chinese hamster ovary cells.

    PubMed

    Xu, Dongsheng; Wan, Aini; Zhang, Jingjing; Peng, Lin; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian

    2016-10-18

    Hepatocyte growth factor (HGF) is a potent multi-functional protein that stimulates proliferation, survival, motility, scattering and differentiation during growth and development, and has been considered to be a potential therapeutic agent for the treatment of a number of intractable diseases. The aim of this study was to enhance the expression of recombinant fusion protein HSA-HGF (R494E) in CHO cells by inhibiting the intracellular ubiquitin ligase activity. The high stable expression sub-clones with different signal peptides were selected by western blot (WB) analysis and used for suspension culture. We found that the expression of fusion protein HSA-HGF (R494E) on day 3 achieved 50 mg/L during the 8 day culture process, a large number of fusion proteins were intracellular degradated by ubiquitination pathway during day 4 to day 8. Furthermore, ubiquitin ligase inhibitor, thalidomide, was added in culture process, and resulted in efficient and stable secretion of HSA-HGF (R494E) in CHO cells. According to biological activity assays, HSA-HGF (R494E) possessed various biological activities similar to native HGF. In conclusion, innhibition of intracellular ubiquitin ligase activity was successfully improve the expression of biologically active fusion protein HSA-HGF (R494E) in CHO cells. Our data may be beneficial to enhance the production of other therapeutic proteins in fed-batch culture.

  2. A Cullin1-Based SCF E3 Ubiquitin Ligase Targets the InR/PI3K/TOR Pathway to Regulate Neuronal Pruning

    PubMed Central

    Wong, Jack Jing Lin; Wang, Cheng; Zhang, Heng; Kirilly, Daniel; Wu, Chunlai; Liou, Yih-Cherng; Wang, Hongyan; Yu, Fengwei

    2013-01-01

    Pruning that selectively eliminates unnecessary axons/dendrites is crucial for sculpting the nervous system during development. During Drosophila metamorphosis, dendrite arborization neurons, ddaCs, selectively prune their larval dendrites in response to the steroid hormone ecdysone, whereas mushroom body γ neurons specifically eliminate their axon branches within dorsal and medial lobes. However, it is unknown which E3 ligase directs these two modes of pruning. Here, we identified a conserved SCF E3 ubiquitin ligase that plays a critical role in pruning of both ddaC dendrites and mushroom body γ axons. The SCF E3 ligase consists of four core components Cullin1/Roc1a/SkpA/Slimb and promotes ddaC dendrite pruning downstream of EcR-B1 and Sox14, but independently of Mical. Moreover, we demonstrate that the Cullin1-based E3 ligase facilitates ddaC dendrite pruning primarily through inactivation of the InR/PI3K/TOR pathway. We show that the F-box protein Slimb forms a complex with Akt, an activator of the InR/PI3K/TOR pathway, and promotes Akt ubiquitination. Activation of the InR/PI3K/TOR pathway is sufficient to inhibit ddaC dendrite pruning. Thus, our findings provide a novel link between the E3 ligase and the InR/PI3K/TOR pathway during dendrite pruning. PMID:24068890

  3. A Small Molecule That Switches a Ubiquitin Ligase From a Processive to a Distributive Enzymatic Mechanism.

    PubMed

    Kathman, Stefan G; Span, Ingrid; Smith, Aaron T; Xu, Ziyang; Zhan, Jennifer; Rosenzweig, Amy C; Statsyuk, Alexander V

    2015-10-07

    E3 ligases are genetically implicated in many human diseases, yet E3 enzyme mechanisms are not fully understood, and there is a strong need for pharmacological probes of E3s. We report the discovery that the HECT E3 Nedd4-1 is a processive enzyme and that disruption of its processivity by biochemical mutations or small molecules switches Nedd4-1 from a processive to a distributive mechanism of polyubiquitin chain synthesis. Furthermore, we discovered and structurally characterized the first covalent inhibitor of Nedd4-1, which switches Nedd4-1 from a processive to a distributive mechanism. To visualize the binding mode of the Nedd4-1 inhibitor, we used X-ray crystallography and solved the first structure of a Nedd4-1 family ligase bound to an inhibitor. Importantly, our study shows that processive Nedd4-1, but not the distributive Nedd4-1:inhibitor complex, is able to synthesize polyubiquitin chains on the substrate in the presence of the deubiquitinating enzyme USP8. Therefore, inhibition of E3 ligase processivity is a viable strategy to design E3 inhibitors. Our study provides fundamental insights into the HECT E3 mechanism and uncovers a novel class of HECT E3 inhibitors.

  4. SUMO-targeted ubiquitin ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin

    PubMed Central

    Ohkuni, Kentaro; Takahashi, Yoshimitsu; Fulp, Alyona; Lawrimore, Josh; Au, Wei-Chun; Pasupala, Nagesh; Levy-Myers, Reuben; Warren, Jack; Strunnikov, Alexander; Baker, Richard E.; Kerscher, Oliver; Bloom, Kerry; Basrai, Munira A.

    2016-01-01

    Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1, since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization than either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability. PMID:26960795

  5. The Oncogenic Role of WWP1 E3 Ubiquitin Ligase in Prostate Cancer Development

    DTIC Science & Technology

    2011-05-01

    Supersignal West Pico enhanced chemiluminescence system (Pierce, Rockford, IL, USA) and a LAS-3000 Fujifilm imaging system. Protein ubiquitination assay...Finally, images were documented by using the Fujifilm Imaging system LAS-3000. WWP1 knockdown by siRNA MCF7 and T47D cells were cultured in phenol red...differences in Crohn’s disease and ulcerative colitis from endoscopic pinch biopsies: insights into distinctive pathogenesis. Inflamm Bowel Dis 13:807

  6. Nse1 RING-like Domain Supports Functions of the Smc5-Smc6 Holocomplex in Genome Stability

    PubMed Central

    Pebernard, Stephanie; Perry, J. Jefferson P.; Tainer, John A.

    2008-01-01

    The Smc5-Smc6 holocomplex plays essential but largely enigmatic roles in chromosome segregation, and facilitates DNA repair. The Smc5-Smc6 complex contains six conserved non-SMC subunits. One of these, Nse1, contains a RING-like motif that often confers ubiquitin E3 ligase activity. We have functionally characterized the Nse1 RING-like motif, to determine its contribution to the chromosome segregation and DNA repair roles of Smc5-Smc6. Strikingly, whereas a full deletion of nse1 is lethal, the Nse1 RING-like motif is not essential for cellular viability. However, Nse1 RING mutant cells are hypersensitive to a broad spectrum of genotoxic stresses, indicating that the Nse1 RING motif promotes DNA repair functions of Smc5-Smc6. We tested the ability of both human and yeast Nse1 to mediate ubiquitin E3 ligase activity in vitro and found no detectable activity associated with full-length Nse1 or the isolated RING domains. Interestingly, however, the Nse1 RING-like domain is required for normal Nse1-Nse3-Nse4 trimer formation in vitro and for damage-induced recruitment of Nse4 and Smc5 to subnuclear foci in vivo. Thus, we propose that the Nse1 RING-like motif is a protein–protein interaction domain required for Smc5-Smc6 holocomplex integrity and recruitment to, or retention at, DNA lesions. PMID:18667531

  7. The Ubiquitin Ligase TRIM62 Regulates CARD9-Mediated Anti-Fungal Immunity and Intestinal Inflammation

    PubMed Central

    Cao, Zhifang; Conway, Kara L.; Heath, Robert J.; Rush, Jason S.; Leshchiner, Elizaveta S.; Ramirez-Ortez, Zaida G.; Nedelsky, Natalia B.; Huang, Hailiang; Ng, Aylwin; Gardet, Agnès; Cheng, Shih-Chin; Shamji, Alykhan F.; Rioux, John D.; Wijmenga, Cisca; Netea, Mihai G.; Means, Terry K.; Daly, Mark J.; Xavier, Ramnik J.

    2015-01-01

    Summary CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 mutations. Here we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigate CARD9 regulation. We showed that the C-terminal truncated CARD9 protective variant acted in a dominant negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that Trim62-deficient mice have increased susceptibility to fungal infection, similar to Card9-deficient mice. This study utilizes a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation. PMID:26488816

  8. The E3 ubiquitin ligase neuregulin receptor degradation protein 1 (Nrdp1) promotes M2 macrophage polarization by ubiquitinating and activating transcription factor CCAAT/enhancer-binding Protein β (C/EBPβ).

    PubMed

    Ye, Shuo; Xu, Hongmei; Jin, Jing; Yang, Mingjin; Wang, Chunmei; Yu, Yizhi; Cao, Xuetao

    2012-08-03

    Macrophage activation, including classical (M1) activation and alternative (M2) activation, plays important roles in host immune response and pathogenesis of diseases. Ubiquitination has been shown to be involved in the differentiation of immune cells and in the regulation of immune responses. However, the role of ubiquitination during M1 versus M2 polarization is poorly explored. Here, we showed that arginase 1 (Arg1), a well recognized marker of M2 macrophages, is highly up-regulated in peritoneal macrophages derived from E3 ubiquitin ligase Nrdp1 transgenic (Nrdp1-TG) mice. Furthermore, other M2 feature markers such as MR, Ym1, and Fizz1, as well as Th2 cytokine IL-10, are also up-regulated in Nrdp1-TG macrophages after IL-4 stimulation. Knockdown of Nrdp1 expression effectively inhibits IL-4-induced expression of M2-related genes in macrophages. Moreover, Nrdp1 inhibits LPS-induced production of inducible NOS and pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in macrophages. Immunoprecipitation assays show that Nrdp1 interacts with and ubiquitinates transcriptional factor C/EBPβ via Lys-63-linked ubiquitination. Nrdp1 enhances C/EBPβ-triggered transcriptional activation of the Arg1 reporter gene in the presence of IL-4 stimulation. Thus, we demonstrate that Nrdp1-mediated ubiquitination and activation of C/EBPβ contributes to a ubiquitin-dependent nonproteolytic pathway that up-regulates Arg1 expression and promotes M2 macrophage polarization.

  9. The E3 Ubiquitin Ligase Neuregulin Receptor Degradation Protein 1 (Nrdp1) Promotes M2 Macrophage Polarization by Ubiquitinating and Activating Transcription Factor CCAAT/Enhancer-binding Protein β (C/EBPβ)*

    PubMed Central

    Ye, Shuo; Xu, Hongmei; Jin, Jing; Yang, Mingjin; Wang, Chunmei; Yu, Yizhi; Cao, Xuetao

    2012-01-01

    Macrophage activation, including classical (M1) activation and alternative (M2) activation, plays important roles in host immune response and pathogenesis of diseases. Ubiquitination has been shown to be involved in the differentiation of immune cells and in the regulation of immune responses. However, the role of ubiquitination during M1 versus M2 polarization is poorly explored. Here, we showed that arginase 1 (Arg1), a well recognized marker of M2 macrophages, is highly up-regulated in peritoneal macrophages derived from E3 ubiquitin ligase Nrdp1 transgenic (Nrdp1-TG) mice. Furthermore, other M2 feature markers such as MR, Ym1, and Fizz1, as well as Th2 cytokine IL-10, are also up-regulated in Nrdp1-TG macrophages after IL-4 stimulation. Knockdown of Nrdp1 expression effectively inhibits IL-4-induced expression of M2-related genes in macrophages. Moreover, Nrdp1 inhibits LPS-induced production of inducible NOS and pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in macrophages. Immunoprecipitation assays show that Nrdp1 interacts with and ubiquitinates transcriptional factor C/EBPβ via Lys-63-linked ubiquitination. Nrdp1 enhances C/EBPβ-triggered transcriptional activation of the Arg1 reporter gene in the presence of IL-4 stimulation. Thus, we demonstrate that Nrdp1-mediated ubiquitination and activation of C/EBPβ contributes to a ubiquitin-dependent nonproteolytic pathway that up-regulates Arg1 expression and promotes M2 macrophage polarization. PMID:22707723

  10. Pepper CaREL1, a ubiquitin E3 ligase, regulates drought tolerance via the ABA-signalling pathway.

    PubMed

    Lim, Chae Woo; Park, Chanmi; Kim, Jung-Hyun; Joo, Hyunhee; Hong, Eunji; Lee, Sung Chul

    2017-03-28

    Drought stress conditions in soil or air hinder plant growth and development. Here, we report that the hot pepper (C apsicum a nnuum) RING type E3 Ligase 1 gene (CaREL1) is essential to the drought stress response. CaREL1 encodes a cytoplasmic- and nuclear-localized protein with E3 ligase activity. CaREL1 expression was induced by abscisic acid (ABA) and drought. CaREL1 contains a C3H2C3-type RING finger motif, which functions in ubiquitination of the target protein. We used CaREL1-silenced pepper plants and CaREL1-overexpressing (OX) transgenic Arabidopsis plants to evaluate the in vivo function of CaREL1 in response to drought stress and ABA treatment. CaREL1-silenced pepper plants displayed a drought-tolerant phenotype characterized by ABA hypersensitivity. In contrast, CaREL1-OX plants exhibited ABA hyposensitivity during the germination, seedling, and adult stages. In addition, plant growth was severely impaired under drought stress conditions, via a high level of transpirational water loss and decreased stomatal closure. Quantitative RT-PCR analyses revealed that ABA-related drought stress responsive genes were more weakly expressed in CaREL1-OX plants than in wild-type plants, indicating that CaREL1 functions in the drought stress response via the ABA-signalling pathway. Taken together, our results indicate that CaREL1 functions as a negative regulator of ABA-mediated drought stress tolerance.

  11. Ubiquitin E3 ligase CRL4(CDT2/DCAF2) as a potential chemotherapeutic target for ovarian surface epithelial cancer.

    PubMed

    Pan, Wei-Wei; Zhou, Jian-Jie; Yu, Chao; Xu, Ying; Guo, Lian-Jun; Zhang, Hai-Yi; Zhou, Dawang; Song, Fang-Zhou; Fan, Heng-Yu

    2013-10-11

    Cullin-RING ubiquitin ligases (CRLs) are the largest family of E3 ligases and require cullin neddylation for their activation. The NEDD8-activating enzyme inhibitor MLN4924 reportedly blocked cullin neddylation and inactivated CRLs, which resulted in apoptosis induction and tumor suppression. However, CRL roles in ovarian cancer cell survival and the ovarian tumor repressing effects of MLN4924 are unknown. We show here that CRL4 components are highly expressed in human epithelial ovarian cancer tissues. MLN4924-induced DNA damage, cell cycle arrest, and apoptosis in ovarian cancer cells in a time- and dose-dependent manner. In addition, MLN4924 sensitized ovarian cancer cells to other chemotherapeutic drug treatments. Depletion of CRL4 components Roc1/2, Cul4a, and DDB1 had inhibitory effects on ovarian cancer cells similar to MLN4924 treatment, which suggested that CRL4 inhibition contributed to the chemotherapeutic effect of MLN4924 in ovarian cancers. We also investigated for key CRL4 substrate adaptors required for ovarian cancer cells. Depleting Vprbp/Dcaf1 did not significantly affect ovarian cancer cell growth, even though it was expressed by ovarian cancer tissues. However, depleting Cdt2/Dcaf2 mimicked the pharmacological effects of MLN4924 and caused the accumulation of its substrate, CDT1, both in vitro and in vivo. MLN4924-induced DNA damage and apoptosis were partially rescued by Cdt1 depletion, suggesting that CRL4(CDT2) repression and CDT1 accumulation were key biochemical events contributing to the genotoxic effects of MLN4924 in ovarian cancer cells. Taken together, these results indicate that CRL4(CDT2) is a potential drug target in ovarian cancers and that MLN4924 may be an effective anticancer agent for targeted ovarian cancer therapy.

  12. The ubiquitin ligase Cullin5SOCS2 regulates NDR1/STK38 stability and NF-κB transactivation

    PubMed Central

    Paul, Indranil; Batth, Tanveer S.; Iglesias-Gato, Diego; Al-Araimi, Amna; Al-Haddabi, Ibrahim; Alkharusi, Amira; Norstedt, Gunnar; Olsen, Jesper V.; Zadjali, Fahad; Flores-Morales, Amilcar

    2017-01-01

    SOCS2 is a pleiotropic E3 ligase. Its deficiency is associated with gigantism and organismal lethality upon inflammatory challenge. However, mechanistic understanding of SOCS2 function is dismal due to our unawareness of its protein substrates. We performed a mass spectrometry based proteomic profiling upon SOCS2 depletion and yield quantitative data for ~4200 proteins. Through this screen we identify a novel target of SOCS2, the serine-threonine kinase NDR1. Over-expression of SOCS2 accelerates turnover, while its knockdown stabilizes, endogenous NDR1 protein. SOCS2 interacts with NDR1 and promotes its degradation through K48-linked ubiquitination. Functionally, over-expression of SOCS2 antagonizes NDR1-induced TNFα-stimulated NF-κB activity. Conversely, depletion of NDR1 rescues the effect of SOCS2-deficiency on TNFα-induced NF-κB transactivation. Using a SOCS2−/− mice model of colitis we show that SOCS2-deficiency is pro-inflammatory and negatively correlates with NDR1 and nuclear p65 levels. Lastly, we provide evidence to suggest that NDR1 acts as an oncogene in prostate cancer. To the best of our knowledge, this is the first report of an identified E3 ligase for NDR1. These results might explain how SOCS2-deficiency leads to hyper-activation of NF-κB and downstream pathological implications and posits that SOCS2 induced degradation of NDR1 may act as a switch in restricting TNFα-NF-κB pathway. PMID:28216640

  13. The Role of Skp1-Cul1-F-box Ubiquitin Ligases in Src-Stimulated Estrogen Receptor Proteolysis and Estrogen Receptor Target Gene Expression

    DTIC Science & Technology

    2014-03-01

    tagged human ERa (or mutant) was purified from insect Sf9 cells infected by recombinant baculoviri harboring the FLAG-tagged ERa-coding sequence...male and female reproductive systems, and it is an important regulator of bone density, brain function and cholesterol mobilization45. Rapid...steroid receptor coactivator-ubiquitin ligase, E6-AP, results in tissue-selective steroid hormone resistance and defects in reproduction . Mol

  14. Development of β-Hairpin Peptides for the Measurement of SCF-Family E3 Ligase Activity in Vitro via Ornithine Ubiquitination

    PubMed Central

    2017-01-01

    Regulation of the ubiquitin–proteasome system (UPS) to treat select types of cancer has become a popular area of drug discovery research. The FDA approval of proteasome inhibitors Bortezomib and Carfilzomib in the treatment of multiple myeloma has led to an increased need for chemical reporters capable of detecting and quantifying protein ubiquitination and the activity of members of the UPS including E3 ubiquitin ligases and the proteasome in the tumor cells of the patients. One limitation of peptide-based reporters is their rapid degradation in the cellular environment by cytosolic peptidases. Conversely, β-hairpin “protectides” exhibit a pronounced secondary structure that significantly increases their lifetime under cellular conditions. The goal of this work was to develop a family of novel, ornithine-rich protectides that could act as primary degrons serving as substrates for in vitro ubiquitination. The fluorescent peptide-based reporters were demonstrated to be highly resistant to degradation in multiple myeloma cell lysates. The most stable β-hairpin primary degron, containing a single ornithine residue at the N-terminus, OWRWR [Ac-OWVRVpGO(FAM)WIRQ-NH2], demonstrated rapid ubiquitination kinetics and a 20-fold increase in stability when compared with an unstructured primary degron. A screen of E1 and E3 enzyme inhibitors in cell lysates showed that ubiquitination of OWRWR was significantly impaired by inhibitors of the SCF family of E3 ligases. Furthermore, this is the first report demonstrating the use of an ornithine residue on a primary degron as a ubiquitination site. This study serves as a strong foundation for the development of stable, fluorescent, peptide-based reporters capable of quantifying protein ubiquitination and the enzymatic activity of members of the UPS. PMID:28393136

  15. IRT1 degradation factor1, a ring E3 ubiquitin ligase, regulates the degradation of iron-regulated transporter1 in Arabidopsis.

    PubMed

    Shin, Lung-Jiun; Lo, Jing-Chi; Chen, Guan-Hong; Callis, Judy; Fu, Hongyong; Yeh, Kuo-Chen

    2013-08-01

    Fe is an essential micronutrient for plant growth and development; plants have developed sophisticated strategies to acquire ferric Fe from the soil. Nongraminaceous plants acquire Fe by a reduction-based mechanism, and graminaceous plants use a chelation-based mechanism. In Arabidopsis thaliana, which uses the reduction-based method, iron-regulated transporter1 (IRT1) functions as the most important transporter for ferrous Fe uptake. Rapid and constitutive degradation of IRT1 allows plants to quickly respond to changing conditions to maintain Fe homeostasis. IRT1 degradation involves ubiquitination. To identify the specific E3 ubiquitin ligases involved in IRT1 degradation, we screened a set of insertional mutants in RING-type E3 ligases and identified a mutant that showed delayed degradation of IRT1 and loss of IRT1-ubiquitin complexes. The corresponding gene was designated IRT1 degradation factor1 (IDF1). Evidence of direct interaction between IDF1 and IRT1 in the plasma membrane supported the role of IDF1 in IRT1 degradation. IRT1 accumulation was reduced when coexpressed with IDF1 in yeast or Xenopus laevis oocytes. IDF1 function was RING domain dependent. The idf1 mutants showed increased tolerance to Fe deficiency, resulting from increased IRT1 levels. This evidence indicates that IDF1 directly regulates IRT1 degradation through its RING-type E3 ligase activity.

  16. GpDSR7, a Novel E3 Ubiquitin Ligase Gene in Grimmia pilifera Is Involved in Tolerance to Drought Stress in Arabidopsis

    PubMed Central

    Li, Mengmeng; Li, Yihao; Zhao, Junyi; Liu, Hai; Jia, Shenghua; Li, Jie; Zhao, Heping; Han, Shengcheng; Wang, Yingdian

    2016-01-01

    The growth and development of plants under drought stress depends mainly on the expression levels of various genes and modification of proteins. To clarify the molecular mechanism of drought-tolerance of plants, suppression subtractive hybridisation cDNA libraries were screened to identify drought-stress-responsive unigenes in Grimmia pilifera, and a novel E3 ubiquitin ligase gene, GpDSR7, was identified among the 240 responsive unigenes. GpDSR7 expression was induced by various abiotic stresses, particularly by drought. GpDSR7 displayed E3 ubiquitin ligase activity in vitro and was exclusively localised on the ER membrane in Arabidopsis mesophyll protoplasts. GpDSR7-overexpressing transgenic Arabidopsis plants showed a high water content and survival ratio under drought stress. Moreover, the expression levels of some marker genes involved in drought stress were higher in the transgenic plants than in wild-type plants. These results suggest that GpDSR7, an E3 ubiquitin ligase, is involved in tolerance to drought stress at the protein modification level. PMID:27228205

  17. IRT1 DEGRADATION FACTOR1, a RING E3 Ubiquitin Ligase, Regulates the Degradation of IRON-REGULATED TRANSPORTER1 in Arabidopsis[C][W][OPEN

    PubMed Central

    Shin, Lung-Jiun; Lo, Jing-Chi; Chen, Guan-Hong; Callis, Judy; Fu, Hongyong; Yeh, Kuo-Chen

    2013-01-01

    Fe is an essential micronutrient for plant growth and development; plants have developed sophisticated strategies to acquire ferric Fe from the soil. Nongraminaceous plants acquire Fe by a reduction-based mechanism, and graminaceous plants use a chelation-based mechanism. In Arabidopsis thaliana, which uses the reduction-based method, IRON-REGULATED TRANSPORTER1 (IRT1) functions as the most important transporter for ferrous Fe uptake. Rapid and constitutive degradation of IRT1 allows plants to quickly respond to changing conditions to maintain Fe homeostasis. IRT1 degradation involves ubiquitination. To identify the specific E3 ubiquitin ligases involved in IRT1 degradation, we screened a set of insertional mutants in RING-type E3 ligases and identified a mutant that showed delayed degradation of IRT1 and loss of IRT1-ubiquitin complexes. The corresponding gene was designated IRT1 DEGRADATION FACTOR1 (IDF1). Evidence of direct interaction between IDF1 and IRT1 in the plasma membrane supported the role of IDF1 in IRT1 degradation. IRT1 accumulation was reduced when coexpressed with IDF1 in yeast or Xenopus laevis oocytes. IDF1 function was RING domain dependent. The idf1 mutants showed increased tolerance to Fe deficiency, resulting from increased IRT1 levels. This evidence indicates that IDF1 directly regulates IRT1 degradation through its RING-type E3 ligase activity. PMID:23995086

  18. The Arabidopsis EDR1 Protein Kinase Negatively Regulates the ATL1 E3 Ubiquitin Ligase to Suppress Cell Death[W

    PubMed Central

    Serrano, Irene; Gu, Yangnan; Qi, Dong; Dubiella, Ullrich

    2014-01-01

    Loss-of-function mutations in the Arabidopsis thaliana ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced programmed cell death under a variety of abiotic and biotic stress conditions. All edr1 mutant phenotypes can be suppressed by missense mutations in the KEEP ON GOING gene, which encodes a trans-Golgi network/early endosome (TGN/EE)-localized E3 ubiquitin ligase. Here, we report that EDR1 interacts with a second E3 ubiquitin ligase, ARABIDOPSIS TOXICOS EN LEVADURA1 (ATL1), and negatively regulates its activity. Overexpression of ATL1 in transgenic Arabidopsis induced severe growth inhibition and patches of cell death, while transient overexpression in Nicotiana benthamiana leaves induced cell death and tissue collapse. The E3 ligase activity of ATL1 was required for both of these processes. Importantly, we found that ATL1 interacts with EDR1 on TGN/EE vesicles and that EDR1 suppresses ATL1-mediated cell death in N. benthamiana and Arabidopsis. Lastly, knockdown of ATL1 expression suppressed cell death phenotypes associated with the edr1 mutant and made Arabidopsis hypersusceptible to powdery mildew infection. Taken together, our data indicate that ATL1 is a positive regulator of programmed cell death and EDR1 negatively regulates ATL1 activity at the TGN/EE and thus controls stress responses initiated by ATL1-mediated ubiquitination events. PMID:25398498

  19. The Anaphase-Promoting Complex (APC) ubiquitin ligase regulates GABA transmission at the C. elegans neuromuscular junction

    PubMed Central

    Kowalski, Jennifer R.; Dube, Hitesh; Touroutine, Denis; Rush, Kristen M.; Goodwin, Patricia R.; Carozza, Marc; Didier, Zachary; Francis, Michael M.; Juo, Peter

    2014-01-01

    Regulation of both excitatory and inhibitory synaptic transmission is critical for proper nervous system function. Aberrant synaptic signaling, including altered excitatory to inhibitory balance, is observed innumerous neurological diseases. The ubiquitin enzyme system controls the abundance of many synaptic proteins and thus plays a key role in regulating synaptic transmission. The Anaphase-Promoting Complex (APC) is a multi-subunit ubiquitin ligase that was originally discovered as a key regulator of protein turnover during the cell cycle. More recently, the APC has been shown to function in postmitotic neurons, where it regulates diverse processes such as synapse development and synaptic transmission at glutamatergic synapses. Here we report that the APC regulates synaptic GABA signaling by acting in motor neurons to control the balance of excitatory (acetylcholine) to inhibitory (GABA) transmission at the Caenorhabditis elegans neuromuscular junction (NMJ). Loss-of-function mutants in multiple APC subunits have increased muscle excitation at the NMJ; this phenotype is rescued by expression of the missing subunit in GABA neurons. Quantitative imaging and electrophysiological analyses indicate that APC mutants have decreased GABA release but normal cholinergic transmission. Consistent with this, APC mutants exhibit convulsions in a seizure assay sensitive to reductions in GABA signaling. Previous studies in other systems showed that the APC can negatively regulate the levels of the active zone protein SYD-2 Liprin-α. Similarly, we found that SYD-2 accumulates in APC mutants at GABAergic presynaptic sites. Finally, we found that the APC subunit EMB-27 CDC16 can localize to presynapses in GABA neurons. Together, our data suggest a model in which the APC acts at GABAergic presynapses to promote GABA release and inhibit muscle excitation. These findings are the first evidence that the APC regulates transmission at inhibitory synapses and have implications for

  20. The E3 ubiquitin ligase HOS1 regulates low ambient temperature-responsive flowering in Arabidopsis thaliana.

    PubMed

    Lee, Jeong Hwan; Kim, Jae Joon; Kim, Soo Hyun; Cho, Hyun Jung; Kim, Joonki; Ahn, Ji Hoon

    2012-10-01

    Ubiquitin-dependent proteolysis regulates multiple aspects of plant growth and development, but little is known about its role in ambient temperature-responsive flowering. In addition to being regulated by daylength, the onset of flowering in many plants can also be delayed by low ambient temperatures. Here, we show that HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1), which encodes an E3 ubiquitin ligase, controls flowering time in response to ambient temperatures (16 and 23°C) and intermittent cold. hos1 mutants flowered early, and were insensitive to ambient temperature, but responded normally to vernalization and gibberellic acid. Genetic analyses suggested that this ambient temperature-insensitive flowering was independent of FLOWERING LOCUS C (FLC). Also, FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) expression was up-regulated in hos1 mutants at both temperatures. The ft tsf mutation almost completely suppressed the early flowering of hos1 mutants at different temperatures, suggesting that FT and TSF are downstream of HOS1 in the ambient temperature response. A lesion in CONSTANS (CO) did not affect the ambient temperature-insensitive flowering phenotype of hos1-3 mutants. In silico analysis showed that FVE was spatiotemporally co-expressed with HOS1. A HOS1-green fluorescent protein (GFP) fusion co-localized with FVE-GFP in the nucleus at both 16 and 23°C. HOS1 physically interacted with FVE and FLK in yeast two-hybrid and co-immunoprecipitation assays. Moreover, hos1 mutants were insensitive to intermittent cold. Collectively, our results suggest that HOS1 acts as a common regulator in the signaling pathways that control flowering time in response to low ambient temperature.

  1. Muscle-specific E3 ubiquitin ligases are involved in muscle atrophy of cancer cachexia: an in vitro and in vivo study.

    PubMed

    Yuan, Lei; Han, Jun; Meng, Qingyang; Xi, Qiulei; Zhuang, Qiulin; Jiang, Yi; Han, Yusong; Zhang, Bo; Fang, Jing; Wu, Guohao

    2015-05-01

    Muscle atrophy F-Box (MAFbx)/atrogin-1 and muscle ring-finger-1 (MuRF-1) have been identified as two muscle-specific E3 ubiquitin ligases that are highly expressed in skeletal muscle during muscle atrophy. However, the role of muscle-specific E3 ubiquitin ligases during the process of muscle atrophy of cancer cachexia remains largely unknown. In the present study, we analyzed the expression of atrogin-1 and MuRF-1 in the skeletal muscle of patients with malignant and benign disease. The possible mechanisms were studied both in a colon 26-induced cancer cachexia mouse model and in tumor necrosis factor-α (TNF-α) induced atrophy C2C12 cells. Our results demonstrated that atrogin-1 and MuRF-1 tended to be increased in the skeletal muscle of patients with malignant disease even before weight loss. Non-tumor body weights and gastrocnemius weights were significantly decreased while expression levels of ubiquitin proteasome pathway associated genes (atrogin-1, MuRF-1, ubiquitin and E2-14K) were upregulated in cancer cachexia mice. Significant myotube atrophy with atrogin-1 overexpression was observed in the C2C12 cells treated with TNF-α. Meanwhile, knockdown of atrogin-1 by small interfering RNA (siRNA) protected C2C12 cells from the adverse effect of TNF-α. In conclusion, muscle-specific E3 ubiquitin ligases were upregulated during cancer cachexia, and atrogin-1 may be a potential molecular target for treating muscle atrophy induced by cancer cachexia.

  2. Diversity in the Architecture of ATLs, a Family of Plant Ubiquitin-Ligases, Leads to Recognition and Targeting of Substrates in Different Cellular Environments

    PubMed Central

    Aguilar-Hernández, Victor; Aguilar-Henonin, Laura; Guzmán, Plinio

    2011-01-01

    Ubiquitin-ligases or E3s are components of the ubiquitin proteasome system (UPS) that coordinate the transfer of ubiquitin to the target protein. A major class of ubiquitin-ligases consists of RING-finger domain proteins that include the substrate recognition sequences in the same polypeptide; these are known as single-subunit RING finger E3s. We are studying a particular family of RING finger E3s, named ATL, that contain a transmembrane domain and the RING-H2 finger domain; none of the member of the family contains any other previously described domain. Although the study of a few members in A. thaliana and O. sativa has been reported, the role of this family in the life cycle of a plant is still vague. To provide tools to advance on the functional analysis of this family we have undertaken a phylogenetic analysis of ATLs in twenty-four plant genomes. ATLs were found in all the 24 plant species analyzed, in numbers ranging from 20–28 in two basal species to 162 in soybean. Analysis of ATLs arrayed in tandem indicates that sets of genes are expanding in a species-specific manner. To get insights into the domain architecture of ATLs we generated 75 pHMM LOGOs from 1815 ATLs, and unraveled potential protein-protein interaction regions by means of yeast two-hybrid assays. Several ATLs were found to interact with DSK2a/ubiquilin through a region at the amino-terminal end, suggesting that this is a widespread interaction that may assist in the mode of action of ATLs; the region was traced to a distinct sequence LOGO. Our analysis provides significant observations on the evolution and expansion of the ATL family in addition to information on the domain structure of this class of ubiquitin-ligases that may be involved in plant adaptation to environmental stress. PMID:21887349

  3. Functional conservation between mammalian MGRN1 and plant LOG2 ubiquitin ligases.

    PubMed

    Guerra, Damian D; Pratelli, Réjane; Kraft, Edward; Callis, Judy; Pilot, Guillaume

    2013-11-01

    Plant LOSS OF GDU 2 (LOG2) and Mammalian Mahogunin Ring Finger 1 (MGRN1) proteins are RING-type E3 ligases sharing similarity N-terminal to the RING domain. Deletion of this region disrupts the interaction of LOG2 with the plant membrane protein GLUTAMINE DUMPER1 (GDU1). Phylogenetic analysis identified two clades of LOG2/MGRN1-like proteins in vertebrates and plants. The ability of MGRN1 to functionally replace LOG2 was tested. MGRN1 ubiquitylates GDU1 in vitro and can partially substitute for LOG2 in the plant, partially restoring amino acid resistance to a GDU1-myc over-expression, log2-2 background. Altogether, these results suggest a conserved function for the N-terminal domain in evolution.

  4. Hey bHLH Proteins Interact with a FBXO45 Containing SCF Ubiquitin Ligase Complex and Induce Its Translocation into the Nucleus.

    PubMed

    Salat, Daniela; Winkler, Anja; Urlaub, Henning; Gessler, Manfred

    2015-01-01

    The Hey protein family, comprising Hey1, Hey2 and HeyL in mammals, conveys Notch signals in many cell types. The helix-loop-helix (HLH) domain as well as the Orange domain, mediate homo- and heterodimerization of these transcription factors. Although distinct interaction partners have been identified so far, their physiological relevance for Hey functions is still largely unclear. Using a tandem affinity purification approach and mass spectrometry analysis we identified members of an ubiquitin E3-ligase complex consisting of FBXO45, PAM and SKP1 as novel Hey1 associated proteins. There is a direct interaction between Hey1 and FBXO45, whereas FBXO45 is needed to mediate indirect Hey1 binding to SKP1. Expression of Hey1 induces translocation of FBXO45 and PAM into the nucleus. Hey1 is a short-lived protein that is degraded by the proteasome, but there is no evidence for FBXO45-dependent ubiquitination of Hey1. On the contrary, Hey1 mediated nuclear translocation of FBXO45 and its associated ubiquitin ligase complex may extend its spectrum to additional nuclear targets triggering their ubiquitination. This suggests a novel mechanism of action for Hey bHLH factors.

  5. LIN-23, an E3 Ubiquitin Ligase Component, Is Required for the Repression of CDC-25.2 Activity during Intestinal Development in Caenorhabditis elegans.

    PubMed

    Son, Miseol; Kawasaki, Ichiro; Oh, Bong-Kyeong; Shim, Yhong-Hee

    2016-11-30

    Caenorhabditis elegans (C. elegans) utilizes two different cell-cycle modes, binucleations during the L1 larval stage and endoreduplications at four larval moltings, for its postembryonic intestinal development. Previous genetic studies indicated that CDC-25.2 is specifically required for binucleations at the L1 larval stage and is repressed before endoreduplications. Furthermore, LIN-23, the C. elegans β-TrCP ortholog, appears to function as a repressor of CDC-25.2 to prevent excess intestinal divisions. We previously reported that intestinal hyperplasia in lin-23(e1883) mutants was effectively suppressed by the RNAi depletion of cdc-25.2. Nevertheless, LIN-23 targeting CDC-25.2 for ubiquitination as a component of E3 ubiquitin ligase has not yet been tested. In this study, LIN-23 is shown to be the major E3 ubiquitin ligase component, recognizing CDC-25.2 to repress their activities for proper transition of cell-cycle modes during the C. elegans postembryonic intestinal development. In addition, for the first time that LIN-23 physically interacts with both CDC-25.1 and CDC-25.2 and facilitates ubiquitination for timely regulation of their activities during the intestinal development.

  6. Protein–Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP)*

    PubMed Central

    Narayan, Vikram; Landré, Vivien; Ning, Jia; Hernychova, Lenka; Muller, Petr; Verma, Chandra; Walkinshaw, Malcolm D.; Blackburn, Elizabeth A.; Ball, Kathryn L.

    2015-01-01

    CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. PMID:26330542

  7. Ubiquitin ligase MARCH 8 cooperates with CD83 to control surface MHC II expression in thymic epithelium and CD4 T cell selection.

    PubMed

    Liu, Haiyin; Jain, Reema; Guan, Jing; Vuong, Vivian; Ishido, Satoshi; La Gruta, Nicole L; Gray, Daniel H; Villadangos, Jose A; Mintern, Justine D

    2016-08-22

    Major histocompatibility complex class II (MHC II) expression is tightly regulated, being subjected to cell type-specific mechanisms that closely control its levels at the cell surface. Ubiquitination by the E3 ubiquitin ligase MARCH 1 regulates MHC II expression in dendritic cells and B cells. In this study, we demonstrate that the related ligase MARCH 8 is responsible for regulating surface MHC II in thymic epithelial cells (TECs). March8(-/-) mice have elevated MHC II at the surface of cortical TECs and autoimmune regulator (AIRE)(-) medullary TECs (mTECs), but not AIRE(+) mTECs. Despite this, thymic and splenic CD4(+) T cell numbers and repertoires remained unaltered in March8(-/-) mice. Notably, the ubiquitination of MHC II by MARCH 8 is controlled by CD83. Mice expressing a mutated form of CD83 (Cd83(anu/anu) mice) have impaired CD4(+) T cell selection, but deleting March8 in Cd83(anu/anu) mice restored CD4(+) T cell selection to normal levels. Therefore, orchestrated regulation of MHC II surface expression in TECs by MARCH 8 and CD83 plays a major role in CD4(+) T cell selection. Our results also highlight the specialized use of ubiquitinating machinery in distinct antigen-presenting cell types, with important functional consequences and implications for therapeutic manipulation.

  8. Ubiquitin ligase MARCH 8 cooperates with CD83 to control surface MHC II expression in thymic epithelium and CD4 T cell selection

    PubMed Central

    Guan, Jing; La Gruta, Nicole L.; Gray, Daniel H.

    2016-01-01

    Major histocompatibility complex class II (MHC II) expression is tightly regulated, being subjected to cell type–specific mechanisms that closely control its levels at the cell surface. Ubiquitination by the E3 ubiquitin ligase MARCH 1 regulates MHC II expression in dendritic cells and B cells. In this study, we demonstrate that the related ligase MARCH 8 is responsible for regulating surface MHC II in thymic epithelial cells (TECs). March8−/− mice have elevated MHC II at the surface of cortical TECs and autoimmune regulator (AIRE)− medullary TECs (mTECs), but not AIRE+ mTECs. Despite this, thymic and splenic CD4+ T cell numbers and repertoires remained unaltered in March8−/− mice. Notably, the ubiquitination of MHC II by MARCH 8 is controlled by CD83. Mice expressing a mutated form of CD83 (Cd83anu/anu mice) have impaired CD4+ T cell selection, but deleting March8 in Cd83anu/anu mice restored CD4+ T cell selection to normal levels. Therefore, orchestrated regulation of MHC II surface expression in TECs by MARCH 8 and CD83 plays a major role in CD4+ T cell selection. Our results also highlight the specialized use of ubiquitinating machinery in distinct antigen-presenting cell types, with important functional consequences and implications for therapeutic manipulation. PMID:27503069

  9. LIN-23, an E3 Ubiquitin Ligase Component, Is Required for the Repression of CDC-25.2 Activity during Intestinal Development in Caenorhabditis elegans

    PubMed Central

    Son, Miseol; Kawasaki, Ichiro; Oh, Bong-Kyeong; Shim, Yhong-Hee

    2016-01-01

    Caenorhabditis elegans (C. elegans) utilizes two different cell-cycle modes, binucleations during the L1 larval stage and endoreduplications at four larval moltings, for its postembryonic intestinal development. Previous genetic studies indicated that CDC-25.2 is specifically required for binucleations at the L1 larval stage and is repressed before endoreduplications. Furthermore, LIN-23, the C. elegans β-TrCP ortholog, appears to function as a repressor of CDC-25.2 to prevent excess intestinal divisions. We previously reported that intestinal hyperplasia in lin-23(e1883) mutants was effectively suppressed by the RNAi depletion of cdc-25.2. Nevertheless, LIN-23 targeting CDC-25.2 for ubiquitination as a component of E3 ubiquitin ligase has not yet been tested. In this study, LIN-23 is shown to be the major E3 ubiquitin ligase component, recognizing CDC-25.2 to repress their activities for proper transition of cell-cycle modes during the C. elegans postembryonic intestinal development. In addition, for the first time that LIN-23 physically interacts with both CDC-25.1 and CDC-25.2 and facilitates ubiquitination for timely regulation of their activities during the intestinal development. PMID:27871172

  10. The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line

    PubMed Central

    Mohiuddin; Kobayashi, Shunsuke; Keka, Islam Shamima; Guilbaud, Guillaume; Sale, Julian; Narita, Takeo; Abdel-Aziz, H. Ismail; Wang, Xin; Ogawa, Saki; Sasanuma, Hiroyuki; Chiu, Roland; Oestergaard, Vibe H.; Lisby, Michael; Takeda, Shunichi

    2017-01-01

    The replicative DNA polymerases are generally blocked by template DNA damage. The resulting replication arrest can be released by one of two post-replication repair (PRR) pathways, translesion DNA synthesis (TLS) and template switching by homologous recombination (HR). The HERC2 ubiquitin ligase plays a role in homologous recombination by facilitating the assembly of the Ubc13 ubiquitin-conjugating enzyme with the RNF8 ubiquitin ligase. To explore the role of HERC2 and RNF8 in PRR, we examined immunoglobulin diversification in chicken DT40 cells deficient in HERC2 and RNF8. Unexpectedly, the HERC2-/- and RNF8-/- cells and HERC2-/-/RNF8-/- double mutant cells exhibit a significant reduction in the rate of immunoglobulin (Ig) hypermutation, compared to wild-type cells. Further, the HERC2-/- and RNF8-/- mutants exhibit defective maintenance of replication fork progression immediately after exposure to UV while retaining proficient post-replicative gap filling. These mutants are both proficient in mono-ubiquitination of PCNA. Taken together, these results suggest that HERC2 and RNF8 promote TLS past abasic sites and UV-lesions at or very close to stalled replication forks. PMID:26994443

  11. The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line.

    PubMed

    Mohiuddin; Kobayashi, Shunsuke; Keka, Islam Shamima; Guilbaud, Guillaume; Sale, Julian; Narita, Takeo; Abdel-Aziz, H Ismail; Wang, Xin; Ogawa, Saki; Sasanuma, Hiroyuki; Chiu, Roland; Oestergaard, Vibe H; Lisby, Michael; Takeda, Shunichi

    2016-04-01

    The replicative DNA polymerases are generally blocked by template DNA damage. The resulting replication arrest can be released by one of two post-replication repair (PRR) pathways, translesion DNA synthesis (TLS) and template switching by homologous recombination (HR). The HERC2 ubiquitin ligase plays a role in homologous recombination by facilitating the assembly of the Ubc13 ubiquitin-conjugating enzyme with the RNF8 ubiquitin ligase. To explore the role of HERC2 and RNF8 in PRR, we examined immunoglobulin diversification in chicken DT40 cells deficient in HERC2 and RNF8. Unexpectedly, the HERC2(-/-) and RNF8(-/-) cells and HERC2(-/-)/RNF8(-/-) double mutant cells exhibit a significant reduction in the rate of immunoglobulin (Ig) hypermutation, compared to wild-type cells. Further, the HERC2(-/-) and RNF8(-/-) mutants exhibit defective maintenance of replication fork progression immediately after exposure to UV while retaining proficient post-replicative gap filling. These mutants are both proficient in mono-ubiquitination of PCNA. Taken together, these results suggest that HERC2 and RNF8 promote TLS past abasic sites and UV-lesions at or very close to stalled replication forks.

  12. Upf1 potentially serves as a RING-related E3 ubiquitin ligase via its association with Upf3 in yeast

    PubMed Central

    Takahashi, Shinya; Araki, Yasuhiro; Ohya, Yuriko; Sakuno, Takeshi; Hoshino, Shin-Ichi; Kontani, Kenji; Nishina, Hiroshi; Katada, Toshiaki

    2008-01-01

    Three Upf proteins are essential to the nonsense-mediated mRNA decay (NMD) pathway. Although these proteins assemble on polysomes for recognition of aberrant mRNAs containing premature termination codons, the significance of this assembly remains to be elucidated. The Cys- and His-rich repeated N terminus (CH domain) of Upf1 has been implicated in its binding to Upf2. Here, we show that CH domain also plays a RING-related role for Upf1 to exhibit E3 ubiquitin ligase activity in yeast. Despite the sequence divergence from typical E3-RING fingers, the CH domain of yeast Upf1 specifically and directly interacted with the yeast E2 Ubc3. Interestingly, Upf1 served as a substrate for the in vitro self-ubiquitination, and the modification required its association with Upf3 rather than Upf2. Substitution of the coordinated Cys and His residues in the CH domain impaired not only self-ubiquitination of Upf1 but also rapid decay of aberrant mRNAs. These results suggest that Upf1 may serve as an E3 ubiquitin ligase upon its association with Upf3 and play an important role in signaling to the NMD pathway. PMID:18676617

  13. Overexpressed ubiquitin ligase Cullin7 in breast cancer promotes cell proliferation and invasion via down-regulating p53

    SciTech Connect

    Guo, Hongsheng; Wu, Fenping; Wang, Yan; Yan, Chong; Su, Wenmei

    2014-08-08

    Highlights: • Cullin7 is overexpressed in human breast cancer samples. • Cullin7 stimulated proliferation and invasion of breast cancer cells. • Inhibition of p53 contributes to Cullin7-induced proliferation and invasion. - Abstract: Ubiquitin ligase Cullin7 has been identified as an oncogene in some malignant diseases such as choriocarcinoma and neuroblastoma. However, the role of Cullin7 in breast cancer carcinogenesis remains unclear. In this study, we compared Cullin7 protein levels in breast cancer tissues with normal breast tissues and identified significantly higher expression of Cullin7 protein in breast cancer specimens. By overexpressing Cullin7 in breast cancer cells HCC1937, we found that Cullin7 could promote cell growth and invasion in vitro. In contrast, the cell growth and invasion was inhibited by silencing Cullin7 in breast cancer cell BT474. Moreover, we demonstrated that Cullin7 promoted breast cancer cell proliferation and invasion via down-regulating p53 expression. Thus, our study provided evidence that Cullin7 functions as a novel oncogene in breast cancer and may be a potential therapeutic target for breast cancer management.

  14. Regulating ehrlich and demethiolation pathways for alcohols production by the expression of ubiquitin-protein ligase gene HUWE1

    PubMed Central

    Zhang, Quan; Jia, Kai-Zhi; Xia, Shi-Tao; Xu, Yang-Hua; Liu, Rui-Sang; Li, Hong-Mei; Tang, Ya-Jie

    2016-01-01

    Ehrlich and demethiolation pathways as two competing branches converted amino acid into alcohols. Controlling both pathways offers considerable potential for industrial applications including alcohols overproduction, flavor-quality control and developing new flavors. While how to regulate ehrlich and demethiolation pathways is still not applicable. Taking the conversion of methionine into methionol and methanethiol for example, we constructed two suppression subtractive cDNA libraries of Clonostachys rosea by using suppression subtractive hybridization (SSH) technology for screening regulators controlling the conversion. E3 ubiquitin-protein ligase gene HUWE1 screened from forward SSH library was validated to be related with the biosynthesis of end products. Overexpressing HUWE1 in C. rosea and S. cerevisiae significantly increased the biosynthesis of methanethiol and its derivatives in demethiolation pathway, while suppressed the biosynthesis of methional and methionol in ehrlich pathway. These results attained the directional regulation of both pathways by overexpressing HUWE1. Thus, HUWE1 has potential to be a key target for controlling and enhancing alcohols production by metabolic engineering. PMID:26860895

  15. HIV-1 Tat Recruits HDM2 E3 Ligase To Target IRF-1 for Ubiquitination and Proteasomal Degradation

    PubMed Central

    Remoli, Anna Lisa; Marsili, Giulia; Perrotti, Edvige; Acchioni, Chiara; Sgarbanti, Marco; Borsetti, Alessandra; Hiscott, John

    2016-01-01

    ABSTRACT In addition to its ability to regulate HIV-1 promoter activation, the viral transactivator Tat also functions as a determinant of pathogenesis and disease progression by directly and indirectly modulating the host anti-HIV response, largely through the capacity of Tat to interact with and modulate the activities of multiple host proteins. We previously demonstrated that Tat modulated both viral and host transcriptional machinery by interacting with the cellular transcription factor interferon regulatory factor 1 (IRF-1). In the present study, we investigated the mechanistic basis and functional significance of Tat−IRF-1 interaction and demonstrate that Tat dramatically decreased IRF-1 protein stability. To accomplish this, Tat exploited the cellular HDM2 (human double minute 2 protein) ubiquitin ligase to accelerate IRF-1 proteasome-mediated degradation, resulting in a quenching of IRF-1 transcriptional activity during HIV-1 infection. These data identify IRF-1 as a new target of Tat-induced modulation of the cellular protein machinery and reveal a new strategy developed by HIV-1 to evade host immune responses. PMID:27795392

  16. The ubiquitin E3 ligase ITCH enhances breast tumor progression by inhibiting the Hippo tumor suppressor pathway.

    PubMed

    Salah, Zaidoun; Itzhaki, Ella; Aqeilan, Rami I

    2014-11-15

    The Hippo kinase pathway is emerging as a conserved signaling pathway that is essential for organ growth and tumorigenesis. Recently, we reported that the ubiquitin E3 ligase ITCH negatively regulates LATS1, thereby increasing YAP activity, which leads to increased cell proliferation and decreased apoptosis. Here, we investigated the role of ITCH in breast tumorigenesis. In particular, we show that ITCH enhances epithelial-to-mesenchymal transition (EMT) through boosting YAP oncogenic function. By contrast, a point mutation in the catalytic domain or WW1 domain of ITCH abolished its EMT-mediated effects. Furthermore, while overexpression of ITCH expression in breast cells is associated with increased incidence of mammary tumor formation and progression, its knockdown inhibited breast cancer cell tumorigenicity and metastasis. Importantly, YAP knockdown was able to attenuate ITCH pro-tumorigenic functions. Lastly, we found that ITCH expression is significantly upregulated in invasive and metastatic breast cancer cases and is associated with worse survival. Together, our results reveal that ITCH pro-tumorigenic functions in breast cancer are mediated, at least in part, through inactivation of the Hippo tumor suppressor pathway.

  17. The ubiquitin E3 ligase ITCH enhances breast tumor progression by inhibiting the Hippo tumor suppressor pathway

    PubMed Central

    Salah, Zaidoun; Itzhaki, Ella; Aqeilan, Rami I

    2014-01-01

    The Hippo kinase pathway is emerging as a conserved signaling pathway that is essential for organ growth and tumorigenesis. Recently, we reported that the ubiquitin E3 ligase ITCH negatively regulates LATS1, thereby increasing YAP activity, which leads to increased cell proliferation and decreased apoptosis. Here, we investigated the role of ITCH in breast tumorigenesis. In particular, we show that ITCH enhances epithelial-to-mesenchymal transition (EMT) through boosting YAP oncogenic function. By contrast, a point mutation in the catalytic domain or WW1 domain of ITCH abolished its EMT-mediated effects. Furthermore, while overexpression of ITCH expression in breast cells is associated with increased incidence of mammary tumor formation and progression, its knockdown inhibited breast cancer cell tumorigenicity and metastasis. Importantly, YAP knockdown was able to attenuate ITCH pro-tumorigenic functions. Lastly, we found that ITCH expression is significantly upregulated in invasive and metastatic breast cancer cases and is associated with worse survival. Together, our results reveal that ITCH pro-tumorigenic functions in breast cancer are mediated, at least in part, through inactivation of the Hippo tumor suppressor pathway. PMID:25350971

  18. E3 ubiquitin ligase Pirh2 enhances tumorigenic properties of human non-small cell lung carcinoma cells

    PubMed Central

    Fedorova, Olga; Shuvalov, Oleg; Merkulov, Valeriy; Vasileva, Elena; Antonov, Alexey; Barlev, Nikolai A.

    2016-01-01

    The product of RCHY1 human gene, Pirh2, is a RING-finger containing E3 ligase that modifies p53 with ubiquitin residues resulting in its subsequent degradation in proteasomes. Transcription of RCHY1 is regulated by p53 itself thus forming a negative regulatory feedback loop. Functionally, by eliminating p53, Pirh2 facilitates tumorigenesis. However, the role of Pirh2 in cancer cells lacking p53 is yet not well understood. Therefore, we decided to elucidate the role of Pirh2 in p53-negative human non-small cell lung carcinoma cells, H1299. We found that ectopic expression of Pirh2 enhanced cell proliferation, resistance to doxorubicin, and increased migration potential. Ablation of Pirh2 by specific shRNA reversed these phenotypes. Mechanistically, Pirh2 increased mRNA and protein levels of the c-Myc oncogene. The bioinformatics data indicate that co-expression of both c-Myc and Pirh2 strongly correlated with poor survival of lung cancer patients. Collectively, our results suggest that Pirh2 can be considered as a potential pharmacological target for developing anticancer therapies to treat p53-negative cancers. PMID:28191284

  19. E3 Ubiquitin Ligase Nedd4 Promotes Japanese Encephalitis Virus Replication by Suppressing Autophagy in Human Neuroblastoma Cells.

    PubMed

    Xu, Qingqiang; Zhu, Naiwei; Chen, Shenglin; Zhao, Ping; Ren, Hao; Zhu, Shiying; Tang, Hailin; Zhu, Yongzhe; Qi, Zhongtian

    2017-03-28

    Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes the most prevalent viral encephalitis in Asia. Since JEV is a neurotropic virus, it is important to identify key molecules that mediate JEV infection in neuronal cells and to investigate their underlying mechanisms. In this study, the critical role of Nedd4, an E3 ubiquitin ligase that is highly expressed in the central nervous system, was examined in JEV propagation. In SK-N-SH neuroblastoma cells, Nedd4 was up-regulated in response to JEV infection. Moreover, down-regulation of Nedd4 resulted in a significant decrease in JEV replication without alterations in virus attachment and internalization or in JEV pseudotyped virus infection, suggesting that Nedd4 participates in the replication but not in the entry stage of JEV infection. Further functional analysis showed that Nedd4 attenuated JEV-induced autophagy, which negatively regulates virus replication during infection. These results suggest that Nedd4 facilitates the replication of JEV by suppressing virus-induced autophagy. Taken together, our results indicate that Nedd4 plays a crucial role in JEV infection of neuronal cells, which provides a potential target for the development of novel treatment to combat JEV infection.

  20. E3 Ubiquitin Ligase Nedd4 Promotes Japanese Encephalitis Virus Replication by Suppressing Autophagy in Human Neuroblastoma Cells

    PubMed Central

    Xu, Qingqiang; Zhu, Naiwei; Chen, Shenglin; Zhao, Ping; Ren, Hao; Zhu, Shiying; Tang, Hailin; Zhu, Yongzhe; Qi, Zhongtian

    2017-01-01

    Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes the most prevalent viral encephalitis in Asia. Since JEV is a neurotropic virus, it is important to identify key molecules that mediate JEV infection in neuronal cells and to investigate their underlying mechanisms. In this study, the critical role of Nedd4, an E3 ubiquitin ligase that is highly expressed in the central nervous system, was examined in JEV propagation. In SK-N-SH neuroblastoma cells, Nedd4 was up-regulated in response to JEV infection. Moreover, down-regulation of Nedd4 resulted in a significant decrease in JEV replication without alterations in virus attachment and internalization or in JEV pseudotyped virus infection, suggesting that Nedd4 participates in the replication but not in the entry stage of JEV infection. Further functional analysis showed that Nedd4 attenuated JEV-induced autophagy, which negatively regulates virus replication during infection. These results suggest that Nedd4 facilitates the replication of JEV by suppressing virus-induced autophagy. Taken together, our results indicate that Nedd4 plays a crucial role in JEV infection of neuronal cells, which provides a potential target for the development of novel treatment to combat JEV infection. PMID:28349961

  1. An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain

    PubMed Central

    Ranjitkar, Prerana; Press, Maximilian O.; Yi, Xianhua; Baker, Richard; MacCoss, Michael J.; Biggins, Sue

    2010-01-01

    Summary Proper centromere function is critical to maintain genomic stability and to prevent aneuploidy, a hallmark of tumors and birth defects. A conserved feature of all eukaryotic centromeres is an essential histone H3 variant called CENP-A that requires a centromere targeting domain (CATD) for its localization. Although proteolysis prevents CENP-A from mislocalizing to euchromatin, regulatory factors have not been identified. Here, we identify an E3 ubiquitin ligase called Psh1 that leads to the degradation of Cse4, the budding yeast CENP-A homolog. Cse4 overexpression is toxic to psh1Δ cells and results in euchromatic localization. Strikingly, the Cse4 centromere targeting domain is a key regulator of its stability and helps Psh1 discriminate Cse4 from histone H3. Taken together, we propose that the CATD has a previously unknown role in maintaining the exclusive localization of Cse4 by preventing its mislocalization to euchromatin via Psh1-mediated degradation. PMID:21070971

  2. Loss of JAK2 regulation via a heterodimeric VHL-SOCS1 E3 ubiquitin ligase underlies Chuvash polycythemia.

    PubMed

    Russell, Ryan C; Sufan, Roxana I; Zhou, Bing; Heir, Pardeep; Bunda, Severa; Sybingco, Stephanie S; Greer, Samantha N; Roche, Olga; Heathcote, Samuel A; Chow, Vinca W K; Boba, Lukasz M; Richmond, Terri D; Hickey, Michele M; Barber, Dwayne L; Cheresh, David A; Simon, M Celeste; Irwin, Meredith S; Kim, William Y; Ohh, Michael

    2011-06-19

    Chuvash polycythemia is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the VHL (von Hippel-Lindau) gene, whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark abnormalities of Chuvash polycythemia, such as hypersensitivity to erythropoietin, are unclear. Here we show that VHL directly binds suppressor of cytokine signaling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated JAK2 (pJAK2) for ubiquitin-mediated destruction. In contrast, Chuvash polycythemia-associated VHL mutants have altered affinity for SOCS1 and do not engage with and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reversed the disease phenotype in Vhl(R200W/R200W) knock-in mice, an experimental model that recapitulates human Chuvash polycythemia. These results show that VHL is a SOCS1-cooperative negative regulator of JAK2 and provide biochemical and preclinical support for JAK2-targeted therapy in individuals with Chuvash polycythemia.

  3. Structural and kinetic analysis of the COP9-Signalosome activation and the cullin-RING ubiquitin ligase deneddylation cycle

    PubMed Central

    Mosadeghi, Ruzbeh; Reichermeier, Kurt M; Winkler, Martin; Schreiber, Anne; Reitsma, Justin M; Zhang, Yaru; Stengel, Florian; Cao, Junyue; Kim, Minsoo; Sweredoski, Michael J; Hess, Sonja; Leitner, Alexander; Aebersold, Ruedi; Peter, Matthias; Deshaies, Raymond J; Enchev, Radoslav I

    2016-01-01

    The COP9-Signalosome (CSN) regulates cullin–RING ubiquitin ligase (CRL) activity and assembly by cleaving Nedd8 from cullins. Free CSN is autoinhibited, and it remains unclear how it becomes activated. We combine structural and kinetic analyses to identify mechanisms that contribute to CSN activation and Nedd8 deconjugation. Both CSN and neddylated substrate undergo large conformational changes upon binding, with important roles played by the N-terminal domains of Csn2 and Csn4 and the RING domain of Rbx1 in enabling formation of a high affinity, fully active complex. The RING domain is crucial for deneddylation, and works in part through conformational changes involving insert-2 of Csn6. Nedd8 deconjugation and re-engagement of the active site zinc by the autoinhibitory Csn5 glutamate-104 diminish affinity for Cul1/Rbx1 by ~100-fold, resulting in its rapid ejection from the active site. Together, these mechanisms enable a dynamic deneddylation-disassembly cycle that promotes rapid remodeling of the cellular CRL network. DOI: http://dx.doi.org/10.7554/eLife.12102.001 PMID:27031283

  4. Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes.

    PubMed

    Le Gallo, Matthieu; O'Hara, Andrea J; Rudd, Meghan L; Urick, Mary Ellen; Hansen, Nancy F; O'Neil, Nigel J; Price, Jessica C; Zhang, Suiyuan; England, Bryant M; Godwin, Andrew K; Sgroi, Dennis C; Hieter, Philip; Mullikin, James C; Merino, Maria J; Bell, Daphne W

    2012-12-01

    Endometrial cancer is the sixth most commonly diagnosed cancer in women worldwide, causing ~74,000 deaths annually. Serous endometrial cancers are a clinically aggressive subtype with a poorly defined genetic etiology. We used whole-exome sequencing to comprehensively search for somatic mutations within ~22,000 protein-encoding genes in 13 primary serous endometrial tumors. We subsequently resequenced 18 genes, which were mutated in more than 1 tumor and/or were components of an enriched functional grouping, from 40 additional serous tumors. We identified high frequencies of somatic mutations in CHD4 (17%), EP300 (8%), ARID1A (6%), TSPYL2 (6%), FBXW7 (29%), SPOP (8%), MAP3K4 (6%) and ABCC9 (6%). Overall, 36.5% of serous tumors had a mutated chromatin-remodeling gene, and 35% had a mutated ubiquitin ligase complex gene, implicating frequent mutational disruption of these processes in the molecular pathogenesis of one of the deadliest forms of endometrial cancer.

  5. The CUL4-DDB1 ubiquitin ligase complex controls adult and embryonic stem cell differentiation and homeostasis

    PubMed Central

    Gao, Jie; Buckley, Shannon M; Cimmino, Luisa; Guillamot, Maria; Strikoudis, Alexandros; Cang, Yong; Goff, Stephen P; Aifantis, Iannis

    2015-01-01

    Little is known on post-transcriptional regulation of adult and embryonic stem cell maintenance and differentiation. Here we characterize the role of Ddb1, a component of the CUL4-DDB1 ubiquitin ligase complex. Ddb1 is highly expressed in multipotent hematopoietic progenitors and its deletion leads to abrogation of both adult and fetal hematopoiesis, targeting specifically transiently amplifying progenitor subsets. However, Ddb1 deletion in non-dividing lymphocytes has no discernible phenotypes. Ddb1 silencing activates Trp53 pathway and leads to significant effects on cell cycle progression and rapid apoptosis. The abrogation of hematopoietic progenitor cells can be partially rescued by simultaneous deletion of Trp53. Conversely, depletion of DDB1 in embryonic stem cell (ESC) leads to differentiation albeit negative effects on cell cycle and apoptosis. Mass spectrometry reveals differing protein interactions between DDB1 and distinct DCAFs, the substrate recognizing components of the E3 complex, between cell types. Our studies identify CUL4-DDB1 complex as a novel post-translational regulator of stem and progenitor maintenance and differentiation. DOI: http://dx.doi.org/10.7554/eLife.07539.001 PMID:26613412

  6. Ubiquitin ligase RNF20/40 facilitates spindle assembly and promotes breast carcinogenesis through stabilizing motor protein Eg5

    PubMed Central

    Duan, Yang; Huo, Dawei; Gao, Jie; Wu, Heng; Ye, Zheng; Liu, Zhe; Zhang, Kai; Shan, Lin; Zhou, Xing; Wang, Yue; Su, Dongxue; Ding, Xiang; Shi, Lei; Wang, Yan; Shang, Yongfeng; Xuan, Chenghao

    2016-01-01

    Whether transcriptional regulators are functionally involved in mitosis is a fundamental question in cell biology. Here we report that the RNF20/40 complex, a major ubiquitin ligase catalysing histone H2B monoubiquitination, interacts with the motor protein Eg5 during mitosis and participates in spindle assembly. We show that the RNF20/40 complex monoubiquitinates and stabilizes Eg5. Loss of RNF20/40 results in spindle assembly defects, cell cycle arrest and apoptosis. Consistently, depletion of either RNF20/40 or Eg5 suppresses breast cancer in vivo. Significantly, RNF20/40 and Eg5 are concurrently upregulated in human breast carcinomas and high Eg5 expression is associated with poorer overall survival of patients with luminal A, or B, breast cancer. Our study uncovers an important spindle assembly role of the RNF20/40 complex, and implicates the RNF20/40-Eg5 axis in breast carcinogenesis, supporting the pursuit of these proteins as potential targets for breast cancer therapeutic interventions. PMID:27557628

  7. Neuroblastoma patient outcomes, tumor differentiation, and ERK activation are correlated with expression levels of the ubiquitin ligase UBE4B

    PubMed Central

    Woodfield, Sarah E.; Guo, Rong Jun; Liu, Yin; Major, Angela M.; Hollingsworth, Emporia Faith; Indiviglio, Sandra; Whittle, Sarah B.; Mo, Qianxing; Bean, Andrew J.; Ittmann, Michael; Lopez-Terrada, Dolores; Zage, Peter E.

    2016-01-01

    Background UBE4B is an E3/E4 ubiquitin ligase whose gene is located in chromosome 1p36.22. We analyzed the associations of UBE4B gene and protein expression with neuroblastoma patient outcomes and with tumor prognostic features and histology. Methods We evaluated the association of UBE4B gene expression with neuroblastoma patient outcomes using the R2 Platform. We screened neuroblastoma tumor samples for UBE4B protein expression using immunohistochemistry. FISH for UBE4B and 1p36 deletion was performed on tumor samples. We then evaluated UBE4B expression for associations with prognostic factors and with levels of phosphorylated ERK in neuroblastoma tumors and cell lines. Results Low UBE4B gene expression is associated with poor outcomes in patients with neuroblastoma and with worse outcomes in all patient subgroups. UBE4B protein expression was associated with neuroblastoma tumor differentiation, and decreased UBE4B protein levels were associated with high-risk features. UBE4B protein levels were also associated with levels of phosphorylated ERK. Conclusions We have demonstrated associations between UBE4B gene expression and neuroblastoma patient outcomes and prognostic features. Reduced UBE4B protein expression in neuroblastoma tumors was associated with high-risk features, a lack of differentiation, and with ERK activation. These results suggest UBE4B may contribute to the poor prognosis of neuroblastoma tumors with 1p36 deletions and that UBE4B expression may mediate neuroblastoma differentiation. PMID:27014418

  8. The Brain Proteome of the Ubiquitin Ligase Peli1 Knock-Out Mouse during Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Lereim, Ragnhild Reehorst; Oveland, Eystein; Xiao, Yichuan; Torkildsen, Øivind; Wergeland, Stig; Myhr, Kjell-Morten; Sun, Shao-Cong; Berven, Frode S

    2016-01-01

    The ubiquitin ligase Peli1 has previously been suggested as a potential treatment target in multiple sclerosis. In the multiple sclerosis disease model, experimental autoimmune encephalomyelitis, Peli1 knock-out led to less activated microglia and less inflammation in the central nervous system. Despite being important in microglia, Peli1 expression has also been detected in glial and neuronal cells. In the present study the overall brain proteomes of Peli1 knock-out mice and wild-type mice were compared prior to experimental autoimmune encephalomyelitis induction, at onset of the disease and at disease peak. Brain samples from the frontal hemisphere, peripheral from the extensive inflammatory foci, were analyzed using TMT-labeling of sample pools, and the discovered proteins were verified in individual mice using label-free proteomics. The greatest proteomic differences between Peli1 knock-out and wild-type mice were observed at the disease peak. In Peli1 knock-out a higher degree of antigen presentation, increased activity of adaptive and innate immune cells and alterations to proteins involved in iron metabolism were observed during experimental autoimmune encephalomyelitis. These results unravel global effects to the brain proteome when abrogating Peli1 expression, underlining the importance of Peli1 as a regulator of the immune response also peripheral to inflammatory foci during experimental autoimmune encephalomyelitis. The proteomics data is available in PRIDE with accession PXD003710. PMID:27746629

  9. Loss of Cbl and Cbl-b ubiquitin ligases abrogates hematopoietic stem cell quiescence and sensitizes leukemic disease to chemotherapy

    PubMed Central

    An, Wei; Nadeau, Scott A.; Mohapatra, Bhopal C.; Feng, Dan; Zutshi, Neha; Storck, Matthew D.; Arya, Priyanka; Talmadge, James E.; Meza, Jane L.; Band, Vimla; Band, Hamid

    2015-01-01

    Cbl and Cbl-b are tyrosine kinase-directed RING finger type ubiquitin ligases (E3s) that negatively regulate cellular activation pathways. E3 activity-disrupting human Cbl mutations are associated with myeloproliferative disorders (MPD) that are reproduced in mice with Cbl RING finger mutant knock-in or hematopoietic Cbl and Cbl-b double knockout. However, the role of Cbl proteins in hematopoietic stem cell (HSC) homeostasis, especially in the context of MPD is unclear. Here we demonstrate that HSC expansion and MPD development upon combined Cbl and Cbl-b deletion are dependent on HSCs. Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion. We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion. In vivo, treatment with 5-fluorouracil hastens DKO HSC exhaustion and protects mice from death due to MPD. Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling. PMID:25871390

  10. Loss of Cbl and Cbl-b ubiquitin ligases abrogates hematopoietic stem cell quiescence and sensitizes leukemic disease to chemotherapy.

    PubMed

    An, Wei; Nadeau, Scott A; Mohapatra, Bhopal C; Feng, Dan; Zutshi, Neha; Storck, Matthew D; Arya, Priyanka; Talmadge, James E; Meza, Jane L; Band, Vimla; Band, Hamid

    2015-04-30

    Cbl and Cbl-b are tyrosine kinase-directed RING finger type ubiquitin ligases (E3s) that negatively regulate cellular activation pathways. E3 activity-disrupting human Cbl mutations are associated with myeloproliferative disorders (MPD) that are reproduced in mice with Cbl RING finger mutant knock-in or hematopoietic Cbl and Cbl-b double knockout. However, the role of Cbl proteins in hematopoietic stem cell (HSC) homeostasis, especially in the context of MPD is unclear. Here we demonstrate that HSC expansion and MPD development upon combined Cbl and Cbl-b deletion are dependent on HSCs. Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion. We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion. In vivo, treatment with 5-fluorouracil hastens DKO HSC exhaustion and protects mice from death due to MPD. Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling.

  11. The E3 ubiquitin ligase TRIM31 attenuates NLRP3 inflammasome activation by promoting proteasomal degradation of NLRP3

    PubMed Central

    Song, Hui; Liu, Bingyu; Huai, Wanwan; Yu, Zhongxia; Wang, Wenwen; Zhao, Jing; Han, Lihui; Jiang, Guosheng; Zhang, Lining; Gao, Chengjiang; Zhao, Wei

    2016-01-01

    The NLRP3 inflammasome has a fundamental role in host defence against microbial pathogens and its deregulation may cause diverse inflammatory diseases. NLRP3 protein expression is a rate-limiting step for inflammasome activation, thus its expression must be tightly controlled to maintain immune homeostasis and avoid detrimental effects. However, how NLRP3 expression is regulated remains largely unknown. In this study, we identify E3 ubiquitin ligase TRIM31 as a feedback suppressor of NLRP3 inflammasome. TRIM31 directly binds to NLRP3, promotes K48-linked polyubiquitination and proteasomal degradation of NLRP3. Consequently, TRIM31 deficiency enhances NLRP3 inflammasome activation and aggravates alum-induced peritonitis in vivo. Furthermore, TRIM31 deficiency attenuates the severity of dextran sodium sulfate (DSS)-induced colitis, an inflammatory bowel diseases model in which NLRP3 possesses protective roles. Thus, our research describes a mechanism by which TRIM31 limits NLRP3 inflammasome activity under physiological conditions and suggests TRIM31 as a potential therapeutic target for the intervention of NLRP3 inflammasome related diseases. PMID:27929086

  12. Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase.

    PubMed

    Weber, Annika; Cohen, Itamar; Popp, Oliver; Dittmar, Gunnar; Reiss, Yuval; Sommer, Thomas; Ravid, Tommer; Jarosch, Ernst

    2016-09-01

    The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system.

  13. Smad3 Couples Pak1 With the Antihypertrophic Pathway Through the E3 Ubiquitin Ligase, Fbxo32.

    PubMed

    Tsui, Hoyee; Zi, Min; Wang, Shunyao; Chowdhury, Sanjoy K; Prehar, Sukhpal; Liang, Qiangrong; Cartwright, Elizabeth J; Lei, Ming; Liu, Wei; Wang, Xin

    2015-12-01

    Pathological cardiac hypertrophy is regarded as a critical intermediate step toward the development of heart failure. Many signal transduction cascades are demonstrated to dictate the induction and progression of pathological hypertrophy; however, our understanding in regulatory mechanisms responsible for the suppression of hypertrophy remains limited. In this study, we showed that exacerbated hypertrophy induced by pressure overload in cardiac-deleted Pak1 mice was attributable to a failure to upregulate the antihypertrophic E3 ligase, Fbxo32, responsible for targeting proteins for the ubiquitin-degradation pathway. Under pressure overload, cardiac overexpression of constitutively active Pak1 mice manifested strong resilience against pathological hypertrophic remodeling. Mechanistic studies demonstrated that subsequent to Pak1 activation, the binding of Smad3 on a critical singular AGAC(-286)-binding site on the FBXO32 promoter was crucial for its transcriptional regulation. Pharmacological upregulation of Fbxo32 by Berberine ameliorated hypertrophic remodeling and improved cardiac performance in cardiac-deficient Pak1 mice under pressure overload. Our findings discover Smad3 and Fbxo32 as novel downstream components of the Pak1-dependent signaling pathway for the suppression of hypertrophy. This discovery opens a new venue for opportunities to identify novel targets for the management of cardiac hypertrophy.

  14. Structural and Functional Impact of Parkinson Disease-Associated Mutations in the E3 Ubiquitin Ligase Parkin.

    PubMed

    Fiesel, Fabienne C; Caulfield, Thomas R; Moussaud-Lamodière, Elisabeth L; Ogaki, Kotaro; Dourado, Daniel F A R; Flores, Samuel C; Ross, Owen A; Springer, Wolfdieter

    2015-08-01

    Mutations in the PARKIN/PARK2 gene that result in loss-of-function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early-onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure-function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility, and protein aggregation. However pathogenic effects can also result from misregulation of Parkin autoinhibition and of its enzymatic functions. In addition, interference of binding to coenzymes, substrates, and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach, we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin.

  15. Tumor cell-specific inhibition of MYC function using small molecule inhibitors of the HUWE1 ubiquitin ligase

    PubMed Central

    Peter, Stefanie; Bultinck, Jennyfer; Myant, Kevin; Jaenicke, Laura A; Walz, Susanne; Müller, Judith; Gmachl, Michael; Treu, Matthias; Boehmelt, Guido; Ade, Carsten P; Schmitz, Werner; Wiegering, Armin; Otto, Christoph; Popov, Nikita; Sansom, Owen; Kraut, Norbert; Eilers, Martin

    2014-01-01

    Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARF-BP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high-throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells. See also: FX Schaub & JL Cleveland (December 2014) PMID:25253726

  16. E3 ubiquitin ligase APC/C-Cdh1 accounts for the Warburg effect by linking glycolysis to cell proliferation

    PubMed Central

    Almeida, Angeles; Bolaños, Juan P.; Moncada, Salvador

    2009-01-01

    Cell proliferation is known to be accompanied by activation of glycolysis. We have recently discovered that the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is degraded by the E3 ubiquitin ligase APC/C-Cdh1, which also degrades cell-cycle proteins. We now show in two different cell types (neoplastic and nonneoplastic) that both proliferation and aerobic glycolysis are prevented by overexpression of Cdh1 and enhanced by its silencing. Furthermore, we have coexpressed Cdh1 with PFKFB3—either wild-type or a mutant form resistant to ubiquitylation by APC/C-Cdh1—or with the glycolytic enzyme 6-phosphofructo-1-kinase and demonstrated that whereas glycolysis is essential for cell proliferation, its initiation in the presence of active Cdh1 does not result in proliferation. Our experiments indicate that the proliferative response, regardless of whether it occurs in normal or neoplastic cells, is dependent on a decrease in the activity of APC/C-Cdh1, which activates both proliferation and glycolysis. These observations have implications for cell proliferation, neoplastic transformation, and the prevention and treatment of cancer. PMID:20080744

  17. SAG/ROC-SCF beta-TrCP E3 ubiquitin ligase promotes pro-caspase-3 degradation as a mechanism of apoptosis protection.

    PubMed

    Tan, Mingjia; Gallegos, Jayme R; Gu, Qingyang; Huang, Yuanhui; Li, Jun; Jin, Yetao; Lu, Hua; Sun, Yi

    2006-12-01

    Skp1-cullin-F-box protein (SCF) is a multicomponent E3 ubiquitin (Ub) ligase that ubiquitinates a number of important biologic molecules such as p27, beta-catenin, and IkappaB for proteasomal degradation, thus regulating cell proliferation and survival. One SCF component, SAG/ROC2/Rbx2/Hrt2, a RING finger protein, was first identified as a redox-inducible protein, which, when overexpressed, inhibited apoptosis both in vitro and in vivo. We report here that sensitive to apoptosis gene (SAG), as well as its family member ROC1/Rbx1, bound to the proinactive form of caspase-3 (pro-caspase-3). Binding was likely mediated through F-box protein, beta-transducin repeat-containing protein (beta-TrCP), which binds to the first 38 amino acids of pro-caspase-3. Importantly, beta-TrCP1 expression significantly shortened the protein half-life of pro-caspase-3, whereas expression of a dominant-negative beta-TrCP1 mutant with the F-box domain deleted extended it. An in vitro ubiquitination assay showed that SAG/ROC-SCF(beta-TrCP) promoted ubiquitination of pro-caspase-3. Furthermore, endogenous levels of pro-caspase-3 were decreased by overexpression of SAG/ROC-SCF(beta-TrCP) E3 Ub ligases, but increased on siRNA silencing of SAG, regulator of cullin-1 (ROC1), or beta-TrCPs, leading to increased apoptosis by etoposide and TNF-related apoptosis-inducing ligand through increased activation of caspase-3. Thus, pro-caspase-3 appears to be a substrate of SAG/ROC-SCF(beta-TrCP) E3 Ub ligase, which protects cells from apoptosis through increased apoptosis threshold by reducing the basal level of pro-caspase-3.

  18. MALT1 cleaves the E3 ubiquitin ligase HOIL-1 in activated T cells, generating a dominant negative inhibitor of LUBAC-induced NF-κB signaling.

    PubMed

    Elton, Lynn; Carpentier, Isabelle; Staal, Jens; Driege, Yasmine; Haegman, Mira; Beyaert, Rudi

    2016-02-01

    Human paracaspase 1 (PCASP1), better known as mucosa associated lymphoid tissue lymphoma translocation 1 (MALT1), plays a key role in immunity and inflammation by regulating gene expression in lymphocytes and other immune cell types. Deregulated MALT1 activity has been implicated in autoimmunity, immunodeficiency and certain types of lymphoma. As a scaffold MALT1 assembles downstream signaling proteins for nuclear factor-κB (NF-κB) activation, while its proteolytic activity further enhances NF-κB activation by cleaving NF-κB inhibitory proteins. MALT1 also processes and inactivates a number of mRNA destabilizing proteins, which further fine-tunes gene expression. MALT1 protease inhibitors are currently developed for therapeutic targeting. Here we show that T cell activation, as well as overexpression of the oncogenic fusion protein API2-MALT1, induces the MALT1-mediated cleavage of haem-oxidized IRP2 ubiquitin ligase 1 (HOIL-1). In addition, to acting as a K48-polyubiquitin specific E3 ubiquitin ligase for different substrates, HOIL-1 co-operates in a catalytic-independent manner with the E3 ubiquitin ligase HOIL-1L interacting protein (HOIP) as part of the linear ubiquitin chain assembly complex (LUBAC). Intriguingly, cleavage of HOIL-1 does not directly abolish its ability to support HOIP-induced NF-κB signaling, which is still mediated by the N-terminal cleavage fragment, but generates a C-terminal fragment with LUBAC inhibitory properties. We propose that MALT1-mediated HOIL-1 cleavage provides a gain-of-function mechanism that is involved in the negative feedback regulation of NF-κB signaling.

  19. The Pepper E3 Ubiquitin Ligase RING1 Gene, CaRING1, Is Required for Cell Death and the Salicylic Acid-Dependent Defense Response1[C][W][OA

    PubMed Central

    Lee, Dong Hyuk; Choi, Hyong Woo; Hwang, Byung Kook

    2011-01-01

    Ubiquitination is essential for ubiquitin/proteasome-mediated protein degradation in plant development and defense. Here, we identified a novel E3 ubiquitin ligase RING1 gene, CaRING1, from pepper (Capsicum annuum). In pepper, CaRING1 expression is induced by avirulent Xanthomonas campestris pv vesicatoria infection. CaRING1 contains an amino-terminal transmembrane domain and a carboxyl-terminal RING domain. In addition, it displays in vitro E3 ubiquitin ligase activity, and the RING domain is essential for E3 ubiquitin ligase activity in CaRING1. CaRING1 also localizes to the plasma membrane. In pepper plants, virus-induced gene silencing of CaRING1 confers enhanced susceptibility to avirulent X. campestris pv vesicatoria infection, which is accompanied by compromised hypersensitive cell death, reduced expression of PATHOGENESIS-RELATED1, and lowered salicylic acid levels in leaves. Transient expression of CaRING1 in pepper leaves induces cell death and the defense response that requires the E3 ubiquitin ligase activity of CaRING1. By contrast, overexpression of CaRING1 in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to hemibiotrophic Pseudomonas syringae pv tomato and biotrophic Hyaloperonospora arabidopsidis infections. Taken together, these results suggest that CaRING1 is involved in the induction of cell death and the regulation of ubiquitination during the defense response to microbial pathogens. PMID:21628629

  20. Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins

    PubMed Central

    Takahashi, Hirotaka; Uematsu, Atsushi; Yamanaka, Satoshi; Imamura, Mei; Nakajima, Tatsuro; Doi, Kousuke; Yasuoka, Saki; Takahashi, Chikako; Takeda, Hiroyuki; Sawasaki, Tatsuya

    2016-01-01

    Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3). Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1) targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3—which there have been no report to bind p53—were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein. PMID:27249653

  1. Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration.

    PubMed

    Del Prete, Dolores; Rice, Richard C; Rajadhyaksha, Anjali M; D'Adamio, Luciano

    2016-08-12

    The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the NH2 terminus of the ACR, and CRL4(CRBN), which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the COOH terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4(CRBN), pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome analysis indicates that these E3 ligases are enzymatically active and ubiquitinate the ACR residues Lys(649/650/651/676/688) Deletion of Crbn reduces ubiquitination of Lys(676) suggesting that Lys(676) is physiologically ubiquitinated by CRL4(CRBN) The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible.

  2. Genomic imprinting of experience-dependent cortical plasticity by the ubiquitin ligase gene Ube3a.

    PubMed

    Sato, Masaaki; Stryker, Michael P

    2010-03-23

    A defect in the maternal copy of a ubiqutin ligase gene Ube3a can produce a neurodevelopmental defect in human children known as Angelman syndrome. We investigated the role of the maternally expressed Ube3a gene in experience-dependent development and plasticity of the mouse visual system. As demonstrated by optical imaging, rapid ocular dominance (OD) plasticity after brief monocular deprivation (MD) was severely impaired during the critical period (CP) in the visual cortex (VC) of Ube3a maternal-deficient (m-/p+) mice. Prolonged MD elicited significant plasticity in m-/p+ mice that never matched the level seen in control animals. In older animals after the CP, 7-day MD elicited mild OD shifts in both control and m-/p+ mice; however, the OD shifts in m-/p+ mice lacked the strengthening of visual responses to the two eyes characteristic of normal adult plasticity. Anatomic effects of the maternal deficiency include reduced spine density on basal, but not apical, dendrites of pyramidal neurons in the binocular region of the VC. Imprinting of Ube3a expression was not fully established in the early postnatal period, consistent with the normal development of cortical retinotopy and visual acuity that we observed in m-/p+ mice, but was fully established by the onset of the CP. These results demonstrate that paternal and maternal genomes are not functionally equivalent for cortical plasticity, and that maternally expressed Ube3a is required for normal experience-dependent modification of cortical circuits during and after the CP.

  3. Haploid Genetic Screens Identify an Essential Role for PLP2 in the Downregulation of Novel Plasma Membrane Targets by Viral E3 Ubiquitin Ligases

    PubMed Central

    Timms, Richard T.; Duncan, Lidia M.; Tchasovnikarova, Iva A.; Antrobus, Robin; Smith, Duncan L.; Dougan, Gordon; Weekes, Michael P.; Lehner, Paul J.

    2013-01-01

    The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2), a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system. PMID:24278019

  4. The Expression of the Ubiquitin Ligase SIAH2 (Seven In Absentia Homolog 2) Is Increased in Human Lung Cancer

    PubMed Central

    Moreno, Paula; Lara-Chica, Maribel; Soler-Torronteras, Rafael; Caro, Teresa; Medina, Manuel; Álvarez, Antonio; Salvatierra, Ángel; Muñoz, Eduardo; Calzado, Marco A.

    2015-01-01

    Objectives Lung cancer is the leading cause of cancer-related deaths worldwide. Overall 5-year survival has shown little improvement over the last decades. Seven in absentia homolog (SIAH) proteins are E3 ubiquitin ligases that mediate proteasomal protein degradation by poly-ubiquitination. Even though SIAH proteins play a key role in several biological processes, their role in human cancer remains controversial. The aim of the study was to document SIAH2 expression pattern at different levels (mRNA, protein level and immunohistochemistry) in human non-small cell lung cancer (NSCLC) samples compared to surrounding healthy tissue from the same patient, and to analyse the association with clinicopathological features. Materials and Methods One hundred and fifty-two samples from a patient cohort treated surgically for primary lung cancer were obtained for the study. Genic and protein expression levels of SIAH2 were analysed and compared with clinic-pathologic variables. Results The present study is the first to analyze the SIAH2 expression pattern at different levels (RNA, protein expression and immunohistochemistry) in non-small cell lung cancer (NSCLC). We found that SIAH2 protein expression is significantly enhanced in human lung adenocarcinoma (ADC) and squamous cell lung cancer (SCC). Paradoxically, non-significant changes at RNA level were found, suggesting a post-traductional regulatory mechanism. More importantly, an increased correlation between SIAH2 expression and tumor grade was detected, suggesting that this protein could be used as a prognostic biomarker to predict lung cancer progression. Likewise, SIAH2 protein expression showed a strong positive correlation with fluorodeoxyglucose (2-deoxy-2(18F)fluoro-D-glucose) uptake in primary NSCLC, which may assist clinicians in stratifying patients at increased overall risk of poor survival. Additionally, we described an inverse correlation between the expression of SIAH2 and the levels of one of its substrates

  5. PJA1, encoding a RING-H2 finger ubiquitin ligase, is a novel human X chromosome gene abundantly expressed in brain.

    PubMed

    Yu, Ping; Chen, Yiwang; Tagle, Danilo A; Cai, Tao

    2002-06-01

    RING-finger proteins contain cysteine-rich, zinc-binding domains and are involved in the formation of macromolecular scaffolds important for transcriptional repression and ubiquitination. In this study, we have identified a RING-H2 finger gene, PJA1 (for praja-1), from a human brain cDNA library and mapped it to human chromosome Xq12 between markers DXS983 and DXS1216, a region implicated in X-linked mental retardation (MRX). Northern blot analysis indicated a 2.7-kb transcript that was abundantly expressed in the brain, including regions of the cerebellum, cerebral cortex, medulla, occipital pole, frontal lobe, temporal lobe, and putamen. Amino acid sequence analysis of the 71-kDa protein PJA1 showed 52.3% identity to human PJA2 (for praja-2, also known as NEURODAP1/KIAA0438) and also a significant identity to its homologs in rat, mouse, and zebrafish. In vitro binding and immunoprecipitation assays demonstrated that both PJA1 and PJA2 are able to bind the ubiquitin-conjugating enzyme UbcH5B. Moreover, the ubiquitination assay indicated that PJA1 and PJA2 have an E2-dependent E3 ubiquitin ligase activity. Thus our findings demonstrate that PJA1 can be involved in protein ubiquitination in the brain and is a suitable candidate gene for MRX.

  6. Regulation of voltage-gated ion channels in excitable cells by the ubiquitin ligases Nedd4 and Nedd4-2.

    PubMed

    Bongiorno, Daria; Schuetz, Friderike; Poronnik, Philip; Adams, David J

    2011-01-01

    The electrical excitability of neurons is mediated primarily by voltage-gated ion channels, particularly voltage-gated Na(+) (Na(v)), K(+) (K(v)) and Cl(-) (ClC) channels. Cells regulate their electrical excitability by controlling not only the activity, but also the number of individual ion channels in the plasma membrane. There exist several mechanisms for regulating levels of voltage-gated ion channels: transcription and translation, retention and export from the endoplasmic reticulum as well as insertion and retrieval from the plasma membrane. Alterations in voltage-gated ion channel activity, composition and distribution can contribute to the pathophysiology of epilepsy, hypertension, neuropathic and inflammatory pain. One mechanism for retrieval is ubiquitination. Here specific ubiquitin ligases bind to membrane proteins to modulate and regulate their cellular fate. In this review, we focus on Nedd4 and Nedd4-2 ubiquitin ligases and the mechanisms by which they regulate voltage-gated ion channels and describe a novel paradigm on the mechanisms that underpin aberrant ion channel function in neurological disorders.

  7. The fate of tandemly duplicated genes assessed by the expression analysis of a group of Arabidopsis thaliana RING-H2 ubiquitin ligase genes of the ATL family.

    PubMed

    Aguilar-Hernández, Victor; Guzmán, Plinio

    2014-03-01

    Gene duplication events exert key functions on gene innovations during the evolution of the eukaryotic genomes. A large portion of the total gene content in plants arose from tandem duplications events, which often result in paralog genes with high sequence identity. Ubiquitin ligases or E3 enzymes are components of the ubiquitin proteasome system that function during the transfer of the ubiquitin molecule to the substrate. In plants, several E3s have expanded in their genomes as multigene families. To gain insight into the consequences of gene duplications on the expansion and diversification of E3s, we examined the evolutionary basis of a cluster of six genes, duplC-ATLs, which arose from segmental and tandem duplication events in Brassicaceae. The assessment of the expression suggested two patterns that are supported by lineage. While retention of expression domains was observed, an apparent absence or reduction of expression was also inferred. We found that two duplC-ATL genes underwent pseudogenization and that, in one case, gene expression is probably regained. Our findings provide insights into the evolution of gene families in plants, defining key events on the expansion of the Arabidopsis Tóxicos en Levadura family of E3 ligases.

  8. The RING Finger Ubiquitin E3 Ligase OsHTAS Enhances Heat Tolerance by Promoting H2O2-Induced Stomatal Closure in Rice1

    PubMed Central

    Liu, Jianping; Zhang, Cuicui; Wei, Chuchu; Liu, Xin; Wang, Mugui; Yu, Feifei; Xie, Qi; Tu, Jumin

    2016-01-01

    Heat stress often results in the generation of reactive oxygen species, such as hydrogen peroxide, which plays a vital role as a secondary messenger in the process of abscisic acid (ABA)-mediated stomatal closure. Here, we characterized the rice (Oryza sativa) HEAT TOLERANCE AT SEEDLING STAGE (OsHTAS) gene, which plays a positive role in heat tolerance at the seedling stage. OsHTAS encodes a ubiquitin ligase localized to the nucleus and cytoplasm. OsHTAS expression was detected in all tissues surveyed and peaked in leaf blade, in which the expression was concentrated in mesophyll cells. OsHTAS was responsive to multiple stresses and was strongly induced by exogenous ABA. In yeast two-hybrid assays, OsHTAS interacted with components of the ubiquitin/26S proteasome system and an isoform of rice ascorbate peroxidase. OsHTAS modulated hydrogen peroxide accumulation in shoots, altered the stomatal aperture status of rice leaves, and promoted ABA biosynthesis. The results suggested that the RING finger ubiquitin E3 ligase OsHTAS functions in leaf blade to enhance heat tolerance through modulation of hydrogen peroxide-induced stomatal closure and is involved in both ABA-dependent and DROUGHT AND SALT TOLERANCE-mediated pathways. PMID:26564152

  9. Deubiquitylase Inhibition Reveals Liver X Receptor-independent Transcriptional Regulation of the E3 Ubiquitin Ligase IDOL and Lipoprotein Uptake.

    PubMed

    Nelson, Jessica Kristine; Cook, Emma Clare Laura; Loregger, Anke; Hoeksema, Marten Anne; Scheij, Saskia; Kovacevic, Igor; Hordijk, Peter Lodewijk; Ovaa, Huib; Zelcer, Noam

    2016-02-26

    Cholesterol metabolism is subject to complex transcriptional and nontranscriptional regulation. Herein, the role of ubiquitylation is emerging as an important post-translational modification that regulates cholesterol synthesis and uptake. Similar to other post-translational modifications, ubiquitylation is reversible in a process dependent on activity of deubiquitylating enzymes (DUBs). Yet whether these play a role in cholesterol metabolism is largely unknown. As a first step to test this possibility, we used pharmacological inhibition of cellular DUB activity. Short term (2 h) inhibition of DUBs resulted in accumulation of high molecular weight ubiquitylated proteins. This was accompanied by a dramatic decrease in abundance of the LDLR and attenuated LDL uptake into hepatic cells. Importantly, this occurred in the absence of changes in the mRNA levels of the LDLR or other SREBP2-regulated genes, in line with this phenotype being a post-transcriptional event. Mechanistically, we identify transcriptional induction of the E3 ubiquitin ligase IDOL in human and rodent cells as the underlying cause for ubiquitylation-dependent lysosomal degradation of the LDLR following DUB inhibition. In contrast to the established transcriptional regulation of IDOL by the sterol-responsive liver X receptor (LXR) transcription factors, induction of IDOL by DUB inhibition is LXR-independent and occurs in Lxrαβ(-/-) MEFs. Consistent with the role of DUBs in transcriptional regulation, we identified a 70-bp region in the proximal promoter of IDOL, distinct from that containing the LXR-responsive element, which mediates the response to DUB inhibition. In conclusion, we identify a sterol-independent mechanism to regulate IDOL expression and IDOL-mediated lipoprotein receptor degradation.

  10. p53 E3 ubiquitin protein ligase homolog regulates p53 in vivo in the adult mouse eye lens

    PubMed Central

    Jaramillo-Rangel, Gilberto; Ortega-Martínez, Marta; Sepúlveda-Saavedra, Julio; Saucedo-Cárdenas, Odila; Montes-de-Oca-Luna, Roberto

    2013-01-01

    Purpose p53 is a transcription factor that plays an important role in preventing cancer development. p53 participates in relevant aspects of cell biology, including apoptosis and cell cycle control and must be strictly regulated to maintain normal tissue homeostasis. p53 E3 ubiquitin protein ligase homolog (Mdm2) is an important negative regulator of p53. The purpose of this study was to determine if Mdm2 regulates p53 in vivo in the adult lens. Methods We analyzed mice expressing human p53 transgene (Tgp53) selectively in the lens in the presence or absence of Mdm2. Mice with the required genotypes were obtained by crossing transgenic, mdm2+/−, and p53−/− mice. Eye phenotype and lens histology and ultrastructure were analyzed in adult mice. Results In a wild-type genetic background (mdm2+/+), lens damage and microphthalmia were observed only in mice homozygous for Tgp53 (t/t). However, in an mdm2 null background, just one allele of Tgp53 (mdm2−/−/Tgp53t/0 mice) was sufficient to cause lens damage and microphthalmia. Furthermore, Mdm2 in only one allele was sufficient to rescue these deleterious effects, since the mdm2+/−/Tgp53t/0 mice had eye size and lens morphology similar to the control mice. Conclusions Mdm2 regulates p53 in the adult lens in vivo. This information may have relevance for analyzing normal and pathological conditions of the lens, and designing cancer therapies targeting Mdm2–p53 interaction. PMID:24339722

  11. Regulation of the histone deacetylase Hst3 by cyclin-dependent kinases and the ubiquitin ligase SCFCdc4.

    PubMed

    Delgoshaie, Neda; Tang, Xiaojing; Kanshin, Evgeny D; Williams, Elizabeth C; Rudner, Adam D; Thibault, Pierre; Tyers, Mike; Verreault, Alain

    2014-05-09

    In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) is a modification of new H3 molecules deposited throughout the genome during S-phase. H3K56ac is removed by the sirtuins Hst3 and Hst4 at later stages of the cell cycle. Previous studies indicated that regulated degradation of Hst3 plays an important role in the genome-wide waves of H3K56 acetylation and deacetylation that occur during each cell cycle. However, little is known regarding the mechanism of cell cycle-regulated Hst3 degradation. Here, we demonstrate that Hst3 instability in vivo is dependent upon the ubiquitin ligase SCF(Cdc4) and that Hst3 is phosphorylated at two Cdk1 sites, threonine 380 and threonine 384. This creates a diphosphorylated degron that is necessary for Hst3 polyubiquitylation by SCF(Cdc4). Mutation of the Hst3 diphospho-degron does not completely stabilize Hst3 in vivo, but it nonetheless results in a significant fitness defect that is particularly severe in mutant cells treated with the alkylating agent methyl methanesulfonate. Unexpectedly, we show that Hst3 can be degraded between G2 and anaphase, a window of the cell cycle where Hst3 normally mediates genome-wide deacetylation of H3K56. Our results suggest an intricate coordination between Hst3 synthesis, genome-wide H3K56 deacetylation by Hst3, and cell cycle-regulated degradation of Hst3 by cyclin-dependent kinases and SCF(Cdc4).

  12. Regulation of the Histone Deacetylase Hst3 by Cyclin-dependent Kinases and the Ubiquitin Ligase SCFCdc4*

    PubMed Central

    Delgoshaie, Neda; Tang, Xiaojing; Kanshin, Evgeny D.; Williams, Elizabeth C.; Rudner, Adam D.; Thibault, Pierre; Tyers, Mike; Verreault, Alain

    2014-01-01

    In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) is a modification of new H3 molecules deposited throughout the genome during S-phase. H3K56ac is removed by the sirtuins Hst3 and Hst4 at later stages of the cell cycle. Previous studies indicated that regulated degradation of Hst3 plays an important role in the genome-wide waves of H3K56 acetylation and deacetylation that occur during each cell cycle. However, little is known regarding the mechanism of cell cycle-regulated Hst3 degradation. Here, we demonstrate that Hst3 instability in vivo is dependent upon the ubiquitin ligase SCFCdc4 and that Hst3 is phosphorylated at two Cdk1 sites, threonine 380 and threonine 384. This creates a diphosphorylated degron that is necessary for Hst3 polyubiquitylation by SCFCdc4. Mutation of the Hst3 diphospho-degron does not completely stabilize Hst3 in vivo, but it nonetheless results in a significant fitness defect that is particularly severe in mutant cells treated with the alkylating agent methyl methanesulfonate. Unexpectedly, we show that Hst3 can be degraded between G2 and anaphase, a window of the cell cycle where Hst3 normally mediates genome-wide deacetylation of H3K56. Our results suggest an intricate coordination between Hst3 synthesis, genome-wide H3K56 deacetylation by Hst3, and cell cycle-regulated degradation of Hst3 by cyclin-dependent kinases and SCFCdc4. PMID:24648511

  13. The E3 ubiquitin ligase APC/C-Cdh1 coordinates neurogenesis and cortical size during development.

    PubMed

    Delgado-Esteban, Maria; Garcia-Higuera, Irene; Moreno, Sergio; Almeida, Angeles

    2014-10-01

    The morphology of the adult brain is the result of a delicate balance between the symmetric divisions to maintain the progenitor cell pool, and the asymmetric divisions to generate a newly differentiated neuron. Neurogenesis is a complex process that relies on an as yet unknown molecular switch that tightly coordinates the cell cycle exit with the start of the differentiation process. The cell cycle length is a key factor that determines the balance between the maintenance of progenitor cells and neuronal differentiation. In fact, neurogenesis in the cerebral cortex is stimulated by lengthening the G1 phase and delayed by shortening it. The anaphase-promoting complex/cyclosome (APC/C) cofactor, Cdh1, regulates mitosis exit and G1-phase length in proliferating cells. Here we assessed whether APC/C-Cdh1 activity would be responsible for the switch from progenitor cells cycling to neurogenesis in the cerebral cortex. We use an embryo-restricted Cdh1 knockout mouse model and show that functional APC/C-Cdh1 ubiquitin ligase activity is required for both terminal differentiation of cortical neurons in vitro and neurogenesis in vivo. Further, genetic ablation of Cdh1 impairs the ability of APC/C to promote neurogenesis by delaying the exit of the progenitor cells from the cell cycle. This causes replicative stress and p53-mediated apoptotic death resulting in decreased number of cortical neurons and cortex size. These results demonstrate that APC/C-Cdh1 coordinates cortical neurogenesis and size, thus posing Cdh1 in the molecular pathogenesis of congenital neurodevelopmental disorders, such as microcephaly.

  14. The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels

    PubMed Central

    Chen, Yi-An; Peng, Yi-Jheng; Hu, Meng-Chun; Huang, Jing-Jia; Chien, Yun-Chia; Wu, June-Tai; Chen, Tsung-Yu; Tang, Chih-Yung

    2015-01-01

    Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels. PMID:26021757

  15. The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels.

    PubMed

    Chen, Yi-An; Peng, Yi-Jheng; Hu, Meng-Chun; Huang, Jing-Jia; Chien, Yun-Chia; Wu, June-Tai; Chen, Tsung-Yu; Tang, Chih-Yung

    2015-05-29

    Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels.

  16. Suppression of Arabidopsis RING E3 ubiquitin ligase AtATL78 increases tolerance to cold stress and decreases tolerance to drought stress.

    PubMed

    Kim, Soo Jin; Kim, Woo Taek

    2013-08-19

    AtATL78 is an Arabidopsis RING E3 ubiquitin ligase. RT-PCR and promoter-GUS assays revealed that AtATL78 was up-regulated by cold stress and down-regulated by drought. AtATL78 was localized at the plasma-membrane. Suppression of AtATL78 increased tolerance to cold stress but decreased tolerance to drought. Our data suggests that AtATL78 is a negative regulator of cold stress response and a positive regulator of drought stress response in Arabidopsis. These results further suggest that AtATL78 plays opposing roles in cold and drought stress responses.

  17. Interactions with DCAF1 and DDB1 in the CRL4 E3 ubiquitin ligase are required for Vpr-mediated G2 arrest

    PubMed Central

    2014-01-01

    Background HIV-1 Vpr-mediated G2 cell cycle arrest is dependent on the interaction of Vpr with an E3 ubiquitin ligase that contains damage-specific DNA binding protein 1 (DDB1), Cullin 4A (Cul4A), DDB1 and Cul4-associated factor 1 (DCAF1), and Rbx1. Vpr is thought to associate directly with DCAF1 in the E3 ubiquitin ligase complex although the exact interaction pattern of the proteins in the complex is not completely defined. The Vpr of SIVagm induces G2 arrest of cognate African Green Monkey (AGM) cells but not human cells. The molecular mechanism by which SIVagm Vpr exhibits its species-specific function remained unknown. Methods Physical interaction of proteins in the E3 ubiquitin ligase complex was assessed by co-immunoprecipitation followed by western blotting. In addition, co-localization of the proteins in cells was investigated by confocal microscopy. The cell cycle was analyzed by propidium iodide staining and flow cytometry. DNA damage response elicited by Vpr was evaluated by detecting phosphorylation of H2AX, a marker for DNA damage response. Results We show that RNAi knock-down of DCAF1 prevented the co-immunoprecipitation of DDB1 with HIV-1 Vpr while DDB1 knock-down did not influence the binding of Vpr to DCAF1. HIV-1 Vpr mutants with a L64P or a R90K mutation maintained the ability to associate with DCAF1 but did not appear to be in a complex with DDB1. SIVagm Vpr associated with AGM DCAF1 and DDB1 while, in human cells, it binds to human DCAF1 but hardly binds to human DDB1, resulting in the reduced activation of H2AX. Conclusions The identification of Vpr mutants which associate with DCAF1 but only poorly with DDB1 suggests that DCAF1 is necessary but the simple binding of Vpr to DCAF1 is not sufficient for the Vpr association with DDB1-containing E3 ligase complex. Vpr may interact both with DCAF1 and DDB1 in the E3 ligase complex. Alternatively, the interaction of Vpr and DCAF1 may induce a conformational change in DCAF1 or Vpr that promotes the

  18. Role of Rsp5 ubiquitin ligase in biogenesis of rRNA, mRNA and tRNA in yeast

    PubMed Central

    Domanska, Anna; Kaminska, Joanna

    2015-01-01

    Rsp5 ubiquitin ligase is required for ubiquitination of a wide variety of proteins involved in essential processes. Rsp5 was shown to be involved in regulation of lipid biosynthesis, intracellular trafficking of proteins, response to various stresses, and many other processes. In this article, we provide a comprehensive review of the nuclear and cytoplasmic functions of Rsp5 with a focus on biogenesis of different RNAs. We also briefly describe the participation of Rsp5 in the regulation of the RNA polymerase II complex, and its potential role in the regulation of other RNA polymerases. Moreover, we emphasize the function of Rsp5 in the coordination of the different steps of rRNA, mRNA and tRNA metabolism in the context of protein biosynthesis. Finally, we highlight the involvement of Rsp5 in controlling diverse cellular mechanisms at multiple levels and in adaptation of the cell to changing growth conditions. PMID:26403176

  19. CRL4Cdt2 E3 ubiquitin ligase and proliferating cell nuclear antigen (PCNA) cooperate to degrade thymine DNA glycosylase in S phase.

    PubMed

    Shibata, Etsuko; Dar, Ashraf; Dutta, Anindya

    2014-08-15

    Thymine DNA glycosylase (TDG) is an essential enzyme playing multiple roles in base excision repair, transcription regulation, and DNA demethylation. TDG mediates the cytotoxicity of the anti-cancer chemotherapeutic drug 5-fluorouracil (5-FU) by prolonging S phase, generating DNA strand breaks, and inducing DNA damage signaling. During S phase of the cell cycle, TDG is degraded via the proteasomal pathway. Here we show that CRL4(Cdt2) E3 ubiquitin ligase promotes ubiquitination and proteasomal degradation of TDG in S phase in a reaction that is dependent on the interaction of TDG with proliferating cell nuclear antigen (PCNA). siRNA-mediated depletion of PCNA or components of CRL4(Cdt2), specifically cullin4A/B or substrate adaptor Cdt2, stabilizes TDG in human cells. Mutations in the PCNA-interacting peptide (PIP) motif of TDG that disrupt the interaction of TDG with PCNA or change critical basic residues essential for the action of the PIP degron prevent the ubiquitination and degradation of TDG. Thus physical interaction of TDG with PCNA through the PIP degron is required for targeting TDG to the CRL4(Cdt2) E3 ubiquitin ligase complex. Compared with forced expression of wild type TDG, CRL4(Cdt2)- resistant TDG (ΔPIP) slows cell proliferation and slightly increases the toxicity of 5-FU. Thus, CRL4(Cdt2)-dependent degradation of TDG occurs in S phase because of the requirement for TDG to interact with chromatin-loaded PCNA, and this degradation is important for preventing toxicity from excess TDG.

  20. The ubiquitin E3 ligase LOSS OF GDU2 is required for GLUTAMINE DUMPER1-induced amino acid secretion in Arabidopsis.

    PubMed

    Pratelli, Réjane; Guerra, Damian D; Yu, Shi; Wogulis, Mark; Kraft, Edward; Frommer, Wolf B; Callis, Judy; Pilot, Guillaume

    2012-04-01

    Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.

  1. Genome-wide identification and characterization of the apple (Malus domestica) HECT ubiquitin-protein ligase family and expression analysis of their responsiveness to abiotic stresses.

    PubMed

    Xu, Jianing; Xing, Shanshan; Cui, Haoran; Chen, Xuesen; Wang, Xiaoyun

    2016-04-01

    The ubiquitin-protein ligases (E3s) directly participate in ubiquitin (Ub) transferring to the target proteins in the ubiquitination pathway. The HECT ubiquitin-protein ligase (UPL), one type of E3s, is characterized as containing a conserved HECT domain of approximately 350 amino acids in the C terminus. Some UPLs were found to be involved in trichome development and leaf senescence in Arabidopsis. However, studies on plant UPLs, such as characteristics of the protein structure, predicted functional motifs of the HECT domain, and the regulatory expression of UPLs have all been limited. Here, we present genome-wide identification of the genes encoding UPLs (HECT gene) in apple. The 13 genes (named as MdUPL1-MdUPL13) from ten different chromosomes were divided into four groups by phylogenetic analysis. Among these groups, the encoding genes in the intron-exon structure and the included additional functional domains were quite different. Notably, the F-box domain was first found in MdUPL7 in plant UPLs. The HECT domain in different MdUPL groups also presented different spatial features and three types of conservative motifs were identified. The promoters of each MdUPL member carried multiple stress-response related elements by cis-acting element analysis. Experimental results demonstrated that the expressions of several MdUPLs were quite sensitive to cold-, drought-, and salt-stresses by qRT-PCR assay. The results of this study helped to elucidate the functions of HECT proteins, especially in Rosaceae plants.

  2. Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2.

    PubMed

    Rosario, Fredrick J; Dimasuay, Kris Genelyn; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

    2016-04-01

    Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

  3. Lys29-linkage of ASK1 by Skp1−Cullin 1−Fbxo21 ubiquitin ligase complex is required for antiviral innate response

    PubMed Central

    Yu, Zhou; Chen, Taoyong; Li, Xuelian; Yang, Mingjin; Tang, Songqing; Zhu, Xuhui; Gu, Yan; Su, Xiaoping; Xia, Meng; Li, Weihua; Zhang, Xuemin; Wang, Qingqing; Cao, Xuetao; Wang, Jianli

    2016-01-01

    Protein ubiquitination regulated by ubiquitin ligases plays important roles in innate immunity. However, key regulators of ubiquitination during innate response and roles of new types of ubiquitination (apart from Lys48- and Lys63-linkage) in control of innate signaling have not been clearly understood. Here we report that F-box only protein Fbxo21, a functionally unknown component of SCF (Skp1–Cul1–F-box protein) complex, facilitates Lys29-linkage and activation of ASK1 (apoptosis signal-regulating kinase 1), and promotes type I interferon production upon viral infection. Fbxo21 deficiency in mice cells impairs virus-induced Lys29-linkage and activation of ASK1, attenuates c-Jun N-terminal kinase (JNK) and p38 signaling pathway, and decreases the production of proinflammatory cytokines and type I interferon, resulting in reduced antiviral innate response and enhanced virus replication. Therefore Fbxo21 is required for ASK1 activation via Lys29-linkage of ASK1 during antiviral innate response, providing mechanistic insights into non-proteolytic roles of SCF complex in innate immune response. DOI: http://dx.doi.org/10.7554/eLife.14087.001 PMID:27063938

  4. The E3 Ubiquitin Ligases, HUWE1 and NEDD4-1, Are Involved in the Post-translational Regulation of the ABCG1 and ABCG4 Lipid Transporters*

    PubMed Central

    Aleidi, Shereen M.; Howe, Vicky; Sharpe, Laura J.; Yang, Alryel; Rao, Geetha; Brown, Andrew J.; Gelissen, Ingrid C.

    2015-01-01

    The ATP-binding cassette transporter ABCG1 has an essential role in cellular cholesterol homeostasis, and dysregulation has been associated with a number of high burden diseases. Previous studies reported that ABCG1 is ubiquitinated and degraded via the ubiquitin proteasome system. However, so far the molecular mechanism, including the identity of any of the rate-limiting ubiquitination enzymes, or E3 ligases, is unknown. Using liquid chromatography mass spectrometry, we identified two HECT domain E3 ligases associated with ABCG1, named HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase) and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), of which the latter is the founding member of the NEDD4 family of ubiquitin ligases. Silencing both HUWE1 and NEDD4-1 in cells overexpressing human ABCG1 significantly increased levels of the ABCG1 monomeric and dimeric protein forms, however ABCA1 protein expression was unaffected. In addition, ligase silencing increased ABCG1-mediated cholesterol export to HDL in cells overexpressing the transporter as well as in THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant reduction in protein levels of both the ABCG1 monomeric and dimeric forms. Like ABCG1, ABCG4 protein levels and cholesterol export activity were significantly increased after silencing both HUWE1 and NEDD4-1 in cells overexpressing this closely related ABC half-transporter. In summary, we have identified for the first time two E3 ligases that are fundamental enzymes in the post-translational regulation of ABCG1 and ABCG4 protein levels and cellular cholesterol export activity. PMID:26296893

  5. Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner

    PubMed Central

    2014-01-01

    Background Smurf2 is a member of the HECT family of E3 ubiquitin ligases that play important roles in determining the competence of cells to respond to TGF- β/BMP signaling pathway. However, besides TGF-β/BMP pathway, Smurf2 regulates a repertoire of other signaling pathways ranging from planar cell polarity during embryonic development to cell proliferation, migration, differentiation and senescence. Expression of Smurf2 is found to be dysregulated in many cancers including breast cancer. The purpose of the present study is to examine the effect of Smurf2 knockdown on the tumorigenic potential of human breast cancer cells emphasizing more on proliferative signaling pathway. Methods siRNAs targeting different regions of the Smurf2 mRNA were employed to knockdown the expression of Smurf2. The biological effects of synthetic siRNAs on human breast cancer cells were investigated by examining the cell proliferation, migration, invasion, focus formation, anchorage-independent growth, cell cycle arrest, and cell cycle and cell proliferation related protein expressions upon Smurf2 silencing. Results Smurf2 silencing in human breast cancer cells resulted in a decreased focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of Smurf2 suppressed cell proliferation. Cell cycle analysis showed that the anti-proliferative effect of Smurf2 siRNA was mediated by arresting cells in the G0/G1 phase, which was caused by decreased expression of cyclin D1and cdk4, followed by upregulation p21 and p27. Furthermore, we demonstrated that silencing of Smurf2 downregulated the proliferation of breast cancer cells by modulating the PI3K- PTEN-AKT-FoxO3a pathway via the scaffold protein CNKSR2 which is involved in RAS-dependent signaling pathways. The present study provides the first evidence that silencing Smurf2 using synthetic siRNAs can regulate the tumorigenic properties of human breast cancer cells in a CNKSR2

  6. The tomato DWD motif-containing protein DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase and plays a pivotal role in abiotic stress responses

    SciTech Connect

    Miao, Min; Zhu, Yunye; Qiao, Maiju; Tang, Xiaofeng; Zhao, Wei; Xiao, Fangming; Liu, Yongsheng

    2014-08-08

    Highlights: • We identify DDI1 as a DAMAGED DNA BINDING PROTEIN1 (DDB1)-interacting protein. • DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase in the nucleus. • DDI1 plays a positive role in regulating abiotic stress response in tomato. - Abstract: CULLIN4(CUL4)–DAMAGED DNA BINDING PROTEIN1 (DDB1)-based ubiquitin ligase plays significant roles in multiple physiological processes via ubiquitination-mediated degradation of relevant target proteins. The DDB1–CUL4-associated factor (DCAF) acts as substrate receptor in the CUL4–DDB1 ubiquitin ligase complex and determines substrate specificity. In this study, we identified a tomato (Solanum lycopersicum) DDB1-interacting (DDI1) protein as a DCAF protein involved in response to abiotic stresses, including UV radiation, high salinity and osmotic stress. Co-immunoprecipitation and bimolecular fluorescence complementation assay indicated that DDI1 associates with CUL4–DDB1 in the nucleus. Quantitative RT-PCR analysis indicated the DDI1 gene is induced by salt, mannitol and UV-C treatment. Moreover, transgenic tomato plants with overexpression or knockdown of the DDI1 gene exhibited enhanced or attenuated tolerance to salt/mannitol/UV-C, respectively. Thus, our data suggest that DDI1 functions as a substrate receptor of the CUL4–DDB1 ubiquitin ligase, positively regulating abiotic stress response in tomato.

  7. Structure/Function Implications in a Dynamic Complex of the Intrinsically Disordered Sic1 with the Cdc4 Subunit of an SCF Ubiquitin Ligase

    SciTech Connect

    Mittag, Tanja; Marsh, Joseph; Grishaev, Alexander; Orlicky, Stephen; Lin, Hong; Sicheri, Frank; Tyers, Mike; Forman-Kay, Julie D.

    2010-11-22

    Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated.

  8. PUB22 and PUB23 U-BOX E3 ligases directly ubiquitinate RPN6, a 26S proteasome lid subunit, for subsequent degradation in Arabidopsis thaliana.

    PubMed

    Cho, Seok Keun; Bae, Hansol; Ryu, Moon Young; Wook Yang, Seong; Kim, Woo TaeK

    2015-09-04

    Drought stress strongly affects plant growth and development, directly connected with crop yields, accordingly. However, related to the function of U-BOX E3 ligases, the underlying molecular mechanisms of desiccation stress response in plants are still largely unknown. Here we report that PUB22 and PUB23, two U-box E3 ligase homologs, tether ubiquitins to 19S proteasome regulatory particle (RP) subunit RPN6, leading to its degradation. RPN6 was identified as an interacting substrate of PUB22 by yeast two-hybrid screening, and in vitro pull-down assay confirmed that RPN6 interacts not only with PUB22, but also with PUB23. Both PUB22 and PUB23 were able to conjugate ubiquitins on RPN6 in vitro. Furthermore, RPN6 showed a shorter protein half-life in PUB22 overexpressing plants than in wild-type, besides RPN6 was significantly stabilized in pub22pub23 double knockout plants. Taken together, these results solidify a notion that PUB22 and PUB23 can alter the activity of 26S proteasome in response to drought stress.

  9. MKRN2 is a novel ubiquitin E3 ligase for the p65 subunit of NF-κB and negatively regulates inflammatory responses

    PubMed Central

    Shin, Chanyoung; Ito, Yuma; Ichikawa, Shota; Tokunaga, Makio; Sakata-Sogawa, Kumiko; Tanaka, Takashi

    2017-01-01

    Activation of NF-κB transcription factor is strictly regulated to prevent excessive inflammatory responses leading to immunopathology. However, it still remains unclear how NF-κB activation is negatively controlled. The PDZ-LIM domain-containing protein PDLIM2 is a nuclear ubiquitin E3 ligase targeting the p65 subunit of NF-κB for degradation, thus terminating NF-κB-mediated inflammation. Using yeast two-hybrid screening, we sought to isolate PDLIM2-interacting proteins that are critical for suppressing NF-κB signaling. Here we identified MKRN2, a RING finger domain-containing protein that belongs to the makorin ring finger protein gene family, as a novel p65 ubiquitin E3 ligase. MKRN2 bound to p65 and promoted the polyubiquitination and proteasome-dependent degradation of p65 through the MKRN2 RING finger domain, thereby suppressing p65-mediated NF-κB transactivation. Notably, MKRN2 and PDLIM2 synergistically promote polyubiquitination and degradation of p65. Consistently, MKRN2 knockdown in dendritic cells resulted in larger amounts of nuclear p65 and augmented production of proinflammatory cytokines in responses to innate stimuli. These results delineate a novel role of MKRN2 in negatively regulating NF-κB-mediated inflammatory responses, cooperatively with PDLIM2. PMID:28378844

  10. Two ubiquitin ligases, APC/C-Cdh1 and SKP1-CUL1-F (SCF)-β-TrCP, sequentially regulate glycolysis during the cell cycle

    PubMed Central

    Tudzarova, Slavica; Colombo, Sergio L.; Stoeber, Kai; Carcamo, Saul; Williams, Gareth H.; Moncada, Salvador

    2011-01-01

    During cell proliferation, the abundance of the glycolysis-promoting enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is controlled by the ubiquitin ligase APC/C-Cdh1 via a KEN box. We now demonstrate in synchronized HeLa cells that PFKFB3, which appears in mid-to-late G1, is essential for cell division because its silencing prevents progression into S phase. In cells arrested by glucose deprivation, progression into S phase after replacement of glucose occurs only when PFKFB3 is present or is substituted by the downstream glycolytic enzyme 6-phosphofructo-1-kinase. PFKFB3 ceases to be detectable during late G1/S despite the absence of Cdh1; this disappearance is prevented by proteasomal inhibition. PFKFB3 contains a DSG box and is therefore a potential substrate for SCF-β-TrCP, a ubiquitin ligase active during S phase. In synchronized HeLa cells transfected with PFKFB3 mutated in the KEN box, the DSG box, or both, we established the breakdown routes of the enzyme at different stages of the cell cycle and the point at which glycolysis is enhanced. Thus, the presence of PFKFB3 is tightly controlled to ensure the up-regulation of glycolysis at a specific point in G1. We suggest that this up-regulation of glycolysis and its associated events represent the nutrient-sensitive restriction point in mammalian cells. PMID:21402913

  11. SAG/RBX2/ROC2 E3 ubiquitin ligase is essential for vascular and neural development by targeting NF1 for degradation.

    PubMed

    Tan, Mingjia; Zhao, Yongchao; Kim, Sun-Jung; Liu, Margaret; Jia, Lijun; Saunders, Thomas L; Zhu, Yuan; Sun, Yi

    2011-12-13

    SAG/RBX2/ROC2 protein is an essential RING component of SCF E3 ubiquitin ligase. The role of SAG during embryogenesis remains unknown. We report a critical role for SAG in controlling vascular and neural development by modulating RAS activity via promoting degradation of neurofibromatosis type 1 (NF1). Mice mutant for Sag died at embryonic day 11.5-12.5 with severe abnormalities in vascular and nervous system. Sag inactivation caused Nf1 accumulation and Ras inhibition, which blocks embryonic stem (ES) cells from undergoing endothelial differentiation and inhibits angiogenesis and proliferation in teratomas. Simultaneous Nf1 deletion fully rescues the differentiation defects in Sag(-/-) ES cells and partially rescues vascular and neural defects in Sag(-/-) embryos, suggesting that the effects of Sag deletion may not be solely explained by Nf1 misregulation. Collectively, our study identifies NF1 as a physiological substrate of SAG-CUL1-FBXW7 E3 ligase and establishes a ubiquitin-dependent regulatory mechanism for the NF1-RAS pathway during embryogenesis.

  12. SAG/RBX2 is a novel substrate of NEDD4-1 E3 ubiquitin ligase and mediates NEDD4-1 induced chemosensitization.

    PubMed

    Zhou, Weihua; Xu, Jie; Zhao, Yongchao; Sun, Yi

    2014-08-30

    Sensitive to apoptosis gene (SAG), also known as RBX2, ROC2, or RNF7, is a RING component of SCF E3 ubiquitin ligases, which regulates cellular functions through ubiquitylation and degradation of many protein substrates. Although our previous studies showed that SAG is transcriptionally induced by redox, mitogen and hypoxia via AP-1 and HIF-1, it is completely unknown whether and how SAG is ubiquitylated and degraded. Here we report that NEDD4-1, a HECT domain-containing E3 ubiquitin ligase, binds via its HECT domain directly with SAG's C-terminal RING domain and ubiquitylates SAG for proteasome-mediated degradation. Consistently, SAG protein half-life is shortened or extended by NEDD4-1 overexpression or silencing, respectively. We also found that SAG bridges NEDD4-1 via its C-terminus and CUL-5 via its N-terminus to form a NEDD4-1/SAG/CUL-5 tri-complex. Biologically, NEDD4-1 overexpression sensitizes cancer cells to etoposide-induced apoptosis by reducing SAG levels through targeted degradation. Thus, SAG is added to a growing list of NEDD4-1 substrates and mediates its biological function.

  13. Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase.

    PubMed

    Mittag, Tanja; Marsh, Joseph; Grishaev, Alexander; Orlicky, Stephen; Lin, Hong; Sicheri, Frank; Tyers, Mike; Forman-Kay, Julie D

    2010-03-14

    Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated.

  14. The atrzf1 mutation of the novel RING-type E3 ubiquitin ligase increases proline contents and enhances drought tolerance in Arabidopsis.

    PubMed

    Ju, Hyun-Woo; Min, Ji-Hee; Chung, Moon-Soo; Kim, Cheol Soo

    2013-04-01

    The covalent attachment of ubiquitin to proteins plays a fundamental role in the regulation of cellular function through biological events involving abiotic or biotic stress responses, immune responses, and apoptosis. Here, we characterize the biological function of the Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1) in dehydration response. AtRZF1 was significantly reduced by drought stress. The atrzf1 mutant was less sensitive to osmotic stress than the wild-type during early seedling development, whereas transgenic plants overexpressing AtRZF1 were hypersensitive, indicating that AtRZF1 negatively regulates drought-mediated control of early seedling development. Moreover, the ectopic expression of the AtRZF1 gene was very significantly influential in drought sensitive parameters including proline content, water loss, membrane ion leakage and the expression of dehydration stress-related genes. AtRZF1 is a functional E3 ubiquitin ligase, and its conserved C3H2C3-type RING domain is likely important for the biological function of AtRZF1 in drought response. Together, these results suggest that the E3 ligase AtRZF1 is an important regulator of water deficit stress during early seedling development.

  15. SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus

    PubMed Central

    Jöhnk, Bastian; Bayram, Özgür; Heinekamp, Thorsten; Mattern, Derek J.; Brakhage, Axel A.; Jacobsen, Ilse D.; Valerius, Oliver; Braus, Gerhard H.

    2016-01-01

    F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus. PMID:27649508

  16. The E3 Ubiquitin Ligase IDOL Induces the Degradation of the Low Density Lipoprotein Receptor Family Members VLDLR and ApoER2*

    PubMed Central

    Hong, Cynthia; Duit, Sarah; Jalonen, Pilvi; Out, Ruud; Scheer, Lilith; Sorrentino, Vincenzo; Boyadjian, Rima; Rodenburg, Kees W.; Foley, Edan; Korhonen, Laura; Lindholm, Dan; Nimpf, Johannes; van Berkel, Theo J. C.; Tontonoz, Peter; Zelcer, Noam

    2010-01-01

    We have previously identified the E3 ubiquitin ligase-inducible degrader of the low density lipoprotein receptor (LDLR) (Idol) as a post-translational modulator of LDLR levels. Idol is a direct target for regulation by liver X receptors (LXRs), and its expression is responsive to cellular sterol status independent of the sterol-response element-binding proteins. Here we demonstrate that Idol also targets two closely related LDLR family members, VLDLR and ApoE receptor 2 (ApoER2), proteins implicated in both neuronal development and lipid metabolism. Idol triggers ubiquitination of the VLDLR and ApoER2 on their cytoplasmic tails, leading to their degradation. We further show that the level of endogenous VLDLR is sensitive to cellular sterol content, Idol expression, and activation of the LXR pathway. Pharmacological activation of the LXR pathway in mice leads to increased Idol expression and to decreased Vldlr levels in vivo. Finally, we establish an unexpected functional link between LXR and Reelin signaling. We demonstrate that LXR activation results in decreased Reelin binding to VLDLR and reduced Dab1 phosphorylation. The identification of VLDLR and ApoER2 as Idol targets suggests potential roles for this LXR-inducible E3 ligase in the central nervous system in addition to lipid metabolism. PMID:20427281

  17. Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas

    PubMed Central

    Kong, Sinyi; Yang, Yi; Xu, Yuanming; Wang, Yajun; Zhang, Yusi; Melo-Cardenas, Johanna; Xu, Xiangping; Gao, Beixue; Thorp, Edward B.; Zhang, Donna D.; Zhang, Bin; Song, Jianxun; Zhang, Kezhong; Zhang, Jianning; Zhang, Jinping; Li, Huabin; Fang, Deyu

    2016-01-01

    Humoral immunity involves multiple checkpoints during B-cell development, maturation, and activation. The cell death receptor CD95/Fas-mediated apoptosis plays a critical role in eliminating the unwanted activation of B cells by self-reactive antigens and in maintaining B-cell homeostasis through activation-induced B-cell death (AICD). The molecular mechanisms controlling AICD remain largely undefined. Herein, we show that the E3 ubiquitin ligase Hrd1 protected B cells from activation-induced cell death by degrading the death receptor Fas. Hrd1-null B cells exhibited high Fas expression during activation and rapidly underwent Fas-mediated apoptosis, which could be largely inhibited by FasL neutralization. Fas mutation in Hrd1 KO mice abrogated the increase in B-cell AICD. We identified Hrd1 as the first E3 ubiquitin ligase of the death receptor Fas and Hrd1-mediated Fas destruction as a molecular mechanism in regulating B-cell immunity. PMID:27573825

  18. A cancer-associated RING finger protein, RNF43, is a ubiquitin ligase that interacts with a nuclear protein, HAP95

    SciTech Connect

    Sugiura, Takeyuki Yamaguchi, Aya; Miyamoto, Kentaro

    2008-04-15

    RNF43 is a recently discovered RING finger protein that is implicated in colon cancer pathogenesis. This protein possesses growth-promoting activity but its mechanism remains unknown. In this study, to gain insight into the biological action of RNF43 we characterized it biochemically and intracellularly. A combination of indirect immunofluorescence analysis and biochemical fractionation experiments suggests that RNF43 resides in the endoplasmic reticulum (ER) as well as in the nuclear envelope. Sucrose density gradient fractionation demonstrates that RNF43 co-exists with emerin, a representative inner nuclear membrane protein in the nuclear subcompartment. The cell-free system with pure components reveals that recombinant RNF43 fused with maltose-binding protein has autoubiquitylation activity. By the yeast two-hybrid screening we identified HAP95, a chromatin-associated protein interfacing the nuclear envelope, as an RNF43-interacting protein and substantiated this interaction in intact cells by the co-immunoprecipitation experiments. HAP95 is ubiquitylated and subjected to a proteasome-dependent degradation pathway, however, the experiments in which 293 cells expressing both RNF43 and HAP95 were treated with a proteasome inhibitor, MG132, show that HAP95 is unlikely to serve as a substrate of RNF43 ubiquitin ligase. These results infer that RNF43 is a resident protein of the ER and, at least partially, the nuclear membrane, with ubiquitin ligase activity and may be involved in cell growth control potentially through the interaction with HAP95.

  19. A forward genetic approach in Arabidopsis thaliana identifies a RING-type ubiquitin ligase as a novel determinant of seed longevity.

    PubMed

    Bueso, Eduardo; Ibañez, Carla; Sayas, Enric; Muñoz-Bertomeu, Jesús; Gonzalez-Guzmán, Miguel; Rodriguez, Pedro L; Serrano, Ramón

    2014-02-01

    Seed longevity is important to preserve crops and wild plants and it is limited by progressive cellular damage (aging) during storage. The induction of cellular stress defenses and the formation of the seed coat are crucial protecting events during seed development, a process mediated in Arabidopsis thaliana by the transcription factors LEC1, LEC2, FUS3 and the abscisic acid-activated ABI3. In order to identify novel determinants of seed longevity we have screened an activation-tagging mutant collection of Arabidopsis and isolated a dominant mutant with increased seed longevity under both natural and accelerated aging conditions. Molecular characterization indicates that the mutant phenotype is caused by over-expression of the At2g26130 gene encoding a RING-type zinc finger putative ubiquitin ligase. Loss of function of this gene in a T-DNA insertion mutant resulted in decreased seed longevity. We named this important gene for seed longevity RSL1 (from Ring finger of Seed Longevity1) and we could demonstrate ubiquitin ligase activity with the recombinant protein. Morphological alterations in shoot tissues of the RSL1 over-expressing plants and analysis of gibberellins levels suggest that RSL1 may increase gibberellins responses by some unknown mechanism. These results validate the forward genetic approach to seed longevity and anticipate the identification of many novel determinants of this important trait.

  20. A Large Complement of the Predicted Arabidopsis ARM Repeat Proteins Are Members of the U-Box E3 Ubiquitin Ligase Family1[w

    PubMed Central

    Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L.; Salt, Jennifer N.; Goring, Daphne R.

    2004-01-01

    The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis. PMID:14657406

  1. Control of the dynamics and homeostasis of the Drosophila Hedgehog receptor Patched by two C2-WW-HECT-E3 Ubiquitin ligases

    PubMed Central

    Brigui, Amira; Hofmann, Line; Argüelles, Camilla; Sanial, Matthieu; Holmgren, Robert A.; Plessis, Anne

    2015-01-01

    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. PMID:26446620

  2. Control of the dynamics and homeostasis of the Drosophila Hedgehog receptor Patched by two C2-WW-HECT-E3 Ubiquitin ligases.

    PubMed

    Brigui, Amira; Hofmann, Line; Argüelles, Camilla; Sanial, Matthieu; Holmgren, Robert A; Plessis, Anne

    2015-10-01

    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.

  3. Core Binding Factor Beta Plays a Critical Role by Facilitating the Assembly of the Vif-Cullin 5 E3 Ubiquitin Ligase

    PubMed Central

    Fribourgh, Jennifer L.; Nguyen, Henry C.; Wolfe, Leslie S.; DeWitt, David C.; Zhang, Wenyan; Yu, Xiao-Fang; Rhoades, Elizabeth

    2014-01-01

    ABSTRACT The HIV-1 virion infectivity factor (Vif) targets the cellular cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) for degradation via the host ubiquitin-proteasome pathway. Vif recruits a cellular E3 ubiquitin ligase to polyubiquitinate A3G/F. The activity of Vif critically depends on the cellular core binding factor beta (CBFβ). In this study, we investigated the Vif-CBFβ interaction and the role of CBFβ in the E3 ligase assembly. Vif-CBFβ interaction requires an extensive region of Vif spanning most of its amino terminus and zinc finger region, and cullin 5 (Cul5) binding enhances the stability of the Vif-CBFβ interaction. Our results further demonstrate that CBFβ plays a critical role in facilitating Cul5 binding to the Vif/elongin B/elongin C complex. Vif, with or without bound substrate, is unable to bind Cul5 in the absence of CBFβ. These studies support the notion that CBFβ serves as a molecular chaperone to facilitate Vif-E3 ligase assembly. IMPORTANCE The host antiviral restriction factors A3G/F inhibit viral replication. The HIV-1 protein Vif targets A3G/F for degradation. This immune evasion activity of Vif is dependent on the cellular factor CBFβ. Multiple regions of Vif are known to be important for Vif function, but the mechanisms are unclear. The studies described here provide important information about the Vif-CBFβ interaction interface and the function of CBFβ in E3 ligase assembly. In particular, our comprehensive Vif-CBFβ interface mapping results help to delineate the role of various Vif regions, determining if they are important for binding CBFβ or A3G/F. Furthermore, our studies reveal an important potential mechanism of CBFβ that has not been shown before. Our results suggest that CBFβ may serve as a molecular chaperone to enable Vif to adopt an appropriate conformation for interaction with the Cul5-based E3 ligase. This study advances our understanding of how CBFβ facilitates the Vif-mediated degradation of

  4. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity

    PubMed Central

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G. Eric; Dobner, Thomas; Branton, Philip E.

    2015-01-01

    ABSTRACT Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new

  5. Spliceosomal introns in the 5′ untranslated region of plant BTL RING-H2 ubiquitin ligases are evolutionary conserved and required for gene expression

    PubMed Central

    2013-01-01

    Background Introns located close to the 5′ end of a gene or in the 5′ untranslated region often exert positive effects on gene expression. This effect, known as intron-mediated enhancement (IME), has been observed in diverse eukaryotic organisms, including plants. The sequences involved in IME seem to be spread across the intron and function in an additive manner. The IMEter algorithm was developed to predict plant introns that may enhance gene expression. We have identified several plant members of the BTL class of E3s, which may have orthologs across eukaryotes, that contain a 5′UTR intron. The RING finger E3 ligases are key enzymes of the ubiquitination system that mediate the transfer of ubiquitin to substrates. Results In this study, we retrieved BTL sequences from several angiosperm species and found that 5′UTR introns showing a strong IMEter score were predicted, suggesting that they may be conserved by lineage. Promoter-GUS fusion lines were used to confirm the IME effect of these 5′UTR introns on gene expression. IMEter scores of BTLs were compared with the 5′UTR introns of two gene families MHX and polyubiquitin genes. Conclusions Analysis performed in two Arabidopsis BTL E3 ligases genes indicated that the 5′UTR introns were essential for gene expression in all the tissues tested. Comparison of the average 5′UTR intron size on three gene families in ten angiosperm species suggests that a prevalent size for a 5′UTR intron is in the range of 600 nucleotides, and that the overall IMEter score within a gene family is preserved across several angiosperms. Our results indicated that gene expression dependent on a 5′UTR intron is an efficient regulatory mechanism in BTL E3 ligases that has been preserved throughout plant evolution. PMID:24228887

  6. Cloning and expression analysis of the mitochondrial ubiquitin ligase activator of NF-κB (MULAN) in Atlantic salmon (Salmo salar).

    PubMed

    Tacchi, Luca; Casadei, Elisa; Bickerdike, Ralph; Secombes, Christopher J; Martin, Samuel A M

    2011-12-01

    Nuclear factor-κB (NF-κB) is a transcription factor involved in the regulation of a large number of genes including many involved in bacterial and viral infections. NF-κB is normally sequestered by inhibitory proteins (IκBs) in the cytoplasm of non-stimulated cells. The degradation of IκBs by the ubiquitin proteasome pathway leads to the rapid translocation of NF-κB to the nucleous where it regulates gene transcription. The Mitochondrial Ubiquitin Ligase Activator of NF-κB, (MULAN), is an E3 ubiquitin ligase believed to be central in controlling activation of NF-κB, and regulating the mitochondrial dynamics and apoptosis process. We report, for the first time in fish, the characterization of a MULAN cDNA in Atlantic salmon and rainbow trout. The salmonid MULAN sequences encode predicted proteins of 352 amino acids. The mRNA of MULAN was expressed in multiple tissues, with the highest abundance in brain and white muscle. An Aeromonas salmonicida bacterial challenge increased expression of this gene in head kidney, liver and gill both at 6 and at 24h following the infection. In vitro experiments using the salmonid cell line RTG-2 indicated MULAN was increased in expression following 4h stimulation with LPS and recombinant trout IL-1β. MULAN expression remained increased 24h post-stimulation with both LPS and IL-1β, but was down regulated by PolyI:C at this time. These results suggest an active role of the MULAN gene in the activation of the NF-κB pathway during piscine immune responses.

  7. MULAN related gene (MRG): a potential novel ubiquitin ligase activator of NF-kB involved in immune response in Atlantic salmon (Salmo salar).

    PubMed

    Tacchi, Luca; Casadei, Elisa; Bickerdike, Ralph; Secombes, Christopher J; Martin, Samuel A M

    2012-12-01

    Nuclear factor-kB (NF-kB) is a transcription factor that plays a central role in the regulation of a variety of genes including many involved in bacterial and viral infections. NF-kB is normally sequestered by inhibitory proteins (IkBs) in the cytoplasm of non-stimulated cells. The degradation of IkBs by the ubiquitin proteasome pathway releases NF-kB allowing its translocation to the nucleus where it regulates gene transcription. The Mitochondrial Ubiquitin Ligase Activator of NF-kB, (MULAN), is an E3 ubiquitin ligase involved in controlling activation of NF-kB, and regulating mitochondrial dynamics and apoptosis. We report the characterisation of a novel piscine-specific MULAN related gene (MRG) sequence, its mRNA tissue distribution and expression following in vivo and in vitro challenges. MRG cDNA was identified in Atlantic salmon and its sequence encodes a predicted protein of 274 amino acids. The mRNA of MRG was expressed in multiple tissues, with the highest abundance head kidney. An Aeromonas salmonicida bacterial challenge increased expression of this gene in head kidney, liver and gill tissue at 6 h and 24 h. In vitro stimulation of a salmonid cell line indicated MRG was increased in expression following stimulation with LPS, PolyI:C and recombinant trout IL-1β for 4 h and 24 h. These results suggest an active role of MRG in the activation of the NF-kB pathway during early immune responses.

  8. Peroxisomal ubiquitin-protein ligases peroxin2 and peroxin10 have distinct but synergistic roles in matrix protein import and peroxin5 retrotranslocation in Arabidopsis.

    PubMed

    Burkhart, Sarah E; Kao, Yun-Ting; Bartel, Bonnie

    2014-11-01

    Peroxisomal matrix proteins carry peroxisomal targeting signals (PTSs), PTS1 or PTS2, and are imported into the organelle with the assistance of peroxin (PEX) proteins. From a microscopy-based screen to identify Arabidopsis (Arabidopsis thaliana) mutants defective in matrix protein degradation, we isolated unique mutations in PEX2 and PEX10, which encode ubiquitin-protein ligases anchored in the peroxisomal membrane. In yeast (Saccharomyces cerevisiae), PEX2, PEX10, and a third ligase, PEX12, ubiquitinate a peroxisome matrix protein receptor, PEX5, allowing the PEX1 and PEX6 ATP-hydrolyzing enzymes to retrotranslocate PEX5 out of the membrane after cargo delivery. We found that the pex2-1 and pex10-2 Arabidopsis mutants exhibited defects in peroxisomal physiology and matrix protein import. Moreover, the pex2-1 pex10-2 double mutant exhibited severely impaired growth and synergistic physiological defects, suggesting that PEX2 and PEX10 function cooperatively in the wild type. The pex2-1 lesion restored the unusually low PEX5 levels in the pex6-1 mutant, implicating PEX2 in PEX5 degradation when retrotranslocation is impaired. PEX5 overexpression altered pex10-2 but not pex2-1 defects, suggesting that PEX10 facilitates PEX5 retrotranslocation from the peroxisomal membrane. Although the pex2-1 pex10-2 double mutant displayed severe import defects of both PTS1 and PTS2 proteins into peroxisomes, both pex2-1 and pex10-2 single mutants exhibited clear import defects of PTS1 proteins but apparently normal PTS2 import. A similar PTS1-specific pattern was observed in the pex4-1 ubiquitin-conjugating enzyme mutant. Our results indicate that Arabidopsis PEX2 and PEX10 cooperate to support import of matrix proteins into plant peroxisomes and suggest that some PTS2 import can still occur when PEX5 retrotranslocation is slowed.

  9. Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 Is Critical for Plant Carbon/Nitrogen Nutrient Balance Response and Controls the Stability of 14-3-3 Proteins*

    PubMed Central

    Yasuda, Shigetaka; Sato, Takeo; Maekawa, Shugo; Aoyama, Shoki; Fukao, Yoichiro; Yamaguchi, Junji

    2014-01-01

    Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr209, Ser247, Ser270, and Ser303 as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr209 and Ser247 on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr209 peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response. PMID:24722992

  10. Differential response between the p53 ubiquitin-protein ligases Pirh2 and MdM2 following DNA damage in human cancer cells

    SciTech Connect

    Duan Wenrui; Gao, Li; Wu Xin; Zhang Yang; Otterson, Gregory A.; Villalona-Calero, Miguel A. . E-mail: Miguel.villalona@osumc.edu

    2006-10-15

    Pirh2, a recently identified ubiquitin-protein ligase, has been reported to promote p53 degradation. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of MDM2. Like MDM2, Pirh2 is thought to participate in an autoregulatory feedback loop that controls p53 function. We have previously reported that Pirh2 was overexpressed in human and murine lung cancers as compared to uninvolved lung tissue. Pirh2 increase could potentially cause degradation of wildtype p53 and reduce its tumor suppression function in the lung tumor cells. Since Pirh2 has been reported to be transactivated by p53, however, the mechanisms by which a high level of Pirh2 expression is maintained in tumor cells despite low level of wildtype p53 protein are unclear. In order to evaluate p53 involvement in the transactivation of Pirh2, we evaluated Pirh2, MDM2, p53 and p21 expression with Western blot analysis and real time PCR after {gamma} irradiation or cisplatin DNA damage treatment using human cancer cell lines containing wildtype (A549, MCF-7), mutant (H719) and null (H1299) p53. Surprisingly, Pirh2 expression was not affected by the presence of wildtype p53 in the cancer cells. In contrast, MDM2 was upregulated by wildtype p53 in A549 and MCF-7 cells and was absent from the H1299 and the H719 cells. We conclude that Pirh2 operates in a distinct manner from MDM2 in response to DNA damage in cancer cells. Pirh2 elevation in p53 null cells indicates the existence of additional molecular mechanisms for Pirh2 upregulation and suggests that p53 is not the sole target of Pirh2 ubiquitin ligase activity.

  11. Ubiquitination by the membrane-associated RING-CH-8 (MARCH-8) ligase controls steady-state cell surface expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptor 1.

    PubMed

    van de Kooij, Bert; Verbrugge, Inge; de Vries, Evert; Gijsen, Merel; Montserrat, Veronica; Maas, Chiel; Neefjes, Jacques; Borst, Jannie

    2013-03-01

    The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.

  12. Heterologous expression and molecular and cellular characterization of CaPUB1 encoding a hot pepper U-Box E3 ubiquitin ligase homolog.

    PubMed

    Cho, Seok Keun; Chung, Hoo Sun; Ryu, Moon Young; Park, Mi Jin; Lee, Myeong Min; Bahk, Young-Yil; Kim, Jungmook; Pai, Hyun Sook; Kim, Woo Taek

    2006-12-01

    The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35S::CaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35SCaPUB1 seedlings

  13. Structural basis of the Cks1-dependent recognition of p27(Kip1) by the SCF(Skp2) ubiquitin ligase

    SciTech Connect

    Hao, B.; Zheng, N.; Schulman, B.A.; Wu, G.; Miller, J.J.; Pagano, M.; Pavletich, N.P.

    2010-07-19

    The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27{sup Kip1} plays a central role in cell cycle progression, and enhanced degradation of p27{sup Kip1} is associated with many common cancers. Proteolysis of p27{sup Kip1} is triggered by Thr187 phosphorylation, which leads to the binding of the SCF{sup Skp2} (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27{sup Kip1} ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27{sup Kip1} phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27{sup Kip1} binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27{sup Kip1} is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27{sup Kip1} to the SCF{sup Skp2}-Cks1 complex.

  14. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1

    PubMed Central

    Dong, Chun-Hai; Agarwal, Manu; Zhang, Yiyue; Xie, Qi; Zhu, Jian-Kang

    2006-01-01

    Plant responses to cold stress are mediated by a transcriptional cascade, in which the transcription factor ICE1 and possibly related proteins activate the expression of C-repeat (CRT)-binding factors (CBFs), leading to the transcription of downstream effector genes. The variant RING finger protein high expression of osmotically responsive gene (HOS)1 was identified genetically as a negative regulator of cold responses. We present evidence here that HOS1 is an E3 ligase required for the ubiquitination of ICE1. HOS1 physically interacts with ICE1 and mediates the ubiquitination of ICE1 both in vitro and in vivo. We found that cold induces the degradation of ICE1 in plants, and this degradation requires HOS1. Consistent with enhanced cold-responsive gene expression in loss-of-function hos1 mutant plants, overexpression of HOS1 represses the expression of CBFs and their downstream genes and confers increased sensitivity to freezing stress. Our results indicate that cold stress responses in Arabidopsis are attenuated by a ubiquitination/proteasome pathway in which HOS1 mediates the degradation of the ICE1 protein. PMID:16702557

  15. The Mid2 X-linked Intellectual Disability Ubiquitin Ligase Associates with Astrin and Regulates Astrin Levels to Promote Cell Division

    PubMed Central

    Gholkar, Ankur A.; Senese, Silvia; Lo, Yu-Chen; Vides, Edmundo; Contreras, Ely; Hodara, Emmanuelle; Capri, Joseph; Whitelegge, Julian P.; Torres, Jorge Z.

    2015-01-01

    SUMMARY Mid1 and Mid2 are ubiquitin ligases that regulate microtubule dynamics and whose mutation is associated with X-linked developmental disorders. We show that Astrin, a microtubule-organizing protein, co-purifies with Mid1 and Mid2, has an overlapping localization with Mid1 and Mid2 at intercellular bridge microtubules, is ubiquitinated by Mid2 on lysine 409 and is degraded during cytokinesis. Mid2 depletion led to Astrin stabilization during cytokinesis, cytokinetic defects, multinucleated cells, and cell death. Similarly, expression of a K409A mutant Astrin in Astrin-depleted cells led to the accumulation of K409A on intercellular bridge microtubules and an increase in cytokinetic defects, multinucleated cells, and cell death. These results indicate that Mid2 regulates cell division through the ubiquitination of Astrin on K409, which is critical for its degradation and proper cytokinesis. These results may help explain how mutation of MID2 leads to misregulation of microtubule organization and the downstream disease pathology associated with X-linked intellectual disabilities. PMID:26748699

  16. A genetic screen for neurite outgrowth mutants in Caenorhabditis elegans reveals a new function for the F-box ubiquitin ligase component LIN-23.

    PubMed Central

    Mehta, Nehal; Loria, Paula M; Hobert, Oliver

    2004-01-01

    Axon pathfinding and target recognition are highly dynamic and tightly regulated cellular processes. One of the mechanisms involved in regulating protein activity levels during axonal and synaptic development is protein ubiquitination. We describe here the isolation of several Caenorhabditis elegans mutants, termed eno (ectopic/erratic neurite outgrowth) mutants, that display defects in axon outgrowth of specific neuron classes. One retrieved mutant is characterized by abnormal termination of axon outgrowth in a subset of several distinct neuron classes, including ventral nerve cord motor neurons, head motor neurons, and mechanosensory neurons. This mutant is allelic to lin-23, which codes for an F-box-containing component of an SCF E3 ubiquitin ligase complex that was previously shown to negatively regulate postembryonic cell divisions. We demonstrate that LIN-23 is a broadly expressed cytoplasmically localized protein that is required autonomously in neurons to affect axon outgrowth. Our newly isolated allele of lin-23, a point mutation in the C-terminal tail of the protein, displays axonal outgrowth defects similar to those observed in null alleles of this gene, but does not display defects in cell cycle regulation. We have thus defined separable activities of LIN-23 in two distinct processes, cell cycle control and axon patterning. We propose that LIN-23 targets distinct substrates for ubiquitination within each process. PMID:15082545

  17. Large-Scale, Lineage-Specific Expansion of a Bric-a-Brac/Tramtrack/Broad Complex Ubiquitin-Ligase Gene Family in Rice[W

    PubMed Central

    Gingerich, Derek J.; Hanada, Kousuke; Shiu, Shin-Han; Vierstra, Richard D.

    2007-01-01

    Selective ubiquitination of proteins is directed by diverse families of ubiquitin-protein ligases (or E3s) in plants. One important type uses Cullin-3 as a scaffold to assemble multisubunit E3 complexes containing one of a multitude of bric-a-brac/tramtrack/broad complex (BTB) proteins that function as substrate recognition factors. We previously described the 80-member BTB gene superfamily in Arabidopsis thaliana. Here, we describe the complete BTB superfamily in rice (Oryza sativa spp japonica cv Nipponbare) that contains 149 BTB domain–encoding genes and 43 putative pseudogenes. Amino acid sequence comparisons of the rice and Arabidopsis superfamilies revealed a near equal repertoire of putative substrate recognition module types. However, phylogenetic comparisons detected numerous gene duplication and/or loss events since the rice and Arabidopsis BTB lineages split, suggesting possible functional specialization within individual BTB families. In particular, a major expansion and diversification of a subset of BTB proteins containing Meprin and TRAF homology (MATH) substrate recognition sites was evident in rice and other monocots that likely occurred following the monocot/dicot split. The MATH domain of a subset appears to have evolved significantly faster than those in a smaller core subset that predates flowering plants, suggesting that the substrate recognition module in many monocot MATH-BTB E3s are diversifying to ubiquitinate a set of substrates that are themselves rapidly changing. Intriguing possibilities include pathogen proteins attempting to avoid inactivation by the monocot host. PMID:17720868

  18. Cancer stem-like cell related protein CD166 degrades through E3 ubiquitin ligase CHIP in head and neck cancer.

    PubMed

    Xiao, Meng; Yan, Ming; Zhang, Jianjun; Xu, Qin; Qi, Shengcai; Wang, Xu; Chen, Wantao

    2017-04-01

    Our previous studies have identified that CD166 works as a cancer stem-like cell (CSC) marker in epithelial cancers with a large repertoire of cellular functions. However, the post-translational regulatory mechanisms underlying CD166 turnover remain elusive. Several independent studies have reported that E3 ubiquitin ligase CHIP revealed significant biological effects through ubiquitin proteasome pathway on some kinds of malignant tumors. With analyzing the effects of CHIP expressions on stem-like cell populations, we found that CHIP represses CSC characteristics mainly targeting the CSC related protein CD166 in head and neck cancer (HNC). To investigate the role and relationship between CD166 and CHIP, HNC tissues and cell lines were used in this study. A significant negative correlation was observed between the expression levels of CHIP and CD166 in HNC patient samples. We also found that CHIP directly regulates the stability of CD166 protein through the ubiquitin proteasome system, which was also identified participating in the regulation of CSC behaviors in HNCs. Our findings demonstrate that CHIP-CD166-proteasome axis participates in regulating CSC properties in HNCs, suggesting that the regulation of CD166 by CHIP could provide new options for diagnosing and treating in the patients with HNCs.

  19. Neuregulin-induced ErbB3 downregulation is mediated by a protein stability cascade involving the E3 ubiquitin ligase Nrdp1.

    PubMed

    Cao, Zhongwei; Wu, Xiuli; Yen, Lily; Sweeney, Colleen; Carraway, Kermit L

    2007-03-01

    The molecular mechanisms underlying epidermal growth factor (EGF) receptor tyrosine kinase down-regulation in response to growth factor binding are coming into focus and involve cbl-mediated receptor ubiquitination followed by lysosomal degradation. However, mechanisms underlying the ligand-stimulated degradation of the related receptor tyrosine kinases of the ErbB family do not involve cbl and remain unexplored. Previous studies have demonstrated that the E3 ubiquitin ligase Nrdp1 contributes to the maintenance of steady-state ErbB3 levels by mediating its growth factor-independent degradation. Here we demonstrate that treatment of cells with the ErbB3 ligand neuregulin-1 (NRG1) stabilizes the deubiquitinating enzyme USP8, which in turn stabilizes Nrdp1. The catalytic activity of USP8 is required for NRG1-induced Nrdp1 stabilization. We provide evidence that Akt-mediated phosphorylation of USP8 threonine residue T907 contributes to USP8 stability. Finally, we demonstrate that Nrdp1 or USP8 knockdown suppresses NRG1-induced ErbB3 ubiquitination and degradation in MCF7 breast cancer cells. We conclude that an NRG1-induced protein stability cascade involving USP8 and Nrdp1 mediates the down-regulation of ErbB3. Our observations raise the possibility that the ligand-induced augmentation of pathways involved in the maintenance of basal levels of receptor tyrosine kinases can contribute to ligand-stimulated down-regulation.

  20. Neuregulin-Induced ErbB3 Downregulation Is Mediated by a Protein Stability Cascade Involving the E3 Ubiquitin Ligase Nrdp1▿

    PubMed Central

    Cao, Zhongwei; Wu, Xiuli; Yen, Lily; Sweeney, Colleen; Carraway, Kermit L.

    2007-01-01

    The molecular mechanisms underlying epidermal growth factor (EGF) receptor tyrosine kinase down-regulation in response to growth factor binding are coming into focus and involve cbl-mediated receptor ubiquitination followed by lysosomal degradation. However, mechanisms underlying the ligand-stimulated degradation of the related receptor tyrosine kinases of the ErbB family do not involve cbl and remain unexplored. Previous studies have demonstrated that the E3 ubiquitin ligase Nrdp1 contributes to the maintenance of steady-state ErbB3 levels by mediating its growth factor-independent degradation. Here we demonstrate that treatment of cells with the ErbB3 ligand neuregulin-1 (NRG1) stabilizes the deubiquitinating enzyme USP8, which in turn stabilizes Nrdp1. The catalytic activity of USP8 is required for NRG1-induced Nrdp1 stabilization. We provide evidence that Akt-mediated phosphorylation of USP8 threonine residue T907 contributes to USP8 stability. Finally, we demonstrate that Nrdp1 or USP8 knockdown suppresses NRG1-induced ErbB3 ubiquitination and degradation in MCF7 breast cancer cells. We conclude that an NRG1-induced protein stability cascade involving USP8 and Nrdp1 mediates the down-regulation of ErbB3. Our observations raise the possibility that the ligand-induced augmentation of pathways involved in the maintenance of basal levels of receptor tyrosine kinases can contribute to ligand-stimulated down-regulation. PMID:17210635

  1. Targeted Disruption of Drosophila Roc1b Reveals Functional Differences in the Roc Subunit of Cullin-dependent E3 Ubiquitin Ligases

    PubMed Central

    Donaldson, Timothy D.; Noureddine, Maher A.; Reynolds, Patrick J.; Bradford, William; Duronio, Robert J.

    2004-01-01

    Cullin-dependent ubiquitin ligases regulate a variety of cellular and developmental processes by recruiting specific proteins for ubiquitin-mediated degradation. Cullin proteins form a scaffold for two functional modules: a catalytic module comprised of a small RING domain protein Roc1/Rbx1 and a ubiquitin-conjugating enzyme (E2), and a substrate recruitment module containing one or more proteins that bind to and bring the substrate in proximity to the catalytic module. Here, we present evidence that the three Drosophila Roc proteins are not functionally equivalent. Mutation of Roc1a causes lethality that cannot be rescued by expression of Roc1b or Roc2 by using the Roc1a promoter. Roc1a mutant cells hyperaccumulate Cubitus interruptus, a transcription factor that mediates Hedgehog signaling. This phenotype is not rescued by expression of Roc2 and only partially by expression of Roc1b. Targeted disruption of Roc1b causes male sterility that is partially rescued by expression of Roc1a by using the Roc1b promoter, but not by similar expression of Roc2. These data indicate that Roc proteins play nonredundant roles during development. Coimmunoprecipitation followed by Western or mass spectrometric analysis indicate that the three Roc proteins preferentially bind certain Cullins, providing a possible explanation for the distinct biological activities of each Drosophila Roc/Rbx. PMID:15331761

  2. The polyglutamine-expanded androgen receptor responsible for spinal and bulbar muscular atrophy inhibits the APC/CCdh1 ubiquitin ligase complex

    PubMed Central

    Bott, Laura C.; Salomons, Florian A.; Maric, Dragan; Liu, Yuhong; Merry, Diane; Fischbeck, Kenneth H.; Dantuma, Nico P.

    2016-01-01

    Polyglutamine expansion in the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA), an X-linked neuromuscular disease that is fully manifest only in males. It has been suggested that proteins with expanded polyglutamine tracts impair ubiquitin-dependent proteolysis due to their propensity to aggregate, but recent studies indicate that the overall activity of the ubiquitin-proteasome system is preserved in SBMA models. Here we report that AR selectively interferes with the function of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), which, together with its substrate adaptor Cdh1, is critical for cell cycle arrest and neuronal architecture. We show that both wild-type and mutant AR physically interact with the APC/CCdh1 complex in a ligand-dependent fashion without being targeted for proteasomal degradation. Inhibition of APC/CCdh1 by mutant but not wild-type AR in PC12 cells results in enhanced neurite outgrowth which is typically followed by rapid neurite retraction and mitotic entry. Our data indicate a role of AR in neuronal differentiation through regulation of APC/CCdh1 and suggest abnormal cell cycle reactivation as a pathogenic mechanism in SBMA. PMID:27312068

  3. Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein

    PubMed Central

    Xu, Shan; Cherok, Edward; Das, Shweta; Li, Sunan; Roelofs, Brian A.; Ge, Shealinna X.; Polster, Brian M.; Boyman, Liron; Lederer, W. Jonathan; Wang, Chunxin; Karbowski, Mariusz

    2016-01-01

    Ubiquitin- and proteasome-dependent outer mitochondrial membrane (OMM)-associated degradation (OMMAD) is critical for mitochondrial and cellular homeostasis. However, the scope and molecular mechanisms of the OMMAD pathways are still not well understood. We report that the OMM-associated E3 ubiquitin ligase MARCH5 controls dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and cell sensitivity to stress-induced apoptosis. MARCH5 knockout selectively inhibited ubiquitination and proteasomal degradation of MiD49, a mitochondrial receptor of Drp1, and consequently led to mitochondrial fragmentation. Mitochondrial fragmentation in MARCH5−/− cells was not associated with inhibition of mitochondrial fusion or bioenergetic defects, supporting the possibility that MARCH5 is a negative regulator of mitochondrial fission. Both MARCH5 re-expression and MiD49 knockout in MARCH5−/− cells reversed mitochondrial fragmentation and reduced sensitivity to stress-induced apoptosis. These findings and data showing MARCH5-dependent degradation of MiD49 upon stress support the possibility that MARCH5 regulation of MiD49 is a novel mechanism controlling mitochondrial fission and, consequently, the cellular response to stress. PMID:26564796

  4. E3 ubiquitin ligase gene CMPG1-V from Haynaldia villosa L. contributes to powdery mildew resistance in common wheat (Triticum aestivum L.).

    PubMed

    Zhu, Yanfei; Li, Yingbo; Fei, Fei; Wang, Zongkuan; Wang, Wei; Cao, Aizhong; Liu, Yuan; Han, Shuang; Xing, Liping; Wang, Haiyan; Chen, Wei; Tang, Sanyuan; Huang, Xiahe; Shen, Qianhua; Xie, Qi; Wang, Xiue

    2015-10-01

    Powdery mildew is one of the most devastating wheat fungal diseases. A diploid wheat relative, Haynaldia villosa L., is highly resistant to powdery mildew, and its genetic resource of resistances, such as the Pm21 locus, is now widely used in wheat breeding. Here we report the cloning of a resistance gene from H. villosa, designated CMPG1-V, that encodes a U-box E3 ubiquitin ligase. Expression of the CMPG1-V gene was induced in the leaf and stem of H. villosa upon inoculation with Blumeria graminis f. sp. tritici (Bgt) fungus, and the presence of Pm21 is essential for its rapid induction of expression. CMPG1-V has conserved key residues for E3 ligase, and possesses E3 ligase activity in vitro and in vivo. CMPG1-V is localized in the nucleus, endoplasmic reticulum, plasma membrane and partially in trans-Golgi network/early endosome vesicles. Transgenic wheat over-expressing CMPG1-V showed improved broad-spectrum powdery mildew resistance at seedling and adult stages, associated with an increase in expression of salicylic acid-responsive genes, H2 O2 accumulation, and cell-wall protein cross-linking at the Bgt infection sites, and the expression of CMPG1-V in H. villosa was increased when treated with salicylic acid, abscisic acid and H2 O2 . These results indicate the involvement of E3 ligase in defense responses to Bgt fungus in wheat, particularly in broad-spectrum disease resistance, and suggest association of reactive oxidative species and the phytohormone pathway with CMPG1-V-mediated powdery mildew resistance.

  5. Quality Control of Plasma Membrane Proteins by Saccharomyces cerevisiae Nedd4-Like Ubiquitin Ligase Rsp5p under Environmental Stress Conditions

    PubMed Central

    Shiga, Takeki; Yoshida, Nobuyuki; Shimizu, Yuko; Suzuki, Etsuko; Sasaki, Toshiya; Watanabe, Daisuke

    2014-01-01

    In Saccharomyces cerevisiae, when a rich nitrogen source such as ammonium is added to the culture medium, the general amino acid permease Gap1p is ubiquitinated by the yeast Nedd4-like ubiquitin ligase Rsp5p, followed by its endocytosis to the vacuole. The arrestin-like Bul1/2p adaptors for Rsp5p specifically mediate this process. In this study, to investigate the downregulation of Gap1p in response to environmental stresses, we determined the intracellular trafficking of Gap1p under various stress conditions. An increase in the extracellular ethanol concentration induced ubiquitination and trafficking of Gap1p from the plasma membrane to the vacuole in wild-type cells, whereas Gap1p remained stable on the plasma membrane under the same conditions in rsp5A401E and Δend3 cells. A 14C-labeled citrulline uptake assay using a nonubiquitinated form of Gap1p (Gap1pK9R/K16R) revealed that ethanol stress caused a dramatic decrease of Gap1p activity. These results suggest that Gap1p is inactivated and ubiquitinated by Rsp5p for endocytosis when S. cerevisiae cells are exposed to a high concentration of ethanol. It is noteworthy that this endocytosis occurs in a Bul1/2p-independent manner, whereas ammonium-triggered downregulation of Gap1p was almost completely inhibited in Δbul1/2 cells. We also found that other environmental stresses, such as high temperature, H2O2, and LiCl, also promoted endocytosis of Gap1p. Similar intracellular trafficking caused by ethanol occurred in other plasma membrane proteins (Agp1p, Tat2p, and Gnp1p). Our findings suggest that stress-induced quality control is a common process requiring Rsp5p for plasma membrane proteins in yeast. PMID:25001409

  6. PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

    PubMed Central

    Zhang, Yong; Mao, Dailing; Roswit, William T.; Jin, Xiaohua; Patel, Anand C.; Patel, Dhara A.; Agapov, Eugene; Wang, Zhepeng; Tidwell, Rose M.; Atkinson, Jeffrey J.; Huang, Guangming; McCarthy, Ronald; Yu, Jinsheng; Yun, Nadezhda E.; Paessler, Slobodan; Lawson, T. Glen; Omattage, Natalie S.; Brett, Tom J.; Holtzman, Michael J.

    2015-01-01

    Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve interferon hyperresponsiveness without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depends on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acts on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to initiate their degradation via the immunoproteasome. Together, PARP9-DTX3L acts on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway. PMID:26479788

  7. Neuronal expression of the ubiquitin E3 ligase APC/C-Cdh1 during development is required for long-term potentiation, behavioral flexibility, and extinction.

    PubMed

    Pick, Joseph E; Wang, Li; Mayfield, Joshua E; Klann, Eric

    2013-02-01

    Cdh1 is a regulatory subunit of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin E3 ligase known to be involved in regulating cell cycle progression. Recent studies have demonstrated a role for Cdh1 in neurons during developmental and adult synaptic plasticity, as well as memory. In order to better characterize the contribution of Cdh1 in synaptic plasticity and memory, we generated conditional knockout mice using a neuron-specific enolase (Nse) promoter where Cdh1 was eliminated in neurons from the onset of differentiation. Although we detected impaired long-term potentiation (LTP) in hippocampal slices from the Nse-Cdh1 knockout (KO) mice, performance on several hippocampus-dependent memory tasks remained intact. However, the Nse-Cdh1 KO mice exhibited impaired behavioral flexibility and extinction of previously consolidated memories. These findings suggest a role for Cdh1 in regulating the updating of consolidated memories.

  8. p38 MAP kinase-dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER-mitochondria association and mitochondria motility.

    PubMed

    Li, Lei; Gao, Guang; Shankar, Jay; Joshi, Bharat; Foster, Leonard J; Nabi, Ivan R

    2015-11-01

    Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER; however, the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an 8-amino acid motif (533-541) in mouse Gp78 isoform 2 that forms part of a highly conserved 41-amino acid region containing 14-3-3- and WW-binding domains and a p38 MAP kinase (p38 MAPK) consensus site on Ser-538 (S538). 3F3A binds selectively to nonphosphorylated S538 Gp78. Using 3F3A as a reporter, we induced Gp78 S538 phosphorylation by serum starvation and showed it to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fission and degrade MFN1 and MFN2 but did not affect in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER-mitochondria interaction, and SB203580 inhibition of p38 MAPK increased ER-mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER-mitochondria association and mitochondria motility.

  9. HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3) Transcriptional Activity

    PubMed Central

    ZeRuth, Gary T.; Williams, Jason G.; Cole, Yasemin C.; Jetten, Anton M.

    2015-01-01

    The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases. PMID:26147758

  10. Ubiquitin Ligases of the N-End Rule Pathway: Assessment of Mutations in UBR1 That Cause the Johanson-Blizzard Syndrome

    PubMed Central

    Hwang, Cheol-Sang; Sukalo, Maja; Batygin, Olga; Addor, Marie-Claude; Brunner, Han; Aytes, Antonio Perez; Mayerle, Julia; Song, Hyun Kyu; Varshavsky, Alexander; Zenker, Martin

    2011-01-01

    Background Johanson-Blizzard syndrome (JBS; OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, facial dysmorphism with the characteristic nasal wing hypoplasia, multiple malformations, and frequent mental retardation. Our previous work has shown that JBS is caused by mutations in human UBR1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. One class of degradation signals (degrons) recognized by UBR1 are destabilizing N-terminal residues of protein substrates. Methodology/Principal Findings Most JBS-causing alterations of UBR1 are nonsense, frameshift or splice-site mutations that abolish UBR1 activity. We report here missense mutations of human UBR1 in patients with milder variants of JBS. These single-residue changes, including a previously reported missense mutation, involve positions in the RING-H2 and UBR domains of UBR1 that are conserved among eukaryotes. Taking advantage of this conservation, we constructed alleles of the yeast Saccharomyces cerevisiae UBR1 that were counterparts of missense JBS-UBR1 alleles. Among these yeast Ubr1 mutants, one of them (H160R) was inactive in yeast-based activity assays, the other one (Q1224E) had a detectable but weak activity, and the third one (V146L) exhibited a decreased but significant activity, in agreement with manifestations of JBS in the corresponding JBS patients. Conclusions/Significance These results, made possible by modeling defects of a human ubiquitin ligase in its yeast counterpart, verified and confirmed the relevance of specific missense UBR1 alleles to JBS, and suggested that a residual activity of a missense allele is causally associated with milder variants of JBS. PMID:21931868

  11. Iron-induced skeletal muscle atrophy involves an Akt-forkhead box O3-E3 ubiquitin ligase-dependent pathway.

    PubMed

    Ikeda, Yasumasa; Imao, Mizuki; Satoh, Akiho; Watanabe, Hiroaki; Hamano, Hirofumi; Horinouchi, Yuya; Izawa-Ishizawa, Yuki; Kihira, Yoshitaka; Miyamoto, Licht; Ishizawa, Keisuke; Tsuchiya, Koichiro; Tamaki, Toshiaki

    2016-05-01

    Skeletal muscle wasting or sarcopenia is a critical health problem. Skeletal muscle atrophy is induced by an excess of iron, which is an essential trace metal for all living organisms. Excessive amounts of iron catalyze the formation of highly toxic hydroxyl radicals via the Fenton reaction. However, the molecular mechanism of iron-induced skeletal muscle atrophy has remained unclear. In this study, 8-weeks-old C57BL6/J mice were divided into 2 groups: vehicle-treated group and the iron-injected group (10 mg iron day(-1)mouse(-1)) during 2 weeks. Mice in the iron-injected group showed an increase in the iron content of the skeletal muscle and serum and ferritin levels in the muscle, along with reduced skeletal muscle mass. The skeletal muscle showed elevated mRNA expression of the muscle atrophy-related E3 ubiquitin ligases, atrogin-1 and muscle ring finger-1(MuRF1), on days 7 and 14 of iron treatment. Moreover, iron-treated mice showed reduced phosphorylation of Akt and forkhea