WorldWideScience

Sample records for e3 ligase machinery

  1. The E3 Ubiquitin Ligase SCF(Cyclin F) Transmits AKT Signaling to the Cell-Cycle Machinery.

    Science.gov (United States)

    Choudhury, Rajarshi; Bonacci, Thomas; Wang, Xianxi; Truong, Andrew; Arceci, Anthony; Zhang, Yanqiong; Mills, Christine A; Kernan, Jennifer L; Liu, Pengda; Emanuele, Michael J

    2017-09-26

    The oncogenic AKT kinase is a key regulator of apoptosis, cell growth, and cell-cycle progression. Despite its important role in proliferation, it remains largely unknown how AKT is mechanistically linked to the cell cycle. We show here that cyclin F, a substrate receptor F-box protein for the SCF (Skp1/Cul1/F-box) family of E3 ubiquitin ligases, is a bona fide AKT substrate. Cyclin F expression oscillates throughout the cell cycle, a rare feature among the 69 human F-box proteins, and all of its known substrates are involved in proliferation. AKT phosphorylation of cyclin F enhances its stability and promotes assembly into productive E3 ligase complexes. Importantly, expression of mutant versions of cyclin F that cannot be phosphorylated by AKT impair cell-cycle entry. Our data suggest that cyclin F transmits mitogen signaling through AKT to the core cell-cycle machinery. This discovery has potential implications for proliferative control in malignancies where AKT is activated. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  2. RING E3 ligases

    DEFF Research Database (Denmark)

    Cho, Seok Keun; Ryu, Moon Young; Kim, Jong Hum

    2017-01-01

    response pathways of plants through various molecular and genetic studies. In particular, it was recently discovered that ubiquitin proteasome system (UPS), a regulatory mechanism for protein turn over, is greatly involved in the stress responsive pathways. In the UPS, many E3 ligases play key roles...... in recognizing and tethering poly-ubiquitins on target proteins for subsequent degradation by the 26S proteasome. Here we discuss the roles of RING ligases that have been defined in related to abiotic stress responses in plants....

  3. E3 Ubiquitin Ligases Neurobiological Mechanisms: Development to Degeneration

    Directory of Open Access Journals (Sweden)

    Arun Upadhyay

    2017-05-01

    Full Text Available Cells regularly synthesize new proteins to replace old or damaged proteins. Deposition of various aberrant proteins in specific brain regions leads to neurodegeneration and aging. The cellular protein quality control system develop various defense mechanisms against the accumulation of misfolded and aggregated proteins. The mechanisms underlying the selective recognition of specific crucial protein or misfolded proteins are majorly governed by quality control E3 ubiquitin ligases mediated through ubiquitin-proteasome system. Few known E3 ubiquitin ligases have shown prominent neurodevelopmental functions, but their interactions with different developmental proteins play critical roles in neurodevelopmental disorders. Several questions are yet to be understood properly. How E3 ubiquitin ligases determine the specificity and regulate degradation of a particular substrate involved in neuronal proliferation and differentiation is certainly the one, which needs detailed investigations. Another important question is how neurodevelopmental E3 ubiquitin ligases specifically differentiate between their versatile range of substrates and timing of their functional modulations during different phases of development. The premise of this article is to understand how few E3 ubiquitin ligases sense major molecular events, which are crucial for human brain development from its early embryonic stages to throughout adolescence period. A better understanding of these few E3 ubiquitin ligases and their interactions with other potential proteins will provide invaluable insight into disease mechanisms to approach toward therapeutic interventions.

  4. Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases

    National Research Council Canada - National Science Library

    Riley, B E; Lougheed, J C; Callaway, K; Velasquez, M; Brecht, E; Nguyen, L; Shaler, T; Walker, D; Yang, Y; Regnstrom, K; Diep, L; Zhang, Z; Chiou, S; Bova, M; Artis, D R; Yao, N; Baker, J; Yednock, T; Johnston, J A

    2013-01-01

    Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson's disease, cancer and mycobacterial infection...

  5. Cyclin F/FBXO1 interacts with HIV-1 Vif and restricts progeny virion infectivity by ubiquitination and proteasomal degradation of Vif through SCF (Cyclin F) E3 ligase machinery.

    Science.gov (United States)

    Augustine, Tracy; Chaudhary, Priyanka; Gupta, Kailash; Islam, Sehbanul; Ghosh, Payel; Santra, Manas Kumar; Mitra, Debashis

    2017-02-09

    Cyclin F, also known as FBXO1, is the largest among all cyclins which oscillates in the cell cycle like other cyclins. Apart from being a G2/M cyclin, Cyclin F functions as the substrate binding subunit of SCFCyclin F E3 ubiquitin ligase. In a gene expression analysis performed to identify novel gene modulations associated with cell cycle dysregulation during HIV-1 infection in CD4+ T cells, we observed down-regulation of Cyclin F (CCNF) gene. Later, using gene over expression and knockdown studies, we identified that Cyclin F negatively influences HIV-1 viral infectivity without any significant impact on virus production. Subsequently, we found that Cyclin F negatively regulates the expression of viral protein, Vif (Viral infectivity factor), at the protein level. We also identified a novel host-pathogen interaction between Cyclin F and Vif protein in T cells during HIV-1 infection. Mutational analysis of a Cyclin F-specific amino acid motif in the C-terminal region of Vif shows rescue of the protein from Cyclin F-mediated down-regulation. Subsequently, we have shown that Vif is a novel substrate of the SCFCyclin F E3 ligase, where Cyclin F mediates ubiquitination and proteasomal degradation of Vif through physical interaction. Finally, we have shown that Cyclin F augments APOBEC3G expression through degradation of Vif to regulate infectivity of progeny virions. Taken together, our results demonstrate Cyclin F as a novel F-box protein which functions as an intrinsic cellular regulator of HIV-1 Vif and imparts a negative regulatory effect on maintenance of viral infectivity by restoring APOBEC3G expression.

  6. Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms.

    Science.gov (United States)

    Dove, Katja K; Stieglitz, Benjamin; Duncan, Emily D; Rittinger, Katrin; Klevit, Rachel E

    2016-08-01

    RING-in-between-RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub-conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT-type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING-type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub-binding site on HHARI RING2 important for its recruitment to RING1-bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.

  7. Ubiquitin E3 ligase FIEL1 regulates fibrotic lung injury through SUMO-E3 ligase PIAS4.

    Science.gov (United States)

    Lear, Travis; McKelvey, Alison C; Rajbhandari, Shristi; Dunn, Sarah R; Coon, Tiffany A; Connelly, William; Zhao, Joe Y; Kass, Daniel J; Zhang, Yingze; Liu, Yuan; Chen, Bill B

    2016-05-30

    The E3 small ubiquitin-like modifier (SUMO) protein ligase protein inhibitor of activated STAT 4 (PIAS4) is a pivotal protein in regulating the TGFβ pathway. In this study, we discovered a new protein isoform encoded by KIAA0317, termed fibrosis-inducing E3 ligase 1 (FIEL1), which potently stimulates the TGFβ signaling pathway through the site-specific ubiquitination of PIAS4. FIEL1 targets PIAS4 using a double locking mechanism that is facilitated by the kinases PKCζ and GSK3β. Specifically, PKCζ phosphorylation of PIAS4 and GSK3β phosphorylation of FIEL1 are both essential for the degradation of PIAS4. FIEL1 protein is highly expressed in lung tissues from patients with idiopathic pulmonary fibrosis (IPF), whereas PIAS4 protein levels are significantly reduced. FIEL1 overexpression significantly increases fibrosis in a bleomycin murine model, whereas FIEL1 knockdown attenuates fibrotic conditions. Further, we developed a first-in-class small molecule inhibitor toward FIEL1 that is highly effective in ameliorating fibrosis in mice. This study provides a basis for IPF therapeutic intervention by modulating PIAS4 protein abundance.

  8. Nuclear targeting of an endosomal E3 ubiquitin ligase.

    Science.gov (United States)

    Bocock, Jeffrey P; Carmicle, Stephanie; Madamba, Egbert; Erickson, Ann H

    2010-06-01

    Ring finger protein 13 (RNF13) is an E3 ubiquitin ligase embedded in endosome membranes. The protein undergoes constitutive post-translational proteolysis, making its detection difficult unless cells are incubated with a proteasome inhibitor to allow biosynthetic forms to accumulate. When cells were treated with phorbol 12-myristate 13-acetate (PMA), RNF13 avoided proteolysis. A similar stabilization was seen on ionomycin treatment of cells. Drug treatment stabilized both the full-length protein and a membrane-embedded C-terminal fragment generated following ectodomain shedding. Immunofluorescence staining revealed that PMA treatment caused the protein to accumulate in recycling endosomes, where it colocalized with transferrin receptor, and on the inner nuclear membrane, where it colocalized with lamin B. Expression of dominant-negative Rab11 inhibited nuclear localization, suggesting RNF13 was targeted to the inner nuclear membrane through recycling endosomes. New protein synthesis was necessary for this targeting. Nuclear localization was confirmed by immunoelectron microscopy and by purification of the inner nuclear membrane. Stress-induced transport of an endosomal protein to the inner nuclear membrane is a novel mechanism for introduction of regulatory proteins to the DNA environment. RNF13, with its ubiquitin ligase-active RING domain, has the potential to turn over key nuclear proteins in response to signals received at the plasma membrane.

  9. Akt is negatively regulated by the MULAN E3 ligase

    Institute of Scientific and Technical Information of China (English)

    Seunghee Bae; Jongdoo Kim; Hong-Duck Um; In-Chul Park; Su-Jae Lee; Seon Young Nam; Young-Woo Jin; Jae Ho Lee; Sungkwan An; Sun-Yong Kim; Jin Hyuk Jung; Yeongmin Yoon; Hwa Jun Cha; Hyunjin Lee; Karam Kim; Jongran Kim; In-Sook An

    2012-01-01

    The serine/threonine kinase Akt functions in multiple cellular processes,including cell survival and tumor development.Studies of the mechanisms that negatively regulate Akt have focused on dephosphorylation-mediated inactivation.In this study,we identified a negative regulator of Akt,MULAN,which possesses both a RING finger domain and E3 ubiquitin ligase activity.Akt was found to directly interact with MULAN and to be ubiquitinated by MULAN in vitro and in vivo.Other molecular assays demonstrated that phosphorylated Akt is a substantive target for both interaction with MULAN and ubiquitination by MULAN.The results of the functional studies suggest that the degradation of Akt by MULAN suppresses cell proliferation and viability.These data provide insight into the Akt ubiquitination signaling network.

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

    Science.gov (United States)

    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.

  11. Overview of the membrane-associated RING-CH (MARCH) E3 ligase family.

    Science.gov (United States)

    Bauer, Johannes; Bakke, Oddmund; Morth, J Preben

    2016-12-14

    E3 ligases are critical checkpoints for protein ubiquitination, a signal that often results in protein sorting and degradation but has also been linked to regulation of transcription and DNA repair. In line with their key role in cellular trafficking and cell-cycle control, malfunction of E3 ligases is often linked to human disease. Thus, they have emerged as prime drug targets. However, the molecular basis of action of membrane-bound E3 ligases is still unknown. Here, we review the current knowledge on the membrane-embedded MARCH E3 ligases (MARCH-1-6,7,8,11) with a focus on how the transmembrane regions can contribute via GxxxG-motifs to the selection and recognition of other membrane proteins as substrates for ubiquitination. Further understanding of the molecular parameters that govern target protein recognition of MARCH E3 ligases will contribute to development of strategies for therapeutic regulation of MARCH-induced ubiquitination.

  12. E3 ubiquitin ligases as drug targets and prognostic biomarkers in melanoma

    Directory of Open Access Journals (Sweden)

    Kristina Bielskienė

    2015-01-01

    E3 ligases are of interest as drug targets for their ability to regulate proteins stability and functions. Compared to the general proteasome inhibitor bortezomib, which blocks the entire protein degradation, drugs that target a particular E3 ligase are expected to have better selectivity with less associated toxicity. Components of different E3 ligases complexes (FBW7, MDM2, RBX1/ROC1, RBX2/ROC2, cullins and many others are known as oncogenes or tumor suppressors in melanomagenesis. These proteins participate in regulation of different cellular pathways and such important proteins in cancer development as p53 and Notch. In this review we summarized published data on the role of known E3 ligases in the development of melanoma and discuss the inhibitors of E3 ligases as a novel approach for the treatment of malignant melanomas.

  13. Structure of the HHARI catalytic domain shows glimpses of a HECT E3 ligase.

    Directory of Open Access Journals (Sweden)

    Donald E Spratt

    Full Text Available The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING, or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT. The RING-inBetweenRING-RING (RBR proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI. These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²⁺-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.

  14. Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases

    DEFF Research Database (Denmark)

    Boomsma, Wouter Krogh; Nielsen, Sofie Vincents; Lindorff-Larsen, Kresten

    2016-01-01

    The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2...... ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions...... of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we...

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

    Science.gov (United States)

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

    2014-03-15

    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.

  16. The centrosomal E3 ubiquitin ligase FBXO31-SCF regulates neuronal morphogenesis and migration.

    Directory of Open Access Journals (Sweden)

    Mayur Vadhvani

    Full Text Available Neuronal development requires proper migration, polarization and establishment of axons and dendrites. Growing evidence identifies the ubiquitin proteasome system (UPS with its numerous components as an important regulator of various aspects of neuronal development. F-box proteins are interchangeable subunits of the Cullin-1 based E3 ubiquitin ligase, but only a few family members have been studied. Here, we report that the centrosomal E3 ligase FBXO31-SCF (Skp1/Cullin-1/F-box protein regulates neuronal morphogenesis and axonal identity. In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth. Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex. Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.

  17. Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity.

    Science.gov (United States)

    Raychaudhuri, Sumana; Espenshade, Peter J

    2015-06-01

    Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1-Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking.

  18. A Bacterial Inhibitor of Host Programmed Cell Death Defenses is an E3 Ubiquitin Ligase

    Energy Technology Data Exchange (ETDEWEB)

    Janjusevic,R.; Abramovitch, R.; Martin, G.; Stebbins, C.

    2005-01-01

    The Pseudomonas syringae protein AvrPtoB is translocated into plant cells, where it inhibits immunity-associated programmed cell death (PCD). The structure of a C-terminal domain of AvrPtoB that is essential for anti-PCD activity reveals an unexpected homology to the U-box and RING-finger components of eukaryotic E3 ubiquitin ligases, and we show that AvrPtoB has ubiquitin ligase activity. Mutation of conserved residues involved in the binding of E2 ubiquitin-conjugating enzymes abolishes this activity in vitro, as well as anti-PCD activity in tomato leaves, which dramatically decreases virulence. These results show that Pseudomonas syringae uses a mimic of host E3 ubiquitin ligases to inactivate plant defenses.

  19. Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases

    DEFF Research Database (Denmark)

    Boomsma, Wouter Krogh; Nielsen, Sofie Vincents; Lindorff-Larsen, Kresten;

    2016-01-01

    conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology...

  20. Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex.

    Science.gov (United States)

    Stewart, Emerson V; Nwosu, Christine C; Tong, Zongtian; Roguev, Assen; Cummins, Timothy D; Kim, Dong-Uk; Hayles, Jacqueline; Park, Han-Oh; Hoe, Kwang-Lae; Powell, David W; Krogan, Nevan J; Espenshade, Peter J

    2011-04-22

    Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

  1. Novel E3 ubiquitin ligases that regulate histone protein levels in the budding yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Rakesh Kumar Singh

    Full Text Available Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3

  2. The substrate binding domains of human SIAH E3 ubiquitin ligases are now crystal clear

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qi; Wang, Zhongduo; Hou, Feng; Harding, Rachel; Huang, Xinyi; Dong, Aiping; Walker, John R.; Tong, Yufeng

    2017-01-01

    Seven in absentia homologs (SIAHs) comprise a family of highly conserved E3 ubiquitin ligases that play an important role in regulating signalling pathways in tumorigenesis, including the DNA damage repair and hypoxia response pathways. SIAH1 and SIAH2 have been found to function as a tumour repressor and a proto-oncogene, respectively, despite the high sequence identity of their substrate binding domains (SBDs). Ubiquitin-specific protease USP19 is a deubiquitinase that forms a complex with SIAHs and counteracts the ligase function. Much effort has been made to find selective inhibitors of the SIAHs E3 ligases. Menadione was reported to inhibit SIAH2 specifically. We used X-ray crystallography, peptide array, bioinformatic analysis, and biophysical techniques to characterize the structure and interaction of SIAHs with deubiquitinases and literature reported compounds. We solved the crystal structures of SIAH1 in complex with a USP19 peptide and of the apo form SIAH2. Phylogenetic analysis revealed the SIAH/USP19 complex is conserved in evolution. We demonstrated that menadione destabilizes both SIAH1 and SIAH2 non-specifically through covalent modification. The SBDs of SIAH E3 ligases are structurally similar with a subtle stability difference. USP19 is the only deubiquitinase that directly binds to SIAHs through the substrate binding pocket. Menadione is not a specific inhibitor for SIAH2. The crystallographic models provide structural insights into the substrate binding of the SIAH family E3 ubiquitin ligases that are critically involved in regulating cancer-related pathways. Our results suggest caution should be taken when using menadione as a specific SIAH2 inhibitor.

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

    Science.gov (United States)

    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.

  4. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis

    Science.gov (United States)

    Plechanovová, Anna; Jaffray, Ellis; Tatham, Michael H.; Naismith, James H.; Hay, Ronald T.

    2012-01-01

    SUMMARY Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING of RNF4 in complex with E2 (UbcH5a) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The C-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilise the consequent tetrahedral transition state intermediate. PMID:22842904

  5. The E3 ubiquitin ligase CTRIP controls CLOCK levels and PERIOD oscillations in Drosophila.

    Science.gov (United States)

    Lamaze, Angélique; Lamouroux, Annie; Vias, Carine; Hung, Hsiu-Cheng; Weber, Frank; Rouyer, François

    2011-06-01

    In the Drosophila circadian clock, the CLOCK/CYCLE complex activates the period and timeless genes that negatively feedback on CLOCK/CYCLE activity. The 24-h pace of this cycle depends on the stability of the clock proteins. RING-domain E3 ubiquitin ligases have been shown to destabilize PERIOD or TIMELESS. Here we identify a clock function for the circadian trip (ctrip) gene, which encodes a HECT-domain E3 ubiquitin ligase. ctrip expression in the brain is mostly restricted to clock neurons and its downregulation leads to long-period activity rhythms in constant darkness. This altered behaviour is associated with high CLOCK levels and persistence of phosphorylated PERIOD during the subjective day. The control of CLOCK protein levels does not require PERIOD. Thus, CTRIP seems to regulate the pace of the oscillator by controlling the stability of both the activator and the repressor of the feedback loop.

  6. HSV-1 ICP0: An E3 Ubiquitin Ligase That Counteracts Host Intrinsic and Innate Immunity

    Directory of Open Access Journals (Sweden)

    Mirna Perusina Lanfranca

    2014-05-01

    Full Text Available The herpes simplex virus type 1 (HSV-1 encoded E3 ubiquitin ligase, infected cell protein 0 (ICP0, is required for efficient lytic viral replication and regulates the switch between the lytic and latent states of HSV-1. As an E3 ubiquitin ligase, ICP0 directs the proteasomal degradation of several cellular targets, allowing the virus to counteract different cellular intrinsic and innate immune responses. In this review, we will focus on how ICP0’s E3 ubiquitin ligase activity inactivates the host intrinsic defenses, such as nuclear domain 10 (ND10, SUMO, and the DNA damage response to HSV-1 infection. In addition, we will examine ICP0’s capacity to impair the activation of interferon (innate regulatory mediators that include IFI16 (IFN γ-inducible protein 16, MyD88 (myeloid differentiation factor 88, and Mal (MyD88 adaptor-like protein. We will also consider how ICP0 allows HSV-1 to evade activation of the NF-κB (nuclear factor kappa B inflammatory signaling pathway. Finally, ICP0’s paradoxical relationship with USP7 (ubiquitin specific protease 7 and its roles in intrinsic and innate immune responses to HSV-1 infection will be discussed.

  7. Identification of Arabidopsis MYB56 as a novel substrate for CRL3(BPM) E3 ligases.

    Science.gov (United States)

    Chen, Liyuan; Bernhardt, Anne; Lee, JooHyun; Hellmann, Hanjo

    2015-02-01

    Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  8. MDM2 is a novel E3 ligase for HIV-1 Vif

    Directory of Open Access Journals (Sweden)

    Tomonaga Mitsunori

    2009-01-01

    Full Text Available Abstract The human immunodeficiency virus type 1 (HIV-1 Vif plays a crucial role in the viral life cycle by antagonizing a host restriction factor APOBEC3G (A3G. Vif interacts with A3G and induces its polyubiquitination and subsequent degradation via the formation of active ubiquitin ligase (E3 complex with Cullin5-ElonginB/C. Although Vif itself is also ubiquitinated and degraded rapidly in infected cells, precise roles and mechanisms of Vif ubiquitination are largely unknown. Here we report that MDM2, known as an E3 ligase for p53, is a novel E3 ligase for Vif and induces polyubiquitination and degradation of Vif. We also show the mechanisms by which MDM2 only targets Vif, but not A3G that binds to Vif. MDM2 reduces cellular Vif levels and reversely increases A3G levels, because the interaction between MDM2 and Vif precludes A3G from binding to Vif. Furthermore, we demonstrate that MDM2 negatively regulates HIV-1 replication in non-permissive target cells through Vif degradation. These data suggest that MDM2 is a regulator of HIV-1 replication and might be a novel therapeutic target for anti-HIV-1 drug.

  9. Arabidopsis HIGH PLOIDY2 sumoylates and stabilizes flowering locus C through its E3 ligase activity

    Directory of Open Access Journals (Sweden)

    Jun Soo eKwak

    2016-04-01

    Full Text Available Flowering Locus C (FLC, a floral repressor, plays an important role in flowering. The mechanisms regulating FLC gene expression and protein function have been studied extensively; however, post-translational regulation of FLC remains unclear. Here, we identified Arabidopsis HIGH PLOIDY2 (HPY2 as an E3 SUMO ligase for FLC. In vitro and vivo pull-down assays showed that FLC physically interacts with HPY2. In vitro assays showed that the stimulation of FLC sumoylation by HPY2 was dependent on SUMO-activating enzyme E1 and -conjugating enzyme E2, indicating that HPY2 was an E3 SUMO ligase for FLC. In transgenic plants, inducible HPY2 overexpression increased the concentration of FLC, indicating that HPY2 stabilized FLC through direct sumoylation. Flowering time in hpy2-2 mutants was shorter than in wild-type plants under long- and short-day conditions, with a greater effect under short-day conditions, and FLC was downregulated in hpy2-2 mutants. These data indicate that HPY2 regulates FLC function and stability at both the transcriptional and post-translational levels through its E3 SUMO ligase activity.

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

    Institute of Scientific and Technical Information of China (English)

    Chuan-Ming Xie; Wenyi Wei; Yi Sun

    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.

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

    Science.gov (United States)

    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.

  12. Self-clearance mechanism of mitochondrial E3 ligase MARCH5 contributes to mitochondria quality control.

    Science.gov (United States)

    Kim, Song-Hee; Park, Yong-Yea; Yoo, Young-Suk; Cho, Hyeseong

    2016-01-01

    MARCH5, a mitochondrial E3 ubiquitin ligase, controls mitochondrial dynamics proteins and misfolded proteins, and has been proposed to play a role in mitochondria quality control. However, it remains unclear how mutant MARCH5 found in cancer tissues is removed from cells. Here, we show that mutation in the MARCH5 ligase domain increased its half-life fourfold, resulting in a drastic increase in its protein level. Abnormal accumulation of the E3 ligase-defective MARCH5 mutants MARCH5(H43W) and MARCH5(C65/68S) was diminished by overexpression of active MARCH5(WT) ; the mutant proteins were degraded through the ubiquitin-proteasome pathway. Coimmunoprecipitation revealed that MARCH5 forms homodimers, and that substitution of Gly to Leu at the first putative GxxxG dimerization motif, but not the second, resulted in a loss of dimeric interaction. Moreover, overexpression of the dimerization-defective mutant MARCH5(4GL) could not decrease the level of accumulated MARCH5(H43W) , suggesting that dimerization of MARCH5 is necessary for self-clearance. Abnormal accumulation of MARCH5(H43W) and mitochondrial hyperfusion led to NF-ĸB activation, which was suppressed by overexpression of MARCH5(WT) . Together, the data reveal a self-protective mechanism involving MARCH5, which can target its own dysfunctional mutant for degradation in order to maintain mitochondrial homeostasis.

  13. RING finger palmitoylation of the endoplasmic reticulum Gp78 E3 ubiquitin ligase.

    Science.gov (United States)

    Fairbank, Maria; Huang, Kun; El-Husseini, Alaa; Nabi, Ivan R

    2012-07-30

    Gp78 is an E3 ubiquitin ligase within the endoplasmic reticulum-associated degradation pathway. We show that Flag-tagged gp78 undergoes sulfhydryl cysteine palmitoylation (S-palmitoylation) within the RING finger motif, responsible for its ubiquitin ligase activity. Screening of 19 palmitoyl acyl transferases (PATs) identified five that increased gp78 RING finger palmitoylation. Endoplasmic reticulum (ER)-localized Myc-DHHC6 overexpression promoted the peripheral ER distribution of Flag-gp78 while RING finger mutation and the palmitoylation inhibitor 2-bromopalmitate restricted gp78 to the central ER. Palmitoylation of RING finger cysteines therefore regulates gp78 distribution to the peripheral ER. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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

  15. Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases

    DEFF Research Database (Denmark)

    Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

    2008-01-01

    The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were...

  16. The Arabidopsis MIEL1 E3 ligase negatively regulates ABA signalling by promoting protein turnover of MYB96

    OpenAIRE

    Lee, Hong Gil; Seo, Pil Joon

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant responses to various environmental challenges. Controlled protein turnover is an important component of ABA signalling. Here we show that the RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (MIEL1) regulates ABA sensitivity by promoting MYB96 turnover in Arabidopsis. Germination of MIEL1-deficient mutant seeds is hypersensitive to ABA, whereas MIEL1-overexpressing transgenic seeds are less sensitive. MIEL1 can interact with MYB96, a regul...

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

    OpenAIRE

    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

    2015-01-01

    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 its teratogenicity, thalidomide and its derivatives lenalidomide and pomalidomide (together known as Immunomodulatory Drugs: IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase and promote the ubiquitination of Ikaros/Aiolos transcription fac...

  18. The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed-Lineage Kinase 3

    OpenAIRE

    Blessing, Natalya A.; Brockman, April L.; Chadee, Deborah N.

    2014-01-01

    Mixed-lineage kinase 3 (MLK3) activates mitogen-activated protein kinase (MAPK) signaling pathways and has important functions in migration, invasion, proliferation, tumorigenesis, and apoptosis. We investigated the role of the E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) in the regulation of MLK3 protein levels. We show that CHIP interacts with MLK3 and, together with the E2 ubiquitin-conjugating enzyme UbcH5 (UbcH5a, -b, -c, or -d), ubiquitinates MLK3 in vitro. CHIP or Hs...

  19. The E3 ubiquitin ligase activity of Trip12 is essential for mouse embryogenesis.

    Directory of Open Access Journals (Sweden)

    Masashi Kajiro

    Full Text Available Protein ubiquitination is a post-translational protein modification that regulates many biological conditions. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12(mt/mt that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12(mt/mt embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16. In contrast, Trip12(mt/mt ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12(mt/mt ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.

  20. E3 ubiquitin ligases Pellinos as regulators of pattern recognition receptor signaling and immune responses.

    Science.gov (United States)

    Medvedev, Andrei E; Murphy, Michael; Zhou, Hao; Li, Xiaoxia

    2015-07-01

    Pellinos are a family of E3 ubiquitin ligases discovered for their role in catalyzing K63-linked polyubiquitination of Pelle, an interleukin-1 (IL-1) receptor-associated kinase homolog in the Drosophila Toll pathway. Subsequent studies have revealed the central and non-redundant roles of mammalian Pellino-1, Pellino-2, and Pelino-3 in signaling pathways emanating from IL-1 receptors, Toll-like receptors, NOD-like receptors, T- and B-cell receptors. While Pellinos ability to interact with many signaling intermediates suggested their scaffolding roles, recent findings in mice expressing ligase-inactive Pellinos demonstrated the importance of Pellino ubiquitin ligase activity. Cell-specific functions of Pellinos have emerged, e.g. Pellino-1 being a negative regulator in T lymphocytes and a positive regulator in myeloid cells, and details of molecular regulation of receptor signaling by various members of the Pellino family have been revealed. In this review, we summarize current information about Pellino-mediated regulation of signaling by pattern recognition receptors, T-cell and B-cell receptors and tumor necrosis factor receptors, and discuss Pellinos roles in sepsis and infectious diseases, as well as in autoimmune, inflammatory, and allergic disorders. We also provide our perspective on the potential of targeting Pellinos with peptide- or small molecule-based drug compounds as a new therapeutic approach for septic shock and autoimmune pathologies.

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

    Science.gov (United States)

    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. Blocking an N-terminal acetylation–dependent protein interaction inhibits an E3 ligase

    Energy Technology Data Exchange (ETDEWEB)

    Scott, Daniel C.; Hammill, Jared T.; Min, Jaeki; Rhee, David Y.; Connelly, Michele; Sviderskiy, Vladislav O.; Bhasin, Deepak; Chen, Yizhe; Ong, Su-Sien; Chai, Sergio C.; Goktug, Asli N.; Huang, Guochang; Monda, Julie K.; Low, Jonathan; Kim, Ho Shin; Paulo, Joao A.; Cannon, Joe R.; Shelat, Anang A.; Chen, Taosheng; Kelsall, Ian R.; Alpi, Arno F.; Pagala, Vishwajeeth; Wang, Xusheng; Peng, Junmin; Singh , Bhuvanesh; Harper, J. Wade; Schulman, Brenda A.; Guy, R. Kip (MSKCC); (Dundee); (SJCH); (Harvard-Med); (MXPL)

    2017-06-05

    N-terminal acetylation is an abundant modification influencing protein functions. Because ~80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.

  3. The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance.

    Science.gov (United States)

    Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L; Abbas, Tarek; Larner, James M

    2017-06-01

    Lung cancer resists radiotherapy, making it one of the deadliest forms of cancer. Here, we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically, ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor, p21 protein, is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene (CDKN1A) in CHIP knockdown cells. Conversely, overexpression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone HSP70. These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity, thus suggesting a novel strategy for the treatment of lung cancer.Implications: The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. Mol Cancer Res; 15(6); 651-9. ©2017 AACR. ©2017 American Association for Cancer Research.

  4. Aβ-Induced Synaptic Alterations Require the E3 Ubiquitin Ligase Nedd4-1.

    Science.gov (United States)

    Rodrigues, Elizabeth M; Scudder, Samantha L; Goo, Marisa S; Patrick, Gentry N

    2016-02-03

    Alzheimer's disease (AD) is a neurodegenerative disease in which patients experience progressive cognitive decline. A wealth of evidence suggests that this cognitive impairment results from synaptic dysfunction in affected brain regions caused by cleavage of amyloid precursor protein into the pathogenic peptide amyloid-β (Aβ). Specifically, it has been shown that Aβ decreases surface AMPARs, dendritic spine density, and synaptic strength, and also alters synaptic plasticity. The precise molecular mechanisms by which this occurs remain unclear. Here we demonstrate a role for ubiquitination in Aβ-induced synaptic dysfunction in cultured rat neurons. We find that Aβ promotes the ubiquitination of AMPARs, as well as the redistribution and recruitment of Nedd4-1, a HECT E3 ubiquitin ligase we previously demonstrated to target AMPARs for ubiquitination and degradation. Strikingly, we show that Nedd4-1 is required for Aβ-induced reductions in surface AMPARs, synaptic strength, and dendritic spine density. Our findings, therefore, indicate an important role for Nedd4-1 and ubiquitin in the synaptic alterations induced by Aβ. Synaptic changes in Alzheimer's disease (AD) include surface AMPAR loss, which can weaken synapses. In a cell culture model of AD, we found that AMPAR loss correlates with increased AMPAR ubiquitination. In addition, the ubiquitin ligase Nedd4-1, known to ubiquitinate AMPARs, is recruited to synapses in response to Aβ. Strikingly, reducing Nedd4-1 levels in this model prevented surface AMPAR loss and synaptic weakening. These findings suggest that, in AD, Nedd4-1 may ubiquitinate AMPARs to promote their internalization and weaken synaptic strength, similar to what occurs in Nedd4-1's established role in homeostatic synaptic scaling. This is the first demonstration of Aβ-mediated control of a ubiquitin ligase to regulate surface AMPAR expression. Copyright © 2016 the authors 0270-6474/16/361590-06$15.00/0.

  5. Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps).

    Science.gov (United States)

    Mark, Kevin G; Loveless, Theresa B; Toczyski, David P

    2016-02-01

    Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d.

  6. An Arabidopsis SUMO E3 Ligase, SIZ1, Negatively Regulates Photomorphogenesis by Promoting COP1 Activity

    KAUST Repository

    Lin, Xiao-Li

    2016-04-29

    COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a ubiquitin E3 ligase, is a central negative regulator of photomorphogenesis. However, how COP1 activity is regulated by post-translational modifications remains largely unknown. Here we show that SUMO (small ubiquitin-like modifier) modification enhances COP1 activity. Loss-of-function siz1 mutant seedlings exhibit a weak constitutive photomorphogenic phenotype. SIZ1 physically interacts with COP1 and mediates the sumoylation of COP1. A K193R substitution in COP1 blocks its SUMO modification and reduces COP1 activity in vitro and in planta. Consistently, COP1 activity is reduced in siz1 and the level of HY5, a COP1 target protein, is increased in siz1. Sumoylated COP1 may exhibits higher transubiquitination activity than does non-sumoylated COP1, but SIZ1-mediated SUMO modification does not affect COP1 dimerization, COP1-HY5 interaction, and nuclear accumulation of COP1. Interestingly, prolonged light exposure reduces the sumoylation level of COP1, and COP1 mediates the ubiquitination and degradation of SIZ1. These regulatory mechanisms may maintain the homeostasis of COP1 activity, ensuing proper photomorphogenic development in changing light environment. Our genetic and biochemical studies identify a function for SIZ1 in photomorphogenesis and reveal a novel SUMO-regulated ubiquitin ligase, COP1, in plants.

  7. Novel roles of Skp2 E3 ligase in cellular senescence, cancer progression, and metastasis

    Institute of Scientific and Technical Information of China (English)

    Guocan Wang; Chia-Hsin Chan; Yuan Gao; Hui-Kuan Lin

    2012-01-01

    S-phase kinase-associated protein 2 (Skp2) belongs to the F-box protein family.It is a component of the SCF E3 ubiquitin ligase complex.Skp2 has been shown to regulate cellular proliferation by targeting several cell cycle-regulated proteins for ubiquitination and degradation,including cyclin-dependent kinase inhibitor p27.Skp2 has also been demonstrated to display an oncogenic function since its overexpression has been observed in many human cancers.This review discusses the recent discoveries on the novel roles of Skp2 in regulating cellular senescence,cancer progression,and metastasis,as well as the therapeutic potential of targeting Skp2 for human cancer treatment.

  8. TRIM E3 ligases interfere with early and late stages of the retroviral life cycle.

    Directory of Open Access Journals (Sweden)

    Pradeep D Uchil

    2008-02-01

    Full Text Available Members of the TRIpartite interaction Motif (TRIM family of E3 ligases have been shown to exhibit antiviral activities. Here we report a near comprehensive screen for antiretroviral activities of 55 TRIM proteins (36 human, 19 mouse. We identified approximately 20 TRIM proteins that, when transiently expressed in HEK293 cells, affect the entry or release of human immunodeficiency virus 1 (HIV, murine leukemia virus (MLV, or avian leukosis virus (ALV. While TRIM11 and 31 inhibited HIV entry, TRIM11 enhanced N-MLV entry by interfering with Ref1 restriction. Strikingly, many TRIM proteins affected late stages of the viral life cycle. Gene silencing of endogenously expressed TRIM 25, 31, and 62 inhibited viral release indicating that they play an important role at late stages of the viral life cycle. In contrast, downregulation of TRIM11 and 15 enhanced virus release suggesting that these proteins contribute to the endogenous restriction of retroviruses in cells.

  9. [Suppression of E3 ubiquitin ligase Cbl-b in interleukin-1 signaling].

    Science.gov (United States)

    Yu, Jiang-Tian; Bu, Xin; Zhao, Hu; Su, Jin

    2015-08-25

    The present study aims to investigate the effect of Cbl-b, a member of E3 ubiquitin ligase family, on interleukin-1 (IL-1) pathway in synoviocytes. The protein expression levels of Cbl-b and IL-1-induced matrix metalloproteinase 13 (MMP-13) in synoviocytes were analyzed by Western blot. Collagen substrates were incubated with the conditioned medium collected from synoviocytes cultures and then subjected to SDS-PAGE for analysis of collagen degradation. The results showed that compared with wild-type cells, Cbl-b-deficient cells expressed more MMP-13 protein and had enhanced ability to degrade collagens under IL-1 stimulation. These data suggest that Cbl-b may negatively regulate IL-1-triggered degradation of collagen matrix in synoviocytes.

  10. Cullin4B/E3-ubiquitin ligase negatively regulates -catenin

    Indian Academy of Sciences (India)

    Rachana Tripathi; Satya Keerthi Kota; Usha K Srinivas

    2007-09-01

    -catenin is the key transducer of Wingless-type MMTV integration site family member (Wnt) signalling, upregulation of which is the cause of cancer of the colon and other tissues. In the absence of Wnt signals, -catenin is targeted to ubiquitin–proteasome-mediated degradation. Here we present the functional characterization of E3-ubiquitin ligase encoded by cul4B. RNAi-mediated knock-down of Cul4B in a mouse cell line C3H T10 (1/2) results in an increase in -catenin levels. Loss-of-function mutation in Drosophila cul4 also shows increased -catenin/Armadillo levels in developing embryos and displays a characteristic naked-cuticle phenotype. Immunoprecipitation experiments suggest that Cul4B and -catenin are part of a signal complex in Drosophila, mouse and human. These preliminary results suggest a conserved role for Cul4B in the regulation of -catenin levels.

  11. Ret Finger Protein: An E3 Ubiquitin Ligase Juxtaposed to the XY Body in Meiosis

    Directory of Open Access Journals (Sweden)

    Isabelle Gillot

    2009-01-01

    Full Text Available During prophase I of male meiosis, the sex chromosomes form a compact structure called XY body that associates with the nuclear membrane of pachytene spermatocytes. Ret Finger Protein is a transcriptional repressor, able to interact with both nuclear matrix-associated proteins and double-stranded DNA. We report the precise and unique localization of Ret Finger Protein in pachytene spermatocytes, in which Ret Finger Protein takes place of lamin B1, between the XY body and the inner nuclear membrane. This localization of Ret Finger Protein does not seem to be associated with O-glycosylation or sumoylation. In addition, we demonstrate that Ret Finger Protein contains an E3 ubiquitin ligase activity. These observations lead to an attractive hypothesis in which Ret Finger Protein would be involved in the positioning and the attachment of XY body to the nuclear lamina of pachytene spermatocytes.

  12. Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase.

    Science.gov (United States)

    Xia, Tian; Dimitropoulou, Christiana; Zeng, Jingmin; Antonova, Galina N; Snead, Connie; Venema, Richard C; Fulton, David; Qian, Shuibing; Patterson, Cam; Papapetropoulos, Andreas; Catravas, John D

    2007-11-01

    The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.

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

    Science.gov (United States)

    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.

  14. A family of Salmonella virulence factors functions as a distinct class of autoregulated E3 ubiquitin ligases

    Science.gov (United States)

    Quezada, Cindy M.; Hicks, Stuart W.; Galán, Jorge E.; Stebbins, C. Erec

    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 E3 ligase [which we have termed NEL for Novel E3 Ligase] C-terminal fold unrelated to previously observed HECT or RING-finger E3 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. PMID:19273841

  15. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors

    NARCIS (Netherlands)

    Koo, B.K.; Spit, M.; Jordens, I.; Low, T.Y.; Stange, D.E.; van de Wetering, M.; van Es, J.H.; Mohammed, S.; Heck, A.J.R.; Maurice, M.M.; Clevers, H.

    2012-01-01

    LGR5+ stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals. Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in LGR5+ stem cells. Simultaneous deletion of the two genes en

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

    DEFF Research Database (Denmark)

    Hao, Zhenyue; Sheng, Yi; Duncan, Gordon S.

    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...

  17. Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

    Institute of Scientific and Technical Information of China (English)

    WANG Pingzhang; WANG Xin; WANG Feng; CAI Tianjing; LUO Ying

    2006-01-01

    MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (eIF4E), although the role of eIF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for eIF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBCVHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBCVHL complex, and is probably one of the new substrates of the CBCVHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor- binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.

  18. The evolutionarily conserved E3 ubiquitin ligase AtCHIP contributes to plant immunity

    Directory of Open Access Journals (Sweden)

    Xin eLi

    2016-03-01

    Full Text Available Plants possess a sophisticated immune system to recognize and respond to microbial threats in their environment. The level of immune signaling must be tightly regulated so that immune responses can be quickly activated in the presence of pathogens, while avoiding autoimmunity. HSP90s, along with their diverse array of co-chaperones, forms chaperone complexes that have been shown to play both positive and negative roles in regulating the accumulation of immune receptors and regulators. In this study, we examined the role of AtCHIP, an evolutionarily conserved E3 ligase that was known to interact with chaperones including HSP90s in multicellular organisms including fruit fly, C. elegans, plants and human. Atchip knockout mutants display enhanced disease susceptibility to a virulent oomycete pathogen, and overexpression of AtCHIP causes enhanced disease resistance at low temperature. Although CHIP was reported to target HSP90 for ubiquitination and degradation, accumulation of HSP90.3 was not affected in Atchip plants. In addition, protein accumulation of nucleotide-binding, leucine-rich repeat domain immune receptor (NLR SNC1 is not altered in Atchip mutant. Thus, while AtCHIP plays a role in immunity, it does not seem to regulate the turnover of HSP90 or SNC1. Further investigation is needed in order to determine the exact mechanism behind AtCHIP’s role in regulating plant immune responses.

  19. BICP0 and its RING finger domain act as ubiquitin E3 ligases in vitro

    Institute of Scientific and Technical Information of China (English)

    DIAO Lirong; QIAO Wentao; CHEN Qimin; WANG Chen; GENG Yunqi

    2005-01-01

    Bovine infected-cell protein 0 (BICP0) encoded by bovine herpes virus 1 (BHV-1) immediate early gene is necessary for efficient productive infection, in a large part because it activates all 3 classes of BHV-1 genes. It also has the ability to efficiently transactivate promoters that are not derived from BHV-1. To investigate the mechanism by which BICP0 achieves these effects, we expressed and purified BICP0 and its different mutants in E. coli. In vitro assays showed that both full-length BICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in BICP0 activity in other assays. Based on these, we conclude that BICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and its RING finger domain is necessary for this function. These strongly support the hypothesis that BICP0 might influence virus infection through its ability to interact with the ubiquitin-proteasome pathway.

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

    Science.gov (United States)

    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.

  1. Natural polymorphisms in C. elegans HECW-1 E3 ligase affect pathogen avoidance behaviour.

    Science.gov (United States)

    Chang, Howard C; Paek, Jennifer; Kim, Dennis H

    2011-11-16

    Heritable variation in behavioural traits generally has a complex genetic basis, and thus naturally occurring polymorphisms that influence behaviour have been defined only in rare instances. The isolation of wild strains of Caenorhabditis elegans has facilitated the study of natural genetic variation in this species and provided insights into its diverse microbial ecology. C. elegans responds to bacterial infection with conserved innate immune responses and, although lacking the immunological memory of vertebrate adaptive immunity, shows an aversive learning response to pathogenic bacteria. Here, we report the molecular characterization of naturally occurring coding polymorphisms in a C. elegans gene encoding a conserved HECT domain-containing E3 ubiquitin ligase, HECW-1. We show that two distinct polymorphisms in neighbouring residues of HECW-1 each affect C. elegans behavioural avoidance of a lawn of Pseudomonas aeruginosa. Neuron-specific rescue and ablation experiments and genetic interaction analysis indicate that HECW-1 functions in a pair of sensory neurons to inhibit P. aeruginosa lawn avoidance behaviour through inhibition of the neuropeptide receptor NPR-1 (ref. 10), which we have previously shown promotes P. aeruginosa lawn avoidance behaviour. Our data establish a molecular basis for natural variation in a C. elegans behaviour that may undergo adaptive changes in response to microbial pathogens.

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

    Directory of Open Access Journals (Sweden)

    Michael L. van de Weijer

    2016-11-01

    Full Text Available 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.

  3. Natural variation and dosage of the HEI10 meiotic E3 ligase control Arabidopsis crossover recombination

    Science.gov (United States)

    Ziolkowski, Piotr A.; Underwood, Charles J.; Lambing, Christophe; Martinez-Garcia, Marina; Lawrence, Emma J.; Ziolkowska, Liliana; Griffin, Catherine; Choi, Kyuha; Franklin, F. Chris H.; Martienssen, Robert A.; Henderson, Ian R.

    2017-01-01

    During meiosis, homologous chromosomes undergo crossover recombination, which creates genetic diversity and balances homolog segregation. Despite these critical functions, crossover frequency varies extensively within and between species. Although natural crossover recombination modifier loci have been detected in plants, causal genes have remained elusive. Using natural Arabidopsis thaliana accessions, we identified two major recombination quantitative trait loci (rQTLs) that explain 56.9% of crossover variation in Col×Ler F2 populations. We mapped rQTL1 to semidominant polymorphisms in HEI10, which encodes a conserved ubiquitin E3 ligase that regulates crossovers. Null hei10 mutants are haploinsufficient, and, using genome-wide mapping and immunocytology, we show that transformation of additional HEI10 copies is sufficient to more than double euchromatic crossovers. However, heterochromatic centromeres remained recombination-suppressed. The strongest HEI10-mediated crossover increases occur in subtelomeric euchromatin, which is reminiscent of sex differences in Arabidopsis recombination. Our work reveals that HEI10 naturally limits Arabidopsis crossovers and has the potential to influence the response to selection. PMID:28223312

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

    Science.gov (United States)

    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.

  5. The HECTD3 E3 Ubiquitin Ligase Suppresses Cisplatin-Induced Apoptosis via Stabilizing MALT1

    Directory of Open Access Journals (Sweden)

    Yi Li

    2013-01-01

    Full Text Available 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 non-degradative 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 overexpression 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.

  6. Myc protein is stabilized by suppression of a novel E3 ligase complex in cancer cells

    Science.gov (United States)

    Choi, Seung H.; Wright, Jason B.; Gerber, Scott A.; Cole, Michael D.

    2010-01-01

    Rapid Myc protein turnover is critical for maintaining basal levels of Myc activity in normal cells and a prompt response to changing growth signals. We characterize a new Myc-interacting factor, TRPC4AP (transient receptor potential cation channel, subfamily C, member 4-associated protein)/TRUSS (tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein), which is the receptor for a DDB1 (damage-specific DNA-binding protein 1)–CUL4 (Cullin 4) E3 ligase complex for selective Myc degradation through the proteasome. TRPC4AP/TRUSS binds specifically to the Myc C terminus and promotes its ubiquitination and destruction through the recognition of evolutionarily conserved domains in the Myc N terminus. TRPC4AP/TRUSS suppresses Myc-mediated transactivation and transformation in a dose-dependent manner. Finally, we found that TRPC4AP/TRUSS expression is strongly down-regulated in most cancer cell lines, leading to Myc protein stabilization. These studies identify a novel pathway targeting Myc degradation that is suppressed in cancer cells. PMID:20551172

  7. RING finger E3 ligase PPP1R11 regulates TLR2 signaling and innate immunity

    Science.gov (United States)

    McKelvey, Alison C; Lear, Travis B; Dunn, Sarah R; Evankovich, John; Londino, James D; Bednash, Joseph S; Zhang, Yingze; McVerry, Bryan J; Liu, Yuan; Chen, Bill B

    2016-01-01

    Toll-like receptor 2 (TLR2) is a pattern recognition receptor that recognizes many types of PAMPs that originate from gram-positive bacteria. Here we describe a novel mechanism regulating TLR2 protein expression and subsequent cytokine release through the ubiquitination and degradation of the receptor in response to ligand stimulation. We show a new mechanism in which an uncharacterized RING finger E3 ligase, PPP1R11, directly ubiquitinates TLR2 both in vitro and in vivo, which leads to TLR2 degradation and disruption of the signaling cascade. Lentiviral gene transfer or knockdown of PPP1R11 in mouse lungs significantly affects lung inflammation and the clearance of Staphylococcus aureus. There is a negative correlation between PPP1R11 and TLR2 levels in white blood cell samples isolated from patients with Staphylococcus aureus infections. These results suggest that PPP1R11 plays an important role in regulating innate immunity and gram-positive bacterial clearance by functioning, in part, through the ubiquitination and degradation of TLR2. DOI: http://dx.doi.org/10.7554/eLife.18496.001 PMID:27805901

  8. Modulation of immune cell functions by the E3 ligase CBL-b

    Directory of Open Access Journals (Sweden)

    Christina eLutz-Nicoladoni

    2015-03-01

    Full Text Available Maintenance of immunological tolerance is a critical hallmark of the immune system. Several signaling checkpoints necessary to balance activating and inhibitory input to immune cells have been described so far, among which the E3 ligase Cbl-b appears to be a central player. Cbl-b is expressed in all leukocyte subsets and regulates several signaling pathways in T cells, NK cells, B cells and different types of myeloid cells. In most cases Cbl-b negatively regulates activation signals through antigen or pattern recognition receptors and co-stimulatory molecules. In line with this function, cblb-deficient immune cells display lower activation thresholds and cblb knockout mice spontaneously develop autoimmunity and are highly susceptible to experimental autoimmunity. Interestingly, genetic association studies link cblb-polymorphisms with autoimmunity also in humans. Vice versa, the increased activation potential of cblb-deficient cells renders them more potent to fight against malignancies or infections. Accordingly, several reports have shown that cblb knockout mice reject tumors, which mainly depends on cytotoxic T and NK cells. Thus targeting Cbl-b may be an interesting strategy to enhance anti-cancer immunity. In this review we summarize the findings on the molecular function of Cbl-b in different cell types and illustrate the potential of Cbl-b as target for immunomodulatory therapies.

  9. Mutation in SUMO E3 ligase, SIZ1, disrupts the mature female gametophyte in Arabidopsis

    KAUST Repository

    Ling, Yu

    2012-01-09

    Female gametophyte is the multicellular haploid structure that can produce embryo and endosperm after fertilization, which has become an attractive model system for investigating molecular mechanisms in nuclei migration, cell specification, cell-to-cell communication and many other processes. Previous reports found that the small ubiquitin-like modifier (SUMO) E3 ligase, SIZ1, participated in many processes depending on particular target substrates and suppression of salicylic acid (SA) accumulation. Here, we report that SIZ1 mediates the reproductive process. SIZ1 showed enhanced expression in female organs, but was not detected in the anther or pollen. A defect in the siz1-2 maternal source resulted in reduced seed-set regardless of high SA concentration within the plant. Moreover, aniline blue staining and scanning electron microscopy revealed that funicular and micropylar pollen tube guidance was arrested in siz1-2 plants. Some of the embryo sacs of ovules in siz1-2 were also disrupted quickly after stage FG7. There was no significant affects of the siz1-2 mutation on expression of genes involved in female gametophyte development- or pollen tube guidance in ovaries. Together, our results suggest that SIZ1 sustains the stability and normal function of the mature female gametophyte which is necessary for pollen tube guidance. © 2012 Ling et al.

  10. MDM2 E3 ubiquitin ligase mediates UT-A1 urea transporter ubiquitination and degradation.

    Science.gov (United States)

    Chen, Guangping; Huang, Haidong; Fröhlich, Otto; Yang, Yuan; Klein, Janet D; Price, S Russ; Sands, Jeff M

    2008-11-01

    UT-A1 is the primary urea transporter in the apical plasma membrane responsible for urea reabsorption in the inner medullary collecting duct. Although the physiological function of UT-A1 has been well established, the molecular mechanisms that regulate its activity are less well understood. Analysis of the UT-A1 amino acid sequence revealed a potential MDM2 E3 ubiquitin ligase-binding motif in the large intracellular loop of UT-A1, suggesting that UT-A1 urea transporter protein may be regulated by the ubiquitin-proteasome pathway. Here, we report that UT-A1 is ubiquitinated and degraded by the proteasome but not the lysosome proteolytic pathway. Inhibition of proteasome activity causes UT-A1 cell surface accumulation and concomitantly increases urea transport activity. UT-A1 interacts directly with MDM2; the binding site is located in the NH2-terminal p53-binding region of MDM2. MDM2 mediates UT-A1 ubiquitination both in vivo and in vitro. Overexpression of MDM2 promotes UT-A1 degradation. The mechanism is likely to be physiologically important as UT-A1 ubiquitination was identified in kidney inner medullary tissue. The ubiquitin-proteasome degradation pathway provides an important novel mechanism for UT-A1 regulation.

  11. Characterization of the Arabidopsis thaliana E3 ubiquitin-ligase AtSINAL7 and identification of the ubiquitination sites.

    Directory of Open Access Journals (Sweden)

    Diego A Peralta

    Full Text Available Protein ubiquitination leading to degradation by the proteasome is an important mechanism in regulating key cellular functions. Protein ubiquitination is carried out by a three step process involving ubiquitin (Ub activation by a E1 enzyme, the transfer of Ub to a protein E2, finally an ubiquitin ligase E3 catalyzes the transfer of the Ub peptide to an acceptor protein. The E3 component is responsible for the specific recognition of the target, making the unveiling of E3 components essential to understand the mechanisms regulating fundamental cell processes through the protein degradation pathways. The Arabidopsis thaliana seven in absentia-like 7 (AtSINAL7 gene encodes for a protein with characteristics from a C3HC4-type E3 ubiquitin ligase. We demonstrate here that AtSINAL7 protein is indeed an E3 protein ligase based on the self-ubiquitination in vitro assay. This activity is dependent of the presence of a Lys residue in position 124. We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development. An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds. Furthermore, UV-B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.

  12. Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling.

    Directory of Open Access Journals (Sweden)

    Wei Li

    Full Text Available Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub ligases. We have annotated approximately 617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein - two transmembrane domains mediate its localization to the organelle's outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-kappaB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.

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

    Science.gov (United States)

    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.

  14. SUMO E3 Ligase AtMMS21 Regulates Drought Tolerance in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Shengchun Zhang; Yanli Qi; Ming Liu; ChengweiYang

    2013-01-01

    Post-translational modifications of proteins by small ubiquitin-like modifiers (SUMOs) play crucial roles in plant growth and development,and in stress responses.The MMS21 is a newly-identified Arabidopsis thaliana L.SUMO E3 ligase gene aside from the SIZ1,and its function requires further elucidation.Here,we show that MMS21 deficient plants display improved drought tolerance,and constitutive expression of MMS21 reduces drought tolerance.The expression of MMS21 was reduced by abscisic acid (ABA),polyethylene glycol (PEG) or drought stress.Under drought conditions,mms21 mutants showed the highest survival rate and the slowest water loss,and accumulated a higher level of free proline compared to wild-type (WT) and MMS21 over-expression plants.Stomatal aperture,seed germination and cotyledon greening analysis indicated that mms21 was hypersensitive to ABA.Molecular genetic analysis revealed that MMS21 deficiency led to elevated expression of a series of ABA-mediated stress-responsive genes,including COR15A,RD22,and P5CS1 The ABA and drought-induced stress-responsive genes,including RAB18,RD29A and RD29B,were inhibited by constitutive expression of MMS21.Moreover,ABA-induced accumulation of SUMO-protein conjugates was blocked in the mms21 mutant.We thus conclude that MMS21 plays a role in the drought stress response,likely through regulation of gene expression in an ABA-dependent pathway.

  15. CHK2 stability is regulated by the E3 ubiquitin ligase SIAH2.

    Science.gov (United States)

    García-Limones, C; Lara-Chica, M; Jiménez-Jiménez, C; Pérez, M; Moreno, P; Muñoz, E; Calzado, M A

    2016-08-18

    The serine threonine checkpoint kinase 2 (CHK2) is a critical protein involved in the DNA damage-response pathway, which is activated by phosphorylation inducing cellular response such as DNA repair, cell-cycle regulation or apoptosis. Although CHK2 activation mechanisms have been amply described, very little is known about degradation control processes. In the present study, we identify the ubiquitin E3 ligase SIAH2 as an interaction partner of CHK2, which mediates its ubiquitination and proteasomal degradation. CHK2 degradation is independent of both its activation and its kinase activity, but also of the phosphorylation in S456. We show that SIAH2-deficient cells present CHK2 accumulation together with lower ubiquitination levels. Accordingly, SIAH2 depletion by siRNA increases CHK2 levels. In response to DNA damage induced by etoposide, interaction between both proteins is disrupted, thus avoiding CHK2 degradation and promoting its stabilization. We also found that CHK2 phosphorylates SIAH2 at three residues (Thr26, Ser28 and Thr119), modifying its ability to regulate certain substrates. Cellular arrest in the G2/M phase induced by DNA damage is reverted by SIAH2 expression through the control of CHK2 levels. We observed that hypoxia decreases CHK2 levels in parallel to SIAH2 induction. Similarly, we provide evidence suggesting that resistance to apoptosis induced by genotoxic agents in cells subjected to hypoxia could be partly explained by the mutual regulation between both proteins. These results indicate that SIAH2 regulates CHK2 basal turnover, with important consequences on cell-cycle control and on the ability of hypoxia to alter the DNA damage-response pathway in cancer cells.

  16. The role of the e3 ligase cbl-B in murine dendritic cells.

    Directory of Open Access Journals (Sweden)

    Stephanie Wallner

    Full Text Available Dendritic cells (DCs are potent antigen-presenting cells with a promising potential in cancer immunotherapy. Cbl proteins are E3 ubiquitin ligases and have been implicated in regulating the functional activity of various immune cells. As an example, c-Cbl negatively affects DC activation. We here describe that another member of the Cbl-protein family (i.e. Cbl-b is highly expressed in murine bone-marrow-derived DCs (BMDCs. Differentiation of cblb-/- bone marrow mononuclear cells into classical BMDCs is unaltered, except enhanced induction of DEC-205 (CD205 expression. When tested in mixed-lymphocyte reaction (MLR, cblb-/- BMDCs exhibit increased allo-stimulatory capacity in vitro. BMDCs were next in vitro stimulated by various toll like receptor (TLR-agonists (LPS, Poly(I:C, CpG and exposed to FITC-labeled dextran. Upon TLR-stimulation, cblb-/- BMDCs produce higher levels of proinflammatory cytokines (IL-1α, IL-6 and TNF-α and exhibit a slightly higher level of FITC-dextran uptake. To further characterize the functional significance of cblb-/- BMDCs we tested them in antigen-specific T cell responses against ovalbumin (OVA protein and peptides, activating either CD8(+ OT-I or CD4(+ OT-II transgenic T cells. However, cblb-/- BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo. The migratory capacity into lymph nodes during inflammation was similarly not affected by the absence of Cbl-b. In line with these observations, cblb-/- peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs. We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo.

  17. The activities of the E3 ubiquitin ligase COP1/SPA, a key repressor in light signaling.

    Science.gov (United States)

    Hoecker, Ute

    2017-06-01

    Light is a critical signal to integrate plant growth and development with the environment. Downstream of photoreceptors, the E3 ubiquitin ligase COP1/SPA is a key repressor of photomorphogenesis which targets many positive regulators of light signaling, mainly transcription factors, for degradation in darkness. In light-grown plants COP1/SPA activity is repressed, allowing light responses to occur. This review provides an overview on our current knowledge on COP1/SPA repressor function, focusing in particular on the roles of the respective protein domains and the mechanisms of light-induced inactivation of COP1/SPA. Moreover, we summarize how COP1 activity is regulated by other interacting proteins, such as a SUMO E3 ligase and Phytochrome-Interacting Factors (PIFs), as well as by hormones. At last, several novel functions of COP1 that were recently revealed are included. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Cyclin F: A component of an E3 ubiquitin ligase complex with roles in neurodegeneration and cancer.

    Science.gov (United States)

    Galper, Jasmin; Rayner, Stephanie L; Hogan, Alison L; Fifita, Jennifer A; Lee, Albert; Chung, Roger S; Blair, Ian P; Yang, Shu

    2017-08-01

    Cyclin F, encoded by CCNF, is the substrate recognition component of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCF(cyclin F). E3 ubiquitin ligases play a key role in ubiquitin-proteasome mediated protein degradation, an essential component of protein homeostatic mechanisms within the cell. By recognising and regulating the availability of several protein substrates, SCF(cyclin F) plays a role in regulating various cellular processes including replication and repair of DNA and cell cycle checkpoint control. Cyclin F dysfunction has been implicated in various forms of cancer and CCNF mutations were recently linked to familial and sporadic amyotrophic lateral sclerosis and frontotemporal dementia, offering a new lead to understanding the pathogenic mechanisms underlying neurodegeneration. In this review, we evaluate the current literature on the function of cyclin F with an emphasis on its roles in cancer and neurodegeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Small, N-terminal tags activate Parkin E3 ubiquitin ligase activity by disrupting its autoinhibited conformation.

    Directory of Open Access Journals (Sweden)

    Lynn Burchell

    Full Text Available Parkin is an E3 ubiquitin ligase, mutations in which cause Autosomal Recessive Parkinson's Disease. Many studies aimed at understanding Parkin function, regulation and dysfunction are performed using N-terminal epitope tags. We report here that the use of small tags such as FLAG, cMyc and HA, influence the physical stability and activity of Parkin in and out of cells, perturbing the autoinhibited native state of Parkin, resulting in an active-for-autoubiquitination species.

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

    Directory of Open Access Journals (Sweden)

    Mingjia Tan

    2015-01-01

    Full Text Available 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.

  1. A non-proteolytic role for ubiquitin in deadenylation of MHC-I mRNA by the RNA-binding E3-ligase MEX-3C

    Science.gov (United States)

    Cano, Florencia; Rapiteanu, Radu; Sebastiaan Winkler, G.; Lehner, Paul J.

    2015-01-01

    The regulation of protein and mRNA turnover is essential for many cellular processes. We recently showed that ubiquitin—traditionally linked to protein degradation—directly regulates the degradation of mRNAs through the action of a newly identified family of RNA-binding E3 ubiquitin ligases. How ubiquitin regulates mRNA decay remains unclear. Here, we identify a new role for ubiquitin in regulating deadenylation, the initial and often rate-limiting step in mRNA degradation. MEX-3C, a canonical member of this family of RNA-binding ubiquitin ligases, associates with the cytoplasmic deadenylation complexes and ubiquitinates CNOT7(Caf1), the main catalytic subunit of the CCR4-NOT deadenylation machinery. We establish a new role for ubiquitin in regulating MHC-I mRNA deadenylation as ubiquitination of CNOT7 by MEX-3C regulates its deadenylation activity and is required for MHC-I mRNA degradation. Since neither proteasome nor lysosome inhibitors rescued MEX-3C-mediated MHC-I mRNA degradation, our findings suggest a new non-proteolytic function for ubiquitin in the regulation of mRNA decay. PMID:26471122

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

    Science.gov (United States)

    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.

  3. Lafora disease E3-ubiquitin ligase malin is related to TRIM32 at both the phylogenetic and functional level

    Directory of Open Access Journals (Sweden)

    Gentry Matthew S

    2011-07-01

    Full Text Available Abstract Background Malin is an E3-ubiquitin ligase that is mutated in Lafora disease, a fatal form of progressive myoclonus epilepsy. In order to perform its function, malin forms a functional complex with laforin, a glucan phosphatase that facilitates targeting of malin to its corresponding substrates. While laforin phylogeny has been studied, there are no data on the evolutionary lineage of malin. Results After an extensive search for malin orthologs, we found that malin is present in all vertebrate species and a cephalochordate, in contrast with the broader species distribution previously reported for laforin. These data suggest that in addition to forming a functional complex, laforin and perhaps malin may also have independent functions. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32, which belongs to the tripartite-motif containing family of proteins. We present experimental evidence that both malin and TRIM32 share some substrates for ubiquitination, although they produce ubiquitin chains with different topologies. However, TRIM32-specific substrates were not reciprocally ubiquitinated by the laforin-malin complex. Conclusions We found that malin and laforin are not conserved in the same genomes. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32. The latter result suggests a common origin for malin and TRIM32 and provides insights into possible functional relationships between both proteins.

  4. FERM-dependent E3 ligase recognition is a conserved mechanism for targeted degradation of lipoprotein receptors.

    Science.gov (United States)

    Calkin, Anna C; Goult, Benjamin T; Zhang, Li; Fairall, Louise; Hong, Cynthia; Schwabe, John W R; Tontonoz, Peter

    2011-12-13

    The E3 ubiquitin ligase IDOL (inducible degrader of the LDL receptor) regulates LDL receptor (LDLR)-dependent cholesterol uptake, but its mechanism of action, including the molecular basis for its stringent specificity, is poorly understood. Here we show that IDOL uses a singular strategy among E3 ligases for target recognition. The IDOL FERM domain binds directly to a recognition sequence in the cytoplasmic tails of lipoprotein receptors. This physical interaction is independent of IDOL's really interesting new gene (RING) domain E3 ligase activity and its capacity for autoubiquitination. Furthermore, IDOL controls its own stability through autoubiquitination of a unique FERM subdomain fold not present in other FERM proteins. Key residues defining the IDOL-LDLR interaction and IDOL autoubiquitination are functionally conserved in their insect homologs. Finally, we demonstrate that target recognition by IDOL involves a tripartite interaction between the FERM domain, membrane phospholipids, and the lipoprotein receptor tail. Our data identify the IDOL-LDLR interaction as an evolutionarily conserved mechanism for the regulation of lipid uptake and suggest that this interaction could potentially be exploited for the pharmacologic modulation of lipid metabolism.

  5. Roles of the TRAF6 and Pellino E3 ligases in MyD88 and RANKL signaling.

    Science.gov (United States)

    Strickson, Sam; Emmerich, Christoph H; Goh, Eddy T H; Zhang, Jiazhen; Kelsall, Ian R; Macartney, Thomas; Hastie, C James; Knebel, Axel; Peggie, Mark; Marchesi, Francesco; Arthur, J Simon C; Cohen, Philip

    2017-04-25

    It is widely accepted that the essential role of TRAF6 in vivo is to generate the Lys63-linked ubiquitin (K63-Ub) chains needed to activate the "master" protein kinase TAK1. Here, we report that TRAF6 E3 ligase activity contributes to but is not essential for the IL-1-dependent formation of K63-Ub chains, TAK1 activation, or IL-8 production in human cells, because Pellino1 and Pellino2 generate the K63-Ub chains required for signaling in cells expressing E3 ligase-inactive TRAF6 mutants. The IL-1-induced formation of K63-Ub chains and ubiquitylation of IRAK1, IRAK4, and MyD88 was abolished in TRAF6/Pellino1/Pellino2 triple-knockout (KO) cells, but not in TRAF6 KO or Pellino1/2 double-KO cells. The reexpression of E3 ligase-inactive TRAF6 mutants partially restored IL-1 signaling in TRAF6 KO cells, but not in TRAF6/Pellino1/Pellino2 triple-KO cells. Pellino1-generated K63-Ub chains activated the TAK1 complex in vitro with similar efficiently to TRAF6-generated K63-Ub chains. The early phase of TLR signaling and the TLR-dependent secretion of IL-10 (controlled by IRAKs 1 and 2) was only reduced modestly in primary macrophages from knockin mice expressing the E3 ligase-inactive TRAF6[L74H] mutant, but the late-phase production of IL-6, IL-12, and TNFα (controlled only by the pseudokinase IRAK2) was abolished. RANKL-induced signaling in macrophages and the differentiation of bone marrow to osteoclasts was similar in TRAF6[L74H] and wild-type cells, explaining why the bone structure and teeth of the TRAF6[L74H] mice was normal, unlike TRAF6 KO mice. We identify two essential roles of TRAF6 that are independent of its E3 ligase activity.

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

    Science.gov (United States)

    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

  7. Hijacking of the host SCF ubiquitin ligase machinery by plant pathogens

    Directory of Open Access Journals (Sweden)

    Shimpei eMagori

    2011-11-01

    Full Text Available The SCF (SKP1-CUL1-F-box protein ubiquitin ligase complex mediates polyubiquitination of proteins targeted for degradation, thereby controlling a plethora of biological processes in eukaryotic cells. Although this ubiquitination machinery is found and functional only in eukaryotes, many non-eukaryotic pathogens also encode F-box proteins, the critical subunits of the SCF complex. Increasing evidence indicates that such non-eukaryotic F-box proteins play an essential role in subverting or exploiting the host ubiquitin/proteasome system for efficient pathogen infection. A recent bioinformatic analysis has identified more than 70 F-box proteins in 22 different bacterial species, suggesting that use of pathogen-encoded F-box effectors in the host cell may be a widespread infection strategy. In this review, we focus on plant pathogen-encoded F-box effectors, such as VirF of Agrobacterium tumefaciens, GALAs of Ralstonia solanacearum, and P0 of Poleroviruses, and discuss the molecular mechanism by which plant pathogens use these factors to manipulate the host cell for their own benefit.

  8. Crystal structure of the substrate-recognition domain of the Shigella E3 ligase IpaH9.8.

    Science.gov (United States)

    Takagi, Kenji; Kim, Minsoo; Sasakawa, Chihiro; Mizushima, Tsunehiro

    2016-04-01

    Infectious diseases caused by bacteria have significant impacts on global public health. During infection, pathogenic bacteria deliver a variety of virulence factors, called effectors, into host cells. The Shigella effector IpaH9.8 functions as an ubiquitin ligase, ubiquitinating the NF-κB essential modulator (NEMO)/IKK-γ to inhibit host inflammatory responses. IpaH9.8 contains leucine-rich repeats (LRRs) involved in substrate recognition and an E3 ligase domain. To elucidate the structural basis of the function of IpaH9.8, the crystal structure of the LRR domain of Shigella IpaH9.8 was determined and this structure was compared with the known structures of other IpaH family members. This model provides insights into the structural features involved in substrate specificity.

  9. Structure of the Siz/PIAS SUMO E3 Ligase Siz1 and Determinants Required for SUMO Modification of PCNA

    Energy Technology Data Exchange (ETDEWEB)

    Yunus, Ali A.; Lima, Christopher D.; (SKI)

    2010-01-12

    Siz1 is a founding member of the Siz/PIAS RING family of SUMO E3 ligases. The X-ray structure of an active Siz1 ligase revealed an elongated tripartite architecture comprised of an N-terminal PINIT domain, a central zinc-containing RING-like SP-RING domain, and a C-terminal domain we term the SP-CTD. Structure-based mutational analysis and biochemical studies show that the SP-RING and SP-CTD are required for activation of the E2SUMO thioester, while the PINIT domain is essential for redirecting SUMO conjugation to the proliferating cell nuclear antigen (PCNA) at lysine 164, a nonconsensus lysine residue that is not modified by the SUMO E2 in the absence of Siz1. Mutational analysis of Siz1 and PCNA revealed surfaces on both proteins that are required for efficient SUMO modification of PCNA in vitro and in vivo.

  10. Classificati,Expression Patter,and E3 Ligase Activity Assay of Rice U-Box-Containing Proteins

    Institute of Scientific and Technical Information of China (English)

    Li-Rong Zeng; Chan Ho Park; R.C.Venu; Julian Gough; Guo-Liang Wang

    2008-01-01

    Ubiquitin ligases play a central role in determining the specificity of the ubiquitination system by selecting a myriad of appropriate candidate proteins for modification.The U-box is a recently identified,ubiquitin ligase activityrelated protein domain that shows greater presence in plants than in other organisms.In this study,we identified 77 putative U-box proteins from the rice genome using a battery of whole genome analysis algorithms.Most of the U-box protein genes are expressed,as supported by the identification of their corresponding expressed sequence tags (ESTs),full-length cDNAs,or massively parallel signature sequencing(MPSS)tags.Using the same algorithms,we identified 61 U-box proteins from the Arabidopsis genome.The rice and Arabidopsis U-box proteins were classified into nine major classes based on their domain compositions.Comparison between rice and Arabidopsis U-box proteins indicates that the majority of rice and Arabidopsis U-box proteins have the same domain organizations.The inferred phylogeny established the homology between rice and Arabidopsis U-box/ARM proteins.Cell death assay using the rice protoplast system suggests that one rice U-box gene,OsPU851,might act as a negative regulator of cell death signaling.In addition,the selected U-box proteins were found to be functional E3 ubiquitin ligases.The identification and analysis of rice U-box proteins hereby at the genomic level will help functionally characterize this class of E3 ubiquitin ligase in the future.

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

    Science.gov (United States)

    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.

  12. Inactivation of SAG E3 ubiquitin ligase blocks embryonic stem cell differentiation and sensitizes leukemia cells to retinoid acid.

    Directory of Open Access Journals (Sweden)

    Mingjia Tan

    Full Text Available 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.

  13. The Nedd4-like ubiquitin E3 ligases target angiomotin/p130 to ubiquitin-dependent degradation.

    Science.gov (United States)

    Wang, Chenji; An, Jian; Zhang, Pingzhao; Xu, Chen; Gao, Kun; Wu, Di; Wang, Dejie; Yu, Hongxiu; Liu, Jun O; Yu, Long

    2012-06-01

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

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

    Science.gov (United States)

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

    OpenAIRE

    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 v...

  16. Overexpression of denticleless E3 ubiquitin protein ligase homolog (DTL) is related to poor outcome in gastric carcinoma

    OpenAIRE

    Kobayashi, Hiroki; Komatsu, Shuhei; Ichikawa, Daisuke; Kawaguchi, Tsutomu; Hirajima, Shoji; Miyamae, Mahito; Okajima, Wataru; Ohashi, Takuma; Kosuga, Toshiyuki; Konishi, Hirotaka; Shiozaki, Atsushi; FUJIWARA, Hitoshi; Okamoto, Kazuma; Tsuda, Hitoshi; Otsuji, Eigo

    2015-01-01

    Background Denticleless E3 ubiquitin protein ligase homolog (DTL) has been identified in amplified region (1q32) of several cancers and has an oncogenic function. In this study, we tested whether DTL acts as a cancer-promoting gene through its activation/overexpression in gastric cancer (GC). Methods We analyzed 7 GC cell lines and 100 primary tumors that were curatively resected in our hospital between 2001 and 2003. Results Overexpression of the DTL protein was detected in GC cell lines (4/...

  17. E3 Ubiquitin Ligase Cbl-b Regulates Pten via Nedd4 in T Cells Independently of Its Ubiquitin Ligase Activity

    Directory of Open Access Journals (Sweden)

    Hui Guo

    2012-05-01

    Full Text Available E3 ubiquitin ligase Cbl-b plays a crucial role in T cell activation and tolerance induction. However, the molecular mechanism by which Cbl-b inhibits T cell activation remains unclear. Here, we report that Cbl-b does not inhibit PI3K but rather suppresses TCR/CD28-induced inactivation of Pten. The elevated Akt activity in Cbl-b−/− T cells is therefore due to heightened Pten inactivation. Suppression of Pten inactivation in T cells by Cbl-b is achieved by impeding the association of Pten with Nedd4, which targets Pten K13 for K63-linked polyubiquitination. Consistent with this finding, introducing Nedd4 deficiency into Cbl-b−/− mice abrogates hyper-T cell responses caused by the loss of Cbl-b. Hence, our data demonstrate that Cbl-b inhibits T cell activation by suppressing Pten inactivation independently of its ubiquitin ligase activity.

  18. DLG1 is an anchor for the E3 ligase MARCH2 at sites of cell-cell contact.

    Science.gov (United States)

    Cao, Zhifang; Huett, Alan; Kuballa, Petric; Giallourakis, Cosmas; Xavier, Ramnik J

    2008-01-01

    PDZ domain containing molecular scaffolds plays a central role in organizing synaptic junctions. Observations in Drosophila and mammalian cells have implicated that ubiquitination and endosomal trafficking, of molecular scaffolds are critical to the development and maintenance of cell-cell junctions and cell polarity. To elucidate if there is a connection between these pathways, we applied an integrative genomic strategy, which combined comparative genomics and proteomics with cell biological assays. Given the importance of ubiquitin in regulating endocytic processes, we first identified the subset of E3 ligases with conserved PDZ binding motifs. Among this subset, the MARCH family ubiquitin ligases account for the largest family and MARCH2 has been previously implicated in endosomal trafficking. Next, we tested in an unbiased fashion, if MARCH2 binds PDZ proteins in vivo using a modified tandem affinity purification strategy followed by mass spectrometry. Of note, DLG1 was co-purified from MARCH2, with subsequent confirmation that MARCH2 interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, we demonstrated that MARCH2 co-localized with DLG1 at sites of cell-cell contact. In addition, loss of the MARCH2 PDZ binding motif led to loss of MARCH2 localization at cell-cell contact sites and MARCH2 appeared to localize away from cell-cell junctions. In in vivo ubiquitination assays we show that MARCH2 promotes DLG1 ubiquitination. Overall, these results suggest that PDZ ligands with E3 ligase activity may link PDZ domain containing tumor suppressors to endocytic pathways and cell polarity determination.

  19. Degradation of phosphorylated p53 by viral protein-ECS E3 ligase complex.

    Directory of Open Access Journals (Sweden)

    Yoshitaka Sato

    2009-07-01

    Full Text Available p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection.

  20. Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors.

    Science.gov (United States)

    Selote, Devarshi; Samira, Rozalynne; Matthiadis, Anna; Gillikin, Jeffrey W; Long, Terri A

    2015-01-01

    Iron uptake and metabolism are tightly regulated in both plants and animals. In Arabidopsis (Arabidopsis thaliana), BRUTUS (BTS), which contains three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain, interacts with basic helix-loop-helix transcription factors that are capable of forming heterodimers with POPEYE (PYE), a positive regulator of the iron deficiency response. BTS has been shown to have E3 ligase capacity and to play a role in root growth, rhizosphere acidification, and iron reductase activity in response to iron deprivation. To further characterize the function of this protein, we examined the expression pattern of recombinant ProBTS::β-GLUCURONIDASE and found that it is expressed in developing embryos and other reproductive tissues, corresponding with its apparent role in reproductive growth and development. Our findings also indicate that the interactions between BTS and PYE-like (PYEL) basic helix-loop-helix transcription factors occur within the nucleus and are dependent on the presence of the RING domain. We provide evidence that BTS facilitates 26S proteasome-mediated degradation of PYEL proteins in the absence of iron. We also determined that, upon binding iron at the HHE domains, BTS is destabilized and that this destabilization relies on specific residues within the HHE domains. This study reveals an important and unique mechanism for plant iron homeostasis whereby an E3 ubiquitin ligase may posttranslationally control components of the transcriptional regulatory network involved in the iron deficiency response.

  1. Ezrin ubiquitylation by the E3 ubiquitin ligase, WWP1, and consequent regulation of hepatocyte growth factor receptor activity.

    Directory of Open Access Journals (Sweden)

    Rania F Zaarour

    Full Text Available The membrane cytoskeleton linker ezrin participates in several functions downstream of the receptor Met in response to Hepatocyte Growth Factor (HGF stimulation. Here we report a novel interaction of ezrin with a HECT E3 ubiquitin ligase, WWP1/Aip5/Tiul1, a potential oncogene that undergoes genomic amplification and overexpression in human breast and prostate cancers. We show that ezrin binds to the WW domains of WWP1 via the consensus motif PPVY(477 present in ezrin's C-terminus. This association results in the ubiquitylation of ezrin, a process that requires an intact PPVY(477 motif. Interestingly ezrin ubiquitylation does not target the protein for degradation by the proteasome. We find that ezrin ubiquitylation by WWP1 in epithelial cells leads to the upregulation of Met level in absence of HGF stimulation and increases the response of Met to HGF stimulation as measured by the ability of the cells to heal a wound. Interestingly this effect requires ubiquitylated ezrin since it can be rescued, after depletion of endogenous ezrin, by wild type ezrin but not by a mutant of ezrin that cannot be ubiquitylated. Taken together our data reveal a new role for ezrin in Met receptor stability and activity through its association with the E3 ubiquitin ligase WWP1. Given the role of Met in cell proliferation and tumorigenesis, our results may provide a mechanistic basis for understanding the role of ezrin in tumor progression.

  2. The U-Box/ARM E3 ligase PUB13 regulates cell death, defense, and flowering time in Arabidopsis.

    Science.gov (United States)

    Li, Wei; Ahn, Il-Pyung; Ning, Yuese; Park, Chan-Ho; Zeng, Lirong; Whitehill, Justin G A; Lu, Haibin; Zhao, Qingzhen; Ding, Bo; Xie, Qi; Zhou, Jian-Min; Dai, Liangying; Wang, Guo-Liang

    2012-05-01

    The components in plant signal transduction pathways are intertwined and affect each other to coordinate plant growth, development, and defenses to stresses. The role of ubiquitination in connecting these pathways, particularly plant innate immunity and flowering, is largely unknown. Here, we report the dual roles for the Arabidopsis (Arabidopsis thaliana) Plant U-box protein13 (PUB13) in defense and flowering time control. In vitro ubiquitination assays indicated that PUB13 is an active E3 ubiquitin ligase and that the intact U-box domain is required for the E3 ligase activity. Disruption of the PUB13 gene by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. The cell death, hydrogen peroxide accumulation, and resistance to necrotrophic pathogens in pub13 are enhanced when plants are pretreated with high humidity. Importantly, pub13 also shows early flowering under middle- and long-day conditions, in which the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and FLOWERING LOCUS T is induced while FLOWERING LOCUS C expression is suppressed. Finally, we found that two components involved in the SA-mediated signaling pathway, SID2 and PAD4, are required for the defense and flowering-time phenotypes caused by the loss of function of PUB13. Taken together, our data demonstrate that PUB13 acts as an important node connecting SA-dependent defense signaling and flowering time regulation in Arabidopsis.

  3. The Salmonella Effector Protein SopA Modulates Innate Immune Responses by Targeting TRIM E3 Ligase Family Members.

    Directory of Open Access Journals (Sweden)

    Jana Kamanova

    2016-04-01

    Full Text Available Salmonella Typhimurium stimulates inflammatory responses in the intestinal epithelium, which are essential for its ability to replicate within the intestinal tract. Stimulation of these responses is strictly dependent on the activity of a type III secretion system encoded within its pathogenicity island 1, which through the delivery of effector proteins, triggers signaling pathways leading to inflammation. One of these effectors is SopA, a HECT-type E3 ligase, which is required for the efficient stimulation of inflammation in an animal model of Salmonella Typhimurium infection. We show here that SopA contributes to the stimulation of innate immune responses by targeting two host E3 ubiquitin ligases, TRIM56 and TRIM65. We also found that TRIM65 interacts with the innate immune receptor MDA5 enhancing its ability to stimulate interferon-β signaling. Therefore, by targeting TRIM56 and TRIM65, SopA can stimulate signaling through two innate immune receptors, RIG-I and MDA5. These findings describe a Salmonella mechanism to modulate inflammatory responses by directly targeting innate immune signaling mechanisms.

  4. Subunit architecture of the Golgi Dsc E3 ligase required for sterol regulatory element-binding protein (SREBP) cleavage in fission yeast.

    Science.gov (United States)

    Lloyd, S Julie-Ann; Raychaudhuri, Sumana; Espenshade, Peter J

    2013-07-19

    The membrane-bound sterol regulatory element-binding protein (SREBP) transcription factors regulate lipogenesis in mammalian cells and are activated through sequential cleavage by the Golgi-localized Site-1 and Site-2 proteases. The mechanism of fission yeast SREBP cleavage is less well defined and, in contrast, requires the Golgi-localized Dsc E3 ligase complex. The Dsc E3 ligase consists of five integral membrane subunits, Dsc1 through Dsc5, and resembles membrane E3 ligases that function in endoplasmic reticulum-associated degradation. Using immunoprecipitation assays and blue native electrophoresis, we determined the subunit architecture for the complex of Dsc1 through Dsc5, showing that the Dsc proteins form subcomplexes and display defined connectivity. Dsc2 is a rhomboid pseudoprotease family member homologous to mammalian UBAC2 and a central component of the Dsc E3 ligase. We identified conservation in the architecture of the Dsc E3 ligase and the multisubunit E3 ligase gp78 in mammals. Specifically, Dsc1-Dsc2-Dsc5 forms a complex resembling gp78-UBAC2-UBXD8. Further characterization of Dsc2 revealed that its C-terminal UBA domain can bind to ubiquitin chains but that the Dsc2 UBA domain is not essential for yeast SREBP cleavage. Based on the ability of rhomboid superfamily members to bind transmembrane proteins, we speculate that Dsc2 functions in SREBP recognition and binding. Homologs of Dsc1 through Dsc4 are required for SREBP cleavage and virulence in the human opportunistic pathogen Aspergillus fumigatus. Thus, these studies advance our organizational understanding of multisubunit E3 ligases involved in endoplasmic reticulum-associated degradation and fungal pathogenesis.

  5. Protein microarrays for the identification of praja1 e3 ubiquitin ligase substrates.

    Science.gov (United States)

    Loch, Christian M; Eddins, Michael J; Strickler, James E

    2011-06-01

    Although they are the primary determinants of substrate specificity, few E3-substrate pairs have been positively identified, and few E3's profiled in a proteomic fashion. Praja1 is an E3 implicated in bone development and highly expressed in brain. Although it has been well studied relative to the majority of E3's, little is known concerning the repertoire of proteins it ubiquitylates. We sought to identify high confidence substrates for Praja1 from an unbiased proteomic profile of thousands of human proteins using protein microarrays. We first profiled Praja1 activity against a panel of E2's to identify its optimal partner in vitro. We then ubiquitylated multiple, identical protein arrays and detected putative substrates with reagents that vary in ubiquitin recognition according to the extent of chain formation. Gene ontology clustering identified putative substrates consistent with information previously known about Praja1 function, and provides clues into novel aspects of this enzyme's function.

  6. Wwp2, an E3 Ubiquitin Ligase That Targets Transcription Factor Oct-4 for Ubiquitination

    Institute of Scientific and Technical Information of China (English)

    HuiMingXu; BingLiao; QianJunZhang; BeiBeiWang; Hui,Li; XiaoMinZhong; HuiZhenSheng; YingXinZhao; YingMingZhao; YingJin

    2005-01-01

    The POU transcription factor Oct-4 is a master regulator affecting the fate of pluripotent embryonic stem cells. However, the precise mechanisms by which the activation and expression of Oct-4 are regulated still remain to be elucidated. We describe here a novel murine ubiquitin ligase, Wwp2, that specifically interacts with Oct-4 and promotes its ubiquitination both in vivo and in vitro. Remarkably, the expression of a catalytically inactive point mutant of Wwp2 abolishes Oct-4 ubiquitination. Moreover, Wwp2 promotes Oct-4 degradation in the presence of overexpressed ubiquitin. The degradation is blocked by treatment with proteasome inhibitor. Fusion of a single ubiquitin to Oct-4 inactivates its transcriptional activity in a heterologous Oct-4-driven reporter system. Furthermore, overexpression of Wwp2 in embryonic stem cells significantly reduces the Oct-4-transcriptional activities. Collectively, we demonstrate for the first time that Oct-4 can be posttranslationatly modified by ubiquitination and that this modification dramatically suppresses its transcriptional activity. These results reveal that the functional status of Oct-4, in addition to its expression level, dictates its transcriptional activity, and the results open up a new avenue to understand how Oct-4 defines the fate of embryonic stem cells.

  7. Rlim, an E3 ubiquitin ligase, influences the stability of Stathmin protein in human osteosarcoma cells.

    Science.gov (United States)

    Chen, Xi; Shen, Jianjun; Li, Xingyu; Wang, Xi; Long, Min; Lin, Fang; Wei, Junxia; Yang, Longfei; Yang, Chinglai; Dong, Ke; Zhang, Huizhong

    2014-07-01

    Stathmin is an oncoprotein and is expressed at high levels in a wide variety of human malignancies, which plays important roles in maintenance of malignant phenotypes. The regulation of Stathmin gene overexpression has been wildly explored, but the exact mechanism still needs to be elucidated. It is believed that regulation of an oncogene protein abundance through post-translational modifications is essential for maintenance of malignant phenotypes. Here we identified the Rlim, a Ring H2 zinc finger protein with intrinsic ubiquitin ligase activity, as a Stathmin-interacting protein that could increase Stathmin turnover through binding with this targeted protein and then induce its degradation by proteasome in a ubiquitin-dependent manner. Inhibition of endogenous Rlim expression by siRNA could increase the level of Stathmin protein, which further led to cell proliferation and cell cycle changes in human osteosarcoma cell lines. On the other hand, forced overexpression of Rlim could decrease the level of Stathmin protein. These results demonstrate that Rlim is involved in the negative regulation of Stathmin protein level through physical interaction and ubiquitin-mediated proteolysis. Hence, Rlim is a novel regulator of Stathmin protein in a ubiquitin-dependent manner, and represents a new pathway for malignant phenotype turnover by modulating the level of Stathmin protein in human osteosarcomas.

  8. E3 Ubiquitin Ligase NEDD4 Promotes Influenza Virus Infection by Decreasing Levels of the Antiviral Protein IFITM3.

    Directory of Open Access Journals (Sweden)

    Nicholas M Chesarino

    2015-08-01

    Full Text Available Interferon (IFN-induced transmembrane protein 3 (IFITM3 is a cell-intrinsic factor that limits influenza virus infections. We previously showed that IFITM3 degradation is increased by its ubiquitination, though the ubiquitin ligase responsible for this modification remained elusive. Here, we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in cells and in vitro. This IFITM3 ubiquitination is dependent upon the presence of a PPxY motif within IFITM3 and the WW domain-containing region of NEDD4. In NEDD4 knockout mouse embryonic fibroblasts, we observed defective IFITM3 ubiquitination and accumulation of high levels of basal IFITM3 as compared to wild type cells. Heightened IFITM3 levels significantly protected NEDD4 knockout cells from infection by influenza A and B viruses. Similarly, knockdown of NEDD4 in human lung cells resulted in an increase in steady state IFITM3 and a decrease in influenza virus infection, demonstrating a conservation of this NEDD4-dependent IFITM3 regulatory mechanism in mouse and human cells. Consistent with the known association of NEDD4 with lysosomes, we demonstrate for the first time that steady state turnover of IFITM3 occurs through the lysosomal degradation pathway. Overall, this work identifies the enzyme NEDD4 as a new therapeutic target for the prevention of influenza virus infections, and introduces a new paradigm for up-regulating cellular levels of IFITM3 independently of IFN or infection.

  9. Actin Cytoskeletal Organization in Drosophila Germline Ring Canals Depends on Kelch Function in a Cullin-RING E3 Ligase.

    Science.gov (United States)

    Hudson, Andrew M; Mannix, Katelynn M; Cooley, Lynn

    2015-11-01

    The Drosophila Kelch protein is required to organize the ovarian ring canal cytoskeleton. Kelch binds and cross-links F-actin in vitro, and it also functions with Cullin 3 (Cul3) as a component of a ubiquitin E3 ligase. How these two activities contribute to cytoskeletal remodeling in vivo is not known. We used targeted mutagenesis to investigate the mechanism of Kelch function. We tested a model in which Cul3-dependent degradation of Kelch is required for its function, but we found no evidence to support this hypothesis. However, we found that mutant Kelch deficient in its ability to interact with Cul3 failed to rescue the kelch cytoskeletal defects, suggesting that ubiquitin ligase activity is the principal activity required in vivo. We also determined that the proteasome is required with Kelch to promote the ordered growth of the ring canal cytoskeleton. These results indicate that Kelch organizes the cytoskeleton in vivo by targeting a protein substrate for degradation by the proteasome.

  10. Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase.

    Science.gov (United States)

    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.

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

    Institute of Scientific and Technical Information of China (English)

    Weihua Zhou; Wenyi Wei; Yi Sun

    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.

  12. Mechanism of replication machinery assembly as revealed by the DNA ligase-PCNA-DNA complex architecture.

    Science.gov (United States)

    Mayanagi, Kouta; Kiyonari, Shinichi; Saito, Mihoko; Shirai, Tsuyoshi; Ishino, Yoshizumi; Morikawa, Kosuke

    2009-03-24

    The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.

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

    Science.gov (United States)

    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.

  14. Ubiquitylation of FACT by the cullin-E3 ligase Rtt101 connects FACT to DNA replication.

    Science.gov (United States)

    Han, Junhong; Li, Qing; McCullough, Laura; Kettelkamp, Charisse; Formosa, Tim; Zhang, Zhiguo

    2010-07-15

    FACT plays important roles in both gene transcription and DNA replication. However, how this protein complex is targeted to these two distinct cellular processes remains largely unknown. Here we show that ubiquitylation of the Spt16 subunit of FACT by Rtt101, the cullin subunit of an E3 ubiquitin ligase in Saccharomyces cerevisiae, links FACT to DNA replication. We find Rtt101 interacts with and ubiquitylates Spt16 in vitro and in vivo. Deletion of RTT101 leads to reduced association of both FACT and the replicative helicase MCM with replication origins. Loss of Rtt101 also reduces binding of FACT to MCM, but not the association of FACT with Leo1 and Spt5, two proteins involved in transcription. Origin function is compromised in cells lacking Rtt101 or with an Spt16 mutation. These findings identify Spt16 as an Rtt101 substrate, and suggest that Spt16 ubiquitylation is important for FACT to function during DNA replication.

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

    Science.gov (United States)

    Baehr, Leslie M.

    2014-01-01

    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. PMID:25096180

  16. PUB13, a U-box/ARM E3 ligase, regulates plant defense, cell death, and flowering time.

    Science.gov (United States)

    Li, Wei; Dai, Liangying; Wang, Guo-Liang

    2012-08-01

    The ubiquitination pathway is involved in a variety of cellular processes in plant growth, development, and immune responses. However, the function of this pathway in connecting plant development and innate immunity is still largely unknown. Recently, we characterized the U-box/ARM E3 ubiquitin ligase PUB13, which regulates both immune responses and flowering time in Arabidopsis. Here, we show that the rice Spl11 gene can complement the cell death and flowering functions of PUB13 in the pub13 mutant. In addition, HFR1, which functions mainly in photomorphogenesis, was identified as one of the PUB13-interacting proteins through yeast two-hybrid screening and pull-down assays. Because the flowering phenotype of pub13 depends on photoperiod, we propose that PUB13 may regulate HFR1 to fine-tune photomorphogenesis and flowering time in Arabidopsis.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. Copyright © 2017 the authors 0270-6474/17/373848-16$15.00/0.

  20. UBR-5, a Conserved HECT-Type E3 Ubiquitin Ligase, Negatively Regulates Notch-Type Signaling in Caenorhabditis elegans

    Science.gov (United States)

    Safdar, Komal; Gu, Anniya; Xu, Xia; Au, Vinci; Taylor, Jon; Flibotte, Stephane; Moerman, Donald G.; Maine, Eleanor M.

    2016-01-01

    Notch-type signaling mediates cell−cell interactions important for animal development. In humans, reduced or inappropriate Notch signaling activity is associated with various developmental defects and disease states, including cancers. Caenorhabditis elegans expresses two Notch-type receptors, GLP-1 and LIN-12. GLP-1 mediates several cell-signaling events in the embryo and promotes germline proliferation in the developing and adult gonad. LIN-12 acts redundantly with GLP-1 in certain inductive events in the embryo and mediates several cell−cell interactions during larval development. Recovery of genetic suppressors and enhancers of glp-1 or lin-12 loss- or gain-of-function mutations has identified numerous regulators of GLP-1 and LIN-12 signaling activity. Here, we report the molecular identification of sog-1, a gene identified in screens for recessive suppressors of conditional glp-1 loss-of-function mutations. The sog-1 gene encodes UBR-5, the sole C. elegans member of the UBR5/Hyd family of HECT-type E3 ubiquitin ligases. Molecular and genetic analyses indicate that the loss of ubr-5 function suppresses defects caused by reduced signaling via GLP-1 or LIN-12. In contrast, ubr-5 mutations do not suppress embryonic or larval lethality associated with mutations in a downstream transcription factor, LAG-1. In the gonad, ubr-5 acts in the receiving cells (germ cells) to limit GLP-1 signaling activity. SEL-10 is the F-box component of SCFSEL-10 E3 ubiquitin–ligase complex that promotes turnover of Notch intracellular domain. UBR-5 acts redundantly with SEL-10 to limit Notch signaling in certain tissues. We hypothesize that UBR-5 activity limits Notch-type signaling by promoting turnover of receptor or limiting its interaction with pathway components. PMID:27185398

  1. UBR-5, a Conserved HECT-Type E3 Ubiquitin Ligase, Negatively Regulates Notch-Type Signaling in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Komal Safdar

    2016-07-01

    Full Text Available Notch-type signaling mediates cell−cell interactions important for animal development. In humans, reduced or inappropriate Notch signaling activity is associated with various developmental defects and disease states, including cancers. Caenorhabditis elegans expresses two Notch-type receptors, GLP-1 and LIN-12. GLP-1 mediates several cell-signaling events in the embryo and promotes germline proliferation in the developing and adult gonad. LIN-12 acts redundantly with GLP-1 in certain inductive events in the embryo and mediates several cell−cell interactions during larval development. Recovery of genetic suppressors and enhancers of glp-1 or lin-12 loss- or gain-of-function mutations has identified numerous regulators of GLP-1 and LIN-12 signaling activity. Here, we report the molecular identification of sog-1, a gene identified in screens for recessive suppressors of conditional glp-1 loss-of-function mutations. The sog-1 gene encodes UBR-5, the sole C. elegans member of the UBR5/Hyd family of HECT-type E3 ubiquitin ligases. Molecular and genetic analyses indicate that the loss of ubr-5 function suppresses defects caused by reduced signaling via GLP-1 or LIN-12. In contrast, ubr-5 mutations do not suppress embryonic or larval lethality associated with mutations in a downstream transcription factor, LAG-1. In the gonad, ubr-5 acts in the receiving cells (germ cells to limit GLP-1 signaling activity. SEL-10 is the F-box component of SCFSEL-10 E3 ubiquitin–ligase complex that promotes turnover of Notch intracellular domain. UBR-5 acts redundantly with SEL-10 to limit Notch signaling in certain tissues. We hypothesize that UBR-5 activity limits Notch-type signaling by promoting turnover of receptor or limiting its interaction with pathway components.

  2. HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase.

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Belzile

    2007-07-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 viral protein R (Vpr has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1, viral protein R binding protein (VPRBP, and cullin 4A (CUL4A--components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4A(VPRBP--were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest-defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4A(VPRBP E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest.

  3. PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

    Science.gov (United States)

    Kondapalli, Chandana; Kazlauskaite, Agne; Zhang, Ning; Woodroof, Helen I.; Campbell, David G.; Gourlay, Robert; Burchell, Lynn; Walden, Helen; Macartney, Thomas J.; Deak, Maria; Knebel, Axel; Alessi, Dario R.; Muqit, Miratul M. K.

    2012-01-01

    Summary Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser65. We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser65. We further show that phosphorylation of Parkin at Ser65 leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser65 or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr257, which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD. PMID:22724072

  4. Distinct and overlapping functions of the cullin E3 ligase scaffolding proteins CUL4A and CUL4B

    Science.gov (United States)

    Hannah, Jeffrey

    2016-01-01

    The cullin 4 subfamily of genes includes CUL4A and CUL4B, which share a mostly identical amino acid sequence aside from the elongated N-terminal region in CUL4B. Both act as scaffolding proteins for modular cullin RING ligase 4 (CRL4) complexes which promote the ubiquitination of a variety of substrates. CRL4 function is vital to cells as loss of both genes or their shared substrate adaptor protein DDB1 halts proliferation and eventually leads to cell death. Due to their high structural similarity, CUL4A and CUL4B share a substantial overlap in function. However, in some cases, differences in subcellular localization, spatiotemporal expression patterns and stress-inducibility preclude functional compensation. In this review, we highlight the most essential functions of the CUL4 genes in: DNA repair and replication, chromatin-remodeling, cell cycle regulation, embryogenesis, hematopoiesis and spermatogenesis. CUL4 genes are also clinically relevant as dysregulation can contribute to the onset of cancer and CRL4 complexes are often hijacked by certain viruses to promote viral replication and survival. Also, mutations in CUL4B have been implicated in a subset of patients suffering from syndromic X-linked intellectual disability (AKA mental retardation). Interestingly, the antitumor effects of immunomodulatory drugs are caused by their binding to the CRL4CRBN complex and re-directing the E3 ligase towards the Ikaros transcription factors IKZF1 and IKZF3. Because of their influence over key cellular functions and relevance to human disease, CRL4s are considered promising targets for therapeutic intervention. PMID:26344709

  5. Control of Formin Distribution and Actin Cable Assembly by the E3 Ubiquitin Ligases Dma1 and Dma2.

    Science.gov (United States)

    Juanes, M Angeles; Piatti, Simonetta

    2016-09-01

    Formins are widespread actin-polymerizing proteins that play pivotal roles in a number of processes, such as cell polarity, morphogenesis, cytokinesis, and cell migration. In agreement with their crucial function, formins are prone to a variety of regulatory mechanisms that include autoinhibition, post-translational modifications, and interaction with formin modulators. Furthermore, activation and function of formins is intimately linked to their ability to interact with membranes. In the budding yeast Saccharomyces cerevisiae, the two formins Bni1 and Bnr1 play both separate and overlapping functions in the organization of the actin cytoskeleton. In addition, they are controlled by both common and different regulatory mechanisms. Here we show that proper localization of both formins requires the redundant E3 ubiquitin ligases Dma1 and Dma2, which were previously involved in spindle positioning and septin organization. In dma1 dma2 double mutants, formin distribution at polarity sites is impaired, thus causing defects in the organization of the actin cable network and hypersensitivity to the actin depolymerizer latrunculin B. Expression of a hyperactive variant of Bni1 (Bni1-V360D) rescues these defects and partially restores proper spindle positioning in the mutant, suggesting that the failure of dma1 dma2 mutant cells to position the spindle is partly due to faulty formin activity. Strikingly, Dma1/2 interact physically with both formins, while their ubiquitin-ligase activity is required for formin function and polarized localization. Thus, ubiquitylation of formin or a formin interactor(s) could promote formin binding to membrane and its ability to nucleate actin. Altogether, our data highlight a novel level of formin regulation that further expands our knowledge of the complex and multilayered controls of these key cytoskeleton organizers.

  6. Arabidopsis BPM proteins function as substrate adaptors to a cullin3-based E3 ligase to affect fatty acid metabolism in plants.

    Science.gov (United States)

    Chen, Liyuan; Lee, Joo Hyun; Weber, Henriette; Tohge, Takayuki; Witt, Sandra; Roje, Sanja; Fernie, Alisdair R; Hellmann, Hanjo

    2013-06-01

    Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity regulation because they play a critical role as key mediators assuring specific expression of target genes. In this work, we show that cullin3-based E3 ligases have the potential to interact with a broad range of ethylene response factor (ERF)/APETALA2 (AP2) transcription factors, mediated by Math-BTB/POZ (for Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog)-Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the wrinkled1 ERF/AP2 protein. Furthermore, loss of Math-BTB/POZ proteins widely affects plant development and causes altered fatty acid contents in mutant seeds. Overall, this work demonstrates a link between fatty acid metabolism and E3 ligase activities in plants and establishes CUL3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members.

  7. Arabidopsis BPM Proteins Function as Substrate Adaptors to a CULLIN3-Based E3 Ligase to Affect Fatty Acid Metabolism in Plants[W

    Science.gov (United States)

    Chen, Liyuan; Lee, Joo Hyun; Weber, Henriette; Tohge, Takayuki; Witt, Sandra; Roje, Sanja; Fernie, Alisdair R.; Hellmann, Hanjo

    2013-01-01

    Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity regulation because they play a critical role as key mediators assuring specific expression of target genes. In this work, we show that CULLIN3-based E3 ligases have the potential to interact with a broad range of ETHYLENE RESPONSE FACTOR (ERF)/APETALA2 (AP2) transcription factors, mediated by MATH-BTB/POZ (for Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog)-Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the WRINKLED1 ERF/AP2 protein. Furthermore, loss of MATH-BTB/POZ proteins widely affects plant development and causes altered fatty acid contents in mutant seeds. Overall, this work demonstrates a link between fatty acid metabolism and E3 ligase activities in plants and establishes CUL3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members. PMID:23792371

  8. The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels.

    Science.gov (United States)

    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.

  9. The E3 ligase RNF43 inhibits Wnt signaling downstream of mutated β-catenin by sequestering TCF4 to the nuclear membrane

    NARCIS (Netherlands)

    Loregger, Anke; Grandl, Martina; Mejías-Luque, Raquel; Allgäuer, Michael; Degenhart, Kathrin; Haselmann, Verena; Oikonomou, Christina; Hatzis, Pantelis; Janssen, Klaus Peter; Nitsche, Ulrich; Gradl, Dietmar; Van Den Broek, Olaf; Destree, Olivier; Ulm, Kurt; Neumaier, Michael; Kalali, Behnam; Jung, Andreas; Varela, Ignacio; Schmid, Roland M.; Rad, Roland; Busch, Dirk H.; Gerhard, Markus

    2015-01-01

    Given its fundamental role in development and cancer, the Wnt-β-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of t

  10. Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC

    NARCIS (Netherlands)

    Luijsterburg, Martijn S.; Goedhart, Joachim; Moser, Jill; Kool, Hanneke; Geverts, Bart; Houtsmuller, Adriaan B.; Mullenders, Leon H. F.; Vermeulen, Wim; van Driel, Roel

    2007-01-01

    Damage DNA binding protein 2 ( DDB2) has a high affinity for UV-damaged DNA and has been implicated in the initial steps of global genome nucleotide excision repair (NER) in mammals. DDB2 binds to CUL4A and forms an E3 ubiquitin ligase. In this study, we have analyzed the properties of DDB2 and

  11. Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC.

    NARCIS (Netherlands)

    Luijsterburg, M.S.; Goedhart, J.; Moser, J.; Kool, H.; Geverts, B.; Houtsmuller, A.B.; Mullenders, L.H.; Vermeulen, W.; van Driel, R.

    2007-01-01

    Damage DNA binding protein 2 (DDB2) has a high affinity for UV-damaged DNA and has been implicated in the initial steps of global genome nucleotide excision repair (NER) in mammals. DDB2 binds to CUL4A and forms an E3 ubiquitin ligase. In this study, we have analyzed the properties of DDB2 and CUL4A

  12. TMEM129 is a Derlin-1 associated ERAD E3 ligase essential for virus-induced degradation of MHC-I

    DEFF Research Database (Denmark)

    van den Boomen, Dick J H; Timms, Richard T; Grice, Guinevere L

    2014-01-01

    role for ubiquitin in this degradation pathway, the responsible E3 ligase is unknown. In a forward genetic screen for host ERAD components hijacked by US11 in near-haploid KBM7 cells, we identified TMEM129, an uncharacterized polytopic membrane protein. TMEM129 is essential and rate-limiting for US11......-mediated MHC-I degradation and acts as a novel ER resident E3 ubiquitin ligase. TMEM129 contains an unusual cysteine-only RING with intrinsic E3 ligase activity and is recruited to US11 via Derlin-1. Together with its E2 conjugase Ube2J2, TMEM129 is responsible for the ubiquitination, dislocation......, and subsequent degradation of US11-associated MHC-I. US11 engages two degradation pathways: a Derlin-1/TMEM129-dependent pathway required for MHC-I degradation and a SEL1L/HRD1-dependent pathway required for "free" US11 degradation. Our data show that TMEM129 is a novel ERAD E3 ligase and the central component...

  13. Heterologous expression of the gourd E3 ubiquitin ligase gene LsRZF1 compromises the drought stress tolerance in Arabidopsis thaliana.

    Science.gov (United States)

    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.

  14. Rice RING protein OSBBI1 with E3 ligase activity confers broad-spectrum resistance against Magnaporthe oryzae by modifying the cell wall defence

    Institute of Scientific and Technical Information of China (English)

    Wei Li; Zuhua He; Sihui Zhong; Guojun Li; Qun Li; Bizeng Mao; Yiwen Deng; Huijuan Zhang; Longjun Zeng; Fengming Song

    2011-01-01

    Emerging evidence suggests that E3 ligases play critical roles in diverse biological processes, including innate immune responses in plants. However, the mechanism of the E3 ligase involvement in plant innate immunity is unclear.We report that a rice gene, OsBBI1, encoding a RING finger protein with E3 ligase activity, mediates broad-spectrum disease resistance. The expression of OSBBI1 was induced by rice blast fungus Magnaporthe oryzae, as well as chemical inducers, benzothiadiazole and salicylic acid. Biochemical analysis revealed that OsBBI1 protein possesses E3ubiquitin ligase activity in vitro. Genetic analysis revealed that the loss of OsBBI1 function in a Tos17-insertion line increased susceptibility, while the overexpression of OsBBI1 in transgenic plants conferred enhanced resistance to multiple races of M.oryzae. This indicates that OsBBI1 modulates broad-spectrum resistance against the blast fungus. The OsBBII-overexpressing plants showed higher levels of H,O, accumulation in cells and higher levels of phenolic compounds and cross-linking of proteins in cell walls at infection sites by M. Oryzae compared with wild-type(WT)plants. The cell walls were thicker in the OsBB11-overexpressing plants and thinner in the mutant plants than in the WT plants. Our results suggest that OsBBH modulates broad-spectrum resistance to blast fungus by modifying cell wall defence responses. The functional characterization of OsBBI1 provides insight into the E3 ligase-mediated innate immunity, and a practical tool for constructing broad-spectrum resistance against the most destructive disease in rice.

  15. Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila.

    Directory of Open Access Journals (Sweden)

    Roland Le Borgne

    2005-04-01

    Full Text Available Signaling by the Notch ligands Delta (Dl and Serrate (Ser regulates a wide variety of essential cell-fate decisions during animal development. Two distinct E3 ubiquitin ligases, Neuralized (Neur and Mind bomb (Mib, have been shown to regulate Dl signaling in Drosophila melanogaster and Danio rerio, respectively. While the neur and mib genes are evolutionarily conserved, their respective roles in the context of a single organism have not yet been examined. We show here that the Drosophila mind bomb (D-mib gene regulates a subset of Notch signaling events, including wing margin specification, leg segmentation, and vein determination, that are distinct from those events requiring neur activity. D-mib also modulates lateral inhibition, a neur- and Dl-dependent signaling event, suggesting that D-mib regulates Dl signaling. During wing development, expression of D-mib in dorsal cells appears to be necessary and sufficient for wing margin specification, indicating that D-mib also regulates Ser signaling. Moreover, the activity of the D-mib gene is required for the endocytosis of Ser in wing imaginal disc cells. Finally, ectopic expression of neur in D-mib mutant larvae rescues the wing D-mib phenotype, indicating that Neur can compensate for the lack of D-mib activity. We conclude that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila.

  16. Mindbomb 1, an E3 ubiquitin ligase, forms a complex with RYK to activate Wnt/β-catenin signaling.

    Science.gov (United States)

    Berndt, Jason D; Aoyagi, Atsushi; Yang, Peitzu; Anastas, Jamie N; Tang, Lan; Moon, Randall T

    2011-09-05

    Receptor-like tyrosine kinase (RYK) functions as a transmembrane receptor for the Wnt family of secreted protein ligands. Although RYK undergoes endocytosis in response to Wnt, the mechanisms that regulate its internalization and concomitant activation of Wnt signaling are unknown. We discovered that RYK both physically and functionally interacts with the E3 ubiquitin ligase Mindbomb 1 (MIB1). Overexpression of MIB1 promotes the ubiquitination of RYK and reduces its steady-state levels at the plasma membrane. Moreover, we show that MIB1 is sufficient to activate Wnt/β-catenin (CTNNB1) signaling and that this activity depends on endogenous RYK. Conversely, in loss-of-function studies, both RYK and MIB1 are required for Wnt-3A-mediated activation of CTNNB1. Finally, we identify the Caenorhabditis elegans orthologue of MIB1 and demonstrate a genetic interaction between ceMIB and lin-18/RYK in vulva development. These findings provide insights into the mechanisms of Wnt/RYK signaling and point to novel targets for the modulation of Wnt signaling.

  17. CRL2(LRR-1 E3-ligase regulates proliferation and progression through meiosis in the Caenorhabditis elegans germline.

    Directory of Open Access Journals (Sweden)

    Julien Burger

    2013-03-01

    Full Text Available The ubiquitin-proteolytic system controls the stability of proteins in space and time. In this study, using a temperature-sensitive mutant allele of the cul-2 gene, we show that CRL2(LRR-1 (CUL-2 RING E3 ubiquitin-ligase and the Leucine Rich Repeat 1 substrate recognition subunit acts at multiple levels to control germline development. CRL2(LRR-1 promotes germ cell proliferation by counteracting the DNA replication ATL-1 checkpoint pathway. CRL2(LRR-1 also participates in the mitotic proliferation/meiotic entry decision, presumably controlling the stability of meiotic promoting factors in the mitotic zone of the germline. Finally, CRL2(LRR-1 inhibits the first steps of meiotic prophase by targeting in mitotic germ cells degradation of the HORMA domain-containing protein HTP-3, required for loading synaptonemal complex components onto meiotic chromosomes. Given its widespread evolutionary conservation, CUL-2 may similarly regulate germline development in other organisms as well.

  18. The FACT complex interacts with the E3 ubiquitin ligase Psh1 to prevent ectopic localization of CENP-A.

    Science.gov (United States)

    Deyter, Gary M R; Biggins, Sue

    2014-08-15

    Centromere identity and its epigenetic maintenance require the incorporation of a histone H3 variant called CENP-A at centromeres. CENP-A mislocalization to ectopic sites may disrupt chromatin-based processes and chromosome segregation, so it is important to uncover the mechanisms by which this variant is exclusively localized to centromeres. Here, we identify a role for the conserved chromatin-modifying complex FACT (facilitates chromatin transcription/transactions) in preventing budding yeast CENP-A(Cse4) mislocalization to euchromatin by mediating its proteolysis. The Spt16 subunit of the FACT complex binds to Psh1 (Pob3/Spt16/histone), an E3 ubiquitin ligase that targets CENP-A(Cse4) for degradation. The interaction between Psh1 and Spt16 is critical for both CENP-A(Cse4) ubiquitylation and its exclusion from euchromatin. We found that Psh1 cannot efficiently ubiquitylate CENP-A(Cse4) nucleosomes in vitro, suggesting that additional factors must facilitate CENP-A(Cse4) removal from chromatin in vivo. Consistent with this, a Psh1 mutant that cannot associate with FACT has a reduced interaction with CENP-A(Cse4) in vivo. Together, our data identify a previously unknown mechanism to maintain centromere identity and genomic stability through the FACT-mediated degradation of ectopically localized CENP-A(Cse4). © 2014 Deyter and Biggins; Published by Cold Spring Harbor Laboratory Press.

  19. E3 Ligase Subunit Fbxo15 and PINK1 Kinase Regulate Cardiolipin Synthase 1 Stability and Mitochondrial Function in Pneumonia

    Directory of Open Access Journals (Sweden)

    Bill B. Chen

    2014-04-01

    Full Text Available Acute lung injury (ALI is linked to mitochondrial injury, resulting in impaired cellular oxygen utilization; however, it is unknown how these events are linked on the molecular level. Cardiolipin, a mitochondrial-specific lipid, is generated by cardiolipin synthase (CLS1. Here, we show that S. aureus activates a ubiquitin E3 ligase component, Fbxo15, that is sufficient to mediate proteasomal degradation of CLS1 in epithelia, resulting in decreased cardiolipin availability and disrupted mitochondrial function. CLS1 is destabilized by the phosphatase and tensin homolog (PTEN-induced putative kinase 1 (PINK1, which binds CLS1 to phosphorylate and regulates CLS1 disposal. Like Fbxo15, PINK1 interacts with and regulates levels of CLS1 through a mechanism dependent upon Thr219. S. aureus infection upregulates this Fbxo15-PINK1 pathway to impair mitochondrial integrity, and Pink1 knockout mice are less prone to S. aureus-induced ALI. Thus, ALI-associated disruption of cellular bioenergetics involves bioeffectors that utilize a phosphodegron to elicit ubiquitin-mediated disposal of a key mitochondrial enzyme.

  20. Structural basis for hijacking CBF-β and CUL5 E3 ligase complex by HIV-1 Vif.

    Science.gov (United States)

    Guo, Yingying; Dong, Liyong; Qiu, Xiaolin; Wang, Yishu; Zhang, Bailing; Liu, Hongnan; Yu, You; Zang, Yi; Yang, Maojun; Huang, Zhiwei

    2014-01-09

    The human immunodeficiency virus (HIV)-1 protein Vif has a central role in the neutralization of host innate defences by hijacking cellular proteasomal degradation pathways to subvert the antiviral activity of host restriction factors; however, the underlying mechanism by which Vif achieves this remains unclear. Here we report a crystal structure of the Vif-CBF-β-CUL5-ELOB-ELOC complex. The structure reveals that Vif, by means of two domains, organizes formation of the pentameric complex by interacting with CBF-β, CUL5 and ELOC. The larger domain (α/β domain) of Vif binds to the same side of CBF-β as RUNX1, indicating that Vif and RUNX1 are exclusive for CBF-β binding. Interactions of the smaller domain (α-domain) of Vif with ELOC and CUL5 are cooperative and mimic those of SOCS2 with the latter two proteins. A unique zinc-finger motif of Vif, which is located between the two Vif domains, makes no contacts with the other proteins but stabilizes the conformation of the α-domain, which may be important for Vif-CUL5 interaction. Together, our data reveal the structural basis for Vif hijacking of the CBF-β and CUL5 E3 ligase complex, laying a foundation for rational design of novel anti-HIV drugs.

  1. NEDD4 E3 ligase inhibits the activity of the Hippo pathway by targeting LATS1 for degradation.

    Science.gov (United States)

    Salah, Zaidoun; Cohen, Sherri; Itzhaki, Ella; Aqeilan, Rami I

    2013-12-15

    Proper regulation of cell proliferation, cell apoptosis, and cell death are vital for the development and survival of living organisms. Failure or dysfunction of any of these processes can have devastating effects, including cancer. The Hippo pathway, first discovered in Drosophila, has been found to be a major growth-regulatory signaling pathway that controls these crucial processes and has been implicated in cell-progress regulation and organ size determination. Abnormal regulation of this pathway has been found in several cancer types. However, the mechanisms that regulate the pathway and its core members yet have to be elucidated. One of the main core components of this pathway is LATS1, a serine/threonine kinase. Therefore, understanding how LATS1 activity is regulated is expected to shed light on new mechanisms that regulate the Hippo pathway. In the current work, we identified several potential LATS1 regulators and proved that NEDD4 E3 ubiquitin ligase controls LATS1 stability. We demonstrate that NEDD4 directly interacts with LATS1, leading to ubiquitination and decreased levels of LATS1 and, thus, increased YAP localization in the nucleus, which subsequently increases the transcriptional activity of YAP. As such, we show that NEDD4 acts as an additional regulator of the Hippo pathway on the protein level via interactions between WW domain-containing and PPxY motif-containing proteins. These findings might be applied in the development of new therapeutic approaches through the activation of LATS1.

  2. Gp78, an E3 ubiquitin ligase acts as a gatekeeper suppressing nonalcoholic steatohepatitis (NASH and liver cancer.

    Directory of Open Access Journals (Sweden)

    Tianpeng Zhang

    Full Text Available Nonalcoholic steatohepatitis (NASH is related to metabolic dysregulation and the perturbation of endoplasmic reticulum (ER homeostasis that frequently develops into hepatocellular carcinoma (HCC. Gp78 is E3 ligase, which regulates endoplasmic reticulum-associated degradation (ERAD by ubiquitinylation of misfolded ER proteins. Here, we report that upon ageing (12 months, gp78-/- mice developed obesity, recapitulating age-related human NASH. Liver histology of gp78-/- mice revealed typical steatosis, hepatic inflammation and fibrosis, followed by progression to hepatocellular tumors. Acute ER stress revealed that loss of gp78 results in up regulation of unfolded protein response (UPR pathways and SREBP-1 regulating de novo lipogenesis, responsible for fatty liver. Tissue array of human hepatocellular carcinoma (HCC demonstrated that the expression of gp78 was inversely correlated with clinical grades of cancer. Here, we have described the generation of the first preclinical experimental model system which spontaneously develops age-related NASH and HCC, linking ERAD to hepatosteatosis, cirrhosis, and cancer. It suggests that gp78 is a regulator of normal liver homeostasis and a tumor suppressor in human liver.

  3. Clomipramine causes osteoporosis by promoting osteoclastogenesis via E3 ligase Itch, which is prevented by Zoledronic acid

    Science.gov (United States)

    Li, Xing; Sun, Wen; Li, Jinbo; Wang, Mengmeng; Zhang, Hengwei; Pei, Lingpeng; Boyce, Brendan F.; Wang, Zhiyu; Xing, Lianping

    2017-01-01

    Patients taking antidepressants, including Clomipramine (CLP), have an increased risk of osteoporotic fracture. However, the effects of CLP on bone metabolism are unknown. Here, we demonstrate that WT mice treated with CLP for 2 weeks had significantly reduced trabecular bone volume and cortical bone thickness, associated with increased osteoclast (OC) numbers, but had no change in osteoblast numbers or bone formation rate. Bone marrow cells from CLP-treated mice had normal OC precursor frequency, but formed significantly more OCs when they were cultured with RANKL and M-CSF. CLP promoted OC formation and bone resorption and expression of OC-associated genes. CLP-induced bone loss was prevented by Zoledronic acid. At the molecular level, CLP inhibited the activity of the ubiquitin E3 ligase Itch. CLP did not promote OC formation from bone marrow cells of Itch−/− mice in vitro nor induce bone loss in Itch−/− mice. Our findings indicate that CLP causes bone loss by enhancing Itch-mediated osteoclastogenesis, which was prevented by Zoledronic acid. Thus, anti-resorptive therapy could be used to prevent bone loss in patients taking antidepressants, such as CLP. PMID:28145497

  4. E3 Ubiquitin Ligase Nedd4 Promotes Japanese Encephalitis Virus Replication by Suppressing Autophagy in Human Neuroblastoma Cells

    Science.gov (United States)

    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

  5. Su(dx) E3 ubiquitin ligase-dependent and -independent functions of polychaetoid, the Drosophila ZO-1 homologue.

    Science.gov (United States)

    Djiane, Alexandre; Shimizu, Hideyuki; Wilkin, Marian; Mazleyrat, Sabine; Jennings, Martin D; Avis, Johanna; Bray, Sarah; Baron, Martin

    2011-01-10

    Zona occludens (ZO) proteins are molecular scaffolds localized to cell junctions, which regulate epithelial integrity in mammals. Using newly generated null alleles, we demonstrate that polychaetoid (pyd), the unique Drosophila melanogaster ZO homologue, regulates accumulation of adherens junction-localized receptors, such as Notch, although it is dispensable for epithelial polarization. Pyd positively regulates Notch signaling during sensory organ development but acts negatively on Notch to restrict the ovary germline stem cell niche. In both contexts, we identify a core antagonistic interaction between Pyd and the WW domain E3 ubiquitin ligase Su(dx). Pyd binds Su(dx) directly, in part through a noncanonical WW-binding motif. Pyd also restricts epithelial wing cell numbers to control adult wing shape, a function associated with the FERM protein Expanded and independent of Su(dx). As both Su(dx) and Expanded regulate trafficking, we propose that a conserved role of ZO proteins is to coordinate receptor trafficking and signaling with junctional organization.

  6. Notch-induced Asb2 expression promotes protein ubiquitination by forming non-canonical E3 ligase complexes

    Institute of Scientific and Technical Information of China (English)

    Lei Nie; Ying Zhao; Wei Wu; Yuan-Zheng Yang; Hong-Cheng Wang; Xiao-Hong Sun

    2011-01-01

    Notch signaling controls multiple developmental processes, thus demanding versatile functions. We have previously shown that this may be partly achieved by accelerating ubiquitin-mediated degradation of important regulators of differentiation. However, the underlying mechanism was unknown. We now find that Notch signaling transcriptionally activates the gene encoding ankyrin-repeat SOCS box-containing protein 2(Asb2). Asb2 promotes the ubiquidnation of Notch targets such as E2A and Janus kinase(Jak)2, and a dominant-negative(DN)mutant of Asb2blocks Notch-induced degradation of these proteins. Asb2 likely binds Jak2 directly but associates with E2A through Skp2. We next provide evidence to suggest that Asb2 bridges the formation of non-canonical cullin-based complexes through interaction with not only ElonginB/C and Cullin(Cul)5, but also the F-box-containing protein, Skp2, which is known to associate with Skpl and Cull. Consistently, ablating the function of Cull or Cu15 using DN mutants or siRNAs protected both E2A and Jak2 from Asb2-mediated or Notch-induced degradation. By shifting monomeric E3ligase complexes to dimeric forms through activation of Asb2 transcription, Notch could effectively control the turnover of a variety of substrates and it exerts diverse effects on cell proliferation and differentiation.

  7. Machinery

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    @@ China Council for the Promotion of International Trade, Machin-ery Sub-Council (referred to as CCPIT MSC) & China Chamber of International Commerce, Machinery Chamber of Commerce was founded in 1988 as one of the first group of industrial trade promotion agencies approved by the governing authorities of China.

  8. Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase

    DEFF Research Database (Denmark)

    Hernández-Muñoz, Inmaculada; Lund, Anders H; van der Stoop, Petra

    2005-01-01

    . This recruitment results in an inactive state that is initially labile but is further locked in by epigenetic marks such as DNA methylation, histone hypoacetylation, and MACROH2A deposition. Here, we report that the E3 ubiquitin ligase consisting of SPOP and CULLIN3 is able to ubiquitinate the Polycomb group...... inactivation in somatic cells. We further demonstrate that MACROH2A1 deposition is regulated by the CULLIN3/SPOP ligase complex and is actively involved in stable X inactivation, likely through the formation of an additional layer of epigenetic silencing....

  9. Inhibition of xanthine oxidase by allopurinol prevents skeletal muscle atrophy: role of p38 MAPKinase and E3 ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Frederic Derbre

    Full Text Available Alterations in muscle play an important role in common diseases and conditions. Reactive oxygen species (ROS are generated during hindlimb unloading due, at least in part, to the activation of xanthine oxidase (XO. The major aim of this study was to determine the mechanism by which XO activation causes unloading-induced muscle atrophy in rats, and its possible prevention by allopurinol, a well-known inhibitor of this enzyme. For this purpose we studied one of the main redox sensitive signalling cascades involved in skeletal muscle atrophy i.e. p38 MAPKinase, and the expression of two well known muscle specific E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFbx; also known as atrogin-1 and Muscle RING (Really Interesting New Gene Finger-1 (MuRF-1. We found that hindlimb unloading induced a significant increase in XO activity and in the protein expression of the antioxidant enzymes CuZnSOD and Catalase in skeletal muscle. The most relevant new fact reported in this paper is that inhibition of XO with allopurinol, a drug widely used in clinical practice, prevents soleus muscle atrophy by ~20% after hindlimb unloading. This was associated with the inhibition of the p38 MAPK-MAFbx pathway. Our data suggest that XO was involved in the loss of muscle mass via the activation of the p38MAPK-MAFbx pathway in unloaded muscle atrophy. Thus, allopurinol may have clinical benefits to combat skeletal muscle atrophy in bedridden, astronauts, sarcopenic, and cachexic patients.

  10. A novel missense mutation of the ubiquitin protein ligase E3A gene in a patient with Angelman syndrome

    Institute of Scientific and Technical Information of China (English)

    BAI Jin-li; QU Yu-jin; ZOU Li-ping; YANG Xin-ying; LIU Li-jun; SONG Fang

    2011-01-01

    Background Angelman syndrome (AS) is a neurogenetic disorder caused by an expression defect of the maternally inherited copy of ubiquitin protein ligase E3A (UBE3A) gene from chromosome 15. Although the most common genetic defects include maternal deletions of chromosome 15q11-13, paternal uniparental disomy and imprinting defect,mutations in the UBE3A gene have been identified in approximately 10% of AS patients.Methods A Chinese girl of 28 months presented clinical manifestation of AS. Genetic diagnosis and molecular genetic defects were studied by methylation-specific PCR (MS-PCR) and linkage analysis by short tandem repeat (STR). We further performed sequence analysis of all the coding exons and flanking sequences of the UBE3A gene. The novel mutation screening was also performed in 100 unrelated healthy individuals to exclude the possibility of identifying a polymorphism variation.Results The MS-PCR analysis of the patient showed biparental inheritance of chromosome 15 with a normal methylation pattern in the 15q11-q13 region. And STR analysis revealed that the patient also inherited biparental alleles for six microsatellites. A novel mutation, cDNA1199 C>A (p. P400H), in exon 9 of the maternal UBE3A gene, was identified in the patient. Meanwhile, the mutation was observed in the patient's mother who had a normal phenotype.Conclusions It is necessary to perform the UBE3A gene mutation analysis in non-deletion/non-UPD/non-ID patients with AS. The clinical picture of the patient is concordant with that observed in previously reported AS patients with UBE3A mutation.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  13. Regulation of Chloroplast Protein Import by the Ubiquitin E3 Ligase SP1 Is Important for Stress Tolerance in Plants.

    Science.gov (United States)

    Ling, Qihua; Jarvis, Paul

    2015-10-05

    Chloroplasts are the organelles responsible for photosynthesis in plants [1, 2]. The chloroplast proteome comprises ∼3,000 different proteins, including components of the photosynthetic apparatus, which are highly abundant. Most chloroplast proteins are nucleus-encoded and imported following synthesis in the cytosol. Such import is mediated by multiprotein complexes in the envelope membranes that surround each organelle [3, 4]. The translocon at the outer envelope membrane of chloroplasts (TOC) mediates client protein recognition and early stages of import. The TOC apparatus is regulated by the ubiquitin-proteasome system (UPS) in a process controlled by the envelope-localized ubiquitin E3 ligase SUPPRESSOR OF PPI1 LOCUS1 (SP1) [5, 6]. Previous work showed that SP1-mediated regulation of chloroplast protein import contributes to the organellar proteome changes that occur during plant development (e.g., during de-etiolation). Here, we reveal a critical role for SP1 in plant responses to abiotic stress, which is a major and increasing cause of agricultural yield losses globally [7]. Arabidopsis plants lacking SP1 are hypersensitive to salt, osmotic, and oxidative stresses, whereas plants overexpressing SP1 are considerably more stress tolerant than wild-type. We present evidence that SP1 acts to deplete the TOC apparatus under stress conditions to limit the import of photosynthetic apparatus components, which may attenuate photosynthetic activity and reduce the potential for reactive oxygen species production and photo-oxidative damage. Our results indicate that chloroplast protein import is responsive to environmental cues, enabling dynamic regulation of the organellar proteome, and suggest new approaches for improving stress tolerance in crops.

  14. Hepatitis B Virus X Protein Sensitizes TRAIL-Induced Hepatocyte Apoptosis by Inhibiting the E3 Ubiquitin Ligase A20.

    Directory of Open Access Journals (Sweden)

    Hang Zhang

    Full Text Available Hepatitis B virus (HBV infection causes hepatocyte death and liver damage, which may eventually lead to cirrhosis and liver cancer. Hepatitis B virus X protein (HBx is a key antigen that is critically involved in HBV-associated liver diseases. However, the molecular basis for its pathogenesis, particularly in liver damage, has not been well defined. Herein, we report that HBx was able to enhance the susceptibility of hepatocytes to TNF-related apoptosis-inducing ligand (TRAIL-induced apoptosis. Increased sensitivity to TRAIL was associated with HBx-induced upregulation of miR-125a, which, in turn, suppressed the expression of its putative target gene, A20 E3 ligase. Importantly, we demonstrate that the defective expression of A20 impaired the K63-linked polyubiquitination of caspase-8, which reciprocally enhanced the activation of caspase-8, the recruitment of Fas-associated death domain (FADD, and the formation of death-inducing signaling complex (DISC, thereby promoting HBx-mediated apoptotic signaling. Accordingly, antagonizing miR-125a or ectopically expressing A20 in hepatocytes abolished the pro-apoptotic effect of HBx. Conversely, the overexpression of miR-125a or knockdown of A20 mimicked HBx to enhance TRAIL susceptibility in hepatocytes. Thus, we establish, for the first time, a miR-125a/A20-initiated and caspase-8-targeted mechanism by which HBx modulates apoptotic signaling and increases hepatic susceptibility to the damaging agent, which might provide novel insight into HBV-related liver pathology.

  15. Centromere architecture breakdown induced by the viral E3 ubiquitin ligase ICP0 protein of herpes simplex virus type 1.

    Directory of Open Access Journals (Sweden)

    Sylvain Gross

    Full Text Available The viral E3 ubiquitin ligase ICP0 protein has the unique property to temporarily localize at interphase and mitotic centromeres early after infection of cells by the herpes simplex virus type 1 (HSV-1. As a consequence ICP0 induces the proteasomal degradation of several centromeric proteins (CENPs, namely CENP-A, the centromeric histone H3 variant, CENP-B and CENP-C. Following ICP0-induced centromere modification cells trigger a specific response to centromeres called interphase Centromere Damage Response (iCDR. The biological significance of the iCDR is unknown; so is the degree of centromere structural damage induced by ICP0. Interphase centromeres are complex structures made of proximal and distal protein layers closely associated to CENP-A-containing centromeric chromatin. Using several cell lines constitutively expressing GFP-tagged CENPs, we investigated the extent of the centromere destabilization induced by ICP0. We show that ICP0 provokes the disappearance from centromeres, and the proteasomal degradation of several CENPs from the NAC (CENP-A nucleosome associated and CAD (CENP-A Distal complexes. We then investigated the nucleosomal occupancy of the centromeric chromatin in ICP0-expressing cells by micrococcal nuclease (MNase digestion analysis. ICP0 expression either following infection or in cell lines constitutively expressing ICP0 provokes significant modifications of the centromeric chromatin structure resulting in higher MNase accessibility. Finally, using human artificial chromosomes (HACs, we established that ICP0-induced iCDR could also target exogenous centromeres. These results demonstrate that, in addition to the protein complexes, ICP0 also destabilizes the centromeric chromatin resulting in the complete breakdown of the centromere architecture, which consequently induces iCDR.

  16. E3 ubiquitin ligase gene CMPG1-V from Haynaldia villosa L. contributes to powdery mildew resistance in common wheat (Triticum aestivum L.).

    Science.gov (United States)

    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.

  17. PUB22 and PUB23 U-BOX E3 ligases directly ubiquitinate RPN6, a 26S proteasome lid subunit, for subsequent degradation in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Cho, Seok Keun; Bae, Hansol; Ryu, Moonyoung

    2015-01-01

    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......, these results solidify a notion that PUB22 and PUB23 can alter the activity of 26S proteasome in response to drought stress....

  18. Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

    OpenAIRE

    Kazlauskaite, Agne; Kelly MR; Johnson, Clare; Baillie, Carla; Hastie, C James; Peggie, Mark; Macartney, Thomas; Woodroof, Helen I.; Alessi, Dario R; Pedrioli, Patrick G.A.; Muqit, Miratul M.K.

    2014-01-01

    Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser65) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser65 phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Mir...

  19. The E3 Ligase APC/C-Cdh1 Is Required for Associative Fear Memory and Long-Term Potentiation in the Amygdala of Adult Mice

    Science.gov (United States)

    Pick, Joseph E.; Malumbres, Marcos; Klann, Eric

    2013-01-01

    The anaphase promoting complex/cyclosome (APC/C) is an E3 ligase regulated by Cdh1. Beyond its role in controlling cell cycle progression, APC/C-Cdh1 has been detected in neurons and plays a role in long-lasting synaptic plasticity and long-term memory. Herein, we further examined the role of Cdh1 in synaptic plasticity and memory by generating…

  20. Erioflorin stabilizes the tumor suppressor Pdcd4 by inhibiting its interaction with the E3-ligase β-TrCP1.

    Science.gov (United States)

    Blees, Johanna S; Bokesch, Heidi R; Rübsamen, Daniela; Schulz, Kathrin; Milke, Larissa; Bajer, Magdalena M; Gustafson, Kirk R; Henrich, Curtis J; McMahon, James B; Colburn, Nancy H; Schmid, Tobias; Brüne, Bernhard

    2012-01-01

    Loss of the tumor suppressor Pdcd4 was reported for various tumor entities and proposed as a prognostic marker in tumorigenesis. We previously characterized decreased Pdcd4 protein stability in response to mitogenic stimuli, which resulted from p70(S6K1)-dependent protein phosphorylation, β-TrCP1-mediated ubiquitination, and proteasomal destruction. Following high-throughput screening of natural product extract libraries using a luciferase-based reporter assay to monitor phosphorylation-dependent proteasomal degradation of the tumor suppressor Pdcd4, we succeeded in showing that a crude extract from Eriophyllum lanatum stabilized Pdcd4 from TPA-induced degradation. Erioflorin was identified as the active component and inhibited not only degradation of the Pdcd4-luciferase-based reporter but also of endogenous Pdcd4 at low micromolar concentrations. Mechanistically, erioflorin interfered with the interaction between the E3-ubiquitin ligase β-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional β-TrCP-targets (such as IκBα and β-catenin), it did not prevent the degradation of targets of other E3-ubiquitin ligases such as p21 (a Skp2-target) and HIF-1α (a pVHL-target), implying selectivity for β-TrCP. Moreover, erioflorin inhibited the tumor-associated activity of known Pdcd4- and IκBα-regulated αtranscription factors, that is, AP-1 and NF-κB, altered cell cycle progression and suppressed proliferation of various cancer cell lines. Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase β-TrCP1. Inhibition of E3-ligase/target-protein interactions may offer the possibility to target degradation of specific proteins only as compared to general proteasome inhibition.

  1. Erioflorin stabilizes the tumor suppressor Pdcd4 by inhibiting its interaction with the E3-ligase β-TrCP1.

    Directory of Open Access Journals (Sweden)

    Johanna S Blees

    Full Text Available Loss of the tumor suppressor Pdcd4 was reported for various tumor entities and proposed as a prognostic marker in tumorigenesis. We previously characterized decreased Pdcd4 protein stability in response to mitogenic stimuli, which resulted from p70(S6K1-dependent protein phosphorylation, β-TrCP1-mediated ubiquitination, and proteasomal destruction. Following high-throughput screening of natural product extract libraries using a luciferase-based reporter assay to monitor phosphorylation-dependent proteasomal degradation of the tumor suppressor Pdcd4, we succeeded in showing that a crude extract from Eriophyllum lanatum stabilized Pdcd4 from TPA-induced degradation. Erioflorin was identified as the active component and inhibited not only degradation of the Pdcd4-luciferase-based reporter but also of endogenous Pdcd4 at low micromolar concentrations. Mechanistically, erioflorin interfered with the interaction between the E3-ubiquitin ligase β-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional β-TrCP-targets (such as IκBα and β-catenin, it did not prevent the degradation of targets of other E3-ubiquitin ligases such as p21 (a Skp2-target and HIF-1α (a pVHL-target, implying selectivity for β-TrCP. Moreover, erioflorin inhibited the tumor-associated activity of known Pdcd4- and IκBα-regulated αtranscription factors, that is, AP-1 and NF-κB, altered cell cycle progression and suppressed proliferation of various cancer cell lines. Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase β-TrCP1. Inhibition of E3-ligase/target-protein interactions may offer the possibility to target degradation of specific proteins only as compared to general proteasome inhibition.

  2. RNF185 is a novel E3 ligase of endoplasmic reticulum-associated degradation (ERAD) that targets cystic fibrosis transmembrane conductance regulator (CFTR).

    Science.gov (United States)

    El Khouri, Elma; Le Pavec, Gwenaëlle; Toledano, Michel B; Delaunay-Moisan, Agnès

    2013-10-25

    In the endoplasmic reticulum (ER), misfolded or improperly assembled proteins are exported to the cytoplasm and degraded by the ubiquitin-proteasome pathway through a process called ER-associated degradation (ERAD). ER-associated E3 ligases, which coordinate substrate recognition, export, and proteasome targeting, are key components of ERAD. Cystic fibrosis transmembrane conductance regulator (CFTR) is one ERAD substrate targeted to co-translational degradation by the E3 ligase RNF5/RMA1. RNF185 is a RING domain-containing polypeptide homologous to RNF5. We show that RNF185 controls the stability of CFTR and of the CFTRΔF508 mutant in a RING- and proteasome-dependent manner but does not control that of other classical ERAD model substrates. Reciprocally, its silencing stabilizes CFTR proteins. Turnover analyses indicate that, as RNF5, RNF185 targets CFTR to co-translational degradation. Importantly, however, simultaneous depletion of RNF5 and RNF185 profoundly blocks CFTRΔF508 degradation not only during translation but also after synthesis is complete. Our data thus identify RNF185 and RNF5 as a novel E3 ligase module that is central to the control of CFTR degradation.

  3. Effects of ageing on expression of the muscle-specific E3 ubiquitin ligases and Akt-dependent regulation of Foxo transcription factors in skeletal muscle.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  5. Both Rbx1 and Rbx2 exhibit a functional role in the HIV-1 Vif-Cullin5 E3 ligase complex in vitro.

    Science.gov (United States)

    Wang, Xiaodan; Wang, Xiaoying; Wang, Weiran; Zhang, Jingyao; Wang, Jiawen; Wang, Chu; Lv, Mingyu; Zuo, Tao; Liu, Donglai; Zhang, Haihong; Wu, Jiaxin; Yu, Bin; Kong, Wei; Wu, Hui; Yu, Xianghui

    2015-06-12

    Rbx1 and Rbx2 are essential components of Cullin-RING E3 Ligases. Vif is generally believed to preferentially recruit the Cul5-Rbx2 module to induce proteasomal degradation of antiretroviral enzyme APOBEC3G, although some investigators have found that the Cul5-Rbx1 module is recruited. Here, to investigate the function of the two Rbx proteins in the Vif-Cul5 complex, we analyzed the performance of Cul5-Rbx1/Cul5-Rbx2 module in the activity of Vif E3 ligase and evaluated the interactions between Rbx1/Rbx2 and Cul5. We found that either Rbx1 or Rbx2 could promote ubiquitination of APOBE3G (A3G) in vitro. We also found that both Rbx1 and Rbx2 could bind Cul5 in cells and Rbx2 could dose-dependently inhibit the interaction of Rbx1 with Cul5. Furthermore, only the decrease of endogenous Rbx2 but not Rbx1 could impair the Vif-induced A3G degradation in cells. These findings indicate that Rbx1 and Rbx2 can both activate Cul5-Vif E3 ligase in vitro, but they may undergo a more delicate selection mechanism in vivo.

  6. The Arabidopsis EDR1 Protein Kinase Negatively Regulates the ATL1 E3 Ubiquitin Ligase to Suppress Cell Death[W

    Science.gov (United States)

    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

  7. PARAQUAT TOLERANCE3 Is an E3 Ligase That Switches off Activated Oxidative Response by Targeting Histone-Modifying PROTEIN METHYLTRANSFERASE4b.

    Directory of Open Access Journals (Sweden)

    Chao Luo

    2016-09-01

    Full Text Available Oxidative stress is unavoidable for aerobic organisms. When abiotic and biotic stresses are encountered, oxidative damage could occur in cells. To avoid this damage, defense mechanisms must be timely and efficiently modulated. While the response to oxidative stress has been extensively studied in plants, little is known about how the activated response is switched off when oxidative stress is diminished. By studying Arabidopsis mutant paraquat tolerance3, we identified the genetic locus PARAQUAT TOLERANCE3 (PQT3 as a major negative regulator of oxidative stress tolerance. PQT3, encoding an E3 ubiquitin ligase, is rapidly down-regulated by oxidative stress. PQT3 has E3 ubiquitin ligase activity in ubiquitination assay. Subsequently, we identified PRMT4b as a PQT3-interacting protein. By histone methylation, PRMT4b upregulates the expression of APX1 and GPX1, encoding two key enzymes against oxidative stress. On the other hand, PRMT4b is recognized by PQT3 for targeted degradation via 26S proteasome. Therefore, we have identified PQT3 as an E3 ligase that acts as a negative regulator of activated response to oxidative stress and found that histone modification by PRMT4b at APX1 and GPX1 loci plays an important role in oxidative stress tolerance.

  8. E3 ligase CHIP and Hsc70 regulate Kv1.5 protein expression and function in mammalian cells.

    Science.gov (United States)

    Li, Peili; Kurata, Yasutaka; Maharani, Nani; Mahati, Endang; Higaki, Katsumi; Hasegawa, Akira; Shirayoshi, Yasuaki; Yoshida, Akio; Kondo, Tatehito; Kurozawa, Youichi; Yamamoto, Kazuhiro; Ninomiya, Haruaki; Hisatome, Ichiro

    2015-09-01

    Kv1.5 confers ultra-rapid delayed-rectifier potassium channel current (IKur) which contributes to repolarization of the atrial action potential. Kv1.5 proteins, degraded via the ubiquitin-proteasome pathway, decreased in some atrial fibrillation patients. Carboxyl-terminus heat shock cognate 70-interacting protein (CHIP), an E3 ubiquitin ligase, is known to ubiquitinate short-lived proteins. Here, we investigated the roles of CHIP in Kv1.5 degradation to provide insights into the mechanisms of Kv1.5 decreases and treatments targeting Kv1.5 for atrial fibrillation. Coexpression of CHIP with Kv1.5 in HEK293 cells increased Kv1.5 protein ubiquitination and decreased the protein level. Immunofluorescence revealed decreases of Kv1.5 proteins in the endoplasmic reticulum and on the cell membrane. A siRNA against CHIP suppressed Kv1.5 protein ubiquitination and increased its protein level. CHIP mutants, lacking either the N-terminal tetratricopeptide region domain or the C-terminal U-box domain, failed to exert these effects on Kv1.5 proteins. Immunoprecipitation showed that CHIP formed complexes with Kv1.5 proteins and heat shock cognate protein 70 (Hsc70). Effects of Hsc70 on Kv1.5 were similar to CHIP by altering interaction of CHIP with Kv1.5 protein. Coexpression of CHIP and Hsc70 with Kv1.5 additionally enhanced Kv1.5 ubiquitination. Kv1.5 currents were decreased by overexpression of CHIP or Hsc70 but were increased by knockdown of CHIP or Hsc70 in HEK 293 cells stably expressing Kv1.5. These effects of CHIP and Hsc70 were also observed on endogenous Kv1.5 in HL-1 mouse cardiomyocytes, decreasing IKur and prolonging action potential duration. These results indicate that CHIP decreases the Kv1.5 protein level and functional channel by facilitating its degradation in concert with chaperone Hsc70.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. 筛选调控Sonic Hedgehog信号转导的泛素连接酶%Identification of E3 ligases that regulate Sonic Hedgehog signaling transduction

    Institute of Scientific and Technical Information of China (English)

    汤颖; 乐珅; 程雁

    2013-01-01

    Objeetive:Screening for HECT E3 ligases that can regulate Sonic Hedgehog (Shh) signaling pathway.Methods:In the first round of screening,siRNAs of the HECT E3 ligases were tested for Shh pathway transduction by GliBS-luciferase assay.And in the second round,these siRNAs were tested for the localization of Patched1-GFP in primary cilia by immunofluorescence staining.Results:After two rounds of screening,5 HECT E3 ligases were identified to regulate Shh pathway,which are Smurf1,Smurf2,Ube3c,Wwp1 and Wwp2.Conclusion:A screening system of new regulators of Shh signaling was setup,and 5 HECT E3 ligases were found from our preliminary screening.%目的:发现调控Sonic Hedgehog (Shh)信号转导的泛素连接酶.方法:第一轮筛选,利用荧光素酶报告基因(8×GliBS-Luc)检测系统,检测相应HECT家族E3泛素连接酶的siRNA对Shh信号通路活性的影响;第二轮筛选,利用细胞免疫荧光激光共聚焦检测系统,检测上述siRNA对Shh的受体Ptch1蛋白原纤毛定位的影响.综合2轮筛选结果,初步发现影响Shh信号通路活性的HECT家族E3泛素连接酶.结果:通过2轮筛选,发现Smurf1、Smurf2、Ube3c、Wwp1、Wwp2共5个泛素连接酶,不仅能调节Ptch1蛋白的原纤毛定位,也可以增加通路下游转录因子Gli的活性.结论:建立了筛选调控Shh信号通路的泛素连接酶的平台.在初步筛选的27个HECT E3泛素连接酶中,有5个成员参与调控Shh信号通路.

  11. Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection.

    Directory of Open Access Journals (Sweden)

    Smita Srivastava

    2008-05-01

    Full Text Available Vpx is a small virion-associated adaptor protein encoded by viruses of the HIV-2/SIVsm lineage of primate lentiviruses that enables these viruses to transduce monocyte-derived cells. This probably reflects the ability of Vpx to overcome an as yet uncharacterized block to an early event in the virus life cycle in these cells, but the underlying mechanism has remained elusive. Using biochemical and proteomic approaches, we have found that Vpx protein of the pathogenic SIVmac 239 strain associates with a ternary protein complex comprising DDB1 and VprBP subunits of Cullin 4-based E3 ubiquitin ligase, and DDA1, which has been implicated in the regulation of E3 catalytic activity, and that Vpx participates in the Cullin 4 E3 complex comprising VprBP. We further demonstrate that the ability of SIVmac as well as HIV-2 Vpx to interact with VprBP and its associated Cullin 4 complex is required for efficient reverse transcription of SIVmac RNA genome in primary macrophages. Strikingly, macrophages in which VprBP levels are depleted by RNA interference resist SIVmac infection. Thus, our observations reveal that Vpx interacts with both catalytic and regulatory components of the ubiquitin proteasome system and demonstrate that these interactions are critical for Vpx ability to enable efficient SIVmac replication in primary macrophages. Furthermore, they identify VprBP/DCAF1 substrate receptor for Cullin 4 E3 ubiquitin ligase and its associated protein complex as immediate downstream effector of Vpx for this function. Together, our findings suggest a model in which Vpx usurps VprBP-associated Cullin 4 ubiquitin ligase to enable efficient reverse transcription and thereby overcome a block to lentivirus replication in monocyte-derived cells, and thus provide novel insights into the underlying molecular mechanism.

  12. A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation

    Directory of Open Access Journals (Sweden)

    Sikorska Marianna

    2008-02-01

    Full Text Available Abstract Background Alterations in multiple cellular pathways contribute to the development of chronic neurodegeneration such as a sporadic Alzheimer's disease (AD. These, in turn, involve changes in gene expression, amongst which are genes regulating protein processing and turnover such as the components of the ubiquitin-proteosome system. Recently, we have identified a cDNA whose expression was altered in AD brains. It contained an open reading frame of 247 amino acids and represented a novel RING finger protein, RNF182. Here we examined its biochemical properties and putative role in brain cells. Results RNF182 is a low abundance cytoplasmic protein expressed preferentially in the brain. Its expression was elevated in post-mortem AD brain tissue and the gene could be up regulated in vitro in cultured neurons subjected to cell death-inducing injuries. Subsequently, we have established that RNF182 protein possessed an E3 ubiquitin ligase activity and stimulated the E2-dependent polyubiquitination in vitro. Yeast two-hybrid screening, overexpression and co-precipitation approaches revealed, both in vitro and in vivo, an interaction between RNF182 and ATP6V0C, known for its role in the formation of gap junction complexes and neurotransmitter release channels. The data indicated that RNF182 targeted ATP6V0C for degradation by the ubiquitin-proteosome pathway. Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis. Conclusion Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults. It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.

  13. Muscle-specific E3 ubiquitin ligases are involved in muscle atrophy of cancer cachexia: an in vitro and in vivo study.

    Science.gov (United States)

    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.

  14. Functional analysis of NopM, a novel E3 ubiquitin ligase (NEL) domain effector of Rhizobium sp. strain NGR234.

    Science.gov (United States)

    Xin, Da-Wei; Liao, Sha; Xie, Zhi-Ping; Hann, Dagmar R; Steinle, Lea; Boller, Thomas; Staehelin, Christian

    2012-01-01

    Type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS) are not only virulence factors of pathogenic bacteria, but also influence symbiotic interactions between nitrogen-fixing nodule bacteria (rhizobia) and leguminous host plants. In this study, we characterized NopM (nodulation outer protein M) of Rhizobium sp. strain NGR234, which shows sequence similarities with novel E3 ubiquitin ligase (NEL) domain effectors from the human pathogens Shigella flexneri and Salomonella enterica. NopM expressed in Escherichia coli, but not the non-functional mutant protein NopM-C338A, showed E3 ubiquitin ligase activity in vitro. In vivo, NopM, but not inactive NopM-C338A, promoted nodulation of the host plant Lablab purpureus by NGR234. When NopM was expressed in yeast, it inhibited mating pheromone signaling, a mitogen-activated protein (MAP) kinase pathway. When expressed in the plant Nicotiana benthamiana, NopM inhibited one part of the plant's defense response, as shown by a reduced production of reactive oxygen species (ROS) in response to the flagellin peptide flg22, whereas it stimulated another part, namely the induction of defense genes. In summary, our data indicate the potential for NopM as a functional NEL domain E3 ubiquitin ligase. Our findings that NopM dampened the flg22-induced ROS burst in N. benthamiana but promoted defense gene induction are consistent with the concept that pattern-triggered immunity is split in two separate signaling branches, one leading to ROS production and the other to defense gene induction.

  15. XBAT35, a Novel Arabidopsis RING E3 Ligase Exhibiting Dual Targeting of Its Splice Isoforms,Is Involved in Ethylene-Mediated Regulation of Apical Hook Curvature

    Institute of Scientific and Technical Information of China (English)

    Sofia D.Carvalho; Rita Saraiva; Teresa M.Maia; Isabel A.Abreu; Paula Duque

    2012-01-01

    The Arabidopsis XBAT35 is one of five structurally related ankyrin repeat-containing Really interesting New Gene (RING) E3 ligases involved in ubiquitin-mediated protein degradation,which plays key roles in a wide range of cellular processes.Here,we show that the XBAT35 gene undergoes alternative splicing,generating two transcripts that are constitutively expressed in all plant tissues.The two splice variants derive from an exon skipping event that excludes an in-frame segment from the XBAT35 precursor mRNA,giving rise to two protein isoforms that differ solely in the presence of a nuclear localization signal (NLS).Transient expression assays indicate that the isoform lacking the NLS localizes in the cytoplasm of plant cells,whereas the other is targeted to the nucleus,accumulating in nuclear speckles.Both isoforms are functional E3 ligases,as assessed by in vitro ubiquitination assays.Two insertion mutant alleles and RNA-interference (RNAi) silencing lines for XBAT35 display no evident phenotypes under normal growth conditions,but exhibit hypersensitivity to the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) during apical hook exaggeration in the dark,which is rescued by an inhibitor of ethylene perception.Independent expression of each XBAT35 splice variant in the mutant background indicates that the two isoforms may differentially contribute to apical hook formation but are both functional in this ethylene-mediated response.Thus,XBAT35 defines a novel player in ethylene signaling involved in negatively regulating apical hook curvature,with alternative splicing controlling dual targeting of this E3 ubiquitin ligase to the nuclear and cytoplasmic compartments.

  16. BTB-BACK Domain Protein POB1 Suppresses Immune Cell Death by Targeting Ubiquitin E3 ligase PUB17 for Degradation

    Science.gov (United States)

    Mesmar, Joelle; McLellan, Hazel; Yang, Chengwei; Craig, Adam; Zhang, Cunjin; Moore, Jonathan David; Tian, Zhendong; Birch, Paul R. J.; Sadanandom, Ari

    2017-01-01

    Hypersensitive response programmed cell death (HR-PCD) is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses. PMID:28056034

  17. Time-of-day- and light-dependent expression of ubiquitin protein ligase E3 component N-recognin 4 (UBR4 in the suprachiasmatic nucleus circadian clock.

    Directory of Open Access Journals (Sweden)

    Harrod H Ling

    Full Text Available Circadian rhythms of behavior and physiology are driven by the biological clock that operates endogenously but can also be entrained to the light-dark cycle of the environment. In mammals, the master circadian pacemaker is located in the suprachiasmatic nucleus (SCN, which is composed of individual cellular oscillators that are driven by a set of core clock genes interacting in transcriptional/translational feedback loops. Light signals can trigger molecular events in the SCN that ultimately impact on the phase of expression of core clock genes to reset the master pacemaker. While transcriptional regulation has received much attention in the field of circadian biology in the past, other mechanisms including targeted protein degradation likely contribute to the clock timing and entrainment process. In the present study, proteome-wide screens of the murine SCN led to the identification of ubiquitin protein ligase E3 component N-recognin 4 (UBR4, a novel E3 ubiquitin ligase component of the N-end rule pathway, as a time-of-day-dependent and light-inducible protein. The spatial and temporal expression pattern of UBR4 in the SCN was subsequently characterized by immunofluorescence microscopy. UBR4 is expressed across the entire rostrocaudal extent of the SCN in a time-of-day-dependent fashion. UBR4 is localized exclusively to arginine vasopressin (AVP-expressing neurons of the SCN shell. Upon photic stimulation in the early subjective night, the number of UBR4-expressing cells within the SCN increases. This study is the first to identify a novel E3 ubiquitin ligase component, UBR4, in the murine SCN and to implicate the N-end rule degradation pathway as a potential player in regulating core clock mechanisms and photic entrainment.

  18. Functional analysis of NopM, a novel E3 ubiquitin ligase (NEL domain effector of Rhizobium sp. strain NGR234.

    Directory of Open Access Journals (Sweden)

    Da-Wei Xin

    Full Text Available Type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS are not only virulence factors of pathogenic bacteria, but also influence symbiotic interactions between nitrogen-fixing nodule bacteria (rhizobia and leguminous host plants. In this study, we characterized NopM (nodulation outer protein M of Rhizobium sp. strain NGR234, which shows sequence similarities with novel E3 ubiquitin ligase (NEL domain effectors from the human pathogens Shigella flexneri and Salomonella enterica. NopM expressed in Escherichia coli, but not the non-functional mutant protein NopM-C338A, showed E3 ubiquitin ligase activity in vitro. In vivo, NopM, but not inactive NopM-C338A, promoted nodulation of the host plant Lablab purpureus by NGR234. When NopM was expressed in yeast, it inhibited mating pheromone signaling, a mitogen-activated protein (MAP kinase pathway. When expressed in the plant Nicotiana benthamiana, NopM inhibited one part of the plant's defense response, as shown by a reduced production of reactive oxygen species (ROS in response to the flagellin peptide flg22, whereas it stimulated another part, namely the induction of defense genes. In summary, our data indicate the potential for NopM as a functional NEL domain E3 ubiquitin ligase. Our findings that NopM dampened the flg22-induced ROS burst in N. benthamiana but promoted defense gene induction are consistent with the concept that pattern-triggered immunity is split in two separate signaling branches, one leading to ROS production and the other to defense gene induction.

  19. Skeletal muscle 11beta-HSD1 controls glucocorticoid-induced proteolysis and expression of E3 ubiquitin ligases atrogin-1 and MuRF-1.

    Directory of Open Access Journals (Sweden)

    Katrin Biedasek

    Full Text Available Recent studies demonstrated expression and activity of the intracellular cortisone-cortisol shuttle 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1 in skeletal muscle and inhibition of 11beta-HSD1 in muscle cells improved insulin sensitivity. Glucocorticoids induce muscle atrophy via increased expression of the E3 ubiquitin ligases Atrogin-1 (Muscle Atrophy F-box (MAFbx and MuRF-1 (Muscle RING-Finger-1. We hypothesized that 11beta-HSD1 controls glucocorticoid-induced expression of atrophy E3 ubiquitin ligases in skeletal muscle. Primary human myoblasts were generated from healthy volunteers. 11beta-HSD1-dependent protein degradation was analyzed by [(3H]-tyrosine release assay. RT-PCR was used to determine mRNA expression of E3 ubiquitin ligases and 11beta-HSD1 activity was measured by conversion of radioactively labeled [(3H]-cortisone to [(3H]-cortisol separated by thin-layer chromatography. We here demonstrate that 11beta-HSD1 is expressed and biologically active in interconverting cortisone to active cortisol in murine skeletal muscle cells (C2C12 as well as in primary human myotubes. 11Beta-HSD1 expression increased during differentiation from myoblasts to mature myotubes (p < 0.01, suggesting a role of 11beta-HSD1 in skeletal muscle growth and differentiation. Treatment with cortisone increased protein degradation by about 20% (p < 0.001, which was paralleled by an elevation of Atrogin-1 and MuRF-1 mRNA expression (p < 0.01, respectively. Notably, pre-treatment with the 11beta-HSD1 inhibitor carbenoxolone (Cbx completely abolished the effect of cortisone on protein degradation as well as on Atrogin-1 and MuRF-1 expression. In summary, our data suggest that 11beta-HSD1 controls glucocorticoid-induced protein degradation in human and murine skeletal muscle via regulation of the E3 ubiquitin ligases Atrogin-1 and MuRF-1.

  20. Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Richard T Timms

    Full Text Available 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.

  1. Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Richard T Timms

    Full Text Available 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.

  2. MALT1 cleaves the E3 ubiquitin ligase HOIL-1 in activated T cells, generating a dominant negative inhibitor of LUBAC-induced NF-κB signaling.

    Science.gov (United States)

    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.

  3. A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Yiyan; Liu, Yaobin; Xu, Guoqiang, E-mail: gux2002@suda.edu.cn

    2015-06-12

    Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions. - Highlights: • Lys27 in NEDD8 is critical for protein neddylation. • NEDD8 K27R mutant impairs the NEDD8 conjugation. • NEDD8 K27R mutant significantly reduces the activity of cullin-RING E3 ligases.

  4. Involvement of NF-κB and muscle specific E3 ubiquitin ligase MuRF1 in cigarette smoke-induced catabolism in C2 myotubes.

    Science.gov (United States)

    Kaisari, Sharon; Rom, Oren; Aizenbud, Dror; Reznick, Abraham Z

    2013-01-01

    Cigarette smoking has been identified as a risk factor for muscular damage and sarcopenia, the age-related loss of muscle mass and strength in old age. Cigarette smoke (CS)-induced oxidative stress and p38 MAPK activation have been shown to be the main cellular mechanisms leading to skeletal muscle catabolism. In order to investigate the involvement of NF-κB as another possible cellular mechanism by which CS promotes muscle catabolism, C2 myotubes, from an in vitro skeletal muscle cell line, were exposed to different time periods of whole vapor phase CS in the presence or absence of NF-κB inhibitor, IMD-0354. The CS-induced reduction in diameter of myotubes and time-dependent degradation of the main contractile protein myosin heavy chain were abolished by NF-κB inhibition. Also, C2 exposure to CS resulted in IκB-α degradation and NF-κB activation, which led to upregulation of the muscle specific E3 ubiquitin ligase MuRF1, but not MAFbx/atrogin-1. In conclusion, our results demonstrate that vapor phase CS exposure to skeletal myotubes triggers NF-κB activation leading to skeletal muscle cell damage and breakdown of muscle proteins mediated by muscle specific E3 ubiquitin ligase MuRF1. Our findings provide another possible molecular mechanism for the catabolic effects of CS in skeletal muscle.

  5. The E3 Ubiquitin Ligase IDOL Induces the Degradation of the Low Density Lipoprotein Receptor Family Members VLDLR and ApoER2*

    Science.gov (United States)

    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

  6. Distinct Functional Domains Contribute to Degradation of the Low Density Lipoprotein Receptor (LDLR) by the E3 Ubiquitin Ligase Inducible Degrader of the LDLR (IDOL)

    Science.gov (United States)

    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

  7. AtPUB 19, a U-Box E3 Ubiquitin Ligase, Negatively Regulates Abscisic Acid and Drought Responses in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Yong-Chang Liu; Yao-Rong Wu; Xia-He Huang; Jie Sun; Qi Xie

    2011-01-01

    Ubiquitination is an important protein post-translational modification,which is involved in various cellular processes in higher plants,and U-box E3 ligases play important roles in diverse functions in eukaryotes.Here,we describe the functions of Arabidopsis thaliana PUB19 (AtPUB19),which we demonstrated in an in vitro assay to encode a U-box type E3 ubiquitin ligase.AtPUB19 was up-regulated by drought,salt,cold,and abscisic acid (ABA).Down-regulation of AtPUB19led to hypersensitivity to ABA,enhanced ABA-induced stomatal closing,and enhanced drought tolerance,while AtPUB 19overexpression resulted in the reverse phenotypes.Molecular analysis showed that the expression levels of a number of ABA and stress marker genes were altered in both AtPUB 19 overexpressing and atpub 19-1 mutant plants.In summary,our data show that AtPUB19 negatively regulates ABA and drought responses in A.thaliana.

  8. Effects of Nephrolithiasis on Serum DNase (Deoxyribonuclease I and II) Activity and E3 SUMO-Protein Ligase NSE2 (NSMCE2) in Malaysian Individuals

    Institute of Scientific and Technical Information of China (English)

    Faridah Yusof; Atheer Awad Mehde; Wesen Adel Mehdi; Raha Ahmed Raus; Hamid Ghazali; Azlina Abd Rahman

    2015-01-01

    Objective Nephrolithiasis is one of the most common disorders of the urinary tract. The aim of this study was to examine a possible relationship between DNase I/II activity and E3 SUMO-protein ligase NSE2 in the sera of nephrolithiasis patients to evaluate the possibility of a new biomarker for evaluating kidney damage. Methods Sixty nephrolithiasis patients and 50 control patients were enrolled in a case-control study. Their blood urea, creatinine, protein levels and DNase I/II activity levels were measured by spectrometry. Serum NSMCE2 levels were measured by ELISA. Blood was collected from patients of the government health clinics in Kuantan-Pahang and fulfilled the inclusion criteria. Results The result indicated that mean levels of sera NSMCE2 have a significantly increase (P Conclusion This study suggests that an increase in serum concentrations of DNase I/II and E3 SUMO-protein ligase NSE2 level can be used as indicators for the diagnosis of kidney injury in patients with nephrolithiasis.

  9. The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule pathway, stabilizes Tex19.1 during spermatogenesis.

    Directory of Open Access Journals (Sweden)

    Fang Yang

    Full Text Available Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation or altered biochemical properties. The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule proteolytic pathway, recognizes proteins with N-terminal destabilizing residues and plays an important role in spermatogenesis. Tex19.1 (also known as Tex19 has been previously identified as a germ cell-specific protein in mouse testis. Here we report that Tex19.1 forms a stable protein complex with Ubr2 in mouse testes. The binding of Tex19.1 to Ubr2 is independent of the second position cysteine of Tex19.1, a putative target for arginylation by the N-end rule pathway R-transferase. The Tex19.1-null mouse mutant phenocopies the Ubr2-deficient mutant in three aspects: heterogeneity of spermatogenic defects, meiotic chromosomal asynapsis, and embryonic lethality preferentially affecting females. In Ubr2-deficient germ cells, Tex19.1 is transcribed, but Tex19.1 protein is absent. Our results suggest that the binding of Ubr2 to Tex19.1 metabolically stabilizes Tex19.1 during spermatogenesis, revealing a new function for Ubr2 outside the conventional N-end rule pathway.

  10. The Arabidopsis U-box E3 Ubiquitin ligase PUB30 Negatively Regulates Salt Tolerance by Facilitating BRI1 KINASE INHIBITOR 1 (BKI1) Degradation.

    Science.gov (United States)

    Zhang, Ming; Zhao, Jinfeng; Li, Long; Gao, Yanan; Zhao, Linlin; Patil, Suyash Bhimgonda; Fang, Jingjing; Zhang, Wenhui; Yang, Yuhong; Li, Ming; Li, Xueyong

    2017-09-02

    The Arabidopsis U-box E3 ubiquitin (Ub) ligases play an important role in the ubiquitin/26S proteasome-mediated protein degradation pathway. Recently PUB30 has been reported to participate in the salt stress response during seed germination stage in ABA-independent manner, but the molecular mechanism remains to be elucidated. Here we displayed that the pub30 mutant was more tolerant to salt stress during seed germination, whereas the mutant of its closest homologue PUB31 showed mild sensitivity to salt stress. PUB30 exhibited E3 ubiquitin ligase activity in vitro. PUB30 specifically interacted with BRI1 KINASE INHIBITOR 1 (BKI1), a regulator playing dual roles in brassinosteroids (BRs) signaling, in vitro and in vivo. We found that BKI1 protein was ubiquitinated and degraded by the 26S proteasome. The degradation of BKI1 was slowed down in the pub30-1 mutant compared with that in the wild-type. The bki1 mutant was sensitive to salt whereas the transgenic plants overexpressing BKI1 showed salt tolerant phenotype. All these results indicate that PUB30 negatively regulates salt tolerance probably through regulating the degradation of BKI1 and BRs signaling in Arabidopsis. This article is protected by copyright. All rights reserved.

  11. TRIM4; a novel mitochondrial interacting RING E3 ligase, sensitizes the cells to hydrogen peroxide (H2O2) induced cell death.

    Science.gov (United States)

    Tomar, Dhanendra; Prajapati, Paresh; Lavie, Julie; Singh, Kritarth; Lakshmi, Sripada; Bhatelia, Khyati; Roy, Milton; Singh, Rochika; Bénard, Giovanni; Singh, Rajesh

    2015-12-01

    The emerging evidences suggest that posttranslational modification of target protein by ubiquitin (Ub) not only regulate its turnover through ubiquitin proteasome system (UPS) but is a critical regulator of various signaling pathways. During ubiquitination, E3 ligase recognizes the target protein and determines the topology of ubiquitin chains. In current study, we studied the role of TRIM4, a member of the TRIM/RBCC protein family of RING E3 ligase, in regulation of hydrogen peroxide (H2O2) induced cell death. TRIM4 is expressed differentially in human tissues and expressed in most of the analyzed human cancer cell lines. The subcellular localization studies showed that TRIM4 forms distinct cytoplasmic speckle like structures which transiently interacts with mitochondria. The expression of TRIM4 induces mitochondrial aggregation and increased level of mitochondrial ROS in the presence of H2O2. It sensitizes the cells to H2O2 induced death whereas knockdown reversed the effect. TRIM4 potentiates the loss of mitochondrial transmembrane potential and cytochrome c release in the presence of H2O2. The analysis of TRIM4 interacting proteins showed its interaction with peroxiredoxin 1 (PRX1), including other proteins involved in regulation of mitochondrial and redox homeostasis. TRIM4 interaction with PRX1 is critical for the regulation of H2O2 induced cell death. Collectively, the evidences in the current study suggest the role of TRIM4 in regulation of oxidative stress induced cell death.

  12. The Medicago truncatula E3 Ubiquitin Ligase PUB1 Interacts with the LYK3 Symbiotic Receptor and Negatively Regulates Infection and Nodulation[W][OA

    Science.gov (United States)

    Mbengue, Malick; Camut, Sylvie; de Carvalho-Niebel, Fernanda; Deslandes, Laurent; Froidure, Solène; Klaus-Heisen, Dörte; Moreau, Sandra; Rivas, Susana; Timmers, Ton; Hervé, Christine; Cullimore, Julie; Lefebvre, Benoit

    2010-01-01

    LYK3 is a lysin motif receptor-like kinase of Medicago truncatula, which is essential for the establishment of the nitrogen-fixing, root nodule symbiosis with Sinorhizobium meliloti. LYK3 is a putative receptor of S. meliloti Nod factor signals, but little is known of how it is regulated and how it transduces these symbiotic signals. In a screen for LYK3-interacting proteins, we identified M. truncatula Plant U-box protein 1 (PUB1) as an interactor of the kinase domain. In planta, both proteins are localized and interact in the plasma membrane. In M. truncatula, PUB1 is expressed specifically in symbiotic conditions, is induced by Nod factors, and shows an overlapping expression pattern with LYK3 during nodulation. Biochemical studies show that PUB1 has a U-box–dependent E3 ubiquitin ligase activity and is phosphorylated by the LYK3 kinase domain. Overexpression and RNA interference studies in M. truncatula show that PUB1 is a negative regulator of the LYK3 signaling pathway leading to infection and nodulation and is important for the discrimination of rhizobia strains producing variant Nod factors. The potential role of PUB E3 ubiquitin ligases in controlling plant–microbe interactions and development through interacting with receptor-like kinases is discussed. PMID:20971894

  13. Effects of Nephrolithiasis on Serum DNase (Deoxyribonuclease I and II) Activity and E3 SUMO-Protein Ligase NSE2 (NSMCE2) in Malaysian Individuals.

    Science.gov (United States)

    Yusof, Faridah; Mehde, Atheer Awad; Mehdi, Wesen Adel; Raus, Raha Ahmed; Ghazali, Hamid; Rahman, Azlina Abd

    2015-09-01

    Nephrolithiasis is one of the most common disorders of the urinary tract. The aim of this study was to examine a possible relationship between DNase I/II activity and E3 SUMO-protein ligase NSE2 in the sera of nephrolithiasis patients to evaluate the possibility of a new biomarker for evaluating kidney damage. Sixty nephrolithiasis patients and 50 control patients were enrolled in a case-control study. Their blood urea, creatinine, protein levels and DNase I/II activity levels were measured by spectrometry. Serum NSMCE2 levels were measured by ELISA. Blood was collected from patients of the government health clinics in Kuantan-Pahang and fulfilled the inclusion criteria. The result indicated that mean levels of sera NSMCE2 have a significantly increase (P<0.01) in patients compared to control group. Compared with control subjects, activities and specific activities of serum DNase I and II were significantly elevated in nephrolithiasis patients (P$lt;0.01). This study suggests that an increase in serum concentrations of DNase I/II and E3 SUMO-protein ligase NSE2 level can be used as indicators for the diagnosis of kidney injury in patients with nephrolithiasis. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  14. A Tale of Two PMLs: Elements Regulating a Differential Substrate Recognition by the ICP0 E3 Ubiquitin Ligase of Herpes Simplex Virus 1.

    Science.gov (United States)

    Zheng, Yi; Samrat, Subodh Kumar; Gu, Haidong

    2016-12-01

    Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an α gene product required for viral replication at low multiplicities of infection. Upon entry, nuclear domain 10 (ND10) converges at the incoming DNA and represses viral gene expression. ICP0 contains a RING-type E3 ubiquitin ligase that degrades the ND10 organizer PML and disperses ND10 to alleviate the repression. In the present study, we focused on understanding the regulation of ICP0 E3 ligase activity in the degradation of different ICP0 substrates. We report the following. (i) A SUMO interaction motif located at ICP0 residues 362 to 364 is required for the degradation of PML isoforms II, IV, and VI but not isoform I. This differentiation mechanism exists in both HEp-2 and U2OS cells, regardless of the cell's permissiveness to the ICP0-null virus. (ii) Physical interaction between SIM362-364 and PML II is necessary but not sufficient for PML II degradation. Both proximal sequences surrounding SIM362-364 and distal sequences located at the ICP0 C terminus enhance the degradation of PML II. (iii) The ICP0 C terminus is dispensable for PML I degradation. Instead, bipartite PML I binding domains located in the N-terminal half of ICP0 coordinate to promote the degradation of PML I. (iv) The stability of ICP0, but not its ND10 fusion ability, affects the rate of PML I degradation. Taken together, our results show that ICP0 uses at least two regulatory mechanisms to differentiate its substrates. The disparate recognition of the ICP0 E3 substrates may be related to the different roles these substrates may play in HSV-1 infection.

  15. The E3 ubiquitin-ligase Bmi1/Ring1A controls the proteasomal degradation of Top2alpha cleavage complex - a potentially new drug target.

    Directory of Open Access Journals (Sweden)

    Iris Alchanati

    Full Text Available BACKGROUND: The topoisomerases Top1, Top2alpha and Top2beta are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2beta are subject to proteasomal degradation, this phenomena was not demonstrated for Top2alpha. METHODOLOGY/PRINCIPAL FINDINGS: We show here that Top2alpha is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2beta. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2alpha degradation, increases the persistence of Top2alpha-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2alpha in-vitro and cellular overexpression of Bmi1 increases drug induced Top2alpha ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2alpha ubiquitination and drug-induced Top2alpha degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner. CONCLUSIONS/SIGNIFICANCE: The discovery that poisoned Top2alpha is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1.

  16. Overexpression of E3 Ubiquitin Ligase Gene AdBiL Contributes to Resistance against Chilling Stress and Leaf Mold Disease in Tomato

    Directory of Open Access Journals (Sweden)

    Shuangchen Chen

    2017-06-01

    Full Text Available Ubiquitination is a common regulatory mechanism, playing a critical role in diverse cellular and developmental processes in eukaryotes. However, a few reports on the functional correlation between E3 ubiquitin ligases and reactive oxygen species (ROS or reactive nitrogen species (RNS metabolism in response to stress are currently available in plants. In the present study, the E3 ubiquitin ligase gene AdBiL (Adi3 Binding E3 Ligase was introduced into tomato line Ailsa Craig via Agrobacterium-mediated method. Transgenic lines were confirmed for integration into the tomato genome using PCR. Transcription of AdBiL in various transgenic lines was determined using real-time PCR. Evaluation of stress tolerance showed that T1 generation of transgenic tomato lines showed only mild symptoms of chilling injury as evident by higher biomass accumulation and chlorophyll content than those of non-transformed plants. Compared with wild-type plants, the contents of AsA, AsA/DHA, GSH and the activity of GaILDH, γ-GCS and GSNOR were increased, while H2O2, O2.−, MDA, NO, SNOs, and GSNO accumulations were significantly decreased in AdBiL overexpressing plants in response to chilling stress. Furthermore, transgenic tomato plants overexpressing AdBiL showed higher activities of enzymes such as G6PDH, 6PGDH, NADP-ICDH, and NADP-ME involved in pentose phosphate pathway (PPP. The transgenic tomato plants also exhibited an enhanced tolerance against the necrotrophic fungus Cladosporium fulvum. Tyrosine nitration protein was activated in the plants infected with leaf mold disease, while the inhibition could be recovered in AdBiL gene overexpressing lines. Taken together, our results revealed a possible physiological role of AdBiL in the activation of the key enzymes of AsA–GSH cycle, PPP and down-regulation of GSNO reductase, thereby reducing oxidative and nitrosative stress in plants. This study demonstrates an optimized transgenic strategy using AdBiL gene for crop

  17. Yeast sterol regulatory element-binding protein (SREBP) cleavage requires Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing subunit of the Golgi Dsc E3 ligase.

    Science.gov (United States)

    Stewart, Emerson V; Lloyd, S Julie-Ann; Burg, John S; Nwosu, Christine C; Lintner, Robert E; Daza, Riza; Russ, Carsten; Ponchner, Karen; Nusbaum, Chad; Espenshade, Peter J

    2012-01-01

    Schizosaccharomyces pombe Sre1 is a membrane-bound transcription factor that controls adaptation to hypoxia. Like its mammalian homolog, sterol regulatory element-binding protein (SREBP), Sre1 activation requires release from the membrane. However, in fission yeast, this release occurs through a strikingly different mechanism that requires the Golgi Dsc E3 ubiquitin ligase complex and the proteasome. The mechanistic details of Sre1 cleavage, including the link between the Dsc E3 ligase complex and proteasome, are not well understood. Here, we present results of a genetic selection designed to identify additional components required for Sre1 cleavage. From the selection, we identified two new components of the fission yeast SREBP pathway: Dsc5 and Cdc48. The AAA (ATPase associated with diverse cellular activities) ATPase Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing protein, interact with known Dsc complex components and are required for SREBP cleavage. These findings provide a mechanistic link between the Dsc E3 ligase complex and the proteasome in SREBP cleavage and add to a growing list of similarities between the Dsc E3 ligase and membrane E3 ligases involved in endoplasmic reticulum-associated degradation.

  18. Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin.

    Science.gov (United States)

    Lazarou, Michael; Jin, Seok Min; Kane, Lesley A; Youle, Richard J

    2012-02-14

    Mutations in the mitochondrial kinase PINK1 and the cytosolic E3 ligase Parkin can cause Parkinson's disease. Damaged mitochondria accumulate PINK1 on the outer membrane where, dependent on kinase activity, it recruits and activates Parkin to induce mitophagy, potentially maintaining organelle fidelity. How PINK1 recruits Parkin is unknown. We show that endogenous PINK1 forms a 700 kDa complex with the translocase of the outer membrane (TOM) selectively on depolarized mitochondria whereas PINK1 ectopically targeted to the outer membrane retains association with TOM on polarized mitochondria. Inducibly targeting PINK1 to peroxisomes or lysosomes, which lack a TOM complex, recruits Parkin and activates ubiquitin ligase activity on the respective organelles. Once there, Parkin induces organelle selective autophagy of peroxisomes but not lysosomes. We propose that the association of PINK1 with the TOM complex allows rapid reimport of PINK1 to rescue repolarized mitochondria from mitophagy, and discount mitochondrial-specific factors for Parkin translocation and activation. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. The Role of E3 Ubiquitin Ligase Cbl Proteins in β-Elemene Reversing Multi-Drug Resistance of Human Gastric Adenocarcinoma Cells

    Directory of Open Access Journals (Sweden)

    Xue-Jun Hu

    2013-05-01

    Full Text Available Recent studies indicate that β-elemene, a compound isolated from the Chinese herbal medicine Curcuma wenyujin, is capable of reversing tumor MDR, although the mechanism remains elusive. In this study, β-Elemene treatment markedly increased the intracellular accumulation of doxorubicin (DOX and rhodamine 123 in both K562/DNR and SGC7901/ADR cells and significantly inhibited the expression of P-gp. Treatment of SGC7901/ADR cells with β-elemene led to downregulation of Akt phosphorylation and significant upregulation of the E3 ubiquitin ligases, c-Cbl and Cbl-b. Importantly, β-elemene significantly enhanced the anti-tumor activity of DOX in nude mice bearing SGC7901/ADR xenografts. Taken together, our results suggest that β-elemene may target P-gp-overexpressing leukemia and gastric cancer cells to enhance the efficacy of DOX treatment.

  20. Shigella IpaH0722 E3 Ubiquitin Ligase Effector Targets TRAF2 to Inhibit PKC–NF-κB Activity in Invaded Epithelial Cells

    Science.gov (United States)

    Ashida, Hiroshi; Nakano, Hiroyasu; Sasakawa, Chihiro

    2013-01-01

    NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella's type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation. PMID:23754945

  1. The homeodomain transcription factor Hoxa2 interacts with and promotes the proteasomal degradation of the E3 ubiquitin protein ligase RCHY1.

    Directory of Open Access Journals (Sweden)

    Isabelle Bergiers

    Full Text Available Hox proteins are conserved homeodomain transcription factors known to be crucial regulators of animal development. As transcription factors, the functions and modes of action (co-factors, target genes of Hox proteins have been very well studied in a multitude of animal models. However, a handful of reports established that Hox proteins may display molecular activities distinct from gene transcription regulation. Here, we reveal that Hoxa2 interacts with 20S proteasome subunits and RCHY1 (also known as PIRH2, an E3 ubiquitin ligase that targets p53 for degradation. We further show that Hoxa2 promotes proteasome-dependent degradation of RCHY1 in an ubiquitin-independent manner. Correlatively, Hoxa2 alters the RCHY1-mediated ubiquitination of p53 and promotes p53 stabilization. Together, our data establish that Hoxa2 can regulate the proteasomal degradation of RCHY1 and stabilization of p53.

  2. Iron-induced skeletal muscle atrophy involves an Akt-forkhead box O3-E3 ubiquitin ligase-dependent pathway.

    Science.gov (United States)

    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 forkhead box O3 (FOXO3a) in skeletal muscles. Inhibition of FOXO3a using siRNA in vitro in C2C12 myotube cells inhibited iron-induced upregulation of atrogin-1 and MuRF1 and reversed the reduction in myotube diameters. Iron-load caused oxidative stress, and an oxidative stress inhibitor abrogated iron-induced muscle atrophy by reactivating the Akt-FOXO3a pathway. Iron-induced skeletal muscle atrophy is suggested to involve the E3 ubiquitin ligase mediated by the reduction of Akt-FOXO3a signaling by oxidative stress.

  3. BPM-CUL3 E3 ligase modulates thermotolerance by facilitating negative regulatory domain-mediated degradation of DREB2A in Arabidopsis.

    Science.gov (United States)

    Morimoto, Kyoko; Ohama, Naohiko; Kidokoro, Satoshi; Mizoi, Junya; Takahashi, Fuminori; Todaka, Daisuke; Mogami, Junro; Sato, Hikaru; Qin, Feng; Kim, June-Sik; Fukao, Yoichiro; Fujiwara, Masayuki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2017-09-18

    DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) acts as a key transcription factor in both drought and heat stress tolerance in Arabidopsis and induces the expression of many drought- and heat stress-inducible genes. Although DREB2A expression itself is induced by stress, the posttranslational regulation of DREB2A, including protein stabilization, is required for its transcriptional activity. The deletion of a 30-aa central region of DREB2A known as the negative regulatory domain (NRD) transforms DREB2A into a stable and constitutively active form referred to as DREB2A CA. However, the molecular basis of this stabilization and activation has remained unknown for a decade. Here we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the Cullin3 (CUL3)-based E3 ligase, as DREB2A-interacting proteins. We observed that DREB2A and BPMs interact in the nuclei, and that the NRD of DREB2A is sufficient for its interaction with BPMs. BPM-knockdown plants exhibited increased DREB2A accumulation and induction of DREB2A target genes under heat and drought stress conditions. Genetic analysis indicated that the depletion of BPM expression conferred enhanced thermotolerance via DREB2A stabilization. Thus, the BPM-CUL3 E3 ligase is likely the long-sought factor responsible for NRD-dependent DREB2A degradation. Through the negative regulation of DREB2A stability, BPMs modulate the heat stress response and prevent an adverse effect of excess DREB2A on plant growth. Furthermore, we found the BPM recognition motif in various transcription factors, implying a general contribution of BPM-mediated proteolysis to divergent cellular responses via an accelerated turnover of transcription factors.

  4. Ubiquitination and degradation of CFTR by the E3 ubiquitin ligase MARCH2 through its association with adaptor proteins CAL and STX6.

    Science.gov (United States)

    Cheng, Jie; Guggino, William

    2013-01-01

    Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent binding, ubiquitination, and degradation of mature CFTR. We found that MARCH2 not only co-immunoprecipitated and co-localized with CAL and STX6, but its binding to CAL was also enhanced by STX6, suggesting a synergistic interaction. In vivo ubiquitination assays demonstrated the ubiquitination of CFTR by MARCH2, and overexpression of MARCH2, like that of CAL and STX6, led to a dose-dependent degradation of mature CFTR that was blocked by bafilomycin A1 treatment. A catalytically dead MARCH2 RING mutant was unable to promote CFTR degradation. In addition, MARCH2 had no effect on a CFTR mutant lacking the PDZ motif, suggesting that binding to the PDZ domain of CAL is required for MARCH2-mediated degradation of CFTR. Indeed, silencing of endogenous CAL ablated the effect of MARCH2 on CFTR. Consistent with its Golgi localization, MARCH2 had no effect on ER-localized ΔF508-CFTR. Finally, siRNA-mediated silencing of endogenous MARCH2 in the CF epithelial cell line CFBE-CFTR increased the abundance of mature CFTR. Taken together, these data suggest that the recruitment of the E3 ubiquitin ligase MARCH2 to the CAL complex and subsequent ubiquitination of CFTR are responsible for the CAL-mediated lysosomal degradation of mature CFTR.

  5. Haploinsufficiency of the E3 ubiquitin ligase C-terminus of heat shock cognate 70 interacting protein (CHIP) produces specific behavioral impairments.

    Science.gov (United States)

    McLaughlin, Bethann; Buendia, Matthew A; Saborido, Tommy P; Palubinsky, Amy M; Stankowski, Jeannette N; Stanwood, Gregg D

    2012-01-01

    The multifunctional E3 ubiquitin ligase CHIP is an essential interacting partner of HSP70, which together promote the proteasomal degradation of client proteins. Acute CHIP overexpression provides neuroprotection against neurotoxic mitochondrial stress, glucocorticoids, and accumulation of toxic amyloid fragments, as well as genetic mutations in other E3 ligases, which have been shown to result in familial Parkinson's disease. These studies have created a great deal of interest in understanding CHIP activity, expression and modulation. While CHIP knockout mice have the potential to provide essential insights into the molecular control of cell fate and survival, the animals have been difficult to characterize in vivo due to severe phenotypic and behavioral dysfunction, which have thus far been poorly characterized. Therefore, in the present study we conducted a battery of neurobehavioral and physiological assays of adult CHIP heterozygotic (HET) mutant mice to provide a better understanding of the functional consequence of CHIP deficiency. We found that CHIP HET mice had normal body and brain weight, body temperature, muscle tone and breathing patterns, but do have a significant elevation in baseline heart rate. Meanwhile basic behavioral screens of sensory, motor, emotional and cognitive functions were normative. We observed no alterations in performance in the elevated plus maze, light-dark preference and tail suspension assays, or two simple cognitive tasks: novel object recognition and spontaneous alternation in a Y maze. Significant deficits were found, however, when CHIP HET mice performed wire hang, inverted screen, wire maneuver, and open field tasks. Taken together, our data indicate a clear subset of behaviors that are altered at baseline in CHIP deficient animals, which will further guide whole animal studies of the effects of CHIP dysregulation on cardiac function, brain circuitry and function, and responsiveness to environmental and cellular stress.

  6. Haploinsufficiency of the E3 ubiquitin ligase C-terminus of heat shock cognate 70 interacting protein (CHIP produces specific behavioral impairments.

    Directory of Open Access Journals (Sweden)

    Bethann McLaughlin

    Full Text Available The multifunctional E3 ubiquitin ligase CHIP is an essential interacting partner of HSP70, which together promote the proteasomal degradation of client proteins. Acute CHIP overexpression provides neuroprotection against neurotoxic mitochondrial stress, glucocorticoids, and accumulation of toxic amyloid fragments, as well as genetic mutations in other E3 ligases, which have been shown to result in familial Parkinson's disease. These studies have created a great deal of interest in understanding CHIP activity, expression and modulation. While CHIP knockout mice have the potential to provide essential insights into the molecular control of cell fate and survival, the animals have been difficult to characterize in vivo due to severe phenotypic and behavioral dysfunction, which have thus far been poorly characterized. Therefore, in the present study we conducted a battery of neurobehavioral and physiological assays of adult CHIP heterozygotic (HET mutant mice to provide a better understanding of the functional consequence of CHIP deficiency. We found that CHIP HET mice had normal body and brain weight, body temperature, muscle tone and breathing patterns, but do have a significant elevation in baseline heart rate. Meanwhile basic behavioral screens of sensory, motor, emotional and cognitive functions were normative. We observed no alterations in performance in the elevated plus maze, light-dark preference and tail suspension assays, or two simple cognitive tasks: novel object recognition and spontaneous alternation in a Y maze. Significant deficits were found, however, when CHIP HET mice performed wire hang, inverted screen, wire maneuver, and open field tasks. Taken together, our data indicate a clear subset of behaviors that are altered at baseline in CHIP deficient animals, which will further guide whole animal studies of the effects of CHIP dysregulation on cardiac function, brain circuitry and function, and responsiveness to environmental and

  7. The Arabidopsis paralogs, PUB46 and PUB48, encoding U-box E3 ubiquitin ligases, are essential for plant response to drought stress.

    Science.gov (United States)

    Adler, Guy; Konrad, Zvia; Zamir, Lyad; Mishra, Amit Kumar; Raveh, Dina; Bar-Zvi, Dudy

    2017-01-11

    Plants respond to abiotic stress on physiological, biochemical and molecular levels. This includes a global change in their cellular proteome achieved by changes in the pattern of their protein synthesis and degradation. The ubiquitin-proteasome system (UPS) is a key player in protein degradation in eukaryotes. Proteins are marked for degradation by the proteasome by coupling short chains of ubiquitin polypeptides in a three-step pathway. The last and regulatory stage is catalyzed by a member of a large family of substrate-specific ubiquitin ligases. We have identified AtPUB46 and AtPUB48-two paralogous genes that encode ubiquitin ligases (E3s)-to have a role in the plant environmental response. The AtPUB46, -47, and -48 appear as tandem gene copies on chromosome 5, and we present a phylogenetic analysis that traces their evolution from an ancestral PUB-ARM gene. Single homozygous T-DNA insertion mutants of AtPUB46 and AtPUB48 displayed hypersensitivity to water stress; this was not observed for similar mutants of AtPUB47. Although the three genes show a similar spatial expression pattern, the steady state levels of their transcripts are differentially affected by abiotic stresses and plant hormones. AtPUB46 and AtPUB48 encode plant U-Box E3s and are involved in the response to water stress. Our data suggest that despite encoding highly homologous proteins, AtPUB46 and AtPUB48 biological activity does not fully overlap.

  8. HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3 Transcriptional Activity.

    Directory of Open Access Journals (Sweden)

    Gary T ZeRuth

    Full Text Available 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.

  9. Arabidopsis RING E3 ubiquitin ligase AtATL80 is negatively involved in phosphate mobilization and cold stress response in sufficient phosphate growth conditions.

    Science.gov (United States)

    Suh, Ji Yeon; Kim, Woo Taek

    2015-08-07

    Phosphate (Pi) remobilization in plants is critical to continuous growth and development. AtATL80 is a plasma membrane (PM)-localized RING E3 ubiquitin (Ub) ligase that belongs to the Arabidopsis Tóxicos en Levadura (ATL) family. AtATL80 was upregulated by long-term low Pi (0-0.02 mM KH2PO4) conditions in Arabidopsis seedlings. AtATL80-overexpressing transgenic Arabidopsis plants (35S:AtATL80-sGFP) displayed increased phosphorus (P) accumulation in the shoots and lower biomass, as well as reduced P-utilization efficiency (PUE) under high Pi (1 mM KH2PO4) conditions compared to wild-type plants. The loss-of-function atatl80 mutant line exhibited opposite phenotypic traits. The atatl80 mutant line bolted earlier than wild-type plants, whereas AtATL80-overexpressors bloomed significantly later and produced lower seed yields than wild-type plants under high Pi conditions. Thus, AtATL80 is negatively correlated not only with P content and PUE, but also with biomass and seed yield in Arabidopsis. In addition, AtATL80-overexpressors were significantly more sensitive to cold stress than wild-type plants, while the atatl80 mutant line exhibited an increased tolerance to cold stress. Taken together, our results suggest that AtATL80, a PM-localized ATL-type RING E3 Ub ligase, participates in the Pi mobilization and cold stress response as a negative factor in Arabidopsis.

  10. Endoplasmic reticulum protein quality control is determined by cooperative interactions between Hsp/c70 protein and the CHIP E3 ligase.

    Science.gov (United States)

    Matsumura, Yoshihiro; Sakai, Juro; Skach, William R

    2013-10-25

    The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligase activity. In a cell-free reconstituted ER-associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I)EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70.

  11. The Pepper RING Finger E3 Ligase, CaDIR1, Regulates the Drought Stress Response via ABA-Mediated Signaling

    Directory of Open Access Journals (Sweden)

    Sang-Wook Han

    2017-04-01

    Full Text Available Drought stress from soil or air limits plant growth and development, leading to a reduction in crop productivity. Several E3 ligases positively or negatively regulate the drought stress response. In the present study, we show that the pepper (Capsicum annuum Drought Induced RING type E3 ligase 1, CaDIR1, regulates the drought stress response via abscisic acid (ABA-mediated signaling. CaDIR1 contains a C3HC4-type RING finger domain in the N-terminal region; this domain functions during protein degradation via attachment of ubiquitins to the substrate target proteins. The expression levels of the CaDIR1 gene were suppressed and induced by ABA and drought treatments, respectively. We conducted loss-of-function and gain-of function genetic studies to examine the in vivo function of CaDIR1 in response to ABA and drought stress. CaDIR1-silenced pepper plants displayed a drought-tolerant phenotype characterized by a low level of transpirational water loss via increased stomatal closure and elevated leaf temperatures. CaDIR1-overexpressing (OX Arabidopsis plants exhibited an ABA-hypersensitive phenotype during the germination stage, but an ABA-hyposensitive phenotype—characterized by decreased stomatal closure and reduced leaf temperatures—at the adult stage. Moreover, adult CaDIR1-OX plants exhibited a drought-sensitive phenotype characterized by high levels of transpirational water loss. Our results indicate that CaDIR1 functions as a negative regulator of the drought stress response via ABA-mediated signaling. Our findings provide a valuable insight into the plant defense mechanism that operates during drought stress.

  12. The E3 Ubiquitin Ligases, HUWE1 and NEDD4-1, Are Involved in the Post-translational Regulation of the ABCG1 and ABCG4 Lipid Transporters*

    Science.gov (United States)

    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

  13. Progressive Purkinje cell degeneration in tambaleante mutant mice is a consequence of a missense mutation in HERC1 E3 ubiquitin ligase.

    Directory of Open Access Journals (Sweden)

    Tomoji Mashimo

    2009-12-01

    Full Text Available The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domains have been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2 and small (HERC3-6. The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a GA transition at position 1448, causing a Gly to Glu substitution (Gly483Glu in the highly conserved N-terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein

  14. Study the effect of kidney stones on serum xanthine oxidase, ecto-5ʹ-nucleotidase activity and E3 SUMO-protein ligase NSE2 (NSMCE2 in Malaysian individuals

    Directory of Open Access Journals (Sweden)

    Faridah Yusof

    2015-08-01

    Conclusions: The present study suggests that the increase in serum of xanthine oxidase,ecto-5ʹ-nucleotidase activities E3 small ubiquitin-like modifier-protein ligase NSE2 concentration can be used as biomarkers for diagnosis of kidney damage in patients with kidney stone, also in developments of change DNA damage and inflammation disorders in these patients.

  15. A cullin E3 ubiquitin ligase complex associates with Rik1 and the Clr4 histone H3-K9 methyltransferase and is required for RNAi-mediated heterochromatin formation.

    Science.gov (United States)

    Hong, Eun-Jin Erica; Villén, Judit; Gerace, Erica L; Gygi, Steven P; Moazed, Danesh

    2005-01-01

    The assembly of heterochromatin in fission yeast and metazoans requires histone H3-lysine 9 (-K9) methylation by the conserved Clr4/Suv39h methyltransferase. In fission yeast, H3-K9 methylation requires components of the RNAi machinery and is initiated by the RNA-Induced Transcriptional Silencing (RITS) complex. Here we report the purification of a novel complex that associates with the Clr4 methyltransferase, termed the CLRC (CLr4-Rik1-Cul4) complex. By affinity purification of the Clr4-associated protein Rik1, we show that, in addition to Clr4, Rik1 is associated with the fission yeast E3 ubiquitin ligase Cullin4 (Cul4, encoded by cul4(+)), the ubiquitin-like protein, Ned8, and two previously uncharacterized proteins, designated Cmc1 and Cmc2. In addition, the complex contains substochiometric amounts of histones H2B and H4, and the 14-3-3 protein, Rad24. Deletion of cul4(+), cmc1(+), cmc2(+) and rad24(+) results in a complete loss of silencing of a ura4(+) reporter gene inserted within centromeric DNA repeats or the silent mating type locus. Each of the above deletions also results in accumulation of noncoding RNAs transcribed from centromeric repeats and telomeric DNA regions, and a corresponding loss of small RNAs that are homologous to centromeric repeats, suggesting a defect in the processing of noncoding RNA to small RNA. Based on these results, we propose that the components of the Clr4-Rik1-Cul4 complex act concertedly at an early step in heterochromatin formation.

  16. U-box E3 ubiquitin ligase PUB17 acts in the nucleus to promote specific immune pathways triggered by Phytophthora infestans.

    Science.gov (United States)

    He, Qin; McLellan, Hazel; Boevink, Petra C; Sadanandom, Ari; Xie, Conghua; Birch, Paul R J; Tian, Zhendong

    2015-06-01

    Ubiquitination regulates many processes in plants, including immunity. The E3 ubiquitin ligase PUB17 is a positive regulator of programmed cell death (PCD) triggered by resistance proteins CF4/9 in tomato. Its role in immunity to the potato late blight pathogen, Phytophthora infestans, was investigated here. Silencing StPUB17 in potato by RNAi and NbPUB17 in Nicotiana benthamiana by virus-induced gene silencing (VIGS) each enhanced P. infestans leaf colonization. PAMP-triggered immunity (PTI) transcriptional responses activated by flg22, and CF4/Avr4-mediated PCD were attenuated by silencing PUB17. However, silencing PUB17 did not compromise PCD triggered by P. infestans PAMP INF1, or co-expression of R3a/AVR3a, demonstrating that not all PTI- and PCD-associated responses require PUB17. PUB17 localizes to the plant nucleus and especially in the nucleolus. Transient over-expression of a dominant-negative StPUB17(V314I,V316I) mutant, which retained nucleolar localization, suppressed CF4-mediated cell death and enhanced P. infestans colonization. Exclusion of the StPUB17(V314I,V316I) mutant from the nucleus abolished its dominant-negative activity, demonstrating that StPUB17 functions in the nucleus. PUB17 is a positive regulator of immunity to late blight that acts in the nucleus to promote specific PTI and PCD pathways.

  17. The ARC1 E3 ligase gene is frequently deleted in self-compatible Brassicaceae species and has a conserved role in Arabidopsis lyrata self-pollen rejection.

    Science.gov (United States)

    Indriolo, Emily; Tharmapalan, Pirashaanthy; Wright, Stephen I; Goring, Daphne R

    2012-11-01

    Self-pollen rejection is an important reproductive regulator in flowering plants, and several different intercellular signaling systems have evolved to elicit this response. In the Brassicaceae, the self-incompatibility system is mediated by the pollen S-locus Cys-Rich/S-locus Protein11 (SCR/SP11) ligand and the pistil S Receptor Kinase (SRK). While the SCR/SP11-SRK recognition system has been identified in several species across the Brassicaceae, less is known about the conservation of the SRK-activated cellular responses in the stigma, following self-pollen contact. The ARM Repeat Containing1 (ARC1) E3 ubiquitin ligase functions downstream of SRK for the self-incompatibility response in Brassica, but it has been suggested that ARC1 is not required in Arabidopsis species. Here, we surveyed the presence of ARC1 orthologs in several recently sequenced genomes from Brassicaceae species that had diversified ∼20 to 40 million years ago. Surprisingly, the ARC1 gene was deleted in several species that had lost the self-incompatibility trait, suggesting that ARC1 may lose functionality in the transition to self-mating. To test the requirement of ARC1 in a self-incompatible Arabidopsis species, transgenic ARC1 RNA interference Arabidopsis lyrata plants were generated, and they exhibited reduced self-incompatibility responses resulting in successful fertilization. Thus, this study demonstrates a conserved role for ARC1 in the self-pollen rejection response within the Brassicaceae.

  18. Aging Triggers Cytoplasmic Depletion and Nuclear Translocation of the E3 Ligase Mahogunin: A Function for Ubiquitin in Neuronal Survival.

    Science.gov (United States)

    Benvegnù, Stefano; Mateo, María Inés; Palomer, Ernest; Jurado-Arjona, Jerónimo; Dotti, Carlos G

    2017-05-04

    A decline in proteasome function is causally connected to neuronal aging and aging-associated neuropathologies. By using hippocampal neurons in culture and in vivo, we show that aging triggers a reduction and a cytoplasm-to-nucleus redistribution of the E3 ubiquitin ligase mahogunin (MGRN1). Proteasome impairment induces MGRN1 monoubiquitination, the key post-translational modification for its nuclear entry. One potential mechanism for MGRN1 monoubiquitination is via progressive deubiquitination at the proteasome of polyubiquitinated MGRN1. Once in the nucleus, MGRN1 potentiates the transcriptional cellular response to proteotoxic stress. Inhibition of MGRN1 impairs ATF3-mediated neuronal responsiveness to proteosomal stress and increases neuronal stress, while increasing MGRN1 ameliorates signs of neuronal aging, including cognitive performance in old animals. Our results imply that, among others, the strength of neuronal survival in a proteasomal deterioration background, like during aging, depends on the fine-tuning of ubiquitination-deubiquitination. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Core-binding factor β increases the affinity between human Cullin 5 and HIV-1 Vif within an E3 ligase complex.

    Science.gov (United States)

    Salter, Jason D; Lippa, Geoffrey M; Belashov, Ivan A; Wedekind, Joseph E

    2012-11-06

    HIV-1 Vif masquerades as a receptor for a cellular E3 ligase harboring Elongin B, Elongin C, and Cullin 5 (EloB/C/Cul5) proteins that facilitate degradation of the antiretroviral factor APOBEC3G (A3G). This Vif-mediated activity requires human core-binding factor β (CBFβ) in contrast to cellular substrate receptors. We observed calorimetrically that Cul5 binds tighter to full-length Vif((1-192))/EloB/C/CBFβ (K(d) = 5 ± 2 nM) than to Vif((95-192))/EloB/C (K(d) = 327 ± 40 nM), which cannot bind CBFβ. A comparison of heat capacity changes supports a model in which CBFβ prestabilizes Vif((1-192)) relative to Vif((95-192)), consistent with a stronger interaction of Cul5 with Vif's C-terminal Zn(2+)-binding motif. An additional interface between Cul5 and an N-terminal region of Vif appears to be plausible, which has therapeutic design implications.

  20. HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains.

    Science.gov (United States)

    Wang, Chong; Long, Wenying; Peng, Chao; Hu, Lin; Zhang, Qiong; Wu, Ailing; Zhang, Xiaoqing; Duan, Xiaotao; Wong, Catherine C L; Tanaka, Yuetsu; Xia, Zongping

    2016-04-01

    The HTLV-1 oncoprotein Tax plays a key role in CD4+ T cell transformation by promoting cell proliferation and survival, mainly through permanent activation of the NK-κB pathway and induction of many NF-κB target genes. Elucidating the underlying molecular mechanism is therefore critical in understanding HTLV-1-mediated transformation. Current studies have suggested multiple but controversial mechanisms regarding Tax-induced IKK activation mainly due to blending of primary Tax-induced IKK activation events and secondary IKK activation events induced by cytokines secreted by the primary Tax-induced IKK-NF-κB activation events. We reconstituted Tax-stimulated IKK activation in a cell-free system to dissect the essential cellular components for primary IKK activation by Tax and studied the underlying biochemical mechanism. We found that Tax is a putative E3 ubiquitin ligase, which, together with UbcH2, UhcH5c, or UbcH7, catalyzes the assembly of free mixed-linkage polyubiquitin chains. These free mixed-linkage polyubiquitin chains are then responsible for direct IKK activation by binding to the NEMO subunit of IKK. Our studies revealed the biochemical function of Tax in the process of IKK activation, which utilizes the minimal cellular ubiquitination components for NF-κB activation.

  1. Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.

    Science.gov (United States)

    Mansur, Daniel S; Maluquer de Motes, Carlos; Unterholzner, Leonie; Sumner, Rebecca P; Ferguson, Brian J; Ren, Hongwei; Strnadova, Pavla; Bowie, Andrew G; Smith, Geoffrey L

    2013-02-01

    The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKβ causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase β-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished β-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

  2. Loss of the E3 ubiquitin ligase LRSAM1 sensitizes peripheral axons to degeneration in a mouse model of Charcot-Marie-Tooth disease

    Directory of Open Access Journals (Sweden)

    Laurent P. Bogdanik

    2013-05-01

    Charcot-Marie-Tooth disease (CMT is a clinically and genetically heterogeneous condition characterized by peripheral axon degeneration with subsequent motor and sensory deficits. Several CMT gene products function in endosomal sorting and trafficking to the lysosome, suggesting that defects in this cellular pathway might present a common pathogenic mechanism for these conditions. LRSAM1 is an E3 ubiquitin ligase that is implicated in this process, and mutations in LRSAM1 have recently been shown to cause CMT. We have generated mouse mutations in Lrsam1 to create an animal model of this form of CMT (CMT2P. Mouse Lrsam1 is abundantly expressed in the motor and sensory neurons of the peripheral nervous system. Both homozygous and heterozygous mice have largely normal neuromuscular performance and only a very mild neuropathy phenotype with age. However, Lrsam1 mutant mice are more sensitive to challenge with acrylamide, a neurotoxic agent that causes axon degeneration, indicating that the axons in the mutant mice are indeed compromised. In transfected cells, LRSAM1 primarily localizes in a perinuclear compartment immediately beyond the Golgi and shows little colocalization with components of the endosome to lysosome trafficking pathway, suggesting that other cellular mechanisms also merit consideration.

  3. Unique Pattern of Component Gene Disruption in the NRF2 Inhibitor KEAP1/CUL3/RBX1 E3-Ubiquitin Ligase Complex in Serous Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Victor D. Martinez

    2014-01-01

    Full Text Available The NFE2-related factor 2 (NRF2 pathway is critical to initiate responses to oxidative stress; however, constitutive activation occurs in different cancer types, including serous ovarian carcinomas (OVCA. The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is a regulator of NRF2 levels. Hence, we investigated the DNA-level mechanisms affecting these genes in OVCA. DNA copy-number loss (CNL, promoter hypermethylation, mRNA expression, and sequence mutation for KEAP1, CUL3, and RBX1 were assessed in a cohort of 568 OVCA from The Cancer Genome Atlas. Almost 90% of cases exhibited loss-of-function alterations in any components of the NRF2 inhibitory complex. CNL is the most prominent mechanism of component disruption, with RBX1 being the most frequently disrupted component. These alterations were associated with reduced mRNA expression of complex components, and NRF2 target gene expression was positively enriched in 90% of samples harboring altered complex components. Disruption occurs through a unique DNA-level alteration pattern in OVCA. We conclude that a remarkably high frequency of DNA and mRNA alterations affects components of the KEAP1/CUL3/RBX1 complex, through a unique pattern of genetic mechanisms. Together, these results suggest a key role for the KEAP1/CUL3/RBX1 complex and NRF2 pathway deregulation in OVCA.

  4. CDK1-dependent inhibition of the E3 ubiquitin ligase CRL4CDT2 ensures robust transition from S Phase to Mitosis.

    Science.gov (United States)

    Rizzardi, Lindsay F; Coleman, Kate E; Varma, Dileep; Matson, Jacob P; Oh, Seeun; Cook, Jeanette Gowen

    2015-01-02

    Replication-coupled destruction of a cohort of cell cycle proteins ensures efficient and precise genome duplication. Three proteins destroyed during replication via the CRL4(CDT2) ubiquitin E3 ligase, CDT1, p21, and SET8 (PR-SET7), are also essential or important during mitosis, making their reaccumulation after S phase a critical cell cycle event. During early and mid-S phase and during DNA repair, proliferating cell nuclear antigen (PCNA) loading onto DNA (PCNA(DNA)) triggers the interaction between CRL4(CDT2) and its substrates, resulting in their degradation. We have discovered that, beginning in late S phase, PCNA(DNA) is no longer sufficient to trigger CRL4(CDT2)-mediated degradation. A CDK1-dependent mechanism that blocks CRL4(CDT2) activity by interfering with CDT2 recruitment to chromatin actively protects CRL4(CDT2) substrates. We postulate that deliberate override of replication-coupled destruction allows anticipatory accumulation in late S phase. We further show that (as for CDT1) de novo SET8 reaccumulation is important for normal mitotic progression. In this manner, CDK1-dependent CRL4(CDT2) inactivation contributes to efficient transition from S phase to mitosis.

  5. Four Closely-related RING-type E3 Ligases, APD1-4,are Involved in Pollen Mitosis Ⅱ Regulation in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Guo Luo; Hongya Gu; Jingjing Liu; Li-Jia Qu

    2012-01-01

    Ubiquitination of proteins is one of the critical regulatory mechanisms in eukaryotes.In higher plants,protein ubiquitination plays an essential role in many biological processes,including hormone signaling,photomorphogenesis,and pathogen defense.However,the roles of protein ubiquitination in the reproductive process are not clear.In this study,we identified four plant-specific RING-finger genes designated (A)berrant (P)ollen (D)evelopment (1) (APD1) to APD4,as regulators of pollen mitosis Ⅱ (PMII) in Arabidopsis thaliana (L.).The apd1 apd2 double mutant showed a significantly increased percentage of bicellular-like pollen at the mature pollen stage.Further downregulation of the APD3 and APD4 transcripts in apd1 apd2 by RNA interference (RNAi) resulted in more severe abnormal bicellular-like pollen phenotypes than in apd1 apd2,suggesting that cell division was defective in male gametogenesis.All of the four genes were expressed in multiple stages at different levels during male gametophyte development.Confocal analysis using green florescence fusion proteins (GFP) GFP-APD1 and GFP-APD2 showed that APDs are associated with intracellular membranes.Furthermore,APD2 had E2-dependent E3 ligase activity in vitro,and five APD2-interacting proteins were identified.Our results suggest that these four genes may be involved,redundantly,in regulating the PMII process during male gametogenesis.

  6. The crystal structure of the Sgt1-Skp1 complex: the link between Hsp90 and both SCF E3 ubiquitin ligases and kinetochores

    Science.gov (United States)

    Willhoft, Oliver; Kerr, Richard; Patel, Dipali; Zhang, Wenjuan; Al-Jassar, Caezar; Daviter, Tina; Millson, Stefan H.; Thalassinos, Konstantinos; Vaughan, Cara K.

    2017-01-01

    The essential cochaperone Sgt1 recruits Hsp90 chaperone activity to a range of cellular factors including SCF E3 ubiquitin ligases and the kinetochore in eukaryotes. In these pathways Sgt1 interacts with Skp1, a small protein that heterodimerizes with proteins containing the F-box motif. We have determined the crystal structure of the interacting domains of Saccharomyces cerevisiae Sgt1 and Skp1 at 2.8 Å resolution and validated the interface in the context of the full-length proteins in solution. The BTB/POZ domain of Skp1 associates with Sgt1 via the concave surface of its TPR domain using residues that are conserved in humans. Dimerization of yeast Sgt1 occurs via an insertion that is absent from monomeric human Sgt1. We identify point mutations that disrupt dimerization and Skp1 binding in vitro and find that the interaction with Skp1 is an essential function of Sgt1 in yeast. Our data provide a structural rationale for understanding the phenotypes of temperature-sensitive Sgt1 mutants and for linking Skp1-associated proteins to Hsp90-dependent pathways. PMID:28139700

  7. p21-Activated kinase 6 (PAK6) inhibits prostate cancer growth via phosphorylation of androgen receptor and tumorigenic E3 ligase murine double minute-2 (Mdm2).

    Science.gov (United States)

    Liu, Tong; Li, Yang; Gu, Hui; Zhu, Ge; Li, Jiabin; Cao, Liu; Li, Feng

    2013-02-01

    The androgen receptor (AR) signaling pathway plays a crucial role in the development and growth of prostate malignancies. Regulation of AR homeostasis in prostate tumorigenesis has not yet been fully characterized. In this study, we demonstrate that p21-activated kinase 6 (PAK6) inhibits prostate tumorigenesis by regulating AR homeostasis. First, we demonstrated that in normal prostate epithelium, AR co-localizes with PAK6 in the cytoplasm and translocates into the nucleus in malignant prostate. Furthermore, AR phosphorylation at Ser-578 by PAK6 promotes AR-E3 ligase murine double minute-2 (Mdm2) association, causing AR degradation upon androgen stimuli. We also showed that PAK6 phosphorylates Mdm2 on Thr-158 and Ser-186, which is critical for AR ubiquitin-mediated degradation. Moreover, we found that Thr-158 collaborates with Ser-186 for AR-Mdm2 association and AR ubiquitin-mediated degradation as it facilitates PAK6-mediated AR homeostasis. PAK6 knockdown promotes prostate tumor growth in vivo. Interestingly, we found a strong inverse correlation between PAK6 and AR expression in the cytoplasm of prostate cancer cells. These observations indicate that PAK6 may be important for the maintenance of androgen-induced AR signaling homeostasis and in prostate malignancy, as well as being a possible new therapeutic target for AR-positive and hormone-sensitive prostate cancer.

  8. Overexpression of the human ubiquitin E3 ligase CUL4A alleviates hypoxia-reoxygenation injury in pheochromocytoma (PC12) cells

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Can [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Zhang, Li-Yang [Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Cancer Research Institute, Central South University, 110 Xiang Ya Road, Changsha 410078 (China); Chen, Hong [Department of Developmental Biology, School of Biological Science and Technology, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Xiao, Ling [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Liu, Xian-Peng, E-mail: xliu@lsuhsc.edu [Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932 (United States); Zhang, Jian-Xiang, E-mail: jianxiangzhang@yahoo.cn [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Department of Developmental Biology, School of Biological Science and Technology, Central South University, 172 Tong Zipo Road, Changsha 410013 (China)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Overexpression of human CUL4A (hCUL4A) in PC12 cells. Black-Right-Pointing-Pointer The effects of hCUL4A on hypoxia-reoxygenation injury were investigated. Black-Right-Pointing-Pointer hCUL4A suppresses apoptosis and DNA damage and thus promotes cell survival. Black-Right-Pointing-Pointer hCUL4A regulates apoptosis-related proteins and cell cycle regulators. -- Abstract: The ubiquitin E3 ligase CUL4A plays important roles in diverse cellular processes including carcinogenesis and proliferation. It has been reported that the expression of CUL4A can be induced by hypoxic-ischemic injury. However, the effect of elevated expression of CUL4A on hypoxia-reoxygenation injury is currently unclear. In this study, human CUL4A (hCUL4A) was expressed in rat pheochromocytoma (PC12) cells using adenoviral vector-mediated gene transfer, and the effects of hCUL4A expression on hypoxia-reoxygenation injury were investigated. In PC12 cells subjected to hypoxia and reoxygenation, we found that hCUL4A suppresses apoptosis and DNA damage by regulating apoptosis-related proteins and cell cycle regulators (Bcl-2, caspase-3, p53 and p27); consequently, hCUL4A promotes cell survival. Taken together, our results reveal the beneficial effects of hCUL4A in PC12 cells upon hypoxia-reoxygenation injury.

  9. Non-degradative ubiquitination of the Notch1 receptor by the E3 ligase MDM2 activates the Notch signalling pathway.

    Science.gov (United States)

    Pettersson, Susanne; Sczaniecka, Matylda; McLaren, Lorna; Russell, Fiona; Gladstone, Karen; Hupp, Ted; Wallace, Maura

    2013-03-15

    The Notch receptor is necessary for modulating cell fate decisions throughout development, and aberrant activation of Notch signalling has been associated with many diseases, including tumorigenesis. The E3 ligase MDM2 (murine double minute 2) plays a role in regulating the Notch signalling pathway through its interaction with NUMB. In the present study we report that MDM2 can also exert its oncogenic effects on the Notch signalling pathway by directly interacting with the Notch 1 receptor through dual-site binding. This involves both the N-terminal and acidic domains of MDM2 and the RAM [RBP-Jκ (recombination signal-binding protein 1 for Jκ)-associated molecule] and ANK (ankyrin) domains of Notch 1. Although the interaction between Notch1 and MDM2 results in ubiquitination of Notch1, this does not result in degradation of Notch1, but instead leads to activation of the intracellular domain of Notch1. Furthermore, MDM2 can synergize with Notch1 to inhibit apoptosis and promote proliferation. This highlights yet another target for MDM2-mediated ubiquitination that results in activation of the protein rather than degradation and makes MDM2 an attractive target for drug discovery for both the p53 and Notch signalling pathways.

  10. Submicroscopic duplications of the hydroxysteroid dehydrogenase HSD17B10 and the E3 ubiquitin ligase HUWE1 are associated with mental retardation.

    Science.gov (United States)

    Froyen, Guy; Corbett, Mark; Vandewalle, Joke; Jarvela, Irma; Lawrence, Owen; Meldrum, Cliff; Bauters, Marijke; Govaerts, Karen; Vandeleur, Lucianne; Van Esch, Hilde; Chelly, Jamel; Sanlaville, Damien; van Bokhoven, Hans; Ropers, Hans-Hilger; Laumonnier, Frederic; Ranieri, Enzo; Schwartz, Charles E; Abidi, Fatima; Tarpey, Patrick S; Futreal, P Andrew; Whibley, Annabel; Raymond, F Lucy; Stratton, Michael R; Fryns, Jean-Pierre; Scott, Rodney; Peippo, Maarit; Sipponen, Marjatta; Partington, Michael; Mowat, David; Field, Michael; Hackett, Anna; Marynen, Peter; Turner, Gillian; Gécz, Jozef

    2008-02-01

    Submicroscopic copy-number imbalances contribute significantly to the genetic etiology of human disease. Here, we report a novel microduplication hot spot at Xp11.22 identified in six unrelated families with predominantly nonsyndromic XLMR. All duplications segregate with the disease, including the large families MRX17 and MRX31. The minimal, commonly duplicated region contains three genes: RIBC1, HSD17B10, and HUWE1. RIBC1 could be excluded on the basis of its absence of expression in the brain and because it escapes X inactivation in females. For the other genes, expression array and quantitative PCR analysis in patient cell lines compared to controls showed a significant upregulation of HSD17B10 and HUWE1 as well as several important genes in their molecular pathways. Loss-of-function mutations of HSD17B10 have previously been associated with progressive neurological disease and XLMR. The E3 ubiquitin ligase HUWE1 has been implicated in TP53-associated regulation of the neuronal cell cycle. Here, we also report segregating sequence changes of highly conserved residues in HUWE1 in three XLMR families; these changes are possibly associated with the phenotype. Our findings demonstrate that an increased gene dosage of HSD17B10, HUWE1, or both contribute to the etiology of XLMR and suggest that point mutations in HUWE1 are associated with this disease too.

  11. E3 ubiquitin ligase CHIP interacts with C-type lectin-like receptor CLEC-2 and promotes its ubiquitin-proteasome degradation.

    Science.gov (United States)

    Shao, Miaomiao; Li, Lili; Song, Shushu; Wu, Weicheng; Peng, Peike; Yang, Caiting; Zhang, Mingming; Duan, Fangfang; Jia, Dongwei; Zhang, Jie; Wu, Hao; Zhao, Ran; Wang, Lan; Ruan, Yuanyuan; Gu, Jianxin

    2016-10-01

    C-type lectin-like receptor 2 (CLEC-2) was originally identified as a member of non-classical C-type lectin-like receptors in platelets and immune cells. Activation of CLEC-2 is involved in thrombus formation, lymphatic/blood vessel separation, platelet-mediated tumor metastasis and immune response. Nevertheless, the regulation of CLEC-2 expression is little understood. In this study, we identified that the C terminus of Hsc70-interacting protein (CHIP) interacted with CLEC-2 by mass spectrometry analysis, and CHIP decreased the protein expression of CLEC-2 through lysine-48-linked ubiquitination and proteasomal degradation. Deleted and point mutation also revealed that CHIP controlled CLEC-2 protein expression via both tetratricopeptide repeats (TPR) domain and Ubox domain in a HSP70/90-independent manner. Moreover, reduced CHIP expression was associated with decreased CLEC-2 polyubiquitination and increased CLEC-2 protein levels in PMA-induced differentiation of THP-1 monocytes into macrophages. These results indicate that CLEC-2 is the target substrate of E3 ubiquitin ligase CHIP, and suggest that the CHIP/CLEC-2 axis may play an important role in the modulation of immune response.

  12. The E3 ligase Ubr3 regulates Usher syndrome and MYH9 disorder proteins in the auditory organs of Drosophila and mammals

    Science.gov (United States)

    Li, Tongchao; Giagtzoglou, Nikolaos; Eberl, Daniel F; Jaiswal, Sonal Nagarkar; Cai, Tiantian; Godt, Dorothea; Groves, Andrew K; Bellen, Hugo J

    2016-01-01

    Myosins play essential roles in the development and function of auditory organs and multiple myosin genes are associated with hereditary forms of deafness. Using a forward genetic screen in Drosophila, we identified an E3 ligase, Ubr3, as an essential gene for auditory organ development. Ubr3 negatively regulates the mono-ubiquitination of non-muscle Myosin II, a protein associated with hearing loss in humans. The mono-ubiquitination of Myosin II promotes its physical interaction with Myosin VIIa, a protein responsible for Usher syndrome type IB. We show that ubr3 mutants phenocopy pathogenic variants of Myosin II and that Ubr3 interacts genetically and physically with three Usher syndrome proteins. The interactions between Myosin VIIa and Myosin IIa are conserved in the mammalian cochlea and in human retinal pigment epithelium cells. Our work reveals a novel mechanism that regulates protein complexes affected in two forms of syndromic deafness and suggests a molecular function for Myosin IIa in auditory organs. DOI: http://dx.doi.org/10.7554/eLife.15258.001 PMID:27331610

  13. SCF(JFK) is a bona fide E3 ligase for ING4 and a potent promoter of the angiogenesis and metastasis of breast cancer.

    Science.gov (United States)

    Yan, Ruorong; He, Lin; Li, Zhongwu; Han, Xiao; Liang, Jing; Si, Wenzhe; Chen, Zhe; Li, Lei; Xie, Guojia; Li, Wanjin; Wang, Peiyan; Lei, Liandi; Zhang, Hongquan; Pei, Fei; Cao, Dengfeng; Sun, Luyang; Shang, Yongfeng

    2015-03-15

    Loss of function/dysregulation of inhibitor of growth 4 (ING4) and hyperactivation of NF-κB are frequent events in many types of human malignancies. However, the molecular mechanisms underlying these remarkable aberrations are not understood. Here, we report that ING4 is physically associated with JFK. We demonstrated that JFK targets ING4 for ubiquitination and degradation through assembly of an Skp1-Cul1-F-box (SCF) complex. We showed that JFK-mediated ING4 destabilization leads to the hyperactivation of the canonical NF-κB pathway and promotes angiogenesis and metastasis of breast cancer. Significantly, the expression of JFK is markedly up-regulated in breast cancer, and the level of JFK is negatively correlated with that of ING4 and positively correlated with an aggressive clinical behavior of breast carcinomas. Our study identified SCF(JFK) as a bona fide E3 ligase for ING4 and unraveled the JFK-ING4-NF-κB axis as an important player in the development and progression of breast cancer, supporting the pursuit of JFK as a potential target for breast cancer intervention.

  14. The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development*♦

    Science.gov (United States)

    Rahman, Kazi; Zhao, Peng; Mandalasi, Msano; van der Wel, Hanke; Wells, Lance; Blader, Ira J.; West, Christopher M.

    2016-01-01

    Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts. PMID:26719340

  15. Phosphorylation of the E3 ubiquitin ligase RNF41 by the kinase Par-1b is required for epithelial cell polarity.

    Science.gov (United States)

    Lewandowski, Katherine T; Piwnica-Worms, Helen

    2014-01-15

    The establishment and maintenance of cell polarity is an essential property governing organismal homeostasis, and loss of polarity is a common feature of cancer cells. The ability of epithelial cells to establish apical-basal polarity depends on intracellular signals generated from polarity proteins, such as the Par-1 family of proteins, as well as extracellular signals generated through cell contacts with the extracellular matrix (ECM). The Par-1 family has a well-established role in regulating cell-cell contacts in the form of tight junctions by phosphorylating Par-3. In addition, Par-1 has been shown to impact on cell-ECM interactions by regulating laminin receptor localization and laminin deposition on the basal surface of epithelial cells. Laminins are major structural and signaling components of basement membrane (BM), a sheet of specialized ECM underlying epithelia. In this study, we identify RNF41, an E3 ubiquitin ligase, as a novel Par-1b (also known as MARK2) effector in the cell-ECM pathway. Par-1b binds to and phosphorylates RNF41 on serine 254. Phosphorylation of RNF41 by Par-1b is required for epithelial cells to localize laminin-111 receptors to their basolateral surfaces and to properly anchor to laminin-111. In addition, phosphorylation of RNF41 is required for epithelial cells to establish apical-basal polarity. Our data suggests that phosphorylation of RNF41 by Par-1b regulates basolateral membrane targeting of laminin-111 receptors, thereby facilitating cell anchorage to laminin-111 and ultimately forming the cell-ECM contacts required for epithelial cells to establish apical-basal cell polarity.

  16. Functional characterization of the SIZ/PIAS-type SUMO E3 ligases, OsSIZ1 and OsSIZ2 in rice

    KAUST Repository

    Park, Hyeongcheol

    2010-06-18

    Sumoylation is a post-translational regulatory process in diverse cellular processes in eukaryotes, involving conjugation/deconjugation of small ubiquitin-like modifier (SUMO) proteins to other proteins thus modifying their function. The PIAS [protein inhibitor of activated signal transducers and activators of transcription (STAT)] and SAP (scaffold attachment factor A/B/acinus/PIAS)/MIZ (SIZ) proteins exhibit SUMO E3-ligase activity that facilitates the conjugation of SUMO proteins to target substrates. Here, we report the isolation and molecular characterization of Oryza sativa SIZ1 (OsSIZ1) and SIZ2 (OsSIZ2), rice homologs of Arabidopsis SIZ1. The rice SIZ proteins are localized to the nucleus and showed sumoylation activities in a tobacco system. Our analysis showed increased amounts of SUMO conjugates associated with environmental stresses such as high and low temperature, NaCl and abscisic acid (ABA) in rice plants. The expression of OsSIZ1 and OsSIZ2 in siz1-2 Arabidopsis plants partially complemented the morphological mutant phenotype and enhanced levels of SUMO conjugates under heat shock conditions. In addition, ABA-hypersensitivity of siz1-2 seed germination was partially suppressed by OsSIZ1 and OsSIZ2. The results suggest that rice SIZ1 and SIZ2 are able to functionally complement Arabidopsis SIZ1 in the SUMO conjugation pathway. Their effects on the Arabidopsis mutant suggest a function for these genes related to stress responses and stress adaptation. © 2010 Blackwell Publishing Ltd.

  17. The putative E3 ubiquitin ligase ECERIFERUM9 regulates abscisic acid biosynthesis and response during seed germination and postgermination growth in arabidopsis

    KAUST Repository

    Zhao, Huayan

    2014-05-08

    The ECERIFERUM9 (CER9) gene encodes a putative E3 ubiquitin ligase that functions in cuticle biosynthesis and the maintenance of plant water status. Here, we found that CER9 is also involved in abscisic acid (ABA) signaling in seeds and young seedlings of Arabidopsis (Arabidopsis thaliana). The germinated embryos of the mutants exhibited enhanced sensitivity to ABA during the transition from reversible dormancy to determinate seedling growth. Expression of the CER9 gene is closely related to ABA levels and displays a similar pattern to that of ABSCISIC ACID-INSENSITIVE5 (ABI5), which encodes a positive regulator of ABA responses in seeds. cer9 mutant seeds exhibited delayed germination that is independent of seed coat permeability. Quantitative proteomic analyses showed that cer9 seeds had a protein profile similar to that of the wild type treated with ABA. Transcriptomics analyses revealed that genes involved in ABA biosynthesis or signaling pathways were differentially regulated in cer9 seeds. Consistent with this, high levels of ABA were detected in dry seeds of cer9. Blocking ABA biosynthesis by fluridone treatment or by combining an ABA-deficient mutation with cer9 attenuated the phenotypes of cer9. Whereas introduction of the abi1-1, abi3-1, or abi4-103 mutation could completely eliminate the ABA hypersensitivity of cer9, introduction of abi5 resulted only in partial suppression. These results indicate that CER9 is a novel negative regulator of ABA biosynthesis and the ABA signaling pathway during seed germination. © 2014 American Society of Plant Biologists. All Rights Reserved.

  18. The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development.

    Science.gov (United States)

    Rahman, Kazi; Zhao, Peng; Mandalasi, Msano; van der Wel, Hanke; Wells, Lance; Blader, Ira J; West, Christopher M

    2016-02-26

    Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.

  19. WW domain containing E3 ubiquitin protein ligase 1 targets the full-length ErbB4 for ubiquitin-mediated degradation in breast cancer.

    Science.gov (United States)

    Li, Y; Zhou, Z; Alimandi, M; Chen, C

    2009-08-20

    ErbB4, a member of the epidermal growth factor receptor family, plays a role in normal breast and breast cancer development by regulating mammary epithelial cell proliferation, survival and differentiation. In this study, we show that WWP1, a C2-WW-HECT type E3 ubiquitin ligase, binds, ubiquitinates and destructs ErbB4-CYT1, but much less efficiently for CYT2, isoforms (both JMa and JMb). The protein-protein interaction occurs primarily between the first and third WW domains of WWP1 and the second PY motif of ErbB4. Knockdown of WWP1 by two different small interfering RNAs increases the endogenous ErbB4 protein levels in both MCF7 and T47D breast cancer cell lines. In addition, overexpression of the wild type, but not the catalytic inactive WWP1, dramatically decreases the endogenous ErbB4 protein levels in MCF7. Importantly, we found that WWP1 negatively regulates the heregulin-beta1-stimulated ErbB4 activity as measured by the serum response element report assay and the BRCA1 mRNA expression. After a systematic screening of all WWP1 family members by small interfering RNA, we found that AIP4/Itch and HECW1/NEDL1 also negatively regulate the ErbB4 protein expression in T47D. Interestingly, the protein expression levels of both WWP1 and ErbB4 are higher in estrogen receptor-alpha-positive than in estrogen receptor-alpha-negative breast cancer cell lines. These data suggest that WWP1 and its family members suppress the ErbB4 expression and function in breast cancer.

  20. O2 sensing associated glycosylation exposes the F-box combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases.

    Science.gov (United States)

    Sheikh, M Osman; Thieker, David; Chalmers, Gordon; Schafer, Christopher M; Ishihara, Mayumi; Azadi, Parastoo; Woods, Robert J; Glushka, John N; Bendiak, Brad; Prestegard, James H; West, Christopher M

    2017-09-19

    Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box binding domain. Prolyl hydroxylation of Skp1 contributes to O2-dependent Dictyostelium development, but full glycosylation at that position is required for optimal O2 sensing. Previous studies have shown that the glycan promotes organization of the F-box binding region in Skp1, and aids in Skp1's association with F-box proteins. Here, nuclear magnetic resonance and mass spectrometry approaches were used to determine the glycan structure, and then a combination of NMR and molecular dynamics simulations were employed to characterize the impact of the glycan on the conformation and motions of the intrinsically flexible F-box binding domain of Skp1. MD trajectories of glycosylated Skp1 whose calculated monosaccharide relaxation kinetics and rotational correlation times agreed with the NMR data indicated that the glycan interacts with the loop connecting two alpha-helices of the F-box combining site. In these trajectories, the helices separated from one another to create a more accessible and dynamic F-box interface. These results offer an unprecedented view of how a glycan modification influences a disordered region of a full-length protein. The increased sampling of an open Skp1 conformation can explain how glycosylation enhances interactions with F-box proteins in cells. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  1. miR-135A regulates preimplantation embryo development through down-regulation of E3 Ubiquitin Ligase Seven In Absentia Homolog 1A (SIAH1A expression.

    Directory of Open Access Journals (Sweden)

    Ronald T K Pang

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are small non-coding RNA molecules capable of regulating transcription and translation. Previously, a cluster of miRNAs that are specifically expressed in mouse zygotes but not in oocytes or other preimplantation stages embryos are identified by multiplex real-time polymerase chain reaction-based miRNA profiling. The functional role of one of these zygote-specific miRNAs, miR-135a, in preimplantation embryo development was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Microinjection of miR-135a inhibitor suppressed first cell cleavage in more than 30% of the zygotes. Bioinformatics analysis identified E3 Ubiquitin Ligase Seven In Absentia Homolog 1A (Siah1a as a predicted target of miR-135a. Western blotting and 3'UTR luciferase functional assays demonstrated that miR-135a down-regulated the expression of Siah1 in HeLa cells and in mouse zygotes. Siah1a was expressed in preimplantation embryos and its expression pattern negatively correlated with that of miR-135a. Co-injection of Siah1a-specific antibody with miR-135a inhibitor partially nullified the effect of miR-135a inhibition. Proteasome inhibition by MG-132 revealed that miR-135a regulated proteasomal degradation and potentially controlled the expression of chemokinesin DNA binding protein (Kid. CONCLUSIONS/SIGNIFICANCE: The present study demonstrated for the first time that zygotic specific miRNA modulates the first cell cleavage through regulating expression of Siah1a.

  2. Tyrosine phosphorylation of the E3 ubiquitin ligase TRIM21 positively regulates interaction with IRF3 and hence TRIM21 activity.

    Directory of Open Access Journals (Sweden)

    Kevin B Stacey

    Full Text Available Patients suffering from Systemic Lupus Erythematous (SLE have elevated type I interferon (IFN levels which correlate with disease activity and severity. TRIM21, an autoantigen associated with SLE, has been identified as an ubiquitin E3 ligase that targets the transcription factor IRF3 in order to turn off and limit type I IFN production following detection of viral and bacterial infection by Toll Like Receptors (TLRs. However, how the activity of TRIM21 is regulated downstream of TLRs is unknown. In this study we demonstrate that TRIM21 is tyrosine phosphorylated following TLR3 and TLR4 stimulation, suggesting that its activity is potentially regulated by tyrosine phosphorylation. Using Netphos, we have identified three key tyrosines that are strongly predicted to be phosphorylated, two of which are conserved between the human and murine forms of TRIM21, at residues 343, 388, and 393, all of which have been mutated from tyrosine to phenylalanine (Y343F, Y388F, and Y393F. We have observed that tyrosine phosphorylation of TRIM21 only occurs in the substrate binding PRY/SPRY domain, and that Y393, and to a lesser extent, Y388 are required for TRIM21 to function as a negative regulator of IFN-β promoter activity. Further studies revealed that mutating Y393 to phenylalanine inhibits the ability of TRIM21 to interact with its substrate, IRF3, thus providing a molecular explanation for the lack of activity of Y393 on the IFN-β promoter. Our data demonstrates a novel role for tyrosine phosphorylation in regulating the activity of TRIM21 downstream of TLR3 and TLR4. Given the pathogenic role of TRIM21 in systemic autoimmunity, these findings have important implications for the development of novel therapeutics.

  3. Cloning and Expression Analysis of E3 Ubiquitin Ligase Nrdp1 from Grass Carp Ctenopharyngodon idella%草鱼E3泛素连接酶Nrdp1基因cDNA克隆和表达分析

    Institute of Scientific and Technical Information of China (English)

    隗黎丽; 杨竹青; 王自蕊; 熊六凤; 周秋白

    2013-01-01

    神经调节素受体降解蛋白1(neuregulin receptor degradation protein-1,Nrdpl)是一种新的E3泛素连接酶,可调节细胞增殖和凋亡等.研究采用RACE方法从草鱼(Ctenopharyngodon idella)肝脏中克隆了Nrdpl的全长cDNA序列;采用半定量RT-PCR方法分析该基因在草鱼不同组织中表达情况.结果表明:该序列全长cDNA大小为1 865 bp(GenBank登录号:JX864048),其中开放阅读框为954 bp,编码318个氨基酸,预测分子量大小为36.09 ku,pH为7.0时理论等电点为5.83;5’和3’非翻译区的长度分别为291 bp和620 bp,在3’非翻译区发现2个mRNA不稳定信号(ATTTA)和1个多聚腺苷酸加尾信号(ATTAAA).该蛋白序列没有信号肽和跨膜结构,其二级结构主要以α-螺旋为主,不含β-折叠.氨基酸序列保守性分析表明,草鱼Nrdp1与斑马鱼、虹鳟及人的同源性分别为98%、94%和89%;该基因在心脏和脑中的表达量较多,在肌肉和血液中表达很少.%Nrdpl ( neuregulin receptor degradation protein - 1) is a newly defined E3 ubiquitin ligase and regulates the cell proliferation and apoptosis. In the present study, the cDNA sequence of grass carp Ctenopha-ryngodon idella Nrdp1 (gcNrdpl) consists of 1865 bp with a 291 bp 5' untranslated region (UTR) and a 620 bp 3' UTR, with an open reading frame of 954 bp encoding the 318 amino acids which is predicted to have no signal petptide and transmembrane helices. The deduced amino acid sequence of gcNrdpl showed 98% , 94% and 89% of identity to zebra fish Danio rerio, rainbow trout Oncorhynchus mykiss and human Homo sapiens Nrdpl, respectively. The domain search revealed that gcNrdpl contains a RING domain (at amino acids 18 -56) and Pfam domain (at amino acids 137 -315). Phylogenetic analysis demonstrated that gcNrdpl is clustered in a same clade with zebra fish and rainbow trout Nrdpl. RT-PCR demonstrated that the mRNA is con-stitutively expressed in all the examined organs examined of healthy fish

  4. Overexpression of the Rice SUMO E3 Ligase Gene OsSIZ1 in Cotton Enhances Drought and Heat Tolerance, and Substantially Improves Fiber Yields in the Field under Reduced Irrigation and Rainfed Conditions

    Science.gov (United States)

    Mishra, Neelam; Sun, Li; Zhu, Xunlu; Smith, Jennifer; Prakash Srivastava, Anurag; Yang, Xiaojie; Pehlivan, Necla; Esmaeili, Nardana; Luo, Hong; Shen, Guoxin; Jones, Don; Auld, Dick; Burke, John

    2017-01-01

    The Arabidopsis SUMO E3 ligase gene AtSIZ1 plays important roles in plant response to abiotic stresses as loss of function in AtSIZ1 leads to increased sensitivity to drought, heat and salt stresses. Overexpression of the AtSIZ1 rice homolog, OsSIZ1, leads to increased heat and drought tolerance in bentgrass, suggesting that the function of the E3 ligase SIZ1 is highly conserved in plants and it plays a critical role in abiotic stress responses. To test the possibility that the SUMO E3 ligase could be used to engineer drought- and heat-tolerant crops, the rice gene OsSIZ1 was overexpressed in cotton. We report here that overexpression of OsSIZ1 in cotton results in higher net photosynthesis and better growth than wild-type cotton under drought and thermal stresses in growth chamber and greenhouse conditions. Additionally, this tolerance to abiotic stresses was correlated with higher fiber yield in both controlled-environment and field trials carried out under reduced irrigation and rainfed conditions. These results suggest that OsSIZ1 is a viable candidate gene to improve crop yields under water-limited and rainfed agricultural production systems. PMID:28340002

  5. Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels.

    Science.gov (United States)

    Hantouche, Christine; Williamson, Brittany; Valinsky, William C; Solomon, Joshua; Shrier, Alvin; Young, Jason C

    2017-02-10

    Cardiac long QT syndrome type 2 is caused by mutations in the human ether a go-go-related gene (hERG) potassium channel, many of which cause misfolding and degradation at the endoplasmic reticulum instead of normal trafficking to the cell surface. The Hsc70/Hsp70 chaperones assist the folding of the hERG cytosolic domains. Here, we demonstrate that the Hsp70 nucleotide exchange factor Bag1 promotes hERG degradation by the ubiquitin-proteasome system at the endoplasmic reticulum to regulate hERG levels and channel activity. Dissociation of hERG complexes containing Hsp70 and the E3 ubiquitin ligase CHIP requires the interaction of Bag1 with Hsp70, but this does not involve the Bag1 ubiquitin-like domain. The interaction with Bag1 then shifts hERG degradation to the membrane-anchored E3 ligase TRC8 and its E2-conjugating enzyme Ube2g2, as determined by siRNA screening. TRC8 interacts through the transmembrane region with hERG and decreases hERG functional expression. TRC8 also mediates degradation of the misfolded hERG-G601S disease mutant, but pharmacological stabilization of the mutant structure prevents degradation. Our results identify TRC8 as a previously unknown Hsp70-independent quality control E3 ligase for hERG. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Regulation of APCCdh1 E3 ligase activity by the Fbw7/cyclin E signaling axis contributes to the tumor suppressor function of Fbw7

    Institute of Scientific and Technical Information of China (English)

    Alan W Lau; Hiroyuki Inuzuka; Hidefumi Fukushima; Lixin Wan; Pengda Liu; Daming Gao; Yi Sun

    2013-01-01

    Fbw7 and Cdh1 are substrate-recognition subunits of the SCF-and APC-type E3 ubiquitin ligases,respectively.There is emerging evidence suggesting that both Fbw7 and Cdh1 function as tumor suppressors by targeting oncoproteins for destruction.Loss of Fbw7,but not Cdh1,is frequently observed in various human tumors.However,it remains largely unknown how Fbw7 mechanistically functions as a tumor suppressor and whether there is a signaling crosstalk between Fbw7 and Cdh1.Here,we report that Fbw7-deficient cells not only display elevated expression levels of SCFFbw7 substrates,including cyclin E,but also have increased expression of various APCdh1 substrates.We further defined cyclin E as the critical signaling link by which Fbw7 governs APCCdh1 activity,as depletion of cyclin E in Fbw7-deficient cells results in decreased expression of APCdh1 substrates to levels comparable to those in wildtype (WT) cells.Conversely,ectopic expression of cyclin E recapitulates the aberrant APCdh1 substrate expression observed in Fbw7-deficient cells.More importantly,4A-Cdh1 that is resistant to Cdk2/cyclin E-mediated phosphorylation,but not WT-Cdh1,reversed the elevated expression of various APCCdh1 substrates in Fbw7-deficient cells.Overexpression of 4A-Cdh1 also resulted in retarded cell growth and decreased anchorage-independent colony formation.Altogether,we have identified a novel regulatory mechanism by which Fbw7 governs Cdh1 activity in a cyclin E-dependent manner.As a result,loss of Fbw7 can lead to aberrant increase in the expression of both SCFFbw7and APCCdh1 substrates.Our study provides a better understanding of the tumor suppressor function of Fbw7,and suggests that Cdk2/cyclin E inhibitors could serve as effective therapeutic agents for treating Fbw7-deficient tumors.

  7. p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1.

    Science.gov (United States)

    Ma-Lauer, Yue; Carbajo-Lozoya, Javier; Hein, Marco Y; Müller, Marcel A; Deng, Wen; Lei, Jian; Meyer, Benjamin; Kusov, Yuri; von Brunn, Brigitte; Bairad, Dev Raj; Hünten, Sabine; Drosten, Christian; Hermeking, Heiko; Leonhardt, Heinrich; Mann, Matthias; Hilgenfeld, Rolf; von Brunn, Albrecht

    2016-08-30

    Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PL(pro)), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95-144 of RCHY1 and 389-652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PL(pro)s from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD-PL(pro) fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PL(pro) alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.

  8. 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.

    Science.gov (United States)

    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.

  9. Genome-Wide Identification of Soybean U-Box E3 Ubiquitin Ligases and Roles of GmPUB8 in Negative Regulation of Drought Stress Response in Arabidopsis.

    Science.gov (United States)

    Wang, Ning; Liu, Yaping; Cong, Yahui; Wang, Tingting; Zhong, Xiujuan; Yang, Shouping; Li, Yan; Gai, Junyi

    2016-06-01

    Plant U-box (PUB) E3 ubiquitin ligases play important roles in hormone signaling pathways and response to abiotic stresses, but little is known about them in soybean, Glycine max. Here, we identified and characterized 125 PUB genes from the soybean genome, which were classified into eight groups according to their protein domains. Soybean PUB genes (GmPUB genes) are broadly expressed in many tissues and are a little more abundant in the roots than in the other tissues. Nine GmPUB genes, GmPUB1-GmPUB9, showed induced expression patterns by drought, and the expression of GmPUB8 was also induced by exogenous ABA and NaCl. GmPUB8 was localized to post-Golgi compartments, interacting with GmE2 protein as demonstrated by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments, and showed E3 ubiquitin ligase activity by in vitro ubiquitination assay. Heterogeneous overexpression of GmPUB8 in Arabidopsis showed decreased drought tolerance, enhanced sensitivity with respect to osmotic and salt stress inhibition of seed germination and seedling growth, and inhibited ABA- and mannitol-mediated stomatal closure. Eight drought stress-related genes were less induced in GmPUB8-overexpressing Arabidopsis after drought treatment compared with the wild type and the pub23 mutant. Taken together, our results suggested that GmPUB8 might negatively regulate plant response to drought stress. In addition, Y2H and BiFC showed that GmPUB8 interacted with soybean COL (CONSTANS LIKE) protein. GmPUB8-overexpressing Arabidopsis flowered earlier under middle- and short-day conditions but later under long-day conditions, indicating that GmPUB8 might regulate flowering time in the photoperiod pathway. This study helps us to understand the functions of PUB E3 ubiquitin ligases in soybean.

  10. The E3 Ubiquitin Ligase Activity Associated with the Adenoviral E1B-55K–E4orf6 Complex Does Not Require CRM1-Dependent Export ▿

    OpenAIRE

    Schmid, Melanie; Kindsmüller, Kathrin; Wimmer, Peter; Groitl, Peter; Gonzalez, Ramon A.; Dobner, Thomas

    2011-01-01

    The adenovirus type 5 (Ad5) E1B-55K and E4orf6 (E1B-55K/E4orf6) proteins are multifunctional regulators of Ad5 replication, participating in many processes required for virus growth. A complex containing the two proteins mediates the degradation of cellular proteins through assembly of an E3 ubiquitin ligase and induces shutoff of host cell protein synthesis through selective nucleocytoplasmic viral late mRNA export. Both proteins shuttle between the nuclear and cytoplasmic compartments via l...

  11. p90RSK targets the ERK5-CHIP ubiquitin E3 ligase activity in diabetic hearts and promotes cardiac apoptosis and dysfunction.

    Science.gov (United States)

    Le, Nhat-Tu; Takei, Yuichiro; Shishido, Tetsuro; Woo, Chang-Hoon; Chang, Eugene; Heo, Kyung-Sun; Lee, Hakjoo; Lu, Yan; Morrell, Craig; Oikawa, Masayoshi; McClain, Carolyn; Wang, Xin; Tournier, Cathy; Molina, Carlos A; Taunton, Jack; Yan, Chen; Fujiwara, Keigi; Patterson, Cam; Yang, Jay; Abe, Jun-ichi

    2012-02-17

    Cardiomyocyte apoptosis is one of the key events in the development and progression of heart failure, and a crucial role for ICER (inducible cAMP early repressor) in this process has been previously reported. ERK5 is known to inhibit cardiac apoptosis after myocardial infarction (MI), especially in hyperglycemic states, via association with CHIP ubiquitin (Ub) ligase and subsequent upregulation of CHIP ligase activity, which induces ICER ubiquitination and subsequent protein degradation. The regulatory mechanism governing ERK5/CHIP interaction is unknown. We previously demonstrated increased p90RSK activation in the diabetic heart. As a logical extension of this work, we now investigate whether p90RSK activation inhibits ERK5-mediated CHIP activation, and subsequently increases ICER levels and apoptosis. p90RSK activation inhibits ERK5/CHIP association and CHIP Ub ligase activity. p90RSK and CHIP share a common binding site in the ERK5 C-terminal domain (aa571-807). Overexpression of either p90RSK or an ERK5 fragment (aa571-807) inhibits ERK5/CHIP association, suggesting that p90RSK and CHIP competes for ERK5 binding and that p90RSK activation is critical for inhibiting ERK5/CHIP interaction. We also identified ERK5-S496 as being directly phosphorylated by p90RSK and demonstrated that an ERK5-S496A mutant significantly impairs Angiotensin II-mediated inhibition of CHIP activity and subsequent increase in ICER levels. In vivo, either cardiac-specific depletion of ERK5 or overexpression of p90RSK inhibits CHIP activity and accelerates cardiac apoptosis after MI-a phenomenon fully reversible by activating ERK5. These data suggest a role for p90RSK in inhibiting CHIP activity and promoting cardiac apoptosis through binding to and phosphorylation of ERK5-S496.

  12. Dsc E3 ligase localization to the Golgi requires the ATPase Cdc48 and cofactor Ufd1 for activation of Sterol Regulatory Element-binding Protein in fission yeast.

    Science.gov (United States)

    Burr, Risa; Ribbens, Diedre; Raychaudhuri, Sumana; Stewart, Emerson V; Ho, Jason; Espenshade, Peter J

    2017-08-18

    Sterol regulatory element-binding proteins (SREBPs) in the fission yeast Schizosaccharomyces pombe regulate lipid homeostasis and the hypoxic response under conditions of low sterol or oxygen availability. SREBPs are cleaved in the Golgi through the combined action of the Dsc E3 ligase complex, the rhomboid protease Rbd2, and the essential ATPases Associated with diverse cellular Activities (AAA+) ATPase Cdc48. The soluble SREBP N-terminal transcription factor domain is then released in the cytosol to enter the nucleus and regulate gene expression. Previously, we reported that Cdc48 binding to Rbd2 is required for Rbd2-mediated SREBP cleavage. Here, using affinity chromatography and mass spectrometry experiments, we identified Cdc48-binding proteins in S. pombe, generating a list of many previously unknown potential Cdc48 binding partners. We show that the established Cdc48 cofactor Ufd1 is required for SREBP cleavage but does not interact with the Cdc48-Rbd2 complex. Cdc48-Ufd1 is instead required at a step prior to Rbd2 function, during Golgi localization of the Dsc E3 ligase complex. Together, these findings demonstrate that two distinct Cdc48 complexes - Cdc48-Ufd1 and Cdc48-Rbd2 - are required for SREBP activation and low-oxygen adaptation in S. pombe. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  13. BRCA1/BARD1 E3 ubiquitin ligase can modify histones H2A and H2B in the nucleosome particle.

    Science.gov (United States)

    Thakar, Amit; Parvin, Jeffrey D; Zlatanova, Jordanka

    2010-02-01

    BRCA1, the protein product of the Breast Cancer Susceptibility Gene (BRCA1) has been implicated in multiple pathways that preserve genome stability, including cell cycle control, DNA repair, transcription, and chromatin remodeling. BRCA1, in complex with another RING-domain protein BARD1, possesses ubiquitin-ligase activity. Only a few targets for this activity have been identified in vivo. Nucleosomal histones may also be targets in vivo since they can be modified by the BRCA1/BARD1 complex in vitro. Here we demonstrate that the BRCA1/BARD1 complex can ubiquitylate both free H2A and H2B histones and histones in the context of nucleosomal particles. These results raise the possibility that BRCA1/BARD1 can directly affect nucleosomal structure, dynamics, and function through its ability to modify nucleosomal histones.

  14. Types of Ubiquitin Ligases.

    Science.gov (United States)

    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.

  15. Lineage-Specific Viral Hijacking of Non-canonical E3 Ubiquitin Ligase Cofactors in the Evolution of Vif Anti-APOBEC3 Activity

    Directory of Open Access Journals (Sweden)

    Joshua R. Kane

    2015-05-01

    Full Text Available HIV-1 encodes the accessory protein Vif, which hijacks a host Cullin-RING ubiquitin ligase (CRL complex as well as the non-canonical cofactor CBFβ, to antagonize APOBEC3 antiviral proteins. Non-canonical cofactor recruitment to CRL complexes by viral factors, to date, has only been attributed to HIV-1 Vif. To further study this phenomenon, we employed a comparative approach combining proteomic, biochemical, structural, and virological techniques to investigate Vif complexes across the lentivirus genus, including primate (HIV-1 and simian immunodeficiency virus macaque [SIVmac] and non-primate (FIV, BIV, and MVV viruses. We find that CBFβ is completely dispensable for the activity of non-primate lentiviral Vif proteins. Furthermore, we find that BIV Vif requires no cofactor and that MVV Vif requires a novel cofactor, cyclophilin A (CYPA, for stable CRL complex formation and anti-APOBEC3 activity. We propose modular conservation of Vif complexes allows for potential exaptation of functions through the acquisition of non-CRL-associated host cofactors while preserving anti-APOBEC3 activity.

  16. Lineage-Specific Viral Hijacking of Non-canonical E3 Ubiquitin Ligase Cofactors in the Evolution of Vif Anti-APOBEC3 Activity.

    Science.gov (United States)

    Kane, Joshua R; Stanley, David J; Hultquist, Judd F; Johnson, Jeffrey R; Mietrach, Nicole; Binning, Jennifer M; Jónsson, Stefán R; Barelier, Sarah; Newton, Billy W; Johnson, Tasha L; Franks-Skiba, Kathleen E; Li, Ming; Brown, William L; Gunnarsson, Hörður I; Adalbjornsdóttir, Adalbjorg; Fraser, James S; Harris, Reuben S; Andrésdóttir, Valgerður; Gross, John D; Krogan, Nevan J

    2015-05-26

    HIV-1 encodes the accessory protein Vif, which hijacks a host Cullin-RING ubiquitin ligase (CRL) complex as well as the non-canonical cofactor CBFβ, to antagonize APOBEC3 antiviral proteins. Non-canonical cofactor recruitment to CRL complexes by viral factors, to date, has only been attributed to HIV-1 Vif. To further study this phenomenon, we employed a comparative approach combining proteomic, biochemical, structural, and virological techniques to investigate Vif complexes across the lentivirus genus, including primate (HIV-1 and simian immunodeficiency virus macaque [SIVmac]) and non-primate (FIV, BIV, and MVV) viruses. We find that CBFβ is completely dispensable for the activity of non-primate lentiviral Vif proteins. Furthermore, we find that BIV Vif requires no cofactor and that MVV Vif requires a novel cofactor, cyclophilin A (CYPA), for stable CRL complex formation and anti-APOBEC3 activity. We propose modular conservation of Vif complexes allows for potential exaptation of functions through the acquisition of non-CRL-associated host cofactors while preserving anti-APOBEC3 activity.

  17. Fission yeast arrestin-related trafficking adaptor, Arn1/Any1, is ubiquitinated by Pub1 E3 ligase and regulates endocytosis of Cat1 amino acid transporter

    Directory of Open Access Journals (Sweden)

    Akio Nakashima

    2014-05-01

    Full Text Available The Tsc1–Tsc2 complex homologous to human tuberous sclerosis complex proteins governs amino acid uptake by regulating the expression and intracellular distribution of amino acid transporters in Schizosaccharomyces pombe. Here, we performed a genetic screening for molecules that are involved in amino acid uptake and found Arn1 (also known as Any1. Arn1 is homologous to ART1, an arrestin-related trafficking adaptor (ART in Saccharomyces cerevisiae, and contains a conserved arrestin motif, a ubiquitination site, and two PY motifs. Overexpression of arn1+ confers canavanine resistance on cells, whereas its disruption causes hypersensitivity to canavanine. We also show that Arn1 regulates endocytosis of the Cat1 amino acid transporter. Furthermore, deletion of arn1+ suppresses a defect of amino acid uptake and the aberrant Cat1 localization in tsc2Δ. Arn1 interacts with and is ubiquitinated by the Pub1 ubiquitin ligase, which is necessary to regulate Cat1 endocytosis. Cat1 undergoes ubiquitinations on lysine residues within the N-terminus, which are mediated, in part, by Arn1 to determine Cat1 localization. Correctively, Arn1 is an ART in S. pombe and contributes to amino acid uptake through regulating Cat1 endocytosis in which Tsc2 is involved.

  18. Reciprocal regulation of very low density lipoprotein receptors (VLDLRs) in neurons by brain-derived neurotrophic factor (BDNF) and Reelin: involvement of the E3 ligase Mylip/Idol.

    Science.gov (United States)

    Do, Hai Thi; Bruelle, Céline; Tselykh, Timofey; Jalonen, Pilvi; Korhonen, Laura; Lindholm, Dan

    2013-10-11

    BDNF positively influences various aspects of neuronal migration, maturation, and survival in the developing brain. Reelin in turn mediates inhibitory signals to migrating neuroblasts, which is crucial for brain development. The interplay between BDNF and Reelin signaling in neurodevelopment is not fully understood. We show here that BDNF increased the levels of the Reelin receptor (VLDL receptor (VLDLR)) in hippocampal neurons by increasing gene expression. In contrast, Reelin decreased VLDLRs, which was accompanied by an increase in the levels of the E3 ligase Mylip/Idol in neurons. Down-regulation of Mylip/Idol using shRNAs abrogated the decrease in VLDLRs induced by Reelin. These results show that VLDLRs are tightly regulated in hippocampal neurons by both transcriptional and post-transcriptional mechanisms. The regulation of VLDLR by BDNF and Reelin may affect the migration of neurons and contribute to neurodevelopmental disorders in the nervous system.

  19. 组蛋白去乙酰化酶HDAC2突变体构建及其SUMO修饰E3连接酶功能研究%Construction Histone Deacetylation Enzyme 2 Mutants and Study their Function on SUMO E3 Ligase Activity

    Institute of Scientific and Technical Information of China (English)

    郭韡; 熊渊; 黄宏; 邢伟; 李向云; 徐祥

    2013-01-01

    Objective: To construct the HDAC2 deletion and point mutants with deacetylation inactivation using double-tube method mutant construction technology and detect SUMO E3 ligase activity of these mutants for investigating on the impact of HDAC2 SUMO E3 ligase on deactylayion function. Methods: Primers were designed for HDAC2 100-322 amino acid lack mutant and 142 amino acid point H→A mutant. HDAC2 pcDNA3.1 eukaryotic expression plasmid was used as the template. Corresponding single DNA in double-tube was amplified by heat efficiency Fusion enzyme. Mutant eukaryotic expression vectors were constructed by the following steps, including annealing, template enzyme cutting, purification of ethanol, screening with resistance flat and sequencing appraisal. The expression of mutants in cells were detected by western blotting and the sumolation E3 ligase activity was measured by LUC report gene. Results: Fragment lack mutant pcDNA3.1/HDAC2 DEL(100-322AA) and point mutant pcDNA3.1/HDAC2(142 H→A) were constructed successfully. The western blotting of C-myc detection showed that these mutants expressed in cells. LUC report gene results showed that the mutants still have the E3 ligase activity. Conclusion: The E3 sumolation ligase activity of HDAC2 had no effect on deacetylation enzyme function. C-terminus of the HDAC2 has the function of E3 ligase activity.%目的:针对人源HDAC2外显子拼接功能区(CDS)基因,运用双管法突变体构建技术,构建HDAC2去乙酰化酶功能失活的片段缺失与关键位点突变体,检测突变体的SUMO化修饰E3连接酶功能活性,探究此HDAC2新功能是否受到其固有的去乙酰化酶功能的影响.方法:设计HDAC2 100-322位氨基酸密码子缺失的片段突变体引物与142位H→A点突变体引物,利用HDAC2的pcDNA3.1真核表达质粒为模板,通过耐热Fusion酶PCR反应双管扩增相应的单链DNA,并经退火、模板酶切、乙醇纯化、感受态转化、抗性平板筛选

  20. In silico analysis identifies a C3HC4-RING finger domain of a putative E3 ubiquitin-protein ligase located at the C-terminus of a polyglutamine-containing protein

    Directory of Open Access Journals (Sweden)

    T. Scior

    2007-03-01

    Full Text Available Almost identical polyglutamine-containing proteins with unknown structures have been found in human, mouse and rat genomes (GenBank AJ277365, AF525300, AY879229. We infer that an identical new gene (RING finger domain of real interest is located in each C-terminal segment. A three-dimensional (3-D model was generated by remote homology modeling and the functional implications are discussed. The model consists of 65 residues from terminal position 707 to 772 of the human protein with a total length of 796 residues. The 3-D model predicts a ubiquitin-protein ligase (E3 as a binding site for ubiquitin-conjugating enzyme (E2. Both enzymes are part of the ubiquitin pathway to label unwanted proteins for subsequent enzymatic degradation. The molecular contact specificities are suggested for both the substrate recognition and the residues at the possible E2-binding surface. The predicted structure, of a ubiquitin-protein ligase (E3, enzyme class number 6.3.2.19, CATH code 3.30.40.10.4 may contribute to explain the process of ubiquitination. The 3-D model supports the idea of a C3HC4-RING finger with a partially new pattern. The putative E2-binding site is formed by a shallow hydrophobic groove on the surface adjacent to the helix and one zinc finger (L722, C739, P740, P741, R744. Solvent-exposed hydrophobic amino acids lie around both zinc fingers (I717, L722, F738, or P765, L766, V767, V733, P734. The 3-D structure was deposited in the protein databank theoretical model repository (2B9G, RCSB Protein Data Bank, NJ.

  1. BRCA1的泛素连接酶活性与肿瘤%E3 Ligase Activity of BRCA1 and Tumor

    Institute of Scientific and Technical Information of China (English)

    汪洋; 詹启敏

    2006-01-01

    乳腺癌易感基因1(BRCA1)是一种抑癌基因表达产物,参与许多重要的细胞生命过程如细胞周期调控、中心体复制、DNA损伤修复等.近来研究表明,BRCA1基因表达产物具有E3泛素连接酶活性,催化底物蛋白FANCD2、NPM、γ-Tubulin、RNAPⅡ等及其自身的泛素化,从而调控众多生命过程的顺利进行,并且与肿瘤的发生发展密切相关.

  2. Green Tea Polyphenol Epigallocatechin-3-gallate Suppresses Toll-like Receptor 4 Expression via Up-regulation of E3 Ubiquitin-protein Ligase RNF216.

    Science.gov (United States)

    Kumazoe, Motofumi; Nakamura, Yuki; Yamashita, Mai; Suzuki, Takashi; Takamatsu, Kanako; Huang, Yuhui; Bae, Jaehoon; Yamashita, Shuya; Murata, Motoki; Yamada, Shuhei; Shinoda, Yuki; Yamaguchi, Wataru; Toyoda, Yui; Tachibana, Hirofumi

    2017-03-10

    Toll-like receptor 4 (TLR4) plays an essential role in innate immunity through inflammatory cytokine induction. Recent studies demonstrated that the abnormal activation of TLR4 has a pivotal role in obesity-induced inflammation, which is associated with several diseases, including hyperinsulinemia, hypertriglyceridemia, and cardiovascular disease. Here we demonstrate that (-)-epigallocatechin-3-O-gallate, a natural agonist of the 67-kDa laminin receptor (67LR), suppressed TLR4 expression through E3 ubiquitin-protein ring finger protein 216 (RNF216) up-regulation. Our data indicate cyclic GMP mediates 67LR agonist-dependent RNF216 up-regulation. Moreover, we show that the highly absorbent 67LR agonist (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me) significantly attenuated TLR4 expression in the adipose tissue. EGCG3″Me completely inhibited the high-fat/high-sucrose (HF/HS)-induced up-regulation of tumor necrosis factor α in adipose tissue and serum monocyte chemoattractant protein-1 increase. Furthermore, this agonist intake prevented HF/HS-induced hyperinsulinemia and hypertriglyceridemia. Taken together, 67LR presents an attractive target for the relief of obesity-induced inflammation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Human liver cytochrome P450 3A4 ubiquitination: molecular recognition by UBC7-gp78 autocrine motility factor receptor and UbcH5a-CHIP-Hsc70-Hsp40 E2-E3 ubiquitin ligase complexes.

    Science.gov (United States)

    Wang, YongQiang; Kim, Sung-Mi; Trnka, Michael J; Liu, Yi; Burlingame, A L; Correia, Maria Almira

    2015-02-06

    CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate

  4. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.).

    Science.gov (United States)

    Gandhi, Anita K; Kang, Jian; Havens, Courtney G; Conklin, Thomas; Ning, Yuhong; Wu, Lei; Ito, Takumi; Ando, Hideki; Waldman, Michelle F; Thakurta, Anjan; Klippel, Anke; Handa, Hiroshi; Daniel, Thomas O; Schafer, Peter H; Chopra, Rajesh

    2014-03-01

    Cereblon (CRBN), the molecular target of lenalidomide and pomalidomide, is a substrate receptor of the cullin ring E3 ubiquitin ligase complex, CRL4(CRBN) . T cell co-stimulation by lenalidomide or pomalidomide is cereblon dependent: however, the CRL4(CRBN) substrates responsible for T cell co-stimulation have yet to be identified. Here we demonstrate that interaction of the transcription factors Ikaros (IKZF1, encoded by the IKZF1 gene) and Aiolos (IKZF3, encoded by the IKZF3 gene) with CRL4(CRBN) is induced by lenalidomide or pomalidomide. Each agent promotes Aiolos and Ikaros binding to CRL4(CRBN) with enhanced ubiquitination leading to cereblon-dependent proteosomal degradation in T lymphocytes. We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression. The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation. Importantly, Aiolos could serve as a proximal pharmacodynamic marker for lenalidomide and pomalidomide, as healthy human subjects administered lenalidomide demonstrated Aiolos degradation in their peripheral T cells. In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4(CRBN) , leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.

  5. The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis[C][W

    Science.gov (United States)

    Doblas, Verónica G.; Amorim-Silva, Vítor; Posé, David; Rosado, Abel; Esteban, Alicia; Arró, Montserrat; Azevedo, Herlander; Bombarely, Aureliano; Borsani, Omar; Valpuesta, Victoriano; Ferrer, Albert; Tavares, Rui M.; Botella, Miguel A.

    2013-01-01

    The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals. PMID:23404890

  6. Defining the interactions and role of DCAF1/VPRBP in the DDB1-cullin4A E3 ubiquitin ligase complex engaged by HIV-1 Vpr to induce a G2 cell cycle arrest.

    Directory of Open Access Journals (Sweden)

    Francine C A Gérard

    Full Text Available HIV viral protein R (Vpr induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/replication stress signalling pathway through engagement of the DDB1-CUL4A-DCAF1 E3 ubiquitin ligase via a direct binding to the substrate specificity receptor DCAF1. Since no high resolution structures of the DDB1-DCAF1-Vpr substrate recognition module currently exist, we used a mutagenesis approach to better define motifs in DCAF1 that are crucial for Vpr and DDB1 binding. Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD. Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1. While we could not identify elements specifically involved in Vpr binding, overall, the mutagenesis data suggest that the predicted β-propeller conformation of DCAF1 is likely to be critical for Vpr association. Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex. Lastly, we show that expression of DCAF1 WD in the absence of endogenous DCAF1 was not sufficient to enable Vpr-mediated G2 arrest activity. Overall, our results reveal that Vpr and DDB1 binding on DCAF1 can be genetically separated and further suggest that DCAF1 contains determinants in addition to the Vpr and DDB1 minimal binding domain, which are required for Vpr to enable the induction of a G2 arrest.

  7. Investigation of the Expression of Myogenic Transcription Factors, microRNAs and Muscle-Specific E3 Ubiquitin Ligases in the Medial Gastrocnemius and Soleus Muscles following Peripheral Nerve Injury.

    Directory of Open Access Journals (Sweden)

    Rebecca Wiberg

    Full Text Available Despite surgical innovation, the sensory and motor outcome after a peripheral nerve injury remains incomplete. One contributing factor to the poor outcome is prolonged denervation of the target organ, leading to apoptosis of both mature myofibres and satellite cells with subsequent replacement of the muscle tissue with fibrotic scar and adipose tissue. In this study, we investigated the expression of myogenic transcription factors, muscle specific microRNAs and muscle-specific E3 ubiquitin ligases at several time points following denervation in two different muscles, the gastrocnemius (containing predominantly fast type fibres and soleus (slow type muscles, since these molecules may influence the degree of atrophy following denervation. Both muscles exhibited significant atrophy (compared with the contra-lateral sides at 7 days following either a nerve transection or crush injury. In the crush model, the soleus muscle showed significantly increased muscle weights at days 14 and 28 which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. This study provides further insights regarding the intracellular regulatory molecules that generate and maintain distinct patterns of gene expression in different fibre types following peripheral nerve injury.

  8. The auto-ubiquitylation of E3 ubiquitin-protein ligase Chfr at G2 phase is required for accumulation of polo-like kinase 1 and mitotic entry in mammalian cells.

    Science.gov (United States)

    Kim, Jo-Sun; Park, Yong-Yea; Park, Sun-Yi; Cho, Hyeseon; Kang, Dongmin; Cho, Hyeseong

    2011-09-01

    The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G(2)/M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G(2)/M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G(2) phase, but not at S phase, were ∼2-3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G(2) phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G(2)/M transition.

  9. (1)H, (15)N and (13)C resonance assignments for free and IEEVD peptide-bound forms of the tetratricopeptide repeat domain from the human E3 ubiquitin ligase CHIP.

    Science.gov (United States)

    Zhang, Huaqun; McGlone, Cameron; Mannion, Matthew M; Page, Richard C

    2017-04-01

    The ubiquitin ligase CHIP catalyzes covalent attachment of ubiquitin to unfolded proteins chaperoned by the heat shock proteins Hsp70/Hsc70 and Hsp90. CHIP interacts with Hsp70/Hsc70 and Hsp90 by binding of a C-terminal IEEVD motif found in Hsp70/Hsc70 and Hsp90 to the tetratricopeptide repeat (TPR) domain of CHIP. Although recruitment of heat shock proteins to CHIP via interaction with the CHIP-TPR domain is well established, alterations in structure and dynamics of CHIP upon binding are not well understood. In particular, the absence of a structure for CHIP-TPR in the free form presents a significant limitation upon studies seeking to rationally design inhibitors that may disrupt interactions between CHIP and heat shock proteins. Here we report the (1)H, (13)C, and (15)N backbone and side chain chemical shift assignments for CHIP-TPR in the free form, and backbone chemical shift assignments for CHIP-TPR in the IEEVD-bound form. The NMR resonance assignments will enable further studies examining the roles of dynamics and structure in regulating interactions between CHIP and the heat shock proteins Hsp70/Hsc70 and Hsp90.

  10. 骨形成蛋白7干预肝纤维化小鼠肝组织E3泛素连接酶Arkadia表达的实验研究%Effects of bone morphogenetic protein-7 therapy on E3 ubiquitin ligase expression in mouse liver with experimentally induced fibrosis

    Institute of Scientific and Technical Information of China (English)

    申春燕; 陈永平; 阳韬; 陆小蒟; 李春艳; 林镯; 宋梅

    2012-01-01

    均<0.01);与12周模型组相比,BMP-7干预组Arkadia、Smad7、TGF β1的蛋白质表达量明显降低(t值分别为23.438、11.667和42.889,P值均<0.01).结论 Arkadia在肝纤维化进展过程中呈上升趋势,BMP-7具有抗肝纤维化作用并可以抑制纤维化过程中Arkadia的表达.%Objective This study explored the dynamic expression of the E3 ubiquitin-protein ligase gene,Arkadia,in response to carbon tetrachloride (CCl4)-induced liver fibrosis in a mouse model and investigated the differential expression that occurs following treatment with the anti-fibrotic bone morphogenetic protein-7 (BMP-7).Methods Thirty healthy male imprinting control region (ICR) mice were randomly assigned to three groups:normal (control; n =6),CCl4-induced model group (model; n =18),and CCl4-induced model with BMP-7 treatment group (treatment; n =6).The model group was further divided into three subgroups (n =6 each) for analysis at 4,8 and 12 weeks after fibrosis induction.Liver fibrosis was induced by hypodermic injections of 60% CCl4/peanut oil (5 mL/kg)to the hind legs of mice two-times per week in alternating legs for a period of 12 weeks.At week 9,the treatment group of CC14-induced mice were given an intraperitoneal injection of BMP-7 (300 pg/g) simultaneously with that day's hypodermic injection of 60% CCl4/peanut oil,and then every other day for a period of four weeks.The pathological changes in liver tissues were observed after staining with hematoxylin-eosin (HE) and Masson's trichrome.Messenger RNA (mRNA) and protein expression of Arkadia in liver were evaluated using reverse transcription-polymerase chain reaction and immunohistochemistry and Western blotting,respectively.Results Mouse models of liver fibrosis were successfully established by CCl4 exposure.Arkadia,Stmad7 and TGF-β1 mRNA levels were up-regulated in the model group in a time-dependent manner (vs.control group),and BMP-7 treatment led to significant down-regulation of the CCl4-induced

  11. Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis.

    Science.gov (United States)

    Maier, Alexander; Schrader, Andrea; Kokkelink, Leonie; Falke, Christian; Welter, Bastian; Iniesto, Elisa; Rubio, Vicente; Uhrig, Joachim F; Hülskamp, Martin; Hoecker, Ute

    2013-05-01

    Anthocyanins are natural pigments that accumulate only in light-grown and not in dark-grown Arabidopsis plants. Repression of anthocyanin accumulation in darkness requires the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) ubiquitin ligase, as cop1 and spa mutants produce anthocyanins also in the dark. Here, we show that COP1 and SPA proteins interact with the myeloblastosis (MYB) transcription factors PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP)1 and PAP2, two members of a small protein family that is required for anthocyanin accumulation and for the expression of structural genes in the anthocyanin biosynthesis pathway. The increased anthocyanin levels in cop1 mutants requires the PAP1 gene family, indicating that COP1 functions upstream of the PAP1 gene family. PAP1 and PAP2 proteins are degraded in the dark and this degradation is dependent on the proteasome and on COP1. Hence, the light requirement for anthocyanin biosynthesis results, at least in part, from the light-mediated stabilization of PAP1 and PAP2. Consistent with this conclusion, moderate overexpression of PAP1 leads to an increase in anthocyanin levels only in the light and not in darkness. Here we show that SPA genes are also required for reducing PAP1 and PAP2 transcript levels in dark-grown seedlings. Taken together, these results indicate that the COP1/SPA complex affects PAP1 and PAP2 both transcriptionally and post-translationally. Thus, our findings have identified mechanisms via which the COP1/SPA complex controls anthocyanin levels in Arabidopsis that may be useful for applications in biotechnology directed towards increasing anthocyanin content in plants. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  12. Post-translational control of IL-1β via the human papillomavirus type 16 E6 oncoprotein: a novel mechanism of innate immune escape mediated by the E3-ubiquitin ligase E6-AP and p53.

    Directory of Open Access Journals (Sweden)

    Martina Niebler

    Full Text Available Infections with high-risk human papillomaviruses (HPVs are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1β which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1β production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1β processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1β regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1β was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1β that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1β is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1β levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1β regulation which ultimately inhibits the secretion of IL-1β in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1

  13. Post-Translational Control of IL-1β via the Human Papillomavirus Type 16 E6 Oncoprotein: A Novel Mechanism of Innate Immune Escape Mediated by the E3-Ubiquitin Ligase E6-AP and p53

    Science.gov (United States)

    Niebler, Martina; Qian, Xu; Höfler, Daniela; Kogosov, Vlada; Kaewprag, Jittranan; Kaufmann, Andreas M.; Ly, Regina; Böhmer, Gerd; Zawatzky, Rainer; Rösl, Frank; Rincon-Orozco, Bladimiro

    2013-01-01

    Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1β) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1β production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1β processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1β regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1β was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1β that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1β is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1β levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1β regulation which ultimately inhibits the secretion of IL-1β in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1β towards cervical

  14. The RING finger domain E3 ubiquitin ligases BRCA1 and the RNF20/RNF40 complex in global loss of the chromatin mark histone H2B monoubiquitination (H2Bub1) in cell line models and primary high-grade serous ovarian cancer.

    Science.gov (United States)

    Dickson, Kristie-Ann; Cole, Alexander J; Gill, Anthony J; Clarkson, Adele; Gard, Gregory B; Chou, Angela; Kennedy, Catherine J; Henderson, Beric R; Fereday, Sian; Traficante, Nadia; Alsop, Kathryn; Bowtell, David D; deFazio, Anna; Clifton-Bligh, Roderick; Marsh, Deborah J

    2016-12-15

    Enzymatic factors driving cancer-associated chromatin remodelling are of increasing interest as the role of the cancer epigenome in gene expression and DNA repair processes becomes elucidated. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is a central histone modification that functions in histone cross-talk, transcriptional elongation, DNA repair, maintaining centromeric chromatin and replication-dependent histone mRNA 3'-end processing, as well as being required for the differentiation of stem cells. The loss of global H2Bub1 is seen in a number of aggressive malignancies and has been linked to tumour progression and/or a poorer prognosis in some cancers. Here, we analyse a large cohort of high-grade serous ovarian cancers (HGSOC) and show loss of global H2Bub1 in 77% (313 of 407) of tumours. Loss of H2Bub1 was seen at all stages (I-IV) of HGSOC, indicating it is a relatively early epigenomic event in this aggressive malignancy. Manipulation of key H2Bub1 E3 ubiquitin ligases, RNF20, RNF40 and BRCA1, in ovarian cancer cell line models modulated H2Bub1 levels, indicative of the role of these RING finger ligases in monoubiquitination of H2Bub1 in vitro. However, in primary HGSOC, loss of RNF20 protein expression was identified in just 6% of tumours (26 of 424) and did not correlate with global H2Bub1 loss. Similarly, germline mutation of BRCA1 did not show a correlation with the global H2Bub1 loss. We conclude that the regulation of tumour-associated H2Bub1 levels is complex. Aberrant expression of alternative histone-associated 'writer' or 'eraser' enzymes are likely responsible for the global loss of H2Bub1 seen in HGSOC. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. E3 Staff Database

    Data.gov (United States)

    US Agency for International Development — E3 Staff database is maintained by E3 PDMS (Professional Development & Management Services) office. The database is Mysql. It is manually updated by E3 staff as...

  16. Pseudosubstrate regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U

    DEFF Research Database (Denmark)

    Davis, Matti; Hatzubai, Ada; Andersen, Jens S

    2002-01-01

    beta-TrCP/E3RS (E3RS) is the F-box protein that functions as the receptor subunit of the SCF(beta-TrCP) ubiquitin ligase (E3). Surprisingly, although its two recognized substrates, IkappaB(alpha) and beta-catenin, are present in the cytoplasm, we have found that E3RS is located predominantly......, and efficacy of a specific protein-ubiquitin ligase....

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

    OpenAIRE

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

    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...

  18. Stealing the spotlight: CUL4-DDB1 ubiquitin ligase docks WD40-repeat proteins to destroy

    Directory of Open Access Journals (Sweden)

    Zhang Hui

    2007-02-01

    Full Text Available Abstract Recent investigation of Cullin 4 (CUL4 has ushered this class of multiprotein ubiquitin E3 ligases to center stage as critical regulators of diverse processes including cell cycle regulation, developmental patterning, DNA replication, DNA damage and repair, and epigenetic control of gene expression. CUL4 associates with DNA Damage Binding protein 1 (DDB1 to assemble an ubiquitin E3 ligase that targets protein substrates for ubiquitin-dependent proteolysis. CUL4 ligase activity is also regulated by the covalent attachment of the ubiquitin-like protein NEDD8 to CUL4, or neddylation, and the COP9 signalosome complex (CSN that removes this important modification. Recently, multiple WD40-repeat proteins (WDR were found to interact with DDB1 and serve as the substrate-recognition subunits of the CUL4-DDB1 ubiquitin ligase. As more than 150–300 WDR proteins exist in the human genome, these findings impact a wide array of biological processes through CUL4 ligase-mediated proteolysis. Here, we review the recent progress in understanding the mechanism of CUL4 ubiquitin E3 ligase and discuss the architecture of CUL4-assembled E3 ubiquitin ligase complexes by comparison to CUL1-based E3s (SCF. Then, we will review several examples to highlight the critical roles of CUL4 ubiquitin ligase in genome stability, cell cycle regulation, and histone lysine methylation. Together, these studies provide insights into the mechanism of this novel ubiquitin ligase in the regulation of important biological processes.

  19. DNA ligase I, the replicative DNA ligase.

    Science.gov (United States)

    Howes, Timothy R L; Tomkinson, Alan E

    2012-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. Improving machinery reliability

    CERN Document Server

    Bloch, Heinz P

    1998-01-01

    This totally revised, updated and expanded edition provides proven techniques and procedures that extend machinery life, reduce maintenance costs, and achieve optimum machinery reliability. This essential text clearly describes the reliability improvement and failure avoidance steps practiced by best-of-class process plants in the U.S. and Europe.

  2. E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection

    OpenAIRE

    2008-01-01

    The ligase IV/XRCC4 complex plays a central role in DNA double-strand break repair by non-homologous end joining (NHEJ). During adenovirus infection, NHEJ is inhibited by viral proteins E4 34k and E1B 55k, which redirect the Cul5/Rbx1/Elongin BC ubiquitin E3 ligase to polyubiquitinate and promote degradation of ligase IV. In cells infected with E1B 55k-deficient adenovirus, ligase IV could not be found in XRCC4-containing complexes and was observed in a novel ligase IV/E4 34k/Cul5/Elongin BC ...

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

    Science.gov (United States)

    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.

  4. TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin-RING ligase complexes.

    Science.gov (United States)

    Kelsall, Ian R; Duda, David M; Olszewski, Jennifer L; Hofmann, Kay; Knebel, Axel; Langevin, Frédéric; Wood, Nicola; Wightman, Melanie; Schulman, Brenda A; Alpi, Arno F

    2013-10-30

    RING (Really Interesting New Gene)-in-between-RING (RBR) enzymes are a distinct class of E3 ubiquitin ligases possessing a cluster of three zinc-binding domains that cooperate to catalyse ubiquitin transfer. The regulation and biological function for most members of the RBR ligases is not known, and all RBR E3s characterized to date are auto-inhibited for in vitro ubiquitylation. Here, we show that TRIAD1 and HHARI, two members of the Ariadne subfamily ligases, associate with distinct neddylated Cullin-RING ligase (CRL) complexes. In comparison to the modest E3 ligase activity displayed by isolated TRIAD1 or HHARI, binding of the cognate neddylated CRL to TRIAD1 or HHARI greatly stimulates RBR ligase activity in vitro, as determined by auto-ubiquitylation, their ability to stimulate dissociation of a thioester-linked UBCH7∼ubiquitin intermediate, and reactivity with ubiquitin-vinyl methyl ester. Moreover, genetic evidence shows that RBR ligase activity impacts both the levels and activities of neddylated CRLs in vivo. Cumulatively, our work proposes a conserved mechanism of CRL-induced Ariadne RBR ligase activation and further suggests a reciprocal role of this special class of RBRs as regulators of distinct CRLs.

  5. Ubiquitin ligase MARCH 8 cooperates with CD83 to control surface MHC II expression in thymic epithelium and CD4 T cell selection.

    Science.gov (United States)

    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.

  6. Cavitation in Hydraulic Machinery

    Energy Technology Data Exchange (ETDEWEB)

    Kjeldsen, M.

    1996-11-01

    The main purpose of this doctoral thesis on cavitation in hydraulic machinery is to change focus towards the coupling of non-stationary flow phenomena and cavitation. It is argued that, in addition to turbulence, superimposed sound pressure fluctuations can have a major impact on cavitation and lead to particularly severe erosion. For the design of hydraulic devices this finding may indicate how to further limit the cavitation problems. Chapter 1 reviews cavitation in general in the context of hydraulic machinery, emphasizing the initial cavitation event and the role of the water quality. Chapter 2 discusses the existence of pressure fluctuations for situations common in such machinery. Chapter 3 on cavitation dynamics presents an algorithm for calculating the nucleation of a cavity cluster. Chapter 4 describes the equipment used in this work. 53 refs., 55 figs.,10 tabs.

  7. Selectivity of E2-E3 interactions in the human ubiquitin system

    NARCIS (Netherlands)

    van Wijk, S.J.L.

    2010-01-01

    Highly selective interactions between ubiquitin-conjugating enzymes and RING-type E3 ligases are crucial for the adequate and efficient action of ubiquitin and ubiquitin-like pathways. Within these cascades, E2 enzymes provide a connecting link between activation and the final covalent conjugation,

  8. Bacterial mitotic machineries

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Ebersbach, Gitte

    2004-01-01

    Here, we review recent progress that yields fundamental new insight into the molecular mechanisms behind plasmid and chromosome segregation in prokaryotic cells. In particular, we describe how prokaryotic actin homologs form mitotic machineries that segregate DNA before cell division. Thus, the P...

  9. The Role of Ubiquitin Ligases in Cardiac Disease

    Science.gov (United States)

    Willis, Monte S.; Bevilacqua, Ariana; Pulinilkunnil, Thomas; Kienesberger, Petra; Tannu, Manasi; Patterson, Cam

    2014-01-01

    Rigorous surveillance of protein quality control is essential for the maintenance of normal cardiac function, while the dysregulation of protein turnover is present in a diverse array of common cardiac diseases. Central to the protein quality control found in all cells is the ubiquitin proteasome system (UPS). The UPS plays a critical role in protein trafficking, cellular signaling, and most prominently, protein degradation. As ubiquitin ligases (E3s) control the specificity of the UPS, their description in the cardiomyocyte has highlighted how ubiquitin ligases are critical to the turnover and function of the sarcomere complex, responsible for the heart’s required continuous contraction. In this review, we provide an overview of the UPS, highlighting a comprehensive overview of the cardiac ubiquitin ligases identified to date. We then focus on recent studies of new cardiac ubiquitin ligases outlining their novel roles in protein turnover, cellular signaling, and the regulation of mitochondrial dynamics and receptor turnover in the pathophysiology of cardiac hypertrophy, cardiac atrophy, myocardial infarction, and heart failure. PMID:24262338

  10. Defects in DNA ligase I trigger PCNA ubiquitylation at Lys 107.

    Science.gov (United States)

    Das-Bradoo, Sapna; Nguyen, Hai Dang; Wood, Jamie L; Ricke, Robin M; Haworth, Justin C; Bielinsky, Anja-Katrin

    2010-01-01

    In all eukaryotes, the ligation of newly synthesized DNA, also known as Okazaki fragments, is catalysed by DNA ligase I (ref. 1). An individual with a DNA ligase I deficiency exhibits growth retardation, sunlight sensitivity and severe immunosuppression, probably due to accumulation of DNA damage. Surprisingly, not much is known about the DNA damage response (DDR) in DNA ligase I-deficient cells. As DNA replication and DDR pathways are highly conserved in eukaryotes, we used Saccharomyces cerevisiae as a model system to address this issue. We uncovered a new pathway, which facilitates ubiquitylation at Lys 107 of proliferating cell nuclear antigen (PCNA). Unlike ubiquitylation at Lys 164 of PCNA in response to UV irradiation, which triggers translesion synthesis, modification of Lys 107 is not dependent on the ubiquitin conjugating enzyme (E2) Rad6 (ref. 4) nor the ubiquitin ligase (E3) Rad18 (ref. 5), but requires the E2 variant Mms2 (ref. 6) in conjunction with Ubc4 (ref. 7) and the E3 Rad5 (Refs 8, 9). Surprisingly, DNA ligase I-deficient S. cerevisiae cdc9-1 cells that carry a PCNAK107R mutation are inviable, because they cannot activate a robust DDR. Furthermore, we show that ubiquitylation of PCNA in response to DNA ligase I deficiency is conserved in humans, yet the lysine residue that is modified remains to be determined. We propose that PCNA ubiquitylation provides a 'DNA damage code' that allows cells to categorize different types of defects that arise during DNA replication.

  11. Vibration of hydraulic machinery

    CERN Document Server

    Wu, Yulin; Liu, Shuhong; Dou, Hua-Shu; Qian, Zhongdong

    2013-01-01

    Vibration of Hydraulic Machinery deals with the vibration problem which has significant influence on the safety and reliable operation of hydraulic machinery. It provides new achievements and the latest developments in these areas, even in the basic areas of this subject. The present book covers the fundamentals of mechanical vibration and rotordynamics as well as their main numerical models and analysis methods for the vibration prediction. The mechanical and hydraulic excitations to the vibration are analyzed, and the pressure fluctuations induced by the unsteady turbulent flow is predicted in order to obtain the unsteady loads. This book also discusses the loads, constraint conditions and the elastic and damping characters of the mechanical system, the structure dynamic analysis, the rotor dynamic analysis and the system instability of hydraulic machines, including the illustration of monitoring system for the instability and the vibration in hydraulic units. All the problems are necessary for vibration pr...

  12. E3 Portfolio Review Database

    Data.gov (United States)

    US Agency for International Development — The E3 Portfolio Review Database houses operational and performance data for all activities that the Bureau funds and/or manages. Activity-level data is collected by...

  13. E3 Sustainable Manufacturing Curriculum

    Science.gov (United States)

    A short E3 course containing three modules on Environmental Sustainability; Lean Manufacturing and Pollution Prevention; and Energy and Carbon. Each module includes slides, a facilitator's guide with handouts, activities, quizzes, and facilitator's notes.

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

    DEFF Research Database (Denmark)

    Hoffmann, Saskia; Smedegaard, Stine; Nakamura, Kyosuke

    2016-01-01

    , 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....

  15. Bacterial mitotic machineries

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Ebersbach, Gitte;

    2004-01-01

    Here, we review recent progress that yields fundamental new insight into the molecular mechanisms behind plasmid and chromosome segregation in prokaryotic cells. In particular, we describe how prokaryotic actin homologs form mitotic machineries that segregate DNA before cell division. Thus, the Par......M protein of plasmid R1 forms F actin-like filaments that separate and move plasmid DNA from mid-cell to the cell poles. Evidence from three different laboratories indicate that the morphogenetic MreB protein may be involved in segregation of the bacterial chromosome....

  16. Overexpression of a soybean ariadne-like ubiquitin ligase gene GmARI1 enhances aluminum tolerance in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Xiaolian Zhang

    Full Text Available 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.

  17. Overexpression of a soybean ariadne-like ubiquitin ligase gene GmARI1 enhances aluminum tolerance in Arabidopsis.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. Identification of a ubiquitin-protein ligase subunit within the CCR4-NOT transcription repressor complex

    NARCIS (Netherlands)

    Albert, TK; Hanzawa, H; Legtenberg, YIA; de Ruwe, MJ; van den Heuvel, FAJ; Collart, MA; Boelens, R; Timmers, HTM

    2002-01-01

    The RING finger protein CNOT4 is a component of the CCR4-NOT complex. This complex is implicated in repression of RNA polymerase II transcription. Here we demonstrate that CNOT4 functions as a ubiquitin-protein ligase (E3). We show that the unique C4C4 RING domain of CNOT4 interacts with a subset of

  20. E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection.

    Science.gov (United States)

    Jayaram, Sumithra; Gilson, Timra; Ehrlich, Elana S; Yu, Xiao-Fang; Ketner, Gary; Hanakahi, Les

    2008-12-20

    The ligase IV/XRCC4 complex plays a central role in DNA double-strand break repair by non-homologous end joining (NHEJ). During adenovirus infection, NHEJ is inhibited by viral proteins E4 34k and E1B 55k, which redirect the Cul5/Rbx1/Elongin BC ubiquitin E3 ligase to polyubiquitinate and promote degradation of ligase IV. In cells infected with E1B 55k-deficient adenovirus, ligase IV could not be found in XRCC4-containing complexes and was observed in a novel ligase IV/E4 34k/Cul5/Elongin BC complex. These observations suggest that dissociation of the ligase IV/XRCC4 complex occurs at an early stage in E4 34k-mediated degradation of ligase IV and indicate a role for E4 34k in dissociation of the ligase IV/XRCCC4 complex. Expression of E4 34k alone was not sufficient to dissociate the ligase IV/XRCC4 complex, which indicates a requirement for an additional, as yet unidentified, factor in E1B 55k-independent dissociation of the ligase IV/XRCC4 complex.

  1. Matrix analysis of electrical machinery

    CERN Document Server

    Hancock, N N

    2013-01-01

    Matrix Analysis of Electrical Machinery, Second Edition is a 14-chapter edition that covers the systematic analysis of electrical machinery performance. This edition discusses the principles of various mathematical operations and their application to electrical machinery performance calculations. The introductory chapters deal with the matrix representation of algebraic equations and their application to static electrical networks. The following chapters describe the fundamentals of different transformers and rotating machines and present torque analysis in terms of the currents based on the p

  2. MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades

    Science.gov (United States)

    Liu, Siqi; Chen, Jueqi; Cai, Xin; Wu, Jiaxi; Chen, Xiang; Wu, You-Tong; Sun, Lijun; Chen, Zhijian J

    2013-01-01

    RNA virus infections are detected by the RIG-I family of receptors, which induce type-I interferons through the mitochondrial protein MAVS. MAVS forms large prion-like polymers that activate the cytosolic kinases IKK and TBK1, which in turn activate NF-κB and IRF3, respectively, to induce interferons. Here we show that MAVS polymers recruit several TRAF proteins, including TRAF2, TRAF5, and TRAF6, through distinct TRAF-binding motifs. Mutations of these motifs that disrupted MAVS binding to TRAFs abrogated its ability to activate IRF3. IRF3 activation was also abolished in cells lacking TRAF2, 5, and 6. These TRAF proteins promoted ubiquitination reactions that recruited NEMO to the MAVS signaling complex, leading to the activation of IKK and TBK1. These results delineate the mechanism of MAVS signaling and reveal that TRAF2, 5, and 6, which are normally associated with NF-κB activation, also play a crucial role in IRF3 activation in antiviral immune responses. DOI: http://dx.doi.org/10.7554/eLife.00785.001 PMID:23951545

  3. Systematic In Vivo RNAi Analysis Identifies IAPs as NEDD8-E3 Ligases

    DEFF Research Database (Denmark)

    Broemer, Meike; Tenev, Tencho; Rigbolt, Kristoffer T G;

    2010-01-01

    The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-li...

  4. The Oncogenic Role of WWP1 E3 Ubiquitin Ligase in Prostate Cancer Development

    Science.gov (United States)

    2011-05-01

    and suppresses its ligand-independent activation. Current Biology, 14, 2228–2236. 42. Chastagner, P., Israel , A., & Brou, C. (2006). Itch/AIP4...proteasome destruction of activated RhoA is impaired in Smurf1−/− cells. Molecular Biology of the Cell, 17, 2489– 2497. 145. Sahai, E., Garcia -Medina...Haga K, Asaka M, Ramirez F, Yoshida T (2003) Downregulation and growth inhibitory effect of epithelial-type Kruppel-like tran- scription factor KLF4

  5. The Role of Ubiquitin E3 Ligase SCF-SKP2 in Prostate Cancer Development

    Science.gov (United States)

    2007-02-01

    that CUL4 was also present in the H3 methylated nucleo - some fractions. Interestingly, CUL4A, but not its paralogue CUL4B, is enriched in methylated...2, e51 (2006). 23. Zou, L., Cortez , D. & Elledge, S. J. Regulation of ATR substrate selection by Rad17- dependent loading of Rad9 complexes onto

  6. Natural Polymorphisms in C. elegans HECW-1 E3 Ligase Affect Pathogen Avoidance Behaviour

    OpenAIRE

    Chang, Howard C.; Paek, Jennifer; Dennis H Kim

    2011-01-01

    Heritable variation in behavioural traits generally has a complex genetic basis 1 , and thus naturally occurring polymorphisms that influence behaviour have been defined in only rare instances 2,3 . The isolation of wild strains of Caenorhabditis elegans has facilitated the study of natural genetic variation in this species 4 and provided insights into its diverse microbial ecology 5 . C. elegans responds to bacterial infection with conserved innate immune responses 6-8 and, while lacking the...

  7. Interplay between the myc oncoprotein, cyclin-dependent kinases and e3 ubiquitin ligases

    OpenAIRE

    Sharifi, Hamid Reza

    2013-01-01

    Mammalian cells grow, divide, and die in a precise and orderly fashion. For cells to grow and maintain their integrity they need to communicate at both intracellular and extracellular level. This communicative circuit is maintained by means of many different factors functioning at different level to for example receive, transmit and respond to the delivered message. Among these factors are transcription factors whose function is to regulate expr...

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

    NARCIS (Netherlands)

    Sargent, Graeme; van Zutphen, Tim; Shatseva, Tatiana; Zhang, Ling; Di Giovanni, Valeria; Bandsma, Robert; Kim, Peter Kijun

    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

  9. Cloning and characterization of mouse cullin4B/E3 ubiquitin ligase

    Indian Academy of Sciences (India)

    Rachana Tripathi; K Seetharama; Satya Keerthi Kota; Usha K Srinivas

    2005-06-01

    Heat induced differentiation of mouse embryonal carcinoma cells PCC4 has been reported earlier. We have further characterized the phenotype of the differentiated cells and by DD-RT-PCR identified several partial cDNAs that are differentially expressed during differentiation. Nucleotide homology search revealed that the genes corresponding to some of the up-regulated partial cDNAs are indeed part of differentiation pathway. 5′ extension of an EST that has homology to one of the partial cDNAs led to the identification of mouse cullin4B. Cullin4B is coded by a separate gene and has a unique and longer amino-terminal end with a putative nuclear localization signal sequence (NLS). We have cloned, expressed and raised antibodies against the amino and carboxy-terminal halves of cullin4B. Immuno staining of differentiated PCC4 cells with N-terminal Cul4B antibody showed enhanced expression of Cul4B and its translocation into the nucleus upon differentiation. Transient transfection of a chimeric gene encoding the N-terminal part of Cul4B fused to green fluorescent protein into PCC4 cells revealed that the protein was localized in the nucleus confirming the functional significance of the putative NLS. Since cullins are involved in recognition of specific proteins for degradation, based on the evidence presented here, we hypothesize that cullin4B is probably involved in differentiation specific degradation/modification of nuclear proteins.

  10. Characterization of an E3 Ubiquitin Ligase that Degrades Neurofibromin in Vitro and Vivo

    Science.gov (United States)

    2012-04-01

    cystic embryoid bodies (cEBs) (Figure 2A) with blood island structures, lined with endothelial cells (Figure 2B, arrows). In contrast, Sag/ ES cells... embryoid bodies: ES cells underwent endothelial differentiation for indicated periods and photographed. Arrows indicate cystic EBs that occurred in...attached onto the Petri dish during suspension culture for differentiation, leading to much fewer numbers of embryoid bodies, although we did observe

  11. Pumping machinery theory and practice

    CERN Document Server

    Badr, Hassan M

    2014-01-01

    Pumping Machinery Theory and Practice comprehensively covers the theoretical foundation and applications of pumping machinery. Key features: Covers characteristics of centrifugal pumps, axial flow pumps and displacement pumpsConsiders pumping machinery performance and operational-type problemsCovers advanced topics in pumping machinery including multiphase flow principles, and two and three-phase flow pumping systemsCovers different methods of flow rate control and relevance to machine efficiency and energy consumptionCovers different methods of flow rate control and relevance to machine effi

  12. The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance.

    Science.gov (United States)

    Bertolin, Giulia; Ferrando-Miguel, Rosa; Jacoupy, Maxime; Traver, Sabine; Grenier, Karl; Greene, Andrew W; Dauphin, Aurélien; Waharte, François; Bayot, Aurélien; Salamero, Jean; Lombès, Anne; Bulteau, Anne-Laure; Fon, Edward A; Brice, Alexis; Corti, Olga

    2013-11-01

    Loss-of-function mutations in PARK2/PARKIN and PINK1 cause early-onset autosomal recessive Parkinson disease (PD). The cytosolic E3 ubiquitin-protein ligase PARK2 cooperates with the mitochondrial kinase PINK1 to maintain mitochondrial quality. A loss of mitochondrial transmembrane potential (ΔΨ) leads to the PINK1-dependent recruitment of PARK2 to the outer mitochondrial membrane (OMM), followed by the ubiquitination and proteasome-dependent degradation of OMM proteins, and by the autophagy-dependent clearance of mitochondrial remnants. We showed here that blockade of mitochondrial protein import triggers the recruitment of PARK2, by PINK1, to the TOMM machinery. PD-causing PARK2 mutations weakened or disrupted the molecular interaction between PARK2 and specific TOMM subunits: the surface receptor, TOMM70A, and the channel protein, TOMM40. The downregulation of TOMM40 or its associated core subunit, TOMM22, was sufficient to trigger OMM protein clearance in the absence of PINK1 or PARK2. However, PARK2 was required to promote the degradation of whole organelles by autophagy. Furthermore, the overproduction of TOMM22 or TOMM40 reversed mitochondrial clearance promoted by PINK1 and PARK2 after ΔΨ loss. These results indicated that the TOMM machinery is a key molecular switch in the mitochondrial clearance program controlled by the PINK1-PARK2 pathway. Loss of functional coupling between mitochondrial protein import and the neuroprotective degradation of dysfunctional mitochondria may therefore be a primary pathogenic mechanism in autosomal recessive PD.

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

    Science.gov (United States)

    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).

  14. Skill Sheets for Agricultural Machinery.

    Science.gov (United States)

    Iowa State Univ. of Science and Technology, Ames. Dept. of Agricultural Education.

    This set of 21 skill sheets for agricultural machinery was developed for use in high school and vocational school agricultural mechanics programs. Each sheet covers a single operational procedure for a piece of agricultural machinery, and includes: (1) a diagram, (2) a step-by-step operational procedure, (3) abilities or understandings taught, (4)…

  15. The α2 helix in the DNA ligase IV BRCT-1 domain is required for targeted degradation of ligase IV during adenovirus infection.

    Science.gov (United States)

    Gilson, Timra; Greer, Amy E; Vindigni, Alessandro; Ketner, Gary; Hanakahi, Leslyn A

    2012-07-05

    In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.

  16. The ubiquitin ligase Cullin5(SOCS2) regulates NDR1/STK38 stability and NF-κB transactivation

    DEFF Research Database (Denmark)

    Paul, Indranil; Batth, Tanveer S; Iglesias-Gato, Diego

    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...... 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...

  17. Cullin-RING Ubiquitin Ligase Family in Plant Abiotic Stress Pathways

    Institute of Scientific and Technical Information of China (English)

    Liquan Guo; Cynthia D.Nezames; Lianxi Sheng; Xingwang Deng; Ning Wei

    2013-01-01

    The ubiquitin-proteasome system is a key mechanism that plants use to generate adaptive responses in coping with various environmental stresses.Cullin-RING (CRL) complexes represent a predominant group of ubiquitin E3 ligases in this system.In this review,we focus on the CRL E3s that have been implicated in abiotic stress signaling pathways in Arabidopsis.By comparing and analyzing these cases,we hope to gain a better understanding on how CRL complexes work under various settings in an attempt to decipher the clues about the regulatory mechanism of CRL E3s.

  18. Structural insight into β-Clamp and its interaction with DNA Ligase in Helicobacter pylori.

    Science.gov (United States)

    Pandey, Preeti; Tarique, Khaja Faisal; Mazumder, Mohit; Rehman, Syed Arif Abdul; Kumari, Nilima; Gourinath, Samudrala

    2016-08-08

    Helicobacter pylori, a gram-negative and microaerophilic bacterium, is the major cause of chronic gastritis, gastric ulcers and gastric cancer. Owing to its central role, DNA replication machinery has emerged as a prime target for the development of antimicrobial drugs. Here, we report 2Å structure of β-clamp from H. pylori (Hpβ-clamp), which is one of the critical components of DNA polymerase III. Despite of similarity in the overall fold of eubacterial β-clamp structures, some distinct features in DNA interacting loops exists that have not been reported previously. The in silico prediction identified the potential binders of β-clamp such as alpha subunit of DNA pol III and DNA ligase with identification of β-clamp binding regions in them and validated by SPR studies. Hpβ-clamp interacts with DNA ligase in micromolar binding affinity. Moreover, we have successfully determined the co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) revealing the region from ligase that interacts with β-clamp.

  19. Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity*

    Science.gov (United States)

    Ranaweera, Ruchira S.; Yang, Xiaolu

    2013-01-01

    The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes. PMID:23671280

  20. Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK.

    Science.gov (United States)

    Costantini, Silvia; Woodbine, Lisa; Andreoli, Lucia; Jeggo, Penny A; Vindigni, Alessandro

    2007-06-01

    DNA non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double-stranded (ds) breaks in mammalian cells. Here, we characterize the interaction between two key components of the NHEJ machinery, the Ku heterodimer and the DNA ligase IV/Xrcc4 complex. Our results demonstrate that Ku interacts with DNA ligase IV via its tandem BRCT domain and that this interaction is enhanced in the presence of Xrcc4 and dsDNA. Moreover, residues 644-748 of DNA ligase IV encompassing the first BRCT motif are necessary for binding. We show that Ku needs to be in its heterodimeric form to bind DNA ligase IV and that the C-terminal tail of Ku80, which mediates binding to DNA-PKcs, is dispensable for DNA ligase IV recognition. Although the interaction between Ku and DNA ligase IV/Xrcc4 occurs in the absence of DNA-PKcs, the presence of the catalytic subunit of DNA-PK kinase enhances complex formation. Previous studies have shown that DNA-PK kinase activity causes disassembly of DNA-PKcs from Ku at the DNA end. Here, we show that DNA-PK kinase activity also results in disassembly of the Ku/DNA ligase IV/Xrcc4 complex. Collectively, our findings provide novel information on the protein-protein interactions that regulate NHEJ in cells.

  1. 46 CFR 169.241 - Machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Machinery. 169.241 Section 169.241 Shipping COAST GUARD... Certification Inspections § 169.241 Machinery. (a) At each inspection for certification and periodic inspection.... Mechanisms are operationally tested and visually examined. (3) Auxiliary machinery. All machinery...

  2. The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation

    DEFF Research Database (Denmark)

    Adhikary, Sovana; Marinoni, Federica; Hock, Andreas

    2005-01-01

    The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linked...

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

    DEFF Research Database (Denmark)

    Hoffmann, Saskia; Smedegaard, Stine; Nakamura, Kyosuke;

    2016-01-01

    , 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......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...... 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...

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

    OpenAIRE

    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 a...

  5. Gln40 deamidation blocks structural reconfiguration and activation of SCF ubiquitin ligase complex by Nedd8

    OpenAIRE

    Yu, Clinton; Mao, Haibin; Novitsky, Eric J.; Tang, Xiaobo; Rychnovsky, Scott D.; Zheng, Ning; Huang, Lan

    2015-01-01

    The full enzymatic activity of the cullin-RING ubiquitin ligases (CRLs) requires a ubiquitin-like protein (that is, Nedd8) modification. By deamidating Gln40 of Nedd8 to glutamate (Q40E), the bacterial cycle-inhibiting factor (Cif) family is able to inhibit CRL E3 activities, thereby interfering with cellular functions. Despite extensive structural studies on CRLs, the molecular mechanism by which Nedd8 Gln40 deamidation affects CRL functions remains unclear. We apply a new quantitative cross...

  6. RNF13: a novel RING-type ubiquitin ligase over-expressed in pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Qiang Zhang; Yunxiao Meng; Lei Zhang; Jie Chen; Dahai Zhu

    2009-01-01

    Protein ubiquitination by E3 ubiquitin ligases plays an important role in cancer development. In this study, we provide experimental evidence that a RING-finger-containing protein RNF13 is an ER/Golgi membrane-associated E3 ubiquitin ligase and its RING finger domain is required for the ubiquitin iigase activity, lmmunohistochemical analysis of pancreatic ductal adenocarcinoma (PDAC) and paracancerous normal tissues from 72 patients documented RNF13 over-expression in 30 tumor samples (41.7%, 30/72), and its expression was significantly associated with histological grading (P= 0.024). In addition, RNFI3 was detected in precancerous lesions: tubular complexes in chronic pancreatitis (CP) and pancreatic intraepithelial neoplasia (PanlN) (79.3%, 23/29 and 62.8%, 22/35, respectively). Moreover, RNF13 staining was significantly correlated with Tenascin-C expression (P = 0.004) in PDAC samples, further supporting the role of RNF13 in cancer progression. Over-expression of wild type but not RING domain-mutant RNF13 in pancreatic MiaPaca-2 cancer cells increased invasive potential and gelatinolytic activity by matrix metalloproteinase-9. Taken together, these findings reveal that RNF13 is a novel E3 ubiquitin ligase involved in pancreatic carcinogenesis; ubiqui-tin-mediated modification of proteins by RNF13 may participate in pancreatic cancer development.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sheren, Jamie E. [Department of Pathology, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045 (United States); Kassenbrock, C. Kenneth, E-mail: ken.kassenbrock@ucdenver.edu [Department of Pathology, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045 (United States); Department of Biology, Colorado State University, Fort Collins, CO 80523-1878 (United States)

    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.

  8. Mechanism of ubiquitylation by dimeric RING ligase RNF4

    Science.gov (United States)

    Plechanovová, Anna; Jaffray, Ellis G.; McMahon, Stephen A.; Johnson, Kenneth A.; Navrátilová, Iva; Naismith, James H.; Hay, Ronald T.

    2012-01-01

    Mammalian RNF4 is a dimeric RING ubiquitin E3 ligase that ubiquitylates poly-SUMOylated proteins. We found that RNF4 bound ubiquitin-charged UbcH5a tightly but free UbcH5a weakly. To provide insight into the mechanism of RING-mediated ubiquitylation we docked the UbcH5~ubiquitin thioester onto the RNF4 RING structure. This revealed that with E2 bound to one monomer of RNF4, the thioester-linked ubiquitin could reach across the dimer to engage the other monomer. In this model the “Ile44 hydrophobic patch” of ubiquitin is predicted to engage a conserved tyrosine located at the dimer interface of the RING and mutation of these residues blocked ubiquitylation activity. Thus, dimeric RING ligases are not simply inert scaffolds that bring substrate and E2-loaded ubiquitin into close proximity. Instead, they facilitate ubiquitin transfer by preferentially binding the E2~ubiquitin thioester across the dimer and activating the thioester bond for catalysis. PMID:21857666

  9. Textile Machinery: Imports Rebound Again

    Institute of Scientific and Technical Information of China (English)

    Zheng Yan

    2007-01-01

    @@ In the year of 2006, the general situation of China's textile machinery equipment imports had shown a clear sign of revival from the downward trend of two years ago, with a total annual import of 4.1 billion USD, an increase of 19.05% against the same period of 2005.

  10. Tractor & Machinery Safety. 1984 Revision.

    Science.gov (United States)

    Montana State Office of Public Instruction, Helena. Dept. of Vocational Education Services.

    This curriculum guide is intended for use in teaching an instructional unit in tractor and machinery safety that is geared toward college freshmen. Addressed in the individual lessons of the unit are the following topics: understanding the importance of safe and efficient tractor operation, understanding the characteristics of tractors, preparing…

  11. Energy Savings Thanks to French Textile Machinery

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    @@ The French Textile Machinery Manufacturers' Association (UCMTF) has presented, during a seminar it organized for textile professionals and students, the spectacular energy savings achieved thanks to state of the art machinery.

  12. Italian Textile Machinery Seminar in Bangladesh

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The Association of Italian Textile Machinery Manufacturers (ACIMIT) and the Italian Trade Commission will hold a technological seminar on "Italian textile machinery: the way to improve Bangladesh textile competitiveness"

  13. 30 CFR 56.14204 - Machinery lubrication.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Machinery lubrication. 56.14204 Section 56.14204 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL... Equipment Safety Practices and Operational Procedures § 56.14204 Machinery lubrication. Machinery...

  14. 30 CFR 57.14204 - Machinery lubrication.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Machinery lubrication. 57.14204 Section 57.14204 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL... Equipment Safety Practices and Operational Procedures § 57.14204 Machinery lubrication. Machinery...

  15. 46 CFR 176.804 - Machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Machinery. 176.804 Section 176.804 Shipping COAST GUARD... CERTIFICATION Material Inspections § 176.804 Machinery. At each initial and subsequent inspection for... ready for inspections of machinery, fuel, and piping systems, including the following: (a) Operation...

  16. 46 CFR 130.450 - Machinery alarms.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Machinery alarms. 130.450 Section 130.450 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.450 Machinery alarms....

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

    Science.gov (United States)

    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.

  18. DNA ligase C1 mediates the LigD-independent nonhomologous end-joining pathway of Mycobacterium smegmatis.

    Science.gov (United States)

    Bhattarai, Hitesh; Gupta, Richa; Glickman, Michael S

    2014-10-01

    Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3' phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo.

  19. Structural basis for c-KIT inhibition by the suppressor of cytokine signaling 6 (SOCS6) ubiquitin ligase

    DEFF Research Database (Denmark)

    Zadjali, Fahad; Pike, Ashley C W; Vesterlund, Mattias

    2011-01-01

    of cytokine signaling 6 (SOCS6) is a member of the SOCS family of E3 ubiquitin ligases that can interact with c-KIT and suppress c-KIT-dependent pathways. Here, we analyzed the molecular mechanisms that determine SOCS6 substrate recognition. Our results show that the SH2 domain of SOCS6 is essential for its...... to substrate residue position pY+6 and envelopes the c-KIT phosphopeptide with a large BG loop insertion that contributes significantly to substrate interaction. We demonstrate that SOCS6 has ubiquitin ligase activity toward c-KIT and regulates c-KIT protein turnover in cells. Our data support a role of SOCS6...... as a feedback inhibitor of SCF-dependent signaling and provides molecular data to account for target specificity within the SOCS family of ubiquitin ligases....

  20. MACHINERY

    African Journals Online (AJOL)

    Data were collected using a portable data acquisition system: SKF Microlog. Data were collected' ... Modelling (HMM) ofvibrational signals. ... trifugal pump designed for a pressure increase of. 6.6 bars at ..... These frequency amplitudes are located at the vertical. axial and .... distribution centre the residual chlorine concen-.

  1. Disconnecting XRCC1 and DNA ligase III.

    Science.gov (United States)

    Katyal, Sachin; McKinnon, Peter J

    2011-07-15

    DNA strand break repair is essential for the prevention of multiple human diseases, particularly those which feature neuropathology. To further understand the pathogenesis of these syndromes, we recently developed animal models in which the DNA single-strand break repair (SSBR) components, XRCC1 and DNA Ligase III (LIG3), were inactivated in the developing nervous system. Although biochemical evidence suggests that inactivation of XRCC1 and LIG3 should share common biological defects, we found profound phenotypic differences between these two models, implying distinct biological roles for XRCC1 and LIG3 during DNA repair. Rather than a key role in nuclear DNA repair, we found LIG3 function was central to mitochondrial DNA maintenance. Instead, our data indicate that DNA Ligase 1 is the main DNA ligase for XRCC1-mediated DNA repair. These studies refine our understanding of DNA SSBR and the etiology of neurological disease.

  2. Disconnecting XRCC1 and DNA ligase III

    Science.gov (United States)

    Katyal, Sachin

    2011-01-01

    DNA strand break repair is essential for the prevention of multiple human diseases, particularly those which feature neuropathology. To further understand the pathogenesis of these syndromes, we recently developed animal models in which the DNA single-strand break repair (SSBR) components, XRCC1 and DNA Ligase III (LIG3), were inactivated in the developing nervous system. Although biochemical evidence suggests that inactivation of XRCC1 and LIG3 should share common biological defects, we found profound phenotypic differences between these two models, implying distinct biological roles for XRCC1 and LIG3 during DNA repair. Rather than a key role in nuclear DNA repair, we found LIG3 function was central to mitochondrial DNA maintenance. Instead, our data indicate that DNA Ligase 1 is the main DNA ligase for XRCC1-mediated DNA repair. These studies refine our understanding of DNA SSBR and the etiology of neurological disease. PMID:21636980

  3. On Generalized Euler Spirals in E^3

    OpenAIRE

    Saracoglu, Semra

    2012-01-01

    The Cornu spirals on plane are the curves whose curvatures are linear. Generalized planar cornu spirals and Euler spirals in E^3, the curves whose curvatures are linear are defined in [1,5]. In this study, these curves are presented as the ratio of two rational linear functions. Also here, generalized Euler spirals in E^3 has been defined and given their some various characterizations. The approach I used in this paper is useful in understanding the role of Euler spirals in E^3 in differentia...

  4. Strategies for Improving Enterprise Standardization Management of Tropical Crop Machinery

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ There are two categories of tropical crop machinery. One comprises operation machinery that is used for planting, managing and harvesting tropical crops, while the other comprises process machinery for processing tropical crops. Tropical crop machinery is distinguished from other agricultural machinery by the special crops that such machinery cultivates and processes.

  5. Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation.

    Science.gov (United States)

    Stolz, Alexandra; Besser, Stefanie; Hottmann, Heike; Wolf, Dieter H

    2013-09-17

    Quality control and degradation of misfolded proteins are essential processes of all cells. The endoplasmic reticulum (ER) is the entry site of proteins into the secretory pathway in which protein folding occurs and terminally misfolded proteins are recognized and retrotranslocated across the ER membrane into the cytosol. Here, proteins undergo polyubiquitination by one of the membrane-embedded ubiquitin ligases, in yeast Hrd1/Der3 (HMG-CoA reductase degradation/degradation of the ER) and Doa10 (degradation of alpha), and are degraded by the proteasome. In this study, we identify cytosolic Ubr1 (E3 ubiquitin ligase, N-recognin) as an additional ubiquitin ligase that can participate in ER-associated protein degradation (ERAD) in yeast. We show that two polytopic ERAD substrates, mutated transporter of the mating type a pheromone, Ste6* (sterile), and cystic fibrosis transmembrane conductance regulator, undergo Ubr1-dependent degradation in the presence and absence of the canonical ER ubiquitin ligases. Whereas in the case of Ste6* Ubr1 is specifically required under stress conditions such as heat or ethanol or in the absence of the canonical ER ligases, efficient degradation of human cystic fibrosis transmembrane conductance regulator requires function of Ubr1 already in wild-type cells under standard growth conditions. Together with the Hsp70 (heat shock protein) chaperone Ssa1 (stress-seventy subfamily A) and the AAA-type ATPase Cdc48 (cell division cycle), Ubr1 directs the substrate to proteasomal degradation. These data unravel another layer of complexity in ERAD.

  6. A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure

    DEFF Research Database (Denmark)

    Luijsterburg, Martijn S; Acs, Klara; Ackermann, Leena

    2012-01-01

    . Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks......, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.......The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation...

  7. E3: Extreme Energy Event monitoring

    CERN Document Server

    CERN. Geneva

    2015-01-01

    World peace through CERN technology: Use of explosives especially in urban areas is the defining phenomenon of the last and current century. E3 aims to reduce such excesses of violence by collecting scientific hard evidence.

  8. Freezing E3-brane instantons with fluxes

    Energy Technology Data Exchange (ETDEWEB)

    Bianchi, M.; Martucci, L. [Dipartimento di Fisica, Universita di Roma Tor Vergata (Italy); I.N.F.N., Sezione di Roma Tor Vergata (Italy); Collinucci, A. [Theory Group, Physics Department, CERN, Geneva (Switzerland); Physique Theorique et Mathematique Universite Libre de Bruxelles (Belgium)

    2012-07-15

    E3-instantons that generate non-perturbative superpotentials in IIB N = 1 compactifications have a much more frequent occurrence than currently believed. Worldvolume fluxes will typically lift the E3-brane geometric moduli and their fermionic superpartners, leaving only the two required universal fermionic zero-modes. We consistently incorporate SL(2,Z) monodromies and world-volume fluxes in the effective theory of the E3-brane fermions and study the resulting zero modes spectrum, highlighting the relation between F-theory and perturbative IIB results. This leads us to a IIB derivation of the index for generation of superpotential terms, which reproduces and generalizes available results. Furthermore, we show how E3 worldvolume fluxes can be explicitly constructed in a one-modulus compactification, such that the instanton has exactly two fermonic zero-modes. This construction is readily applicable to numerous scenarios. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  9. E3 Travel & Mission Support System

    Data.gov (United States)

    US Agency for International Development — ETRAMS is a travel data collection system developed by the CKM team in E3 that collects information on both the basic details of an employee's trips (destination,...

  10. E3: Economy, Energy and Environment

    Science.gov (United States)

    E3 is a technical assistance framework helping communities, manufacturers, and manufacturing supply chains adapt and thrive in today's green economy. Find information on pollution prevention, sustainable business practices, and energy efficiency.

  11. The MLLE domain of the ubiquitin ligase UBR5 binds to its catalytic domain to regulate substrate binding.

    Science.gov (United States)

    Muñoz-Escobar, Juliana; Matta-Camacho, Edna; Kozlov, Guennadi; Gehring, Kalle

    2015-09-11

    E3 ubiquitin ligases catalyze the transfer of ubiquitin from an E2-conjugating enzyme to a substrate. UBR5, homologous to the E6AP C terminus (HECT)-type E3 ligase, mediates the ubiquitination of proteins involved in translation regulation, DNA damage response, and gluconeogenesis. In addition, UBR5 functions in a ligase-independent manner by prompting protein/protein interactions without ubiquitination of the binding partner. Despite recent functional studies, the mechanisms involved in substrate recognition and selective ubiquitination of its binding partners remain elusive. The C terminus of UBR5 harbors the HECT catalytic domain and an adjacent MLLE domain. MLLE domains mediate protein/protein interactions through the binding of a conserved peptide motif, termed PAM2. Here, we characterize the binding properties of the UBR5 MLLE domain to PAM2 peptides from Paip1 and GW182. The crystal structure with a Paip1 PAM2 peptide reveals the network of hydrophobic and ionic interactions that drive binding. In addition, we identify a novel interaction of the MLLE domain with the adjacent HECT domain mediated by a PAM2-like sequence. Our results confirm the role of the MLLE domain of UBR5 in substrate recruitment and suggest a potential role in regulating UBR5 ligase activity.

  12. HECT E3s and human disease

    Directory of Open Access Journals (Sweden)

    Staub Olivier

    2007-11-01

    Full Text Available Abstract In a simplified view, members of the HECT E3 family have a modular structure consisting of the C-terminal HECT domain, which is catalytically involved in the attachment of ubiquitin to substrate proteins, and N-terminal extensions of variable length and sequence that mediate the substrate specificity of the respective HECT E3. Although the physiologically relevant substrates of most HECT E3s have remained elusive, it is becoming increasingly clear that HECT E3s play an important role in sporadic and hereditary human diseases including cancer, cardiovascular (Liddle's syndrome and neurological (Angelman syndrome disorders, and/or in disease-relevant processes including bone homeostasis, immune response and retroviral budding. Thus, molecular approaches to target the activity of distinct HECT E3s, regulators thereof, and/or of HECT E3 substrates could prove valuable in the treatment of the respective diseases. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com.

  13. Tyrosine phosphorylation of NEDD4 activates its ubiquitin ligase activity.

    Science.gov (United States)

    Persaud, Avinash; Alberts, Philipp; Mari, Sara; Tong, Jiefei; Murchie, Ryan; Maspero, Elena; Safi, Frozan; Moran, Michael F; Polo, Simona; Rotin, Daniela

    2014-10-07

    Ligand binding to the receptor tyrosine kinase fibroblast growth factor (FGF) receptor 1 (FGFR1) causes dimerization and activation by transphosphorylation of tyrosine residues in the kinase domain. FGFR1 is ubiquitylated by the E3 ligase NEDD4 (also known as NEDD4-1), which promotes FGFR1 internalization and degradation. Although phosphorylation of FGFR1 is required for NEDD4-dependent endocytosis, NEDD4 directly binds to a nonphosphorylated region of FGFR1. We found that activation of FGFR1 led to activation of c-Src kinase-dependent tyrosine phosphorylation of NEDD4, enhancing the ubiquitin ligase activity of NEDD4. Using mass spectrometry, we identified several FGF-dependent phosphorylated tyrosines in NEDD4, including Tyr(43) in the C2 domain and Tyr(585) in the HECT domain. Mutating these tyrosines to phenylalanine to prevent phosphorylation inhibited FGF-dependent NEDD4 activity and FGFR1 endocytosis and enhanced cell proliferation. Mutating the tyrosines to glutamic acid to mimic phosphorylation enhanced NEDD4 activity. Moreover, the NEDD4 C2 domain bound the HECT domain, and the presence of phosphomimetic mutations inhibited this interaction, suggesting that phosphorylation of NEDD4 relieves an inhibitory intra- or intermolecular interaction. Accordingly, activation of FGFR1 was not required for activation of NEDD4 that lacked its C2 domain. Activation of c-Src by epidermal growth factor (EGF) also promoted tyrosine phosphorylation and enhanced the activity of NEDD4. Thus, we identified a feedback mechanism by which receptor tyrosine kinases promote catalytic activation of NEDD4 and that may represent a mechanism of receptor crosstalk.

  14. Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase.

    Science.gov (United States)

    Nohales, María-Ángeles; Flores, Ricardo; Daròs, José-Antonio

    2012-08-21

    Viroids are a unique class of noncoding RNAs: composed of only a circular, single-stranded molecule of 246-401 nt, they manage to replicate, move, circumvent host defenses, and frequently induce disease in higher plants. Viroids replicate through an RNA-to-RNA rolling-circle mechanism consisting of transcription of oligomeric viroid RNA intermediates, cleavage to unit-length strands, and circularization. Though the host RNA polymerase II (redirected to accept RNA templates) mediates RNA synthesis and a type-III RNase presumably cleavage of Potato spindle tuber viroid (PSTVd) and closely related members of the family Pospiviroidae, the host enzyme catalyzing the final circularization step, has remained elusive. In this study we propose that PSTVd subverts host DNA ligase 1, converting it to an RNA ligase, for the final step. To support this hypothesis, we show that the tomato (Solanum lycopersicum L.) DNA ligase 1 specifically and efficiently catalyzes circularization of the genuine PSTVd monomeric linear replication intermediate opened at position G95-G96 and containing 5'-phosphomonoester and 3'-hydroxyl terminal groups. Moreover, we also show a decreased PSTVd accumulation and a reduced ratio of monomeric circular to total monomeric PSTVd forms in Nicotiana benthamiana Domin plants in which the endogenous DNA ligase 1 was silenced. Thus, in a remarkable example of parasitic strategy, viroids reprogram for their replication the template and substrate specificity of a DNA-dependent RNA polymerase and a DNA ligase to act as RNA-dependent RNA polymerase and RNA ligase, respectively.

  15. Ubiquitin Ligase HUWE1 Regulates Axon Branching through the Wnt/beta-Catenin Pathway in a Drosophila Model for Intellectual Disability

    NARCIS (Netherlands)

    Vandewalle, J.; Langen, M.; Zschaetzsch, M.; Nijhof, B.; Kramer, J.M.; Brems, H.; Bauters, M.; Lauwers, E.; Srahna, M.; Marynen, P.; Verstreken, P.; Schenck, A.; Hassan, B.A.; Froyen, G.

    2013-01-01

    We recently reported that duplication of the E3 ubiquitin ligase HUWE1 results in intellectual disability (ID) in male patients. However, the underlying molecular mechanism remains unknown. We used Drosophila melanogaster as a model to investigate the effect of increased HUWE1 levels on the developi

  16. Developing Process of Tropical Crop Machinery Standardization

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    @@ General Situation Tropical crop machinery is a new special mechanical profession, which began to develop from 1950s to 1960s in China. Because the weather, soil and farm crops varieties in tropical region are greatly different from those in the other regions, most of the traditional farm machinery can't be directly used in tropical region or on the tropical crops. Tropical crop machinery needs a special design and manufacture. So some professional research institutes and education units were set up and some enterprises were built at that time, and the profession of tropical crop machinery was formed.

  17. Membrane manipulations by the ESCRT machinery.

    Science.gov (United States)

    Odorizzi, Greg

    2015-01-01

    The endosomal sorting complexes required for transport (ESCRTs) collectively comprise a machinery that was first known for its function in the degradation of transmembrane proteins in the endocytic pathway of eukaryotic cells. Since their discovery, however, ESCRTs have been recognized as playing important roles at the plasma membrane, which appears to be the original site of function for the ESCRT machinery. This article reviews some of the major research findings that have shaped our current understanding of how the ESCRT machinery controls membrane dynamics and considers new roles for the ESCRT machinery that might be driven by these mechanisms.

  18. Oils degradation in agricultural machinery

    Directory of Open Access Journals (Sweden)

    Vojtěch Kumbár

    2013-01-01

    Full Text Available Evaluating of oils condition in agricultural machinery is very important. With monitoring and evaluating we can prevent technical and economic losses. In this paper there were monitored the liquid lubricants taken from mobile thresher New Holland CX 860. Chemical and viscosity degradation of the lubricants were evaluated. Temperature dependence dynamic viscosity was observed in the range of temperature from −10 °C to 80 °C (for all oils. Considerable temperature dependence dynamic viscosity was found and demonstrated in case of all samples, which is in accordance with theoretical assumptions and literature data. Mathematical models were developed and tested. Temperature dependence dynamic viscosity was modeled using a polynomial 6th degree. The proposed models can be used for prediction of flow behavior of oils.

  19. The E3-ligase TRIM family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors.

    Science.gov (United States)

    Versteeg, Gijs A; Rajsbaum, Ricardo; Sánchez-Aparicio, Maria Teresa; Maestre, Ana M; Valdiviezo, Julio; Shi, Mude; Inn, Kyung-Soo; Fernandez-Sesma, Ana; Jung, Jae; García-Sastre, Adolfo

    2013-02-21

    Innate immunity conferred by the type I interferon is critical for antiviral defense. To date only a limited number of tripartite motif (TRIM) proteins have been implicated in modulation of innate immunity and anti-microbial activity. Here we report the complementary DNA cloning and systematic analysis of all known 75 human TRIMs. We demonstrate that roughly half of the 75 TRIM-family members enhanced the innate immune response and that they do this at multiple levels in signaling pathways. Moreover, messenger RNA levels and localization of most of these TRIMs were found to be altered during viral infection, suggesting that their regulatory activities are highly controlled at both pre- and posttranscriptional levels. Taken together, our data demonstrate a very considerable dedication of this large protein family to the positive regulation of the antiviral response, which supports the notion that this family of proteins evolved as a component of innate immunity.

  20. BRCC36, a Novel Subunit of a BRCA1 E3 Ubiquitin Ligase Complex: Candidates for BRCA3

    Science.gov (United States)

    2008-06-01

    J, Dahia PL, Wang SI, Zheng Z, Bose S, Call KM, Tsou HC, Peacocke M and others. 1997. Germline mutations of the PTEN gene in Cowden disease, an...Hum Mutat 20:65–73. Arnold N, Peper H, Bandick K, Kreikemeier M, Karow D, Teegen B, Jonat W. 2002. Establishing a control population to screen for

  1. BRCC36, A Novel Subunit of a BRCA1 E3 Ubiquitin Ligase Complex, Candidates for BRCA3

    Science.gov (United States)

    2006-06-01

    Lymphocytes isolated from FRAP blood samples were infected with Epstein-Barr virus ( EBV ) to establish the immortal lymphoblastoid cell lines (LCLs...BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures . Genes Dev 2000;14:927–39. 27...inhibitor, puromycin (Sigma, St. Louis, MO; www.sigmaaldrich.com), was added to the EBV cells at the concentration of 200 mg/ml for 14 hr before total RNAs

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

    OpenAIRE

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

    2015-01-01

    eLife digest The world is facing a global epidemic of obesity, which also increases the risk for diabetes and heart disease. Obesity is caused when excess fat is stored in fat cells, and overweight individuals have larger fat cells compared to healthy weight people. Therefore understanding how fat cells are created in the body can provide new ways to combat obesity. Fat cells, also known as adipocytes, arise from precursor cells via a process called adipogenesis. This requires the activity of...

  3. Mouse RING finger protein Rnf133 is a testis-specific endoplasmic reticulum-associated E3 ubiquitin ligase

    Institute of Scientific and Technical Information of China (English)

    Hong Nian; Chunsheng Han; Wei Zhang; Hexin Shi; Qingzhen Zhao; Qi Xie; Shangying Liao; Yan Zhang; Zhuqiang Zhang; Chen Wang

    2008-01-01

    @@ Dear Editor, Spermatogenesis, the process by which sperms are generated within the male gonads, involves a number of events that occur only in the testis. Examples include the nucleus condensation as a result of histone sequential replacement by transition proteins 1, 2 and protamin, the formation of sperm-specific organelles such as acrosomes and sperm tails, and the shedding of majority of the cytoplasm as residual bodies.

  4. The Olympic of Textile Machinery closed

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    The five days 2012 China International Textile Machinery Exhibition ITMA Asia Exhibition, which attracted much attention from the industry, was closed at the Shanghai New International Expo Center on June 16, more than 1300 textile enterprises from nearly 30 countries around the world gathered on the exhibition, the world’s latest textile machinery technologies, crafts and equipments were also presented one by one.

  5. 46 CFR 115.804 - Machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Machinery. 115.804 Section 115.804 Shipping COAST GUARD....804 Machinery. At each initial and subsequent inspection for certification of a vessel, the owner or managing operator shall be prepared to conduct tests and have the vessel ready for inspections of...

  6. China Textile Machinery Expresses Self-Surpassing

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    It’s difficult to imagine that any sector of the textile industry has benefited more from innovations in the past 10 years than textile machinery. Advanced textile machinery has brought new life to the production segment of the business and fulfills the essential preconditions for economically efficient textile production.

  7. 46 CFR 58.01-40 - Machinery, angles of inclination.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Machinery, angles of inclination. 58.01-40 Section 58.01... AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-40 Machinery, angles of inclination. (a) Propulsion machinery and all auxiliary machinery essential to the propulsion and safety of the vessel must...

  8. 46 CFR 58.01-45 - Machinery space, ventilation.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Machinery space, ventilation. 58.01-45 Section 58.01-45... MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-45 Machinery space, ventilation. Each machinery space must be ventilated to ensure that, when machinery or boilers are operating at full power in...

  9. The APC/C Ubiquitin Ligase: from Cell Biology to Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Clara ePenas

    2012-01-01

    Full Text Available The ubiquitin proteasome system (UPS is required for normal cell proliferation, vertebrate development, and cancer cell transformation. The UPS consists of multiple proteins that work in concert to target a protein for degradation via the 26S proteasome. Chains of an 8.5 kDa protein called ubiquitin are attached to substrates, thus allowing recognition by the 26S proteasome. Enzymes called ubiquitin ligases or E3s mediate specific attachment to substrates. Although there are over 600 different ubiquitin ligases, the Skp1-Cullin-F-box proteins (SCF ubiquitin ligases and the Anaphase Promoting Complex/cyclosome (APC/C are the most studied. SCF involvement in cancer has been known for some time while APC/C’s cancer role has recently emerged. In this review we will discuss the importance of APC/C to normal cell proliferation and development, thus underscoring its possible contribution to transformation. We will also put forth the hypothesis that modulating a specific interaction of the APC/C may be therapeutically attractive in specific cancer subtypes. Finally, given that the APC/C pathway is relatively new as a cancer target, therapeutic interventions affecting APC/C activity may be beneficial in cancers that are resistant to classical chemotherapy.

  10. DNA ligase IV syndrome; a review.

    Science.gov (United States)

    Altmann, Thomas; Gennery, Andrew R

    2016-10-07

    DNA ligase IV deficiency is a rare primary immunodeficiency, LIG4 syndrome, often associated with other systemic features. DNA ligase IV is part of the non-homologous end joining mechanism, required to repair DNA double stranded breaks. Ubiquitously expressed, it is required to prevent mutagenesis and apoptosis, which can result from DNA double strand breakage caused by intracellular events such as DNA replication and meiosis or extracellular events including damage by reactive oxygen species and ionising radiation.Within developing lymphocytes, DNA ligase IV is required to repair programmed DNA double stranded breaks induced during lymphocyte receptor development.Patients with hypomorphic mutations in LIG4 present with a range of phenotypes, from normal to severe combined immunodeficiency. All, however, manifest sensitivity to ionising radiation. Commonly associated features include primordial growth failure with severe microcephaly and a spectrum of learning difficulties, marrow hypoplasia and a predisposition to lymphoid malignancy. Diagnostic investigations include immunophenotyping, and testing for radiosensitivity. Some patients present with microcephaly as a predominant feature, but seemingly normal immunity. Treatment is mainly supportive, although haematopoietic stem cell transplantation has been used in a few cases.

  11. Stochastic noise in splicing machinery.

    Science.gov (United States)

    Melamud, Eugene; Moult, John

    2009-08-01

    The number of known alternative human isoforms has been increasing steadily with the amount of available transcription data. To date, over 100 000 isoforms have been detected in EST libraries, and at least 75% of human genes have at least one alternative isoform. In this paper, we propose that most alternative splicing events are the result of noise in the splicing process. We show that the number of isoforms and their abundance can be predicted by a simple stochastic noise model that takes into account two factors: the number of introns in a gene and the expression level of a gene. The results strongly support the hypothesis that most alternative splicing is a consequence of stochastic noise in the splicing machinery, and has no functional significance. The results are also consistent with error rates tuned to ensure that an adequate level of functional product is produced and to reduce the toxic effect of accumulation of misfolding proteins. Based on simulation of sampling of virtual cDNA libraries, we estimate that error rates range from 1 to 10% depending on the number of introns and the expression level of a gene.

  12. Structural Basis of DNA Ligase IV-Artemis Interaction in Nonhomologous End-Joining

    Directory of Open Access Journals (Sweden)

    Pablo De Ioannes

    2012-12-01

    Full Text Available DNA ligase IV (LigIV and Artemis are central components of the nonhomologous end-joining (NHEJ machinery that is required for V(DJ recombination and the maintenance of genomic integrity in mammalian cells. We report here crystal structures of the LigIV DNA binding domain (DBD in both its apo form and in complex with a peptide derived from the Artemis C-terminal region. We show that LigIV interacts with Artemis through an extended hydrophobic surface. In particular, we find that the helix α2 in LigIV-DBD is longer than in other mammalian ligases and presents residues that specifically interact with the Artemis peptide, which adopts a partially helical conformation on binding. Mutations of key residues on the LigIV-DBD hydrophobic surface abolish the interaction. Together, our results provide structural insights into the specificity of the LigIV-Artemis interaction and how the enzymatic activities of the two proteins may be coordinated during NHEJ.

  13. Structural basis of DNA ligase IV-Artemis interaction in nonhomologous end-joining.

    Science.gov (United States)

    De Ioannes, Pablo; Malu, Shruti; Cortes, Patricia; Aggarwal, Aneel K

    2012-12-27

    DNA ligase IV (LigIV) and Artemis are central components of the nonhomologous end-joining (NHEJ) machinery that is required for V(D)J recombination and the maintenance of genomic integrity in mammalian cells. We report here crystal structures of the LigIV DNA binding domain (DBD) in both its apo form and in complex with a peptide derived from the Artemis C-terminal region. We show that LigIV interacts with Artemis through an extended hydrophobic surface. In particular, we find that the helix α2 in LigIV-DBD is longer than in other mammalian ligases and presents residues that specifically interact with the Artemis peptide, which adopts a partially helical conformation on binding. Mutations of key residues on the LigIV-DBD hydrophobic surface abolish the interaction. Together, our results provide structural insights into the specificity of the LigIV-Artemis interaction and how the enzymatic activities of the two proteins may be coordinated during NHEJ. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology

    Directory of Open Access Journals (Sweden)

    Cecilia R. Chambers

    2015-01-01

    Full Text Available With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and biotechnology. Currently, the nucleic acid ligases from bacteriophage T4 are used extensively in these protocols. In this review, we argue that the nucleic acid ligases from Archaea represent a largely untapped pool of enzymes with diverse and potentially favourable properties for new and emerging biotechnological applications. We summarise the current state of knowledge on archaeal DNA and RNA ligases, which makes apparent the relative scarcity of information on in vitro activities that are of most relevance to biotechnologists (such as the ability to join blunt- or cohesive-ended, double-stranded DNA fragments. We highlight the existing biotechnological applications of archaeal DNA ligases and RNA ligases. Finally, we draw attention to recent experiments in which protein engineering was used to modify the activities of the DNA ligase from Pyrococcus furiosus and the RNA ligase from Methanothermobacter thermautotrophicus, thus demonstrating the potential for further work in this area.

  15. DNA Ligase IV regulates XRCC4 nuclear localization.

    Science.gov (United States)

    Francis, Dailia B; Kozlov, Mikhail; Chavez, Jose; Chu, Jennifer; Malu, Shruti; Hanna, Mary; Cortes, Patricia

    2014-09-01

    DNA Ligase IV, along with its interacting partner XRCC4, are essential for repairing DNA double strand breaks by non-homologous end joining (NHEJ). Together, they complete the final ligation step resolving the DNA break. Ligase IV is regulated by XRCC4 and XLF. However, the mechanism(s) by which Ligase IV control the NHEJ reaction and other NHEJ factor(s) remains poorly characterized. Here, we show that a C-terminal region of Ligase IV (aa 620-800), which encompasses a NLS, the BRCT I, and the XRCC4 interacting region (XIR), is essential for nuclear localization of its co-factor XRCC4. In Ligase IV deficient cells, XRCC4 showed deregulated localization remaining in the cytosol even after induction of DNA double strand breaks. DNA Ligase IV was also required for efficient localization of XLF into the nucleus. Additionally, human fibroblasts that harbor hypomorphic mutations within the Ligase IV gene displayed decreased levels of XRCC4 protein, implicating that DNA Ligase IV is also regulating XRCC4 stability. Our results provide evidence for a role of DNA Ligase IV in controlling the cellular localization and protein levels of XRCC4.

  16. Structure and function of the DNA ligases encoded by the mammalian LIG3 gene

    OpenAIRE

    Tomkinson, Alan E.; Sallmyr, Annahita

    2013-01-01

    Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA lig...

  17. Cellular DNA ligase I is recruited to cytoplasmic vaccinia virus factories and masks the role of the vaccinia ligase in viral DNA replication.

    Science.gov (United States)

    Paran, Nir; De Silva, Frank S; Senkevich, Tatiana G; Moss, Bernard

    2009-12-17

    Vaccinia virus (VACV) encodes DNA polymerase and additional proteins that enable cytoplasmic replication. We confirmed the ability of VACV DNA ligase mutants to replicate and tested the hypothesis that cellular ligases compensate for loss of viral gene expression. RNA silencing of human DNA ligase I expression and a small molecule inhibitor of human DNA ligase I [corrected] severely reduced replication of viral DNA in cells infected with VACV ligase-deficient mutants, indicating that the cellular enzyme plays a complementary role. Replication of ligase-deficient VACV was greatly reduced and delayed in resting primary cells, correlating with initial low levels of ligase I and subsequent viral induction and localization of ligase I in virus factories. These studies indicate that DNA ligation is essential for poxvirus replication and explain the ability of ligase deletion mutants to replicate in dividing cells but exhibit decreased pathogenicity in mice. Encoding its own ligase might allow VACV to "jump-start" DNA synthesis.

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

    Science.gov (United States)

    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.

  19. Scaling laws in (e,3e) processes

    Energy Technology Data Exchange (ETDEWEB)

    Gasaneo, G; Rodriguez, K V [Departamento de Fisica - Universidad Nacional del Sur and CONICET, 8000 BahIa Blanca, Buenos Aires (Argentina); Ancarani, L U; Cappello, C Dal [Laboratoire de Physique Moleculaire et des Collisions, Universite Paul Verlaine - Metz, 57078 Metz (France); Charpentier, I [Laboratoire de Physique et Mecanique des Materiaux, UMR CNRS 7554, Ile du Saulcy, 57045 Metz (France)

    2009-11-01

    We study the double ionization of helium-like ions by impact of electrons with high incident energy. Within the isoelectronic sequence, an approximate scaling law for (e,3e) differential cross sections is proposed and confirmed by calculations. The latter are performed using 14-parameters Hylleraas-like wave functions to represent the bound electrons in the initial channel, plane waves for the fast incoming and scattered electrons, and a continuum distorted wave approach for the two ejected electrons in the final channel.

  20. Textile Machinery:Imports Rebound Again

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In the year of 2006,the general situation of China’s textile machinery equipment imports had shown a clear sign of revival from the downward trend of two years ago,with a total annual import of 4.1 billion USD,an increase of 19.05% against the same period of 2005. Continuingly,the year of 2007 has witnessed a sustainable growth trend of textile machinery equipment imports in the first quarter and the trend definitely will be maintained through the whole year.According to statistics released from China Customs,the imports of textile machinery reached 1.098 billion USD in the first three months of 2007, up by 35.26% year-on-year. Then,why China’s textile machinery imports warm up again after two years’ cool down?

  1. A comprehensive overview of hybrid construction machinery

    Directory of Open Access Journals (Sweden)

    Jixin Wang

    2016-03-01

    Full Text Available With the increasing attention of energy saving and emission reduction technology, the recent application of hybrid powertrain technology affects the development of construction machinery industry. This article reviews these publications and provides comprehensive references. This article reviews the state-of-art for the hybrid wheel loader and excavator, which focuses on powertrain configuration, energy storage devices, and energy management strategies. The basis of classification and characteristic of each powertrain configuration are described. Advantages and disadvantages of batteries, supercapacitors, hydraulic accumulators, and flywheel used in hybrid construction machinery are summarized. The existing energy management strategies for hybrid construction machinery are also elaborated. The technological challenges and developing trends in the near future for hybrid construction machinery are discussed.

  2. Energy Savings Thanks to French Textile Machinery

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The French Textile Machinery Manufacturers’ Association (UCMTF) has presented,during a seminar it organized for textile professionals and students,the spectacular energy savings achieved thanks to state of the art

  3. Textile Machinery Import and Export in 2011

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    Along with the rebounded international market, in the year of 2011, the foreign trade of textile machinery industry preserved a stable development: the import amount saw a slightly decrease, while the total import and export value kept an increasing trend. According to the Customs, the export and import of textile machinery totalized 2.245 billion USD and 5.364 billion USD, increasing by 27.81% and 24.70%, respectively, comparing with the same period of time in 2010.

  4. Origin and diversification of TRIM ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Ignacio Marín

    Full Text Available 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. Expression and biochemical characterization of Plasmodium falciparum DNA ligase I.

    Science.gov (United States)

    Buguliskis, Jeffrey S; Casta, Louis J; Butz, Charles E; Matsumoto, Yoshihiro; Taraschi, Theodore F

    2007-10-01

    We report that Plasmodium falciparum (Pf) encodes a 912 amino acid ATP-dependent DNA ligase. Protein sequence analysis of Pf DNA ligase I indicates a strong sequence similarity, particularly in the C-terminal region, to DNA ligase I homologues. The activity of recombinant Pf DNA ligase I (PfLigI) was investigated using protein expressed in HEK293 cells. The PfLigI gene product is approximately 94kDa and catalyzes phosphodiester bond formation on a singly nicked DNA substrate. The enzyme is most active at alkaline pH (8.5) and with Mg(2+) or Mn(2+) and ATP as cofactors. Kinetic studies of PfLigI revealed that the enzyme has similar substrate affinity (K(m) 2.6nM) as compared to human DNA ligase I and k(cat) (2.3x10(-3)s(-1)) and k(cat)/K(m) (8.8x10(5)M(-1)s(-1)) which are similar to other ATP-dependent DNA ligases. PfLigI was able to join RNA-DNA substrates only when the RNA sequence was upstream of the nick, confirming that it is DNA ligase I and has no associated DNA ligase III like activity.

  6. KF-1 ubiquitin ligase: an anxiety suppressor

    Directory of Open Access Journals (Sweden)

    Tamotsu Hashimoto-Gotoh

    2009-05-01

    Full Text Available Anxiety is an instinct that may have developed to promote adaptive survival by evading unnecessary danger. However, excessive anxiety is disruptive and can be a basic disorder of other psychiatric diseases such as depression. The KF-1, a ubiquitin ligase located to the endoplasmic reticulum (ER, may prevent excessive anxiety; kf-1−/− mice exhibit selectively elevated anxiety-like behavior against light or heights. Thus, KF-1 may degrade some target proteins, responsible for promoting anxiety, through the ER-associated degradation pathway, similar to Parkin in Parkinson's disease (PD. Parkin, another ER-ubiquitin ligase, prevents the degeneration of dopaminergic neurons by degrading the target proteins responsible for PD. Molecular phylogenetic studies have revealed that the prototype of kf-1 appeared in the very early phase of animal evolution but was lost, unlike parkin, in the lineage leading up to Drosophila. Therefore, kf-1−/− mice, be a powerful tool for elucidating the molecular mechanisms involved in emotional regulation, and for screening novel anxiolytic/antidepressant compounds.

  7. 46 CFR 58.20-15 - Installation of refrigerating machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Installation of refrigerating machinery. 58.20-15... AND AUXILIARY MACHINERY AND RELATED SYSTEMS Refrigeration Machinery § 58.20-15 Installation of refrigerating machinery. (a) Where refrigerating machines are installed in which anhydrous ammonia is used as...

  8. 46 CFR 169.625 - Compartments containing diesel machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Compartments containing diesel machinery. 169.625... SCHOOL VESSELS Machinery and Electrical Ventilation § 169.625 Compartments containing diesel machinery. (a) Spaces containing machinery must be fitted with adequate dripproof ventilators, trunks,...

  9. 46 CFR 174.195 - Bulkheads in machinery spaces.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Bulkheads in machinery spaces. 174.195 Section 174.195... in machinery spaces. (a) The bulkhead in each machinery space of each OSV must be watertight to the bulkhead deck. (b) Each penetration of, and each opening in, a bulkhead in a machinery space must— (1)...

  10. 46 CFR 58.01-35 - Main propulsion auxiliary machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Main propulsion auxiliary machinery. 58.01-35 Section 58... AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-35 Main propulsion auxiliary machinery. Auxiliary machinery vital to the main propulsion system must be provided in duplicate unless the...

  11. 46 CFR 58.01-25 - Means of stopping machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Means of stopping machinery. 58.01-25 Section 58.01-25 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-25 Means of stopping machinery. Machinery...

  12. 46 CFR 42.15-35 - Machinery space openings.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Machinery space openings. 42.15-35 Section 42.15-35... BY SEA Conditions of Assignment of Freeboard § 42.15-35 Machinery space openings. (a) Machinery space..., funnel, or machinery space ventilators in an exposed position on the freeboard or superstructure...

  13. 46 CFR 45.149 - Machinery space openings.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Machinery space openings. 45.149 Section 45.149 Shipping... Assignment § 45.149 Machinery space openings. (a) Machinery space openings in position 1 or 2 must be framed... funnel or machinery space ventilator that must be kept open for the essential operations of the ship...

  14. Overexpression of ligase defective E6-associated protein, E6-AP, results in mammary tumorigenesis.

    Science.gov (United States)

    Ramamoorthy, Sivapriya; Tufail, Rozina; Hokayem, Jimmy El; Jorda, Mercy; Zhao, Wei; Reis, Zizi; Nawaz, Zafar

    2012-02-01

    E6-associated protein (E6-AP) is a dual function protein. It acts as an E3 ubiquitin-protein ligase enzyme and coactivator of steroid hormone receptors such as estrogen (ERα) and progesterone (PR) receptors. It promotes the degradation of ERα and PR through the ubiquitin-proteasome pathway. Furthermore, it has been shown that the levels of E6-AP are inversely associated with that of ERα in human breast tumors. But the role of wild-type human E6-AP and its ubiquitin-protein ligase activity in mammary tumorigenesis is still unknown. To investigate this role, the authors utilized transgenic mice lines that specifically overexpress either the wild-type human E6-AP (E6-AP(WT)) or the ubiquitin-protein ligase defective E6-AP that contains C833S mutation (E6-AP(C833S)) in the mammary gland. To further substantiate the role of E6-AP in the development of breast tumorigenesis, it was also examined the expression of E6-AP in a large cohort of human breast cancer samples. The transgenic mice that overexpress wild-type E6-AP (E6-AP(WT)) fail to develop mammary tumors. Unlike the E6-AP(WT) mice, the E6-AP(C833S) mice that overexpress ubiquitin-protein ligase defective E6-AP protein develop mammary hyperplasia with a median latency of 18 months. These observations suggest that the inactivation of the ubiquitin-protein ligase function of E6-AP is sufficient to initiate the process of mammary tumor development. Furthermore, the data also suggests that E6-AP exerts its effects on target cells by modulating the protein levels and functions of ERα and PR. In addition, it was found in human breast cancer patients that the level of E6-AP is decreased in invasive breast tumors compared to normal breast tissue. Moreover, the authors also show that the survival patterns for E6-AP negative patients were worse compared to E6-AP positive patients. Taken together, these data suggests that E6-AP may act as a tumor suppressor in breast.

  15. ADJUSTMENT, MAINTENANCE, AND REPAIR OF CROP HARVESTING MACHINERY. AGRICULTURAL MACHINERY--SERVICE OCCUPATIONS, MODULE NUMBER 11.

    Science.gov (United States)

    Ohio State Univ., Columbus. Center for Vocational and Technical Education.

    ONE OF A SERIES DESIGNED FOR HELPING TEACHERS PREPARE POSTSECONDARY-LEVEL STUDENTS FOR AGRICULTURAL MACHINERY SERVICE OCCUPATIONS AS PARTS MEN, MECHANICS, MECHANIC'S HELPERS, AND SERVICE SUPERVISORS, THIS GUIDE AIMS TO DEVELOP STUDENT COMPETENCY IN ADJUSTING, REPAIRING, AND MAINTAINING CROP HARVESTING MACHINERY. SUGGESTIONS FOR INTRODUCTION OF THE…

  16. Expression, purification and biochemical characterization of Methanocaldococcus jannaschii DNA ligase.

    Science.gov (United States)

    Wang, You; Xie, Juan-Juan; Han, Zhong; Liu, Jian-Hua; Liu, Xi-Peng

    2013-02-01

    We describe the biochemical characterization of Methanocaldococcus jannaschii (M. jannaschii) DNA ligase and its potential application in single nucleotide polymorphism (SNP) genotyping. The recombinant M. jannaschii DNA ligase is an ATP-dependent ligase. The ligase activity was dependent on metal ions of Mg(2+) and Mn(2+). The optimal concentrations of ATP cofactor and Mg(2+) ion were 0.01-2 and 10 mM, respectively. The optimal pH value for DNA ligation was 8.5. High concentrations of NaCl inhibited DNA ligation. The effects of mismatches on joining short oligonucleotides by M. jannaschii DNA ligase were fully characterized. The mismatches at the first position 5' to the nick inhibited ligation more than those at the first position 3' to the nick. The mismatches at other positions 5' to the nick (3rd to 7th sites) exhibited less inhibition on ligation. However, the introduction of a C/C mismatch at the third position 5' to the nick could completely inhibit the ligation of the terminal-mismatched nick of an oligonucleotide duplex by M. jannaschii DNA ligase. Therefore, introducing an additional mismatch at the third position 5' to the SNP site is a more effective approach in genotyping by M. jannaschii DNA ligase.

  17. Mitochondrial Machineries for Protein Import and Assembly.

    Science.gov (United States)

    Wiedemann, Nils; Pfanner, Nikolaus

    2017-03-15

    Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. Most of the >1,000 different mitochondrial proteins are synthesized as precursors in the cytosol and are imported into mitochondria by five transport pathways. The protein import machineries of the mitochondrial membranes and aqueous compartments reveal a remarkable variability of mechanisms for protein recognition, translocation, and sorting. The protein translocases do not operate as separate entities but are connected to each other and to machineries with functions in energetics, membrane organization, and quality control. Here, we discuss the versatility and dynamic organization of the mitochondrial protein import machineries. Elucidating the molecular mechanisms of mitochondrial protein translocation is crucial for understanding the integration of protein translocases into a large network that controls organelle biogenesis, function, and dynamics. Expected final online publication date for the Annual Review of Biochemistry Volume 86 is June 20, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  18. The RNA polymerase I transcription machinery.

    Science.gov (United States)

    Russell, Jackie; Zomerdijk, Joost C B M

    2006-01-01

    The rRNAs constitute the catalytic and structural components of the ribosome, the protein synthesis machinery of cells. The level of rRNA synthesis, mediated by Pol I (RNA polymerase I), therefore has a major impact on the life and destiny of a cell. In order to elucidate how cells achieve the stringent control of Pol I transcription, matching the supply of rRNA to demand under different cellular growth conditions, it is essential to understand the components and mechanics of the Pol I transcription machinery. In this review, we discuss: (i) the molecular composition and functions of the Pol I enzyme complex and the two main Pol I transcription factors, SL1 (selectivity factor 1) and UBF (upstream binding factor); (ii) the interplay between these factors during pre-initiation complex formation at the rDNA promoter in mammalian cells; and (iii) the cellular control of the Pol I transcription machinery.

  19. Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae.

    OpenAIRE

    Ramos, W; Tappe, N; Talamantez, J; Friedberg, E C; Tomkinson, A E

    1997-01-01

    Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomyces cerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism. In the present study we have identified a second DNA ligase activity in mitotic extracts of S. cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joi...

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

    Science.gov (United States)

    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.

  1. Machinery condition monitoring principles and practices

    CERN Document Server

    Mohanty, Amiya Ranjan

    2015-01-01

    Find the Fault in the MachinesDrawing on the author's more than two decades of experience with machinery condition monitoring and consulting for industries in India and abroad, Machinery Condition Monitoring: Principles and Practices introduces the practicing engineer to the techniques used to effectively detect and diagnose faults in machines. Providing the working principle behind the instruments, the important elements of machines as well as the technique to understand their conditions, this text presents every available method of machine fault detection occurring in machines in general, an

  2. Italian Textile Machinery Industry Focuses on Sustainability

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    As for fashion industry,Italian brands always play as one of the leaders influencing the global trends.This time,in Shanghai,the Italian textile machinery manufacturers highlight the latest proposals on sustainability and eco-friendly technology.

  3. Italian Textite Machinery Industry Focuses on Sustainability

    Institute of Scientific and Technical Information of China (English)

    Wang Ting

    2010-01-01

    @@ At the ITMA Asia+CITME 2010,which was held on June 22at the Shanghai New International Expo Centre in Shanghai,Italian textile machinery manufacturers represented one of the largest foreign delegations: 115exhibitors occupying a total surface area of about 4,000 sq.meters.

  4. Active Vibration Dampers For Rotating Machinery

    Science.gov (United States)

    Kascack, Albert F.; Ropchock, John J.; Lakatos, Tomas F.; Montague, Gerald T.; Palazzolo, Alan; Lin, Reng Rong

    1994-01-01

    Active dampers developed to suppress vibrations in rotating machinery. Essentially feedback control systems and reciprocating piezoelectric actuators. Similar active damper containing different actuators described in LEW-14488. Concept also applicable to suppression of vibrations in stationary structures subject to winds and earthquakes. Active damper offers adjustable suppression of vibrations. Small and lightweight and responds faster to transients.

  5. CCPIT Machinery Exhibition Succeeded in Kuala Lumpur

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

      From August 18 to 20, 2005, China Council for the Promotion of International Trade(CCPIT) held China Machinery and Electronics Trade Exhibition, CME 2005 in Kuala Lumpur, the capital of Malaysia on behalf of China, a good job has been done.……

  6. CCPIT Machinery Exhibition Succeeded in Kuala Lumpur

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    @@ From August 18 to 20, 2005, China Council for the Promotion of International Trade(CCPIT) held China Machinery and Electronics Trade Exhibition, CME 2005 in Kuala Lumpur, the capital of Malaysia on behalf of China, a good job has been done.

  7. Brownian machinery in physics and biology

    OpenAIRE

    Hänggi, Peter

    2001-01-01

    Brownian machinery in physics and biology. - In: Noise in physical systems and 1/f fluctuations : proceedings of the 16th internat. conference, Gainesville, Fl., 22-25 Oct. 2001 / Ed.: Gijs Bosman. - New Jersey u.a. : World Scientific, 2001. - S. 397-399

  8. Computational design of hydrogen fluid machinery

    Energy Technology Data Exchange (ETDEWEB)

    Kaupert, K.A. [Ebara Cryodynamics Div., Sparks, NV (United States)

    2001-06-01

    This paper presented computational design methods for liquid hydrogen fluid machinery for which the aerospace industry holds a particular interest. The main design points are reliability, efficiency and predictability. The methods described here were based on experience gained in the liquefied natural gas sector. The main design validation tools presented in this paper were computational fluid dynamics, computational rotordynamics, and computational stress analysis. Recent advances have made it possible to incorporate the influence of unsteady phenomena. These new design techniques make it possible to increase the reliability, efficiency and predictability of fluid machinery. A design example for an Ebara Cryodynamics multi-stage impeller/diffuser-return vane combination operating in liquefied natural gas was presented. The fluid machinery characteristics were compared for liquid hydrogen and liquefied natural gas to demonstrate the technical feasibility of industrial liquid hydrogen fluid machinery. Currently, no significant demand exists for liquid hydrogen turbomachinery except for the rocket engine industry. But the emergence of the hydrogen economy will promote the growth in liquefied natural gas and or liquid hydrogen cryogenic turbomachinery. 11 refs., 3 tabs., 6 figs.

  9. DNA Ligase I Is Not Essential for Mammalian Cell Viability

    Directory of Open Access Journals (Sweden)

    Li Han

    2014-04-01

    Full Text Available Of the three DNA ligases present in all vertebrates, DNA ligase I (Lig1 has been considered essential for ligating Okazaki fragments during DNA replication and thereby essential for cell viability. Here, we report the striking finding that a Lig1-null murine B cell line is viable. Surprisingly, the Lig1-null cells exhibit normal proliferation and normal immunoglobulin heavy chain class switch recombination and are not hypersensitive to a wide variety of DNA damaging agents. These findings demonstrate that Lig1 is not absolutely required for cellular DNA replication and repair and that either Lig3 or Lig4 can substitute for the role of Lig1 in joining Okazaki fragments. The establishment of a Lig1-null cell line will greatly facilitate the characterization of DNA ligase function in mammalian cells, but the finding alone profoundly reprioritizes the role of ligase I in DNA replication, repair, and recombination.

  10. DNA ligase I is not essential for mammalian cell viability.

    Science.gov (United States)

    Han, Li; Masani, Shahnaz; Hsieh, Chih-lin; Yu, Kefei

    2014-04-24

    Of the three DNA ligases present in all vertebrates, DNA ligase I (Lig1) has been considered essential for ligating Okazaki fragments during DNA replication and thereby essential for cell viability. Here, we report the striking finding that a Lig1-null murine B cell line is viable. Surprisingly, the Lig1-null cells exhibit normal proliferation and normal immunoglobulin heavy chain class switch recombination and are not hypersensitive to a wide variety of DNA damaging agents. These findings demonstrate that Lig1 is not absolutely required for cellular DNA replication and repair and that either Lig3 or Lig4 can substitute for the role of Lig1 in joining Okazaki fragments. The establishment of a Lig1-null cell line will greatly facilitate the characterization of DNA ligase function in mammalian cells, but the finding alone profoundly reprioritizes the role of ligase I in DNA replication, repair, and recombination.

  11. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III

    Directory of Open Access Journals (Sweden)

    Hiroshi Arakawa

    2015-06-01

    Full Text Available Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1, DNA ligase 3 (Lig3 and DNA ligase 4 (Lig4. While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER, homologous recombination repair (HRR and nucleotide excision repair (NER. Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ. Lig3 is implicated in a short-patch base excision repair (BER pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche-ligase

  12. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III.

    Science.gov (United States)

    Arakawa, Hiroshi; Iliakis, George

    2015-06-23

    Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER), homologous recombination repair (HRR) and nucleotide excision repair (NER). Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs) by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ). Lig3 is implicated in a short-patch base excision repair (BER) pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche-ligase to a

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

    Science.gov (United States)

    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.

  14. Zn-binding AZUL domain of human ubiquitin protein ligase Ube3A

    Energy Technology Data Exchange (ETDEWEB)

    Lemak, Alexander; Yee, Adelinda [University of Toronto, and Northeast Structural Genomics Consortium, Ontario Cancer Institute, Campbell Family Cancer Research Institute and Department of Medical Biophysics (Canada); Bezsonova, Irina, E-mail: bezsonova@uchc.edu [University of Connecticut Health Center, Department of Molecular Microbial and Structural Biology (United States); Dhe-Paganon, Sirano, E-mail: sirano.dhepaganon@utoronto.ca [University of Toronto, Structural Genomics Consortium (Canada); Arrowsmith, Cheryl H., E-mail: carrow@uhnresearch.ca [University of Toronto, and Northeast Structural Genomics Consortium, Ontario Cancer Institute, Campbell Family Cancer Research Institute and Department of Medical Biophysics (Canada)

    2011-09-15

    Ube3A (also referred to as E6AP for E6 Associated Protein) is a E3 ubiquitin-protein ligase implicated in the development of Angelman syndrome by controlling degradation of synaptic protein Arc and oncogenic papilloma virus infection by controlling degradation of p53. This article describe the solution NMR structure of the conserved N-terminal domain of human Ube3A (residues 24-87) that contains two residues (Cys44 and Arg62) found to be mutated in patients with Angelman syndrome. The structure of this domain adopts a novel Zn-binding fold we called AZUL (Amino-terminal Zn-finger of Ube3a Ligase). The AZUL domain has a helix-loop-helix architecture with a Zn ion coordinated by four Cys residues arranged in Cys-X{sub 4}-Cys-X{sub 4}-Cys-X{sub 28}-Cys motif. Three of the Zn-bound residues are located in a 23-residue long and well structured loop that connects two {alpha}-helicies.

  15. Ubiquitin ligase UBE3C promotes melanoma progression by increasing epithelial-mesenchymal transition in melanoma cells

    OpenAIRE

    TANG, Li; Yi, Xue-Mei; Chen, Jia; Chen, Fu-Juan; Lou, Wei; Gao, Yun-Lu; Zhou, Jing; Su, Li-Na; Xu, Xin; Lu, Jia-Qing; Ma, Jun; Yu, Ning; Ding, Yang-Feng

    2016-01-01

    Melanoma is the most aggressive type of skin cancer, exhibiting extensive local invasion and early distant metastasis. Aberrant expression of ubiquitin-protein ligase E3C (UBE3C) plays a key role in tumor development and progression. In the present study, we analyzed UBE3C expression in samples of cancerous and normal skin tissue. Levels of UBE3C expression were much higher in primary and metastatic melanoma tissues than in normal skin, cutaneous squamous cell carcinoma or basal cell carcinom...

  16. Targeting RING domains of Mdm2-MdmX E3 complex activates apoptotic arm of the p53 pathway in leukemia/lymphoma cells.

    Science.gov (United States)

    Wu, W; Xu, C; Ling, X; Fan, C; Buckley, B P; Chernov, M V; Ellis, L; Li, F; Muñoz, I G; Wang, X

    2015-12-31

    Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2-p53 interface or MdmX ((MDM4), mouse double minute 4)-p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2-MdmX really interesting new gene (RING)-RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2-MdmX RING-RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2-MdmX RING domain inhibitors)) that specifically inhibit Mdm2-MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2-MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2-MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.

  17. Single-nucleotide polymorphisms of microRNA processing machinery genes and risk of colorectal cancer

    Directory of Open Access Journals (Sweden)

    Zhao Y

    2015-02-01

    Full Text Available Yufei Zhao, Yanming Du, Shengnan Zhao, Zhanjun GuoDepartment of Gastroenterology and Hepatology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, People’s Republic of ChinaObjective: MicroRNA (miRNA-related single-nucleotide polymorphisms (miR-SNPs in miRNA processing machinery genes can affect cancer risk, treatment efficacy, and patient prognosis. We genotyped 6 miR-SNPs of miRNA processing machinery genes including XPO5 (rs11077, RAN (rs14035, Dicer (rs3742330, TNRC6B (rs9623117, GEMIN3 (rs197412, and GEMIN4 (rs2740348 in a case-control study to evaluate their impact on colorectal cancer (CRC risk.Materials and methods: miR-SNPs were genotyped using the polymerase chain reaction–ligase detection reaction. The Χ2 test was used to analyze dichotomous values, such as the presence or absence of any individual SNP in CRC patients and healthy controls.Results: Two of these SNPs were identified for their association with cancer risk in the Dicer and GEMIN3 genes. The AA allele of rs3742330 located in the Dicer gene exhibited a significantly increased risk of CRC (odds ratio, 2.11; 95% confidence interval: 1.33–3.34; P=0.001; the TT allele of rs197412 located in GEMIN3 also exhibited a significantly increased risk of CRC (odds ratio, 1.68; 95% confidence interval: 1.07–2.65; P=0.024.Conclusion: Our results suggest that the specific genetic variants in miRNA machinery genes may affect CRC susceptibility.Keywords: miR-SNP, CRC, GEMIN3, Dicer

  18. Mdm2 ligase dead mutants did not act in a dominant negative manner to re-activate p53, but promoted tumor cell growth.

    Science.gov (United States)

    Swaroop, Manju; Sun, Yi

    2003-01-01

    Mdm2 (murine double minute 2) is an oncogene, first identified in BALB/c 3T3 cells. Over-expression and gene amplification of Mdm2 were found in a variety of human cancers. Recently, Mdm2 was found to be an E3 ubiquitin ligase that promotes degradation of p53, which contributes significantly to its oncogenic activity. In this study, we test a hypothesis that Mdm2 ligase dead mutants, which retained p53 binding activity but lost degradation activity, would act in a dominant negative manner to re-activate p53, especially upon stressed conditions. Five Mdm2 constructs expressing wild-type and E3 ligase-dead Mdm2 proteins were generated in a Tet-Off system and transfected into MCF-7 breast cancer cells (p53+/+ with Mdm2 overexpression) as well as MCF10A immortalized breast cells (p53+/+ without Mdm2 overexpression) as a normal control. We found that expression of Mdm2 mutants were tightly regulated by doxycycline. Withdrawal of doxycycline in culture medium triggered overexpression of Mdm2 mutants. However, expression of ligase dead mutants in MCF7 and MCF10A cells did not reactivate p53 as shown by a luciferase-reporter transcription assay and Western blot of p53 and its downstream target p21 under either unstressed condition or after exposure to DNA damaging agents. Biologically, over-expression of Mdm2 mutants had no effect on p53-induced apoptosis following DNA damage. Interestingly, over-expression of Mdm2 mutants promoted growth of MCF7 tumor cells probably via a p53-independent mechanism. Over-expression of Mdm2 mutants, however, had no effect on the growth of normal MCF10A cells and did not cause their transformation. Thus, ligase dead mutants of Mdm2 did not act in a dominant negative manner to reactivate p53 and they are not oncogenes in MCF10A cells.

  19. DNA ligases from rat liver. Purification and partial characterization of two molecular forms

    Energy Technology Data Exchange (ETDEWEB)

    Elder, R.H.; Rossignol, J.M. (Laboratoire de Biologie Moleculaire de la Replication, UPR 272-CNRS, IRSC, Villejuif (France))

    1990-06-26

    The differential ability of mammalian DNA ligases to use oligo(dT).poly(rA) as a substrate has been used to detect, and thereby extensively purify, two immunologically distinct forms of DNA ligase from rat liver. The activity of DNA ligase I, which is unable to use this template, is uniquely increased during liver regeneration, while that of DNA ligase II remains at a low level. Both enzymes require ATP and Mg2+ for activity and form an adenylylated intermediate which is stable and reactive. After SDS-PAGE, such radiolabeled complexes correspond to polypeptides of 130,000 and 80,000 Da for DNA ligase I and to 100,000 Da for DNA ligase II. That these labeled polypeptides do indeed correspond to active polypeptides of two different forms of DNA ligase is shown by the removal of the radiolabeled AMP, only when the intermediate is incubated with an appropriate substrate. In contrast to other eukaryotic DNA ligases, rat liver DNA ligase II has a lower Km for ATP (1.2 X 10(-5) M) than DNA ligase I (6 X 10(-5) M). Also, DNA ligase II can use ATP alpha S as a cofactor in the ligation reaction much more efficiently than DNA ligase I, further discriminating the ATP binding sites of these enzymes. Finally, antibodies raised against the 130,000-Da polypeptide of DNA ligase I specifically recognize this species in an immunoblot and inhibit only the activity of DNA ligase I.

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

    Directory of Open Access Journals (Sweden)

    Julia Wang

    2016-05-01

    Full Text Available 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

  1. A Self-Replicating Ligase Ribozyme

    Science.gov (United States)

    Paul, Natasha; Joyce, Gerald F.

    2002-01-01

    A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of Itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 per min, whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 x 10(exp -11) M per min. Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication.

  2. Fbxw5 suppresses nuclear c-Myb activity via DDB1-Cul4-Rbx1 ligase-mediated sumoylation

    Energy Technology Data Exchange (ETDEWEB)

    Kanei-Ishii, Chie; Nomura, Teruaki; Egoh, Ayako [Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074 (Japan); Ishii, Shunsuke, E-mail: sishii@rtc.riken.jp [Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074 (Japan)

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Fbxw5 enhances sumoylation of c-Myb. Black-Right-Pointing-Pointer The DDB1-Cul4A-Rbx1 complex mediates c-Myb sumoylation. Black-Right-Pointing-Pointer The Fbxw5-DDB1-Cul4A-Rdx1 complex is a dual SUMO/ubiquitin ligase. Black-Right-Pointing-Pointer Fbxw5 suppresses the c-Myb trans-activating capacity. -- Abstract: The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling. In this process, Fbxw7{alpha}, the F-box protein of the SCF complex, binds to c-Myb via its C-terminal WD40 domain, and induces the ubiquitination of c-Myb. Here, we report that Fbxw5, another F-box protein, enhances sumoylation of nuclear c-Myb. Fbxw5 enhanced c-Myb sumoylation via the DDB1-Cul4A-Rbx1 complex. Since the Fbxw5-DDB1-Cul4A-Rbx1 complex was shown to act as a ubiquitin ligase for tumor suppressor TSC2, our results suggest that this complex can function as a dual SUMO/ubiquitin ligase. Fbxw5, which is localized to both nucleus and cytosol, enhanced sumoylation of nuclear c-Myb and induced the localization of c-Myb to nuclear dot-like domains. Co-expression of Fbxw5 suppressed the trans-activation of c-myc promoter by wild-type c-Myb, but not by v-Myb, which lacks the sumoylation sites. These results suggest that multiple E3 ligases suppress c-Myb activity through sumoylation or ubiquitination, and that v-Myb is no longer subject to these negative regulations.

  3. Present Situation of Petroleum Machinery Manufacturers in China

    Institute of Scientific and Technical Information of China (English)

    Li Xilu

    2008-01-01

    Since China joined the WTO, the environment for the development of petroleum machinery industry in the country has changed a lot. As international petroleum machinery manufacturers enter into Chinese market, petroleum machinery manufacturers of the country are facing fierce competition both at home and abroad.

  4. 30 CFR 56.14205 - Machinery, equipment, and tools.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Machinery, equipment, and tools. 56.14205... NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-SURFACE METAL AND NONMETAL MINES Machinery and Equipment Safety Practices and Operational Procedures § 56.14205 Machinery, equipment, and tools....

  5. 46 CFR 58.01-20 - Machinery guards.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Machinery guards. 58.01-20 Section 58.01-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-20 Machinery guards. Gears, couplings, flywheels...

  6. 46 CFR 109.205 - Inspection of boilers and machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Inspection of boilers and machinery. 109.205 Section 109... OPERATIONS Tests, Drills, and Inspections § 109.205 Inspection of boilers and machinery. The chief engineer or engineer in charge, before he assumes charge of the boilers and machinery of a unit shall...

  7. 29 CFR 1915.165 - Ship's deck machinery.

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 7 2010-07-01 2010-07-01 false Ship's deck machinery. 1915.165 Section 1915.165 Labor... (CONTINUED) OCCUPATIONAL SAFETY AND HEALTH STANDARDS FOR SHIPYARD EMPLOYMENT Ship's Machinery and Piping Systems § 1915.165 Ship's deck machinery. (a) Before work is performed on the anchor windlass or any...

  8. 46 CFR 122.208 - Accidents to machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Accidents to machinery. 122.208 Section 122.208 Shipping... Voyage Records § 122.208 Accidents to machinery. The owner, managing operator, or master shall report damage to a boiler, unfired pressure vessel, or machinery that renders further use of the item...

  9. 29 CFR 1915.164 - Ship's propulsion machinery.

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 7 2010-07-01 2010-07-01 false Ship's propulsion machinery. 1915.164 Section 1915.164 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION... Machinery and Piping Systems § 1915.164 Ship's propulsion machinery. (a) Before work is performed on...

  10. 46 CFR 109.419 - Report of unsafe machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Report of unsafe machinery. 109.419 Section 109.419... OPERATIONS Reports, Notifications, and Records Reports and Notifications § 109.419 Report of unsafe machinery. If a boiler, unfired pressure vessel, or other machinery on a unit is unsafe to operate, the...

  11. 30 CFR 57.14205 - Machinery, equipment, and tools.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Machinery, equipment, and tools. 57.14205... NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Machinery and Equipment Safety Practices and Operational Procedures § 57.14205 Machinery, equipment, and...

  12. 46 CFR 252.33 - Hull and machinery insurance.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Hull and machinery insurance. 252.33 Section 252.33... Subsidy Rates § 252.33 Hull and machinery insurance. (a) Subsidy items. The fair and reasonable net premium costs (including stamp taxes) of hull and machinery, increased value, excess general...

  13. 29 CFR 1910.214 - Cooperage machinery. [Reserved

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 5 2010-07-01 2010-07-01 false Cooperage machinery. 1910.214 Section 1910.214 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.214 Cooperage machinery....

  14. 46 CFR 196.30-5 - Accidents to machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Accidents to machinery. 196.30-5 Section 196.30-5... Reports of Accidents, Repairs, and Unsafe Equipment § 196.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use...

  15. 46 CFR 97.30-5 - Accidents to machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Accidents to machinery. 97.30-5 Section 97.30-5 Shipping... Reports of Accidents, Repairs, and Unsafe Equipment § 97.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use...

  16. 46 CFR 185.208 - Accidents to machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Accidents to machinery. 185.208 Section 185.208 Shipping...) OPERATIONS Marine Casualties and Voyage Records § 185.208 Accidents to machinery. The owner, managing operator, or master shall report damage to a boiler, unfired pressure vessel, or machinery that...

  17. 46 CFR 78.33-5 - Accidents to machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 3 2010-10-01 2010-10-01 false Accidents to machinery. 78.33-5 Section 78.33-5 Shipping... Accidents, Repairs, and Unsafe Equipment § 78.33-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use of the...

  18. 46 CFR 185.352 - Ventilation of gasoline machinery spaces.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Ventilation of gasoline machinery spaces. 185.352 Section 185.352 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS... machinery spaces. The mechanical exhaust for the ventilation of a gasoline machinery space, required...

  19. 46 CFR 130.460 - Placement of machinery alarms.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Placement of machinery alarms. 130.460 Section 130.460..., AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.460 Placement of machinery alarms. (a) Visible and audible alarms must be installed at the pilothouse to...

  20. 46 CFR 171.095 - Machinery space bulkhead.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Machinery space bulkhead. 171.095 Section 171.095... PERTAINING TO VESSELS CARRYING PASSENGERS Additional Subdivision Requirements § 171.095 Machinery space... transverse watertight bulkheads to separate the machinery space from the remainder of the vessel....

  1. 46 CFR 111.103-3 - Machinery space ventilation.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Machinery space ventilation. 111.103-3 Section 111.103-3...-GENERAL REQUIREMENTS Remote Stopping Systems § 111.103-3 Machinery space ventilation. (a) Each machinery space ventilation system must have two controls to stop the ventilation, one of which may be the...

  2. 33 CFR 157.39 - Machinery space bilges.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Machinery space bilges. 157.39... Vessel Operation § 157.39 Machinery space bilges. (a) A tank vessel may discharge an oily mixture from a machinery space bilge that is combined with an oil cargo residue if the vessel discharges in compliance...

  3. OPTIMAL MAINTENANCE AND REPLACEMENT OF EXTRACTION MACHINERY

    Institute of Scientific and Technical Information of China (English)

    Suresh P.SETHI; Hong-Mo YEH; Rong ZHANG; Andrew K.S.JARDINE

    2008-01-01

    This paper considers a problem of optimal preventive maintenance and replacement schedule ofequipment devoted to extracting resources from known deposits. Typical examples are oil drills, mine shovels, etc. At most one replacement of the existing machinery by a new one is allowed. The problem is formulated as an optimal control problem subject to the state constraint that the remaining deposit at any given time is nonnegative. We show that the optimal preventive maintenance, production rates, and the replacement and salvage times of the existing machinery and the new one, if required, can be obtained by solving sequentially a series of free-end-point optimal control problems. Moreover, an algorithm based on this result is developed and used to solve two illustrative examples.

  4. The Autophagic Machinery in Enterovirus Infection

    Directory of Open Access Journals (Sweden)

    Jeffrey K. F. Lai

    2016-01-01

    Full Text Available The Enterovirus genus of the Picornaviridae family comprises many important human pathogens, including polioviruses, rhinovirus, enterovirus A71, and enterovirus D68. They cause a wide variety of diseases, ranging from mild to severe life-threatening diseases. Currently, no effective vaccine is available against enteroviruses except for poliovirus. Enteroviruses subvert the autophagic machinery to benefit their assembly, maturation, and exit from host. Some enteroviruses spread between cells via a process described as autophagosome-mediated exit without lysis (AWOL. The early and late phases of autophagy are regulated through various lipids and their metabolizing enzymes. Some of these lipids and enzymes are specifically regulated by enteroviruses. In the present review, we summarize the current understanding of the regulation of autophagic machinery by enteroviruses, and provide updates on recent developments in this field.

  5. Measurement of the Radiative $K_{e3}$ Branching Ratio

    CERN Document Server

    Lai, A; Bevan, A; Dosanjh, R S; Gershon, T J; Hay, B; Kalmus, George Ernest; Lazzeroni, C; Munday, D J; Olaiya, E; Parker, M A; White, T O; Wotton, S A; Barr, G; Bocquet,; Ceccucci, A; Çuhadar-Dönszelmann, T; Cundy, Donald C; D'Agostini, G; Doble, Niels T; Falaleev, V; Gatignon, L; Gonidec, A; Gorini, B; Govi, G; Grafström, P; Kubischta, Werner; Mikulec, I; Lacourt, A; Norton, A; Palestini, S; Panzer-Steindel, B; Taureg, H; Velasco, M; Wahl, H; Cheshkov, C; Khristov, P Z; Kekelidze, Vladimir D; Litov, L; Madigozhin, D T; Molokanova, N A; Potrebenikov, Yu K; Stoynev, S; Zinchenko, A I; Knowles, I; Martin, V; Sacco, R; Walker, A; Contalbrigo, M; Dalpiaz, Pietro; Duclos, J; Frabetti, P L; Gianoli, A; Martini, M; Petrucci, F; Savrié, M; Bizzeti, A; Calvetti, M; Collazuol, G; Graziani, G; Iacopini, E; Lenti, M; Martelli, F; Veltri, M; Becker, H G; Eppard, K; Eppard, M; Fox, H; Kalter, A; Kleinknecht, K; Koch, U; Köpke, L; Lopes da Silva, P; Marouelli, P; Pellmann, I A; Peters, A; Renk, B; Schmidt, S A; Schönharting, V; Schué, Yu; Wanke, R; Winhart, A; Wittgen, M; Chollet, J C; Fayard, L; Iconomidou-Fayard, L; Ocariz, J; Unal, G; Wingerter-Seez, I; Anzivino, Giuseppina; Cenci, P; Imbergamo, E; Lubrano, P; Mestvirishvili, A; Nappi, A; Pepé, M; Piccini, M; Bertanza, L; Carosi, R; Casali, R; Cerri, C; Cirilli, M; Costantini, F; Fantechi, R; Giudici, Sergio; Mannelli, I; Pierazzini, G M; Sozzi, M; Chèze, J B; Cogan, J; De Beer, M; Debu, P; Formica, A; Granier de Cassagnac, R; Mazzucato, E; Peyaud, B; Turlay, René; Vallage, B; Holder, M; Maier, A; Ziolkowski, M; Arcidiacono, R; Biino, C; Cartiglia, N; Marchetto, F; Menichetti, E; Pastrone, N; Nassalski, J P; Rondio, Ewa; Szleper, M; Wislicki, W; Wronka, S; Dibon, Heinz; Fischer, G; Jeitler, Manfred; Markytan, Manfred; Neuhofer, G; Pernicka, M; Taurok, A; Widhalm, L

    2005-01-01

    We present a measurement of the relative branching ratio of the decay {K}^0 -> pi+-e-+ nu gamma ({K}_e3 gamma) with respect to {K}^0 -> pi+- e-+ nu (gamma) ({k}_e3 + k}_e3 gamma) decay. The result is based on observation of 19 000 {K}_e3 gamma and 5.6 . {10}^6 {K}_e3 decays. The value of branching ratio is Br({K}^0 _e3 gamma{E}^*_gamma > 30 MeV, {theta}^*_e gamma > 20 deg) / Br({K}^0_e3 = (0.964 +- {0.008}^+0.011_0.009)%. This result agrees with the theoretical predictions but is at variance with a recently published result.

  6. Machinery prognostics and prognosis oriented maintenance management

    CERN Document Server

    Yan, Jihong

    2014-01-01

    This book gives a complete presentatin of the basic essentials of machinery prognostics and prognosis oriented maintenance management, and takes a look at the cutting-edge discipline of intelligent failure prognosis technologies for condition-based maintenance.  Latest research results and application methods are introduced for signal processing, reliability moelling, deterioration evaluation, residual life prediction and maintenance-optimization as well as applications of these methods.

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

    Science.gov (United States)

    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.

  8. Property Analysis of the Agricultural Machinery Lubricants

    Directory of Open Access Journals (Sweden)

    Tone Ploj

    2000-03-01

    Full Text Available We need to produce enough healthy and cheap food as well as to preserve the ecologic equilibrium. This can be achived by using modern machinery and up- to-date knowledge and technology. Agricultural machinery, in which 40-60% of all funds are invested, is poorly maintained and underused. The main causes for this are poor knowledge and extensive farm land fragmentation. The fact that over 140,000 tractors in Slovenia are on average 9.6 years old, i.e. that more than 80% of overall agricultural machinery is obsolete, should be a matter of serious concern. In the paper we follow tribological conditions in particular tractor assemblies. In the first part of the paper we have treated the required conditions of tractor manufacturers in Europe and primarily in Slovenia, what has served us in the final phase of the research for elaboration of the model. In this way we have got data about the presence of particular tractor types. We have separately elaborated the necessary specifications of engine lubricants, transmission, gears, hydraulics and wet breaks. We have carried out chemical and mechanical analyses of all accessible lubricants in agricultural mechanisation. The results of the new oils were coordinated with the required specifications of tractor manufacturers and so we have got such a model, that certainly meet all lubricating requirements of our tractors.

  9. The exportomer: the peroxisomal receptor export machinery.

    Science.gov (United States)

    Platta, Harald W; Hagen, Stefanie; Erdmann, Ralf

    2013-04-01

    Peroxisomes constitute a dynamic compartment of almost all eukaryotic cells. Depending on environmental changes and cellular demands peroxisomes can acquire diverse metabolic roles. The compartmentalization of peroxisomal matrix enzymes is a prerequisite to carry out their physiologic function. The matrix proteins are synthesized on free ribosomes in the cytosol and are ferried to the peroxisomal membrane by specific soluble receptors. Subsequent to cargo release into the peroxisomal matrix, the receptors are exported back to the cytosol to facilitate further rounds of matrix protein import. This dislocation step is accomplished by a remarkable machinery, which comprises enzymes required for the ubiquitination as well as the ATP-dependent extraction of the receptor from the membrane. Interestingly, receptor ubiquitination and dislocation are the only known energy-dependent steps in the peroxisomal matrix protein import process. The current view is that the export machinery of the receptors might function as molecular motor not only in the dislocation of the receptors but also in the import step of peroxisomal matrix protein by coupling ATP-dependent removal of the peroxisomal import receptor with cargo translocation into the organelle. In this review we will focus on the architecture and function of the peroxisomal receptor export machinery, the peroxisomal exportomer.

  10. Mammalian DNA ligase III: Molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jingwen; Danehower, S.; Besterman, J.M.; Husain, I. [Glaxo Research Inst., Research Triangle Park, NC (United States)] [and others

    1995-10-01

    Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating supermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replications. In contrast, elevated levels of DNA ligase III mRNA were observed in primary supermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells. 62 refs., 7 figs.

  11. Biochemical characterization of the DNA ligase I from Entamoeba histolytica.

    Science.gov (United States)

    Cardona-Felix, Cesar S; Pastor-Palacios, Guillermo; Cardenas, Helios; Azuara-Liceaga, Elisa; Brieba, Luis G

    2010-11-01

    DNA ligases play an essential role in DNA replication and repair. Herein, we report the cloning and biochemical characterization of DNA ligase I from the protozoan parasite Entamoeba histolytica (EhDNAligI). EhDNAligI is an ATP-dependent DNA ligase of 685 amino acids with 35% identity to human DNA ligase I. This report shows that heterologous expressed EhDNAligI is able to perform the three conserved steps of a DNA ligation reaction: adenylation, binding to a 5'-phosphorylated nicked DNA substrate and sealing of the nick. EhDNAligI is strongly inhibited by NaCl and displays optimal activity at pH 7.5. EhDNAligI uses Mn2+ or Mg2+ as metal cofactors and ATP as nucleotide cofactor. EhDNAligI has a nicked DNA binding constant of 6.6microM and follows Michaelis-Menten steady-state kinetics with a K(m) ATP of 64nM and a k(cat) of 2.4min(-1). Accordingly to its properties as a family I DNA ligase, EhDNAligI is able to ligate a RNA strand upstream of a nucleic acid nick, but not in the downstream or the template position. We propose that EhDNAligI is involved in sealing DNA nicks during lagging strand synthesis and may have a role in base excision repair in E. histolytica.

  12. The plastid-dividing machinery: formation, constriction and fission.

    Science.gov (United States)

    Yoshida, Yamato; Miyagishima, Shin-ya; Kuroiwa, Haruko; Kuroiwa, Tsuneyoshi

    2012-12-01

    Plastids divide by constriction of the plastid-dividing (PD) machinery, which encircles the division site. The PD machinery consists of the stromal inner machinery which includes the inner PD and filamenting temperature-sensitive mutant Z (FtsZ) rings and the cytosolic outer machinery which includes the outer PD and dynamin rings. The major constituent of the PD machinery is the outer PD ring, which consists of a bundle of polyglucan filaments. In addition, recent proteomic studies suggest that the PD machinery contains additional proteins that have not been characterized. The PD machinery forms from the inside to the outside of the plastid. The constriction seems to occur by sliding of the polyglucan filaments of the outer PD ring, aided by dynamin. The final fission of the plastid is probably promoted by the 'pinchase' activity of dynamin.

  13. E3: Economy - Energy - Environment; Supporting Manufacturing Leadership through Sustainability

    Science.gov (United States)

    The E3 initiative is designed to help you thrive in a new business era focused on sustainability and, working together, to promote sustainable manufacturing and economic growth throughout the United States. Within the E3 framework, we can: - Drive Innovation - Increase Manufacturing Productivity - Boost Local Economies - Reduce Environmental Impacts - Foster Development - Conserve Energy and Resources This website provides information and tools for E3, including fact sheets, contacts, and calculators.

  14. One-step assay for the quantification of T4 DNA ligase.

    Science.gov (United States)

    Franke, Steffi; Kreisig, Thomas; Buettner, Karin; Zuchner, Thole

    2015-02-01

    As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5 %) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays.

  15. 46 CFR 30.10-42 - Machinery space-TB/ALL.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Machinery space-TB/ALL. 30.10-42 Section 30.10-42...-42 Machinery space—TB/ALL. The term machinery space means any space that contains machinery and related equipment including Category A machinery spaces, propelling machinery, boilers, oil fuel...

  16. A wild-type DNA ligase I gene is expressed in Bloom's syndrome cells.

    OpenAIRE

    Petrini, J H; Huwiler, K G; Weaver, D T

    1991-01-01

    Alteration of DNA ligase I activity is a consistent biochemical feature of Bloom's syndrome (BS) cells. DNA ligase I activity in BS cells either is reduced and abnormally thermolabile or is present in an anomalously dimeric form. To assess the role of DNA ligase function in the etiology of BS, we have cloned the DNA ligase I cDNA from normal human cells by a PCR strategy using degenerate oligonucleotide primers based on conserved regions of the Saccharomyces cerevisiae and Schizosaccharomyces...

  17. 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.

    Science.gov (United States)

    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.

  18. Analysis of electric machinery and drive systems

    CERN Document Server

    Krause, Paul C; Sudhoff, Scott D; Pekarek, Steven

    2013-01-01

    Introducing a new edition of the popular reference on machine analysis Now in a fully revised and expanded edition, this widely used reference on machine analysis boasts many changes designed to address the varied needs of engineers in the electric machinery, electric drives, and electric power industries. The authors draw on their own extensive research efforts, bringing all topics up to date and outlining a variety of new approaches they have developed over the past decade. Focusing on reference frame theory that has been at the core of this work since the first edition, th

  19. The RNA polymerase I transcription machinery

    OpenAIRE

    Russell, Jackie; Zomerdijk, Joost C. B. M.

    2006-01-01

    The rRNAs constitute the catalytic and structural components of the ribosome, the protein synthesis machinery of cells. The level of rRNA synthesis, mediated by Pol I (RNA polymerase I), therefore has a major impact on the life and destiny of a cell. In order to elucidate how cells achieve the stringent control of Pol I transcription, matching the supply of rRNA to demand under different cellular growth conditions, it is essential to understand the components and mechanics of the Pol I transc...

  20. Rotating Machinery Predictive Maintenance Through Expert System

    Directory of Open Access Journals (Sweden)

    M. Sarath Kumar

    2000-01-01

    Full Text Available Modern rotating machines such as turbomachines, either produce or absorb huge amount of power. Some of the common applications are: steam turbine-generator and gas turbine-compressor-generator trains produce power and machines, such as pumps, centrifugal compressors, motors, generators, machine tool spindles, etc., are being used in industrial applications. Condition-based maintenance of rotating machinery is a common practice where the machine's condition is monitored constantly, so that timely maintenance can be done. Since modern machines are complex and the amount of data to be interpreted is huge, we need precise and fast methods in order to arrive at the best recommendations to prevent catastrophic failure and to prolong the life of the equipment. In the present work using vibration characteristics of a rotor-bearing system, the condition of a rotating machinery (electrical rotor is predicted using an off-line expert system. The analysis of the problem is carried out in an Object Oriented Programming (OOP framework using the finite element method. The expert system which is also developed in an OOP paradigm gives the type of the malfunctions, suggestions and recommendations. The system is implemented in C++.

  1. Evolution of the chloroplast division machinery

    Institute of Scientific and Technical Information of China (English)

    Hongbo GAO; Fuli GAO

    2011-01-01

    Chloroplasts are photosynthetic organelles derived from endosymbiotic cyanobacteria during evolution.Dramatic changes occurred during the process of the formation and evolution of chloroplasts,including the large-scale gene transfer from chloroplast to nucleus.However,there are still many essential characters remaining.For the chloroplast division machinery,FtsZ proteins,Ftn2,SulA and part of the division site positioning system- MinD and MinE are still conserved.New or at least partially new proteins,such as FtsZ family proteins FtsZl and ARC3,ARC6H,ARC5,PDV1,PDV2 and MCD1,were introduced for the division of chloroplasts during evolution.Some bacterial cell division proteins,such as FtsA,MreB,Ftn6,FtsW and Ftsl,probably lost their function or were gradually lost.Thus,the chloroplast division machinery is a dynamically evolving structure with both conservation and innovation.

  2. On Development of Agricultural Machinery Operating Service in Chongqing

    Institute of Scientific and Technical Information of China (English)

    Chongjing; TAN; Shi; YANG

    2015-01-01

    Development of agricultural machinery operating service in Chongqing takes on rapid increase in number of service organizations,diversified service methods,improvement in service level,and constant service income. However,there are some problems,including unreasonable composition and small scale of service organization,imbalanced development of four service methods,low service level,and low operating income of agricultural machinery households. To accelerate development of agricultural machinery operating service in Chongqing,it is recommended to take following measures: adjusting subsidy for purchase and operation of agricultural machinery; improving fiscal and taxation and financial system; speeding up infrastructure construction,establishing agricultural machinery information network,and improving organizational form and methods of agricultural machinery operating service.

  3. ABOUT ETHICS IN PLANT, MACHINERY AND EQUIPMENT VALUATION DOMAIN

    Directory of Open Access Journals (Sweden)

    Sorin Adrian Achim

    2015-09-01

    Full Text Available In this paper we put in discussion some ethical aspects of plant, machinery and equipment valuation. After a presentation of the general issues of ethics in valuation domain, we analyze the application of the fundamental ethical principles in plant, machinery and equipment valuation domain. To support our conclusions we use some findings of a study that we conducted to identify the particularities of the plant, machinery and equipment valuation activity in Romania.

  4. Ancient origin of animal U-box ubiquitin ligases

    Directory of Open Access Journals (Sweden)

    Marín Ignacio

    2010-10-01

    Full Text Available Abstract Background The patterns of emergence and diversification of the families of ubiquitin ligases provide insights about the evolution of the eukaryotic ubiquitination system. U-box ubiquitin ligases (UULs are proteins characterized by containing a peculiar protein domain known as U box. In this study, the origin of the animal UUL genes is described. Results Phylogenetic and structural data indicate that six of the seven main UUL-encoding genes found in humans (UBE4A, UBE4B, UIP5, PRP19, CHIP and CYC4 were already present in the ancestor of all current metazoans and the seventh (WDSUB1 is found in placozoans, cnidarians and bilaterians. The fact that only 4 - 5 genes orthologous to the human ones are present in the choanoflagellate Monosiga brevicollis suggests that several animal-specific cooptions of the U box to generate new genes occurred. Significantly, Monosiga contains five additional UUL genes that are not present in animals. One of them is also present in distantly-related protozoans. Along animal evolution, losses of UUL-encoding genes are rare, except in nematodes, which lack three of them. These general patterns are highly congruent with those found for other two families (RBR, HECT of ubiquitin ligases. Conclusions Finding that the patterns of emergence, diversification and loss of three unrelated families of ubiquitin ligases (RBR, HECT and U-box are parallel indicates that there are underlying, linage-specific evolutionary forces shaping the complexity of the animal ubiquitin system.

  5. Plasmid containing a DNA ligase gene from Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Griffin, K.; Setlow, J.K.

    1984-05-01

    A ligase gene from Haemophilus influenzae was cloned into the shuttle vector pDM2. Although the plasmid did not affect X-ray sensitivity, it caused an increase in UV sensitivity of the wild-type but not excision-defective H. influenzae and a decrease in UV sensitivity of the rec-1 mutant. 14 references, 2 figures.

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

    Science.gov (United States)

    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

  7. Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

    OpenAIRE

    Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

    2013-01-01

    Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of M...

  8. General Course of Failure Distributions at Complex Machineries

    Directory of Open Access Journals (Sweden)

    Naqib Daneshjo

    2017-02-01

    Full Text Available In the process of maintenance management of machinery and devices there is necessity to keep in the mind "construction technologicity” (ability of construction technology. This term is used in evaluating of machinery construction, their groups and components in terms of production. The aim of management and planning maintenance of machinery is the failure-free operation in the application process. The range of machinery maintenance machines from routine one and inspection to general repairs is important to organize in a way to prevent unplanned idle time and failures or very likely to accidents.

  9. Successful conversion of the Bacillus subtilis BirA Group II biotin protein ligase into a Group I ligase

    National Research Council Canada - National Science Library

    Henke, Sarah K; Cronan, John E

    2014-01-01

    ...: bioWAFDBI, yuiG and yhfUTS. Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function...

  10. Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining.

    Science.gov (United States)

    Lu, Guangqing; Duan, Jinzhi; Shu, Sheng; Wang, Xuxiang; Gao, Linlin; Guo, Jing; Zhang, Yu

    2016-02-02

    In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated. Surprisingly, we found that both Lig1- and Lig3-containing complexes could efficiently catalyze A-EJ for class switching recombination (CSR) in the IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes. However, only deletion of nuclear Lig3, but not Lig1, could significantly reduce the interchromosomal translocations in Lig4(-/-) cells, suggesting the unique role of Lig3 in catalyzing chromosome translocation. Additional sequence analysis of chromosome translocation junction microhomology revealed the specificity of different ligase-containing complexes. The data suggested the existence of multiple DNA ligase-containing complexes in A-EJ.

  11. Accessorizing the human mitochondrial transcription machinery.

    Science.gov (United States)

    Bestwick, Megan L; Shadel, Gerald S

    2013-06-01

    The human genome comprises large chromosomes in the nucleus and mitochondrial DNA (mtDNA) housed in the dynamic mitochondrial network. Human cells contain up to thousands of copies of the double-stranded, circular mtDNA molecule that encodes essential subunits of the oxidative phosphorylation complexes and the rRNAs and tRNAs needed to translate these in the organelle matrix. Transcription of human mtDNA is directed by a single-subunit RNA polymerase, POLRMT, which requires two primary transcription factors, TFB2M (transcription factor B2, mitochondrial) and TFAM (transcription factor A, mitochondrial), to achieve basal regulation of the system. Here, we review recent advances in understanding the structure and function of the primary human transcription machinery and the other factors that facilitate steps in transcription beyond initiation and provide more intricate control over the system. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. On the mechanochemical machinery underlying chromatin remodeling

    Science.gov (United States)

    Yusufaly, Tahir I.

    This dissertation discuss two recent efforts, via a unique combination of structural bioinformatics and density functional theory, to unravel some of the details concerning how molecular machinery within the eukaryotic cell nucleus controls chromatin architecture. The first, a study of the 5-methylation of cytosine in 5'-CG-3' : 5'-CG-3' base-pair steps, reveals that the methyl groups roughen the local elastic energy landscape of the DNA. This enhances the probability of the canonical B-DNA structure transitioning into the undertwisted A-like and overtwisted C-like forms seen in nucleosomes, or looped segments of DNA bound to histones. The second part focuses on the formation of salt bridges between arginine residues in histones and phosphate groups on the DNA backbone. The arginine residues are ob- served to apply a tunable mechanical load to the backbone, enabling precision-controlled activation of DNA deformations.

  13. The SNARE machinery in mast cell secretion

    Directory of Open Access Journals (Sweden)

    Axel eLorentz

    2012-06-01

    Full Text Available Mast cells are known as inflammatory cells which exert their functions in allergic and anaphylactic reactions by secretion of numerous inflammatory mediators. During an allergic response, the high-affinity IgE receptor, FcεRI, becomes cross-linked by receptor-bound IgE and antigen resulting in immediate release of pre-synthesized mediators – stored in granules – as well as in de novo synthesis of various mediators like cytokines and chemokines. Soluble N-ethylmaleimide-Sensitive Factor (NSF Attachment Protein (SNAP Receptors (SNARE proteins were found to play a central role in regulating membrane fusion events during exocytosis. In addition, several accessory regulators like Munc13, Munc18, Rab GTPases, SCAMPs, complexins or synaptotagmins were found to be involved in membrane fusion. In this review we summarize our current knowledge about the SNARE machinery and its mechanism of action in mast cell secretion.

  14. Numerical Noise Prediction in Fluid Machinery

    Institute of Scientific and Technical Information of China (English)

    Iris PANTLE; Franco MAGAGNATO; Martin GABI

    2005-01-01

    Numerical methods successively became important in the design and optimization of fluid machinery. However,as noise emission is considered, one can hardly find standardized prediction methods combining flow and acoustical optimization. Several numerical field methods for sound calculations have been developed. Due to the complexity of the considered flow, approaches must be chosen to avoid exhaustive computing. In this contribution the noise of a simple propeller is investigated. The configurations of the calculations comply with an existing experimental setup chosen for evaluation. The used in-house CFD solver SPARC contains an acoustic module based on Ffowcs Williams-Hawkings Acoustic Analogy. From the flow results of the time dependent Large Eddy Simulation the time dependent acoustic sources are extracted and given to the acoustic module where relevant sound pressure levels are calculated. The difficulties, which arise while proceeding from open to closed rotors and from gas to liquid are discussed.

  15. E3: Economy - Energy - Environment; Supporting Manufacturing Leadership through Sustainability

    Data.gov (United States)

    U.S. Environmental Protection Agency — The E3 initiative is designed to help you thrive in a new business era focused on sustainability and, working together, to promote sustainable manufacturing and...

  16. About E3: Economy – Energy – Environment

    Science.gov (United States)

    This page explains EPA's E3 – Economy, Energy, and Environment – program, a federal and local technical assistance initiative, helping manufacturers adapt and thrive with a focus on sustainability.

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

    Science.gov (United States)

    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.

  18. Machinery failure analysis and troubleshooting practical machinery management for process plants

    CERN Document Server

    Bloch, Heinz P

    2012-01-01

    Solve the machinery failure problems costing you time and money with this classic, comprehensive guide to analysis and troubleshooting  Provides detailed, complete and accurate information on anticipating risk of component failure and avoiding equipment downtime Includes numerous photographs of failed parts to ensure you are familiar with the visual evidence you need to recognize Covers proven approaches to failure definition and offers failure identification and analysis methods that can be applied to virtually all problem situations Demonstr

  19. Industrial Machinery Maintenance and Repair. Florida Vocational Program Guide.

    Science.gov (United States)

    University of South Florida, Tampa. Dept. of Adult and Vocational Education.

    This vocational program guide is intended to assist in the organization, operation, and evaluation of a program in industrial machinery maintenance and repair in school districts, area vocational centers, and community colleges. The following topics are covered: job duties of millwrights, maintenance mechanics, and machinery erectors; program…

  20. Stepwise evolution of the Sec machinery in Proteobacteria

    NARCIS (Netherlands)

    van der Sluis, EO; Driessen, AJM; Sluis, Eli O. van der

    2006-01-01

    The Sec machinery facilitates the translocation of proteins across and into biological membranes. In several of the Proteobacteria, this machinery contains accessory features that are not present in any other bacterial division. The genomic distribution of these features in the context of bacterial

  1. Recession Hits China’s Textile Machinery Markets

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    China’s textile machinery industry saw a continued decline intotaI profit and a hefty slump in imports and exports in the first twomonths this year.Analysts anticipated a continued weakening ofmomentum for China’s textile machinery markets owing to weakerconsumer spending and easing export growth.

  2. ITALIAN TEXTILE MACHINERY WORKSHOP IN SUZHOU(CHINA)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The Association of Italian Textile Machinery Manufacturers (ACIMIT)and the Italian Trade Commission(ICE)held a technical workshop on Italian textile machinery for the production of technical textiles and nonwovens in China.The workshop occurred in Suzhou(Juangsu Province),on May 24th-25th,2007.

  3. 46 CFR 282.23 - Hull and machinery insurance.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Hull and machinery insurance. 282.23 Section 282.23... COMMERCE OF THE UNITED STATES Calculation of Subsidy Rates § 282.23 Hull and machinery insurance. (a) Subsidy items. The fair and reasonable net premium costs (including stamp taxes) of hull and...

  4. 46 CFR 111.103-9 - Machinery stop stations.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Machinery stop stations. 111.103-9 Section 111.103-9 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Remote Stopping Systems § 111.103-9 Machinery stop stations. (a) Each forced...

  5. Occupational incidents with self-propelled machinery in Austrian agriculture.

    Science.gov (United States)

    Mayrhofer, Hannes; Quendler, Elisabeth; Boxberger, Josef

    2013-01-01

    Tractors, self-propelled harvesting machinery, and material handling machinery are the most commonly used self-propelled machineries in Austrian agriculture, and they have similarities in main accident scenarios. Statistical data of all occupational incidents with these machines reported between 2008 and 2010 were analyzed to obtain information about the circumstances of the incidents, and about the victims and their work environments. Criteria of recognized occupational incidents provided by the Austrian Social Insurance Institution for Farmers were analyzed according to machinery category by means of cross-tabulation and chi-square tests. The results were discussed and evaluated in the context of the literature. The results of the analysis of the databases show that 786 occupational incidents with tractors, self-propelled harvesting machinery, and material handling machinery occurred in Austrian agriculture between 2008 and 2010. There were 231 occupational incidents in 2008; the number rose to 268 in 2009 and to 286 in 2010. A total of 41 incidents were fatal. For the machinery categories analyzed, the majority of injured victims were male, older than 40 years, Austrian citizens, and managers of a mixed-agricultural farm. A large number of the incidents occurred in all machinery categories by loss of control during operating a vehicle.

  6. Evolution and diversification of the basal transcription machinery.

    Science.gov (United States)

    Duttke, Sascha H C

    2015-03-01

    Transcription initiation was once thought to be regulated primarily by sequence-specific transcription factors with the basal transcription machinery being largely invariant. Gradually it became apparent that the basal transcription machinery greatly diversified during evolution and new studies now demonstrate that diversification of the TATA-binding protein (TBP) family yielded specialized and largely independent transcription systems.

  7. 46 CFR 119.465 - Ventilation of spaces containing diesel machinery.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Ventilation of spaces containing diesel machinery. 119... MACHINERY INSTALLATION Specific Machinery Requirements § 119.465 Ventilation of spaces containing diesel machinery. (a) A space containing diesel machinery must be fitted with adequate means, such as...

  8. Dynamic organization of the mitochondrial protein import machinery.

    Science.gov (United States)

    Straub, Sebastian P; Stiller, Sebastian B; Wiedemann, Nils; Pfanner, Nikolaus

    2016-11-01

    Mitochondria contain elaborate machineries for the import of precursor proteins from the cytosol. The translocase of the outer mitochondrial membrane (TOM) performs the initial import of precursor proteins and transfers the precursors to downstream translocases, including the presequence translocase and the carrier translocase of the inner membrane, the mitochondrial import and assembly machinery of the intermembrane space, and the sorting and assembly machinery of the outer membrane. Although the protein translocases can function as separate entities in vitro, recent studies revealed a close and dynamic cooperation of the protein import machineries to facilitate efficient transfer of precursor proteins in vivo. In addition, protein translocases were found to transiently interact with distinct machineries that function in the respiratory chain or in the maintenance of mitochondrial membrane architecture. Mitochondrial protein import is embedded in a regulatory network that ensures protein biogenesis, membrane dynamics, bioenergetic activity and quality control.

  9. The E3 ubiquitin ligase ARIH1 protects against genotoxic stress by initiating a 4EHP-mediated mRNA translation arrest

    DEFF Research Database (Denmark)

    von Stechow, Louise; Typas, Dimitris; Carreras Puigvert, Jordi

    2015-01-01

    /ISGylase, ariadne homologue 1 (ARIH1). Silencing ARIH1 sensitized ES and cancer cells to genotoxic compounds and ionizing radiation, irrespective of their p53 or caspase-3 status. Expression of wild-type but not ubiquitinase-defective ARIH1 constructs prevented sensitization caused by ARIH1 knockdown. ARIH1 protein...

  10. Submicroscopic duplications of the hydroxysteroid dehydrogenase HSD17B10 and the E3 ubiquitin ligase HUWE1 are associated with mental retardation.

    NARCIS (Netherlands)

    Froyen, G.; Corbett, M.; Walle, J. van de; Jarvela, I.; Lawrence, O.; Meldrum, C.; Bauters, M.; Govaerts, K.; Vandeleur, L.; Esch, H. van; Chelly, J.; Sanlaville, D.; Bokhoven, H. van; Ropers, H.H.; Laumonnier, F.; Ranieri, E.; Schwartz, C.E.; Abidi, F.; Tarpey, P.S.; Futreal, P.A.; Whibley, A.; Raymond, F.L.; Stratton, M.R.; Fryns, J.P.; Scott, R.; Peippo, M.; Sipponen, M.; Partington, M.; Mowat, D.; Field, M.; Hackett, A.; Marynen, P.; Turner, G.; Gecz, J.

    2008-01-01

    Submicroscopic copy-number imbalances contribute significantly to the genetic etiology of human disease. Here, we report a novel microduplication hot spot at Xp11.22 identified in six unrelated families with predominantly nonsyndromic XLMR. All duplications segregate with the disease, including the

  11. Submicroscopic duplications of the hydroxysteroid dehydrogenase HSD17B10 and the E3 ubiquitin ligase HUWE1 are associated with mental retardation

    NARCIS (Netherlands)

    Froyen, Guy; Corbett, Mark; Vandewalle, Joke; Jarvela, Irma; Lawrence, Owen; Meldrum, Cliff; Bauters, Marijke; Govaerts, Karen; Vandeleur, Lucianne; van Esch, Hilde; Chelly, Jamel; Sanlaville, Damien; van Bokhoven, Hans; Ropers, Hans-Hilger; Laumonnier, Frederic; Ranieri, Enzo; Schwartz, Charles E.; Abidi, Fatima; Tarpey, Patrick S.; Futreal, P. Andrew; Whibley, Annabel; Raymond, F. Lucy; Stratton, Michael R.; Fryns, Jean-Pierre; Scott, Rodney; Peippo, Maarit; Sipponen, Marjatta; Partington, Michael; Mowat, David; Field, Michael; Hackett, Anna; Marynen, Peter; Turner, Gillian; Gecz, Jozef

    Submicroscopic copy-number imbalances contribute significantly to the genetic etiology of human disease. Here, we report a novel microduplication hot spot at Xp11.22 identified in six unrelated families with predominantly nonsyndromic XLMR. All duplications segregate with the disease, including the

  12. Submicroscopic duplications of the hydroxysteroid dehydrogenase HSD17B10 and the E3 ubiquitin ligase HUWE1 are associated with mental retardation

    NARCIS (Netherlands)

    Froyen, Guy; Corbett, Mark; Vandewalle, Joke; Jarvela, Irma; Lawrence, Owen; Meldrum, Cliff; Bauters, Marijke; Govaerts, Karen; Vandeleur, Lucianne; van Esch, Hilde; Chelly, Jamel; Sanlaville, Damien; van Bokhoven, Hans; Ropers, Hans-Hilger; Laumonnier, Frederic; Ranieri, Enzo; Schwartz, Charles E.; Abidi, Fatima; Tarpey, Patrick S.; Futreal, P. Andrew; Whibley, Annabel; Raymond, F. Lucy; Stratton, Michael R.; Fryns, Jean-Pierre; Scott, Rodney; Peippo, Maarit; Sipponen, Marjatta; Partington, Michael; Mowat, David; Field, Michael; Hackett, Anna; Marynen, Peter; Turner, Gillian; Gecz, Jozef

    2008-01-01

    Submicroscopic copy-number imbalances contribute significantly to the genetic etiology of human disease. Here, we report a novel microduplication hot spot at Xp11.22 identified in six unrelated families with predominantly nonsyndromic XLMR. All duplications segregate with the disease, including the

  13. The E3 ubiquitin ligase Ro52 negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3

    National Research Council Canada - National Science Library

    Higgs, Rowan; Ní Gabhann, Joan; Ben Larbi, Nadia; Breen, Eamon P; Fitzgerald, Katherine A; Jefferies, Caroline A

    2008-01-01

    .... The role of the transcription factor IRF3 is well established in driving this process. However, equally as important are cellular mechanisms for turning off type I IFN production to limit this response...

  14. The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line

    DEFF Research Database (Denmark)

    Mohiuddin, Mohammed; Kobayashi, Shunsuke; Keka, Islam Shamima

    2016-01-01

    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....

  15. Genome-wide RNAi screen reveals the E3 SUMO-protein ligase gene SIZ1 as a novel determinant of furfural tolerance in Saccharomyces cerevisiae

    OpenAIRE

    Xiao, Han; Zhao, Huimin

    2014-01-01

    Background Furfural is a major growth inhibitor in lignocellulosic hydrolysates and improving furfural tolerance of microorganisms is critical for rapid and efficient fermentation of lignocellulosic biomass. In this study, we used the RNAi-Assisted Genome Evolution (RAGE) method to select for furfural resistant mutants of Saccharomyces cerevisiae, and identified a new determinant of furfural tolerance. Results By using a genome-wide RNAi (RNA-interference) screen in S. cerevisiae for genes in...

  16. A molecular triad governing adult stem cells activation : Crystallographic studies of LGR5, R-spondin 1 and E3 ligase ZNRF3

    NARCIS (Netherlands)

    Peng, W.C.

    2014-01-01

    The small intestinal tissue is divided into two compartments: the ‘villus’ (with finger-like projection) and the ‘crypt’ (invagination between villi). The small intestine is responsible for absorption of nutrients in the food ingested and is constantly damaged due to exposure to biological and

  17. Exploring the Structural Requirements for Inhibition of the Ubiquitin E3 Ligase Breast Cancer Associated Protein 2 (BCA2)a as a Treatment for Breast Cancer

    OpenAIRE

    Brahemi, Ghali; Kona, Fathima R; Fiasella, Annalisa; Buac, Daniela; Soukupová, Jitka; Brancale, Andrea; Burger, Angelika M; Westwell, Andrew D.

    2010-01-01

    The zinc-ejecting aldehyde dehydrogenase (ALDH) inhibitory drug disulfiram (DSF) was found to be a breast cancer-associated protein 2 (BCA2) inhibitor with potent antitumor activity. We herein describe our work in the synthesis and evaluation of new series of zinc-affinic molecules to explore the structural requirements for selective BCA2-inhibitory antitumor activity. An N(C=S)S-S motif was found to be required, based on selective activity in BCA2-expressing breast cancer cell lines and agai...

  18. Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation

    DEFF Research Database (Denmark)

    Emdal, Kristina B; Pedersen, Anna-Kathrine; Bekker-Jensen, Dorte B

    2015-01-01

    SH-SY5Y neuroblastoma cells respond to nerve growth factor (NGF)-mediated activation of the tropomyosin-related kinase A (TrkA) with neurite outgrowth, thereby providing a model to study neuronal differentiation. We performed a time-resolved analysis of NGF-TrkA signaling in neuroblastoma cells...... then becomes phosphorylated and ubiquitylated and decreases in abundance. We also found that recruitment of Cbl-b promotes TrkA ubiquitylation and degradation. Furthermore, the amount of phosphorylation of the kinase ERK and neurite outgrowth increased upon Cbl-b depletion in several neuroblastoma cell lines...

  19. Ribosome evolution: Emergence of peptide synthesis machinery

    Indian Academy of Sciences (India)

    Koji Tamura

    2011-12-01

    Proteins, the main players in current biological systems, are produced on ribosomes by sequential amide bond (peptide bond) formations between amino-acid-bearing tRNAs. The ribosome is an exquisite super-complex of RNA-proteins, containing more than 50 proteins and at least 3 kinds of RNAs. The combination of a variety of side chains of amino acids (typically 20 kinds with some exceptions) confers proteins with extraordinary structure and functions. The origin of peptide bond formation and the ribosome is crucial to the understanding of life itself. In this article, a possible evolutionary pathway to peptide bond formation machinery (proto-ribosome) will be discussed, with a special focus on the RNA minihelix (primordial form of modern tRNA) as a starting molecule. Combining the present data with recent experimental data, we can infer that the peptidyl transferase center (PTC) evolved from a primitive system in the RNA world comprising tRNA-like molecules formed by duplication of minihelix-like small RNA.

  20. Occupational Accidents with Agricultural Machinery in Austria.

    Science.gov (United States)

    Kogler, Robert; Quendler, Elisabeth; Boxberger, Josef

    2016-01-01

    The number of recognized accidents with fatalities during agricultural and forestry work, despite better technology and coordinated prevention and trainings, is still very high in Austria. The accident scenarios in which people are injured are very different on farms. The common causes of accidents in agriculture and forestry are the loss of control of machine, means of transport or handling equipment, hand-held tool, and object or animal, followed by slipping, stumbling and falling, breakage, bursting, splitting, slipping, fall, and collapse of material agent. In the literature, a number of studies of general (machine- and animal-related accidents) and specific (machine-related accidents) agricultural and forestry accident situations can be found that refer to different databases. From the database Data of the Austrian Workers Compensation Board (AUVA) about occupational accidents with different agricultural machinery over the period 2008-2010 in Austria, main characteristics of the accident, the victim, and the employer as well as variables on causes and circumstances by frequency and contexts of parameters were statistically analyzed by employing the chi-square test and odds ratio. The aim of the study was to determine the information content and quality of the European Statistics on Accidents at Work (ESAW) variables to evaluate safety gaps and risks as well as the accidental man-machine interaction.