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Sample records for animal hect ubiquitin

  1. Evolution of Plant HECT Ubiquitin Ligases

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    Ignacio Marín

    2013-01-01

    HECT ubiquitin ligases are key components of the ubiquitin-proteasome system, which is present in all eukaryotes. In this study, the patterns of emergence of HECT genes in plants are described. Phylogenetic and structural data indicate that viridiplantae have six main HECT subfamilies, which arose before the split that separated green algae from the rest of plants. It is estimated that the common ancestor of all plants contained seven HECT genes. Contrary to what happened in animals, the numb...

  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-linke...

  3. Hippo Stabilises Its Adaptor Salvador by Antagonising the HECT Ubiquitin Ligase Herc4.

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    Birgit L Aerne

    Full Text Available Signalling through the Hippo (Hpo pathway involves a kinase cascade, which leads to the phosphorylation and inactivation of the pro-growth transcriptional co-activator Yorkie (Yki. Despite the identification of a large number of pathway members and modulators, our understanding of the molecular events that lead to activation of Hpo and the downstream kinase Warts (Wts remain incomplete. Recently, targeted degradation of several Hpo pathway components has been demonstrated as a means of regulating pathway activity. In particular, the stability of scaffold protein Salvador (Sav, which is believed to promote Hpo/Wts association, is crucially dependent on its binding partner Hpo. In a cell-based RNAi screen for ubiquitin regulators involved in Sav stability, we identify the HECT domain protein Herc4 (HECT and RLD domain containing E3 ligase as a Sav E3 ligase. Herc4 expression promotes Sav ubiquitylation and degradation, while Herc4 depletion stabilises Sav. Interestingly, Hpo reduces Sav/Herc4 interaction in a kinase-dependent manner. This suggests the existence of a positive feedback loop, where Hpo stabilises its own positive regulator by antagonising Herc4-mediated degradation of Sav.

  4. Insights into Ubiquitin Transfer Cascades from a Structure of a UbcH5B[is equivalent to]Ubiquitin-HECT[superscript NEDD4L] Complex

    Energy Technology Data Exchange (ETDEWEB)

    Kamadurai, Hari B.; Souphron, Judith; Scott, Daniel C.; Duda, David M.; Miller, Darcie J.; Stringer, Daniel; Piper, Robert C.; Schulman, Brenda A.; (SJCH)

    2010-02-23

    In E1-E2-E3 ubiquitin (Ub) conjugation cascades, the E2 first forms a transient E2 {approx} Ub covalent complex and then interacts with an E3 for Ub transfer. For cascades involving E3s in the HECT class, Ub is transferred from an associated E2 to the acceptor cysteine in the HECT domain C lobe. To gain insights into this process, we determined the crystal structure of a complex between the HECT domain of NEDD4L and the E2 UbcH5B bearing a covalently linked Ub at its active site (UbcH5B {approx} Ub). Noncovalent interactions between UbcH5B and the HECT N lobe and between Ub and the HECT domain C lobe lead to an overall compact structure, with the Ub C terminus sandwiched between UbcH5B and HECT domain active sites. The structure suggests a model for E2-to-HECT Ub transfer, in which interactions between a donor Ub and an acceptor domain constrain upstream and downstream enzymes for conjugation.

  5. Ancient origin of animal U-box ubiquitin ligases

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    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.

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

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    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

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

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    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.

  8. A HECT ubiquitin-protein ligase as a novel candidate gene for altered quinine and quinidine responses in Plasmodium falciparum.

    Science.gov (United States)

    Sanchez, Cecilia P; Liu, Chia-Hao; Mayer, Sybille; Nurhasanah, Astutiati; Cyrklaff, Marek; Mu, Jianbing; Ferdig, Michael T; Stein, Wilfred D; Lanzer, Michael

    2014-05-01

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

  9. Genome-wide identification and characterization of the apple (Malus domestica) HECT ubiquitin-protein ligase family and expression analysis of their responsiveness to abiotic stresses.

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    Xu, Jianing; Xing, Shanshan; Cui, Haoran; Chen, Xuesen; Wang, Xiaoyun

    2016-04-01

    The ubiquitin-protein ligases (E3s) directly participate in ubiquitin (Ub) transferring to the target proteins in the ubiquitination pathway. The HECT ubiquitin-protein ligase (UPL), one type of E3s, is characterized as containing a conserved HECT domain of approximately 350 amino acids in the C terminus. Some UPLs were found to be involved in trichome development and leaf senescence in Arabidopsis. However, studies on plant UPLs, such as characteristics of the protein structure, predicted functional motifs of the HECT domain, and the regulatory expression of UPLs have all been limited. Here, we present genome-wide identification of the genes encoding UPLs (HECT gene) in apple. The 13 genes (named as MdUPL1-MdUPL13) from ten different chromosomes were divided into four groups by phylogenetic analysis. Among these groups, the encoding genes in the intron-exon structure and the included additional functional domains were quite different. Notably, the F-box domain was first found in MdUPL7 in plant UPLs. The HECT domain in different MdUPL groups also presented different spatial features and three types of conservative motifs were identified. The promoters of each MdUPL member carried multiple stress-response related elements by cis-acting element analysis. Experimental results demonstrated that the expressions of several MdUPLs were quite sensitive to cold-, drought-, and salt-stresses by qRT-PCR assay. The results of this study helped to elucidate the functions of HECT proteins, especially in Rosaceae plants.

  10. Rescue of HIV-1 release by targeting widely divergent NEDD4-type ubiquitin ligases and isolated catalytic HECT domains to Gag.

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    Eric R Weiss

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

  11. HECT E3s and human disease

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    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.

  12. Ubiquitin

    DEFF Research Database (Denmark)

    Vinther-Jensen, T.; Simonsen, A. H.; Budtz-Jorgensen, E.;

    2015-01-01

    BACKGROUND: Finding early and dynamic biomarkers in Huntington's disease is a key to understanding the early pathology of Huntington's disease and potentially to tracking disease progression. This would benefit the future evaluation of potential neuroprotective and disease-modifying therapies......, as well as aid in identifying an optimal time point for initiating a potential therapeutic intervention. METHODS: This explorative proteomics study evaluated cerebrospinal fluid from 94 Huntington's disease gene-expansion carriers (39 premanifest and 55 manifest) and 27 Huntington's disease gene...... and controls. One of them identified as ubiquitin was shown to be dependent on the Unified Huntington Disease Rating Scale Total Functional Capacity, a pseudo-measure of disease severity (P = 0.001), and the Symbol Digit Modalities Test (0.04) in manifest and CAG-age product score (P = 0.019) in all gene...

  13. Basic Tilted Helix Bundle – A new protein fold in human FKBP25/FKBP3 and HectD1

    Energy Technology Data Exchange (ETDEWEB)

    Helander, Sara; Montecchio, Meri [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden); Lemak, Alexander [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Farès, Christophe [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Almlöf, Jonas [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden); Li, Yanjun [Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Yee, Adelinda [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Arrowsmith, Cheryl H. [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Dhe-Paganon, Sirano [Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Sunnerhagen, Maria, E-mail: maria.sunnerhagen@liu.se [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden)

    2014-04-25

    Highlights: • We describe the structure of a novel fold in FKBP25 and HectD. • The new fold is named the Basic Tilted Helix Bundle (BTHB) domain. • A conserved basic surface patch is presented, suggesting a functional role. - Abstract: In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP25{sub 1–73}, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains.

  14. Basic Tilted Helix Bundle – A new protein fold in human FKBP25/FKBP3 and HectD1

    International Nuclear Information System (INIS)

    Highlights: • We describe the structure of a novel fold in FKBP25 and HectD. • The new fold is named the Basic Tilted Helix Bundle (BTHB) domain. • A conserved basic surface patch is presented, suggesting a functional role. - Abstract: In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP251–73, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains

  15. In Vitro Ubiquitination: Self-Ubiquitination, Chain Formation, and Substrate Ubiquitination Assays.

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    Maspero, Elena; Polo, Simona

    2016-01-01

    Ubiquitination of proteins in vitro has evolved as an indispensable tool for the functional analysis of this posttranslational modification. In vitro ubiquitination is particularly helpful to study conjugation mechanisms. The efficiency of the ubiquitination reaction depends in part on the quality of the enzymes utilized. Here we introduce the assay developed in our lab to study HECT E3 ligases. It involves bacterially expressed E1, His-tagged Ube2D3 (also called UbcH5c, the best E2 for Nedd4), untagged Nedd4, and untagged ubiquitin (Ub). As tags may impair specific activity of the enzymes or even interfere with the enzymatic reaction, they should be avoided, removed, or kept to a minimal size whenever possible, unless proven to be without consequence. The protocol described here is suitable for other E3 ligases capable of forming Ub chains as pseudo-product of the enzyme reaction. It is also adapted to include substrates. In this case, substrates should be tagged and purified after the reaction is completed to allow the detection of the ubiquitinated products. PMID:27613033

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

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    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.

  17. Regulation of plant immune receptors by ubiquitination

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    Giulia eFurlan

    2012-10-01

    Full Text Available From pathogen perception and the activation of signal transduction cascades to the deployment of defense responses, protein ubiquitination plays a key role in the modulation of plant immunity. Ubiquitination is mediated by three enzymes, of which the E3 ubiquitin ligases, the substrate determinants, have been the major focus of attention. Accumulating evidence suggests that ubiquitination modulates signaling mediated by pattern recognition receptors (PRRs and is important for the accumulation of nucleotide-binding leucine-rich repeat (NB-LRR type intracellular immune sensors. Recent studies also indicate that ubiquitination directs vesicle trafficking, a function that has been clearly established for immune signaling in animals. In this mini review, we discuss these and other recent advances and highlight important open questions.

  18. Regulation of plant immune receptors by ubiquitination.

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    Furlan, Giulia; Klinkenberg, Jörn; Trujillo, Marco

    2012-01-01

    From pathogen perception and the activation of signal transduction cascades to the deployment of defense responses, protein ubiquitination plays a key role in the modulation of plant immunity. Ubiquitination is mediated by three enzymes, of which the E3 ubiquitin ligases, the substrate determinants, have been the major focus of attention. Accumulating evidence suggests that ubiquitination modulates signaling mediated by pattern recognition receptors and is important for the accumulation of nucleotide-binding leucine-rich repeat type intracellular immune sensors. Recent studies also indicate that ubiquitination directs vesicle trafficking, a function that has been clearly established for immune signaling in animals. In this mini review, we discuss these and other recent advances and highlight important open questions. PMID:23109936

  19. [Progress in ubiquitin, ubiquitin chain and protein ubiquitination].

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    Lan, Qiuyan; Gao, Yuan; Li, Yanchang; Hong, Xuechuan; Xu, Ping

    2016-01-01

    Protein ubiquitination is one of the most important and widely exist protein post-translational modifications in eukaryotic cells, which takes the ubiquitin and ubiquitin chains as signal molecules to covalently modify other protein substrates. It plays an important roles in the control of almost all of the life processes, including gene transcription and translation, signal transduction and cell-cycle progression, besides classical 26S protesome degradation pathway. Varied modification sites in the same substrates as well as different types of ubiquitin linkages in the same modification sites contain different structural information, which conduct different signal or even determine the fate of the protein substrates in the cell. Any abnormalities in ubiquitin chain formation or its modification process may cause severe problem in maintaining the balance of intracellular environment and finally result in serious health problem of human being. In this review, we discussed the discovery, genetic characteristics and the crystal structure of the ubiquitin. We also emphasized the recent progresses of the assembly processes, structure and their biological function of ubiquitin chains. The relationship between the disregulation and related human diseases has also been discussed. These progress will shed light on the complexity of proteome, which may also provide tools in the new drug research and development processes. PMID:27363196

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

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    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.

  1. In the family with ubiquitin

    OpenAIRE

    Alexandru, Gabriela; Pariente, Nonia; Xirodimas, Dimitris

    2011-01-01

    The Cold Spring Harbor meeting on ‘The Ubiquitin family', held in May 2011, brought together scientists from a wide range of fields, all under the common umbrella of ubiquitin and ubiquitin-like protein structure, function and regulation.

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

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    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.

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

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    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.

  4. Ubiquitination of plant immune receptors.

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    Zhou, Jinggeng; He, Ping; Shan, Libo

    2014-01-01

    Ubiquitin is a highly conserved regulatory protein consisting of 76 amino acids and ubiquitously expressed in all eukaryotic cells. The reversible ubiquitin conjugation to a wide variety of target proteins, a process known as ubiquitination or ubiquitylation, serves as one of the most important and prevalent posttranslational modifications to regulate the myriad actions of protein cellular functions, including protein degradation, vesicle trafficking, and subcellular localization. Protein ubiquitination is an ATP-dependent stepwise covalent attachment of one or more ubiquitin molecules to target proteins mediated by a hierarchical enzymatic cascade consisting of an E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. The plant plasma membrane resident receptor-like kinase Flagellin Sensing 2 (FLS2) recognizes bacterial flagellin and initiates innate immune signaling to defend against pathogen attacks. We have recently shown that two plant U-box E3 ubiquitin ligases PUB12 and PUB13 directly ubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, which in turn attenuates FLS2 signaling to prevent excessive or prolonged activation of immune responses. Here, we use FLS2 as an example to describe a protocol for detection of protein ubiquitination in plant cells in vivo and in test tubes in vitro. In addition, we elaborate the approach to identify different types of ubiquitin linkages by using various lysine mutants of ubiquitin. The various in vivo and in vitro ubiquitination assays will provide researchers with the tools to address how ubiquitination regulates diverse cellular functions of target proteins. PMID:25117287

  5. Animals

    International Nuclear Information System (INIS)

    The radionuclides of most concern with respect to contamination of animals after a nuclear accident are radioiodine, radiocaesium and radiostrontium (ICRP 30, 1979). Of the other significant anthropogenic radionuclides likely to be released in most accidents, only small proportions of that ingested will be absorbed in an animals gut, and the main animal products, milk and meat, will not normally be contaminated to a significant extent. Animal products will mostly be contaminated as a result of ingestion of contaminated feed and possibly, but to a much lesser extent, from inhalation (for radioiodine only). Direct external contamination of animals is of little or no consequence in human food production. Radioiodine and radiostrontium are important with respect to contamination of milk; radiocaesium contaminates both milk and meat. The physical and chemical form of a radionuclide can influence its absorption in the animal gut. For example, following the Chernobyl accident radiocaesium incorporated into vegetation by root uptake was more readily absorbed than that associated with the original deposit. The transfer of radiocaesium and radiostrontium to animals will be presented both as transfer coefficients and aggregated transfer coefficients. For most animal meat products, only radiocaesium is important as other radionuclides do not significantly contaminate muscle. Farm animal products are the most important foodstuff determining radiocaesium intake by the average consumer in the Nordic countries. The major potential source of radioiodine and radiostrontium to humans is milk and milk products. Of the different species, the smaller animals have the highest transfer of radiocaesium from fodder to meat and milk. (EG)

  6. Animals

    Energy Technology Data Exchange (ETDEWEB)

    Skuterud, L.; Strand, P. [Norwegian Radiation Protection Authority (Norway); Howard, B.J. [Inst. of Terrestrial Ecology (United Kingdom)

    1997-10-01

    The radionuclides of most concern with respect to contamination of animals after a nuclear accident are radioiodine, radiocaesium and radiostrontium (ICRP 30, 1979). Of the other significant anthropogenic radionuclides likely to be released in most accidents, only small proportions of that ingested will be absorbed in an animals gut, and the main animal products, milk and meat, will not normally be contaminated to a significant extent. Animal products will mostly be contaminated as a result of ingestion of contaminated feed and possibly, but to a much lesser extent, from inhalation (for radioiodine only). Direct external contamination of animals is of little or no consequence in human food production. Radioiodine and radiostrontium are important with respect to contamination of milk; radiocaesium contaminates both milk and meat. The physical and chemical form of a radionuclide can influence its absorption in the animal gut. For example, following the Chernobyl accident radiocaesium incorporated into vegetation by root uptake was more readily absorbed than that associated with the original deposit. The transfer of radiocaesium and radiostrontium to animals will be presented both as transfer coefficients and aggregated transfer coefficients. For most animal meat products, only radiocaesium is important as other radionuclides do not significantly contaminate muscle. Farm animal products are the most important foodstuff determining radiocaesium intake by the average consumer in the Nordic countries. The major potential source of radioiodine and radiostrontium to humans is milk and milk products. Of the different species, the smaller animals have the highest transfer of radiocaesium from fodder to meat and milk. (EG). 68 refs.

  7. Ubiquitin domain proteins in disease

    DEFF Research Database (Denmark)

    Klausen, Louise Kjær; Schulze, Andrea; Seeger, Michael;

    2007-01-01

    The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite...... and cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com)....

  8. 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.

  9. Comprehensive database of human E3 ubiquitin ligases: application to aquaporin-2 regulation.

    Science.gov (United States)

    Medvar, Barbara; Raghuram, Viswanathan; Pisitkun, Trairak; Sarkar, Abhijit; Knepper, Mark A

    2016-07-01

    Aquaporin-2 (AQP2) is regulated in part via vasopressin-mediated changes in protein half-life that are in turn dependent on AQP2 ubiquitination. Here we addressed the question, "What E3 ubiquitin ligase is most likely to be responsible for AQP2 ubiquitination?" using large-scale data integration based on Bayes' rule. The first step was to bioinformatically identify all E3 ligase genes coded by the human genome. The 377 E3 ubiquitin ligases identified in the human genome, consisting predominant of HECT, RING, and U-box proteins, have been used to create a publically accessible and downloadable online database (https://hpcwebapps.cit.nih.gov/ESBL/Database/E3-ligases/). We also curated a second database of E3 ligase accessory proteins that included BTB domain proteins, cullins, SOCS-box proteins, and F-box proteins. Using Bayes' theorem to integrate information from multiple large-scale proteomic and transcriptomic datasets, we ranked these 377 E3 ligases with respect to their probability of interaction with AQP2. Application of Bayes' rule identified the E3 ligases most likely to interact with AQP2 as (in order of probability): NEDD4 and NEDD4L (tied for first), AMFR, STUB1, ITCH, ZFPL1. Significantly, the two E3 ligases tied for top rank have also been studied extensively in the reductionist literature as regulatory proteins in renal tubule epithelia. The concordance of conclusions from reductionist and systems-level data provides strong motivation for further studies of the roles of NEDD4 and NEDD4L in the regulation of AQP2 protein turnover. PMID:27199454

  10. Phospho-ubiquitin: upending the PINK-Parkin-ubiquitin cascade.

    Science.gov (United States)

    Matsuda, Noriyuki

    2016-04-01

    Mitochondria with decreased membrane potential are characterized by defects in protein import into the matrix and impairments in high-efficiency synthesis of ATP. These low-quality mitochondria are marked with ubiquitin for selective degradation. Key factors in this mechanism are PTEN-induced putative kinase 1 (PINK1, a mitochondrial kinase) and Parkin (a ubiquitin ligase), disruption of which has been implicated in predisposition to Parkinson's disease. Previously, the clearance of damaged mitochondria had been thought to be the end result of a simple cascading reaction of PINK1-Parkin-ubiquitin. However, in the past year, several research groups including ours unexpectedly revealed that Parkin regulation is mediated by PINK1-dependent phosphorylation of ubiquitin. These results overturned the simple hierarchy that posited PINK1 and ubiquitin as the upstream and downstream factors of Parkin, respectively. Although ubiquitylation is well-known as a post-translational modification, it has recently become clear that ubiquitin itself can be modified, and that this modification unexpectedly converts ubiquitin to a factor that functions in retrograde signalling.

  11. Animals

    Institute of Scientific and Technical Information of China (English)

    杨光

    2000-01-01

    The largest animal ever to live on the earth is the blue whale(蓝鲸)It weighs about 80 tons--more than 24 elephants. It is more than 30 metres long. A newborn baby whale weighs as much as a big elephant.

  12. ANIMALS

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Mammals(哺乳动物)Mammals are the world's most dominant(最占优势的)animal.They are extremely(非常)diverse(多种多样的)creatures(生物,动物)that include(包括)the biggest ever animal (the blue whale鲸,which eats up to 6 tons every day),the smallest(leaf-nosed bat小蹄蝠) and the laziest(sloth树獭,who spends 80% of their time sleeping).There are over 4,600 kinds of mammals and they live in very different environments(环境)—oceans(海洋),rivers,the jungle(丛林),deserts,and plains(平原).

  13. The ubiquitin-proteasome system

    Indian Academy of Sciences (India)

    Dipankar Nandi; Pankaj Tahiliani; Anujith Kumar; Dilip Chandu

    2006-03-01

    The 2004 Nobel Prize in chemistry for the discovery of protein ubiquitination has led to the recognition of cellular proteolysis as a central area of research in biology. Eukaryotic proteins targeted for degradation by this pathway are first ‘tagged’ by multimers of a protein known as ubiquitin and are later proteolyzed by a giant enzyme known as the proteasome. This article recounts the key observations that led to the discovery of ubiquitin-proteasome system (UPS). In addition, different aspects of proteasome biology are highlighted. Finally, some key roles of the UPS in different areas of biology and the use of inhibitors of this pathway as possible drug targets are discussed.

  14. The WW-HECT protein Smurf2 interacts with the Docking Protein NEDD9/HEF1 for Aurora A activation

    Directory of Open Access Journals (Sweden)

    Moore Finola E

    2010-09-01

    Full Text Available Abstract The multi-functional adaptor protein NEDD9/HEF1/Cas-L regulates cell motility, invasion and cell cycle progression, and plays key roles in cancer progression and metastasis. NEDD9 is localized to the centrosome and is required for activation of Aurora A kinase in mitosis. Here we demonstrate that the HECT-WW protein Smurf2 physically associates with NEDD9 and is required for the stability of NEDD9 protein. Smurf2 depletion results in a marked decrease in NEDD9 protein levels, by facilitating polyubiquitination and proteasomal degradation of NEDD9. Conversely, forced overexpression of Smurf2 results in upregulation of endogenous NEDD9 protein, confirming the role for Smurf2 in NEDD9 stability. Cells with Smurf2 depletion fail to activate Aurora A at the G2/M boundary, leading to a marked delay in mitotic entry. These observations suggest that the stable complex of Smurf2 and NEDD9 is required for timely entry into mitosis via Aurora A activation.

  15. Ubiquitin and proteasomes in transcription.

    Science.gov (United States)

    Geng, Fuqiang; Wenzel, Sabine; Tansey, William P

    2012-01-01

    Regulation of gene transcription is vitally important for the maintenance of normal cellular homeostasis. Failure to correctly regulate gene expression, or to deal with problems that arise during the transcription process, can lead to cellular catastrophe and disease. One of the ways cells cope with the challenges of transcription is by making extensive use of the proteolytic and nonproteolytic activities of the ubiquitin-proteasome system (UPS). Here, we review recent evidence showing deep mechanistic connections between the transcription and ubiquitin-proteasome systems. Our goal is to leave the reader with a sense that just about every step in transcription-from transcription initiation through to export of mRNA from the nucleus-is influenced by the UPS and that all major arms of the system--from the first step in ubiquitin (Ub) conjugation through to the proteasome-are recruited into transcriptional processes to provide regulation, directionality, and deconstructive power. PMID:22404630

  16. Non-degradative Ubiquitination of Protein Kinases.

    OpenAIRE

    K Aurelia Ball; Johnson, Jeffrey R.; Lewinski, Mary K; John Guatelli; Erik Verschueren; Krogan, Nevan J.; Matthew P Jacobson

    2016-01-01

    Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichm...

  17. 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.

  18. 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.

  19. The ubiquitin ligase HERC3 attenuates NF-κB-dependent transcription independently of its enzymatic activity by delivering the RelA subunit for degradation.

    Science.gov (United States)

    Hochrainer, Karin; Pejanovic, Nadja; Olaseun, Victoria A; Zhang, Sheng; Iadecola, Costantino; Anrather, Josef

    2015-11-16

    Activation of NF-κB-dependent transcription represents an important hallmark of inflammation. While the acute inflammatory response is per se beneficial, it can become deleterious if its spatial and temporal profile is not tightly controlled. Classically, NF-κB activity is limited by cytoplasmic retention of the NF-κB dimer through binding to inhibitory IκB proteins. However, increasing evidence suggests that NF-κB activity can also be efficiently contained by direct ubiquitination of NF-κB subunits. Here, we identify the HECT-domain ubiquitin ligase HERC3 as novel negative regulator of NF-κB activity. We find that HERC3 restricts NF-κB nuclear import and DNA binding without affecting IκBα degradation. Instead HERC3 indirectly binds to the NF-κB RelA subunit after liberation from IκBα inhibitor leading to its ubiquitination and protein destabilization. Remarkably, the regulation of RelA activity by HERC3 is independent of its inherent ubiquitin ligase activity. Rather, we show that HERC3 and RelA are part of a multi-protein complex containing the proteasome as well as the ubiquitin-like protein ubiquilin-1 (UBQLN1). We present evidence that HERC3 and UBQLN1 provide a link between NF-κB RelA and the 26S proteasome, thereby facilitating RelA protein degradation. Our findings establish HERC3 as novel candidate regulating the inflammatory response initiated by NF-κB. PMID:26476452

  20. 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

  1. Non-degradative Ubiquitination of Protein Kinases.

    Directory of Open Access Journals (Sweden)

    K Aurelia Ball

    2016-06-01

    Full Text Available Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

  2. Non-degradative Ubiquitination of Protein Kinases.

    Science.gov (United States)

    Ball, K Aurelia; Johnson, Jeffrey R; Lewinski, Mary K; Guatelli, John; Verschueren, Erik; Krogan, Nevan J; Jacobson, Matthew P

    2016-06-01

    Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

  3. Enhancement of CIITA transcriptional function by ubiquitin.

    Science.gov (United States)

    Greer, Susanna F; Zika, Eleni; Conti, Brian; Zhu, Xin-Sheng; Ting, Jenny P-Y

    2003-11-01

    Although increasing evidence indicates that there is a direct link between ubiquitination and mono-ubiquitination and transcription in yeast, this link has not been demonstrated in higher eukaryotes. Here we show that the major histocompatibility complex (MHC) class II transactivator (CIITA), which is required for expression of genes encoding MHC class II molecules, is ubiquitinated. This ubiquitination enhanced the association of CIITA with both MHC class II transcription factors and the MHC class II promoter, resulting in an increase in transactivation function and in the expression of MHC class II mRNA. The degree of CIITA ubiquitination was controlled by histone acetylases (HATs) and deacetylases (HDACs), indicating that the crucial cellular processes mediated by these enzymes are linked to regulate transcription. Thus, ubiquitin positively regulates a mammalian coactivator by enhancing its assembly at the promoter.

  4. Crystal structures of two bacterial HECT-like E3 ligases in complex with a human E2 reveal atomic details of pathogen-host interactions

    Energy Technology Data Exchange (ETDEWEB)

    Lin, David Yin-wei; Diao, Jianbo; Chen, Jue (Purdue); (Fudan)

    2012-12-10

    In eukaryotes, ubiquitination is an important posttranslational process achieved through a cascade of ubiquitin-activating (E1), conjugating (E2), and ligase (E3) enzymes. Many pathogenic bacteria deliver virulence factors into the host cell that function as E3 ligases. How these bacterial 'Trojan horses' integrate into the eukaryotic ubiquitin system has remained a mystery. Here we report crystal structures of two bacterial E3s, Salmonella SopA and Escherichia coli NleL, both in complex with human E2 UbcH7. These structures represent two distinct conformational states of the bacterial E3s, supporting the necessary structural rearrangements associated with ubiquitin transfer. The E2-interacting surface of SopA and NleL has little similarity to those of eukaryotic E3s. However, both bacterial E3s bind to the canonical surface of E2 that normally interacts with eukaryotic E3s. Furthermore, we show that a glutamate residue on E3 is involved in catalyzing ubiquitin transfer from E3 to the substrate, but not from E2 to E3. Together, these results provide mechanistic insights into the ubiquitin pathway and a framework for understanding molecular mimicry in bacterial pathogenesis.

  5. Ubiquitin and Ubiquitin-Like Modifications of the p53 Family

    Directory of Open Access Journals (Sweden)

    Ian R. Watson

    2006-08-01

    Full Text Available Regulation of p53 by the ubiquitin-proteasomal pathway has been studied considerably. Studies have also demonstrated that the ubiquitin-like proteins SUMO-1 and NEDD8 modify p53. Similarly, p63 and p73 are subject to regulation by ubiquitin and ubiquitin-like modifications, and perturbations of these pathways in the regulation of the p53 family have been implicated in tumorigenesis and developmental abnormalities. Here, we provide an overview of the current understanding of the regulation of the p53 family by covalent modification by ubiquitin, SUMO-1, and NEDD8.

  6. Pink1, the first ubiquitin kinase

    OpenAIRE

    Zheng, Xinde; Hunter, Tony

    2014-01-01

    Pink1 and Parkin, identified through studies of hereditary early onset Parkinson's disease, are involved in mitochondria quality control. Parkin E3 ubiquitin ligase activity is activated by Pink1 kinase activity, although the mechanism is still elusive. Three recent reports uncover a surprising mechanism in which Pink1 directly phosphorylates ubiquitin to boost Parkin activity.

  7. Met1-linked Ubiquitination in Immune Signalling

    DEFF Research Database (Denmark)

    Fiil, Berthe Katrine; Gyrd-Hansen, Mads

    2014-01-01

    Methionine 1-linked ubiquitin chains (Met1-Ub), or linear ubiquitin, has emerged as a central post-translational modification in innate immune signalling. Molecular machinery that assembles, senses and, more recently, disassembles Met1-Ub has been identified, and technical advances have enabled...

  8. Regulation of nucleotide excision repair through ubiquitination

    Institute of Scientific and Technical Information of China (English)

    Jia Li; Audesh Bhat; Wei Xiao

    2011-01-01

    Nucleotide excision repair (NER) is the most versatile DNA-repair pathway in all organisms.While bacteria require only three proteins to complete the incision step of NER,eukaryotes employ about 30 proteins to complete the same step.Here we summarize recent studies demonstrating that ubiquitination,a post-translational modification,plays critical roles in regulating the NER activity either dependent on or independent of ubiquitin-proteolysis.Several NER components have been shown as targets of ubiquitination while others are actively involved in the ubiquitination process.We argue through this analysis that ubiquitination serves to coordinate various steps of NER and meanwhile connect NER with other related pathways to achieve the efficient global DNA-damage response.

  9. 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

  10. Mutation in E1, the ubiquitin activating enzyme, reduces Drosophila lifespan and results in motor impairment.

    Science.gov (United States)

    Liu, Hsiu-Yu; Pfleger, Cathie M

    2013-01-01

    Neurodegenerative diseases cause tremendous suffering for those afflicted and their families. Many of these diseases involve accumulation of mis-folded or aggregated proteins thought to play a causal role in disease pathology. Ubiquitinated proteins are often found in these protein aggregates, and the aggregates themselves have been shown to inhibit the activity of the proteasome. These and other alterations in the Ubiquitin Pathway observed in neurodegenerative diseases have led to the question of whether impairment of the Ubiquitin Pathway on its own can increase mortality or if ongoing neurodegeneration alters Ubiquitin Pathway function as a side-effect. To address the role of the Ubiquitin Pathway in vivo, we studied loss-of-function mutations in the Drosophila Ubiquitin Activating Enzyme, Uba1 or E1, the most upstream enzyme in the Ubiquitin Pathway. Loss of only one functional copy of E1 caused a significant reduction in adult lifespan. Rare homozygous hypomorphic E1 mutants reached adulthood. These mutants exhibited further reduced lifespan and showed inappropriate Ras activation in the brain. Removing just one functional copy of Ras restored the lifespan of heterozygous E1 mutants to that of wild-type flies and increased the survival of homozygous E1 mutants. E1 homozygous mutants also showed severe motor impairment. Our findings suggest that processes that impair the Ubiquitin Pathway are sufficient to cause early mortality. Reduced lifespan and motor impairment are seen in the human disease X-linked Infantile Spinal Muscular Atrophy, which is associated with mutation in human E1 warranting further analysis of these mutants as a potential animal model for study of this disease.

  11. The natural history of ubiquitin and ubiquitin-related domains.

    Science.gov (United States)

    Burroughs, Alexander Maxwell; Iyer, Lakshminarayan M; Aravind, L

    2012-01-01

    The ubiquitin (Ub) system is centered on conjugation and deconjugation of Ub and Ub-like (Ubls) proteins by a system of ligases and peptidases, respectively. Ub/Ubls contain the beta-grasp fold, also found in numerous proteins with biochemically distinct roles unrelated to the conventional Ub-system. The beta-GF underwent an early radiation spawning at least seven clades prior to the divergence of extant organisms from their last universal common ancestor, first emerging in the context of translation-related RNA-interactions and subsequently exploding to occupy various functional niches. Most beta-GF diversification occurred in prokaryotes, with the Ubl clade showing dramatic expansion in the eukaryotes. Diversification of Ubl families in eukaryotes played a major role in emergence of characteristic eukaryotic cellular sub-structures and systems. Recent comparative genomics studies indicate precursors of the eukaryotic Ub-system emerged in prokaryotes. The simplest of these combine an Ubl and an E1-like enzyme in metabolic pathways. Sampylation in archaea and Urmylation in eukaryotes appear to represent recruitment of such systems as simple protein-tagging apparatuses. However, other prokaryotic systems incorporated further components and mirror the eukaryotic condition in possessing an E2, a RING-type E3 or both of these components. Additionally, prokaryotes have evolved conjugation systems independent of Ub ligases, such as the Pup system.

  12. The natural history of ubiquitin and ubiquitin-related domains.

    Science.gov (United States)

    Burroughs, Alexander Maxwell; Iyer, Lakshminarayan M; Aravind, L

    2012-01-01

    The ubiquitin (Ub) system is centered on conjugation and deconjugation of Ub and Ub-like (Ubls) proteins by a system of ligases and peptidases, respectively. Ub/Ubls contain the beta-grasp fold, also found in numerous proteins with biochemically distinct roles unrelated to the conventional Ub-system. The beta-GF underwent an early radiation spawning at least seven clades prior to the divergence of extant organisms from their last universal common ancestor, first emerging in the context of translation-related RNA-interactions and subsequently exploding to occupy various functional niches. Most beta-GF diversification occurred in prokaryotes, with the Ubl clade showing dramatic expansion in the eukaryotes. Diversification of Ubl families in eukaryotes played a major role in emergence of characteristic eukaryotic cellular sub-structures and systems. Recent comparative genomics studies indicate precursors of the eukaryotic Ub-system emerged in prokaryotes. The simplest of these combine an Ubl and an E1-like enzyme in metabolic pathways. Sampylation in archaea and Urmylation in eukaryotes appear to represent recruitment of such systems as simple protein-tagging apparatuses. However, other prokaryotic systems incorporated further components and mirror the eukaryotic condition in possessing an E2, a RING-type E3 or both of these components. Additionally, prokaryotes have evolved conjugation systems independent of Ub ligases, such as the Pup system. PMID:22201813

  13. Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

    Energy Technology Data Exchange (ETDEWEB)

    Reidick, Christina [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany); El Magraoui, Fouzi; Meyer, Helmut E. [Biomedical Research, Human Brain Proteomics II, Leibniz-Institut für Analytische Wissenschaften-ISAS, Dortmund 44139 (Germany); Stenmark, Harald [Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Montebello, Oslo 0310 (Norway); Platta, Harald W., E-mail: harald.platta@rub.de [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany)

    2014-12-23

    The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept.

  14. Role of ubiquitination in meiotic recombination repair

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Programmed and unprogrammed double-strand breaks (DSBs) often arise from such physiological requirements as meiotic recombination, and exogenous insults, such as ionizing radiation (IR). Due to deleterious impacts on genome stability, DSBs must be appropriately processed and repaired in a regulatory manner. Recent investigations have indicated that ubiquitination is a critical factor in DNA damage response and meiotic recombination repair. This review summarizes the effects of proteins and complexes associated with ubiquitination with regard to homologous recombination (HR)-dependent DSB repair.

  15. Cdh1 inhibits WWP2-mediated ubiquitination of PTEN to suppress tumorigenesis in an APC-independent manner.

    Science.gov (United States)

    Liu, Jia; Wan, Lixin; Liu, Jing; Yuan, Zhu; Zhang, Jinfang; Guo, Jianfeng; Malumbres, Marcos; Liu, Jiankang; Zou, Weiguo; Wei, Wenyi

    2016-01-01

    Anaphase-promoting complex/cyclosome/Cdh1 is a multi-subunit ubiquitin E3 ligase that drives M to G1 cell cycle progression through primarily earmarking various substrates for ubiquitination and subsequent degradation by the 26S proteasome. Notably, emerging evidence suggested that Cdh1 could also function in various cellular processes independent of anaphase-promoting complex/cyclosome. To this end, we recently identified an anaphase-promoting complex/cyclosome-independent function of Cdh1 in modulating osteoblast differentiation through activating Smurf1, one of the NEDD4 family of HECT domain-containing E3 ligases. However, it remains largely unknown whether Cdh1 could exert its tumor suppressor role through similarly modulating the E3 ligase activities of other NEDD4 family members, most of which have characterized important roles in tumorigenesis. Here we report that in various tumor cells, Cdh1, conversely, suppresses the E3 ligase activity of WWP2, another NEDD4 family protein, in an anaphase-promoting complex/cyclosome-independent manner. As such, loss of Cdh1 activates WWP2, leading to reduced abundance of WWP2 substrates including PTEN, which subsequently activates PI3K/Akt oncogenic signaling to facilitate tumorigenesis. This study expands the non-anaphase-promoting complex/cyclosome function of Cdh1 in regulating the NEDD4 family E3 ligases, and further suggested that enhancing Cdh1 to inhibit the E3 ligase activity of WWP2 could be a promising strategy for treating human cancers.

  16. Análisis Financiero del Estudio de Factibilidad para la Siembra de 1990 Hectáreas de Caucho (Hevea brasiliensis

    Directory of Open Access Journals (Sweden)

    González González Humberto

    1995-09-01

    Full Text Available El análisis financiero del estudio de factibilidad para la siembra de 1990 hectáreas de caucho (Hevea Brasiliensis, presenta en los costos del cultivo claras complementariedades en las distintas actividades agronómicas, análogas al concepto de valor añadido. La construcción de los precios del látex no centrifugado se hizo mediante un método indirecto; los de la lámina y del látex centrifugado a partir de datos del mercado y del INCORA. La TIR y el VPN se calcularon para cuatro escenarios de ingresos y para dos modalidades: proyecto puro y proyecto financiado. También se determinó el precio del producto que hace el VPN igual a cero. Finalmente, el proyecto presenta claros beneficios así: 1 en la producción nacional; 2 para la reducción de importaciones; 3 en la generación de empleo en la zona; 4 en la conservación de los recursos agua y suelo y ; 5 para el adelanto de investigaciones sobre este cultivo.

  17. The recognition of ubiquitinated proteins by the proteasome.

    Science.gov (United States)

    Grice, Guinevere L; Nathan, James A

    2016-09-01

    The ability of ubiquitin to form up to eight different polyubiquitin chain linkages generates complexity within the ubiquitin proteasome system, and accounts for the diverse roles of ubiquitination within the cell. Understanding how each type of ubiquitin linkage is correctly interpreted by ubiquitin binding proteins provides important insights into the link between chain recognition and cellular fate. A major function of ubiquitination is to signal degradation of intracellular proteins by the 26S proteasome. Lysine-48 (K48) linked polyubiquitin chains are well established as the canonical signal for proteasomal degradation, but recent studies show a role for other ubiquitin linked chains in facilitating degradation by the 26S proteasome. Here, we review how different types of polyubiquitin linkage bind to ubiquitin receptors on the 26S proteasome, how they signal degradation and discuss the implications of ubiquitin chain linkage in regulating protein breakdown by the proteasome. PMID:27137187

  18. Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers

    DEFF Research Database (Denmark)

    Schwertman, Petra; Bekker-Jensen, Simon; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs...

  19. Ubiquitination of specific mitochondrial matrix proteins.

    Science.gov (United States)

    Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G; Ciechanover, Aaron

    2016-06-17

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems - at least partially - in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. PMID:27157140

  20. Dengue Virus Genome Uncoating Requires Ubiquitination

    Directory of Open Access Journals (Sweden)

    Laura A. Byk

    2016-06-01

    Full Text Available The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process.

  1. Cellular contractility requires ubiquitin mediated proteolysis.

    Directory of Open Access Journals (Sweden)

    Yuval Cinnamon

    Full Text Available BACKGROUND: Cellular contractility, essential for cell movement and proliferation, is regulated by microtubules, RhoA and actomyosin. The RhoA dependent kinase ROCK ensures the phosphorylation of the regulatory Myosin II Light Chain (MLC Ser19, thereby activating actomyosin contractions. Microtubules are upstream inhibitors of contractility and their depolymerization or depletion cause cells to contract by activating RhoA. How microtubule dynamics regulates RhoA remains, a major missing link in understanding contractility. PRINCIPAL FINDINGS: We observed that contractility is inhibited by microtubules not only, as previously reported, in adherent cells, but also in non-adhering interphase and mitotic cells. Strikingly we observed that contractility requires ubiquitin mediated proteolysis by a Cullin-RING ubiquitin ligase. Inhibition of proteolysis, ubiquitination and neddylation all led to complete cessation of contractility and considerably reduced MLC Ser19 phosphorylation. CONCLUSIONS: Our results imply that cells express a contractility inhibitor that is degraded by ubiquitin mediated proteolysis, either constitutively or in response to microtubule depolymerization. This degradation seems to depend on a Cullin-RING ubiquitin ligase and is required for cellular contractions.

  2. Dengue Virus Genome Uncoating Requires Ubiquitination

    Science.gov (United States)

    Byk, Laura A.; Iglesias, Néstor G.; De Maio, Federico A.; Gebhard, Leopoldo G.; Rossi, Mario

    2016-01-01

    ABSTRACT The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process. PMID:27353759

  3. Ubiquitination of specific mitochondrial matrix proteins.

    Science.gov (United States)

    Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G; Ciechanover, Aaron

    2016-06-17

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems - at least partially - in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins.

  4. Heat shock induced change in protein ubiquitination in Chlamydomonas

    International Nuclear Information System (INIS)

    Ubiquitin was purified from pea (Pisum sativum L.) and its antibody was produced. Western blot analysis showed that the antibody cross-reacted with ubiquitins from a green alga Chlamydomonas reinhardtii, a brown alga Laminaria angustata and a red alga Porphyridium cruentum but not with ubiquitin from a blue-green alga Synechococcus sp. In Chlamydomonas, the antibody also reacted with some ubiquitinated proteins including 28- and 31-kDa polypeptides. The isoelectric points of Chlamydomonas ubiquitin and the 28- and 31-kDa ubiquitinated proteins were 8.0, 8.9 and 10.3, respectively. The ubiquitinated proteins, including the 28- and 31-kDa polypeptides were detected after in vitro ATP-dependent ubiquitination of Chlamydomonas cell extract with l25I-labeled bovine ubiquitin. Heat treatment of Chlamydomonas cells (>40°C) caused drastic increase of ubiquitinated proteins with high mol wt (>60kDa), and coordinated redistribution or decrease of other ubiquitinated proteins and free ubiquitin. Quantitative analysis revealed that the 28- and 31-kDa ubiquitinated proteins showed different responses against heat stress, i.e. the former being more sensitive than the latter. (author)

  5. Estudio florístico - estructural de dos hectáreas de bosque de tierra firme en la Amazonía colombia (Parque Nacional Natural Amacayacu

    Directory of Open Access Journals (Sweden)

    Villegas Carolina

    2002-06-01

    Full Text Available La zona de estudio está ubicada al norte del Parque en el sector del río Cotuhé, extremo sur de la Amazonía colombiana. Donde se delimitaron dos levantamientos de 1 Ha en los cuales se tuvieron en cuenta los individuos con DAP≥10 cm y dos transectos de 0,1 Ha en los cuales
    se tuvieron en cuenta los individuos con DAP≥2,5 cm. Se midieron el DAP y la altura de todos los individuos; determinando la familia, género y especie de cada uno de ellos. Las familias más importantes en cuanto a número de especies se refiere fueron: Leguminosae, Annonaceae, Meliaceae, Moraceae, Euphorbiaceae, Sapotaceae, Myristicaceae, Lauraceae, Burseraceae y Chrysobalanaceae; las cuales hacen parte de las familias que más contribuyen en cantidad de
    especies en las tierras bajas del neotrópico. Se encontraron 255-257 especies/Ha (DAP≥10 cm y 196-199 especies/0,1 ha (DAP≥2,5 cm; altos valores de riqueza que confirman la hipótesis que los define cómo los bosques más ricos del mundo. El índice de dominancia presentó valores muy cercanos a cero; indicando que no hay predominio de ninguna especie en la comunidad debido a que la mayoría de las especies son raras; puesto que no hay ninguna especie que esté monopolizando los recursos. La curva de especies/área no se estabilizó teniendo en cuenta las dos hectáreas estudiadas y todas las especies encontradas, sin embargo, teniendo en cuenta sólo las especies que aparecieron más de 1 vez, la curva tendió a estabilizarse hacia las 1.5 hectáreas. Estructuralmente, en los levantamientos se encontró una distribución de alturas en forma de campana máxima hacia los 15 m; mientras que en los transectos al disminuir el rango mínimo del DAP a 2,5 cm, aumentó la importancia de los primeros estratos y disminuyó la importancia de los estratos medios y altos. El comportamiento del DAP tanto para los levantamientos como para los transectos fue similar ya que mostró la típica j invertida de los

  6. The Molecular Basis for Ubiquitin and Ubiquitin-like Specificities in Bacterial Effector Proteases.

    Science.gov (United States)

    Pruneda, Jonathan N; Durkin, Charlotte H; Geurink, Paul P; Ovaa, Huib; Santhanam, Balaji; Holden, David W; Komander, David

    2016-07-21

    Pathogenic bacteria rely on secreted effector proteins to manipulate host signaling pathways, often in creative ways. CE clan proteases, specific hydrolases for ubiquitin-like modifications (SUMO and NEDD8) in eukaryotes, reportedly serve as bacterial effector proteins with deSUMOylase, deubiquitinase, or, even, acetyltransferase activities. Here, we characterize bacterial CE protease activities, revealing K63-linkage-specific deubiquitinases in human pathogens, such as Salmonella, Escherichia, and Shigella, as well as ubiquitin/ubiquitin-like cross-reactive enzymes in Chlamydia, Rickettsia, and Xanthomonas. Five crystal structures, including ubiquitin/ubiquitin-like complexes, explain substrate specificities and redefine relationships across the CE clan. Importantly, this work identifies novel family members and provides key discoveries among previously reported effectors, such as the unexpected deubiquitinase activity in Xanthomonas XopD, contributed by an unstructured ubiquitin binding region. Furthermore, accessory domains regulate properties such as subcellular localization, as exemplified by a ubiquitin-binding domain in Salmonella Typhimurium SseL. Our work both highlights and explains the functional adaptations observed among diverse CE clan proteins. PMID:27425412

  7. Homocysteine thiolactone affects protein ubiquitination in yeast.

    Science.gov (United States)

    Bretes, Ewa; Zimny, Jarosław

    2013-01-01

    The formation of homocysteine thiolactone (HcyTl) from homocysteine occurs in all examined so far organisms including bacteria, yeast, and humans. Protein N-homocysteinylation at the ε-amino group of lysine is an adverse result of HcyTl accumulation. Since tagging of proteins by ubiquitination before their proteasomal degradation takes place at the same residue, we wondered how N-homocysteinylation may affect the ubiquitination of proteins. We used different yeast strains carrying mutations in genes involved in the homocysteine metabolism. We found positive correlation between the concentration of endogenous HcyTl and the concentration of ubiquitinated proteins. This suggests that N-homocysteinylation of proteins apparently does not preclude but rather promotes their decomposition. PMID:24051443

  8. Cytochrome P450 3A Conjugation to Ubiquitin in a Process Distinct from Classical Ubiquitination Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zangar, Richard C.(BATTELLE (PACIFIC NW LAB)); Kimzey, Amy L.(ASSOC WESTERN UNIVERSITY); Okita, Janice R.(Washington State University); Wunschel, David S.(BATTELLE (PACIFIC NW LAB)); Edwards, Robert J.(Imperial College School of Medical, Hammersmith Campus); Kim, Hyesook (Wayne State University); Okita, Richard T.(Washington State University)

    2001-12-01

    We characterize a novel microsome system that forms high-molecular-mass (HMM) CYP3A, CYP2E1, and ubiquitin conjugates, but does not alter CYP4A or most other microsomal proteins. The formation of the HMM bands was observed in hepatic microsomes isolated from rats treated 1 week or more with high doses (50 mg/kg/day) of nicardipine, clotrimazole, or pregnenolone 16alpha-carbonitrile, but not microsomes from control, dexamethasone-, nifedipine-, or diltiazem-treated rats. Extensive washing of the microsomes to remove loosely attached proteins or cytosolic contaminants did not prevent the conjugation reaction. In contrast to prototypical ubiquitination pathways, this reaction did not require addition of ubiquitin, ATP, Mg(2+), or cytosol. Addition of cytosol did result in the degradation of the HMM CYP3A bands in a process that was not blocked by proteasome inhibitors. Immunoprecipitated CYP3A contained HMM ubiquitin. Even so, mass spectrometric analysis of tryptic peptides indicated that the HMM CYP3A was in molar excess to ubiquitin, suggesting that the formation of the HMM CYP3A may have resulted from conjugation to itself or a diffuse pool of ubiquitinated proteins already present in the microsomes. Addition of CYP3A substrates inhibited the formation of the HMM CYP3A and the cytosol-dependent degradation of HMM CYP3A. These results suggest that after extended periods of elevated CYP3A expression, microsomal factors are induced that catalyze the formation of HMM CYP3A conjugates that contain ubiquitin. This conjugation reaction, however, seems to be distinct from the classical ubiquitination pathway but may be related to the substrate-dependent stabilization of CYP3A observed in vivo.

  9. 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.

  10. Ubiquitin-mediated response to microsporidia and virus infection in C. elegans.

    Directory of Open Access Journals (Sweden)

    Malina A Bakowski

    2014-06-01

    Full Text Available Microsporidia comprise a phylum of over 1400 species of obligate intracellular pathogens that can infect almost all animals, but little is known about the host response to these parasites. Here we use the whole-animal host C. elegans to show an in vivo role for ubiquitin-mediated response to the microsporidian species Nematocida parisii, as well to the Orsay virus, another natural intracellular pathogen of C. elegans. We analyze gene expression of C. elegans in response to N. parisii, and find that it is similar to response to viral infection. Notably, we find an upregulation of SCF ubiquitin ligase components, such as the cullin ortholog cul-6, which we show is important for ubiquitin targeting of N. parisii cells in the intestine. We show that ubiquitylation components, the proteasome, and the autophagy pathway are all important for defense against N. parisii infection. We also find that SCF ligase components like cul-6 promote defense against viral infection, where they have a more robust role than against N. parisii infection. This difference may be due to suppression of the host ubiquitylation system by N. parisii: when N. parisii is crippled by anti-microsporidia drugs, the host can more effectively target pathogen cells for ubiquitylation. Intriguingly, inhibition of the ubiquitin-proteasome system (UPS increases expression of infection-upregulated SCF ligase components, indicating that a trigger for transcriptional response to intracellular infection by N. parisii and virus may be perturbation of the UPS. Altogether, our results demonstrate an in vivo role for ubiquitin-mediated defense against microsporidian and viral infections in C. elegans.

  11. The role of ubiquitin-proteasome pathway in spermatogenesis.

    Science.gov (United States)

    Lianhua, Dong; Maoliang, Ran; Zhi, Li; Fuzhi, Peng; Bin, Chen

    2016-09-01

    Ubiquitin-proteasome pathway (UPP) is the main pathway of protein degradation in eukaryotic cells. The UPP plays very important roles in cell cycle progression, apoptosis, stress response and growth and development through regulating protein interaction, protein activity, protein localization and signal transduction. Previous studies have shown that the UPP is essential for regulating acrosome and tail biogenesis during spermatogenesis in human and animals. The dysregulation of UPP during spermatogenesis results in sperm deformity and reduced sperm motility and leads to reproductive system diseases such as oligospermatism, infertility and testicular tumors. In this review, we summarized the signal transduction and regulation mechanism of UPP in spermatogenesis, which may provide references for future studies. PMID:27644740

  12. Cooperativity of the SUMO and Ubiquitin Pathways in Genome Stability

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    Minghua Nie

    2016-02-01

    Full Text Available Covalent attachment of ubiquitin (Ub or SUMO to DNA repair proteins plays critical roles in maintaining genome stability. These structurally related polypeptides can be viewed as distinct road signs, with each being read by specific protein interaction motifs. Therefore, via their interactions with selective readers in the proteome, ubiquitin and SUMO can elicit distinct cellular responses, such as directing DNA lesions into different repair pathways. On the other hand, through the action of the SUMO-targeted ubiquitin ligase (STUbL family proteins, ubiquitin and SUMO can cooperate in the form of a hybrid signal. These mixed SUMO-ubiquitin chains recruit “effector” proteins such as the AAA+ ATPase Cdc48/p97-Ufd1-Npl4 complex that contain both ubiquitin and SUMO interaction motifs. This review will summarize recent key findings on collaborative and distinct roles that ubiquitin and SUMO play in orchestrating DNA damage responses.

  13. Advantages of Extracellular Ubiquitin in Modulation of Immune Responses.

    Science.gov (United States)

    Sujashvili, Rusudan

    2016-01-01

    T and B lymphocytes play a central role in protecting the human body from infectious pathogens but occasionally they can escape immune tolerance, become activated, and induce autoimmune diseases. All deregulated cellular processes are associated with improper functioning of the ubiquitin-proteasome system (UPS) in eukaryotic cells. The role of ubiquitin in regulation of immune responses and in autoimmune diseases is only beginning to emerge. Ubiquitin is found in intra- and extracellular fluids and is involved in regulation of numerous cellular processes. Extracellular ubiquitin ascribed a role in lymphocyte differentiation. It regulates differentiation and maturation of hematopoietic cell lines. Ubiquitination is involved in initiation, propagation, and termination of immune responses. Disrupted ubiquitination can lead to autoimmunity. Recent observations showed that it can suppress immune response and prevent inflammation. Exogenous ubiquitin may provide good potential as a new tool for targeted therapy for immune mediated disorders of various etiologies. PMID:27642236

  14. Perturbation of host ubiquitin systems by plant pathogen/pest effector proteins

    OpenAIRE

    Banfield, Mark J.

    2014-01-01

    Microbial pathogens and pests of animals and plants secrete effector proteins into host cells, altering cellular physiology to the benefit of the invading parasite. Research in the past decade has delivered significant new insights into the molecular mechanisms of how these effector proteins function, with a particular focus on modulation of host immunity-related pathways. One host system that has emerged as a common target of effectors is the ubiquitination system in which substrate proteins...

  15. Ubiquitin reference technique and its use in ubiquitin-lacking prokaryotes.

    Directory of Open Access Journals (Sweden)

    Konstantin Piatkov

    Full Text Available In a pulse-chase assay, the in vivo degradation of a protein is measured through a brief labeling of cells with, for example, a radioactive amino acid, followed by cessation of labeling and analysis of cell extracts prepared at different times afterward ("chase", using immunoprecipitation, electrophoresis and autoradiography of a labeled protein of interest. A conventional pulse-chase assay is fraught with sources of data scatter, as the efficacy of labeling and immunoprecipitation can vary, and sample volumes can vary as well. The ubiquitin reference technique (URT, introduced in 1996, addresses these problems. In eukaryotes, a DNA-encoded linear fusion of ubiquitin to another protein is cleaved by deubiquitylases at the ubiquitin-protein junction. A URT assay uses a fusion in which the ubiquitin moiety is located between a downstream polypeptide (test protein and an upstream polypeptide (a long-lived reference protein. The cotranslational cleavage of a URT fusion by deubiquitylases after the last residue of ubiquitin produces, at the initially equimolar ratio, a test protein with a desired N-terminal residue and a reference protein containing C-terminal ubiquitin moiety. In addition to being more accurate than pulse-chases without a reference, URT makes it possible to detect and measure the degradation of a test protein during the pulse (before the chase. Because prokaryotes, including Gram-negative bacteria such as, for example, Escherichia coli and Vibrio vulnificus, lack the ubiquitin system, the use of URT in such cells requires ectopic expression of a deubiquitylase. We describe designs and applications of plasmid vectors that coexpress, in bacteria, both a URT-type fusion and Ubp1, a deubiquitylase of the yeast Saccharomyces cerevisiae. This single-plasmid approach extends the accuracy-enhancing URT assay to studies of protein degradation in prokaryotes.

  16. Radiation inhibits proteasomes and increases ubiquitinated proteins

    International Nuclear Information System (INIS)

    Full text: Exposure of cells to ionizing radiation results in accumulation of a number of short lived proteins that mediate cell survival/death, proliferation, repair, and differentiation. Expression of most of these proteins, including p53, mdm2, p21, c-jun, IkB-a, bcl-2, bax, cyclins A, B, E, Cdc25A, DNA-PKcs, and caspase-3 is regulated at the post-transcriptional level through ubiquitin/26S proteasome pathway. Several previous studies have shown that inhibition of proteasome activity by drugs leads to accumulation of ubiquitinated proteins. In this study we show that irradiation can do the same due to its inhibitory effect on 26S, but not 20S, proteasome activity. Two prostate cancer cell lines, murine TRAMP-C1 and human PC3, were used to examine the effect of ionizing radiation on the catalytic activity of the 26S proteasome. Cells were irradiated with different doses ranging from 0.25 to 20 Gy and lysed at different time points after irradiation. Crude extracts of both cell lines showed a rapid 30-50% decrease in chymotryptic activity of the 26S proteasome, as measured by a fluorogenic assay. The same level of inhibition was observed if purified 26S proteasomes were themselves irradiated, indicating that radiation has direct effects on this multicatalytic enzyme complex. Neither direct irradiation of proteasomes or cells had effect on 20S catalytic activity, suggesting that radiation selectively acts on 26S structure. Next, we examined whether this partial inhibition had any effect on ability of 26S proteasome to efficiently remove ubiquitinated proteins. Cells were irradiated with 10Gy and lysed at different time points. Ubiquitinated proteins were precipitated and examined by Western blot. Levels of ubiquitinated conjugates slowly increased over time and peaked at 7h post-irradiation. Accumulation of ubiquitinated conjugates has been shown to lead to formation of protein aggregates which can induce cell death. It has also been shown that monoubiquitination

  17. The Ubiquitin System and Jasmonate Signaling

    Directory of Open Access Journals (Sweden)

    Astrid Nagels Durand

    2016-01-01

    Full Text Available The ubiquitin (Ub system is involved in most, if not all, biological processes in eukaryotes. The major specificity determinants of this system are the E3 ligases, which bind and ubiquitinate specific sets of proteins and are thereby responsible for target recruitment to the proteasome or other cellular processing machineries. The Ub system contributes to the regulation of the production, perception and signal transduction of plant hormones. Jasmonic acid (JA and its derivatives, known as jasmonates (JAs, act as signaling compounds regulating plant development and plant responses to various biotic and abiotic stress conditions. We provide here an overview of the current understanding of the Ub system involved in JA signaling.

  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. The increasing complexity of the ubiquitin code.

    Science.gov (United States)

    Yau, Richard; Rape, Michael

    2016-05-27

    Ubiquitylation is essential for signal transduction as well as cell division and differentiation in all eukaryotes. Substrate modifications range from a single ubiquitin molecule to complex polymeric chains, with different types of ubiquitylation often eliciting distinct outcomes. The recent identification of novel chain topologies has improved our understanding of how ubiquitylation establishes precise communication within cells. Here, we discuss how the increasing complexity of ubiquitylation is employed to ensure robust and faithful signal transduction in eukaryotic cells. PMID:27230526

  20. Degradation of Activated Protein Kinases by Ubiquitination

    OpenAIRE

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases.

  1. Sch9 regulates intracellular protein ubiquitination by controlling stress responses

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    Beibei Qie

    2015-08-01

    Full Text Available Protein ubiquitination and the subsequent degradation are important means by which aberrant proteins are removed from cells, a key requirement for long-term survival. In this study, we found that the overall level of ubiquitinated proteins dramatically decreased as yeast cell grew from log to stationary phase. Deletion of SCH9, a gene encoding a key protein kinase for longevity control, decreased the level of ubiquitinated proteins in log phase and this effect could be reversed by restoring Sch9 function. We demonstrate here that the decrease of ubiquitinated proteins in sch9Δ cells in log phase is not caused by changes in ubiquitin expression, proteasome activity, or autophagy, but by enhanced expression of stress response factors and a decreased level of oxidative stress. Our results revealed for the first time how Sch9 regulates the level of ubiquitinated proteins and provides new insight into how Sch9 controls longevity.

  2. Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin-3

    OpenAIRE

    Todi, Sokol V.; Winborn, Brett J; Scaglione, K Matthew; Blount, Jessica R.; Travis, Sue M.; Paulson, Henry L.

    2009-01-01

    Deubiquitinating enzymes (DUBs) control the ubiquitination status of proteins in various cellular pathways. Regulation of the activity of DUBs, which is critically important to cellular homoeostasis, can be achieved at the level of gene expression, protein complex formation, or degradation. Here, we report that ubiquitination also directly regulates the activity of a DUB, ataxin-3, a polyglutamine disease protein implicated in protein quality control pathways. Ubiquitination enhances ubiquiti...

  3. Promoters active in interphase are bookmarked during mitosis by ubiquitination

    OpenAIRE

    Arora, Mansi; Jie ZHANG; Heine, George F.; Ozer, Gulcin; Liu, Hui-Wen; Huang, Kun; Parvin, Jeffrey D.

    2012-01-01

    We analyzed modification of chromatin by ubiquitination in human cells and whether this mark changes through the cell cycle. HeLa cells were synchronized at different stages and regions of the genome with ubiquitinated chromatin were identified by affinity purification coupled with next-generation sequencing. During interphase, ubiquitin marked the chromatin on the transcribed regions of ∼70% of highly active genes and deposition of this mark was sensitive to transcriptional inhibition. Promo...

  4. N-Methylcysteine-Mediated Total Chemical Synthesis of Ubiquitin Thioester

    OpenAIRE

    Erlich, Lesly A.; Ajish Kumar, K. S.; Haj-Yahya, Mahmood; Dawson, Philip E.; Brik, Ashraf

    2010-01-01

    Ubiquitin thioester is a key intermediate in the ubiquitylation of proteins and is formed enzymatically through the activation of α-COOH of ubiquitin in an ATP dependent manner using the E1 enzyme. The current methods used for the preparation of ubiquitin thioester rely on either the enzymatic machinery or on expressed protein ligation technology. In this article, we report a new chemical strategy, combining native chemical ligation and N-methylcysteine containing peptides, to chemically prep...

  5. Ubiquitin at the crossroad of cell death and survival

    Institute of Scientific and Technical Information of China (English)

    Yu-Shan Chen; Xiao-Bo Qiu

    2013-01-01

    Ubiquitination is crucial for cellular processes, such as protein degradation, apoptosis, autophagy, and cell cycle progression. Dysregulation of the ubiquitination network accounts for the development of numerous diseases, including cancer. Thus, targeting ubiquitination is a promising strategy in cancer therapy. Both apoptosis and autophagy are involved in tumorigenesis and response to cancer therapy. Although both are categorized as types of celldeath, autophagy is general y considered to have protective functions, including protecting cells from apoptosis under certain cellular stress conditions. This review highlights recent advances in understanding the regulation of apoptosis and autophagy by ubiquitination.

  6. HUWE1 and TRIP12 collaborate in degradation of ubiquitin-fusion proteins and misframed ubiquitin.

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    Esben G Poulsen

    Full Text Available In eukaryotic cells an uncleavable ubiquitin moiety conjugated to the N-terminus of a protein signals the degradation of the fusion protein via the proteasome-dependent ubiquitin fusion degradation (UFD pathway. In yeast the molecular mechanism of the UFD pathway has been well characterized. Recently the human E3 ubiquitin-protein ligase TRIP12 was connected with the UFD pathway, but little is otherwise known about this system in mammalian cells. In the present work, we utilized high-throughput imaging on cells transfected with a targeted siRNA library to identify components involved in degradation of the UFD substrate Ub(G76V-YFP. The most significant hits from the screen were the E3 ubiquitin-protein ligase HUWE1, as well as PSMD7 and PSMD14 that encode proteasome subunits. Accordingly, knock down of HUWE1 led to an increase in the steady state level and a retarded degradation of the UFD substrate. Knock down of HUWE1 also led to a stabilization of the physiological UFD substrate UBB(+1. Precipitation experiments revealed that HUWE1 is associated with both the Ub(G76V-YFP substrate and the 26S proteasome, indicating that it functions late in the UFD pathway. Double knock down of HUWE1 and TRIP12 resulted in an additive stabilization of the substrate, suggesting that HUWE1 and TRIP12 function in parallel during UFD. However, even when both HUWE1 and TRIP12 are downregulated, ubiquitylation of the UFD substrate was still apparent, revealing functional redundancy between HUWE1, TRIP12 and yet other ubiquitin-protein ligases.

  7. Ubiquitination-dependent mechanisms regulate synaptic growth and function.

    Science.gov (United States)

    DiAntonio, A; Haghighi, A P; Portman, S L; Lee, J D; Amaranto, A M; Goodman, C S

    2001-07-26

    The covalent attachment of ubiquitin to cellular proteins is a powerful mechanism for controlling protein activity and localization. Ubiquitination is a reversible modification promoted by ubiquitin ligases and antagonized by deubiquitinating proteases. Ubiquitin-dependent mechanisms regulate many important processes including cell-cycle progression, apoptosis and transcriptional regulation. Here we show that ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila neuromuscular junction (NMJ). Neuronal overexpression of the deubiquitinating protease fat facets leads to a profound disruption of synaptic growth control; there is a large increase in the number of synaptic boutons, an elaboration of the synaptic branching pattern, and a disruption of synaptic function. Antagonizing the ubiquitination pathway in neurons by expression of the yeast deubiquitinating protease UBP2 (ref. 5) also produces synaptic overgrowth and dysfunction. Genetic interactions between fat facets and highwire, a negative regulator of synaptic growth that has structural homology to a family of ubiquitin ligases, suggest that synaptic development may be controlled by the balance between positive and negative regulators of ubiquitination.

  8. The Ubiquitin-Proteasome Pathway and Synaptic Plasticity

    Science.gov (United States)

    Hegde, Ashok N.

    2010-01-01

    Proteolysis by the ubiquitin-proteasome pathway (UPP) has emerged as a new molecular mechanism that controls wide-ranging functions in the nervous system, including fine-tuning of synaptic connections during development and synaptic plasticity in the adult organism. In the UPP, attachment of a small protein, ubiquitin, tags the substrates for…

  9. Lysine Ubiquitination and Acetylation of Human Cardiac 20S Proteasomes

    Science.gov (United States)

    Lau, Edward; Choi, Howard JH; Ng, Dominic CM; Meyer, David; Fang, Caiyun; Li, Haomin; Wang, Ding; Zelaya, Ivette M; Yates, John R; Lam, Maggie PY

    2016-01-01

    Purpose Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets poly-ubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. Experimental design Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. Results We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. Conclusion and clinical relevance This is the most comprehensive characterization of cardiac proteasome ubiquitination to-date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes. PMID:24957502

  10. COMMD1-mediated ubiquitination regulates CFTR trafficking.

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    Loïc Drévillon

    Full Text Available The CFTR (cystic fibrosis transmembrane conductance regulator protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.

  11. Activity Based Profiling of Deubiquitylating Enzymes and Inhibitors in Animal Tissues.

    Science.gov (United States)

    McLellan, Lauren; Forder, Cassie; Cranston, Aaron; Harrigan, Jeanine; Jacq, Xavier

    2016-01-01

    The attachment of ubiquitin or ubiquitin-like modifiers to proteins is an important signal for the regulation of a variety of biological processes including the targeting of substrates for degradation, receptor internalization, regulation of gene expression, and DNA repair. Posttranslational modification of proteins by ubiquitin controls many cellular processes, and aberrant ubiquitylation can contribute to cancer, immunopathologies, and neurodegeneration. Thus, deubiquitylating enzymes (DUBs) that remove ubiquitin from proteins have become attractive therapeutic targets. Monitoring the activity of DUBs in cells or in tissues is critical for understanding the biological function of DUBs in particular pathways and is essential for determining the physiological specificity and potency of small-molecule DUB inhibitors. Here, we describe a method for the homogenization of animal tissues and incubation of tissue lysates with ubiquitin-based activity probes to monitor DUB activity in mouse tissues and target engagement following treatment of animals with small-molecule DUB inhibitors. PMID:27613053

  12. Lipid raft-dependent FcepsilonRI ubiquitination regulates receptor endocytosis through the action of ubiquitin binding adaptors.

    Directory of Open Access Journals (Sweden)

    Rosa Molfetta

    Full Text Available The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcepsilonRI expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcepsilonRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcepsilonRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.

  13. PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination.

    Directory of Open Access Journals (Sweden)

    Susana P Barrera

    Full Text Available Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1. Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40-50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications.

  14. Mechanistic studies on activation of ubiquitin and di-ubiquitin-like protein, FAT10, by ubiquitin-like modifier activating enzyme 6, Uba6.

    Science.gov (United States)

    Gavin, James M; Chen, Jesse J; Liao, Hua; Rollins, Neil; Yang, Xiaofeng; Xu, Qing; Ma, Jingya; Loke, Huay-Keng; Lingaraj, Trupti; Brownell, James E; Mallender, William D; Gould, Alexandra E; Amidon, Benjamin S; Dick, Lawrence R

    2012-05-01

    Uba6 is a homolog of the ubiquitin-activating enzyme, Uba1, and activates two ubiquitin-like proteins (UBLs), ubiquitin and FAT10. In this study, biochemical and biophysical experiments were performed to understand the mechanisms of how Uba6 recognizes two distinct UBLs and catalyzes their activation and transfer. Uba6 is shown to undergo a three-step activation process and form a ternary complex with both UBLs, similar to what has been observed for Uba1. The catalytic mechanism of Uba6 is further supported by inhibition studies using a mechanism-based E1 inhibitor, Compound 1, which forms covalent adducts with both ubiquitin and FAT10. In addition, pre-steady state kinetic analysis revealed that the rates of UBL-adenylate (step 1) and thioester (step 2) formation are similar between ubiquitin and FAT10. However, distinct kinetic behaviors were also observed for ubiquitin and FAT10. FAT10 binds Uba6 with much higher affinity than ubiquitin while demonstrating lower catalytic activity in both ATP-PP(i) exchange and E1-E2 transthiolation assays. Also, Compound 1 is less potent with FAT10 as the UBL compared with ubiquitin in ATP-PP(i) exchange assays, and both a slow rate of covalent adduct formation and weak adduct binding to Uba6 contribute to the diminished potency observed for FAT10. Together with expression level analysis in IM-9 cells, this study sheds light on the potential role of cytokine-induced FAT10 expression in regulating Uba6 pathways.

  15. Characterization of the Ubiquitin E2 Enzyme Variant Gene Family in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Yu Zhang; Pei Wen; On-Sun Lau; Xing-Wang Deng

    2007-01-01

    Ubiquitin E2 enzyme variant (UEV) proteins are similar to ubiquitin-conjugating enzyme (E2) in both sequence and structure, but the lack of a catalytic cysteine residue renders them incapable of forming a thiolester linkage with ubiquitin. While the functional roles of several UEVs have been defined in yeast and animal systems, Arabidopsis COP10, a photomorphogenesis repressor, is the only UEV characterized in plants. Phylogenetic analysis revealed that the eight Arabidopsis UEV genes belong to three subfamilies.The expression of those genes is supported by either the presence of ESTs or RT-PCR analysis. We also characterized the other members of the COP10 subfamily, UEV2. Semi-quantitative RT-PCR analysis indicated that the UEV2 transcripts can be detected in most organs of Arabidopsis. Analysis of UEV2::GUS transgenic lines also showed its ubiquitous expression in nearly all the developmental stages of Arabidopsis.Transient expression analysis indicated that the sGFP-UEV2 fusion protein can localize to both the cytoplasm and nucleus. A T-DNA insertion mutant, uev2-1, which abolished the transcription of UEV2, displays no visible phenotype. Further, the cop10-4 uev2-1 double mutant exhibits the same phenotype as the cop10-4mutant in darkness. UEV2 is therefore not functionally redundant with COP10.

  16. The ubiquitin proteasome system and efficacy of proteasome inhibitors in diseases.

    Science.gov (United States)

    Chitra, Selvarajan; Nalini, Ganesan; Rajasekhar, Gopalakrishnan

    2012-06-01

    In eukaryotes the ubiquitin proteasome pathway plays an important role in cellular homeostasis and also it exerts a critical role in regulating a wide variety of cellular pathways, including cell growth and proliferation, apoptosis, DNA repair, transcription and immune response. Defects in these pathways have been implicated in a number of human pathologies. Inhibition of the ubiquitin proteasome pathway by proteasome inhibitors may be a rational therapeutic approach for various diseases, such as cancer and inflammatory diseases. Many of the critical cytokine and chemokine mediators of the progression of rheumatoid arthritis are regulated by nuclear factor kappa B (NF-κB). In peptidoglycan/polysaccharide-induced polyarthritis, proteasome inhibitors limit the overall inflammation, reduce NF-κB activation, decrease cellular adhesion molecule expression, inhibit nitric oxide synthase, attenuate circulating levels of proinflammatory cytokine interleukin-6 and reduce the arthritis index and swelling in the joints of the animals. Since proteasome inhibitors exhibit anti-inflammatory and anti proliferative effects, diseases characterized by both of these processes such as rheumatoid arthritis might also represent clinical opportunities for such drugs. The regulation of the proteasomal complex by proteasome inhibitors also has implications and potential benefits for the treatment of rheumatoid arthritis. This review summarizes the ubiquitin proteasome pathway, the structure of 26S proteasomes and types of proteasome inhibitors, with their actions, and clinical applications of proteasome inhibitors in various diseases. PMID:22709487

  17. Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition.

    Science.gov (United States)

    Matsumoto, Hotaru; Saitoh, Hisato

    2016-07-29

    We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3. As removal of the drugs from the medium led to disappearance of the SUMO-2/3-ubiquitin nuclear foci, our findings indicated that nuclear assembly/disassembly of SUMO-2/3 and ubiquitin was pharmacologically manipulable, supporting our previous observation on OP-Puro, which predicted the ubiquitin family function in sequestrating aberrant proteins to the nucleus. PMID:27181354

  18. A cotranslational ubiquitination pathway for quality control of misfolded proteins.

    Science.gov (United States)

    Wang, Feng; Durfee, Larissa A; Huibregtse, Jon M

    2013-05-01

    Previous studies have indicated that 6%-30% of newly synthesized proteins are rapidly degraded by the ubiquitin-proteasome system; however, the relationship of ubiquitination to translation for these proteins has been unclear. We report that cotranslational ubiquitination (CTU) is a robust process, with 12%-15% of nascent polypeptides being ubiquitinated in human cells. CTU products contained primarily K48-linked polyubiquitin chains, consistent with a proteasomal targeting function. While nascent chains have been shown previously to be ubiquitinated within stalled complexes (CTU(S)), the majority of nascent chain ubiquitination occurred within active translation complexes (CTU(A)). CTU(A) was increased in response to agents that induce protein misfolding, while CTU(S) was increased in response to agents that lead to translational errors or stalling. These results indicate that ubiquitination of nascent polypeptides occurs in two contexts and define CTU(A) as a component of a quality control system that marks proteins for destruction while they are being synthesized. PMID:23583076

  19. Mcl-1 Ubiquitination: Unique Regulation of an Essential Survival Protein

    Directory of Open Access Journals (Sweden)

    Barbara Mojsa

    2014-05-01

    Full Text Available Mcl-1 is an anti-apoptotic protein of the Bcl-2 family that is essential for the survival of multiple cell lineages and that is highly amplified in human cancer. Under physiological conditions, Mcl-1 expression is tightly regulated at multiple levels, involving transcriptional, post-transcriptional and post-translational processes. Ubiquitination of Mcl-1, that targets it for proteasomal degradation, allows for rapid elimination of the protein and triggering of cell death, in response to various cellular events. In the last decade, a number of studies have elucidated different pathways controlling Mcl-1 ubiquitination and degradation. Four different E3 ubiquitin-ligases (e.g., Mule, SCFβ-TrCP, SCFFbw7 and Trim17 and one deubiquitinase (e.g., USP9X, that respectively mediate and oppose Mcl-1 ubiquitination, have been formerly identified. The interaction between Mule and Mcl-1 can be modulated by other Bcl-2 family proteins, while recognition of Mcl-1 by the other E3 ubiquitin-ligases and deubiquitinase is influenced by phosphorylation of specific residues in Mcl-1. The protein kinases and E3 ubiquitin-ligases that are involved in the regulation of Mcl-1 stability vary depending on the cellular context, highlighting the complexity and pivotal role of Mcl-1 regulation. In this review, we attempt to recapitulate progress in understanding Mcl-1 regulation by the ubiquitin-proteasome system.

  20. Enhanced detection of ubiquitin isopeptides using reductive methylation.

    Science.gov (United States)

    Chicooree, Navin; Connolly, Yvonne; Tan, Chong-Teik; Malliri, Angeliki; Li, Yaoyong; Smith, Duncan L; Griffiths, John R

    2013-03-01

    Identification of ubiquitination (Ub) sites is of great interest due to the critical roles that the modification plays in cellular regulation. Current methods using mass spectrometry rely upon tryptic isopeptide diglycine tag generation followed by database searching. We present a novel approach to ubiquitin detection based upon the dimethyl labeling of isopeptide N-termini glycines. Ubiquitinated proteins were digested with trypsin and the resulting peptide mixture was derivatized using formaldehyde-D2 solution and sodium cyanoborohydride. The dimethylated peptide mixtures were next separated by liquid chromatography and analyzed on a quadrupole-TOF based mass spectrometer. Diagnostic b2' and a1' ions released from the isopeptide N-terminus upon collision-induced dissociation (CID) were used to spectrally improve the identification of ubiquitinated isopeptides. Proof of principle was established by application to a ubiquitinated protein tryptic digest spiked into a six-protein mix digest background. Extracted ion chromatograms of the a1' and b2' diagnostic product ions from the diglycine tag resulted in a significant reduction in signal complexity and demonstrated a selectivity towards the identification of diglycine branched isopeptides. The method was further shown to be capable of identifying diglycine isopeptides resulting from in-gel tryptic digests of ubiquitin enriched material from a His-Ub transfected cell line. We envisage that these ions may be utilized in global ubiquitination studies with post-acquisition MS/MS (or MSe) data interrogation on high resolution hybrid mass spectrometers. ᅟ PMID:23361369

  1. Water Evaporation and Conformational Changes from Partially Solvated Ubiquitin

    Directory of Open Access Journals (Sweden)

    Saravana Prakash Thirumuruganandham

    2010-01-01

    Full Text Available Using molecular dynamics simulation, we study the evaporation of water molecules off partially solvated ubiquitin. The evaporation and cooling rates are determined for a molecule at the initial temperature of 300 K. The cooling rate is found to be around 3 K/ns, and decreases with water temperature in the course of the evaporation. The conformation changes are monitored by studying a variety of intermediate partially solvated ubiquitin structures. We find that ubiquitin shrinks with decreasing hydration shell and exposes more of its hydrophilic surface area to the surrounding.

  2. Genome-Wide Survey of Gut Fungi (Harpellales) Reveals the First Horizontally Transferred Ubiquitin Gene from a Mosquito Host

    Science.gov (United States)

    Wang, Yan; White, Merlin M.; Kvist, Sebastian; Moncalvo, Jean-Marc

    2016-01-01

    Harpellales, an early-diverging fungal lineage, is associated with the digestive tracts of aquatic arthropod hosts. Concurrent with the production and annotation of the first four Harpellales genomes, we discovered that Zancudomyces culisetae, one of the most widely distributed Harpellales species, encodes an insect-like polyubiquitin chain. Ubiquitin and ubiquitin-like proteins are universally involved in protein degradation and regulation of immune response in eukaryotic organisms. Phylogenetic analyses inferred that this polyubiquitin variant has a mosquito origin. In addition, its amino acid composition, animal-like secondary structure, as well as the fungal nature of flanking genes all further support this as a horizontal gene transfer event. The single-copy polyubiquitin gene from Z. culisetae has lower GC ratio compared with homologs of insect taxa, which implies homogenization of the gene since its putatively ancient transfer. The acquired polyubiquitin gene may have served to improve important functions within Z. culisetae, by perhaps exploiting the insect hosts’ ubiquitin-proteasome systems in the gut environment. Preliminary comparisons among the four Harpellales genomes highlight the reduced genome size of Z. culisetae, which corroborates its distinguishable symbiotic lifestyle. This is the first record of a horizontally transferred ubiquitin gene from disease-bearing insects to the gut-dwelling fungal endobiont and should invite further exploration in an evolutionary context. PMID:27343289

  3. A role for PCNA ubiquitination in immunoglobulin hypermutation.

    Directory of Open Access Journals (Sweden)

    Hiroshi Arakawa

    2006-11-01

    Full Text Available Proliferating cell nuclear antigen (PCNA is a DNA polymerase cofactor and regulator of replication-linked functions. Upon DNA damage, yeast and vertebrate PCNA is modified at the conserved lysine K164 by ubiquitin, which mediates error-prone replication across lesions via translesion polymerases. We investigated the role of PCNA ubiquitination in variants of the DT40 B cell line that are mutant in K164 of PCNA or in Rad18, which is involved in PCNA ubiquitination. Remarkably, the PCNA(K164R mutation not only renders cells sensitive to DNA-damaging agents, but also strongly reduces activation induced deaminase-dependent single-nucleotide substitutions in the immunoglobulin light-chain locus. This is the first evidence, to our knowledge, that vertebrates exploit the PCNA-ubiquitin pathway for immunoglobulin hypermutation, most likely through the recruitment of error-prone DNA polymerases.

  4. Substrate specificity of the ubiquitin and Ubl proteases

    Science.gov (United States)

    Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

    2016-01-01

    Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

  5. Ubiquitin Metabolism Affects Cellular Response to Volatile Anesthetics in Yeast

    OpenAIRE

    Wolfe, Darren; Reiner, Thomas; Keeley, Jessica L.; Pizzini, Mark; Keil, Ralph L.

    1999-01-01

    To investigate the mechanism of action of volatile anesthetics, we are studying mutants of the yeast Saccharomyces cerevisiae that have altered sensitivity to isoflurane, a widely used clinical anesthetic. Several lines of evidence from these studies implicate a role for ubiquitin metabolism in cellular response to volatile anesthetics: (i) mutations in the ZZZ1 gene render cells resistant to isoflurane, and the ZZZ1 gene is identical to BUL1 (binds ubiquitin ligase), which appears to be invo...

  6. Reconstitution of CHIP E3 Ubiquitin Ligase Activity

    OpenAIRE

    Ren, Hong Yu; Patterson, Cam; Cyr, Douglas M.; Rosser, Meredith F. N.

    2011-01-01

    CHIP, the carboxyl-terminus of Hsp70 interacting protein, is both an E3 ubiquitin ligase and an Hsp70 co-chaperone and is implicated in the degradation of cytosolic quality control and numerous disease substrates. CHIP has been shown to monitor the folding status of the CFTR protein, and we have successfully reconstituted this activity using a recombinant CFTR fragment consisting of the cytosolic NBD1 and R domains. We have found that efficient ubiquitination of substrates requires chaperone ...

  7. Detection of ubiquitinated huntingtin species in intracellular aggregates

    Directory of Open Access Journals (Sweden)

    Katrin eJuenemann

    2015-01-01

    Full Text Available Protein conformation diseases, including polyglutamine diseases, result from the accumulation and aggregation of misfolded proteins. Huntington’s disease is one of nine diseases caused by an expanded polyglutamine repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated huntingtin such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated huntingtin fragments offers an important possibility to understand huntingtin degradation and aggregation processes within the cell. For the identification of aggregated huntingtin and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant huntingtin. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

  8. In silico elucidation of the recognition dynamics of ubiquitin.

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    Dong Long

    2011-04-01

    Full Text Available Elucidation of the mechanism of biomacromolecular recognition events has been a topic of intense interest over the past century. The inherent dynamic nature of both protein and ligand molecules along with the continuous reshaping of the energy landscape during the binding process renders it difficult to characterize this process at atomic detail. Here, we investigate the recognition dynamics of ubiquitin via microsecond all-atom molecular dynamics simulation providing both thermodynamic and kinetic information. The high-level of consistency found with respect to experimental NMR data lends support to the accuracy of the in silico representation of the conformational substates and their interconversions of free ubiquitin. Using an energy-based reweighting approach, the statistical distribution of conformational states of ubiquitin is monitored as a function of the distance between ubiquitin and its binding partner Hrs-UIM. It is found that extensive and dense sampling of conformational space afforded by the µs MD trajectory is essential for the elucidation of the binding mechanism as is Boltzmann sampling, overcoming inherent limitations of sparsely sampled empirical ensembles. The results reveal a population redistribution mechanism that takes effect when the ligand is at intermediate range of 1-2 nm from ubiquitin. This mechanism, which may be depicted as a superposition of the conformational selection and induced fit mechanisms, also applies to other binding partners of ubiquitin, such as the GGA3 GAT domain.

  9. Resistance to UV-induced apoptosis by β-HPV5 E6 involves targeting of activated BAK for proteolysis by recruitment of the HERC1 ubiquitin ligase.

    Science.gov (United States)

    Holloway, Amy; Simmonds, Mark; Azad, Abul; Fox, Joanna L; Storey, Alan

    2015-06-15

    UV exposure is the main etiological agent in the development of non-melanoma skin cancer (NMSC), but mounting evidence suggests a co-factorial role for β-genus HPV types early in tumor initiation or progression. UV damage initiates an apoptotic response, driven at the mitochondrial level by BCL-2 family proteins, that eliminates damaged cells that may accumulate deleterious mutations and acquire tumorigenic properties. BAK is a pro-apoptotic BCL-2 protein that functions ultimately to form pores that permeabilize the mitochondrial outer membrane, thereby committing a cell to death, a process involving changes in BAK phosphorylation and conformation. The E6 protein of β-type HPV5 signals BAK for proteasomal degradation, a function that confers protection from UV-induced apoptosis. We find that HPV5 E6 does not constitutively target BAK for proteolysis, but targets the latter stages of BAK activation, following changes in phosphorylation and conformation. A mutational analysis identified the lysine residue on BAK required for proteolysis, and a functional siRNA screen identified the HECT domain E3 ubiquitin ligase HERC1 as being required for E6-mediated BAK degradation. We show that HERC1 interacts with BAK in E6-expressing cells that have been damaged by UV, and provide evidence that the interaction of HERC1 with BAK requires access to a hydrophobic surface on BAK that binds BH3 domains of BCL-2 proteins. We also show that HERC1 contains a putative BH3 domain that can bind to BAK. These findings reveal a specific and unique mechanism used by the HPV5 E6 protein to target BAK.

  10. Protein Camouflage: Supramolecular Anion Recognition by Ubiquitin.

    Science.gov (United States)

    Mallon, Madeleine; Dutt, Som; Schrader, Thomas; Crowley, Peter B

    2016-04-15

    Progress in the field of bio-supramolecular chemistry, the bottom-up assembly of protein-ligand systems, relies on a detailed knowledge of molecular recognition. To address this issue, we have characterised complex formation between human ubiquitin (HUb) and four supramolecular anions. The ligands were: pyrenetetrasulfonic acid (4PSA), p-sulfonato-calix[4]arene (SCLX4), bisphosphate tweezers (CLR01) and meso-tetrakis (4-sulfonatophenyl)porphyrin (TPPS), which vary in net charge, size, shape and hydrophobicity. All four ligands induced significant changes in the HSQC spectrum of HUb. Chemical shift perturbations and line-broadening effects were used to identify binding sites and to quantify affinities. Supporting data were obtained from docking simulations. It was found that these weakly interacting ligands bind to extensive surface patches on HUb. A comparison of the data suggests some general indicators for the protein-binding specificity of supramolecular anions. Differences in binding were observed between the cavity-containing and planar ligands. The former had a preference for the arginine-rich, flexible C terminus of HUb. PMID:26818656

  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. Fluorescent Tools for In Vivo Studies on the Ubiquitin-Proteasome System.

    Science.gov (United States)

    Matilainen, Olli; Jha, Sweta; Holmberg, Carina I

    2016-01-01

    The ubiquitin-proteasome system (UPS) plays a key role in maintaining proteostasis by degrading most of the cellular proteins. Traditionally, UPS activity is studied in vitro, in yeast, or in mammalian cell cultures by using short-lived GFP-based UPS reporters. Here, we present protocols for two fluorescent tools facilitating real-time imaging of UPS activity in living animals. We have generated transgenic Caenorhabditis elegans (C. elegans) expressing a photoconvertible UbG76V-Dendra2 UPS reporter, which permits measurement of reporter degradation by the proteasome independently of reporter protein synthesis, and a fluorescent polyubiquitin-binding reporter for detection of the endogenous pool of Lys48-linked polyubiquitinated proteasomal substrates. These reporter systems facilitate cell- and tissue-specific analysis of UPS activity especially in young adult animals, but can also be used for studies during development, aging, and for example stress conditions. PMID:27613038

  13. UHRF2, another E3 ubiquitin ligase for p53

    Energy Technology Data Exchange (ETDEWEB)

    Bai, Lu; Wang, Xiaohui; Jin, Fangmin; Yang, Yan; Qian, Guanhua [Department of Cell Biology and Medical Genetics, Chongqing Medical University, Chongqing (China); Duan, Changzhu, E-mail: duanchzhu@cqmu.edu.cn [Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education, Faculty of Laboratory Medicine, Chongqing Medical University, Chongqing (China); Department of Cell Biology and Medical Genetics, Chongqing Medical University, Chongqing (China)

    2012-09-07

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

  14. Ubiquitin and Autophagy%泛素与自噬

    Institute of Scientific and Technical Information of China (English)

    冯梅; 王莉新; 王易

    2011-01-01

    Protein degradation mediated by ubiquitin and autophagy are the basic mechanisms involved in cellular self-regulation. Ubiquitin may be involved in the process of autophagy by serving as a umversal recognition signal. Induction of autophagy can promote ubiquitination, thereby enhancing the degradation of substrate. This paper mainly focuses on the relation and the potential mutual regulation between ubiquitination and autophagy, as well as the phenomenon of programmed cell death that is associated with both ubiquitination and autophagy processes.%泛素调节的蛋白质降解过程和细胞的自噬现象都是细胞自我调节的基本机制.其中,泛素可能作为一种普遍的识别信号参与了自噬过程;而自噬的诱导又能促进泛素化作用,从而增强对底物的降解.本文着重探讨这两者间的关系及可能存在的相互调节作用,并兼及两者共同涉及的细胞程序性死亡现象.

  15. Roles of protein ubiquitination and degradation kinetics in biological oscillations.

    Directory of Open Access Journals (Sweden)

    Lida Xu

    Full Text Available Protein ubiquitination and degradation play important roles in many biological functions and are associated with many human diseases. It is well known that for biochemical oscillations to occur, proper degradation rates of the participating proteins are needed. In most mathematical models of biochemical reactions, linear degradation kinetics has been used. However, the degradation kinetics in real systems may be nonlinear, and how nonlinear degradation kinetics affects biological oscillations are not well understood. In this study, we first develop a biochemical reaction model of protein ubiquitination and degradation and calculate the degradation rate against the concentration of the free substrate. We show that the protein degradation kinetics mainly follows the Michaelis-Menten formulation with a time delay caused by ubiquitination and deubiquitination. We then study analytically how the Michaelis-Menten degradation kinetics affects the instabilities that lead to oscillations using three generic oscillation models: 1 a positive feedback mediated oscillator; 2 a positive-plus-negative feedback mediated oscillator; and 3 a negative feedback mediated oscillator. In all three cases, nonlinear degradation kinetics promotes oscillations, especially for the negative feedback mediated oscillator, resulting in much larger oscillation amplitudes and slower frequencies than those observed with linear kinetics. However, the time delay due to protein ubiquitination and deubiquitination generally suppresses oscillations, reducing the amplitude and increasing the frequency of the oscillations. These theoretical analyses provide mechanistic insights into the effects of specific proteins in the ubiquitination-proteasome system on biological oscillations.

  16. The Challenge of Producing Ubiquitinated Proteins for Structural Studies

    Directory of Open Access Journals (Sweden)

    Serena Faggiano

    2014-06-01

    Full Text Available Protein ubiquitination is an important post-translational modification involved in several essential signalling pathways. It has different effects on the target protein substrate, i.e., it can trigger the degradation of the protein in the proteasome, change the interactions of the modified protein with its partners, or affect its localization and activity. In order to understand the molecular mechanisms underlying the consequences of protein ubiquitination, scientists have to face the challenging task of producing ubiquitinated proteins for structural characterization with X-ray crystallography and/or nuclear magnetic resonance (NMR spectroscopy. These techniques require milligrams of homogeneous samples of high purity. The strategies proposed so far for the production of ubiquitinated proteins can be divided into two groups, i.e., chemical (or non-enzymatic and enzymatic methodologies. In this review, we summarize the still very sparse examples available in the literature that describe successful production of ubiquitinated proteins amenable for biochemical and structural studies, and discuss advantages and disadvantages of the techniques proposed. We also give a perspective of the direction in which the field might evolve.

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

    Science.gov (United States)

    Wang, Julia; Jennings, Alexandra K; Kowalski, Jennifer R

    2016-01-01

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

  18. Dynamics of ubiquitin-mediated signalling: insights from mathematical modelling and experimental studies.

    Science.gov (United States)

    Nguyen, Lan K

    2016-05-01

    Post-translational modification of cellular proteins by ubiquitin is a pivotal regulatory event that controls not only protein degradation, but also a variety of non-proteolytic functions. Ubiquitination is involved in a broad array of physiological processes, and its dysregulation has been associated with many human diseases, including neuronal disorders and cancers. Ubiquitin-mediated signalling has thus come to the forefront of biomedical research. It is increasingly apparent that ubiquitination is a highly complex and dynamic process, evidenced by a myriad of ways of ubiquitin chain formation, tightly regulatory mechanisms involving E3 ligases and deubiquitinating enzymes and extensive crosstalk with other post-translational modifications. To unravel the complexity of ubiquitination and understand the dynamic properties of ubiquitin-mediated signalling are challenging, but critical topics in ubiquitin research, which will undoubtedly benefit our effort in developing strategies that could target ubiquitin signalling for therapeutics. Computational modelling and model-based approaches are emerging as promising tools that help tackle the complexity and provide useful frameworks for quantitative and dynamical analysis of ubiquitin signalling. In this article, I will discuss recent advances in our understanding of the dynamic behaviour of ubiquitination from both theoretical and experimental studies, and aspects of ubiquitin signalling that may have major dynamical consequences. It is expected the discussed issues will be of relevant interest to both the ubiquitin and systems biology fields.

  19. The Role of Ubiquitine Proteasome Pathway in Carcinogenesis

    Directory of Open Access Journals (Sweden)

    N.Ceren Sumer Turanligil

    2010-02-01

    Full Text Available Ubiquitin works as a marker protein which targets misfolded or injured proteins to cellular degradation. It brings the abnormal proteins to a subcellular organelle named proteasome and it maintains the degradation of proteins in limited lenghts of peptides by leaving the process withuout being changed. Mistakes in ubiquitin-dependent proteolysis in various steps of carcinogenesis is known. In this review, we dealed with the effects of ubiquitin-proteasome pathway (UPP on carcinogenesis via intercellular signaling molecules like Ras, transcription factors like NF-kB, cytokines like TNF-alfa Tumor necrosis factor, protooncogenes like p53 and MDM2(murine double minute 2, components of cell cycle and DNA repair proteins like BRCA1. We also focused on the relationship of UPP on antigen presentation which is active in immune response and its place in the aetiology of colon cancer to provide a specific example. [Archives Medical Review Journal 2010; 19(1.000: 36-55

  20. Alimento animal y energía de la caña de azúcar

    Directory of Open Access Journals (Sweden)

    José Villar

    2010-01-01

    Full Text Available La situación actual, caracterizada por una profunda crisis económica, en la que no obstante se mantienen relativamente altos los precios de los combustibles, los cereales empleados en la alimentación animal, los fertilizantes, plaguicidas y herbicidas necesarios para la producción nacional de granos, y la existencia de una agroindustria azucarera, con potencialidad para proporcionar energía y productos para la alimentación animal, son el fundamento de este trabajo. Se propone la producción de meladura invertida y bagazo hidrolizado, conjuntamente con la de azúcar y energía eléctrica, que se compara positivamente con la variante tradicional de producir azúcar y melaza. Se determina que una hectárea de caña procesada por la variante propuesta, cuando se emplea la meladura para la alimentación de cerdos, produce un valor 53% mayor que una hectárea de maíz utilizada con el mismo propósito.

  1. In silico analysis of ubiquitin/ubiquitin-like modifiers and their conjugating enzymes in Entamoeba species.

    Science.gov (United States)

    Arya, Shweta; Sharma, Gaurav; Gupta, Preeti; Tiwari, Swati

    2012-07-01

    Covalent modification of proteins by ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) regulates many cellular functions in eukaryotes. These modifications are likely to be associated with pathogenesis, growth, and development of many protozoan parasites but molecular details about this pathway are unavailable for most protozoa. This study presents an analysis of the Ub pathway in three members of the Entamoeba species. Using bioinformatics tools we have identified all Ub and Ubl genes along with their corresponding activating, conjugating, and ligating enzymes (E1, E2s, and E3s) in three Entamoeba species, Entamoeba histolytica, Entamoeba dispar, and Entamoeba invadens. Phylogenetic trees were established for the identified E2s and RING finger E3s using maximum-likelihood method to infer the relationship among these proteins. In silico co-domain analysis of RING finger E3s implicates these proteins in a variety of functions. Several known and putative regulatory motifs were identified in the upstream regions of RING finger domain containing E3 genes. All E2 and E3 genes were analyzed in genomic context in E. histolytica and E. dispar. Most E2s and E3s were in syntenic positions in the two genomes. Association of these genes with transposable elements (TEs) was compared between E. histolytica and E. dispar. A closer association was found between RING finger E3s with TEs in E. histolytica. In summary, our analyses suggests that the complexity of the Ub pathway in Entamoeba species is close to that observed in higher eukaryotes. This study provides important data for further understanding the role of Ub pathway in the biology of these organisms.

  2. The ubiquitin conjugating enzyme UbcH7, controls cell migration

    Science.gov (United States)

    Post translational modification by ubiquitination can target proteins for degradation, allow the interaction of proteins to form complexes or direct relocalization of proteins to different subcellular compartments. As such, ubiquitin controls a variety of essential cellular processes. Previously we ...

  3. The ubiquitin proteasome system in glia and its role in neurodegenerative diseases

    NARCIS (Netherlands)

    Jansen, Anne H P; Reits, Eric A J; Hol, Elly M

    2014-01-01

    The ubiquitin proteasome system (UPS) is crucial for intracellular protein homeostasis and for degradation of aberrant and damaged proteins. The accumulation of ubiquitinated proteins is a hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's, Parkinson's,

  4. The Ubiquitin-like modifier FAT10 in tolerance induction

    OpenAIRE

    Bürger, Stefanie

    2013-01-01

    HLA-F adjacent transcript 10 (FAT10) is a ubiquitin-like modifier which is predominantly expressed in lymphoid tissue like thymus, lymph node and spleen. The expression of FAT10 is inducible in various cell types with pro-inflammatory cytokines tumor necrosis factor-alpha and interferon-gamma. FAT10 is able to target proteins for proteasomal degradation, but unlike ubiquitin, it is degraded along with its substrates. Only a few biological functions of FAT10 have been described, for instance, ...

  5. FAT10, a Ubiquitin-Independent Signal for Proteasomal Degradation

    OpenAIRE

    Hipp, Mark Steffen; Kalveram, Birte; Raasi, Shahri; Groettrup, Marcus; Schmidtke, Gunter

    2005-01-01

    FAT10 is a small ubiquitin-like modifier that is encoded in the major histocompatibility complex and is synergistically inducible by tumor necrosis factor alpha and gamma interferon. It is composed of two ubiquitin-like domains and possesses a free C-terminal diglycine motif that is required for the formation of FAT10 conjugates. Here we show that unconjugated FAT10 and a FAT10 conjugate were rapidly degraded by the proteasome at a similar rate. Fusion of FAT10 to the N terminus of very long-...

  6. " Animal, trop animal "

    OpenAIRE

    Potestà, Andréa

    2010-01-01

    Dans la tradition philosophique, on trouve plusieurs définitions de l’homme. La célèbre définition aristotélicienne, zoon logon echon (animal doué du langage ou animal rationnel) fournit le paradigme ainsi que la méthode de toutes les définitions successives. Il s’agit d’ajouter au vivant, à l’animal, quelque chose d’autre, quelque chose de plus, qui permette de le caractériser et le fasse entendre comme différent des bêtes. Cette diversité peut être conçue différemment : en tant qu’élévation...

  7. Extralysosomal turnover of cellular proteins: Targeting substrates in the ubiquitin, ATP-dependent degradation system

    Energy Technology Data Exchange (ETDEWEB)

    Marriott, D.

    1988-01-01

    Calmodulin derived from a cloned chicken gene can be ubiquitinated and degraded by an in vitro reticulocyte lysate system. The chemical reactivity and the surface accessibility of the {epsilon}-amino group on lysine 115 in the calmodulin polypeptide chain were studied by trace labeling with acetic anhydride and with a ubiquitin derivative containing an azido group at the C-terminal glycine residue. Fractionation of reticulocyte lysate proteins separated the activity which degrades the calmodulin moiety of ubiquitin-calmodulin conjugates from that which acts on the isopeptide linkage. Neither of these two activities act on a synthetic isopeptide, which mimics the junction of ubiquitin-calmodulin, indicating the importance of the folding of ubiquitin for recognition. Based on recent findings that the ubiquitin moieties linked to {beta}galactosidase exist as a single multiubiquitin chain, studies were carried out to determine the structure of the ubiquitin-ubiquitin linkage. Ubiquitin was in vivo labeled with ({sup 3}H) and conjugated to {beta}galactosidase. Individual conjugates were isolated and subjected to peptide mapping by trypsin digestion, and tryptic fragments were analyzed of HPLC. The results indicated that the ubiquitin-ubiquitin linkage involves lysine residue 48 in the ubiquitin sequence.

  8. UBPY-mediated epidermal growth factor receptor (EGFR) de-ubiquitination promotes EGFR degradation

    NARCIS (Netherlands)

    Alwan, H.A.J.; Leeuwen, J.E.M. van

    2007-01-01

    Whereas poly-ubiquitination targets protein substrates for proteasomal degradation, mono-ubiquitination is known to regulate protein trafficking in the endosomal system and to target cargo proteins for lysosomal degradation. The role of the de-ubiquitinating enzymes AMSH and UBPY in endosomal traffi

  9. 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,

  10. A Ubl/ubiquitin switch in the activation of Parkin.

    Science.gov (United States)

    Sauvé, Véronique; Lilov, Asparouh; Seirafi, Marjan; Vranas, Marta; Rasool, Shafqat; Kozlov, Guennadi; Sprules, Tara; Wang, Jimin; Trempe, Jean-François; Gehring, Kalle

    2015-10-14

    Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86-130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding. PMID:26254305

  11. Strategies to Detect Endogenous Ubiquitination of a Target Mammalian Protein.

    Science.gov (United States)

    Sigismund, Sara; Polo, Simona

    2016-01-01

    Different biochemical techniques are well established to investigate target's ubiquitination in mammals without overexpressing a tagged version of ubiquitin (Ub). The simplest and more direct approach is to immunoprecipitate (IP) your target protein from cell lysate (stimulated and/or properly treated), followed by western blot analysis utilizing specific antibodies against Ub (see Subheading 3.1). This approach requires a good antibody against the target working in IP; alternatively, one could express a tagged version of the protein, possibly at the endogenous level. Another approach consists in IP ubiquitinated proteins from total cell lysate followed by detection with the antibody against the protein of interest. This second method relies on the availability of specific and very efficient antibodies against Ub (see Subheading 3.2). A more quantitative approach is the DELFIA assay (Perkin Elmer), an ELISA-based assay, which allows comparing more samples and conditions (see Subheading 3.3). Cross-validation with more than one approach is usually recommended in order to prove that your protein is modified by ubiquitin.Here we will use the EGFR as model system but protocols can be easily modified according to the protein of interest. PMID:27613032

  12. Mechanism for the selective conjugation of ubiquitin to phytochrome

    Energy Technology Data Exchange (ETDEWEB)

    Vierstra, R.D.

    1990-01-01

    The goal of this project is to understand at the molecular level how phytochrome functions and how intracellular proteins are degraded. Phytochrome is marked for degradation by covalent attachment of ubiquitin. Ubiquitin-phytochrome conjugates (UbP) were characterized with respect to formation kinetics, subcellular localization and site of ubiquitin attachment. UbP appears to be a general phenomenon during phytochrome degradation in a variety of species. UbP was isolated from oat seedlings and characterized. Residues 747-830 of phytochrome have been identified as a possible attachment site for ubiquitin. By placing the gene for etiolated phytochrome in tobacco we have created a transgenic system for over expressing phytochrome. The effects of this over expression are described, and it appears that tobacco degrades this foreign protein through formation of UbP. We have created a series of site-directed mutants of the oat phytochrome gene, and are in the process of characterizing them to determine sequence requirements for ubiquination. 8 refs., 1 fig. (MHB)

  13. How the ubiquitin proteasome system regulates the regulators of transcription.

    Science.gov (United States)

    Ee, Gary; Lehming, Norbert

    2012-01-01

    The ubiquitin proteasome system plays an important role in transcription. Monoubiquitination of activators is believed to aid their function, while the 26S proteasomal degradation of repressors is believed to restrict their function. What remains controversial is the question of whether the degradation of activators aids or restricts their function.

  14. Aerobic exercise training improves oxidative stress and ubiquitin proteasome system activity in heart of spontaneously hypertensive rats.

    Science.gov (United States)

    de Andrade, Luiz Henrique Soares; de Moraes, Wilson Max Almeida Monteiro; Matsuo Junior, Eduardo Hiroshi; de Orleans Carvalho de Moura, Elizabeth; Antunes, Hanna Karen Moreira; Montemor, Jairo; Antonio, Ednei Luiz; Bocalini, Danilo Sales; Serra, Andrey Jorge; Tucci, Paulo José Ferreira; Brum, Patricia Chakur; Medeiros, Alessandra

    2015-04-01

    The activity of the ubiquitin proteasome system (UPS) and the level of oxidative stress contribute to the transition from compensated cardiac hypertrophy to heart failure in hypertension. Moreover, aerobic exercise training (AET) is an important therapy for the treatment of hypertension, but its effects on the UPS are not completely known. The aim of this study was to evaluate the effect of AET on UPS's activity and oxidative stress level in heart of spontaneously hypertensive rats (SHR). A total of 53 Wistar and SHR rats were randomly divided into sedentary and trained groups. The AET protocol was 5×/week in treadmill for 13 weeks. Exercise tolerance test, non-invasive blood pressure measurement, echocardiographic analyses, and left ventricle hemodynamics were performed during experimental period. The expression of ubiquitinated proteins, 4-hydroxynonenal (4-HNE), Akt, phospho-Akt(ser473), GSK3β, and phospho-GSK3β(ser9) were analyzed by western blotting. The evaluation of lipid hydroperoxide concentration was performed using the xylenol orange method, and the proteasomal chymotrypsin-like activity was measured by fluorimetric assay. Sedentary hypertensive group presented cardiac hypertrophy, unaltered expression of total Akt, phospho-Akt, total GSK3β and phospho-GSK3β, UPS hyperactivity, increased lipid hydroperoxidation as well as elevated expression of 4-HNE but normal cardiac function. In contrast, AET significantly increased exercise tolerance, decreased resting systolic blood pressure and heart rate in hypertensive animals. In addition, the AET increased phospho-Akt expression, decreased phospho-GSK3β, and did not alter the expression of total Akt, total GSK3β, and ubiquitinated proteins, however, significantly attenuated 4-HNE levels, lipid hydroperoxidation, and UPS's activity toward normotensive group levels. Our results provide evidence for the main effect of AET on attenuating cardiac ubiquitin proteasome hyperactivity and oxidative stress in SHR

  15. Roles of the ubiquitin proteasome system in the effects of drugs of abuse

    Directory of Open Access Journals (Sweden)

    Nicolas eMassaly

    2015-01-01

    Full Text Available Because of its ability to regulate the abundance of selected proteins the ubiquitin proteasome system (UPS plays an important role in neuronal and synaptic plasticity. As a result various stages of learning and memory depend on UPS activity. Drug addiction, another phenomenon that relies on neuroplasticity, shares molecular substrates with memory processes. However the necessity of proteasome-dependent protein degradation for the development of addiction has been poorly studied. Here we first review evidences from the literature that drugs of abuse regulate the expression and activity of the UPS system in the brain. We then provide a list of proteins which have been shown to be targeted to the proteasome following drug treatment and could thus be involved in neuronal adaptations underlying behaviors associated with drug use and abuse. Finally we describe the few studies that addressed the need for UPS-dependent protein degradation in animal models of addiction-related behaviors.

  16. Final Report. The Role of RUB (related to ubiquitin) Family of Proteins in the Hormone Response

    Energy Technology Data Exchange (ETDEWEB)

    Callis, Judy [Univ. of California, Davis, CA (United States)

    2013-03-22

    The Rub pathway is a conserved protein modification pathway. RUB (called Rubp1 in budding yeast, Nedd8 in animals and RUB in plants) is a ubiquitin-like 76-amino acid protein. It covalently attaches to protein using an enzymatic machinery analogous to the enzymes that attach ubiquitin to its substrate proteins. However, the nature of the complement of Rub-modified proteins in organisms was not clear. From bioinformatics analyses, one can identify a Rub activating enzymes and Rub conjugating enzymes. However, in many cases, their biochemical properties were not described. In DOE-funded work, we made major advances in our understanding of the Rub pathway in yeast and plants, work that is applicable to other organisms as well. There is a multi-subunit enzyme called SCF in all eukaryotes. The SCF consists of several subunits that serve as a scaffold (the cullin, SKP and RBX subunits) and one subunit that interacts with the substrate. This cullin protein (called Cdc53p in yeast and CULLIN 1 in plants and animals) was a known Rub target. In this work, we identified additional Rub targets in yeast as the other cullin-like proteins Cul3p and Rtt101p. Additionally we described the conservation of the Rub pathway because plant RUB1 can conjugated to yeast Cdc53p- in yeast. In the model plant Arabidopsis thaliana, we characterized the Rub activating enzymes and showed that they are not biochemically equivalent. We also showed that the Rub pathway is essential in plants and characterized plants with reduced levels of rub proteins. These plants are affected in multiple developmental processes. We discovered that they over-produce ethylene as dark-grown seedlings. We characterized a mutant allele of CULLIN1 in Arabidopsis with impaired interaction with RBX and showed that it is unstable in vivo. We used our knowledge of monitoring protein degradation to map the degradation determinants in a plant transcription factor. Finally, we took a mass spectrometric approach to identify

  17. A Co-Translational Ubiquitination Pathway For Quality Control of Misfolded Proteins

    Science.gov (United States)

    Wang, Feng; Durfee, Larissa A.; Huibregtse, Jon M.

    2013-01-01

    Previous studies have indicated that 6–30% of all newly synthesized proteins are rapidly degraded by the ubiquitin-proteasome system, however the relationship of ubiquitination to translation for these proteins has been unclear. We report that co-translational ubiquitination (CTU) is a robust process, with ~12–15% of nascent polypeptides being ubiquitinated in human cells. CTU products contained primarily K48-linked polyubiquitin chains, consistent with a proteasomal targeting function. While nascent chains have been shown previously to be ubiquitinated within stalled complexes (CTUS), the majority of nascent chain ubiquitination occurred within active translation complexes (CTUA). CTUA was increased in response to agents that induce protein misfolding, while CTUS was increased in response to agents that lead to translational errors or stalling. These results indicate that ubiquitination of nascent polypeptides occurs in two contexts, and define CTUA as a component of a quality control system that marks proteins for destruction while they are being synthesized. PMID:23583076

  18. Amazing Animals

    Science.gov (United States)

    Al-Kuwari, Najat Saad

    2007-01-01

    "Animals" is a three-part lesson plan for young learners with a zoo animal theme. The first lesson is full of activities to describe animals, with Simon Says, guessing games, and learning stations. The second lesson is about desert animals, but other types of animals could be chosen depending on student interest. This lesson teaches…

  19. Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation

    Energy Technology Data Exchange (ETDEWEB)

    Boudreaux, David A.; Maiti, Tushar K.; Davies, Christopher W.; Das, Chittaranjan (Purdue)

    2010-07-06

    Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 {angstrom} from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarily at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal {beta}-hairpin at the distal site - a surface-exposed hydrophobic crevice 17 {angstrom} away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 {angstrom} of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.

  20. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors.

    Science.gov (United States)

    Qiu, Jiazhang; Sheedlo, Michael J; Yu, Kaiwen; Tan, Yunhao; Nakayasu, Ernesto S; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

    2016-05-01

    Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP.

  1. Características de la caña de azúcar asociadas con toneladas de caña por hectárea y sacarosa (% caña

    Directory of Open Access Journals (Sweden)

    Carlos Arturo Viveros Valens

    2015-07-01

    Full Text Available En las etapas iníciales del proceso de selección clonal en caña de azúcar (Saccharum spp. se realiza la selección indirecta para las variables toneladas de caña por hectárea (TCH y porcentaje de sacarosa. Esta selección indirecta puede aumentar su eficiencia en la medida que se precise mejor el conocimiento acerca de la naturaleza y la magnitud de las asociaciones existentes entre las características de interés y entre éstas y los indicadores de productividad. El objetivo del presente trabajo fue estimar las correlaciones genéticas entre las variables de tipo agronómico (factores causales y las de rendimiento (variables de respuesta, además descomponer su magnitud mediante análisis de sendero. Se evaluaron cinco caracteres de interés en caña de azúcar (altura, diámetro de tallos, población de tallos por metro, TCH y sacarosa (% caña en cinco localidades de la zona semiseca del valle del río Cauca para 17 variedades y dos testigos (CC 85-92 y MZC 74-275 en plantilla utilizando un diseño experimental Latice. Para estimar los coeficientes de correlación genética y de sendero (‘path coefficient’ se utilizó el software GENES. El análisis mostró que para obtener variedades con alto TCH y alta sacarosa (% caña, primero se deben seleccionar clones con altura superior que la variedad testigo CC 85-92 (334 cm para asegurar un contenido alto de sacarosa y posteriormente hacer un segundo tamizado por tallos gruesos de diámetro mayor que el testigo (32 mm y alta población de tallos igual o superiores que el testigo (14 tallos/m

  2. Animal research

    DEFF Research Database (Denmark)

    Olsson, I.A.S.; Sandøe, Peter

    2012-01-01

    in research is analyzed from the viewpoint of three distinct ethical approaches: contractarianism, utilitarianism, and animal rights view. On a contractarian view, research on animals is only an ethical issue to the extent that other humans as parties to the social contract care about how research animals...... are faring. From the utilitarian perspective, the use of sentient animals in research that may harm them is an ethical issue, but harm done to animals can be balanced by benefit generated for humans and other animals. The animal rights view, when thoroughgoing, is abolitionist as regards the use of animals......This article presents the ethical issues in animal research using a combined approach of ethical theory and analysis of scientific findings with bearing on the ethical analysis. The article opens with a general discussion of the moral acceptability of animal use in research. The use of animals...

  3. Mastermind-Like 1 Is Ubiquitinated: Functional Consequences for Notch Signaling.

    Directory of Open Access Journals (Sweden)

    Mozhgan Farshbaf

    Full Text Available Early studies demonstrated the involvement of ubiquitination of the Notch intracellular domain for rapid turnover of the transcriptional complex at Notch target genes. It was shown that this ubiquitination was promoted by the co-activator Mastermind like 1 (MAML1. MAML1 also contains numerous lysine residues that may also be ubiquitinated and necessary for protein regulation. In this study, we show that over-expressed MAML1 is ubiquitinated and identify eight conserved lysine residues which are required for ubiquitination. We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination. Furthermore, we show that a mutant MAML1 that has decreased ubiquitination shows increased output from a HES1 reporter gene assay. Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation. In summary, this study identifies that MAML1 is ubiquitinated in the absence of Notch signaling to maintain low levels of MAML1 in the cell. Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.

  4. Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting

    Science.gov (United States)

    Wing, S. S.; Goldberg, A. L.; Goldberger, A. L. (Principal Investigator)

    1993-01-01

    Glucocorticoids are essential for the increase in protein breakdown in skeletal muscle normally seen during fasting. To determine which proteolytic pathway(s) are activated upon fasting, leg muscles from fed and fasted normal rats were incubated under conditions that block or activate different proteolytic systems. After food deprivation (1 day), the nonlysosomal ATP-dependent process increased by 250%, as shown in experiments involving depletion of muscle ATP. Also, the maximal capacity of the lysosomal process increased 60-100%, but no changes occurred in the Ca(2+)-dependent or the residual energy-independent proteolytic processes. In muscles from fasted normal and adrenalectomized (ADX) rats, the protein breakdown sensitive to inhibitors of the lysosomal or Ca(2+)-dependent pathways did not differ. However, the ATP-dependent process was 30% slower in muscles from fasted ADX rats. Administering dexamethasone to these animals or incubating their muscles with dexamethasone reversed this defect. During fasting, when the ATP-dependent process rises, muscles show a two- to threefold increase in levels of ubiquitin (Ub) mRNA. However, muscles of ADX animals failed to show this response. Injecting dexamethasone into the fasted ADX animals increased muscle Ub mRNA within 6 h. Thus glucocorticoids activate the ATP-Ub-dependent proteolytic pathway in fasting apparently by enhancing the expression of components of this system such as Ub.

  5. Ataxia, Dementia, and Hypogonadotropism Caused by Disordered Ubiquitination

    DEFF Research Database (Denmark)

    Margolin, David H.; Kousi, Maria; Chan, Yee-Ming;

    2013-01-01

    affected patients. Neurologic and reproductive endocrine phenotypes were characterized in detail. The effects of sequence variants and the presence of an epistatic interaction were tested in a zebrafish model. RESULTS Digenic homozygous mutations in RNF216 and OTUD4, which encode a ubiquitin E3 ligase...... in zebrafish embryos induced defects in the eye, optic tectum, and cerebellum; combinatorial suppression of both genes exacerbated these phenotypes, which were rescued by nonmutant, but not mutant, human RNF216 or OTUD4 messenger RNA. All patients had progressive ataxia and dementia. Neuronal loss was observed...... in cerebellar pathways and the hippocampus; surviving hippocampal neurons contained ubiquitin-immunoreactive intranuclear inclusions. Defects were detected at the hypothalamic and pituitary levels of the reproductive endocrine axis. CONCLUSIONS The syndrome of hypogonadotropic hypogonadism, ataxia, and dementia...

  6. Role of the Ubiquitin Proteasome System in Regulating Skin Pigmentation

    Directory of Open Access Journals (Sweden)

    Hideya Ando

    2009-10-01

    Full Text Available Pigmentation of the skin, hair and eyes is regulated by tyrosinase, the critical rate-limiting enzyme in melanin synthesis by melanocytes. Tyrosinase is degraded endogenously, at least in part, by the ubiquitin proteasome system (UPS. Several types of inherited hypopigmentary diseases, such as oculocutaneous albinism and Hermansky-Pudlak syndrome, involve the aberrant processing and/or trafficking of tyrosinase and its subsequent degradation which can occur due to the quality-control machinery. Studies on carbohydrate modifications have revealed that tyrosinase in the endoplasmic reticulum (ER is proteolyzed via ER-associated protein degradation and that tyrosinase degradation can also occur following its complete maturation in the Golgi. Among intrinsic factors that regulate the UPS, fatty acids have been shown to modulate tyrosinase degradation in contrasting manners through increased or decreased amounts of ubiquitinated tyrosinase that leads to its accelerated or decelerated degradation by proteasomes.

  7. Cell fate determination by ubiquitin-dependent regulation of translation

    Science.gov (United States)

    Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T.; Rape, Michael

    2015-01-01

    Metazoan development depends on accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates 1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell fate determination is less well understood. Here, we have identified the vertebrate-specific ubiquitin ligase CUL3KBTBD8 as an essential regulator of neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralog TCOF1, whose mutation underlies the neurocristopathy Treacher Collins Syndrome 2,3. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favor of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell fate determination. PMID:26399832

  8. Direct observation of silver nanoparticle-ubiquitin corona formation

    CERN Document Server

    Ding, Feng; Choudhary, Poonam; Chen, Ran; Brown, Jared M; Ke, Pu Chun

    2012-01-01

    Upon entering physiological environments, nanoparticles readily assume the form of a nanoparticle-protein corona that dictates their biological identity. Understanding the structure and dynamics of nanoparticle-protein corona is essential for predicting the fate, transport, and toxicity of nanomaterials in living systems and for enabling the vast applications of nanomedicine. We combined multiscale molecular dynamics simulations and complementary experiments to characterize the silver nanoparticle-ubiquitin corona formation. Specifically, ubiquitins competed with citrates for the nanoparticle surface and bound to the particle in a specific manner. Under a high protein/nanoparticle stoichiometry, ubiquitions formed a multi-layer corona on the particle surface. The binding exhibited an unusual stretched-exponential behavior, suggesting a rich kinetics originated from protein-protein, protein-citrate, and protein-nanoparticle interactions. Furthermore, the binding destabilized the {\\alpha}-helices while increasi...

  9. Ubiquitin-dependent trafficking and turnover of ionotropic glutamate receptors

    Directory of Open Access Journals (Sweden)

    Marisa S Goo

    2015-10-01

    Full Text Available Changes in synaptic strength underlie the basis of learning and memory and are controlled, in part, by the insertion or removal of AMPA-type glutamate receptors at the postsynaptic membrane of excitatory synapses. Once internalized, these receptors may be recycled back to the plasma membrane by subunit-specific interactions with other proteins or by post-translational modifications such as phosphorylation. Alternatively, these receptors may be targeted for destruction by multiple degradation pathways in the cell. Ubiquitination, another post-translational modification, has recently emerged as a key signal that regulates the recycling and trafficking of glutamate receptors. In this review, we will discuss recent findings on the role of ubiquitination in the trafficking and turnover of ionotropic glutamate receptors and plasticity of excitatory synapses.

  10. Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy.

    Science.gov (United States)

    Lork, Marie; Delvaeye, Mieke; Gonçalves, Amanda; Van Hamme, Evelien; Beyaert, Rudi

    2016-01-01

    Salmonella is a gram-negative facultative intracellular pathogen that is capable of infecting a variety of hosts. Inside host cells, most Salmonella bacteria reside and replicate within Salmonella-containing vacuoles. They use virulence proteins to manipulate the host cell machinery for their own benefit and hijack the host cytoskeleton to travel toward the perinuclear area. However, a fraction of bacteria escapes into the cytosol where they get decorated with a dense layer of polyubiquitin, which labels the bacteria for clearance by autophagy. More specifically, autophagy receptor proteins recognize the ubiquitinated bacteria and deliver them to autophagosomes, which subsequently fuse to lysosomes. Here, we describe methods used to infect HeLa cells with Salmonella bacteria and to detect their ubiquitination via immunofluorescence and laser scanning confocal microscopy. PMID:27613040

  11. Ubiquitination of inducible nitric oxide synthase is required for its degradation

    Science.gov (United States)

    Kolodziejski, Pawel J.; Musial, Aleksandra; Koo, Ja-Seok; Eissa, N. Tony

    2002-01-01

    Inducible nitric oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from l-arginine in response to inflammatory mediators. We have previously shown that iNOS is degraded through the 26S proteasome. Targeting of proteins for proteasomal degradation may or may not require their covalent linkage to multiubiquitin chains (ubiquitination). In addition, ubiquitination of a protein can serve functions other than signaling proteolysis. In this context, it is not known whether iNOS is subject to ubiquitination or whether ubiquitination is required for its degradation. In this study, we show that iNOS, expressed in HEK293 cells or induced in primary bronchial epithelial cells, A549 cells, or murine macrophages, is subject to ubiquitination. To investigate whether iNOS ubiquitination is required for its degradation, HEK293T cells were cotransfected with plasmids containing cDNAs of human iNOS and of the dominant negative ubiquitin mutant K48R. Disruption of ubiquitination by K48R ubiquitin resulted in inhibition of iNOS degradation. ts20 is a mutant cell line that contains a thermolabile ubiquitin-activating enzyme (E1) that is inactivated at elevated temperature, preventing ubiquitination. Incubation of ts20 cells, stably expressing human iNOS, at the nonpermissive temperature (40°C) resulted in inhibition of iNOS degradation and marked accumulation of iNOS. These studies indicate that iNOS is subject to ubiquitination and that ubiquitination is required for its degradation. PMID:12221289

  12. Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Jennifer Hurst-Kennedy

    2012-01-01

    Full Text Available Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1, aka PGP9.5 is an abundant, neuronal deubiquitinating enzyme that has also been suggested to possess E3 ubiquitin-protein ligase activity and/or stabilize ubiquitin monomers in vivo. Recent evidence implicates dysregulation of UCH-L1 in the pathogenesis and progression of human cancers. Although typically only expressed in neurons, high levels of UCH-L1 have been found in many nonneuronal tumors, including breast, colorectal, and pancreatic carcinomas. UCH-L1 has also been implicated in the regulation of metastasis and cell growth during the progression of nonsmall cell lung carcinoma, colorectal cancer, and lymphoma. Together these studies suggest UCH-L1 has a potent oncogenic role and drives tumor development. Conversely, others have observed promoter methylation-mediated silencing of UCH-L1 in certain tumor subtypes, suggesting a potential tumor suppressor role for UCH-L1. In this paper, we provide an overview of the evidence supporting the involvement of UCH-L1 in tumor development and discuss the potential mechanisms of action of UCH-L1 in oncogenesis.

  13. Conservation and developmental expression of ubiquitin isopeptidases in Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Roberta Verciano Pereira

    2014-02-01

    Full Text Available Several genes related to the ubiquitin (Ub-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs proteins found in the database of the parasite’s genome. An in silico ana- lysis (GeneDB and MEROPS identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and Sm BAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.

  14. Novel RING E3 Ubiquitin Ligases in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Angelika Burger

    2006-08-01

    Full Text Available Defects in ubiquitin E3 ligases are implicated in the pathogenesis of several human diseases, including cancer, because of their central role in the control of diverse signaling pathways. RING E3 ligases promote the ubiquitination of proteins that are essential to a variety of cellular events. Identification of which ubiquitin ligases specifically affect distinct cellular processes is essential to the development of targeted therapeutics for these diseases. Here we discuss two novel RING E3 ligases, BCA2 and RNF11, that are closely linked to human breast cancer. BCA2 E3 ligase is coregulated with estrogen receptor and plays a role in the regulation of epidermal growth factor receptor (EGF-R trafficking. RNF11 is a small RING E3 ligase that affects transforming growth factorβ and EGF-R signaling and is overexpressed in invasive breast cancers. These two proteins demonstrate the complexity of RING E3 ligase interactions in breast cancer and are potential targets for therapeutic interventions.

  15. Enhancing ubiquitin crystallization through surface-entropy reduction.

    Science.gov (United States)

    Loll, Patrick J; Xu, Peining; Schmidt, John T; Melideo, Scott L

    2014-10-01

    Ubiquitin has many attributes suitable for a crystallization chaperone, including high stability and ease of expression. However, ubiquitin contains a high surface density of lysine residues and the doctrine of surface-entropy reduction suggests that these lysines will resist participating in packing interactions and thereby impede crystallization. To assess the contributions of these residues to crystallization behavior, each of the seven lysines of ubiquitin was mutated to serine and the corresponding single-site mutant proteins were expressed and purified. The behavior of these seven mutants was then compared with that of the wild-type protein in a 384-condition crystallization screen. The likelihood of obtaining crystals varied by two orders of magnitude within this set of eight proteins. Some mutants crystallized much more readily than the wild type, while others crystallized less readily. X-ray crystal structures were determined for three readily crystallized variants: K11S, K33S and the K11S/K63S double mutant. These structures revealed that the mutant serine residues can directly promote crystallization by participating in favorable packing interactions; the mutations can also exert permissive effects, wherein crystallization appears to be driven by removal of the lysine rather than by addition of a serine. Presumably, such permissive effects reflect the elimination of steric and electrostatic barriers to crystallization.

  16. Downregulation of Smurf2, a tumor-suppressive ubiquitin ligase, in triple-negative breast cancers: Involvement of the RB-microRNA axis

    International Nuclear Information System (INIS)

    The HECT family ubiquitin ligase Smurf2 regulates cell polarity, migration, division, differentiation and death, by targeting diverse substrates that are critical for receptor signaling, cytoskeleton, chromatin remodeling and transcription. Recent studies suggest that Smurf2 functions as a tumor suppressor in mice. However, no inactivating mutation of SMURF2 has been reported in human, and information about Smurf2 expression in human cancer remains limited or complicated. Here we demonstrate that Smurf2 expression is downregulated in human breast cancer tissues, especially of the triple-negative subtype, and address the mechanism of Smurf2 downregulation in triple-negative breast cancer cells. Human breast cancer tissues (47 samples expressing estrogen receptor (ER) and 43 samples with triple-negative status) were examined by immunohistochemistry for the expression of Smurf2. Ten widely-studied human breast cancer cell lines were examined for the expression of Smurf2. Furthermore, microRNA-mediated regulation of Smurf2 was investigated in triple-negative cancer cell lines. Immunohistochemical analysis showed that benign mammary epithelial cells expressed high levels of Smurf2, so did cells in ductal carcinomas in situ. In contrast, invasive ductal carcinomas showed focal or diffuse decrease in Smurf2 expression, which was observed more frequently in triple-negative tumors than in ER-positive tumors. Consistently, human triple-negative breast cancer cell lines such as BT549, MDA-MB-436, DU-4475 and MDA-MB-468 cells showed significantly lower expression of Smurf2 protein, compared to ER + or HER2+ cell lines. Studies using quantitative PCR and specific microRNA inhibitors indicated that increased expression of miR-15a, miR-15b, miR-16 and miR-128 was involved in Smurf2 downregulation in those triple-negative cancer cell lines, which have mutations in the retinoblastoma (RB) gene. Forced expression of RB increased levels of Smurf2 protein with concomitant decreases in

  17. 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.

  18. Characterization of ubiquitination dependent dynamics in growth factor receptor signaling by quantitative proteomics

    DEFF Research Database (Denmark)

    Akimov, Vyacheslav; Rigbolt, Kristoffer T G; Nielsen, Mogens M;

    2011-01-01

    or chains of ubiquitin molecules of various types and lengths to targeted proteins is known to alter proteins' lifespan, localization and function and to modulate protein interactions. Despite its central importance in various aspects of cellular life and function there are only a limited number of reports...... investigating ubiquitination on a proteomic scale, mainly due to the inherited complexity and heterogeneity of ubiquitination. We describe here a quantitative proteomics strategy based on the specificity of ubiquitin binding domains (UBDs) and Stable Isotope Labeling by Amino acids in Cell culture (SILAC......) for selectively decoding ubiquitination-driven processes involved in the regulation of cellular signaling networks. We applied this approach to characterize the temporal dynamics of ubiquitination events accompanying epidermal growth factor receptor (EGFR) signal transduction. We used recombinant UBDs derived...

  19. The ubiquitin ligase RPM-1 and the p38 MAPK PMK-3 regulate AMPA receptor trafficking.

    Directory of Open Access Journals (Sweden)

    Eun Chan Park

    Full Text Available Ubiquitination occurs at synapses, yet its role remains unclear. Previous studies demonstrated that the RPM-1 ubiquitin ligase organizes presynaptic boutons at neuromuscular junctions in C. elegans motorneurons. Here we find that RPM-1 has a novel postsynaptic role in interneurons, where it regulates the trafficking of the AMPA-type glutamate receptor GLR-1 from synapses into endosomes. Mutations in rpm-1 cause the aberrant accumulation of GLR-1 in neurites. Moreover, rpm-1 mutations enhance the endosomal accumulation of GLR-1 observed in mutants for lin-10, a Mint2 ortholog that promotes GLR-1 recycling from Syntaxin-13 containing endosomes. As in motorneurons, RPM-1 negatively regulates the pmk-3/p38 MAPK pathway in interneurons by repressing the protein levels of the MAPKKK DLK-1. This regulation of PMK-3 signaling is critical for RPM-1 function with respect to GLR-1 trafficking, as pmk-3 mutations suppress both lin-10 and rpm-1 mutations. Positive or negative changes in endocytosis mimic the effects of rpm-1 or pmk-3 mutations, respectively, on GLR-1 trafficking. Specifically, RAB-5(GDP, an inactive mutant of RAB-5 that reduces endocytosis, mimics the effect of pmk-3 mutations when introduced into wild-type animals, and occludes the effect of pmk-3 mutations when introduced into pmk-3 mutants. By contrast, RAB-5(GTP, which increases endocytosis, suppresses the effect of pmk-3 mutations, mimics the effect of rpm-1 mutations, and occludes the effect of rpm-1 mutations. Our findings indicate a novel specialized role for RPM-1 and PMK-3/p38 MAPK in regulating the endosomal trafficking of AMPARs at central synapses.

  20. Decreased outer membrane permeability protects mycobacteria from killing by ubiquitin-derived peptides

    OpenAIRE

    Purdy, Georgiana E.; Niederweis, Michael; Russell, David G.

    2009-01-01

    Ubiquitin-derived peptides are bactericidal in vitro and contribute to the mycobactericidal activity of the lysosome. To further define interactions of ubiquitin-derived peptides with mycobacteria, we screened for mutants with increased resistance to the bactericidal activity of the synthetic ubiquitin-derived peptide Ub2. The four Ub2-resistant M. smegmatis mutants were also resistant to the bactericidal action of other antimicrobial peptides and macrophages. Two mutants were in the mspA gen...

  1. Expression of TRAF6 and ubiquitin mRNA in skeletal muscle of gastric cancer patients

    Directory of Open Access Journals (Sweden)

    Sun Yuan-Shui

    2012-09-01

    Full Text Available Abstract Objective To investigate the prognostic significance of tumor necrosis factor receptor (TNFR,-associated factor 6 (TRAF6,-and ubiquitin in gastric cancer patients. Methods Biopsies of the rectus abdominis muscle were obtained intra operatively from 102 gastric cancer patients and 29 subjects undergoing surgery for benign abdominal diseases, and muscle TRAF6 and ubiquitin mRNA expression and proteasome proteolytic activities were assessed. Results TRAF6 was significantly upregulated in muscle of gastric cancer compared with the control muscles. TRAF6 was upregulated in 67.65% (69/102 muscle of gastric cancer. Over expression of TRAF6 in muscles of gastric cancer were associated with TNM stage, level of serum albumin and percent of weight loss. Ubiquitin was significantly upregulated in muscle of gastric cancer compared with the control muscles. Ubiquitin was upregulated in 58.82% (60/102 muscles of gastric cancer. Over expression of ubiquitin in muscles of gastric cancer were associated with TNM (Tumor-Node-Metastasis stage and weight loss. There was significant relation between TRAF6 and ubiquitin expression. Conclusions We found a positive correlation between TRAF6 and ubiquitin expression, suggesting that TRAF6 may up regulates ubiquitin activity in cancer cachexia. While more investigations are required to understand its mechanisms of TRAF6 and ubiquitin in skeletal muscle. Correct the catabolic-anabolic imbalance is essential for the effective treatment of cancer cachexia.

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

    Science.gov (United States)

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

    2016-07-01

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

  3. Animal Bites

    Science.gov (United States)

    ... and complications from bites Never pet, handle, or feed unknown animals Leave snakes alone Watch your children closely around animals Vaccinate your cats, ferrets, and dogs against rabies Spay or neuter ...

  4. Animal Bites

    Science.gov (United States)

    Wild animals usually avoid people. They might attack, however, if they feel threatened, are sick, or are protecting their ... or territory. Attacks by pets are more common. Animal bites rarely are life-threatening, but if they ...

  5. Animal Farm

    Institute of Scientific and Technical Information of China (English)

    徐蓉蓉

    2015-01-01

    This essay first introduce the background of Animal Farm and a brief introduction of the author.Then it discuss three thesis about this novel and briefly discussed about it.At last it give highly review on Animal Farm.

  6. Animal Farm

    Institute of Scientific and Technical Information of China (English)

    徐蓉蓉

    2015-01-01

    This essayfirst introduce the background of Animal Farm and a brief introduction of the author.Then it discuss three thesis about this novel and briefly discussed about it.At last it give highly review on Animal Farm.

  7. Animal ethics

    OpenAIRE

    Palmer, Clare; Sandøe, Peter

    2011-01-01

    This chapter describes and discusses different views concerning our duties towards animals. First, we explain why it is necessary to engage in thinking about animal ethics and why it is not enough to rely on feelings alone. Secondly, we present and discuss five different kinds of views about the nature of our duties to animals. They are: contractarianism, utilitarianism, the animal rights view, contextual views, and a respect for nature view. Finally, we briefly consider whether it is possibl...

  8. Animal Deliberation

    NARCIS (Netherlands)

    Driessen, C.P.G.

    2014-01-01

    While much has been written on environmental politics on the one hand, and animal ethics and welfare on the other, animal politics, as the interface of the two, is underexamined. There are key political implications in the increase of animal protection laws, the rights of nature, and political parti

  9. Animal models

    DEFF Research Database (Denmark)

    Gøtze, Jens Peter; Krentz, Andrew

    2014-01-01

    In this issue of Cardiovascular Endocrinology, we are proud to present a broad and dedicated spectrum of reviews on animal models in cardiovascular disease. The reviews cover most aspects of animal models in science from basic differences and similarities between small animals and the human...

  10. Unfolding Ubiquitin by force: water mediated H-bond destabilization

    Directory of Open Access Journals (Sweden)

    Germán Pabón

    2012-12-01

    Full Text Available Using the “pull and wait” (PNW simulation protocol at 300 K, we studied the unfolding by force of an ubiquitin molecule. PNW was implemented in the CHARMM program using an integration time step of 1 fs and a uniform dielectric constant of 1. The ubiquitin molecule, initially solvated, was put under mechanical stress, exerting forces from different directions. The rupture of five hydrogen bonds between parallel strands β1 and β5 takes place during the extension from 13 to 15 Å, defines a mechanical barrier for unfolding and dominates the point of maximum unfolding force. The simulations described here show that given adequate time, a small applied force can destabilize those five H-bonds relative to the bonds that can be created to water molecules; allowing the formation of stable H-bonds between a single water molecule and the donor and acceptor groups of the interstrand H-bonds. Thus, simulations run with PNW show that the force is not responsible for “ripping apart” the backbone H-bonds; it merely destabilizes them making them less stable than the H-bonds they can make with water. Additional simulations show that the force necessary to destabilize the H-bonds and allow them to be replaced by H-bonds to water molecules depends strongly on the pulling direction. By using a simulation protocol that allows equilibration at each extension we have been able to observe the details of the events leading to the unfolding of ubiquitin by mechanical force.

  11. Incorporating distant sequence features and radial basis function networks to identify ubiquitin conjugation sites.

    Science.gov (United States)

    Lee, Tzong-Yi; Chen, Shu-An; Hung, Hsin-Yi; Ou, Yu-Yen

    2011-03-09

    Ubiquitin (Ub) is a small protein that consists of 76 amino acids about 8.5 kDa. In ubiquitin conjugation, the ubiquitin is majorly conjugated on the lysine residue of protein by Ub-ligating (E3) enzymes. Three major enzymes participate in ubiquitin conjugation. They are E1, E2 and E3 which are responsible for activating, conjugating and ligating ubiquitin, respectively. Ubiquitin conjugation in eukaryotes is an important mechanism of the proteasome-mediated degradation of a protein and regulating the activity of transcription factors. Motivated by the importance of ubiquitin conjugation in biological processes, this investigation develops a method, UbSite, which uses utilizes an efficient radial basis function (RBF) network to identify protein ubiquitin conjugation (ubiquitylation) sites. This work not only investigates the amino acid composition but also the structural characteristics, physicochemical properties, and evolutionary information of amino acids around ubiquitylation (Ub) sites. With reference to the pathway of ubiquitin conjugation, the substrate sites for E3 recognition, which are distant from ubiquitylation sites, are investigated. The measurement of F-score in a large window size (-20∼+20) revealed a statistically significant amino acid composition and position-specific scoring matrix (evolutionary information), which are mainly located distant from Ub sites. The distant information can be used effectively to differentiate Ub sites from non-Ub sites. As determined by five-fold cross-validation, the model that was trained using the combination of amino acid composition and evolutionary information performs best in identifying ubiquitin conjugation sites. The prediction sensitivity, specificity, and accuracy are 65.5%, 74.8%, and 74.5%, respectively. Although the amino acid sequences around the ubiquitin conjugation sites do not contain conserved motifs, the cross-validation result indicates that the integration of distant sequence features of Ub

  12. Incorporating distant sequence features and radial basis function networks to identify ubiquitin conjugation sites.

    Directory of Open Access Journals (Sweden)

    Tzong-Yi Lee

    Full Text Available Ubiquitin (Ub is a small protein that consists of 76 amino acids about 8.5 kDa. In ubiquitin conjugation, the ubiquitin is majorly conjugated on the lysine residue of protein by Ub-ligating (E3 enzymes. Three major enzymes participate in ubiquitin conjugation. They are E1, E2 and E3 which are responsible for activating, conjugating and ligating ubiquitin, respectively. Ubiquitin conjugation in eukaryotes is an important mechanism of the proteasome-mediated degradation of a protein and regulating the activity of transcription factors. Motivated by the importance of ubiquitin conjugation in biological processes, this investigation develops a method, UbSite, which uses utilizes an efficient radial basis function (RBF network to identify protein ubiquitin conjugation (ubiquitylation sites. This work not only investigates the amino acid composition but also the structural characteristics, physicochemical properties, and evolutionary information of amino acids around ubiquitylation (Ub sites. With reference to the pathway of ubiquitin conjugation, the substrate sites for E3 recognition, which are distant from ubiquitylation sites, are investigated. The measurement of F-score in a large window size (-20∼+20 revealed a statistically significant amino acid composition and position-specific scoring matrix (evolutionary information, which are mainly located distant from Ub sites. The distant information can be used effectively to differentiate Ub sites from non-Ub sites. As determined by five-fold cross-validation, the model that was trained using the combination of amino acid composition and evolutionary information performs best in identifying ubiquitin conjugation sites. The prediction sensitivity, specificity, and accuracy are 65.5%, 74.8%, and 74.5%, respectively. Although the amino acid sequences around the ubiquitin conjugation sites do not contain conserved motifs, the cross-validation result indicates that the integration of distant sequence

  13. Entry, Descent, Landing Animation (Animation)

    Science.gov (United States)

    2005-01-01

    [figure removed for brevity, see original site] Click on the image for Entry, Descent, Landing animation This animation illustrates the path the Stardust return capsule will follow once it enters Earth's atmosphere.

  14. Redox control of the ubiquitin-proteasome system

    DEFF Research Database (Denmark)

    Kriegenburg, Franziska; Poulsen, Esben G; Koch, Annett;

    2011-01-01

    is characteristic of many diseases and during aging. To counter the adverse effects of oxidative stress, cells can initiate an antioxidative response in an attempt to repair the damage, or rapidly channel the damaged proteins for degradation by the ubiquitin-proteasome system (UPS). Recent studies...... associated with a thioredoxin and other cofactors that may adjust the particle's response during an oxidative challenge. Here, we give an overview of the UPS and a detailed description of the degradation of oxidized proteins and of the crosstalk between oxidative stress and protein degradation in health and...

  15. Gclust Server: 94699 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available milarity to hect domain E3 ubiquitin-protein ligases, not essential for viability 1 1.00e-90 0.0 0.0 0.0 0.0...ative annotation Protein with similarity to hect domain E3 ubiquitin-protein ligases, not essential for viab

  16. Gclust Server: 94681 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available milarity to hect domain E3 ubiquitin-protein ligases, not essential for viability 1 1.00e-25 0.0 0.0 0.0 0.0...ative annotation Protein with similarity to hect domain E3 ubiquitin-protein ligases, not essential for viab

  17. 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.

  18. The BAH domain of BAF180 is required for PCNA ubiquitination

    Energy Technology Data Exchange (ETDEWEB)

    Niimi, Atsuko [Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601 (Japan); Hopkins, Suzanna R; Downs, Jessica A [Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ (United Kingdom); Masutani, Chikahide, E-mail: masutani@riem.nagoya-u.ac.jp [Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601 (Japan)

    2015-09-15

    Highlights: • The expression of BAF180 promotes UV-induced PCNA ubiquitination during S phase. • The BAH domains of BAF180 alone are sufficient to promote PCNA ubiquitination. • The BAH domains are not assembled into the PBAF in the absence of the C-terminus. - Abstract: Monoubiquitination of proliferating cell nuclear antigen (PCNA) is a critical regulator of post replication repair (PRR). The depletion of BAF180, a unique subunit of the PBAF chromatin remodeling complex in human cells results in reduced PCNA ubiquitination leading to less efficient fork progression following DNA damage, but little is known about the mechanism. Here, we report that the expression of exogenous BAF180 in cells promotes PCNA ubiquitination during S-phase after UV irradiation and it persists for many hours. No correlation was observed between the protein level of ubiquitin-specific protease 1 (USP1) and ubiquitinated PCNA in BAF180 expressing cells. Analysis of cells expressing BAF180 deletion mutants showed that the bromo-adjacent homology (BAH) domains are responsible for this effect. Surprisingly, a deletion construct encoding only the BAH domain region is able to increase the level of ubiquitinated PCNA, even though it is unable to be assembled into the PBAF complex. These results suggest that the ATPase-dependent chromatin remodeling activity of PBAF is not necessary, but instead the BAH domains are sufficient to promote PCNA ubiquitination.

  19. Silencing of unpaired chromatin and histone H2A ubiquitination in mammalian meiosis.

    NARCIS (Netherlands)

    Baarends, W.M.; Wassenaar, E.; Laan, R. van der; Hoogerbrugge, J.W.; Sleddens-Linkels, E.; Hoeijmakers, J.H.; Boer, P. de; Grootegoed, J.A.

    2005-01-01

    During meiotic prophase in male mammals, the X and Y chromosomes are incorporated in the XY body. This heterochromatic body is transcriptionally silenced and marked by increased ubiquitination of histone H2A. This led us to investigate the relationship between histone H2A ubiquitination and chromati

  20. Silencing of unpaired chromatin and histone H2A ubiquitination in mammalian meiosis.

    NARCIS (Netherlands)

    W.M. Baarends (Willy); E. Wassenaar (Evelyne); R. van der Laan (Roald); J.W. Hoogerbrugge (Jos); E. Sleddens-Linkels (Esther); J.H.J. Hoeijmakers (Jan); P. de Boer (Peter); J.A. Grootegoed (Anton)

    2005-01-01

    textabstractDuring meiotic prophase in male mammals, the X and Y chromosomes are incorporated in the XY body. This heterochromatic body is transcriptionally silenced and marked by increased ubiquitination of histone H2A. This led us to investigate the relationship between histone H2A ubiquitination

  1. Ubiquitination Accomplished: E1 and E2 Enzymes Were Not Necessary.

    Science.gov (United States)

    Nakasone, Mark A; Huang, Danny T

    2016-06-16

    Qiu et al. (2016) show that a mono-ADP-ribosyltransferase, SdeA, from Legionella pneumophila catalyzes ADP-ribosylation of ubiquitin, allowing SdeA to modify substrate with ubiquitin in the absence of E1 and E2 enzymes. PMID:27315555

  2. Combined Infrared Multiphoton Dissociation with Ultraviolet Photodissociation for Ubiquitin Characterization

    Science.gov (United States)

    Halim, Mohammad A.; Girod, Marion; MacAleese, Luke; Lemoine, Jérôme; Antoine, Rodolphe; Dugourd, Philippe

    2016-06-01

    Herein we report the successful implementation of the consecutive and simultaneous photodissociation with high (213 nm) and low (10.6 μm) energy photons (HiLoPD, high-low photodissociation) on ubiquitin in a quadrupole-Orbitrap mass spectrometer. Absorption of high-energy UV photon is dispersed over the whole protein and stimulates extensive C-Cα backbone fragmentation, whereas low-energy IR photon gradually increases the internal energy and thus preferentially dissociates the most labile amide (C-N) bonds. We noticed that simultaneous irradiation of UV and IR lasers on intact ubiquitin in a single MS/MS experiment provides a rich and well-balanced fragmentation array of a/x, b/y, and z ions. Moreover, secondary fragmentation from a/x and z ions leads to the formation of satellite side-chain ions (d, v, and w) and can help to distinguish isomeric residues in a protein. Implementation of high-low photodissociation in a high-resolution mass spectrometer may offer considerable benefits to promote a comprehensive portrait of protein characterization.

  3. Animal Shelter

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Beijing activist Zhang Luping gives up a lucrative business career to provide a home for stray and abandoned pets "I have never been married, but I have I hundreds of children," said Zhang Luping, founder of the Beijing Human and Animal Environment Education Center (the Animal Center). "God sent me to this planet and gave me the mission of taking care of helpless and homeless dogs and cats. I will never let Him down." The Animal Center, one of a few non-

  4. Animal ethics

    DEFF Research Database (Denmark)

    Palmer, Clare; Sandøe, Peter

    2011-01-01

    the nature of our duties to animals. They are: contractarianism, utilitarianism, the animal rights view, contextual views, and a respect for nature view. Finally, we briefly consider whether it is possible to combine elements from the presented views, and how to make up one’s mind.......This chapter describes and discusses different views concerning our duties towards animals. First, we explain why it is necessary to engage in thinking about animal ethics and why it is not enough to rely on feelings alone. Secondly, we present and discuss five different kinds of views about...

  5. Animated Asphalt

    DEFF Research Database (Denmark)

    Paldam, Camilla Skovbjerg

    2015-01-01

    “animation”, defined as “an innate (and learnable) ability of our bodies to discover life in inanimate images” (Belting 2012, 188). In this essay I investigate the animation of pictures in dialogue with Mitchell, both by addressing general questions such as: how is animation of otherwise static pictures...... to be understood? How does animation differ in different media? And in particular by focusing on and questioning the gender positions inherent in Mitchell’s theory. Animation has an erotic component of seduction and desire, and what pictures want, becomes for Mitchell, what women want. There is of course no simple...

  6. Enrichment and analysis of rice seedling ubiquitin-related proteins using four UBA domains (GST-qUBAs).

    Science.gov (United States)

    Meng, Qingshi; Rao, Liqun; Pan, Yinghong

    2014-12-01

    Protein ubiquitination is a common posttranslational modification that often occurs on lysine residues. It controls the half-life, interaction and trafficking of intracellular proteins and is involved in different plant development stages and responses to environment stresses. Four Ubiquitin-Associated (UBA) domains were sequentially fused with Glutathione S-transferase (GST) tag (GST-qUBA) as bait protein in this study. A two-step affinity protocol was successfully developed and the identification of ubiquitinated proteins and their interaction proteins increased almost threefold compared to methods that directly identify ubiquitinated proteins from crude samples. A total of 170 ubiquitin-related proteins were identified in GST-qUBAs enriched samples taken from rice seedlings. There were 134 ubiquitinated proteins, 5 ubiquitin-activating enzymes (E1s), 5 ubiquitin-conjugating enzymes (E2s), 19 ubiquitin ligases (E3s) and 7 deubiquitinating enzymes (DUBs), which all contained various key factors that regulated a wide range of biological processes. Moreover, a series of novel ubiquitinated proteins and E3s were identified that had not been previously reported. This study investigated a high-efficiency method for identifying novel ubiquitinated proteins involved in biological processes and a primary mapping of the ubiquitylome during rice seedling development, which could extend our understanding of how ubiquitin modification regulates plant proteins, pathways and cellular processes.

  7. DMPD: Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16982211 Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Wullaer...vg) (.html) (.csml) Show Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. PubmedID 1698221...1 Title Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Author

  8. Animal Detectives

    Science.gov (United States)

    Mulvey, Bridget; Warnock, Carly

    2015-01-01

    During a two-week inquiry-based 5E learning cycle unit, children made observations and inferences to guide their explorations of animal traits and habitats (Bybee 2014). The children became "animal detectives" by studying a live-feed webcam and digital images of wolves in their natural habitat, reading books and online sources about…

  9. Kindergarten Animation

    Science.gov (United States)

    Hinshaw, Craig

    2012-01-01

    Animation is one of the last lessons that come to mind when thinking of kindergarten art. The necessary understanding of sequencing, attention to small, often detailed drawings, and the use of technology all seem more suitable to upper elementary. With today's emphasis on condensing and integrating curriculum, consider developing animation lessons…

  10. Animal cytomegaloviruses.

    OpenAIRE

    Staczek, J.

    1990-01-01

    Cytomegaloviruses are agents that infect a variety of animals. Human cytomegalovirus is associated with infections that may be inapparent or may result in severe body malformation. More recently, human cytomegalovirus infections have been recognized as causing severe complications in immunosuppressed individuals. In other animals, cytomegaloviruses are often associated with infections having relatively mild sequelae. Many of these sequelae parallel symptoms associated with human cytomegalovir...

  11. ANIMAL code

    International Nuclear Information System (INIS)

    This report describes ANIMAL, a two-dimensional Eulerian magnetohydrodynamic computer code. ANIMAL's physical model also appears. Formulated are temporal and spatial finite-difference equations in a manner that facilitates implementation of the algorithm. Outlined are the functions of the algorithm's FORTRAN subroutines and variables

  12. Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

    Directory of Open Access Journals (Sweden)

    Scheuring David

    2012-09-01

    Full Text Available Abstract Background In yeast and mammals, many plasma membrane (PM proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. Results Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. Conclusions Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route

  13. Animal learning.

    Science.gov (United States)

    Castro, Leyre; Wasserman, Edward A

    2010-01-01

    Pavlov and Thorndike pioneered the experimental study of animal learning and provided psychologists with powerful tools to unveil its underlying mechanisms. Today's research developments and theoretical analyses owe much to the pioneering work of these early investigators. Nevertheless, in the evolution of our knowledge about animal learning, some initial conceptions have been challenged and revised. We first review the original experimental procedures and findings of Pavlov and Thorndike. Next, we discuss critical research and consequent controversies which have greatly shaped animal learning theory. For example, although contiguity seemed to be the only condition that is necessary for learning, we now know that it is not sufficient; the conditioned stimulus (CS) also has to provide information about the occurrence of the unconditioned stimulus (US). Also, animals appear to learn different things about the same stimuli when circumstances vary. For instance, when faced with situations in which the meaning of a CS changes, as in the case of acquisition and later extinction, animals seem to preserve the original knowledge (CS-US) in addition to learning about the new conditions (CS-noUS). Finally, we discuss how parallels among Pavlovian conditioning, operant conditioning, and human causal judgment suggest that causal knowledge may lie at the root of both human and animal learning. All of these empirical findings and theoretical developments prove that animal learning is more complex and intricate than was once imagined. Copyright © 2009 John Wiley & Sons, Ltd. For further resources related to this article, please visit the WIREs website. PMID:26272842

  14. Involvement of protein degradation by the ubiquitin proteasome system in opiate addictive behaviors.

    Science.gov (United States)

    Massaly, Nicolas; Dahan, Lionel; Baudonnat, Mathieu; Hovnanian, Caroline; Rekik, Khaoula; Solinas, Marcello; David, Vincent; Pech, Stéphane; Zajac, Jean-Marie; Roullet, Pascal; Mouledous, Lionel; Frances, Bernard

    2013-03-01

    Plastic changes in the nucleus accumbens (NAcc), a structure occupying a key position in the neural circuitry related to motivation, are among the critical cellular processes responsible for drug addiction. During the last decade, it has been shown that memory formation and related neuronal plasticity may rely not only on protein synthesis but also on protein degradation by the ubiquitin proteasome system (UPS). In this study, we assess the role of protein degradation in the NAcc in opiate-related behaviors. For this purpose, we coupled behavioral experiments to intra-accumbens injections of lactacystin, an inhibitor of the UPS. We show that protein degradation in the NAcc is mandatory for a full range of animal models of opiate addiction including morphine locomotor sensitization, morphine conditioned place preference, intra-ventral tegmental area morphine self-administration and intra-venous heroin self-administration but not for discrimination learning rewarded by highly palatable food. This study provides the first evidence of a specific role of protein degradation by the UPS in addiction.

  15. Wild Animals

    Institute of Scientific and Technical Information of China (English)

    宁静

    2005-01-01

    Many of us think that all wild animals are dangerous. In fact, very few of them will eat a man if he leaves them alone. If you meet a tiger, I'm sure you will run away, but even a tiger doesn't like meeting a man if it isn't hungry. Tigers only kill and eat man when they are too old to catch their food, such as sheep and other small animals. Some animals get frightened when they only smell a man. Some of themst and and look at a man for a short time before they run away.

  16. Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock.

    Science.gov (United States)

    Li, Guang-Qi; Zang, Xiao-Nan; Zhang, Xue-Cheng; Lu, Ning; Ding, Yan; Gong, Le; Chen, Wen-Chao

    2014-03-15

    To study the response of Gracilaria lemaneiformis to heat stress, two key enzymes - ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) - of the Ubiquitin/26S proteasome pathway (UPP) were studied in three strains of G. lemaneiformis-wild type, heat-tolerant cultivar 981 and heat-tolerant cultivar 07-2. The full length DNA sequence of E1 contained only one exon. The open reading frame (ORF) sequence was 981 nucleotides encoding 326 amino acids, which contained conserved ATP binding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVCAI) and the ubiquitin-activating domains (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The gene sequence of E2 contained four exons and three introns. The sum of the four exons gave an open reading frame sequence of 444 nucleotides encoding 147 amino acids, which contained a conserved ubiquitin-activating domain (GSICLDIL), ubiquitin-conjugating domains (RIYHPNIN, KVLLSICSLL, DDPLV) and ubiquitin-ligase (E3) recognition sites (KRI, YPF, WSP). Real-time-PCR analysis of transcription levels of E1 and E2 under heat shock conditions (28°C and 32°C) showed that in wild type, transcriptions of E1 and E2 were up-regulated at 28°C, while at 32°C, transcriptions of the two enzymes were below the normal level. In cultivar 981 and cultivar 07-2 of G. lemaneiformis, the transcription levels of the two enzymes were up-regulated at 32°C, and transcription level of cultivar 07-2 was even higher than that of cultivar 981. These results suggest that the UPP plays an important role in high temperature resistance of G. lemaneiformis and the bioactivity of UPP is directly related to the heat-resistant ability of G. lemaneiformis.

  17. Shigella flexneri suppresses NF-κB activation by inhibiting linear ubiquitin chain ligation.

    Science.gov (United States)

    de Jong, Maarten F; Liu, Zixu; Chen, Didi; Alto, Neal M

    2016-01-01

    The linear ubiquitin chain assembly complex (LUBAC) is a multimeric E3 ligase that catalyses M1 or linear ubiquitination of activated immune receptor signalling complexes (RSCs). Mutations that disrupt linear ubiquitin assembly lead to complex disease pathologies including immunodeficiency and autoinflammation in both humans and mice, but microbial toxins that target LUBAC function have not yet been discovered. Here, we report the identification of two homologous Shigella flexneri type III secretion system effector E3 ligases IpaH1.4 and IpaH2.5, which directly interact with LUBAC subunit Heme-oxidized IRP2 ubiquitin ligase-1 (HOIL-1L) and conjugate K48-linked ubiquitin chains to the catalytic RING-between-RING domain of HOIL-1-interacting protein (HOIP). Proteasomal degradation of HOIP leads to irreversible inactivation of linear ubiquitination and blunting of NF-κB nuclear translocation in response to tumour-necrosis factor (TNF), IL-1β and pathogen-associated molecular patterns. Loss of function studies in mammallian cells in combination with bacterial genetics explains how Shigella evades a broad spectrum of immune surveillance systems by cooperative inhibition of receptor ubiquitination and reveals the critical importance of LUBAC in host defence against pathogens. PMID:27572974

  18. Acetylation stimulates the epithelial sodium channel by reducing its ubiquitination and degradation.

    Science.gov (United States)

    Butler, Phillip L; Staruschenko, Alexander; Snyder, Peter M

    2015-05-15

    The epithelial Na(+) channel (ENaC) functions as a pathway for Na(+) absorption in the kidney and lung, where it is crucial for Na(+) homeostasis and blood pressure regulation. ENaC is regulated in part through signaling pathways that control the ubiquitination state of ENaC lysines. A defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. Here we determined that α-, β-, and γENaC are also substrates for lysine acetylation. Trichostatin A (TSA), a histone deacetylase inhibitor, enhanced ENaC acetylation and increased ENaC abundance in the total cell lysate and at the cell surface. Moreover, TSA increased ENaC current in Fischer rat thyroid and kidney collecting duct epithelia. We found that HDAC7 is expressed in the kidney collecting duct, supporting a potential role for this histone deacetylase in ENaC regulation. HDAC7 overexpression reduced ENaC abundance and ENaC current, whereas ENaC abundance and current were increased by silencing of HDAC7. ENaC and HDAC7 form a complex, as detected by coimmunoprecipitation. We observed a reciprocal relationship between acetylation and ubiquitination; TSA reduced ENaC ubiquitination, whereas HDAC7 increased ubiquitination. By reducing ENaC ubiquitination, TSA decreased the rate of ENaC degradation. Thus, acetylation increases epithelial Na(+) absorption by antagonizing ENaC ubiquitination. This stabilizes ENaC, and hence, increases its abundance at the cell surface. PMID:25787079

  19. Microsecond molecular dynamics simulation of guanidinium chloride induced unfolding of ubiquitin.

    Science.gov (United States)

    Mandal, Manoj; Mukhopadhyay, Chaitali

    2014-10-21

    An all atom molecular dynamics simulation was used to explore the atomic detail mechanism of guanidinium induced unfolding of the protein ubiquitin. Ubiquitin unfolds through pre-unfolded (intermediate) states, i.e. guanidinium induced unfolding of ubiquitin appears to be a multi-step process, and loss of hydrophobic contacts of C-terminal residues is crucial for ubiquitin unfolding. Free-energy landscapes show that barrier separation between folded and unfolded basins is ∼5.0 kcal mol(-1), and both the basins are of comparable energy. It was observed that guanidinium ions interact directly with ubiquitin. Favorable electrostatic interaction is the main driving force for such accumulation of guanidinium ions near protein, but van der Waals energy also contributes. RDF plots show that accumulation of guanidinium ions near specific residues is the main cause for destabilization of intra-residue interactions crucial to maintain the three-dimensional fold of the protein. One salt-bridge interaction between Lys11 and Glu34 appears to be important to maintain the crystal structure of ubiquitin and this salt-bridge can map the unfolding process of ubiquitin. PMID:25197836

  20. The ubiquitin-proteasome system in glioma cell cycle control

    Directory of Open Access Journals (Sweden)

    Vlachostergios Panagiotis J

    2012-07-01

    Full Text Available Abstract A major determinant of cell fate is regulation of cell cycle. Tight regulation of this process is lost during the course of development and progression of various tumors. The ubiquitin-proteasome system (UPS constitutes a universal protein degradation pathway, essential for the consistent recycling of a plethora of proteins with distinct structural and functional roles within the cell, including cell cycle regulation. High grade tumors, such as glioblastomas have an inherent potential of escaping cell cycle control mechanisms and are often refractory to conventional treatment. Here, we review the association of UPS with several UPS-targeted proteins and pathways involved in regulation of the cell cycle in malignant gliomas, and discuss the potential role of UPS inhibitors in reinstitution of cell cycle control.

  1. Structure And Function of the Yeast U-Box-Containing Ubiquitin Ligase Ufd2p

    Energy Technology Data Exchange (ETDEWEB)

    Tu, D.; Li, W.; Ye, Y.; Brunger, A.T.

    2009-06-04

    Proteins conjugated by Lys-48-linked polyubiquitin chains are preferred substrates of the eukaryotic proteasome. Polyubiquitination requires an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). Occasionally, these enzymes only assemble short ubiquitin oligomers, and their extension to full length involves a ubiquitin elongating factor termed E4. Ufd2p, as the first E4 identified to date, is involved in the degradation of misfolded proteins of the endoplasmic reticulum and of a ubiquitin-{beta}-GAL fusion substrate in Saccharomyces cerevisiae. The mechanism of action of Ufd2p is unknown. Here we describe the crystal structure of the full-length yeast Ufd2p protein. Ufd2p has an elongated shape consisting of several irregular Armadillo-like repeats with two helical hairpins protruding from it and a U-box domain flexibly attached to its C terminus. The U-box of Ufd2p has a fold similar to that of the RING (Really Interesting New Gene) domain that is present in certain ubiquitin ligases. Accordingly, Ufd2p has all of the hallmarks of a RING finger-containing ubiquitin ligase: it associates with its cognate E2 Ubc4p via its U-box domain and catalyzes the transfer of ubiquitin from the E2 active site to Ufd2p itself or to an acceptor ubiquitin molecule to form unanchored diubiquitin oligomers. Thus, Ufd2p can function as a bona fide E3 ubiquitin ligase to promote ubiquitin chain elongation on a substrate.

  2. Animation & Neurocinematics*

    DEFF Research Database (Denmark)

    Carpe Pérez, Inmaculada Concepción

    2016-01-01

    machines that think”-(Damasio, A. Descartes error). Such feelings come from the interpretation of the emotions in our bodies. Emotions are our universal language, the motivation of living, the key to what makes a movie successful and truly an art piece that you will remember because moves you. Animation......, indeed, can be considered a social/ emotional learning media, which goes beyond the limitations of live action movies. This is due to the diversity of techniques, and its visual plasticity that constructs the impossible. Animators are not real actors but more like the midwife who brings the anima...... into aliveness, which requires knowing how emotions work. Ed Hooks as an expert in training animators and actors, always remarks: “emotions tend to lead to action”. In this paper we want to argue that by producing animated films, as we watch them, cause a stronger effect, not only in our brains, but also in our...

  3. Animal performance

    OpenAIRE

    Abaye, A. O. (Azenegashe Ozzie); Rotz, Jonathan Daniel; Scaglia Alonso, Guillermo, 1963-; Fike, John Herschel; Smith, Ray Lee, 1962-

    2009-01-01

    Any forage crop that stretches the grazing season by providing additional feed in early spring, mid-summer, and late fall will provide the livestock producer with lower feed costs and boost animal performance.

  4. Groundwater animals

    OpenAIRE

    Maurice, Louise; Bloomfield, John; Robertson, Anne; Allen, Debbie

    2010-01-01

    Groundwater animals are adapted to live in environments with no light and limited nutrients, They can provide insights into fundamental questions of evolution, ecology and biodiversity. They also have an important role to play in informing the reconstruction of past changes in geomorphology and climate, and can be used for characterising aquifers. The BGS is undertaking a systematic survey of selected areas and lithologies in the UK where groundwater animals have not been inves...

  5. RNF111/Arkadia is a SUMO-targeted ubiquitin ligase that facilitates the DNA damage response

    DEFF Research Database (Denmark)

    Poulsen, Sara L; Hansen, Rebecca K; Wagner, Sebastian A;

    2013-01-01

    Protein modifications by ubiquitin and small ubiquitin-like modifier (SUMO) play key roles in cellular signaling pathways. SUMO-targeted ubiquitin ligases (STUbLs) directly couple these modifications by selectively recognizing SUMOylated target proteins through SUMO-interacting motifs (SIMs......)-induced SUMOylation and ubiquitylation. Moreover, we show that RNF111 facilitated NER by regulating the recruitment of XPC to UV-damaged DNA. Our findings establish RNF111 as a new STUbL that directly links nonproteolytic ubiquitylation and SUMOylation in the DNA damage response....

  6. Regulation of MHC Class II-Peptide Complex Expression by Ubiquitination

    Directory of Open Access Journals (Sweden)

    Kyung Jin eCho

    2013-11-01

    Full Text Available MHC class II (MHC-II molecules are present on antigen presenting cells (APCs and these molecules function by binding antigenic peptides and presenting these peptides to antigen-specific CD4+ T cells. APCs continuously generate and degrade MHC-II molecules, and ubiquitination of MHC-II has recently been shown to be a key regulator of MHC-II expression in dendritic cells (DCs. In this mini-review we will examine the mechanism by which the E3 ubiquitin ligase March-I regulates MHC-II expression on APCs and will discuss the functional consequences of altering MHC-II ubiquitination.

  7. Exploiting the ubiquitin and phosphoinositide pathways by the Legionella pneumophila effector, SidC.

    Science.gov (United States)

    Wasilko, David J; Mao, Yuxin

    2016-02-01

    Intracellular bacterial pathogens use secreted effector proteins to alter host cellular processes, with the goal of subverting host defenses and allowing the infection to progress. One such pathogen, Legionella pneumophila, secretes ~300 proteins into its host to alter a number of pathways including intracellular trafficking, phosphoinositide metabolism, and cell signaling. The Legionella effector SidC was previously found to bind to PI(4)P and was responsible for the enrichment of ER proteins and ubiquitinated species on the Legionella-containing vacuoles. Through our recent work, we have discovered that SidC contains a unique N-terminal E3 ubiquitin ligase domain and a C-terminal novel PI(4)P-binding domain. Our results demonstrate that SidC serves to link two distinct cellular pathways, ubiquitin and phosphoinositide. However, how the ubiquitin ligase activity regulates host membrane trafficking events remains to be investigated. PMID:26433729

  8. Multiple sclerosis autoantigen myelin basic protein escapes control by ubiquitination during proteasomal degradation.

    Science.gov (United States)

    Belogurov, Alexey; Kudriaeva, Anna; Kuzina, Ekaterina; Smirnov, Ivan; Bobik, Tatyana; Ponomarenko, Natalia; Kravtsova-Ivantsiv, Yelena; Ciechanover, Aaron; Gabibov, Alexander

    2014-06-20

    The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.

  9. Animated symbols

    DEFF Research Database (Denmark)

    Frølunde, Lisbeth

    2008-01-01

    This paper is based on data about animation film production by 18-year-old students in a Danish upper secondary school. The optic is the on-going potential for learning and development of reflection. The purpose is to clarify what might support young people's reflection on media. I propose...... an analytic working model called Animated Symbols concerning critical reflection in a dialogic learning process. The model shows dialogue as interactions that involve two types of transformation: inner ‘learning processes' and outer signs and symbols. The classroom-based research study is part of a Ph...

  10. Biotecnologia animal

    OpenAIRE

    Luiz Lehmann Coutinho; Millor Fernandes do Rosário; Erika Cristina Jorge

    2010-01-01

    A biotecnologia animal tem fornecido novas ferramentas para os programas de melhoramento e, dessa forma, contribuído para melhorar a eficiência da produção dos produtos de origem animal. No entanto, os avanços têm sido mais lentos do que antecipados, especialmente em razão da dificuldade na identificação dos genes responsáveis pelas características fenotípicas de interesse zootécnico. Três estratégias principais têm sido utilizadas para identificar esses genes - mapeamento de QTL, genes candi...

  11. 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.

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

    OpenAIRE

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

    2015-01-01

    Objective Chronic, low-grade adipose tissue inflammation associated with adipocyte hypertrophy is an important link in the relationship between obesity and insulin resistance. Although ubiquitin ligases regulate inflammatory processes, the role of these enzymes in metabolically driven adipose tissue inflammation is relatively unexplored. Herein, we examined the effect of the ubiquitin ligase Siah2 on obesity-related adipose tissue inflammation. Methods Wild-type and Siah2KO mice were fed a lo...

  13. E3Miner: a text mining tool for ubiquitin-protein ligases

    OpenAIRE

    Lee, Hodong; Yi, Gwan-Su; Park, Jong C.

    2008-01-01

    Ubiquitination is a regulatory process critically involved in the degradation of >80% of cellular proteins, where such proteins are specifically recognized by a key enzyme, or a ubiquitin-protein ligase (E3). Because of this important role of E3s, a rapidly growing body of the published literature in biology and biomedical fields reports novel findings about various E3s and their molecular mechanisms. However, such findings are neither adequately retrieved by general text-mining tools nor sys...

  14. The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immunity

    DEFF Research Database (Denmark)

    Damgaard, Rune Busk; Nachbur, Ueli; Yabal, Monica;

    2012-01-01

    -linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly...... signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis....

  15. Fluorogenic ATP Analogues for Online Monitoring of ATP Consumption : Observing Ubiquitin Activation in Real Time

    OpenAIRE

    Hacker, Stephan; Pagliarini, Dana; Tischer, Thomas; Hardt, Normann; Schneider, Daniel; Mex, Martin; Mayer, Thomas; Scheffner, Martin; Marx, Andreas

    2013-01-01

    Many enzymes use ATP in signal-transducing processes or as an energy source. New fluorogenic ATP analogues signal ATP consumption by ubiquitin-like protein-activating enzymes in real time. Thus the inhibition and stimulation of these ATP-processing enzymes can be studied without auxiliary enzymes and reagents. beta-Lapachone was identified as an inhibitor of the ubiquitin-activating enzyme UBA1 (see scheme; A=acceptor, D=donor).

  16. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

    Science.gov (United States)

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-07-22

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

  17. Temporal separation of aggregation and ubiquitination during early inclusion formation in transgenic mice carrying the Huntington's disease mutation.

    Directory of Open Access Journals (Sweden)

    Belvin Gong

    Full Text Available Abnormal insoluble ubiqitinated protein aggregates are found in the brains of Huntington's disease (HD patients and in mice transgenic for the HTT mutation. Here, we describe the earliest stages of visible NII formation in brains of R6/2 mice killed between 2 and 6 weeks of age. We found that huntingtin-positive aggregates formed rapidly (within 24-48 hours in a spatiotemporal manner similar to that we described previously for ubiquitinated inclusions. However, in most neurons, aggregates are not ubiquitinated when they first form. It has always been assumed that mutant huntingtin is recognised as 'foreign' and consequently ubiquitinated and targeted for degradation by the ubiquitin-proteasome system pathway. Our data, however, suggest that aggregation and ubiquitination are separate processes, and that mutant huntingtin fragment is not recognized as 'abnormal' by the ubiquitin-proteasome system before aggregation. Rather, mutant Htt appears to aggregate before it is ubiquitinated, and then either aggregated huntingtin is ubiquitinated or ubiquitinated proteins are recruited into aggregates. Our findings have significant implications for the role of the ubiquitin-proteasome system in the formation of aggregates, as they suggest that this system is not involved until after the first aggregates form.

  18. Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

    Directory of Open Access Journals (Sweden)

    Scaife Jes R

    2008-02-01

    Full Text Available Abstract Background Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose (to stimulate insulin and essential amino acids (EAA would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of

  19. Animal house

    OpenAIRE

    Turka, Laurence A.

    2008-01-01

    While the JCI was originally conceived as a journal that would integrate various scientific approaches to the examination of human physiology and pathophysiology, we now find many of its pages filled with animal models of human disease. Is this a good thing?

  20. Animated war

    DEFF Research Database (Denmark)

    Frølunde, Lisbeth

    2012-01-01

    production: Gzim Rewind (Sweden, 2011) by Knutte Wester, and In-World War (USA, expected 2011) by DJ Bad Vegan. These films have themes of war and include film scenes that are ‘machinima’ (real-time animation made in 3D graphic environments) within live action film scenes. Machinima harnesses the...

  1. Animated Symbols

    DEFF Research Database (Denmark)

    Frolunde, Lisbeth

    ' processer af fem udvalgte elever er gennemgået i forhold til tre opdelinger: filmskabere, filmskabelse processen og film. Den teoretiske tilgang er pragmatisme, social semiotik og diskursanalyse. Modellen "Animating Symbols" er udviklet og diskuteret som forsøg på at forstå reflektion og design som en slags...

  2. Transgenic Animals.

    Science.gov (United States)

    Jaenisch, Rudolf

    1988-01-01

    Describes three methods and their advantages and disadvantages for introducing genes into animals. Discusses the predictability and tissue-specificity of the injected genes. Outlines the applications of transgenic technology for studying gene expression, the early stages of mammalian development, mutations, and the molecular nature of chromosomes.…

  3. Characterization and Structural Studies of the Plasmodium falciparum Ubiquitin and Nedd8 Hydrolase UCHL3

    Energy Technology Data Exchange (ETDEWEB)

    Artavanis-Tsakonas, Katerina; Weihofen, Wilhelm A.; Antos, John M.; Coleman, Bradley I.; Comeaux, Christy A.; Duraisingh, Manoj T.; Gaudet, Rachelle; Ploegh, Hidde L. (Whitehead); (Harvard); (Harvard-SPH)

    2010-03-29

    Like their human hosts, Plasmodium falciparum parasites rely on the ubiquitin-proteasome system for survival. We previously identified PfUCHL3, a deubiquitinating enzyme, and here we characterize its activity and changes in active site architecture upon binding to ubiquitin. We find strong evidence that PfUCHL3 is essential to parasite survival. The crystal structures of both PfUCHL3 alone and in complex with the ubiquitin-based suicide substrate UbVME suggest a rather rigid active site crossover loop that likely plays a role in restricting the size of ubiquitin adduct substrates. Molecular dynamics simulations of the structures and a model of the PfUCHL3-PfNedd8 complex allowed the identification of shared key interactions of ubiquitin and PfNedd8 with PfUCHL3, explaining the dual specificity of this enzyme. Distinct differences observed in ubiquitin binding between PfUCHL3 and its human counterpart make it likely that the parasitic DUB can be selectively targeted while leaving the human enzyme unaffected.

  4. Structural Basis for Ubiquitin Recognition by the Otu1 Ovarian Tumor Domain Protein

    Energy Technology Data Exchange (ETDEWEB)

    T Messick; N Russel; A Iwata; K Sarachan; R Shiekhattar; I Shanks; F Reyes-Turcu; K Wilkinson; R Marmorstein

    2011-12-31

    Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of {approx}130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys{sup 48} linkages, having little or no activity on Lys{sup 63}- and Lys{sup 29}-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.

  5. Interference with ubiquitination in CFTR modifies stability of core glycosylated and cell surface pools.

    Science.gov (United States)

    Lee, Seakwoo; Henderson, Mark J; Schiffhauer, Eric; Despanie, Jordan; Henry, Katherine; Kang, Po Wei; Walker, Douglas; McClure, Michelle L; Wilson, Landon; Sorscher, Eric J; Zeitlin, Pamela L

    2014-07-01

    It is recognized that both wild-type and mutant CFTR proteins undergo ubiquitination at multiple lysines in the proteins and in one or more subcellular locations. We hypothesized that ubiquitin is added to specific sites in wild-type CFTR to stabilize it and at other sites to signal for proteolysis. Mass spectrometric analysis of wild-type CFTR identified ubiquitinated lysines 68, 710, 716, 1041, and 1080. We demonstrate that the ubiquitinated K710, K716, and K1041 residues stabilize wild-type CFTR, protecting it from proteolysis. The polyubiquitin linkage is predominantly K63. N-tail mutants, K14R and K68R, lead to increased mature band CCFTR, which can be augmented by proteasomal (but not lysosomal) inhibition, allowing trafficking to the surface. The amount of CFTR in the K1041R mutant was drastically reduced and consisted of bands A/B, suggesting that the site in transmembrane 10 (TM10) was critical to further processing beyond the proteasome. The K1218R mutant increases total and cell surface CFTR, which is further accumulated by proteasomal and lysosomal inhibition. Thus, ubiquitination at residue 1218 may destabilize wild-type CFTR in both the endoplasmic reticulum (ER) and recycling pools. Small molecules targeting the region of residue 1218 to block ubiquitination or to preserving structure at residues 710 to 716 might be protein sparing for some forms of cystic fibrosis.

  6. Cullin-RING Ubiquitin Ligases in Salicylic Acid-Mediated Plant Immune Signaling

    Directory of Open Access Journals (Sweden)

    James J. Furniss

    2015-03-01

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

  7. Stressing the ubiquitin-proteasome system without 20S proteolytic inhibition selectively kills cervical cancer cells.

    Directory of Open Access Journals (Sweden)

    Ravi K Anchoori

    Full Text Available Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine.

  8. USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites.

    Science.gov (United States)

    Lee, Byung-Hoon; Lu, Ying; Prado, Miguel A; Shi, Yuan; Tian, Geng; Sun, Shuangwu; Elsasser, Suzanne; Gygi, Steven P; King, Randall W; Finley, Daniel

    2016-04-21

    USP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes. Its effects on protein turnover are substrate-specific, for unknown reasons. We report that USP14 shows a marked preference for ubiquitin-cyclin B conjugates that carry more than one ubiquitin modification or chain. This specificity is conserved from yeast to humans and is independent of chain linkage type. USP14 has been thought to cleave single ubiquitin groups from the distal tip of a chain, but we find that it removes chains from cyclin B en bloc, proceeding until a single chain remains. The suppression of degradation by USP14's catalytic activity reflects its capacity to act on a millisecond time scale, before the proteasome can initiate degradation of the substrate. In addition, single-molecule studies showed that the dwell time of ubiquitin conjugates at the proteasome was reduced by USP14-dependent deubiquitination. In summary, the specificity of the proteasome can be regulated by rapid ubiquitin chain removal, which resolves substrates based on a novel aspect of ubiquitin conjugate architecture. PMID:27074503

  9. The Ubiquitin-Conjugating System: Multiple Roles in Viral Replication and Infection

    Directory of Open Access Journals (Sweden)

    Arianna Calistri

    2014-05-01

    Full Text Available Through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiquitin to a target protein acts as a reversible post-translational modification functionally similar to phosphorylation. Indeed, ubiquitination is more and more recognized as a central process for the fine regulation of many cellular pathways. Due to their nature as obligate intracellular parasites, viruses rely on the most conserved host cell machineries for their own replication. Thus, it is not surprising that members from almost every viral family are challenged by ubiquitin mediated mechanisms in different steps of their life cycle and have evolved in order to by-pass or exploit the cellular ubiquitin conjugating system to maximize their chance to establish a successful infection. In this review we will present several examples of the complex interplay that links viruses and the ubiquitin conjugation machinery, with a special focus on the mechanisms evolved by the human immunodeficiency virus to escape from cellular restriction factors and to exit from infected cells.

  10. A phylogenetic analysis of the ubiquitin superfamily based on sequence and structural information.

    Science.gov (United States)

    Yang, Zhen; Chen, Haikui; Yang, Xiaobo; Wan, Xueshuai; He, Lian; Miao, Ruoyu; Yang, Huayu; Zhong, Yang; Wang, Li; Zhao, Haitao

    2014-09-01

    Ubiquitin belongs to an important class of protein modifier and gene expression regulator proteins that participates in various cellular processes. A large number of ubiquitin-related proteins have been identified during the last two decades. However, the evolutionary history of this ancient gene family remains largely unknown. We analyzed the members of the superfamily using both sequence- and structure-based methodology to better understand the evolution of ubiquitin-related proteins. As a part of these analyses we used the MEME algorithm to extract common sequence motifs across the superfamily, and we inferred the phylogeny and distribution of the superfamily members across multiple species. A total of 23 families were identified in the gene family. Several common sequence motifs were revealed and evaluated. We also found that the number of genes for ubiquitin-related proteins encoded within a specific genome correlates with the biological complexity of that particular species. This analysis should provide valuable insight into the sequence/function relationships and evolutionary history of ubiquitin and ubiquitin-related proteins. PMID:24997693

  11. CLONING AND CHARACTERIZATION OF HUMAN UBIQUITIN BINDING ENZYME 2 cDNA

    Institute of Scientific and Technical Information of China (English)

    李光涛; 吕鸿雁; 周严; 金鉴; 蒋科艺; 彭小忠; 袁建刚; 强伯勤

    2002-01-01

    Objective.To clone and identify the gene encoding human ubiquitin binding enzyme 2 and study its expression pattern. Methods.According to the sequence of human EST,which is highly homologous to the mouse ubiquitin binding/conjugating enzyme (E2),primers were synthesized to screen the human fetal brain cDNA library.The gene was analyzed by bioinformatics technique and its expression pattern was studied by using multiple tissue Northern blot. Results.Two cDNA clones encoding human ubiquitin conjugating enzyme have been isolated and identified.Both containing the ubiquitin conjugating domain,the 2 cDNA clones are 88% identical in amino acid sequences and splicing isoforms to each other only with an exon excised to form the short sequence.They belong to a highly conserved and widely expressed E2 enzyme family.Northern blot shows that they are expressed exclusively in adult human heart,placenta,and pancreas but no transcripts can be detected in brain,lung,liver,skeletal muscle or kidney.Conclusions.The gene encoding human ubiquitin binding enzyme is expressed under temporal control.As a key enzyme in the degradation of proteins,ubiquitin conjugating enzymes play a central role in the expression regulation on the level of post translation.

  12. RNF8-dependent histone ubiquitination during DNA damage response and spermatogenesis

    Institute of Scientific and Technical Information of China (English)

    Teng Ma; Jennifer A.Keller; Xiaochun Yu

    2011-01-01

    Histone ubiquitination regulates the chromatin structure that is important for many biological processes. Recently,ubiquitination of histones was observed during the DNA damage response (DDR), and this modification is controlled by really interesting new gene (RING) domain E3 ligase, RNF8. Together with the E2 conjugating enzyme UBC13, RNF8 catalyzes ubiquitination of the histones H2A and H2AX during the DDR, thus facilitating downstream recruitment of DDR factors, such as p53 binding protein 1 (53BP1) and breast cancer type 1 susceptibility protein (BRCA1), to the damage site.Accordingly, the RNF8 knockout mice display phenotypes associated with failed DDR, including hypersensitivity to ionizing radiation, V(D)J recombination deficiency, and a predisposition to cancer. In addition to the DDR phenotypes, RNF8 knockout mice fail to generate mature sperm during spermatogenesis, resulting in male sterility. The RNF8 knockout mice also have a drastic reduction in histone ubiquitination in the testes. These findings indicate that the role of histone ubiquitination during chromatin remodeling in two different biological events could be linked by an RNF8-dependent mechanism. Here, we review the molecular mechanism of RNF8-dependent histone ubiquitination both in DDR and spermatogenesis.

  13. Biotecnologia animal

    Directory of Open Access Journals (Sweden)

    Luiz Lehmann Coutinho

    2010-01-01

    Full Text Available A biotecnologia animal tem fornecido novas ferramentas para os programas de melhoramento e, dessa forma, contribuído para melhorar a eficiência da produção dos produtos de origem animal. No entanto, os avanços têm sido mais lentos do que antecipados, especialmente em razão da dificuldade na identificação dos genes responsáveis pelas características fenotípicas de interesse zootécnico. Três estratégias principais têm sido utilizadas para identificar esses genes - mapeamento de QTL, genes candidatos e sequenciamento de DNA e mRNA - e cada uma tem suas vantagens e limitações. O mapeamento de QTL permite determinar as regiões genômicas que contêm genes, mas o intervalo de confiança do QTL pode ser grande e conter muitos genes. A estratégia de genes candidatos é limitada por causa do conhecimento ainda restrito das funções de todos os genes. Os sequenciamentos de genomas e de sequências expressas podem auxiliar na identificação da posição de genes e de vias metabólicas associadas à característica de interesse. A integração dessas estratégias por meio do desenvolvimento de programas de bioinformática permitirá a identificação de novos genes de interesse zootécnico. Assim, os programas de melhoramento genético se beneficiarão pela inclusão da informação obtida diretamente do DNA na avaliação do mérito genético dos plantéis disponíveis.Animal biotechnology is providing new tools for animal breeding and genetics and thus contributing to advances in production efficiency and quality of animal products. However, the progress is slower than anticipated, mainly because of the difficulty involved in identifying genes that control phenotypic characteristics of importance to the animal industry. Three main strategies: QTL mapping, candidate genes and DNA and mRNA sequencing have been used to identify genes of economic interest to animal breeding and each has advantages and disadvantages. QTL mapping allows

  14. Arachidonate 15-lipoxygenase and ubiquitin as fertility markers in boars.

    Science.gov (United States)

    Lovercamp, K W; Safranski, T J; Fischer, K A; Manandhar, G; Sutovsky, M; Herring, W; Sutovsky, P

    2007-03-01

    Accurate semen analysis is an important issue in the swine industry. We evaluated two candidate fertility marker proteins associated with sperm cytoplasmic droplet (CD), including 15-lipoxygenase (15-LOX) and ubiquitin (UBI) in a controlled single-sire artificial insemination (AI) trial. Ejaculates (n=116) were collected from 18 fertile Large White boars monthly for 8 mo, and analyzed by semi-quantitative, densitometry-based Western blotting and flow cytometry with antibodies against 15-LOX and UBI. Data were correlated with farrowing rates (FR) and total numbers of piglets born (TNB) from 1754 AI services by 13 of 18 boars, and compared with a conventional microscopic semen analysis. In semi-quantitative Western blotting, both 15-LOX and UBI were correlated with seasonal changes in the percentage of normal (r=-0.38, Pflow cytometry, UBI and 15-LOX levels showed seasonal changes coinciding with seasonal changes of FR and TNB, representing 13 boars, 88 ejaculates and 1,232 AI services. There were correlations between flow cytometric values of UBI and FR (r=0.31; PFlow cytometric measurements of 15-LOX correlated negatively with TNB (r=-0.33; Pboar fertility estimation could be achieved within a group of fertile boars by the use of objectively measurable fertility markers. Flow cytometry appeared more informative and more practical than semi-quantitative Western blotting. This technology could be further optimized for the selection of the most fertile sires in an artificial insemination program. PMID:17116325

  15. Animal facilities

    International Nuclear Information System (INIS)

    The animal facilities in the Division are described. They consist of kennels, animal rooms, service areas, and technical areas (examining rooms, operating rooms, pathology labs, x-ray rooms, and 60Co exposure facilities). The computer support facility is also described. The advent of the Conversational Monitor System at Argonne has launched a new effort to set up conversational computing and graphics software for users. The existing LS-11 data acquisition systems have been further enhanced and expanded. The divisional radiation facilities include a number of gamma, neutron, and x-ray radiation sources with accompanying areas for related equipment. There are five 60Co irradiation facilities; a research reactor, Janus, is a source for fission-spectrum neutrons; two other neutron sources in the Chicago area are also available to the staff for cell biology studies. The electron microscope facilities are also described

  16. Animal Locomotion

    CERN Document Server

    Taylor, Graham K; Tropea, Cameron

    2010-01-01

    This book provides a wide-ranging snapshot of the state-of-the-art in experimental research on the physics of swimming and flying animals. The resulting picture reflects not only upon the questions that are of interest in current pure and applied research, but also upon the experimental techniques that are available to answer them. Doubtless, many new questions will present themselves as the scope and performance of our experimental toolbox develops over the coming years.

  17. Expression and cellular distribution of ubiquitin in response to injury in the developing spinal cord of Monodelphis domestica

    DEFF Research Database (Denmark)

    Noor, Natassya M; Møllgård, Kjeld; Wheaton, Benjamin J;

    2013-01-01

    Ubiquitin, an 8.5 kDa protein associated with the proteasome degradation pathway has been recently identified as differentially expressed in segment of cord caudal to site of injury in developing spinal cord. Here we describe ubiquitin expression and cellular distribution in spinal cord up to pos...... changes in ubiquitin expression and cellular distribution in development and response to spinal injury suggest an intricate regulatory system that modulates these responses which, when better understood, may lead to potential therapeutic targets....

  18. Redox regulation of E3 ubiquitin ligases and their role in skeletal muscle atrophy.

    Science.gov (United States)

    Olaso-Gonzalez, Gloria; Ferrando, Beatriz; Derbre, Frederic; Salvador-Pascual, Andrea; Cabo, Helena; Pareja-Galeano, Helios; Sabater-Pastor, Frederic; Gomez-Cabrera, Mari Carmen; Vina, Jose

    2014-10-01

    Muscle atrophy is linked to reactive oxygen species (ROS) production during hindlimb-unloading due, at least in part, to the activation of xanthine oxidase (XO). The major aim of our study was to determine the mechanism by which ROS cause muscle atrophy and its possible prevention by allopurinol, a well-known inhibitor of XO widely used in clinical practice, and indomethacin, a nonsteroidal anti-inflammatory drug. We studied the activation of p38 MAP Kinase and NF-?B pathways, and the expression of two E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFb) and Muscle RING Finger-1 (MuRF-1). Male Wistar rats (3 mold) conditioned by 14 days of hindlimb unloading (n=18), with or without the treatment, were compared with freely ambulating controls (n=18). After the experimental intervention, soleus muscles were removed, weighted and analyzed to determine oxidative stress and inflammatory parameters. We found that hindlimb unloading induced a significant increase in XO activity in plasma (39%, p=0.001) and in the protein expression of CuZnSOD and Catalase in skeletal muscle. Inhibitionof XO partially prevented protein carbonylation, both in plasma and in soleus muscle, in the unloaded animals. The most relevant new fact reported is that allopurinol prevents soleus muscle atrophy by ~20% after hindlimb unloading. Combining allopurinol and indomethacin we found a further prevention in the atrophy process. This is mediated by the inhibition of the p38 MAPK-MAFbx and NF-?B -MuRF-1 pathways. Our data point out the potential benefit of allopurinol and indomethacin administration for bedridden, astronauts, sarcopenic and cachexic patients. PMID:26461377

  19. Animal Drug Safety FAQs

    Science.gov (United States)

    ... Vaccines, Blood & Biologics Animal & Veterinary Cosmetics Tobacco Products Animal & Veterinary Home Animal & Veterinary Safety & Health Frequently Asked Questions Animal Drug Safety Frequently Asked Questions Share Tweet Linkedin ...

  20. It Is All about (U)biquitin: Role of Altered Ubiquitin-Proteasome System and UCHL1 in Alzheimer Disease

    Science.gov (United States)

    Tramutola, Antonella; Di Domenico, Fabio; Barone, Eugenio; Perluigi, Marzia; Butterfield, D. Allan

    2016-01-01

    Free radical-mediated damage to macromolecules and the resulting oxidative modification of different cellular components are a common feature of aging, and this process becomes much more pronounced in age-associated pathologies, including Alzheimer disease (AD). In particular, proteins are particularly sensitive to oxidative stress-induced damage and these irreversible modifications lead to the alteration of protein structure and function. In order to maintain cell homeostasis, these oxidized/damaged proteins have to be removed in order to prevent their toxic accumulation. It is generally accepted that the age-related accumulation of “aberrant” proteins results from both the increased occurrence of damage and the decreased efficiency of degradative systems. One of the most important cellular proteolytic systems responsible for the removal of oxidized proteins in the cytosol and in the nucleus is the proteasomal system. Several studies have demonstrated the impairment of the proteasome in AD thus suggesting a direct link between accumulation of oxidized/misfolded proteins and reduction of this clearance system. In this review we discuss the impairment of the proteasome system as a consequence of oxidative stress and how this contributes to AD neuropathology. Further, we focus the attention on the oxidative modifications of a key component of the ubiquitin-proteasome pathway, UCHL1, which lead to the impairment of its activity. PMID:26881020

  1. HIV-1 Nef down-modulates C-C and C-X-C chemokine receptors via ubiquitin and ubiquitin-independent mechanism.

    Directory of Open Access Journals (Sweden)

    Prabha Chandrasekaran

    Full Text Available Human and Simian Immunodeficiency virus (HIV-1, HIV-2, and SIV encode an accessory protein, Nef, which is a pathogenesis and virulence factor. Nef is a multivalent adapter that dysregulates the trafficking of many immune cell receptors, including chemokine receptors (CKRs. Physiological endocytic itinerary of agonist occupied CXCR4 involves ubiquitinylation of the phosphorylated receptor at three critical lysine residues and dynamin-dependent trafficking through the ESCRT pathway into lysosomes for degradation. Likewise, Nef induced CXCR4 degradation was critically dependent on the three lysines in the C-terminal -SSLKILSKGK- motif. Nef directly recruits the HECT domain E3 ligases AIP4 or NEDD4 to CXCR4 in the resting state. This mechanism was confirmed by ternary interactions of Nef, CXCR4 and AIP4 or NEDD4; by reversal of Nef effect by expression of catalytically inactive AIP4-C830A mutant; and siRNA knockdown of AIP4, NEDD4 or some ESCRT-0 adapters. However, ubiquitinylation dependent lysosomal degradation was not the only mechanism by which Nef downregulated CKRs. Agonist and Nef mediated CXCR2 (and CXCR1 degradation was ubiquitinylation independent. Nef also profoundly downregulated the naturally truncated CXCR4 associated with WHIM syndrome and engineered variants of CXCR4 that resist CXCL12 induced internalization via an ubiquitinylation independent mechanism.

  2. Food vacuole associated enolase in plasmodium undergoes multiple post-translational modifications: evidence for atypical ubiquitination.

    Directory of Open Access Journals (Sweden)

    Saudamini Shevade

    Full Text Available Plasmodium enolase localizes to several sub-cellular compartments viz. cytosol, nucleus, cell membrane, food vacuole (FV and cytoskeleton, without having any organelle targeting signal sequences. This enzyme has been shown to undergo multiple post-translational modifications (PTMs giving rise to several variants that show organelle specific localization. It is likely that these PTMs may be responsible for its diverse distribution and moonlighting functions. While most variants have a MW of ~50 kDa and are likely to arise due to changes in pI, food vacuole (FV associated enolase showed three forms with MW~50, 65 and 75 kDa. Evidence from immuno-precipitation and western analysis indicates that the 65 and 75 kDa forms of FV associated enolase are ubiquitinated. Using mass spectrometry (MS, definitive evidence is obtained for the nature of PTMs in FV associated variants of enolase. Results showed several modifications, viz. ubiquitination at K147, phosphorylation at Y148 and acetylation at K142 and K384. MS data also revealed the conjugation of three ubiquitin (Ub molecules to enolase through K147. Trimeric ubiquitin has a linear peptide linkage between the NH2-terminal methionine of the first ubiquitin (Ub1 and the C-terminal G76 of the second (Ub2. Ub2 and third ubiquitin (Ub3 were linked through an atypical isopeptide linkage between K6 of Ub2 and G76 of Ub3, respectively. Further, the tri-ubiquitinated form was found to be largely associated with hemozoin while the 50 and 65 kDa forms were present in the NP-40 soluble fraction of FV. Mass spectrometry results also showed phosphorylation of S42 in the cytosolic enolase from P. falciparum and T337 in the cytoskeleton associated enolase from P. yoelii. The composition of food vacuolar proteome and likely interactors of enolase are also being reported.

  3. Ubiquitin-Related Roles of β-Arrestins in Endocytic Trafficking and Signal Transduction.

    Science.gov (United States)

    Jean-Charles, Pierre-Yves; Rajiv, Vishwaesh; Shenoy, Sudha K

    2016-10-01

    The non-visual arrestins, β-arrestin1, and β-arrestin2 were originally identified as proteins that bind to seven-transmembrane receptors (7TMRs, also called G protein-coupled receptors, GPCRs) and block heterotrimeric G protein activation, thus leading to desensitization of transmembrane signaling. However, as subsequent discoveries have continually demonstrated, their functionality is not constrained to desensitization. They are now recognized for their critical roles in mediating intracellular trafficking of 7TMRs, growth factor receptors, ion transporters, ion channels, nuclear receptors, and non-receptor proteins. Additionally, they function as crucial mediators of ubiquitination of 7TMRs as well as other receptors and non-receptor proteins. Recently, emerging studies suggest that a class of proteins with predicted structural features of β-arrestins regulate substrate ubiquitination in yeast and higher mammals, lending support to the idea that the adaptor role of β-arrestins in protein ubiquitination is evolutionarily conserved. β-arrestins also function as scaffolds for kinases and transduce signals from 7TMRs through pathways that do not require G protein activation. Remarkably, the endocytic and scaffolding functions of β-arrestin are intertwined with its ubiquitination status; the dynamic and site specific ubiquitination on β-arrestin plays a critical role in stabilizing β-arrestin-7TMR association and the formation of signalosomes. This review summarizes the current findings on ubiquitin-dependent regulation of 7TMRs as well as β-arrestins and the potential role of reversible ubiquitination as a "biological switch" in signal transduction. J. Cell. Physiol. 231: 2071-2080, 2016. © 2016 Wiley Periodicals, Inc.

  4. Breast cancer cells: Modulation by melatonin and the ubiquitin-proteasome system--a review.

    Science.gov (United States)

    Vriend, Jerry; Reiter, Russel J

    2015-12-01

    Melatonin inhibits human breast cancer cells stimulated with estrogen. This antiproliferative action depends on the presence of the estrogen receptor alpha (ERα) in the human MCF-7 cell line and is strictly dose-dependent. Since researchers concerned with melatonin and breast cancer have not considered the relevance of the ubiquitin-proteasome system to this research in this review we do so. The fact that the first breast cancer susceptibility gene to be identified, Brca1, functions as a ubiquitin ligase indicates that the ubiquitin-proteasome system has a role in regulating susceptibility to breast cancer. While mutations of this gene increase the incidence of breast cancer, the wild type gene suppresses estrogen-dependent transcriptional events relying on the estrogen receptor ERα. Three other ubiquitin ligases, SCF(Skp2), E6AP and APC, interact directly with ERα at the ERE and AP-1 promoters of ERα target genes. Melatonin, like proteasome inhibitors, decreases estrogen-induced gene transcription. Indeed, it has been reported that melatonin specifically inhibits estrogen-induced transcription mediated by ERα at the ERE and AP1 gene promoters. Herein, we present a model in which the inhibitory action of melatonin on MCF-7 cells is mediated, directly or indirectly, by the ubiquitin-proteasome system. In this model ERα, apoptotic proteins, and cell cycle proteins, all influenced by melatonin, are substrates of key ubiquitin ligases including SCF(Skp2), E6AP, and SCF(B-TrCP). Since dysfunction of the ubiquitin-proteasome system is a risk factor for breast cancer, this model provides a context in which to test the clinical potential, and limitations, of melatonin and proteasome inhibitors. PMID:26363225

  5. Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks

    DEFF Research Database (Denmark)

    Poulsen, Maria; Lukas, Claudia; Lukas, Jiri;

    2012-01-01

    Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid double-strand breaks (DSBs), mediated by the RNF8/RNF168 ubiquitin ligases, plays a key role in recruiting repair factors, including 53BP1 and BRCA1, to reestablish genome integrity. In this paper, we show that human RNF......169, an uncharacterized E3 ubiquitin ligase paralogous to RNF168, accumulated in DSB repair foci through recognition of RNF168-catalyzed ubiquitylation products by its motif interacting with ubiquitin domain. Unexpectedly, RNF169 was dispensable for chromatin ubiquitylation and ubiquitin...

  6. Animal Testing

    Science.gov (United States)

    Moretto, Johnny; Chauffert, Bruno; Bouyer, Florence

    The development of a new anticancer drug is a long, complex and multistep process which is supervised by regulatory authorities from the different countries all around the world [1]. Application of a new drug for admission to the market is supported by preclinical and clinical data, both including the determination of pharmacodynamics, toxicity, antitumour activity, therapeutic index, etc. As preclinical studies are associated with high cost, optimization of animal experiments is crucial for the overall development of a new anticancer agent. Moreover, in vivo efficacy studies remain a determinant panel for advancement of agents to human trials and thus, require cautious design and interpretation from experimental and ethical point of views.

  7. Animated war

    DEFF Research Database (Denmark)

    Frølunde, Lisbeth

    2012-01-01

    in production: Gzim Rewind (Sweden, 2011) by Knutte Wester, and In-World War (USA, expected 2011) by DJ Bad Vegan. These films have themes of war and include film scenes that are ‘machinima’ (real-time animation made in 3D graphic environments) within live action film scenes. Machinima harnesses...... DIY multimedia storytellers explore new ways to tell and to ‘animate’ stories. The article contains four parts: introduction to machinima and the notions of resemiosis and authorial practice, presentation of DIY filmmaking as a practice that intertwines with new networked economics, analysis...

  8. Behavioral characteristics of ubiquitin-specific peptidase 46-deficient mice.

    Directory of Open Access Journals (Sweden)

    Saki Imai

    Full Text Available We have previously identified Usp46, which encodes for ubiquitin-specific peptidase 46, as a quantitative trait gene affecting the immobility time of mice in the tail suspension test (TST and forced swimming test. The mutation that we identified was a 3-bp deletion coding for lysine (Lys 92, and mice with this mutation (MT mice, as well as Usp46 KO mice exhibited shorter TST immobility times. Behavioral pharmacology suggests that the gamma aminobutyric acid A (GABAA receptor is involved in regulating TST immobility time. In order to understand how far Usp46 controls behavioral phenotypes, which could be related to mental disorders in humans, we subjected Usp46 MT and KO mice to multiple behavioral tests, including the open field test, ethanol preference test, ethanol-induced loss of righting reflex test, sucrose preference test, novelty-suppressed feeding test, marble burying test, and novel object recognition test. Although behavioral phenotypes of the Usp46 MT and KO mice were not always identical, deficiency of Usp46 significantly affected performance in all these tests. In the open field test, activity levels were lower in Usp46 KO mice than wild type (WT or MT mice. Both MT and KO mice showed lower ethanol preference and shorter recovery times after ethanol administration. Compared to WT mice, Usp46 MT and KO mice exhibited decreased sucrose preference, took longer latency periods to bite pellets, and buried more marbles in the sucrose preference test, novelty-suppressed feeding test, and marble burying test, respectively. In the novel object recognition test, neither MT nor KO mice showed an increase in exploration of a new object 24 hours after training. These findings indicate that Usp46 regulates a wide range of behavioral phenotypes that might be related to human mental disorders and provides insight into the function of USP46 deubiquitinating enzyme in the neural system.

  9. The Ubiquitin-Proteasome System and Microvascular Complications of Diabetes

    Directory of Open Access Journals (Sweden)

    Saeed Yadranji Aghdam

    2013-01-01

    Full Text Available The ubiquitin-proteasome system (UPS is the mainstay of protein quality control which regulates cell cycle, differentiation and various signal transduction pathways in eukaryotic cells. The timely and selective degradation of surplus and/or aberrant proteins by the UPS is essential for normal cellular physiology. Any disturbance, delay or exaggeration in the process of selection, sequestration, labeling for degradation and degradation of target proteins by the UPS will compromise cellular and tissue homeostasis. High blood glucose or hyperglycemia caused by diabetes disrupts normal vascular function in several target organs including the retina and kidney resulting in the development of diabetic retinopathy (DR and diabetic nephropathy (DN. We and others have shown that hyperglycemia and oxidative stress modulate UPS activity in the retina and kidney. The majority of studies have focused on the kidney and provided insights into the contribution of dysregulated UPS to microvascular damage in DN. The eye is a unique organ in which a semi-fluid medium, the vitreous humor, separates the neural retina and its anastomosed blood vessels from the semi-solid lens tissue. The complexity of the cellular and molecular components of the eye may require a normal functioning and well tuned UPS for healthy vision. Altered UPS activity may contribute to the development of retinal microvascular complications of diabetes. A better understanding of the molecular nature of the ocular UPS function under normal and diabetic conditions is essential for development of novel strategies targeting its activity. This review will discuss the association of retinal vascular cell UPS activity with microvascular damage in DR with emphasis on alterations of the PA28 subunits of the UPS.

  10. Cdh1 regulates craniofacial development via APC-dependent ubiquitination and activation of Goosecoid.

    Science.gov (United States)

    Shao, Rui; Liu, Jia; Yan, Guang; Zhang, Jinfang; Han, Yujiao; Guo, Jianfeng; Xu, Zhan; Yuan, Zhu; Liu, Jiankang; Malumbres, Marcos; Wan, Lixin; Wei, Wenyi; Zou, Weiguo

    2016-06-01

    Craniofacial anomalies (CFAs) characterized by birth defects of skull and facial bones are the most frequent congenital disease. Genomic analysis has identified multiple genes responsible for CFAs; however, the underlying genetic mechanisms for the majority of CFAs remain largely unclear. Our previous study revealed that the Wwp2 E3 ubiquitin ligase facilitates craniofacial development in part through inducing monoubiquitination and activation of the paired-like homeobox transcription factor, Goosecoid (Gsc). Here we report that Gsc is also ubiquitinated and activated by the APC(Cdh1) E3 ubiquitin ligase, leading to transcriptional activation of various Gsc target genes crucial for craniofacial development. Consistenly, neural crest-specific Cdh1-knockout mice display similar bone malformation as Wwp2-deficient mice in the craniofacial region, characterized by a domed skull, a short snout and a twisted nasal bone. Mechanistically, like Wwp2-deficient mice, mice with Cdh1 deficiency in neural crest cells exhibit reduced Gsc/Sox6 transcriptional activities. Simultaneous deletion of Cdh1 and Wwp2 results in a more severe craniofacial defect compared with single gene deletion, suggesting a synergistic augmentation of Gsc activity by these two E3 ubiquitin ligases. Hence, our study reveals a novel role for Cdh1 in craniofacial development through promoting APC-dependent non-proteolytic ubiquitination and activation of Gsc.

  11. It's all about talking: two-way communication between proteasomal and lysosomal degradation pathways via ubiquitin.

    Science.gov (United States)

    Liebl, Martina P; Hoppe, Thorsten

    2016-08-01

    Selective degradation of proteins requires a fine-tuned coordination of the two major proteolytic pathways, the ubiquitin-proteasome system (UPS) and autophagy. Substrate selection and proteolytic activity are defined by a plethora of regulatory cofactors influencing each other. Both proteolytic pathways are initiated by ubiquitylation to mark substrate proteins for degradation, although the size and/or topology of the modification are different. In this context E3 ubiquitin ligases, ensuring the covalent attachment of activated ubiquitin to the substrate, are of special importance. The regulation of E3 ligase activity, competition between different E3 ligases for binding E2 conjugation enzymes and substrates, as well as their interplay with deubiquitylating enzymes (DUBs) represent key events in the cross talk between the UPS and autophagy. The coordination between both degradation routes is further influenced by heat shock factors and ubiquitin-binding proteins (UBPs) such as p97, p62, or optineurin. Mutations in enzymes and ubiquitin-binding proteins or a general decline of both proteolytic systems during aging result in accumulation of damaged and aggregated proteins. Thus further mechanistic understanding of how UPS and autophagy communicate might allow therapeutic intervention especially against age-related diseases. PMID:27225656

  12. Role of ubiquitin C-terminal hydrolase-L1 in antipolyspermy defense of mammalian oocytes.

    Science.gov (United States)

    Susor, Andrej; Liskova, Lucie; Toralova, Tereza; Pavlok, Antonin; Pivonkova, Katerina; Karabinova, Pavla; Lopatarova, Miloslava; Sutovsky, Peter; Kubelka, Michal

    2010-06-01

    The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

  13. 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.

  14. What do we really know about the ubiquitin-proteasome pathway in muscle atrophy?

    Science.gov (United States)

    Jagoe, R. T.; Goldberg, A. L.

    2001-01-01

    Studies of many different rodent models of muscle wasting have indicated that accelerated proteolysis via the ubiquitin-proteasome pathway is the principal cause of muscle atrophy induced by fasting, cancer cachexia, metabolic acidosis, denervation, disuse, diabetes, sepsis, burns, hyperthyroidism and excess glucocorticoids. However, our understanding about how muscle proteins are degraded, and how the ubiquitin-proteasome pathway is activated in muscle under these conditions, is still very limited. The identities of the important ubiquitin-protein ligases in skeletal muscle, and the ways in which they recognize substrates are still largely unknown. Recent in-vitro studies have suggested that one set of ubquitination enzymes, E2(14K) and E3(alpha), which are responsible for the 'N-end rule' system of ubiquitination, plays an important role in muscle, especially in catabolic states. However, their functional significance in degrading different muscle proteins is still unclear. This review focuses on the many gaps in our understanding of the functioning of the ubiquitin-proteasome pathway in muscle atrophy, and highlights the strengths and limitations of the different experimental approaches used in such studies.

  15. Decreased outer membrane permeability protects mycobacteria from killing by ubiquitin-derived peptides.

    Science.gov (United States)

    Purdy, Georgiana E; Niederweis, Michael; Russell, David G

    2009-09-01

    Ubiquitin-derived peptides are bactericidal in vitro and contribute to the mycobactericidal activity of the lysosome. To further define interactions of ubiquitin-derived peptides with mycobacteria, we screened for mutants with increased resistance to the bactericidal activity of the synthetic ubiquitin-derived peptide Ub2. The four Ub2-resistant Mycobacterium smegmatis mutants were also resistant to the bactericidal action of other antimicrobial peptides and macrophages. Two mutants were in the mspA gene encoding the main M. smegmatis porin. Using a translocation-deficient MspA point mutant, we showed that susceptibility of M. smegmatis to Ub2 was independent of MspA channel activity. Instead, the M. smegmatis Ub2-resistant mutants shared a common phenotype of decreased cell wall permeability compared with wild-type bacteria. Expression of mspA rendered Mycobacterium tuberculosis CDC1551 more susceptible both to ubiquitin-derived peptides in vitro and to lysosomal killing in macrophages. Finally, biochemical assays designed to assess membrane integrity indicated that Ub2 treatment impairs membrane function of M. smegmatis and M. tuberculosis cells. The M. smegmatis Ub2-resistant mutants were more resistant than wild-type M. smegmatis to this damage. We conclude that Ub2 targets mycobacterial membranes and that reduced membrane permeability provides mycobacteria intrinsic resistance against antimicrobial compounds including bactericidal ubiquitin-derived peptides. PMID:19682257

  16. New Insights Into Roles of Ubiquitin Modification in Regulating Plastids and Other Endosymbiotic Organelles.

    Science.gov (United States)

    Broad, W; Ling, Q; Jarvis, P

    2016-01-01

    Recent findings have revealed important and diverse roles for the ubiquitin modification of proteins in the regulation of endosymbiotic organelles, which include the primary plastids of plants as well as complex plastids: the secondary endosymbiotic organelles of cryptophytes, alveolates, stramenopiles, and haptophytes. Ubiquitin modifications have a variety of potential consequences, both to the modified protein itself and to cellular regulation. The ubiquitin-proteasome system (UPS) can target individual proteins for selective degradation by the cytosolic 26S proteasome. Ubiquitin modifications can also signal the removal of whole endosymbiotic organelles, for example, via autophagy as has been well characterized in mitochondria. As plastids must import over 90% of their proteins from the cytosol, the observation that the UPS selectively targets the plastid protein import machinery is particularly significant. In this way, the UPS may influence the development and interconversions of different plastid types, as well as plastid responses to stress, by reconfiguring the organellar proteome. In complex plastids, the Symbiont-derived ERAD-Like Machinery (SELMA) has coopted the protein transport capabilities of the ER-Associated Degradation (ERAD) system, whereby misfolded proteins are retrotranslocated from ER for proteasomal degradation, uncoupling them from proteolysis: SELMA components have been retargeted to the second outermost plastid membrane to mediate protein import. In spite of this wealth of new information, there still remain a large number of unanswered questions and a need to define the roles of ubiquitin modification further in the regulation of plastids. PMID:27241217

  17. Animal Intuitions.

    Science.gov (United States)

    Kaebnick, Gregory E

    2016-07-01

    As described by Lori Gruen in the Perspective column at the back of this issue, federally supported biomedical research conducted on chimpanzees has now come to an end in the United States, although the wind-down has taken longer than expected. The process began with a 2011 Institute of Medicine report that set up several stringent criteria that sharply limited biomedical research. The National Institutes of Health accepted the recommendations and formed a committee to determine how best to implement them. The immediate question raised by this transition was whether the IOM restrictions should be extended in some form to other nonhuman primates-and beyond them to other kinds of animals. In the lead article in this issue, Anne Barnhill, Steven Joffe, and Franklin Miller consider the status of other nonhuman primates. PMID:27417859

  18. Bioethical Problems: Animal Welfare, Animal Rights.

    Science.gov (United States)

    March, B. E.

    1984-01-01

    Discusses various bioethical issues and problems related to animal welfare and animal rights. Areas examined include: Aristotelian views; animal welfare legislation; Darwin and evolutionary theory; animal and human behavior; and vegetarianism. A 14-point universal declaration of the rights of animals is included. (JN)

  19. Roles of Ubiquitination and SUMOylation on Prostate Cancer: Mechanisms and Clinical Implications

    Directory of Open Access Journals (Sweden)

    Zhenbang Chen

    2015-02-01

    Full Text Available The initiation and progression of human prostate cancer are highly associated with aberrant dysregulations of tumor suppressors and proto-oncogenes. Despite that deletions and mutations of tumor suppressors and aberrant elevations of oncogenes at the genetic level are reported to cause cancers, emerging evidence has revealed that cancer progression is largely regulated by posttranslational modifications (PTMs and epigenetic alterations. PTMs play critical roles in gene regulation, cellular functions, tissue development, diseases, malignant progression and drug resistance. Recent discoveries demonstrate that ubiquitination and SUMOylation are complicated but highly-regulated PTMs, and make essential contributions to diseases and cancers by regulation of key factors and signaling pathways. Ubiquitination and SUMOylation pathways can be differentially modulated under various stimuli or stresses in order to produce the sustained oncogenic potentials. In this review, we discuss some new insights about molecular mechanisms on ubiquitination and SUMOylation, their associations with diseases, oncogenic impact on prostate cancer (PCa and clinical implications for PCa treatment.

  20. Data in support of UbSRD: The Ubiquitin Structural Relational Database.

    Science.gov (United States)

    Harrison, Joseph S; Jacobs, Tim M; Houlihan, Kevin; Van Doorslaer, Koenraad; Kuhlman, Brian

    2015-12-01

    This article provides information to support the database article titled "UbSRD: The Ubiquitin Structural Relational Database" (Harrison et al., 2015) [1] . The ubiquitin-like homology fold (UBL) represents a large family that encompasses both post-translational modifications, like ubiquitin (UBQ) and SUMO, and functional domains on many biologically important proteins like Parkin, UHRF1 (ubiquitin-like with PDB and RING finger domains-1), and Usp7 (ubiquitin-specific protease-7) (Zhang et al., 2015; Rothbart et al., 2013; Burroughs et al., 2012; Wauer et al., 2015) [2], [3], [4], [5]. The UBL domain can participate in several unique protein-protein interactions (PPI) since protein adducts can be attached to and removed from amino groups of lysine side chains and the N-terminus of proteins. Given the biological significance of UBL domains, many have been characterized with high-resolution techniques, and for UBQ and SUMO, many protein complexes have been characterized. We identified all the UBL domains in the PDB and created a relational database called UbSRD (Ubiquitin Structural Relational Database) by using structural analysis tools in the Rosetta (Leaver et al., 2013; O'Meara et al., 2015; Leaver-fay et al., 2011) [1], [6], [7], [8]. Querying UbSRD permitted us to report many quantitative properties of UBQ and SUMO recognition at different types interfaces (noncovalent: NC, conjugated: CJ, and deubiquitanse: DB). In this data article, we report the average number of non-UBL neighbors, secondary structure of interacting motifs, and the type of inter-molecular hydrogen bonds for each residue of UBQ and SUMO. Additionally, we used PROMALS3D to generate a multiple sequence alignment used to construct a phylogram for the entire set of UBLs (Pei and Grishin, 2014) [9]. The data described here will be generally useful to scientists studying the molecular basis for recognition of UBQ or SUMO. PMID:26958617

  1. Secondary Structures of Ubiquitin Ions Soft-Landed onto Self-Assembled Monolayer Surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Qichi; Laskin, Julia

    2016-06-09

    The secondary structures of multiply charged ubiquitin ions soft-landed onto self-assembled monolayer (SAM) surfaces were studied using in situ infrared reflection-absorption spectroscopy (IRRAS). Two charge states of ubiquitin, 5+ and 13+, were mass selected separately from a mixture of different charge states produced by electrospray ionization (ESI). The low 5+ charge state represents a native-like folded state of ubiquitin, while the high 13+ charge state assumes an extended, almost linear conformation. Each of the two charge states was soft-landed onto a CH3- and COOH-terminated SAM of alkylthiols on gold (HSAM and COOH-SAM). HSAM is a hydrophobic surface known to stabilize helical conformations of soft-landed protonated peptides, whereas COOH-SAM is a hydrophilic surface that preferentially stabilizes β-sheet conformations. IRRAS spectra of the soft-landed ubiquitin ions were acquired as a function of time during and after ion soft-landing. Similar to smaller peptide ions, helical conformations of ubiquitin are found to be more abundant on HSAM, while the relative abundance of β-sheet conformations increases on COOH-SAM. The initial charge state of ubiquitin also has a pronounced effect on its conformation on the surface. Specifically, on both surfaces, a higher relative abundance of helical conformations and lower relative abundance of β-sheet conformations is observed for the 13+ charge state compared to the 5+ charge state. Time-resolved experiments indicate that the α-helical band in the spectrum of the 13+ charge state slowly increases with time on the HSAM surface and decreases in the spectrum of the 13+ charge state on COOH-SAM. These results further support the preference of the hydrophobic HSAM surface toward helical conformations and demonstrate that soft-landed protein ions may undergo slow conformational changes during and after deposition.

  2. Data in support of UbSRD: The Ubiquitin Structural Relational Database.

    Science.gov (United States)

    Harrison, Joseph S; Jacobs, Tim M; Houlihan, Kevin; Van Doorslaer, Koenraad; Kuhlman, Brian

    2015-12-01

    This article provides information to support the database article titled "UbSRD: The Ubiquitin Structural Relational Database" (Harrison et al., 2015) [1] . The ubiquitin-like homology fold (UBL) represents a large family that encompasses both post-translational modifications, like ubiquitin (UBQ) and SUMO, and functional domains on many biologically important proteins like Parkin, UHRF1 (ubiquitin-like with PDB and RING finger domains-1), and Usp7 (ubiquitin-specific protease-7) (Zhang et al., 2015; Rothbart et al., 2013; Burroughs et al., 2012; Wauer et al., 2015) [2], [3], [4], [5]. The UBL domain can participate in several unique protein-protein interactions (PPI) since protein adducts can be attached to and removed from amino groups of lysine side chains and the N-terminus of proteins. Given the biological significance of UBL domains, many have been characterized with high-resolution techniques, and for UBQ and SUMO, many protein complexes have been characterized. We identified all the UBL domains in the PDB and created a relational database called UbSRD (Ubiquitin Structural Relational Database) by using structural analysis tools in the Rosetta (Leaver et al., 2013; O'Meara et al., 2015; Leaver-fay et al., 2011) [1], [6], [7], [8]. Querying UbSRD permitted us to report many quantitative properties of UBQ and SUMO recognition at different types interfaces (noncovalent: NC, conjugated: CJ, and deubiquitanse: DB). In this data article, we report the average number of non-UBL neighbors, secondary structure of interacting motifs, and the type of inter-molecular hydrogen bonds for each residue of UBQ and SUMO. Additionally, we used PROMALS3D to generate a multiple sequence alignment used to construct a phylogram for the entire set of UBLs (Pei and Grishin, 2014) [9]. The data described here will be generally useful to scientists studying the molecular basis for recognition of UBQ or SUMO.

  3. The E2-25K ubiquitin-associated (UBA) domain aids in polyubiquitin chain synthesis and linkage specificity

    International Nuclear Information System (INIS)

    Research highlights: → We examine the role of a ubiquitin-associated (UBA) domain in an E2 enzyme. → The E2-25K UBA domain directs polyubiquitin chain linkage specificity. → The E2-25K UBA domain regulates length of polyubiquitin chains synthesized. -- Abstract: E2-25K is an ubiquitin-conjugating enzyme with the ability to synthesize Lys48-linked polyubiquitin chains. E2-25K and its homologs represent the only known E2 enzymes which contain a C-terminal ubiquitin-associated (UBA) domain as well as the conserved catalytic ubiquitin-conjugating (UBC) domain. As an additional non-covalent binding surface for ubiquitin, the UBA domain must provide some functional specialization. We mapped the protein-protein interface involved in the E2-25K UBA/ubiquitin complex by solution nuclear magnetic resonance (NMR) spectroscopy and subsequently modeled the structure of the complex. Domain-domain interactions between the E2-25K catalytic UBC domain and the UBA domain do not induce significant structural changes in the UBA domain or alter the affinity of the UBA domain for ubiquitin. We determined that one of the roles of the C-terminal UBA domain, in the context of E2-25K, is to increase processivity in Lys48-linked polyubiquitin chain synthesis, possibly through increased binding to the ubiquitinated substrate. Additionally, we see evidence that the UBA domain directs specificity in polyubiquitin chain linkage.

  4. 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

    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 decondensatio...

  5. The Ubiquitin-Specific Protease 14 (USP14) Is a Critical Regulator of Long-Term Memory Formation

    Science.gov (United States)

    Jarome, Timothy J.; Kwapis, Janine L.; Hallengren, Jada J.; Wilson, Scott M.; Helmstetter, Fred J.

    2014-01-01

    Numerous studies have suggested a role for ubiquitin-proteasome-mediated protein degradation in learning-dependent synaptic plasticity; however, very little is known about how protein degradation is regulated at the level of the proteasome during memory formation. The ubiquitin-specific protease 14 (USP14) is a proteasomal deubiquitinating enzyme…

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

    Science.gov (United States)

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

  7. Animal welfare: an animal science approach.

    Science.gov (United States)

    Koknaroglu, H; Akunal, T

    2013-12-01

    Increasing world population and demand for animal-derived protein puts pressure on animal production to meet this demand. For this purpose animal breeding efforts were conducted to obtain the maximum yield that the genetic makeup of the animals permits. Under the influence of economics which is the driving force behind animal production, animal farming became more concentrated and controlled which resulted in rearing animals under confinement. Since more attention was given on economics and yield per animal, animal welfare and behavior were neglected. Animal welfare which can be defined as providing environmental conditions in which animals can display all their natural behaviors in nature started gaining importance in recent years. This does not necessarily mean that animals provided with good management practices would have better welfare conditions as some animals may be distressed even though they are in good environmental conditions. Consumers are willing to pay more for welfare-friendly products (e.g.: free range vs caged egg) and this will change the animal production practices in the future. Thus animal scientists will have to adapt themselves for the changing animal welfare rules and regulations that differ for farm animal species and countries. In this review paper, animal welfare is discussed from an animal science standpoint.

  8. Rictor forms a complex with Cullin-1 to promote SGK1 ubiquitination and destruction

    OpenAIRE

    Gao, Daming; Wan, Lixin; Inuzuka, Hiroyuki; Berg, Anders H.; Tseng, Alan; Zhai, Bo; Shaik, Shavali; Bennett, Eric; Tron, Adriana E.; Gasser, Jessica A.; Lau, Alan; Gygi, Steven; Harper, J. Wade; DeCaprio, James A.; Toker, Alex

    2010-01-01

    The Rictor/mTOR complex (also known as mTORC2) plays a critical role in cellular homeostasis by phosphorylating AGC kinases such as Akt and SGK at their hydrophobic motifs to activate downstream signaling. However, the regulation of mTORC2 and whether it has additional function(s), remains largely unknown. Here we report that Rictor associates with Cullin-1 to form a functional E3 ubiquitin ligase. Rictor, but not Raptor or mTOR alone promotes SGK1 ubiquitination. Loss of Rictor/Cullin-1-medi...

  9. Smad ubiquitination regulatory factor 2 expression is enhanced in hypertrophic scar fibroblasts from burned children

    OpenAIRE

    Zhang, Zhi; Finnerty, Celeste C.; He, Jing; Herndon, David N

    2011-01-01

    Transforming growth factor-β1 (TGF-β1) plays a key role in hypertrophic scar formation. A lot of studies have shown that TGF-β1 stimulates fibroblast proliferation, collagen production, and α-smooth muscle actin (α-SMA) expression, inhibits matrix degradation and eventually leads to scar formation. Smad proteins are important intracellular mediators of TGF-β1 signaling, and Smad ubiquitination regulatory factor 2 (Smurf2), an ubiquitin ligase for Smads, plays critical roles in the regulation ...

  10. RNF2 is recruited by WASH to ubiquitinate AMBRA1 leading to downregulation of autophagy

    OpenAIRE

    Xia, Pengyan; Wang, Shuo; Huang, Guanling; Du, Ying; Zhu, Pingping; Li, Man; Fan, Zusen

    2014-01-01

    WASH (Wiskott-Aldrich syndrome protein (WASP) and SCAR homolog) was identified to function in endosomal sorting via Arp2/3 activation. We previously demonstrated that WASH is a new interactor of BECN1 and present in the BECN1-PIK3C3 complex with AMBRA1. The AMBRA1-DDB1-CUL4A complex is an E3 ligase for K63-linked ubiquitination of BECN1, which is required for starvation-induced autophagy. WASH suppresses autophagy by inhibition of BECN1 ubiquitination. However, how AMBRA1 is regulated during ...

  11. Plant ubiquitin-proteasome pathway and its role in gibberellin signaling

    Institute of Scientific and Technical Information of China (English)

    Feng Wang; Xing Wang Deng

    2011-01-01

    The ubiquitin-proteasome system (UPS) in plants,like in other eukaryotes,targets numerous intracellular regulators and thus modulates almost every aspect of growth and development.The well-known and best-characterized outcome of ubiquitination is mediating target protein degradation via the 26S proteasome,which represents the major selective protein degradation pathway conserved among eukaryotes.In this review,we will discuss the molecular composition,regulation and function of plant UPS,with a major focus on how DELLA protein degradation acts as a key in gibberellin signal transduction and its implication in the regulation of plant growth.

  12. 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.

  13. The ubiquitin-proteasome system is a key component of the SUMO-2/3 cycle

    DEFF Research Database (Denmark)

    Schimmel, Joost; Larsen, Katja M; Matic, Ivan;

    2008-01-01

    proteomics experiments enabled the identification of 73 SUMO-2 conjugated proteins that accumulated in cells treated with proteasome inhibitors. Crosstalk between SUMO-2/3 and the ubiquitin-proteasome system controls many target proteins that regulate all aspects of nucleic acid metabolism. Surprisingly......, the relative abundance of 40 SUMO-2 conjugated proteins was reduced by proteasome inhibitors, possibly due to a lack of recycled SUMO-2. We conclude that SUMO-2/3 conjugation and the ubiquitin-proteasome system are tightly integrated and act in a cooperative manner....

  14. Covalent modifier NEDD8 is essential for SCF ubiquitin-ligase in fission yeast

    OpenAIRE

    Osaka, Fumio; Saeki, Mihoro; Katayama, Satoshi; Aida, Noriko; Toh-e, Akio; Kominami, Kin-ichiro; Toda, Takashi; Suzuki, Toshiaki; Chiba, Tomoki; Tanaka, Keiji; Kato, Seishi

    2000-01-01

    A ubiquitin-like modifier, NEDD8, is covalently attached to cullin-family proteins, but its physiological role is poorly understood. Here we report that the NEDD8-modifying pathway is essential for cell viability and function of Pcu1 (cullin-1 orthologue) in fission yeast. Pcu1 assembled on SCF ubiquitin-ligase was completely modified by NEDD8. Pcu1K713R defective for NEDD8 conjugation lost the ability to complement lethality due to pcu1 deletion. Forced expression of Pcu1K713R or depletion o...

  15. Opposite Regulation of CD36 Ubiquitination by Fatty Acids and Insulin: EFFECTS ON FATTY ACID UPTAKE*

    OpenAIRE

    Smith, Jill; Su, Xiong; El-Maghrabi, Raafat; Stahl, Philip D.; Abumrad, Nada A.

    2008-01-01

    FAT/CD36 is a membrane scavenger receptor that facilitates long chain fatty acid uptake by muscle. Acute increases in membrane CD36 and fatty acid uptake have been reported in response to insulin and contraction. In this study we have explored protein ubiquitination as one potential mechanism for the regulation of CD36 level. CD36 expressed in Chinese hamster ovary (CHO) or HEK 293 cells was found to be polyubiquitinated via a process involving both lysines 48 and 63 of ubiquitin. Using CHO c...

  16. Animated nature

    International Nuclear Information System (INIS)

    Animated nature is educational-training project pronounced by the Slovak Environmental Agency (SAZP) in cooperation with Field Studies Council form Great Britain and financial support of Darwin Initiative and Slovensky plynarensky priemysel, s.p. In the present time this is ultimate and the most successful children's project aimed on mapping and protection of biodiversity in Europe. Activity in project is spare-time and therefore is voluntary. The interest territory is a natural as well as cultural landscape in vicinity of a school or other organisation, habitation and so on. In the project work schoolchildren at the age from 10 till 15 years. Leaders of work-groups are student of secondary schools and universities, teachers, professional workers of state and non-governmental organisation and parents. In one group works approximately 10 children. Each group which has send to SAZP result of biodiversity mapping, cost free obtained data base CD - Detske mapy biodiverzity (Children's maps of biodiversity) and so they were informed about results of all groups frame: within the frame of Slovakia. Results of activities of this project in 2001-2004 and perspectives for 2005-2006 years are discussed

  17. Animating Brains

    Science.gov (United States)

    Borck, Cornelius

    2016-01-01

    A recent paper famously accused the rising field of social neuroscience of using faulty statistics under the catchy title ‘Voodoo Correlations in Social Neuroscience’. This Special Issue invites us to take this claim as the starting point for a cross-cultural analysis: in which meaningful ways can recent research in the burgeoning field of functional imaging be described as, contrasted with, or simply compared to animistic practices? And what light does such a reading shed on the dynamics and effectiveness of a century of brain research into higher mental functions? Reviewing the heated debate from 2009 around recent trends in neuroimaging as a possible candidate for current instances of ‘soul catching’, the paper will then compare these forms of primarily image-based brain research with older regimes, revolving around the deciphering of the brain’s electrical activity. How has the move from a decoding paradigm to a representational regime affected the conceptualisation of self, psyche, mind and soul (if there still is such an entity)? And in what ways does modern technoscience provide new tools for animating brains? PMID:27292322

  18. A novel injectable BRET-based in vivo imaging probe for detecting the activity of hypoxia-inducible factor regulated by the ubiquitin-proteasome system

    Science.gov (United States)

    Kuchimaru, Takahiro; Suka, Tomoya; Hirota, Keisuke; Kadonosono, Tetsuya; Kizaka-Kondoh, Shinae

    2016-01-01

    The ubiquitin-proteasome system (UPS) is a selective protein degradation system that plays a critical role in many essential biological processes by regulating the existence of various cellular proteins. The target proteins of UPS are recognized and tagged with polyubiquitin chains by E3 ubiquitin ligases, which have high substrate-specific activities. Here we present a novel injectable imaging probe POL-N that can detect the UPS-regulated hypoxia-inducible factor (HIF) activity in vivo. Because the luciferase is fused to the E3 ligase-recognition domain of the HIF-1α, POL-N is intact only in the HIFα-overexpressing cells, that is, HIF-active cells, generating signals via an intramolecular bioluminescence resonance energy transfer (BRET) between luciferase and a near-infrared (NIR) fluorescent dye at the C-terminal end of the probe. Off-target signals of the NIR-BRET were so low that we could achieve highly sensitive and fast detection of intratumoral HIF-activity. Notably, we successfully detected hypoxic liver metastasis, which is extremely difficult to detect by injectable imaging probes due to strong off-target signals, as early as 1 h after systemic injection of POL-N. Our probe design can be widely adapted to UPS-target proteins and may contribute to the exploration of their roles in animal disease models. PMID:27698477

  19. The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks

    DEFF Research Database (Denmark)

    Meerang, Mayura; Ritz, Danilo; Paliwal, Shreya;

    2011-01-01

    Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signalling and repair proteins to the site of lesion. Protein modification with ubiquitin is crucial for the signalling...... cascade, but how ubiquitylation coordinates the dynamic assembly of these complexes is poorly understood. Here, we show that the human ubiquitin-selective protein segregase p97 (also known as VCP; valosin-containing protein) cooperates with the ubiquitin ligase RNF8 to orchestrate assembly of signalling...... complexes and efficient DSB repair after exposure to ionizing radiation. p97 is recruited to DNA lesions by its ubiquitin adaptor UFD1-NPL4 and Lys-48-linked ubiquitin (K48-Ub) chains, whose formation is regulated by RNF8. p97 subsequently removes K48-Ub conjugates from sites of DNA damage to orchestrate...

  20. Ubiquitination and Degradation of CFTR by the E3 Ubiquitin Ligase MARCH2 through Its Association with Adaptor Proteins CAL and STX6

    OpenAIRE

    Jie Cheng; William Guggino

    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 bindin...

  1. Organic Chemistry Applied to Synthetic Proteins: Modifying the Vicinity of the Isopeptide Bond Revealed Differential Behavior of Ubiquitin Chains with Interacting Proteins

    Science.gov (United States)

    Haj-Yahya, Najat; Haj-Yahya, Mahmood; Castañeda, Carlos A.; Spasser, Liat; Hemantha, Hosahalli P.; Jbara, Muhammad; Penner, Marlin; Ciechanover, Aaron; Fushman, David

    2013-01-01

    In Every Direction Chemical synthesis of proteins allowed the synthesis of ubiquitin chains modified in the vicinity of the isopeptide peptide to examine their behavior with deubiquitinases and ubiquitin binding domains. Our results set the ground for the generation of unique probes for studying the interactions of these chains with various ubiquitin-interacting proteins. PMID:24006204

  2. Targeted ubiquitination and degradation of G-protein-coupled receptor kinase 5 by the DDB1-CUL4 ubiquitin ligase complex.

    Directory of Open Access Journals (Sweden)

    Ziyan Wu

    Full Text Available The G protein-coupled receptor kinases (GRKs phosphorylate agonist occupied G protein-coupled receptors (GPCRs and desensitize GPCR-mediated signaling. Recent studies indicate they also function non-catalytically via interaction with other proteins. In this study, a proteomic approach was used to screen interacting proteins of GRK5 in MDA-MB-231 cells and HUVEC cells. Mass spectrometry analysis reveals several proteins in the GRK5 immunocomplex including damaged DNA-binding protein 1 (DDB1, an adaptor subunit of the CUL4-ROC1 E3 ubiquitin ligase complex. Co-immunoprecipitation experiments confirmed the association of GRK5 with DDB1-CUL4 complex, and reveal that DDB1 acts as an adapter to link GRK5 to CUL4 to form the complex. Overexpression of DDB1 promoted, whereas knockdown of DDB1 inhibited the ubiquitination of GRK5, and the degradation of GRK5 was reduced in cells deficient of DDB1. Furthermore, the depletion of DDB1 decreased Hsp90 inhibitor-induced GRK5 destabilization and UV irradiation-induced GRK5 degradation. Thus, our study identified potential GRK5 interacting proteins, and reveals the association of GRK5 with DDB1 in cell and the regulation of GRK5 level by DDB1-CUL4 ubiquitin ligase complex-dependent proteolysis pathway.

  3. The wild animal as a research animal

    NARCIS (Netherlands)

    Swart, JAA

    2004-01-01

    Most discussions on animal experimentation refer to domesticated animals and regulations are tailored to this class of animals. However, wild animals are also used for research, e. g., in biological field research that is often directed to fundamental ecological-evolutionary questions or to conserva

  4. The ubiquitin ligase RNF5 regulates antiviral responses by mediating degradation of the adaptor protein MITA.

    Science.gov (United States)

    Zhong, Bo; Zhang, Lu; Lei, Caoqi; Li, Ying; Mao, Ai-Ping; Yang, Yan; Wang, Yan-Yi; Zhang, Xiao-Lian; Shu, Hong-Bing

    2009-03-20

    Viral infection activates transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. MITA (also known as STING) has recently been identified as an adaptor that links virus-sensing receptors to IRF3 activation. Here, we showed that the E3 ubiquitin ligase RNF5 interacted with MITA in a viral-infection-dependent manner. Overexpression of RNF5 inhibited virus-triggered IRF3 activation, IFNB1 expression, and cellular antiviral response, whereas knockdown of RNF5 had opposite effects. RNF5 targeted MITA at Lys150 for ubiquitination and degradation after viral infection. Both MITA and RNF5 were located at the mitochondria and endoplasmic reticulum (ER) and viral infection caused their redistribution to the ER and mitochondria, respectively. We further found that virus-induced ubiquitination and degradation of MITA by RNF5 occurred at the mitochondria. These findings suggest that RNF5 negatively regulates virus-triggered signaling by targeting MITA for ubiquitination and degradation at the mitochondria.

  5. Rictor forms a complex with Cullin-1 to promote SGK1 ubiquitination and destruction.

    Science.gov (United States)

    Gao, Daming; Wan, Lixin; Inuzuka, Hiroyuki; Berg, Anders H; Tseng, Alan; Zhai, Bo; Shaik, Shavali; Bennett, Eric; Tron, Adriana E; Gasser, Jessica A; Lau, Alan; Gygi, Steven P; Harper, J Wade; DeCaprio, James A; Toker, Alex; Wei, Wenyi

    2010-09-10

    The Rictor/mTOR complex (also known as mTORC2) plays a critical role in cellular homeostasis by phosphorylating AGC kinases such as Akt and SGK at their hydrophobic motifs to activate downstream signaling. However, the regulation of mTORC2 and whether it has additional function(s) remain largely unknown. Here, we report that Rictor associates with Cullin-1 to form a functional E3 ubiquitin ligase. Rictor, but not Raptor or mTOR alone, promotes SGK1 ubiquitination. Loss of Rictor/Cullin-1-mediated ubiquitination leads to increased SGK1 protein levels as detected in Rictor null cells. Moreover, as part of a feedback mechanism, phosphorylation of Rictor at T1135 by multiple AGC kinases disrupts the interaction between Rictor and Cullin-1 to impair SGK1 ubiquitination. These findings indicate that the Rictor/Cullin-1 E3 ligase activity is regulated by a specific signal relay cascade and that misregulation of this mechanism may contribute to the frequent overexpression of SGK1 in various human cancers. PMID:20832730

  6. Further insights into the ubiquitin pathway: understanding the scarlet letter code.

    Science.gov (United States)

    Pastore, Annalisa

    2010-08-11

    Understanding the machinery that decides proteins' fate by tagging them with ubiquitin is an important goal of structural biology. Benirschke et al. (2010) have solved the structure of human E4B (or UFD2a), a U-box-containing protein that functions both as an E3 Ub ligase and as an E4 polyUb chain elongation factor. PMID:20696387

  7. SINAT5 promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals.

    Science.gov (United States)

    Xie, Qi; Guo, Hui-Shan; Dallman, Geza; Fang, Shengyun; Weissman, Allan M; Chua, Nam-Hai

    2002-09-12

    The plant hormone indole-3 acetic acid (IAA or auxin) controls many aspects of plant development, including the production of lateral roots. Ubiquitin-mediated proteolysis has a central role in this process. The genes AXR1 and TIR1 aid the assembly of an active SCF (Skp1/Cullin/F-box) complex that probably promotes degradation of the AUX/IAA transcriptional repressors in response to auxin. The transcription activator NAC1, a member of the NAM/CUC family of transcription factors, functions downstream of TIR1 to transduce the auxin signal for lateral root development. Here we show that SINAT5, an Arabidopsis homologue of the RING-finger Drosophila protein SINA, has ubiquitin protein ligase activity and can ubiquitinate NAC1. This activity is abolished by mutations in the RING motif of SINAT5. Overexpressing SINAT5 produces fewer lateral roots, whereas overexpression of a dominant-negative Cys49 --> Ser mutant of SINAT5 develops more lateral roots. These lateral root phenotypes correlate with the expression of NAC1 observed in vivo. Low expression of NAC1 in roots can be increased by treatment with a proteasome inhibitor, which indicates that SINAT5 targets NAC1 for ubiquitin-mediated proteolysis to downregulate auxin signals in plant cells.

  8. Ubiquitination as a Mechanism To Transport Soluble Mycobacterial and Eukaryotic Proteins to Exosomes.

    Science.gov (United States)

    Smith, Victoria L; Jackson, Liam; Schorey, Jeffrey S

    2015-09-15

    Exosomes are extracellular vesicles of endocytic origin that function in intercellular communication. Our previous studies indicate that exosomes released from Mycobacterium tuberculosis-infected macrophages contain soluble mycobacterial proteins. However, it was unclear how these secreted proteins were targeted to exosomes. In this study, we determined that exosome production by the murine macrophage cell line RAW264.7 requires the endosomal sorting complexes required for transport and that trafficking of mycobacterial proteins from phagocytosed bacilli to exosomes was dependent on protein ubiquitination. Moreover, soluble mycobacterial proteins, when added exogenously to RAW264.7 or human HEK293 cells, were endocytosed, ubiquitinated, and released via exosomes. This suggested that endocytosed proteins could be recycled from cells through exosomes. This hypothesis was supported using the tumor-associated protein He4, which, when endocytosed by RAW264.7 or HEK293 cells, was transported to exosomes in a ubiquitin-dependent manner. Our data suggest that ubiquitination is a modification sufficient for trafficking soluble proteins within the phagocytic/endocytic network to exosomes.

  9. The Ubiquitin-Proteasome System and Its Role in Inflammatory and Autoimmune Diseases

    Institute of Scientific and Technical Information of China (English)

    Jingsong Wang; Michael A. Maldonado

    2006-01-01

    Protein degradation through the ubiquitin-proteasome system is the major pathway of non-lysosomal proteolysis of intracellular proteins. It plays important roles in a variety of fundamental cellular processes such as regulation of cell cycle progression, division, development and differentiation, apoptosis, cell trafficking, and modulation of the immune and inflammatory responses. The central element of this system is the covalent linkage of ubiquitin to targeted proteins, which are then recognized by the 26S proteasome, an adenosine triphosphate-dependent,multi-catalytic protease. Damaged, oxidized, or misfolded proteins as well as regulatory proteins that control many critical cellular functions are among the targets of this degradation process. Aberration of this system leads to the dysregulation of cellular homeostasis and the development of multiple diseases. In this review, we described the basic biochemistry and molecular biology of the ubiquitin-proteasome system, and its complex role in the development of inflammatory and autoimmune diseases. In addition, therapies and potential therapeutic targets related to the ubiquitin-proteasome system are discussed as well. Cellular & Molecular Immunology. 2006;3(4):255-261.

  10. 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.

  11. Quantifying protein-protein interactions in the ubiquitin pathway by surface plasmon resonance

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Gordon, Colin

    2005-01-01

    The commercial availability of instruments, such as Biacore, that are capable of monitoring surface plasmon resonance (SPR) has greatly simplified the quantification of protein-protein interactions. Already, this technique has been used for some studies of the ubiquitin-proteasome system. Here we...

  12. Ubiquitin-SUMO Circuitry Controls Activated Fanconi Anemia ID Complex Dosage in Response to DNA Damage

    DEFF Research Database (Denmark)

    Gibbs-Seymour, Ian; Oka, Yasuyoshi; Rajendra, Eeson;

    2015-01-01

    We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase...

  13. Protein Degradation by Ubiquitin-Proteasome System in Formation and Labilization of Contextual Conditioning Memory

    Science.gov (United States)

    Fustiñana, María Sol; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

    2014-01-01

    The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this…

  14. SUMO and ubiquitin-dependent XPC exchange drives nucleotide excision repair

    DEFF Research Database (Denmark)

    Van Cuijk, Loes; Van Belle, Gijsbert J.; Turkyilmaz, Yasemin;

    2015-01-01

    initiation, and is needed for stable incorporation of the NER endonucleases XPG and ERCC1/XPF. Our data suggest that RNF111, together with the CRL4DDB2 ubiquitin ligase complex, is responsible for sequential XPC ubiquitylation, which regulates the recruitment and release of XPC and is crucial...

  15. Frequency of ubiquitin and FUS-positive, TDP-43-negative frontotemporal lobar degeneration

    NARCIS (Netherlands)

    H. Seelaar (Harro); K.Y. Klijnsma (Kirsten); I. de Koning (Inge); A. van der Lugt (Aad); W.Z. Chiu (Wang Zheng); A. Azmani (Asma); A.J.M. Rozemuller (Annemieke); J.C. van Swieten (John)

    2010-01-01

    textabstractFrontotemporal lobar degeneration (FTLD) is a clinically, genetically and pathologically heterogeneous disorder. Within FTLD with ubiquitin-positive inclusions (FTLD-U), a new pathological subtype named FTLD-FUS was recently found with fused in sarcoma (FUS) positive, TDP-43-negative inc

  16. Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN

    Energy Technology Data Exchange (ETDEWEB)

    Stroschein, Shannon L.; Bonni, Shirin; Wrana, Jeffrey L.; Luo, Kunxin

    2001-09-11

    Smad proteins mediate transforming growth factor-b signaling to regulate cell growth and differentiation. SnoN is an important negative regulator of TGFb signaling that functions to maintain the repressed state of TGFb target genes in the absence of ligand. Upon TGFb stimulation, Smad3 and Smad2 translocate into the nucleus and induce a rapid degradation of SnoN, allowing activation of TGFb target genes. Here we show that Smad2- or Smad3-induced degradation of SnoN requires the ubiquitin-dependent proteasome and can be mediated by the anaphase promoting complex (APC) and the UbcH5 family of ubiquitin conjugating enzymes. Smad3 and to a lesser extent, Smad2, interact with both the APC and SnoN, resulting in the recruitment of the APC to SnoN and subsequent ubiquitination of SnoN in a destruction box-dependent manner. In addition to the destruction box, efficient degradation of SnoN also requires the Smad3 binding site in SnoN as well as key lysine residues necessary for ubiquitin attachment. Mutation of either the Smad3 binding site or lysine residues results in stabilization of SnoN and in enhanced antagonism of TGFb signaling. Our studies elucidate an important pathway for the degradation of SnoN and reveal a novel role of the APC in regulation of TGFb signaling.

  17. A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies

    NARCIS (Netherlands)

    Reggiori, Fulvio; Pelham, Hugh R B; Reggiori, Fulvio

    2002-01-01

    Membrane proteins with transmembrane domains (TMDs) that contain polar residues exposed to the lipid bilayer are selectively sorted into multivesicular bodies (MVBs) and delivered to the yeast vacuole. Sorting of some, although not all, proteins into these structures is mediated by ubiquitination. W

  18. Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage

    DEFF Research Database (Denmark)

    Thorslund, Tina; Ripplinger, Anita; Hoffmann, Saskia;

    2015-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that trigger non-proteolytic ubiquitylation of adjacent chromatin areas to generate binding sites for DNA repair factors. This depends on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 (refs 1-6), and UBC13 (also...

  19. 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

    2014-08-01

    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 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.

  20. Linear ubiquitination is involved in the pathogenesis of optineurin-associated amyotrophic lateral sclerosis

    Science.gov (United States)

    Nakazawa, Seshiru; Oikawa, Daisuke; Ishii, Ryohei; Ayaki, Takashi; Takahashi, Hirotaka; Takeda, Hiroyuki; Ishitani, Ryuichiro; Kamei, Kiyoko; Takeyoshi, Izumi; Kawakami, Hideshi; Iwai, Kazuhiro; Hatada, Izuho; Sawasaki, Tatsuya; Ito, Hidefumi; Nureki, Osamu; Tokunaga, Fuminori

    2016-01-01

    Optineurin (OPTN) mutations cause neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and glaucoma. Although the ALS-associated E478G mutation in the UBAN domain of OPTN reportedly abolishes its NF-κB suppressive activity, the precise molecular basis in ALS pathogenesis still remains unclear. Here we report that the OPTN-UBAN domain is crucial for NF-κB suppression. Our crystal structure analysis reveals that OPTN-UBAN binds linear ubiquitin with homology to NEMO. TNF-α-mediated NF-κB activation is enhanced in OPTN-knockout cells, through increased ubiquitination and association of TNF receptor (TNFR) complex I components. Furthermore, OPTN binds caspase 8, and OPTN deficiency accelerates TNF-α-induced apoptosis by enhancing complex II formation. Immunohistochemical analyses of motor neurons from OPTN-associated ALS patients reveal that linear ubiquitin and activated NF-κB are partially co-localized with cytoplasmic inclusions, and that activation of caspases is elevated. Taken together, OPTN regulates both NF-κB activation and apoptosis via linear ubiquitin binding, and the loss of this ability may lead to ALS. PMID:27552911

  1. The ubiquitin conjugating enzyme, TaU4 regulates wheat defence against the phytopathogen Zymoseptoria tritici

    Science.gov (United States)

    Millyard, Linda; Lee, Jack; Zhang, Cunjin; Yates, Gary; Sadanandom, Ari

    2016-01-01

    Mycosphaerella graminicola (Zymoseptoria tritici commonly known as Septoria), the causal agent of Septoria Leaf Blotch (STB), is considered one of the major threats to European wheat production. Previous studies have shown the importance of ubiquitination in plant defence against a multitude of pathogens. However the ubiquitination machinery in wheat is under studied, particularly E2 enzymes that have the ability to control the ubiquitination and thereby the fate of many different target proteins. In this study we identify an E2 enzyme, Triticum aestivum Ubiquitin conjugating enzyme 4 (TaU4) that functions in wheat defence against Septoria. We demonstrate TaU4 to be a bona fide E2 enzyme through an E2 charging assay. TaU4 localises in both the cytoplasm and nucleus, therefore potentially interacting with E3 ligases and substrate proteins in multiple compartments. Virus Induced Gene Silencing of TaU4 in wheat leaves resulted in delayed development of disease symptoms, reduced Septoria growth and reproduction. We conclude that TaU4 is a novel negative regulator of defence against Septoria. PMID:27759089

  2. Animation of Antimicrobial Resistance

    Science.gov (United States)

    ... Veterinary Home Animal & Veterinary Safety & Health Antimicrobial Resistance Animation of Antimicrobial Resistance Share Tweet Linkedin Pin it ... Veterinary Medicine is cited as the corporate author. Animation Animation of Antimicrobial Resistance (WMV - 19.2MB) 9: ...

  3. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... Radiation-Emitting Products Vaccines, Blood & Biologics Animal & Veterinary Cosmetics Tobacco Products Animal & Veterinary Home Animal & Veterinary Safety & ... Radiation-Emitting Products Vaccines, Blood & Biologics Animal & Veterinary Cosmetics Tobacco Products

  4. Learning Anime Studio

    CERN Document Server

    Troftgruben, Chad

    2014-01-01

    Anime Studio is your complete animation program to help you create 2D movies, cartoons, anime, and cut out animations. You can create your own animated shorts and use Anime Studio to produce cartoon animations for film, video, or streaming over the Web, which can be enjoyed on YouTube, Vimeo, and other popular sites. Anime Studio is great for hobbyists and professionals alike, combining tools for both illustration and animation. With Anime Studio's easy-to-use interface, you will be creating an animated masterpiece in no time. This practical, step-by-step guide will provide you with a structur

  5. 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.

  6. Regulation of Mitochondrial Genome Inheritance by Autophagy and Ubiquitin-Proteasome System: Implications for Health, Fitness, and Fertility

    Directory of Open Access Journals (Sweden)

    Won-Hee Song

    2014-01-01

    Full Text Available Mitochondria, the energy-generating organelles, play a role in numerous cellular functions including adenosine triphosphate (ATP production, cellular homeostasis, and apoptosis. Maternal inheritance of mitochondria and mitochondrial DNA (mtDNA is universally observed in humans and most animals. In general, high levels of mitochondrial heteroplasmy might contribute to a detrimental effect on fitness and disease resistance. Therefore, a disposal of the sperm-derived mitochondria inside fertilized oocytes assures normal preimplantation embryo development. Here we summarize the current research and knowledge concerning the role of autophagic pathway and ubiquitin-proteasome-dependent proteolysis in sperm mitophagy in mammals, including humans. Current data indicate that sperm mitophagy inside the fertilized oocyte could occur along multiple degradation routes converging on autophagic clearance of paternal mitochondria. The influence of assisted reproductive therapies (ART such as intracytoplasmic sperm injection (ICSI, mitochondrial replacement (MR, and assisted fertilization of oocytes from patients of advanced reproductive age on mitochondrial function, inheritance, and fitness and for the development and health of ART babies will be of particular interest to clinical audiences. Altogether, the study of sperm mitophagy after fertilization has implications in the timing of evolution and developmental and reproductive biology and in human health, fitness, and management of mitochondrial disease.

  7. Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants.

    Directory of Open Access Journals (Sweden)

    Katarzyna Jarmoszewicz

    Full Text Available Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

  8. 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. PMID:27197154

  9. An Armadillo Motif in Ufd3 Interacts with Cdc48 and is Involved in Ubiquitin Homeostasis and Protein Degradation

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, G.; Li, G; Schindelin, H; Lennarz, W

    2009-01-01

    The yeast AAA-ATPase Cdc48 and the ubiquitin fusion degradation (UFD) proteins play important, evolutionarily conserved roles in ubiquitin dependent protein degradation. The N-terminal domain of Cdc48 interacts with substrate-recruiting cofactors, whereas the C terminus of Cdc48 binds to proteins such as Ufd3 that process substrates. Ufd3 is essential for efficient protein degradation and for maintaining cellular ubiquitin levels. This protein contains an N-terminal WD40 domain, a central ubiquitin-binding domain, and a C-terminal Cdc48-binding PUL domain. The crystal structure of the PUL domain reveals an Armadillo repeat with high structural similarity to importin-a, and the Cdc48-binding site could be mapped to the concave surface of the PUL domain by biochemical studies. Alterations of the Cdc48 binding site of Ufd3 by site-directed mutagenesis resulted in a depletion of cellular ubiquitin pools and reduced activity of the ubiquitin fusion degradation pathway. Therefore, our data provide direct evidence that the functions of Ufd3 in ubiquitin homeostasis and protein degradation depend on its interaction with the C terminus of Cdc48.

  10. Sent to destroy: the ubiquitin proteasome system regulates cell signaling and protein quality control in cardiovascular development and disease.

    Science.gov (United States)

    Willis, Monte S; Townley-Tilson, W H Davin; Kang, Eunice Y; Homeister, Jonathon W; Patterson, Cam

    2010-02-19

    The ubiquitin proteasome system (UPS) plays a crucial role in biological processes integral to the development of the cardiovascular system and cardiovascular diseases. The UPS prototypically recognizes specific protein substrates and places polyubiquitin chains on them for subsequent destruction by the proteasome. This system is in place to degrade not only misfolded and damaged proteins, but is essential also in regulating a host of cell signaling pathways involved in proliferation, adaptation to stress, regulation of cell size, and cell death. During the development of the cardiovascular system, the UPS regulates cell signaling by modifying transcription factors, receptors, and structural proteins. Later, in the event of cardiovascular diseases as diverse as atherosclerosis, cardiac hypertrophy, and ischemia/reperfusion injury, ubiquitin ligases and the proteasome are implicated in protecting and exacerbating clinical outcomes. However, when misfolded and damaged proteins are ubiquitinated by the UPS, their destruction by the proteasome is not always possible because of their aggregated confirmations. Recent studies have discovered how these ubiquitinated misfolded proteins can be destroyed by alternative "specific" mechanisms. The cytosolic receptors p62, NBR, and histone deacetylase 6 recognize aggregated ubiquitinated proteins and target them for autophagy in the process of "selective autophagy." Even the ubiquitination of multiple proteins within whole organelles that drive the more general macro-autophagy may be due, in part, to similar ubiquitin-driven mechanisms. In summary, the crosstalk between the UPS and autophagy highlight the pivotal and diverse roles the UPS plays in maintaining protein quality control and regulating cardiovascular development and disease. PMID:20167943

  11. Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation.

    Science.gov (United States)

    Yamaguchi, Masaya; VanderLinden, Ryan; Weissmann, Florian; Qiao, Renping; Dube, Prakash; Brown, Nicholas G; Haselbach, David; Zhang, Wei; Sidhu, Sachdev S; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A

    2016-08-18

    The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly and conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCC's CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation. PMID:27522463

  12. Ubiquitin and stromal cell-derived factor-1α in bronchoalveolar lavage fluid after burn and inhalation injury.

    Science.gov (United States)

    Baker, Todd A; Davis, Christopher S; Bach, Harold H; Romero, Jacqueline; Burnham, Ellen L; Kovacs, Elizabeth J; Gamelli, Richard L; Majetschak, Matthias

    2012-01-01

    The objective of the study was to determine whether the CXC chemokine receptor (CXCR) 4 ligands ubiquitin and stromal cell-derived factor (SDF)-1α are detectable in bronchoalveolar lavage fluid (BALF) after burn and inhalation injury and whether their concentrations in BALF are associated with injury severity, physiological variables, or clinical outcomes. BALF was obtained on hospital admission from 51 patients (48 ± 18 years) with burn (TBSA: 23 ± 24%) and inhalation injury (controls: 10 healthy volunteers, 42 ± 8 years). BALF was analyzed for total protein and for ubiquitin and SDF-1α by enzyme-linked immunosorbent assay. Ubiquitin/SDF-1α levels were normalized to total BALF protein content. The extent of inhalation injury was determined during bronchoscopy using a standardized scoring system. Percent TBSA, Baux scores, revised Baux scores, and clinical variables were documented. Ubiquitin and SDF-1α were detectable in 40% of normal BALF specimens. After injury, ubiquitin was detectable in 90% (P patients (P burn and inhalation injury. Increases in BALF ubiquitin after inhalation injury may maintain CXCR4-mediated lung protection and repair processes. The finding that BALF ubiquitin decreased with higher grades of inhalation injury may provide a biological correlate for an insufficient local inflammatory response after severe inhalation injury.

  13. Insulin alleviates degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system in septic rats

    Directory of Open Access Journals (Sweden)

    Gao Tao

    2011-06-01

    Full Text Available Abstract Hypercatabolism is common under septic conditions. Skeletal muscle is the main target organ for hypercatabolism, and this phenomenon is a vital factor in the deterioration of recovery in septic patients. In skeletal muscle, activation of the ubiquitin-proteasome system plays an important role in hypercatabolism under septic status. Insulin is a vital anticatabolic hormone and previous evidence suggests that insulin administration inhibits various steps in the ubiquitin-proteasome system. However, whether insulin can alleviate the degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system under septic condition is unclear. This paper confirmed that mRNA and protein levels of the ubiquitin-proteasome system were upregulated and molecular markers of skeletal muscle proteolysis (tyrosine and 3-methylhistidine simultaneously increased in the skeletal muscle of septic rats. Septic rats were infused with insulin at a constant rate of 2.4 mU.kg-1.min-1 for 8 hours. Concentrations of mRNA and proteins of the ubiquitin-proteasome system and molecular markers of skeletal muscle proteolysis were mildly affected. When the insulin infusion dose increased to 4.8 mU.kg-1.min-1, mRNA for ubiquitin, E2-14 KDa, and the C2 subunit were all sharply downregulated. At the same time, the levels of ubiquitinated proteins, E2-14KDa, and the C2 subunit protein were significantly reduced. Tyrosine and 3-methylhistidine decreased significantly. We concluded that the ubiquitin-proteasome system is important skeletal muscle hypercatabolism in septic rats. Infusion of insulin can reverse the detrimental metabolism of skeletal muscle by inhibiting the ubiquitin-proteasome system, and the effect is proportional to the insulin infusion dose.

  14. Co-expression of Ubiquitin gene and capsid protein gene enhances the potency of DNA immunization of PCV2 in mice

    Directory of Open Access Journals (Sweden)

    Zhou Yanjun

    2011-05-01

    Full Text Available Abstract A recombinant plasmid that co-expressed ubiquitin and porcine circovirus type 2 (PCV2 virus capsid protein (Cap, denoted as pc-Ub-Cap, and a plasmid encoding PCV2 virus Cap alone, denoted as pc-Cap, were transfected into 293T cells. Indirect immunofluorescence (IIF and confocal microscopy were performed to measure the cellular expression of Cap. Three groups of mice were then vaccinated once every three weeks for a total of three doses with pc-Ub-Cap, pc-Cap or the empty vector pCAGGS, followed by challenging all mice intraperitoneally with 0.5 mL 106.5 TCID50/mL PCV2. To characterize the protective immune response against PCV2 infection in mice, assays of antibody titer (including different IgG isotypes, flow cytometric analysis (FCM, lymphocyte proliferation, cytokine production and viremia were evaluated. The results showed that pc-Ub-Cap and pc-Cap were efficiently expressed in 293T cells. However, pc-Ub-Cap-vaccinated animals had a significantly higher level of Cap-specific antibody and induced a stronger Th1 type cellular immune response than did pc-Cap-vaccinated animals, suggesting that ubiquitin conjugation improved both the cellular and humoral immune responses. Additionally, viral replication in blood was lower in the pc-Ub-Cap-vaccinated group than in the pc-Cap and empty vector groups, suggesting that the protective immunity induced by pc-Ub-Cap is superior to that induced by pc-Cap.

  15. Deregulation of the ubiquitin-proteasome system is the predominant molecular pathology in OPMD animal models and patients

    DEFF Research Database (Denmark)

    Anvar, Seyed Yahya; hoen, Peter Ac; Venema, Andrea;

    2011-01-01

    Oculopharyngeal muscular dystrophy (OPMD) is a late-onset progressive muscle disorder caused by a poly-alanine expansion mutation in the Poly(A) Binding Protein Nuclear 1 (PABPN1). The molecular mechanisms that regulate disease onset and progression are largely unknown. In order to identify molec...

  16. Animal welfare assessment

    OpenAIRE

    Vučinić Marijana; Lazić Ivana

    2008-01-01

    The paper deals with animal welfare definitions and animal welfare assessment. Animal welfare is a prolonged mental state, resulting from how the animal experiences its environment over time. There are different methods for animal welfare assessment. The four basic criteria for animal welfare assessment are feeding, housing, health and appropriate behavior. Therefore, criteria used to assess animal welfare are not direct measures of the mental state but only parameters that need to be interpr...

  17. Animal Protection and Animal 'Rights' in Hungary

    OpenAIRE

    Toth, Zoltan J.

    2012-01-01

    In Hungary, the first Act on Animal Protection, which aimed at handling and respecting animals as living creatures capable of feelings and suffering and thus deserving and entitled to protection, was adopted in 1998. Based on this, the Act contains several regulations which ensure that animals are protected against all possible kinds of avoidable physical or mental harm. Furthermore, it prohibits and imposes sanctions for any treatment that causes animals unnecessary suffering. The present st...

  18. Animal rights, animal minds, and human mindreading

    OpenAIRE

    Mameli, M.; Bortolotti, L

    2006-01-01

    Do non‐human animals have rights? The answer to this question depends on whether animals have morally relevant mental properties. Mindreading is the human activity of ascribing mental states to other organisms. Current knowledge about the evolution and cognitive structure of mindreading indicates that human ascriptions of mental states to non‐human animals are very inaccurate. The accuracy of human mindreading can be improved with the help of scientific studies of animal minds. However, the s...

  19. [Animal experimentation, animal welfare and scientific research].

    Science.gov (United States)

    Tal, H

    2013-10-01

    Hundreds of thousands of laboratory animals are being used every year for scientific experiments held in Israel, mostly mice, rats, rabbits, guinea pigs, and a few sheep, cattle, pigs, cats, dogs, and even a few dozen monkeys. In addition to the animals sacrificed to promote scientific research, millions of animals slain every year for other purposes such as meat and fine leather fashion industries. While opening a front against all is an impossible and perhaps an unjustified task, the state of Israel enacted the Animal Welfare (Animal Experimentation) Law (1994). The law aims to regulate scientific animal experiments and to find the appropriate balance between the need to continue to perform animal experiments for the advancement of research and medicine, and at the same time to avoid unnecessary trials and minimize animal suffering. Among other issues the law deals with the phylogenetic scale according to which experimental animals should be selected, experiments for teaching and practicing, and experiments for the cosmetic industry. This article discusses bioethics considerations in animal experiments as well as the criticism on the scientific validity of such experiments. It further deals with the vitality of animal studies and the moral and legal obligation to prevent suffering from laboratory animals. PMID:24660572

  20. A comparative analysis of the ubiquitination kinetics of multiple degrons to identify an ideal targeting sequence for a proteasome reporter.

    Science.gov (United States)

    Melvin, Adam T; Woss, Gregery S; Park, Jessica H; Dumberger, Lukas D; Waters, Marcey L; Allbritton, Nancy L

    2013-01-01

    The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins. The conjugation of a polyubiquitin chain, or polyubiquitination, to a target protein requires an increasingly diverse cascade of enzymes culminating with the E3 ubiquitin ligases. Protein recognition by an E3 ligase occurs through a specific sequence of amino acids, termed a degradation sequence or degron. Recently, degrons have been incorporated into novel reporters to monitor proteasome activity; however only a limited few degrons have successfully been incorporated into such reporters. The goal of this work was to evaluate the ubiquitination kinetics of a small library of portable degrons that could eventually be incorporated into novel single cell reporters to assess proteasome activity. After an intensive literary search, eight degrons were identified from proteins recognized by a variety of E3 ubiquitin ligases and incorporated into a four component degron-based substrate to comparatively calculate ubiquitination kinetics. The mechanism of placement of multiple ubiquitins on the different degron-based substrates was assessed by comparing the data to computational models incorporating first order reaction kinetics using either multi-monoubiquitination or polyubiquitination of the degron-based substrates. A subset of three degrons was further characterized to determine the importance of the location and proximity of the ubiquitination site lysine with respect to the degron. Ultimately, this work identified three candidate portable degrons that exhibit a higher rate of ubiquitination compared to peptidase-dependent degradation, a desired trait for a proteasomal targeting motif. PMID:24205101

  1. Insulin alleviates degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system in septic rats

    OpenAIRE

    Gao Tao; Yu Wenkui; Tang Shaoqiu; Li Weiqin; Zhu Weiming; Li Ning; Chen Qiyi; Zhang Juanjuan; Li Jieshou

    2011-01-01

    Abstract Hypercatabolism is common under septic conditions. Skeletal muscle is the main target organ for hypercatabolism, and this phenomenon is a vital factor in the deterioration of recovery in septic patients. In skeletal muscle, activation of the ubiquitin-proteasome system plays an important role in hypercatabolism under septic status. Insulin is a vital anticatabolic hormone and previous evidence suggests that insulin administration inhibits various steps in the ubiquitin-proteasome sys...

  2. The Transmembrane E3 Ligase GRAIL Ubiquitinates and Degrades CD83 on CD4 T Cells1

    OpenAIRE

    Su, Leon L.; Iwai, Hideyuki; Lin, Jack T; Fathman, C. Garrison

    2009-01-01

    Ubiquitination of eukaryotic proteins regulates a broad range of cellular processes, including T cell activation and tolerance. We have previously demonstrated that GRAIL (gene related to anergy in lymphocytes), a transmembrane RING finger ubiquitin E3 ligase, initially described as induced during the induction of CD4 T cell anergy, is also expressed in resting CD4 T cells. In this study, we show that GRAIL can down-modulate the expression of CD83 (previously described as a cell surface marke...

  3. ASPM regulates symmetric stem cell division by tuning Cyclin E ubiquitination

    Science.gov (United States)

    Capecchi, Mario R.; Pozner, Amir

    2016-01-01

    We generate a mouse model for the human microcephaly syndrome by mutating the ASPM locus, and demonstrate a premature exhaustion of the neuronal progenitor pool due to dysfunctional self-renewal processes. Earlier studies have linked ASPM mutant progenitor excessive cell cycle exit to a mitotic orientation defect. Here, we demonstrate a mitotic orientation-independent effect of ASPM on cell cycle duration. We pinpoint the cell fate-determining factor to the length of time spent in early G1 before traversing the restriction point. Characterization of the molecular mechanism reveals an interaction between ASPM and the Cdk2/Cyclin E complex, regulating the Cyclin activity by modulating its ubiquitination, phosphorylation and localization into the nucleus, before the cell is fated to transverse the restriction point. Thus, we reveal a novel function of ASPM in mediating the tightly coordinated Ubiquitin- Cyclin E- Retinoblastoma- E2F bistable-signalling pathway controlling restriction point progression and stem cell maintenance. PMID:26581405

  4. Role of the ubiquitin system and tumor viruses in AIDS-related cancer

    Directory of Open Access Journals (Sweden)

    Pagano Joseph S

    2007-11-01

    Full Text Available Abstract Tumor viruses are linked to approximately 20% of human malignancies worldwide. This review focuses on examples of human oncogenic viruses that manipulate the ubiquitin system in a subset of viral malignancies; those associated with AIDS. The viruses include Kaposi's sarcoma herpesvirus, Epstein-Barr virus and human papilloma virus, which are causally linked to Kaposi's sarcoma, certain B-cell lymphomas and cervical cancer, respectively. We discuss the molecular mechanisms by which these viruses subvert the ubiquitin system and potential viral targets for anti-cancer therapy from the perspective of this system. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com.

  5. Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity

    Science.gov (United States)

    Lu, Dongping; Lin, Wenwei; Gao, Xiquan; Wu, Shujing; Cheng, Cheng; Avila, Julian; Heese, Antje; Devarenne, Timothy P.; He, Ping; Shan, Libo

    2011-01-01

    Innate immune responses are triggered by the activation of pattern-recognition receptors (PRRs). The Arabidopsis PRR FLS2 senses bacterial flagellin and initiates immune signaling by association with BAK1. The molecular mechanisms underlying the attenuation of FLS2 activation are largely unknown. We report that flagellin induces recruitment of two closely related U-box E3 ubiquitin ligases PUB12 and PUB13 to FLS2 receptor complex in Arabidopsis. BAK1 phosphorylates PUB12/13 and is required for FLS2-PUB12/13 association. PUB12/13 polyubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, and the pub12 and pub13 mutants displayed elevated immune responses to flagellin treatment. Our study has revealed a unique regulatory circuit of direct ubiquitination and turnover of FLS2 by BAK1-mediated phosphorylation and recruitment of specific E3 ligases for attenuation of immune signaling. PMID:21680842

  6. The Ubiquitin-Like SUMO System and Heart Function: From Development to Disease.

    Science.gov (United States)

    Mendler, Luca; Braun, Thomas; Müller, Stefan

    2016-01-01

    SUMOylation is a ubiquitin-related transient posttranslational modification pathway catalyzing the conjugation of small ubiquitin-like modifier (SUMO) proteins (SUMO1, SUMO2, and SUMO3) to lysine residues of proteins. SUMOylation targets a wide variety of cellular regulators and thereby affects a multitude of different cellular processes. SUMO/sentrin-specific proteases are able to remove SUMOs from targets, contributing to a tight control of SUMOylated proteins. Genetic and cell biological experiments indicate a critical role of balanced SUMOylation/deSUMOylation for proper cardiac development, metabolism, and stress adaptation. Here, we review the current knowledge about SUMOylation/deSUMOylation in the heart and provide an integrated picture of cardiac functions of the SUMO system under physiologic or pathologic conditions. We also describe potential therapeutic approaches targeting the SUMO machinery to combat heart disease.

  7. Nutritional conditions regulate transcriptional activity of SF-1 by controlling sumoylation and ubiquitination.

    Science.gov (United States)

    Lee, Jiwon; Yang, Dong Joo; Lee, Syann; Hammer, Gary D; Kim, Ki Woo; Elmquist, Joel K

    2016-01-11

    Steroidogenic factor 1 (SF-1) is a transcription factor expressed in the ventral medial nucleus of the hypothalamus that regulates energy homeostasis. However, the molecular mechanisms of SF-1 in the control of energy balance are largely unknown. Here, we show that nutritional conditions, such as the presence or absence of serum, affect SF-1 action. Serum starvation significantly decreased hypothalamic SF-1 levels by promoting ubiquitin-dependent degradation, and sumoylation was required for this process. SF-1 transcriptional activity was also differentially regulated by nutritional status. Under normal conditions, the transcriptional activity of hypothalamic SF-1 was activated by SUMO, but this was attenuated during starvation. Taken together, these results indicate that sumoylation and ubiquitination play crucial roles in the regulation of SF-1 function and that these effects are dependent on nutritional conditions, further supporting the importance of SF-1 in the control of energy homeostasis.

  8. Itch inhibits IL-17-mediated colon inflammation and tumorigenesis by ROR-γt ubiquitination.

    Science.gov (United States)

    Kathania, Mahesh; Khare, Prashant; Zeng, Minghui; Cantarel, Brandi; Zhang, Haiying; Ueno, Hideki; Venuprasad, K

    2016-08-01

    Dysregulated expression of interleukin 17 (IL-17) in the colonic mucosa is associated with colonic inflammation and cancer. However, the cell-intrinsic molecular mechanisms by which IL-17 expression is regulated remain unclear. We found that deficiency in the ubiquitin ligase Itch led to spontaneous colitis and increased susceptibility to colon cancer. Itch deficiency in the TH17 subset of helper T cells, innate lymphoid cells and γδ T cells resulted in the production of elevated amounts of IL-17 in the colonic mucosa. Mechanistically, Itch bound to the transcription factor ROR-γt and targeted ROR-γt for ubiquitination. Inhibition or genetic inactivation of ROR-γt attenuated IL-17 expression and reduced spontaneous colonic inflammation in Itch(-/-) mice. Thus, we have identified a previously unknown role for Itch in regulating IL-17-mediated colonic inflammation and carcinogenesis. PMID:27322655

  9. Mitochondrial and Ubiquitin Proteasome System Dysfunction in Ageing and Disease: Two Sides of the Same Coin?

    Directory of Open Access Journals (Sweden)

    Jaime M. Ross

    2015-08-01

    Full Text Available Mitochondrial dysfunction and impairment of the ubiquitin proteasome system have been described as two hallmarks of the ageing process. Additionally, both systems have been implicated in the etiopathogenesis of many age-related diseases, particularly neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Interestingly, these two systems are closely interconnected, with the ubiquitin proteasome system maintaining mitochondrial homeostasis by regulating organelle dynamics, the proteome, and mitophagy, and mitochondrial dysfunction impairing cellular protein homeostasis by oxidative damage. Here, we review the current literature and argue that the interplay of the two systems should be considered in order to better understand the cellular dysfunction observed in ageing and age-related diseases. Such an approach may provide valuable insights into molecular mechanisms underlying the ageing process, and further discovery of treatments to counteract ageing and its associated diseases. Furthermore, we provide a hypothetical model for the heterogeneity described among individuals during ageing.

  10. Non-26S Proteasome Proteolytic Role of Ubiquitin in Plant Endocytosis and Endosomal Trafficking

    Institute of Scientific and Technical Information of China (English)

    Miaomiao Tian; Qi Xie

    2013-01-01

    The 76 amino acid protein ubiquitin (Ub) is highly conserved in all eukaryotic species.It plays important roles in many cellular processes by covalently attaching to the target proteins.The best known function of Ub is marking substrate proteins for degradation by the 26S proteasome.In fact,other consequences of ubiquitination have been discovered in yeast and mammals,such as membrane trafficking,DNA repair,chromatin modification,and protein kinase activation.The common mechanism underlying these processes is that Ub serves as a signal to sort proteins to the vacuoles or lysosomes for degradation as opposed to 26S proteasome-dependent degradation.To date,several reports have indicated that a similar function of Ub also exists in plants.This review focuses on a summary and analysis of the recent research progress on Ub acting as a signal to mediate endocytosis and endosomal trafficking in plants.

  11. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... Home Food Drugs Medical Devices Radiation-Emitting Products Vaccines, Blood & Biologics Animal & Veterinary Cosmetics Tobacco Products Animal & ... back Food Drugs Medical Devices Radiation-Emitting Products Vaccines, Blood & Biologics Animal & Veterinary Cosmetics Tobacco Products

  12. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... Animal & Veterinary Cosmetics Tobacco Products Animal & Veterinary ... The Food and Drug Administration's (FDA's) Center for Veterinary Medicine (CVM) produced a nine-minute animation explaining how ...

  13. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... 08 Animation of Antimicrobial Resistance (text version) Arabic Translation - Animation of Antimicrobial Resistance (WMV - 19.2MB) Chinese Translation - Animation of Antimicrobial Resistance (WMV - 19.2MB) French ...

  14. 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.

  15. Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane.

    LENUS (Irish Health Repository)

    Knodler, Leigh A

    2009-11-01

    The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella-containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono-ubiquitinated. Substitution of these six lysine residues with arginine, SopB-K(6)R, almost completely eliminated SopB ubiquitination. We found that ubiquitination does not affect SopB stability or membrane association, or SopB-dependent events in SCV biogenesis. However, two spatially and temporally distinct events are dependent on ubiquitination, downregulation of SopB activity at the plasma membrane and prolonged retention of SopB on the SCV. Activation of the mammalian pro-survival kinase Akt\\/PKB, a downstream target of SopB, was intensified and prolonged after infection with the SopB-K(6)R mutant. At later times, fewer SCV were decorated with SopB-K(6)R compared with SopB. Instead SopB-K(6)R was present as discrete vesicles spread diffusely throughout the cell. Altogether, our data show that ubiquitination of SopB is not related to its intracellular stability but rather regulates its enzymatic activity at the plasma membrane and intracellular localization.

  16. 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. PMID:27443248

  17. 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.

  18. Ret finger protein mediates Pax7-induced ubiquitination of MyoD in skeletal muscle atrophy.

    Science.gov (United States)

    Joung, Hosouk; Eom, Gwang Hyeon; Choe, Nakwon; Lee, Hye Mi; Ko, Jeong-Hyeon; Kwon, Duk-Hwa; Nam, Yoon Seok; Min, Hyunki; Shin, Sera; Kook, Jeewon; Cho, Young Kuk; Kim, Jeong Chul; Seo, Sang Beom; Baik, Yung Hong; Nam, Kwang-Il; Kook, Hyun

    2014-10-01

    Skeletal muscle atrophy results from the net loss of muscular proteins and organelles and is caused by pathologic conditions such as nerve injury, immobilization, cancer, and other metabolic diseases. Recently, ubiquitination-mediated degradation of skeletal-muscle-specific transcription factors was shown to be involved in muscle atrophy, although the mechanisms have yet to be defined. Here we report that ret finger protein (RFP), also known as TRIM27, works as an E3 ligase in Pax7-induced degradation of MyoD. Muscle injury induced by sciatic nerve transection up-regulated RFP and RFP physically interacted with both Pax7 and MyoD. RFP and Pax7 synergistically reduced the protein amounts of MyoD but not the mRNA. RFP-induced reduction of MyoD protein was blocked by proteasome inhibitors. The Pax7-induced reduction MyoD was attenuated by RFP siRNA and by MG132, a proteasome inhibitor. RFPΔR, an RFP construct that lacks the RING domain, failed to reduce MyoD amounts. RFP ubiquitinated MyoD, but RFPΔR failed to do so. Forced expression of RFP, but not RFPΔR, enhanced Pax7-induced ubiquitination of MyoD, whereas RFP siRNA blocked the ubiquitination. Sciatic nerve injury-induced muscle atrophy as well the reduction in MyoD was attenuated in RFP knockout mice. Taken together, our results show that RFP works as a novel E3 ligase in the Pax7-mediated degradation of MyoD in response to skeletal muscle atrophy. PMID:25025573

  19. Datasets from an interaction proteomics screen for substrates of the SCFβTrCP ubiquitin ligase

    NARCIS (Netherlands)

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

    2015-01-01

    An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCFβTrCP ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCFβTrCP, was used as bait. βTrCP2 wild type and the two mutants βTrCP2-R44

  20. Choreographing the double strand break response: Ubiquitin and SUMO control of nuclear architecture

    Directory of Open Access Journals (Sweden)

    Shane M Harding

    2016-06-01

    Full Text Available The cellular response to DNA double strand breaks (DSBs is a multifaceted signaling program that centers on post-translational modifications including phosphorylation, ubiquitylation and SUMOylation. In this review we discuss how ubiquitin and SUMO orchestrate the recognition of DSBs and explore how this influences chromatin organization. We discuss functional outcomes of this response including transcriptional silencing and how pre-existing chromatin states may control the DSB response and the maintenance of genomic stability.

  1. Protein breakdown in muscle wasting: Role of autophagy-lysosome and ubiquitin-proteasome

    OpenAIRE

    Sandri, Marco

    2013-01-01

    Skeletal muscle adapts its mass as consequence of physical activity, metabolism and hormones. Catabolic conditions or inactivity induce signaling pathways that regulate the process of muscle loss. Muscle atrophy in adult tissue occurs when protein degradation rates exceed protein synthesis. Two major protein degradation pathways, the ubiquitin-proteasome and the autophagy-lysosome systems, are activated during muscle atrophy and variably contribute to the loss of muscle mass. These degradatio...

  2. Drosophila Parkin requires PINK1 for mitochondrial translocation and ubiquitinates Mitofusin

    OpenAIRE

    Ziviani, E.; Tao, R.N.; Whitworth, A. J.

    2010-01-01

    Loss of the E3 ubiquitin ligase Parkin causes early onset Parkinson's disease, a neurodegenerative disorder of unknown etiology. Parkin has been linked to multiple cellular processes including protein degradation, mitochondrial homeostasis, and autophagy; however, its precise role in pathogenesis is unclear. Recent evidence suggests that Parkin is recruited to damaged mitochondria, possibly affecting mitochondrial fission and/or fusion, to mediate their autophagic turnover. The precise mechan...

  3. Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination

    OpenAIRE

    Qiao, Bo; Sugianto, Priscilla; Fung, Eileen; del-Castillo-Rueda, Alejandro; Moran-Jimenez, Maria-Josefa; Ganz, Tomas; Nemeth, Elizabeta

    2012-01-01

    Ferroportin exports iron into plasma from absorptive enterocytes, erythrophagocytosing macrophages, and hepatic stores. The hormone hepcidin controls cellular iron export and plasma iron concentrations by binding to ferroportin and causing its internalization and degradation. We explored the mechanism of hepcidin-induced endocytosis of ferroportin, the key molecular event in systemic iron homeostasis. Hepcidin binding caused rapid ubiquitination of ferroportin in cell lines overexpressing fer...

  4. Ubiquitin-Specific Protease 4 Is an Endogenous Negative Regulator of Pathological Cardiac Hypertrophy.

    Science.gov (United States)

    He, Ben; Zhao, Yi-Chao; Gao, Ling-Chen; Ying, Xiao-Ying; Xu, Long-Wei; Su, Yuan-Yuan; Ji, Qing-Qi; Lin, Nan; Pu, Jun

    2016-06-01

    Dysregulation of the ubiquitin proteasome system components ubiquitin ligases and proteasome plays an important role in the pathogenesis of cardiac hypertrophy. However, little is known about the role of another ubiquitin proteasome system component, the deubiquitinating enzymes, in cardiac hypertrophy. Here, we revealed a crucial role of ubiquitin specific protease 4 (USP4), a deubiquitinating enzyme prominently expressed in the heart, in attenuating pathological cardiac hypertrophy and dysfunction. USP4 levels were consistently decreased in human failing hearts and in murine hypertrophied hearts. Adenovirus-mediated gain- and loss-of-function approaches indicated that deficiency of endogenous USP4 promoted myocyte hypertrophy induced by angiotensin II in vitro, whereas restoration of USP4 significantly attenuated the prohypertrophic effect of angiotensin II. To corroborate the role of USP4 in vivo, we generated USP4 global knockout mice and mice with cardiac-specific overexpression of USP4. Consistent with the in vitro study, USP4 depletion exacerbated the hypertrophic phenotype and cardiac dysfunction in mice subjected to pressure overload, whereas USP4 transgenic mice presented ameliorated pathological cardiac hypertrophy compared with their control littermates. Molecular analysis revealed that USP4 deficiency augmented the activation of the transforming growth factor β-activated kinase 1 (TAK1)-(JNK1/2)/P38 signaling in response to hypertrophic stress, and blockage of TAK1 activation abolished the pathological effects of USP4 deficiency in vivo. These findings provide the first evidence for the involvement of USP4 in cardiac hypertrophy, and shed light on the therapeutic potential of targeting USP4 in the treatment of cardiac hypertrophy.

  5. Three-dimensional structure of ubiquitin at 2.8 A resolution.

    OpenAIRE

    Vijay-Kumar, S; Bugg, C E; Wilkinson, K. D.; Cook, W. J.

    1985-01-01

    The three-dimensional structure of ubiquitin has been determined at 2.8 A resolution. X-ray diffraction data for the native protein and derivatives were collected with an automated diffractometer. Phases were obtained by use of a single isomorphous mercuric acetate derivative. The molecule contains a pronounced hydrophobic core. Prominent secondary structural features include three and one-half turns of alpha-helix, a mixed beta-sheet that contains four strands, and seven reverse turns. The h...

  6. Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers

    Directory of Open Access Journals (Sweden)

    Peter eRuethemann

    2016-04-01

    Full Text Available Global-genome nucleotide excision repair (GG-NER prevents genome instability by excising a wide range of structurally unrelated DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV radiation or intracellular metabolic by-products. As a versatile damage sensor, xeroderma pigmentosum group C (XPC protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4DDB2 and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4DDB2 or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin.

  7. Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers.

    Science.gov (United States)

    Rüthemann, Peter; Balbo Pogliano, Chiara; Naegeli, Hanspeter

    2016-01-01

    Global-genome nucleotide excision repair (GG-NER) prevents genome instability by excising a wide range of different DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV) light or intracellular side products of metabolism. As a versatile damage sensor, xeroderma pigmentosum group C (XPC) protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4(DDB2) and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4(DDB2) or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin. PMID:27200078

  8. Quantitative proteomics and terminomics to elucidate the role of ubiquitination and proteolysis in adaptive immunity.

    Science.gov (United States)

    Klein, Theo; Viner, Rosa I; Overall, Christopher M

    2016-10-28

    Adaptive immunity is the specialized defence mechanism in vertebrates that evolved to eliminate pathogens. Specialized lymphocytes recognize specific protein epitopes through antigen receptors to mount potent immune responses, many of which are initiated by nuclear factor-kappa B activation and gene transcription. Most, if not all, pathways in adaptive immunity are further regulated by post-translational modification (PTM) of signalling proteins, e.g. phosphorylation, citrullination, ubiquitination and proteolytic processing. The importance of PTMs is reflected by genetic or acquired defects in these pathways that lead to a dysfunctional immune response. Here we discuss the state of the art in targeted proteomics and systems biology approaches to dissect the PTM landscape specifically regarding ubiquitination and proteolysis in B- and T-cell activation. Recent advances have occurred in methods for specific enrichment and targeted quantitation. Together with improved instrument sensitivity, these advances enable the accurate analysis of often rare PTM events that are opaque to conventional proteomics approaches, now rendering in-depth analysis and pathway dissection possible. We discuss published approaches, including as a case study the profiling of the N-terminome of lymphocytes of a rare patient with a genetic defect in the paracaspase protease MALT1, a key regulator protease in antigen-driven signalling, which was manifested by elevated linear ubiquitination.This article is part of the themed issue 'Quantitative mass spectrometry'. PMID:27644975

  9. 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.

  10. E3 ubiquitin-ligases and their target proteins during the regulation of plant innate immunity

    Directory of Open Access Journals (Sweden)

    Vincent eDuplan

    2014-02-01

    Full Text Available Reversible protein ubiquitination plays a crucial role during the regulation of plant immune signaling. E3 ubiquitin (Ub-ligase enzymes, which are classified into different families depending on their structural and functional features, confer the specificity of substrate and are the best characterized components of the ubiquitination cascade. E3 Ub-ligases of different families have been shown to be involved in all steps of plant immune responses. Indeed, they have been involved in the first steps of pathogen perception, as they appear to modulate perception of pathogen-associated molecular patterns by pattern-recognition receptors at the plasma membrane and to regulate the accumulation of nucleotide-binding leucine-rich repeat-type intracellular immune receptors. In addition, E3 Ub-ligase proteins are also involved in the regulation of the signaling responses downstream of pathogen perception through targeting vesicle trafficking components or nuclear transcription factors, for instance. Finally, we also discuss the case of microbial effector proteins that are able to target host E3 Ub-ligases, or to act themselves as E3 Ub-ligases, in their attempt to subvert the host proteasome to promote disease.

  11. E3 ubiquitin-ligases and their target proteins during the regulation of plant innate immunity.

    Science.gov (United States)

    Duplan, Vincent; Rivas, Susana

    2014-01-01

    Reversible protein ubiquitination plays a crucial role during the regulation of plant immune signaling. E3 ubiquitin (Ub)-ligase enzymes, which are classified into different families depending on their structural and functional features, confer the specificity of substrate and are the best characterized components of the ubiquitination cascade. E3 Ub-ligases of different families have been shown to be involved in all steps of plant immune responses. Indeed, they have been involved in the first steps of pathogen perception, as they appear to modulate perception of pathogen-associated molecular patterns by pattern-recognition receptors at the plasma membrane and to regulate the accumulation of nucleotide-binding leucine-rich repeat-type intracellular immune receptors. In addition, E3 Ub-ligase proteins are also involved in the regulation of the signaling responses downstream of pathogen perception through targeting vesicle trafficking components or nuclear transcription factors, for instance. Finally, we also discuss the case of microbial effector proteins that are able to target host E3 Ub-ligases, or to act themselves as E3 Ub-ligases, in their attempt to subvert the host proteasome to promote disease. PMID:24592270

  12. A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Eli Arama

    2007-10-01

    Full Text Available In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, we screened a large collection of sterile male flies for mutants that block effector caspase activation at the onset of spermatid individualization. Here, we describe the identification and characterization of a testis-specific, Cullin-3-dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3(Testis, the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC domain of Cul3(Testis that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis-like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation.

  13. Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system

    Directory of Open Access Journals (Sweden)

    Lalonde Sylvie

    2003-03-01

    Full Text Available Abstract Background The Arabidopsis genome contains nine sucrose transporter paralogs falling into three clades: SUT1-like, SUT2 and SUT4. The carriers differ in their kinetic properties. Many transport proteins are known to exist as oligomers. The yeast-based split ubiquitin system can be used to analyze the ability of membrane proteins to interact. Results Promoter-GUS fusions were used to analyze the cellular expression of the three transporter genes in transgenic Arabidopsis plants. All three fusion genes are co-expressed in companion cells. Protein-protein interactions between Arabidopsis sucrose transporters were tested using the split ubiquitin system. Three paralogous sucrose transporters are capable of interacting as either homo- or heteromers. The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters. Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters. Conclusions The split-ubiquitin system provides a powerful tool to detect potential interactions between plant membrane proteins by heterologous expression in yeast, and can be used to screen for interactions with membrane proteins as baits. Like other membrane proteins, the Arabidopsis sucrose transporters are able to form oligomers. The biochemical approaches are required to confirm the in planta interaction.

  14. Genetic immunization based on the ubiquitin-fusion degradation pathway against Trypanosoma cruzi

    Energy Technology Data Exchange (ETDEWEB)

    Chou, Bin [Department of Microbiology and Immunology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan); Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan); Hiromatsu, Kenji, E-mail: khiromatsu@fukuoka-u.ac.jp [Department of Microbiology and Immunology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan); Hisaeda, Hajime; Duan, Xuefeng; Imai, Takashi [Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan); Murata, Shigeo; Tanaka, Keiji [Department of Molecular Oncology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613 (Japan); Himeno, Kunisuke [Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan)

    2010-02-12

    Cytotoxic CD8{sup +} T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8{sup +} T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8{sup +} T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4{sup +} T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8{sup +} T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.

  15. Genetic immunization based on the ubiquitin-fusion degradation pathway against Trypanosoma cruzi

    International Nuclear Information System (INIS)

    Cytotoxic CD8+ T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8+ T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8+ T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4+ T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8+ T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.

  16. Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase

    Energy Technology Data Exchange (ETDEWEB)

    Orlicky, Steve; Tang, Xiaojing; Willems, Andrew; Tyers, Mike; Sicheri, Frank

    2010-12-01

    Cell cycle progression depends on precise elimination of cyclins and cyclin-dependent kinase (CDK) inhibitors by the ubiquitin system. Elimination of the CDK inhibitor Sic1 by the SCF{sup Cdc4} ubiquitin ligase at the onset of S phase requires phosphorylation of Sic1 on at least six of its nine Cdc4-phosphodegron (CPD) sites. A 2.7 {angstrom} X-ray crystal structure of a Skp1-Cdc4 complex bound to a high-affinity CPD phosphopeptide from human cyclin E reveals a core CPD motif, Leu-Leu-pThr-Pro, bound to an eight-bladed WD40 propeller domain in Cdc4. The low affinity of each CPD motif in Sic1 reflects structural discordance with one or more elements of the Cdc4 binding site. Reengineering of Cdc4 to reduce selection against Sic1 sequences allows ubiquitination of lower phosphorylated forms of Sic1. These features account for the observed phosphorylation threshold in Sic1 recognition and suggest an equilibrium binding mode between a single receptor site in Cdc4 and multiple low-affinity CPD sites in Sic1.

  17. The ubiquitin ligase Praja1 reduces NRAGE expression and inhibits neuronal differentiation of PC12 cells.

    Science.gov (United States)

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

    2013-01-01

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

  18. The ubiquitin ligase Praja1 reduces NRAGE expression and inhibits neuronal differentiation of PC12 cells.

    Directory of Open Access Journals (Sweden)

    Jan Teuber

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

  19. XIAP inhibits mature Smac-induced apoptosis by degrading it through ubiquitination in NSCLC

    Science.gov (United States)

    Qin, Sida; Yang, Chengcheng; Zhang, Boxiang; Li, Xiang; Sun, Xin; Li, Gang; Zhang, Jing; Xiao, Guodong; Gao, Xiao; Huang, Guanghong; Wang, Peili; Ren, Hong

    2016-01-01

    X-linked inhibitor of apoptosis protein (XIAP) and second mitochondrial-derived activator of caspase (Smac) are two important prognostic biomarkers for cancer. They are negatively correlated in many types of cancer. However, their relationship is still unknown in lung cancer. In the present study, we found that there was a negative correlation between Smac and XIAP at the level of protein but not mRNA in NSCLC patients. However, XIAP overexpression had no effect on degrading endogenous Smac in lung cancer cell lines. Therefore, we constructed plasmids with full length of Smac (fSmac) and mature Smac (mSmac) which located in cytoplasm instead of original mitochondrial location, and was confirmed by immunofluorescence. Subsequently, we found that mSmac rather than fSmac was degraded by XIAP and inhibited cell viability. CHX chase assay and ubiquitin assay were performed to illustrate XIAP degraded mSmac through ubiquitin pathway. Overexpression of XIAP partially reverted apoptotic induction and cell viability inhibition by mSmac, which was due to inhibiting caspase-3 activation. In nude mouse xenograft experiments, mSmac inhibited Ki-67 expression and slowed down lung cancer growth, while XIAP partially reversed the effect of mSmac by degrading it. In conclusion, XIAP inhibits mature Smac-induced apoptosis by degrading it through ubiquitination in NSCLC. PMID:27498621

  20. Solution structure of the ubiquitin-binding domain in Swa2p from Saccharomyces cerevisiae.

    Science.gov (United States)

    Chim, Nicholas; Gall, Walter E; Xiao, Jing; Harris, Mark P; Graham, Todd R; Krezel, Andrzej M

    2004-03-01

    The SWA2/AUX1 gene has been proposed to encode the Saccharomyces cerevisiae ortholog of mammalian auxilin. Swa2p is required for clathrin assembly/dissassembly in vivo, thereby implicating it in intracellular protein and lipid trafficking. While investigating the 287-residue N-terminal region of Swa2p, we found a single stably folded domain between residues 140 and 180. Using binding assays and structural analysis, we established this to be a ubiquitin-associated (UBA) domain, unidentified by bioinformatics of the yeast genome. We determined the solution structure of this Swa2p domain and found a characteristic three-helix UBA fold. Comparisons of structures of known UBA folds reveal that the position of the third helix is quite variable. This helix in Swa2p UBA contains a bulkier tyrosine in place of smaller residues found in other UBAs and cannot pack as close to the second helix. The molecular surface of Swa2p UBA has a mostly negative potential, with a single hydrophobic surface patch found also in the UBA domains of human protein, HHR23A. The presence of a UBA domain implicates Swa2p in novel roles involving ubiquitin and ubiquitinated substrates. We propose that Swa2p is a multifunctional protein capable of recognizing several proteins through its protein-protein recognition domains. PMID:14997574

  1. Structure and Ubiquitination-Dependent Activation of TANK-Binding Kinase 1

    Directory of Open Access Journals (Sweden)

    Daqi Tu

    2013-03-01

    Full Text Available Upon stimulation by pathogen-associated inflammatory signals, TANK-binding kinase 1 (TBK1 induces type I interferon expression and modulates nuclear factor κB (NF-κB signaling. Here, we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a dimeric assembly created by an extensive network of interactions among the kinase, ubiquitin-like, and scaffold/dimerization domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on lysines 30 and 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog inhibitor of κB kinase ∊ (IKK∊ but not in IKKβ, a canonical IKK that assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IKKs in normal and disease physiology and further the development of more specific inhibitors that may be useful as anticancer or anti-inflammatory agents.

  2. Observing a late folding intermediate of Ubiquitin at atomic resolution by NMR.

    Science.gov (United States)

    Surana, Parag; Das, Ranabir

    2016-08-01

    The study of intermediates in the protein folding pathway provides a wealth of information about the energy landscape. The intermediates also frequently initiate pathogenic fibril formations. While observing the intermediates is difficult due to their transient nature, extreme conditions can partially unfold the proteins and provide a glimpse of the intermediate states. Here, we observe the high resolution structure of a hydrophobic core mutant of Ubiquitin at an extreme acidic pH by nuclear magnetic resonance (NMR) spectroscopy. In the structure, the native secondary and tertiary structure is conserved for a major part of the protein. However, a long loop between the beta strands β3 and β5 is partially unfolded. The altered structure is supported by fluorescence data and the difference in free energies between the native state and the intermediate is reflected in the denaturant induced melting curves. The unfolded region includes amino acids that are critical for interaction with cofactors as well as for assembly of poly-Ubiquitin chains. The structure at acidic pH resembles a late folding intermediate of Ubiquitin and indicates that upon stabilization of the protein's core, the long loop converges on the core in the final step of the folding process. PMID:27111887

  3. Structural Basis of Selective Ubiquitination of TRF1 by SCF[superscript Fbx4

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Zhixiong; Wang, Wei; Yang, Yuting; Chen, Yong; Yang, Xiaomei; Diehl, J. Alan; Liu, Xuedong; Lei, Ming (UPENN); (Michigan-Med); (Colorado)

    2010-09-03

    TRF1 is a critical regulator of telomere length. As such, TRF1 levels are regulated by ubiquitin-dependent proteolysis via an SCF E3 ligase where Fbx4 contributes to substrate specification. Here, we report the crystal structure of the Fbx4-TRF1 complex at 2.4 {angstrom} resolution. Fbx4 contains an unusual substrate-binding domain that adopts a small GTPase fold. Strikingly, this atypical GTPase domain of Fbx4 binds to a globular domain of TRF1 through an intermolecular {beta} sheet, instead of recognizing short peptides/degrons as often seen in other F-box protein-substrate complexes. Importantly, mutations in this interface abrogate Fbx4-dependent TRF1 binding and ubiquitination. Furthermore, the data demonstrate that recognition of TRF1 by SCFFbx4 is regulated by another telomere protein, TIN2. Our results reveal an atypical small GTPase domain within Fbx4 as a substrate-binding motif for SCFFbx4 and uncover a mechanism for selective ubiquitination and degradation of TRF1 in telomere homeostasis control.

  4. Involvement of the Ubiquitin-Proteasome System in the Formation of Experimental Postsurgical Peritoneal Adhesions

    Directory of Open Access Journals (Sweden)

    Clara Di Filippo

    2012-01-01

    Full Text Available We investigated the Ubiquitin-Proteasome System (UPS, major nonlysosomal intracellular protein degradation system, in the genesis of experimental postsurgical peritoneal adhesions. We assayed the levels of UPS within the adhered tissue along with the development of peritoneal adhesions and used the specific UPS inhibitor bortezomib in order to assess the effect of the UPS blockade on the peritoneal adhesions. We found a number of severe postsurgical peritoneal adhesions at day 5 after surgery increasing until day 10. In the adhered tissue an increased values of ubiquitin and the 20S proteasome subunit, NFkB, IL-6, TNF-α and decreased values of IkB-beta were found. In contrast, bortezomib-treated rats showed a decreased number of peritoneal adhesions, decreased values of ubiquitin and the 20S proteasome, NFkB, IL-6, TNF-α, and increased levels of IkB-beta in the adhered peritoneal tissue. The UPS system, therefore, is primarily involved in the formation of post-surgical peritoneal adhesions in rats.

  5. Transfer RNA is an essential component of the ubiquitin- and ATP-dependent proteolytic system

    Energy Technology Data Exchange (ETDEWEB)

    Ciechanover, A.; Wolin, S.L.. Steitz, J.A.; Lodish, H.F.

    1985-03-01

    Protein degradation via the nonlysosomal ATP-dependent pathway in rabbit reticulocytes involves a number of components. In the initial event, ubiquitin, an abundant 76-residue polypeptide, becomes covalently linked to the protein substrate in an ATP-requiring reaction. Once marked in this way, the conjugated protein is proteolyzed in a reaction that also requires ATP. Here the authors show that tRNA is another essential component of the system. Ribonucleases strongly inhibit the ubiquitin- and ATP-dependent degradation of /sup 125/I-labeled bovine serum albumin in the reticulocyte system in vitro. RNAs extracted from fractions of the reticulocyte extract or from mouse cells restore proteolytic activity. When the RNA is fractionated by gel electrophoresis, only the tRNA fraction is active in restoring proteolysis. Furthermore, pure mouse tRNA/sup His/, isolated by immunoprecipitation with patient autoimmune sera, restores the proteolytic activity. The possibility that the level of uncharged tRNA in mammalian cells regulates the ubiquitin- and ATP-dependent proteolytic system is discussed.

  6. Ubiquitin-Mediated Regulation of Endocytosis by Proteins of the Arrestin Family

    Directory of Open Access Journals (Sweden)

    Michel Becuwe

    2012-01-01

    Full Text Available In metazoans, proteins of the arrestin family are key players of G-protein-coupled receptors (GPCRS signaling and trafficking. Following stimulation, activated receptors are phosphorylated, thus allowing the binding of arrestins and hence an “arrest” of receptor signaling. Arrestins act by uncoupling receptors from G proteins and contribute to the recruitment of endocytic proteins, such as clathrin, to direct receptor trafficking into the endocytic pathway. Arrestins also serve as adaptor proteins by promoting the recruitment of ubiquitin ligases and participate in the agonist-induced ubiquitylation of receptors, known to have impact on their subcellular localization and stability. Recently, the arrestin family has expanded following the discovery of arrestin-related proteins in other eukaryotes such as yeasts or fungi. Surprisingly, most of these proteins are also involved in the ubiquitylation and endocytosis of plasma membrane proteins, thus suggesting that the role of arrestins as ubiquitin ligase adaptors is at the core of these proteins' functions. Importantly, arrestins are themselves ubiquitylated, and this modification is crucial for their function. In this paper, we discuss recent data on the intricate connections between arrestins and the ubiquitin pathway in the control of endocytosis.

  7. Ubiquitin Ligase TRIM62 Regulates CARD9-Mediated Anti-fungal Immunity and Intestinal Inflammation.

    Science.gov (United States)

    Cao, Zhifang; Conway, Kara L; Heath, Robert J; Rush, Jason S; Leshchiner, Elizaveta S; Ramirez-Ortiz, Zaida G; Nedelsky, Natalia B; Huang, Hailiang; Ng, Aylwin; Gardet, Agnès; Cheng, Shih-Chin; Shamji, Alykhan F; Rioux, John D; Wijmenga, Cisca; Netea, Mihai G; Means, Terry K; Daly, Mark J; Xavier, Ramnik J

    2015-10-20

    CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 alterations. Here, we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigating CARD9 regulation. We showed that the protective variant of CARD9, which is C-terminally truncated, acted in a dominant-negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that similar to Card9-deficient mice, Trim62-deficient mice had increased susceptibility to fungal infection. In this study, we utilized a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation. PMID:26488816

  8. Seeing the animal

    DEFF Research Database (Denmark)

    Harfeld, Jes Lynning; Cornou, Cecile; Kornum, Anna;

    2016-01-01

    This article discusses the notion that the invisibility of the animalness of the animal constitutes a fundamental obstacle to change within current production systems. It is discussed whether housing animals in environments that resemble natural habitats could lead to a re-animalization...... of the animals, a higher appreciation of their moral significance, and thereby higher standards of animal welfare. The basic claim is that experiencing the animals in their evolutionary and environmental context would make it harder to objectify animals as mere bioreactors and production systems. It is argued...... that the historic objectification of animals within intensive animal production can only be reversed if animals are given the chance to express themselves as they are and not as we see them through the tunnel visions of economy and quantifiable welfare assessment parameters....

  9. Animal rights, animal minds, and human mindreading.

    Science.gov (United States)

    Mameli, M; Bortolotti, L

    2006-02-01

    Do non-human animals have rights? The answer to this question depends on whether animals have morally relevant mental properties. Mindreading is the human activity of ascribing mental states to other organisms. Current knowledge about the evolution and cognitive structure of mindreading indicates that human ascriptions of mental states to non-human animals are very inaccurate. The accuracy of human mindreading can be improved with the help of scientific studies of animal minds. However, the scientific studies do not by themselves solve the problem of how to map psychological similarities (and differences) between humans and animals onto a distinction between morally relevant and morally irrelevant mental properties. The current limitations of human mindreading-whether scientifically aided or not-have practical consequences for the rational justification of claims about which rights (if any) non-human animals should be accorded.

  10. Refining Animal Models to Enhance Animal Welfare

    Institute of Scientific and Technical Information of China (English)

    Patricia V.Turner

    2012-01-01

    The use of animals in research will be necessary for scientific advances in the basic and biomedical sciences for the foreseeable future.As we learn more about the ability of animals to experience pain,suffering,and distress,and particularly for mammals,it becomes the responsibility of scientists,institutions,animal caregivers,and veterinarians to seek ways to improve the lives of research animals and refine their care and use.Refinement is one of the three R's emphasized by Russell and Burch,and refers to modification of procedures to minimise the potential for pain,suffering and distress. It may also refer to procedures used to enhance animal comfort. This paper summarizes considerations for refinements in research animal.

  11. Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein.

    Directory of Open Access Journals (Sweden)

    Ricardo Rajsbaum

    Full Text Available Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1 proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04, avian (HK156, swine (SwTx98 and mouse-adapted (PR8 influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production.

  12. Animal Images and Metaphors in Animal Farm

    OpenAIRE

    Ping Sun

    2015-01-01

    In literary works animal images are frequently used as the “source domain” of a metaphor to disclose the natures of the “target domain”, human beings. This is called “cross-domain mapping” or “conceptual metaphor” in cognitive linguistics, which is based on the similar qualities between animals and human beings. Thus the apparent descriptions of the animals are really the deep revelations of the human beings. Animal Farm is one exemplary product of this special expressing way. Diversified ani...

  13. Ian Ingram: Next Animals

    DEFF Research Database (Denmark)

    2015-01-01

    Ian Ingram: Next Animals is an exhibition catalogue presenting research on the work by Ian Ingram in relation to his exhibition Next Animals at Nikolaj Kunsthal in 2015.......Ian Ingram: Next Animals is an exhibition catalogue presenting research on the work by Ian Ingram in relation to his exhibition Next Animals at Nikolaj Kunsthal in 2015....

  14. FARM ANIMAL WELFARE ECONOMICS

    Directory of Open Access Journals (Sweden)

    L.T. CZISZTER

    2013-07-01

    Full Text Available This paper reviews the literature regarding the economics of the farm animal welfare. The following issues are addressed: productions costs and savings of the animal welfare regulations, benefits of improved animal welfare, and consumers’ willingness to pay for animal-friendly products.

  15. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... En Español Search FDA Submit search Popular Content Home Food Drugs Medical Devices Radiation-Emitting Products Vaccines, ... Biologics Animal & Veterinary Cosmetics Tobacco Products Animal & Veterinary Home Animal & Veterinary Safety & Health Antimicrobial Resistance Animation of ...

  16. Physics for Animation Artists

    Science.gov (United States)

    Chai, David; Garcia, Alejandro L.

    2011-01-01

    Animation has become enormously popular in feature films, television, and video games. Art departments and film schools at universities as well as animation programs at high schools have expanded in recent years to meet the growing demands for animation artists. Professional animators identify the technological facet as the most rapidly advancing…

  17. Carotenoids in Marine Animals

    Directory of Open Access Journals (Sweden)

    Takashi Maoka

    2011-02-01

    Full Text Available Marine animals contain various carotenoids that show structural diversity. These marine animals accumulate carotenoids from foods such as algae and other animals and modify them through metabolic reactions. Many of the carotenoids present in marine animals are metabolites of β-carotene, fucoxanthin, peridinin, diatoxanthin, alloxanthin, and astaxanthin, etc. Carotenoids found in these animals provide the food chain as well as metabolic pathways. In the present review, I will describe marine animal carotenoids from natural product chemistry, metabolism, food chain, and chemosystematic viewpoints, and also describe new structural carotenoids isolated from marine animals over the last decade.

  18. Memory formation for trace fear conditioning requires ubiquitin-proteasome mediated protein degradation in the prefrontal cortex.

    Directory of Open Access Journals (Sweden)

    David S Reis

    2013-10-01

    Full Text Available The cellular mechanisms supporting plasticity during memory consolidation have been a subject of considerable interest. De novo protein and mRNA synthesis in several brain areas are critical, and more recently protein degradation, mediated by the ubiquitin-proteasome system (UPS, has been shown to be important. Previous work clearly establishes a relationship between protein synthesis and protein degradation in the amygdala, but it is unclear whether cortical mechanisms of memory consolidation are similar to those in the amygdala. Recent work demonstrating a critical role for prefrontal cortex (PFC in the acquisition and consolidation of fear memory allows us to address this question. Here we use a PFC-dependent fear conditioning protocol to determine whether UPS mediated protein degradation is necessary for memory consolidation in PFC. Groups of rats were trained with auditory delay or trace fear conditioning and sacrificed 60 min after training. PFC tissue was then analyzed to quantify the amount of polyubiquinated protein. Other animals were trained with similar procedures but were infused with either a proteasome inhibitor (clasto-lactacystin β-lactone or a translation inhibitor (anisomycin in the PFC immediately after training. Our results show increased UPS-mediated protein degradation in the PFC following trace but not delay fear conditioning. Additionally, post-training proteasome or translation inhibition significantly impaired trace but not delay fear memory when tested the next day. Our results further support the idea that the PFC is critical for trace but not delay fear conditioning highlight the role of UPS-mediated degradation as critical for synaptic plasticity.

  19. Ethics in Animal Experimentation

    Directory of Open Access Journals (Sweden)

    Yusuf Ergun

    2010-08-01

    Full Text Available Experimental animals are frequently used to obtain information for primarily scientific reasons. In the present review, ethics in animal experimentation is examined. At first, the history of animal experimentation and animal rights is outlined. Thereafter, the terms in relation with the topic are defined. Finally, prominent aspects of 3Rs constituting scientific and ethical basis in animal experimentation are underlined. [Archives Medical Review Journal 2010; 19(4.000: 220-235

  20. Ethics in Animal Experimentation

    OpenAIRE

    Yusuf Ergun

    2010-01-01

    Experimental animals are frequently used to obtain information for primarily scientific reasons. In the present review, ethics in animal experimentation is examined. At first, the history of animal experimentation and animal rights is outlined. Thereafter, the terms in relation with the topic are defined. Finally, prominent aspects of 3Rs constituting scientific and ethical basis in animal experimentation are underlined. [Archives Medical Review Journal 2010; 19(4.000): 220-235

  1. Carotenoids in Marine Animals

    OpenAIRE

    Takashi Maoka

    2011-01-01

    Marine animals contain various carotenoids that show structural diversity. These marine animals accumulate carotenoids from foods such as algae and other animals and modify them through metabolic reactions. Many of the carotenoids present in marine animals are metabolites of β-carotene, fucoxanthin, peridinin, diatoxanthin, alloxanthin, and astaxanthin, etc. Carotenoids found in these animals provide the food chain as well as metabolic pathways. In the present review, I will describe marine a...

  2. Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, James M.; Korzhnev, Dmitry M.; Ceccarelli, Derek F.; Briant, Douglas J.; Zarrine-Afsar, Arash; Sicheri, Frank; Kay, Lewis E.; Pawson, Tony (Mount Sinai Hospital); (Toronto)

    2012-10-23

    The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studies of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for mono ubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the N- and C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.

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

    Science.gov (United States)

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

    2016-09-01

    Ubiquitination, a post-translational modification, involves the covalent attachment of ubiquitin to the target protein. The ubiquitin-proteasome pathway and the endosome-lysosome pathway control the degradation of the majority of eukaryotic proteins. Our previous study illustrated that δ-catenin ubiquitination occurs in a glycogen synthase kinase-3 (GSK-3) phosphorylation-dependent manner. However, the molecular mechanism of δ-catenin ubiquitination is still unknown. Here, we show that the lysine residues required for ubiquitination are located mainly in the C-terminal portion of δ-catenin. In addition, we provide evidence that β-TrCP-1 interacts with δ-catenin and functions as an E3 ligase, mediating δ-catenin ubiquitin-proteasome degradation. Furthermore, we prove that both the ubiquitin-proteasome pathway and the lysosome degradation pathway are involved in δ-catenin degradation. Our novel findings on the mechanism of δ-catenin ubiquitination will add a new perspective to δ-catenin degradation and the effects of δ-catenin on E-cadherin involved in epithelial cell-cell adhesion, which is implicated in prostate cancer progression. PMID:27316454

  4. Identification of lysines within membrane-anchored Mga2p120 that are targets of Rsp5p ubiquitination and mediate mobilization of tethered Mga2p90

    OpenAIRE

    Bhattacharya, Sabyasachi; Shcherbik, Natalia; Vasilescu, Julian; Smith, Jeffrey C.; Figeys, Daniel; Haines, Dale S.

    2008-01-01

    Mga2p90 is an endoplasmic reticulum (ER) localized transcription factor that is released from the ER membrane by a unique ubiquitin-dependent mechanism. Mga2p90 mobilization requires poly-ubiquitination of its associating membrane-bound Mga2p120 anchor and subsequent Mga2p120-Mga2p90 complex disassembly that is mediated by ATPase Cdc48p and its heteromeric ubiquitin-binding adaptor Npl4p-Ufd1p. Although previous studies have identified the ubiquitin ligase (i.e. Rsp5p) and ligase binding site...

  5. Dynamic ubiquitination of the mitogen-activated protein kinase kinase (MAPKK) Ste7 determines mitogen-activated protein kinase (MAPK) specificity.

    Science.gov (United States)

    Hurst, Jillian H; Dohlman, Henrik G

    2013-06-28

    Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF(Cdc4) and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF(Cdc4) ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF(Cdc4) and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF(Cdc4) is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK. PMID:23645675

  6. 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.

  7. Structure of full-length ubiquitin-conjugating enzyme E2-25K (huntingtin-interacting protein 2)

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Randall C.; Hughes, Ronny C.; Flatt, Justin W.; Meehan, Edward J.; Ng, Joseph D.; Twigg, Pamela D.; (UAH)

    2009-08-07

    The ubiquitin-conjugating enzyme E2-25K has been identified as a huntingtin (the key protein in Huntington's disease) interacting protein and has been shown to play a role in mediating the toxicity of A{beta}, the principal protein involved in Alzheimer's disease pathogenesis. E2-25K is a dual-domain protein with an ubiquitin-associated (UBA) domain as well as a conserved ubiquitin-conjugating (UBC) domain which catalyzes the formation of a covalent bond between the C-terminal glycine of an ubiquitin molecule and the {var_epsilon}-amine of a lysine residue on the acceptor protein as part of the ubiquitin-proteasome pathway. The crystal structures of E2-25K M172A mutant protein at pH 6.5 and pH 8.5 were determined to 1.9 and 2.2 {angstrom} resolution, respectively. Examination of the structures revealed domain-domain interactions between the UBC and UBA domains which have not previously been reported

  8. Cadmium interferes with the degradation of ATF5 via a post-ubiquitination step of the proteasome degradation pathway

    International Nuclear Information System (INIS)

    ATF5 is a member of the CREB/ATF family of transcription factors. In the current study, using a transient transfection system to express FLAG epitope fusion proteins of ATF5, we have shown that CdCl2 or NaAsO3 increases the protein levels of ATF5 in cells, and that cadmium stabilizes the ATF5 protein. Proteasome inhibitors had a similar effect to cadmium on the cellular accumulation of ATF5. Proteasome inhibition led to an increase in ubiquitinated ATF5, while cadmium did not appear to reduce the extent of ATF5 ubiquitination. ATF5 contains a putative nuclear export signal within its N-terminus. We demonstrated that whereas deletion of N-terminal region resulted in a increase of ATF5 levels, this region does not appear to be involved in the ubiquitination of ATF5. These results indicate that ATF5 is targeted for degradation by the ubiquitin-proteasome pathway, and that cadmium slows the rate of ATF5 degradation via a post-ubiquitination mechanism.

  9. Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network

    Science.gov (United States)

    Markson, Gabriel; Kiel, Christina; Hyde, Russell; Brown, Stephanie; Charalabous, Panagoula; Bremm, Anja; Semple, Jennifer; Woodsmith, Jonathan; Duley, Simon; Salehi-Ashtiani, Kourosh; Vidal, Marc; Komander, David; Serrano, Luis; Lehner, Paul; Sanderson, Christopher M.

    2009-01-01

    In eukaryotic cells the stability and function of many proteins are regulated by the addition of ubiquitin or ubiquitin-like peptides. This process is dependent upon the sequential action of an E1-activating enzyme, an E2-conjugating enzyme, and an E3 ligase. Different combinations of these proteins confer substrate specificity and the form of protein modification. However, combinatorial preferences within ubiquitination networks remain unclear. In this study, yeast two-hybrid (Y2H) screens were combined with true homology modeling methods to generate a high-density map of human E2/E3-RING interactions. These data include 535 experimentally defined novel E2/E3-RING interactions and >1300 E2/E3-RING pairs with more favorable predicted free-energy values than the canonical UBE2L3–CBL complex. The significance of Y2H predictions was assessed by both mutagenesis and functional assays. Significantly, 74/80 (>92%) of Y2H predicted complexes were disrupted by point mutations that inhibit verified E2/E3-RING interactions, and a ∼93% correlation was observed between Y2H data and the functional activity of E2/E3-RING complexes in vitro. Analysis of the high-density human E2/E3-RING network reveals complex combinatorial interactions and a strong potential for functional redundancy, especially within E2 families that have undergone evolutionary expansion. Finally, a one-step extended human E2/E3-RING network, containing 2644 proteins and 5087 edges, was assembled to provide a resource for future functional investigations. PMID:19549727

  10. The Rewiring of Ubiquitination Targets in a Pathogenic Yeast Promotes Metabolic Flexibility, Host Colonization and Virulence.

    Science.gov (United States)

    Childers, Delma S; Raziunaite, Ingrida; Mol Avelar, Gabriela; Mackie, Joanna; Budge, Susan; Stead, David; Gow, Neil A R; Lenardon, Megan D; Ballou, Elizabeth R; MacCallum, Donna M; Brown, Alistair J P

    2016-04-01

    Efficient carbon assimilation is critical for microbial growth and pathogenesis. The environmental yeast Saccharomyces cerevisiae is "Crabtree positive", displaying a rapid metabolic switch from the assimilation of alternative carbon sources to sugars. Following exposure to sugars, this switch is mediated by the transcriptional repression of genes (carbon catabolite repression) and the turnover (catabolite inactivation) of enzymes involved in the assimilation of alternative carbon sources. The pathogenic yeast Candida albicans is Crabtree negative. It has retained carbon catabolite repression mechanisms, but has undergone posttranscriptional rewiring such that gluconeogenic and glyoxylate cycle enzymes are not subject to ubiquitin-mediated catabolite inactivation. Consequently, when glucose becomes available, C. albicans can continue to assimilate alternative carbon sources alongside the glucose. We show that this metabolic flexibility promotes host colonization and virulence. The glyoxylate cycle enzyme isocitrate lyase (CaIcl1) was rendered sensitive to ubiquitin-mediated catabolite inactivation in C. albicans by addition of a ubiquitination site. This mutation, which inhibits lactate assimilation in the presence of glucose, reduces the ability of C. albicans cells to withstand macrophage killing, colonize the gastrointestinal tract and cause systemic infections in mice. Interestingly, most S. cerevisiae clinical isolates we examined (67%) have acquired the ability to assimilate lactate in the presence of glucose (i.e. they have become Crabtree negative). These S. cerevisiae strains are more resistant to macrophage killing than Crabtree positive clinical isolates. Moreover, Crabtree negative S. cerevisiae mutants that lack Gid8, a key component of the Glucose-Induced Degradation complex, are more resistant to macrophage killing and display increased virulence in immunocompromised mice. Thus, while Crabtree positivity might impart a fitness advantage for yeasts in

  11. The ubiquitin ligase tripartite-motif-protein 32 is induced in Duchenne muscular dystrophy.

    Science.gov (United States)

    Assereto, Stefania; Piccirillo, Rosanna; Baratto, Serena; Scudieri, Paolo; Fiorillo, Chiara; Massacesi, Manuela; Traverso, Monica; Galietta, Luis J; Bruno, Claudio; Minetti, Carlo; Zara, Federico; Gazzerro, Elisabetta

    2016-08-01

    Activation of the proteasome pathway is one of the secondary processes of cell damage, which ultimately lead to muscle degeneration and necrosis in Duchenne muscular dystrophy (DMD). In mdx mice, the proteasome inhibitor bortezomib up-regulates the membrane expression of members of the dystrophin complex and reduces the inflammatory reaction. However, chronic inhibition of the 26S proteasome may be toxic, as indicated by the systemic side-effects caused by this drug. Therefore, we sought to determine the components of the ubiquitin-proteasome pathway that are specifically activated in human dystrophin-deficient muscles. The analysis of a cohort of patients with genetically determined DMD or Becker muscular dystrophy (BMD) unveiled a selective up-regulation of the ubiquitin ligase tripartite motif-containing protein 32 (TRIM32). The induction of TRIM32 was due to a transcriptional effect and it correlated with disease severity in BMD patients. In contrast, atrogin1 and muscle RING-finger protein-1 (MuRF-1), which are strongly increased in distinct types of muscular atrophy, were not affected by the DMD dystrophic process. Knock-out models showed that TRIM32 is involved in ubiquitination of muscle cytoskeletal proteins as well as of protein inhibitor of activated STAT protein gamma (Piasγ) and N-myc downstream-regulated gene, two inhibitors of satellite cell proliferation and differentiation. Accordingly, we showed that in DMD/BMD muscle tissue, TRIM32 induction was more pronounced in regenerating myofibers rather than in necrotic muscle cells, thus pointing out a role of this protein in the regulation of human myoblast cell fate. This finding highlights TRIM32 as a possible therapeutic target to favor skeletal muscle regeneration in DMD patients.

  12. Accessing ns-{mu}s side chain dynamics in ubiquitin with methyl RDCs

    Energy Technology Data Exchange (ETDEWEB)

    Fares, Christophe [University of Toronto, University Health Network, Max Bell Research Center (Canada); Lakomek, Nils-Alexander [National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Walter, Korvin F. A.; Frank, Benedikt T. C. [Max-Planck Institute for Biophysical Chemistry, Department of NMR-based Structural Biology (Germany); Meiler, Jens [Vanderbilt University, Department of Chemistry, Center of Structural Biology (United States); Becker, Stefan; Griesinger, Christian [Max-Planck Institute for Biophysical Chemistry, Department of NMR-based Structural Biology (Germany)], E-mail: cigr@nmr.mpibpc.mpg.de

    2009-09-15

    This study presents the first application of the model-free analysis (MFA) (Meiler in J Am Chem Soc 123:6098-6107, 2001; Lakomek in J Biomol NMR 34:101-115, 2006) to methyl group RDCs measured in 13 different alignment media in order to describe their supra-{tau}{sub c} dynamics in ubiquitin. Our results indicate that methyl groups vary from rigid to very mobile with good correlation to residue type, distance to backbone and solvent exposure, and that considerable additional dynamics are effective at rates slower than the correlation time {tau}{sub c}. In fact, the average amplitude of motion expressed in terms of order parameters S{sup 2} associated with the supra-{tau}{sub c} window brings evidence to the existence of fluctuations contributing as much additional mobility as those already present in the faster ps-ns time scale measured from relaxation data. Comparison to previous results on ubiquitin demonstrates that the RDC-derived order parameters are dominated both by rotameric interconversions and faster libration-type motions around equilibrium positions. They match best with those derived from a combined J-coupling and residual dipolar coupling approach (Chou in J Am Chem Soc 125:8959-8966, 2003) taking backbone motion into account. In order to appreciate the dynamic scale of side chains over the entire protein, the methyl group order parameters are compared to existing dynamic ensembles of ubiquitin. Of those recently published, the broadest one, namely the EROS ensemble (Lange in Science 320:1471-1475, 2008), fits the collection of methyl group order parameters presented here best. Last, we used the MFA-derived averaged spherical harmonics to perform highly-parameterized rotameric searches of the side chains conformation and find expanded rotamer distributions with excellent fit to our data. These rotamer distributions suggest the presence of concerted motions along the side chains.

  13. The Rewiring of Ubiquitination Targets in a Pathogenic Yeast Promotes Metabolic Flexibility, Host Colonization and Virulence.

    Directory of Open Access Journals (Sweden)

    Delma S Childers

    2016-04-01

    Full Text Available Efficient carbon assimilation is critical for microbial growth and pathogenesis. The environmental yeast Saccharomyces cerevisiae is "Crabtree positive", displaying a rapid metabolic switch from the assimilation of alternative carbon sources to sugars. Following exposure to sugars, this switch is mediated by the transcriptional repression of genes (carbon catabolite repression and the turnover (catabolite inactivation of enzymes involved in the assimilation of alternative carbon sources. The pathogenic yeast Candida albicans is Crabtree negative. It has retained carbon catabolite repression mechanisms, but has undergone posttranscriptional rewiring such that gluconeogenic and glyoxylate cycle enzymes are not subject to ubiquitin-mediated catabolite inactivation. Consequently, when glucose becomes available, C. albicans can continue to assimilate alternative carbon sources alongside the glucose. We show that this metabolic flexibility promotes host colonization and virulence. The glyoxylate cycle enzyme isocitrate lyase (CaIcl1 was rendered sensitive to ubiquitin-mediated catabolite inactivation in C. albicans by addition of a ubiquitination site. This mutation, which inhibits lactate assimilation in the presence of glucose, reduces the ability of C. albicans cells to withstand macrophage killing, colonize the gastrointestinal tract and cause systemic infections in mice. Interestingly, most S. cerevisiae clinical isolates we examined (67% have acquired the ability to assimilate lactate in the presence of glucose (i.e. they have become Crabtree negative. These S. cerevisiae strains are more resistant to macrophage killing than Crabtree positive clinical isolates. Moreover, Crabtree negative S. cerevisiae mutants that lack Gid8, a key component of the Glucose-Induced Degradation complex, are more resistant to macrophage killing and display increased virulence in immunocompromised mice. Thus, while Crabtree positivity might impart a fitness

  14. The ubiquitin ligase tripartite-motif-protein 32 is induced in Duchenne muscular dystrophy.

    Science.gov (United States)

    Assereto, Stefania; Piccirillo, Rosanna; Baratto, Serena; Scudieri, Paolo; Fiorillo, Chiara; Massacesi, Manuela; Traverso, Monica; Galietta, Luis J; Bruno, Claudio; Minetti, Carlo; Zara, Federico; Gazzerro, Elisabetta

    2016-08-01

    Activation of the proteasome pathway is one of the secondary processes of cell damage, which ultimately lead to muscle degeneration and necrosis in Duchenne muscular dystrophy (DMD). In mdx mice, the proteasome inhibitor bortezomib up-regulates the membrane expression of members of the dystrophin complex and reduces the inflammatory reaction. However, chronic inhibition of the 26S proteasome may be toxic, as indicated by the systemic side-effects caused by this drug. Therefore, we sought to determine the components of the ubiquitin-proteasome pathway that are specifically activated in human dystrophin-deficient muscles. The analysis of a cohort of patients with genetically determined DMD or Becker muscular dystrophy (BMD) unveiled a selective up-regulation of the ubiquitin ligase tripartite motif-containing protein 32 (TRIM32). The induction of TRIM32 was due to a transcriptional effect and it correlated with disease severity in BMD patients. In contrast, atrogin1 and muscle RING-finger protein-1 (MuRF-1), which are strongly increased in distinct types of muscular atrophy, were not affected by the DMD dystrophic process. Knock-out models showed that TRIM32 is involved in ubiquitination of muscle cytoskeletal proteins as well as of protein inhibitor of activated STAT protein gamma (Piasγ) and N-myc downstream-regulated gene, two inhibitors of satellite cell proliferation and differentiation. Accordingly, we showed that in DMD/BMD muscle tissue, TRIM32 induction was more pronounced in regenerating myofibers rather than in necrotic muscle cells, thus pointing out a role of this protein in the regulation of human myoblast cell fate. This finding highlights TRIM32 as a possible therapeutic target to favor skeletal muscle regeneration in DMD patients. PMID:27295345

  15. NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses.

    Directory of Open Access Journals (Sweden)

    Jie Zhou

    Full Text Available Plant autophagy plays an important role in delaying senescence, nutrient recycling, and stress responses. Functional analysis of plant autophagy has almost exclusively focused on the proteins required for the core process of autophagosome assembly, but little is known about the proteins involved in other important processes of autophagy, including autophagy cargo recognition and sequestration. In this study, we report functional genetic analysis of Arabidopsis NBR1, a homolog of mammalian autophagy cargo adaptors P62 and NBR1. We isolated two nbr1 knockout mutants and discovered that they displayed some but not all of the phenotypes of autophagy-deficient atg5 and atg7 mutants. Like ATG5 and ATG7, NBR1 is important for plant tolerance to heat, oxidative, salt, and drought stresses. The role of NBR1 in plant tolerance to these abiotic stresses is dependent on its interaction with ATG8. Unlike ATG5 and ATG7, however, NBR1 is dispensable in age- and darkness-induced senescence and in resistance to a necrotrophic pathogen. A selective role of NBR1 in plant responses to specific abiotic stresses suggest that plant autophagy in diverse biological processes operates through multiple cargo recognition and delivery systems. The compromised heat tolerance of atg5, atg7, and nbr1 mutants was associated with increased accumulation of insoluble, detergent-resistant proteins that were highly ubiquitinated under heat stress. NBR1, which contains an ubiquitin-binding domain, also accumulated to high levels with an increasing enrichment in the insoluble protein fraction in the autophagy-deficient mutants under heat stress. These results suggest that NBR1-mediated autophagy targets ubiquitinated protein aggregates most likely derived from denatured or otherwise damaged nonnative proteins generated under stress conditions.

  16. Photosensitivity to Triflusal: Formation of a Photoadduct with Ubiquitin Demonstrated by Photophysical and Proteomic Techniques

    Science.gov (United States)

    Nuin, Edurne; Pérez-Sala, Dolores; Lhiaubet-Vallet, Virginie; Andreu, Inmaculada; Miranda, Miguel A.

    2016-01-01

    Triflusal is a platelet aggregation inhibitor chemically related to acetylsalicylic acid, which is used for the prevention and/or treatment of vascular thromboembolisms, which acts as a prodrug. Actually, after oral administration it is absorbed primarily in the small intestine, binds to plasma proteins (99%) and is rapidly biotransformed in the liver into its deacetylated active metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB). In healthy humans, the half-life of triflusal is ca. 0.5 h, whereas for HTB it is ca. 35 h. From a pharmacological point of view, it is interesting to note that HTB is itself highly active as a platelet anti-aggregant agent. Indeed, studies on the clinical profile of both drug and metabolite have shown no significant differences between them. It has been evidenced that HTB displays ability to induce photoallergy in humans. This phenomenon involves a cell-mediated immune response, which is initiated by covalent binding of a light-activated photosensitizer (or a species derived therefrom) to a protein. In this context, small proteins like ubiquitin could be appropriate models for investigating covalent binding by means of MS/MS and peptide fingerprint analysis. In previous work, it was shown that HTB forms covalent photoadducts with isolated lysine. Interestingly, ubiquitin contains seven lysine residues that could be modified by a similar reaction. With this background, the aim of the present work is to explore adduct formation between the triflusal metabolite and ubiquitin as model protein upon sunlight irradiation, combining proteomic and photophysical (fluorescence and laser flash photolysis) techniques. Photophysical and proteomic analysis demonstrates monoadduct formation as the major outcome of the reaction. Interestingly, addition can take place at any of the ε-amino groups of the lysine residues of the protein and involves replacement of the trifluoromethyl moiety with a new amide function. This process can in principle

  17. 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.

  18. The ubiquitin proteasome system plays a role in venezuelan equine encephalitis virus infection.

    Directory of Open Access Journals (Sweden)

    Moushimi Amaya

    Full Text Available Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV and Western equine encephalitis virus (WEEV demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections.

  19. Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating activity of SARS-CoV papain-like protease.

    Directory of Open Access Journals (Sweden)

    Kiira Ratia

    2014-05-01

    Full Text Available Severe acute respiratory syndrome coronavirus (SARS-CoV encodes a papain-like protease (PLpro with both deubiquitinating (DUB and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15 specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a "ridge" region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response.

  20. RETHINKING THE ANIMATE, RE-ANIMATING THOUGHT

    Directory of Open Access Journals (Sweden)

    Tim Ingold

    2013-12-01

    Full Text Available Animism is often described as the imputation of life to inert objects. Such imputation is more typical of people in western societies who dream of finding life on other planets than of indigenous peoples to whom the label of animism has classically been applied. These peoples are united not in their beliefs but in a way of being that is alive and open to a world in continuous birth. In this animic ontology, beings do not propel themselves across a ready-made world but rather issue forth through a world-in-formation, along the lines of their relationships. To its inhabitants this weather-world, embracing both sky and earth, is a source of astonishment but not surprise. Re-animating the ‘western’ tradition of thought means recovering the sense of astonishment banished from offi cial science.

  1. OTULIN Restricts Met1-Linked Ubiquitination to Control Innate Immune Signaling

    DEFF Research Database (Denmark)

    Fiil, Berthe Katrine; Damgaard, Rune Busk; Wagner, Sebastian Alexander;

    2013-01-01

    Conjugation of Met1-linked polyubiquitin (Met1-Ub) by the linear ubiquitin chain assembly complex (LUBAC) is an important regulatory modification in innate immune signaling. So far, only few Met1-Ub substrates have been described, and the regulatory mechanisms have remained elusive. We recently...... identified that the ovarian tumor (OTU) family deubiquitinase OTULIN specifically disassembles Met1-Ub. Here, we report that OTULIN is critical for limiting Met1-Ub accumulation after nucleotide-oligomerization domain-containing protein 2 (NOD2) stimulation, and that OTULIN depletion augments signaling...

  2. The possible role of the ubiquitin proteasome system in the development of atherosclerosis in diabetes

    Directory of Open Access Journals (Sweden)

    sasso Ferdinando

    2007-10-01

    Full Text Available Abstract We have reviewed the impact of the ubiquitin proteasome system (UPS on atherosclerosis progression of diabetic patients. A puzzle of many pieces of evidence suggests that UPS, in addition to its role in the removal of damaged proteins, is involved in a number of biological processes including inflammation, proliferation and apoptosis, all of which constitute important characteristics of atherosclerosis. From what can be gathered from the very few studies on the UPS in diabetic cardiovascular diseases published so far, the system seems to be functionally active to a different extent in the initiation, progression, and complication stage of atherosclerosis in the diabetic people. Further evidence for this theory, however, has to be given, for instance by specifically targeted antagonism of the UPS. Nonetheless, this hypothesis may help us understand why diverse therapeutic interventions, which have in common the ability to reduce ubiquitin-proteasome activity, can impede or delay the onset of diabetes and cardiovascular diseases (CVD. People with type 2 diabetes are disproportionately affected by CVD, compared with those without diabetes 1. The prevalence, incidence, and mortality from all forms of CVD (myocardial infarction, cerebro-vascular disease and congestive heart failure are strikingly increased in persons with diabetes compared with those withoutdiabetes 2. Furthermore, diabetic patients have not benefited by the advances in the management of obesity, dyslipidemia, and hypertension that have resulted in a decrease in mortality for coronary heart disease (CHD patients without diabetes 3. Nevertheless, these risk factors do not fully explain the excess risk for CHD associated with diabetes 45. Thus, the determinants of progression of atherosclerosis in persons with diabetes must be elucidated. Beyond the major risk factors, several studies have demonstrated that such factors, strictly related to diabetes, as insulin

  3. The E3 ubiquitin ligase RNF8 stabilizes TPP1 to promote telomere end protection

    OpenAIRE

    Rai, Rekha; Li, Ju-mei; Zheng, Hong; Lok, Gabriel Tsz-Mei; Deng, Yu; Huen, Michael; Chen, Junjie; Jin, Jianping; Chang, Sandy

    2011-01-01

    TPP1, a component of the mammalian shelterin complex, plays essential roles in telomere maintenance. It forms a heterodimer with POT1 to repress ATR-dependent DNA damage signaling at telomeres, and recruits telomerase to chromosome ends. Here we show that the E3 ubiquitin ligase RNF8 localizes to and promotes the accumulation of DNA damage proteins 53BP1 and γ-H2AX to uncapped telomeres. TPP1 is unstable in the absence of RNF8, resulting in telomere shortening and chromosome fusions via the a...

  4. Small ubiquitin-like modifier protein-specific protease 1 and prostate cancer

    Institute of Scientific and Technical Information of China (English)

    Yong Zuo; Jin-Ke Cheng

    2009-01-01

    Small ubiquitin-like modifier protein (SUMO) modification is a highly dynamic process,catalyzed by SUMO-specific activating (E1),conjugating (E2) and ligating (E3) enzymes,and reversed by a family of SUMO-specific proteases (SENPs).There are six members of the human SENP family,and each SENP has different cellular locations and substrate specificities.However,the precise roles of SENPs in cellular processes have not been elucidated to date.This brief review will focus on recent advances pertaining to the identified targets of SENP 1 and its potential role in prostate cancer.

  5. Ubiquitin is a Novel Substrate for Human Insulin-Degrading Enzyme

    OpenAIRE

    Ralat, Luis A.; Kalas, Vasilios; Zheng, Zhongzhou; Goldman, Robert D.; Sosnick, Tobin R.; Tang, Wei-Jen

    2010-01-01

    Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β (Aβ), peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two C-terminal glycines (kcat = 2 sec-1) followed by a slow cleavage between residues 72-73 (kcat = 0.07 sec-1), thereby...

  6. E3 ubiquitin ligase GRAIL controls primary T cell activation and oral tolerance

    OpenAIRE

    Kriegel, Martin A.; Rathinam, Chozhavendan; Richard A Flavell

    2009-01-01

    T cell unresponsiveness or anergy is one of the mechanisms that maintain inactivity of self-reactive lymphocytes. E3 ubiquitin ligases are important mediators of the anergic state. The RING finger E3 ligase GRAIL is thought to selectively function in anergic T cells but its mechanism of action and its role in vivo are largely unknown. We show here that genetic deletion of Grail in mice leads not only to loss of an anergic phenotype in various models but also to hyperactivation of primary CD4+...

  7. The Inherited Blindness Protein AIPL1 Regulates the Ubiquitin-Like FAT10 Pathway

    OpenAIRE

    Bett, John S.; Naheed Kanuga; Emma Richet; Gunter Schmidtke; Marcus Groettrup; Cheetham, Michael E.; Jacqueline van der Spuy

    2012-01-01

    Mutations in AIPL1 cause the inherited blindness Leber congenital amaurosis (LCA). AIPL1 has previously been shown to interact with NUB1, which facilitates the proteasomal degradation of proteins modified with the ubiquitin-like protein FAT10. Here we report that AIPL1 binds non-covalently to free FAT10 and FAT10ylated proteins and can form a ternary complex with FAT10 and NUB1. In addition, AIPL1 antagonised the NUB1-mediated degradation of the model FAT10 conjugate, FAT10-DHFR, and pathogen...

  8. Interaction between animal personality and animal cognition

    OpenAIRE

    Claudio CARERE, Charles LOCURTO

    2011-01-01

    The study of animal personality has attracted considerable attention, as it has revealed a number of similarities in personality between humans and several nonhuman species. At the same time the adaptive value and evolutionary maintenance of different personalities are the subject of debate. Since Pavlov’s work on dogs, students of comparative cognition have been aware that animals display vast individual differences on cognitive tasks, and that these differences may not be entirely accounted...

  9. Animals in Education.

    Science.gov (United States)

    Rowan, Andrew N.

    1981-01-01

    Summarizes viewpoints on the use of animals in science experiments in the biology classroom, including those of teachers, education researchers, biomedical scientists, science education administrators, and animal welfare advocates. (Author/CS)

  10. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... FDA Submit search Popular Content Home Food Drugs Medical Devices Radiation-Emitting Products Vaccines, Blood & Biologics Animal & ... by Product Area Product Areas back Food Drugs Medical Devices Radiation-Emitting Products Vaccines, Blood & Biologics Animal & ...

  11. "Name" that Animal

    Science.gov (United States)

    Laird, Shirley

    2010-01-01

    In this article, the author describes a texture and pattern project. Students started by doing an outline contour drawing of an animal. With the outline drawn, the students then write one of their names to fit "inside" the animal.

  12. Morris Animal Foundation

    Science.gov (United States)

    ... the transmission of serious illnesses. Read more » Morris Animal Foundation Receives $750,000 Grant for Cancer Studies. ... Give Partners Become a Partner Meet Our Partners Animal Lovers Our Work Ways to Give Pet Health ...

  13. Generic Face Animation

    OpenAIRE

    Cerda, Mauricio; Valenzuela, Renato; Hitschfeld-Kahler, Nancy; Terissi, Lucas; Gomez, Juan C.

    2010-01-01

    International audience In computer vision, the animation of objects has attracted a lot attention, specially the animations of 3D face models. The animation of face models requires in general to manually adapt each generic movement (open/close mouth) to each specific head geometry. In this work we propose a technique for the animation of any face model avoiding most of the manual intervention. In order to achieve this we assume that: (1) faces, despite obvious differences are quite similar...

  14. Biopolitics: Animals, meat, food

    OpenAIRE

    Janović Nikola

    2009-01-01

    The general idea of this text is to reflect biopolitical constitution of the society and its implications related to the issues of animal welfare. Since animal in biopolitical formation is technically reduced to an object - commodity for contentment of the industry and of the people needs - critical public advisories are calling from moral, ethical and legal standpoint for attention to the fact that is necessary to protect animals from the unnecessary exploitation. It is obvious that animal p...

  15. Bioethics in animal experimentation

    OpenAIRE

    Popa V.I.; Lascar I.; Valcu M.; Sebe Ioana Teona; Caraban B.; Margina Arina Cristiana

    2015-01-01

    Animal experiments are used on a large scale worldwide in order to develop or to refine new medicines, medicinal products or surgical procedures. It is morally wrong to cause animals to suffer, this is why animal experimentation causes serious moral problems.

  16. Animal Models for imaging

    OpenAIRE

    Croft, Barbara Y.

    2002-01-01

    Animal models can be used in the study of disease. This chapter discusses imaging animal models to elucidate the process of human disease. The mouse is used as the primary model. Though this choice simplifies many research choices, it necessitates compromises for in vivo imaging. In the future, we can expect improvements in both animal models and imaging techniques.

  17. Animal violence demystified

    NARCIS (Netherlands)

    Natarajan, Deepa; Caramaschi, Doretta

    2010-01-01

    Violence has been observed in humans and animals alike, indicating its evolutionary/biological significance. However, violence in animals has often been confounded with functional forms of aggressive behavior. Currently, violence in animals is identified primarily as either a quantitative behavior (

  18. I like animals

    Institute of Scientific and Technical Information of China (English)

    官健

    2008-01-01

    @@ Animals are our friends.We should protect them and we mustn't hurtthem. Do you like animals?My answer is"yes".Maybe you may ask me why.I will tell you they are very lovely.I like many animals,such as pandas,monkeys and elephants.

  19. F-box protein FBXL2 inhibits gastric cancer proliferation by ubiquitin-mediated degradation of forkhead box M1.

    Science.gov (United States)

    Li, Liang-qing; Pan, Dun; Chen, Hui; Zhang, Lin; Xie, Wen-jun

    2016-02-01

    F-box/LRR-repeat protein 2 (FBXL2), a component of Skp-Cullin-F box (SCF) ubiquitin E3 ligase, has been shown to inhibit tumorigenesis by targeting and ubiquitinating several oncoproteins. However, its role in gastric cancer remains poorly understood. Here, by tandem mass spectrometry, we show that FBXL2 interacts with forkhead box M1 (FoxM1) transcription factor. As a result, FBXL2 promotes ubiquitination and degradation of FoxM1 in gastric cancer cells. Furthermore, overexpression of FBXL2 inhibits, while its deficiency promotes cell proliferation and invasion. Expression levels of cell-cycle regulators (Cdc25B and p27), which are down-stream target effectors of FoxM1, are also regulated by FBXL2. Therefore, our results uncover a previous unknown network involving FBXL2 and FoxM1 in the regulation of gastric cancer growth.

  20. Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

    Energy Technology Data Exchange (ETDEWEB)

    Imura, Yoshiyuki, E-mail: imura@brs.nihon-u.ac.jp; Molho, Melissa; Chuang, Chingkai; Nagy, Peter D., E-mail: pdnagy2@uky.edu

    2015-10-15

    Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement both defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.

  1. Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export

    Directory of Open Access Journals (Sweden)

    Marriott Susan J

    2007-12-01

    Full Text Available Abstract Background The HTLV-I oncoprotein, Tax, is a pleiotropic protein whose activity is partially regulated by its ability to interact with, and perturb the functions of, numerous cellular proteins. Tax is predominantly a nuclear protein that localizes to nuclear foci known as Tax Speckled Structures (TSS. We recently reported that the localization of Tax and its interactions with cellular proteins are altered in response to various forms of genotoxic and cellular stress. The level of cytoplasmic Tax increases in response to stress and this relocalization depends upon the interaction of Tax with CRM1. Cellular pathways and signals that regulate the subcellular localization of Tax remain to be determined. However, post-translational modifications including sumoylation and ubiquitination are known to influence the subcellular localization of Tax and its interactions with cellular proteins. The sumoylated form of Tax exists predominantly in the nucleus while ubiquitinated Tax exists predominantly in the cytoplasm. Therefore, we hypothesized that post-translational modifications of Tax that occur in response to DNA damage regulate the localization of Tax and its interactions with cellular proteins. Results We found a significant increase in mono-ubiquitination of Tax in response to UV irradiation. Mutation of specific lysine residues (K280 and K284 within Tax inhibited DNA damage-induced ubiquitination. In contrast to wild-type Tax, which undergoes transient nucleocytoplasmic shuttling in response to DNA damage, the K280 and K284 mutants were retained in nuclear foci following UV irradiation and remained co-localized with the cellular TSS protein, sc35. Conclusion This study demonstrates that the localization of Tax, and its interactions with cellular proteins, are dynamic following DNA damage and depend on the post-translational modification status of Tax. Specifically, DNA damage induces the ubiquitination of Tax at K280 and K284

  2. IAPs contain an evolutionarily conserved ubiquitin-binding domain that regulates NF-kappaB as well as cell survival and oncogenesis

    DEFF Research Database (Denmark)

    Gyrd-Hansen, Mads; Darding, Maurice; Miasari, Maria;

    2008-01-01

    -MALT1 stimulates NF-kappaB signalling by binding to polyubiquitylated NEMO. Significantly, 98% of all cIAP2-MALT1 fusion proteins retain the UBA domain, suggesting that ubiquitin-binding contributes to the oncogenic potential of cIAP2-MALT1 in MALT lymphoma. Our data identify IAPs as ubiquitin...

  3. Ubiquitin, a central component of selective cytoplasmic proteolysis, is linked to proteins residing at the locus of non-selective proteolysis, the vacuole

    NARCIS (Netherlands)

    Simeon, Angela; Klei, Ida J. van der; Veenhuis, Marten; Wolf, Dieter H.

    1992-01-01

    Ubiquitin, an evolutionary highly conserved protein, is known to be involved in selective proteolysis in the cytoplasm. Here we show that ubiquitin-protein conjugates are also found in the yeast vacuole. Mutants defective in the major vacuolar endopeptidases, proteinase yscA and yscB, lead to accumu

  4. 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

  5. DMPD: A pervasive role of ubiquitin conjugation in activation and termination ofIkappaB kinase pathways. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15809659 A pervasive role of ubiquitin conjugation in activation and termination ofIkappaB kinase path...csml) Show A pervasive role of ubiquitin conjugation in activation and termination ofIkappaB kinase pathways... and termination ofIkappaB kinase pathways. Authors Krappmann D, Scheidereit C. Publication EMBO Rep. 2005 A

  6. Animal models of dementia

    DEFF Research Database (Denmark)

    Olsson, I. Anna S.; Sandøe, Peter

    2011-01-01

    This chapter aims to encourage scientists and others interested in the use of animal models of disease – specifically, in the study of dementia – to engage in ethical reflection. It opens with a general discussion of the moral acceptability of animal use in research. Three ethical approaches...... are here distinguished. These serve as points of orientation in the following discussion of four more specific ethical questions: Does animal species matter? How effective is disease modelling in delivering the benefits claimed for it? What can be done to minimize potential harm to animals in research? Who...... bears responsibility for the use of animals in disease models?...

  7. Requirement of the SCFPop1/Pop2 Ubiquitin Ligase for Degradation of the Fission Yeast S Phase Cyclin Cig2

    OpenAIRE

    Yamano, H; Kominami, K; Harrison, C; Kitamura, K.; Katayama, S; Dhut, S.; Hunt, T; Toda, T.

    2004-01-01

    Two multiprotein E3 (ubiquitin-protein ligase) ubiquitin ligases, the SCF (Skp1-Cullin-1-F-box) and the APC/C (anaphase promoting complex/cyclosome), are vital in ensuring the temporal order of the cell cycle. Particularly, timely destruction of cyclins via these two E3s is essential for down-regulation of cyclin-dependent kinase. In general, G(1) and S phase cyclins are ubiquitylated by the SCF, whereas ubiquitylation of mitotic cyclins is catalyzed by the APC/C. Here we show that fission ye...

  8. Dyrk1A phosphorylates parkin at Ser-131 and negatively regulates its ubiquitin E3 ligase activity.

    Science.gov (United States)

    Im, Eunju; Chung, Kwang Chul

    2015-08-01

    Mutations of parkin are associated with the occurrence of autosomal recessive familial Parkinson's disease (PD). Parkin acts an E3 ubiquitin ligase, which ubiquitinates target proteins and subsequently regulates either their steady-state levels through the ubiquitin-proteasome system or biochemical properties. In this study, we identify a novel regulatory mechanism of parkin by searching for new regulatory factors. After screening human fetal brain using a yeast two hybrid assay, we found dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (Dyrk1A) as a novel binding partner of parkin. We also observed that parkin interacts and co-localizes with Dyrk1A in mammalian cells. In addition, Dyrk1A directly phosphorylated parkin at Ser-131, causing the inhibition of its E3 ubiquitin ligase activity. Moreover, Dyrk1A-mediated phosphorylation reduced the binding affinity of parkin to its ubiquitin-conjugating E2 enzyme and substrate, which could be the underlying inhibitory mechanism of parkin activity. Furthermore, Dyrk1A-mediated phosphorylation inhibited the neuroprotective action of parkin against 6-hydroxydopamine toxicity in dopaminergic SH-SY5Y cells. These findings suggest that Dyrk1A acts as a novel functional modulator of parkin. Parkin phosphorylation by Dyrk1A suppresses its E3 ubiquitin ligase activity potentially contributing to the pathogenesis of PD under PD-inducing pathological conditions. Mutations of parkin are linked to autosomal recessive forms of familial Parkinson's disease (PD). According to its functional relevance in abnormal protein aggregation and neuronal cell death, a number of post-translational modifications regulate the ubiquitin E3 ligase activity of parkin. Here we propose a novel inhibitory mechanism of parkin E3 ubiquitin ligase through dual-specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A)-mediated phosphorylation as well as its neuroprotective action against 6-hydroxydopamine (6-OHDA)-induced cell death

  9. 1H, 13C, and 15N resonance assignment of the ubiquitin-like domain from Dsk2p

    OpenAIRE

    Chen, Tony; Zhang, Daoning; Matiuhin, Yulia; Glickman, Michael; Fushman, David

    2008-01-01

    The ubiquitin-like domain (UBL) of yeast protein Dsk2p is widely believed to recognize and bind to ubiquitin receptors on the proteasome and, as part of Dsk2p, to bridge polyubiquitinated substrates and proteasomal degradation machinery. Here we report NMR resonance assignment for 1H, 15N, and 13C nuclei in the backbone and side chains of the UBL domain of Dsk2p. This assignment will aid in NMR studies focused on understanding of Dsk2’s interactions with proteasomal receptors and its role as ...

  10. [Animal experimentation in Israel].

    Science.gov (United States)

    Epstein, Yoram; Leshem, Micah

    2002-04-01

    In 1994 the Israeli parliament (Knesset) amended the Cruelty to Animals Act to regulate the use of experimental animals. Accordingly, animal experiments can only be carried out for the purposes of promoting health and medical science, reducing suffering, advancing scientific research, testing or production of materials and products (excluding cosmetics and cleaning products) and education. Animal experiments are only permitted if alternative methods are not possible. The National Board for Animal Experimentation was established to implement the law. Its members are drawn from government ministries, representatives of doctors, veterinarians, and industry organizations, animal rights groups, and academia. In order to carry out an animal experiment, the institution, researchers involved, and the specific experiment, all require approval by the Board. To date the Board has approved some 35 institutions, about half are public institutions (universities, hospitals and colleges) and the rest industrial firms in biotechnology and pharmaceutics. In 2000, 250,000 animals were used in research, 85% were rodents, 11% fowls, 1,000 other farm animals, 350 dogs and cats, and 39 monkeys. Academic institutions used 74% of the animals and industry the remainder. We also present summarized data on the use of animals in research in other countries.

  11. Importin-11, a nuclear import receptor for the ubiquitin-conjugating enzyme, UbcM2.

    Science.gov (United States)

    Plafker, S M; Macara, I G

    2000-10-16

    Importins are members of a family of transport receptors (karyopherins) that mediate the nucleocytoplasmic transport of protein and RNA cargoes. We identified importin-11 as a potential new human member of this family, on the basis of limited similarity to the Saccharomyces cerevisiae protein, Lph2p, and cloned the complete open reading frame. Importin-11 interacts with the Ran GTPase, and constitutively shuttles between the nuclear and cytoplasmic compartments. A yeast dihybrid screen identified UbcM2, an E2-type ubiquitin-conjugating enzyme, as a binding partner and potential transport cargo for importin-11. Importin-11 and UbcM2 interact directly, and the complex is disassembled by Ran:GTP but not by Ran:GDP. UbcM2 is constitutively nuclear and shuttles between the nuclear and cytoplasmic compartments. Nuclear import of UbcM2 requires Ran and importin-11, and is inhibited by wheatgerm agglutinin, energy depletion or dominant interfering mutants of Ran and importin-beta. These data establish importin-11 as a new member of the karyopherin family of transport receptors, and identify UbcM2 as a nuclear member of the E2 ubiquitin-conjugating enzyme family. PMID:11032817

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

    Science.gov (United States)

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

    2016-07-14

    Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4(CRBN) E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumour activity. The anti-tumour activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degradation of the translation termination factor GSPT1. Patient-derived acute myeloid leukaemia tumour cells exhibit high sensitivity to CC-885, indicating the clinical potential of this mechanism. Crystallographic studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn containing a glycine residue at a key position, interacting with both CC-885 and a 'hotspot' on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homology to previously known cereblon substrates, mutational analysis and modelling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon 'neosubstrate' selectivity, and identify an anti-tumour target rendered druggable by cereblon modulation. PMID:27338790

  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. Datasets from an interaction proteomics screen for substrates of the SCFβTrCP ubiquitin ligase

    Directory of Open Access Journals (Sweden)

    Roberto Magliozzi

    2015-09-01

    Full Text Available An affinity purification-mass spectrometry (AP-MS method was employed to identify novel substrates of the SCFβTrCP ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCFβTrCP, was used as bait. βTrCP2 wild type and the two mutants βTrCP2-R447A and βTrCP2-ΔF were expressed and purified from HEK293T cells to be able to discriminate between potential substrates of SCFβTrCP and unspecific binders. Affinity-purified samples were analyzed by mass spectrometry-based proteomics, applying ultra-high performance liquid chromatography (UHPLC coupled to high-resolution tandem mass spectrometry. The raw mass spectrometry data have been deposited to the PRIDE partner repository with the identifiers PXD001088 and PXD001224. The present dataset is associated with a research resource published in T.Y. Low, M. Peng, R. Magliozzi, S. Mohammed, D. Guardavaccaro, A.J.R. Heck, A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase. Sci. Signal. 7 (2014 rs8–rs8, 10.1126/scisignal.2005882.

  15. Ubiquitin-dependent distribution of the transcriptional coactivator p300 in cytoplasmic inclusion bodies.

    Science.gov (United States)

    Chen, Jihong; Halappanavar, Sabina; Th' ng, John P H; Li, Qiao

    2007-01-01

    The protein level of transcriptional coactivator p300, an essential nuclear protein, is critical to a broad array of cellular activities including embryonic development, cell differentiation and proliferation. We have previously established that histone deacetylase inhibitor such as valproic acid induces p300 degradation through the 26S proteasome pathway. Here, we report the roles of cellular trafficking and spatial redistribution in valproic acid-induced p300 turnover. Our study demonstrates that p300 is redistributed to the cytoplasm prior to valproic acid-induced turnover. Inhibition of proteasome-dependent protein degradation, does not prevent nucleo-cytoplasmic shuttling of p300, rather sequesters the cytoplasmic p300 to a distinct perinuclear region. In addition, the formation of p300 aggregates in the perinuclear region depends on functional microtubule networks and correlates with p300 ubiquitination. Our work establishes, for the first time, that p300 is also a substrate of the cytoplasmic ubiquitin-proteasome system and provides insight on how cellular trafficking and spatial redistribution regulate the availability and activity of transcriptional coactivator p300.

  16. Histone deacetylase 2 is phosphorylated, ubiquitinated, and degraded by cigarette smoke.

    Science.gov (United States)

    Adenuga, David; Yao, Hongwei; March, Thomas H; Seagrave, Jeanclare; Rahman, Irfan

    2009-04-01

    Cigarette smoke (CS)-induced lung inflammation involves the reduction of histone deacetylase 2 (HDAC2) abundance, which is associated with steroid resistance in patients with chronic obstructive pulmonary disease and in individuals with severe asthma who smoke cigarettes. However, the molecular mechanism of CS-mediated reduction of HDAC2 is not clearly known. We hypothesized that HDAC2 is phosphorylated and subsequently degraded by the proteasome in vitro in macrophages (MonoMac6), human bronchial and primary small airway epithelial cells, and in vivo in mouse lungs in response to chronic CS exposure. Cigarette smoke extract (CSE) exposure in MonoMac6 and in bronchial and airway epithelial cells led to phosphorylation of HDAC2 on serine/threonine residues by a protein kinase CK2-mediated mechanism, decreased HDAC2 activity, and increased ubiquitin-proteasome-dependent HDAC2 degradation. CK2 and proteasome inhibitors reversed CSE-mediated HDAC2 degradation, whereas serine/threonine phosphatase inhibitor, okadaic acid, caused phosphorylation and subsequent ubiquitination of HDAC2. CS-induced HDAC2 phosphorylation was detected in mouse lungs from 2 weeks to 4 months of CS exposure, and mice showed significantly lower lung HDAC2 levels. Thus, CS-mediated down-regulation of HDAC2 in human macrophages and lung epithelial cells in vitro and in mouse lung in vivo involves the induction of serine/threonine phosphorylation and proteasomal degradation, which may have implications for steroid resistance and abnormal inflammation caused by cigarette smoke. PMID:18927347

  17. The ubiquitin hydrolase USP22 contributes to 3'-end processing of JAK-STAT-inducible genes.

    Science.gov (United States)

    Chipumuro, Edmond; Henriksen, Melissa A

    2012-02-01

    The JAK-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway drives cellular growth, differentiation, and the immune response. STAT-activated gene expression is both rapid and transient and requires dynamic post-translational modification of the chromatin template. We previously showed that monoubiquitination of histone H2B (ubH2B) is highly dynamic at the STAT1 target gene, interferon regulatory factor 1 (IRF1), suggesting that a deubiquitinase is recruited during gene activation. Here, we report that RNAi-mediated knockdown of the ubiquitin hydrolase, USP22, results in 2-fold higher ubH2B, and 2-fold lower transcriptional elongation at IRF1. We also demonstrate that USP22 depletion diminishes 3'-end cleavage/polyadenylation by 2- to 3-fold. Furthermore, the polyadenylation factor CPSF73 is not effectively recruited, and serine 2 phosphorylation (Ser2P) of the C-terminal domain of RNA polymerase II is also disrupted. The transcriptional and processing defects observed in the USP22-knockdown cells are reversed by transient USP22 overexpression. Together, these results suggest that ubH2B helps recruit polyadenylation factors to STAT1-activated genes. We propose a working model, wherein a cycle of H2B ubiquitination/deubiquitination specifies Ser2P to regulate elongation and 3'-end processing of JAK-STAT-inducible mRNAs. These results further elaborate USP22 function and its role as a putative cancer stem cell marker.

  18. A novel ubiquitin carboxyl terminal hydrolase is involved in toad oocyte maturation

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    p28, a 28kD protein from toad (Bufo bufo gargarizans) oocytes, was identified by using p13suc1-agaroseaffinity chromatography. Sequence homology analysis of the full-length cDNA of p28 (Gene Bank accessionnumber: AF 314091) indicated that it encodes a protein containing 224 amino-acids with about 55% iden-tities and more than 70% positives to human, rat or mouse UCH-L1, and contains homological functionaldomains of UCH family. Anti-p28 monoclonal antibody, on injecting into the oocytes, could inhibit theprogesterone-induced resumption of meiotic division in a dose-dependent manner. The recombinant proteinp28 showed similar SDS/PAGE behaviors to the native one, and promoted ubiquitin ethyl ester hydrolysis,a classical catalytic reaction for ubiquitin carboxyl terminai hydrolases (UCHs). The results in this paperreveal that a novel protein, p28, exists in the toad oocytes, is a UCH L1 homolog, was engaged in theprocess of progesterone-induced oocyte maturation possibly through an involvement in protein turnover anddegradation.

  19. Roles of Ubiquitination in the Control of Phosphate Starvation Responses in Plants

    Institute of Scientific and Technical Information of China (English)

    Mónica Rojas-Triana; Regla Bustos; Ana Espinosa-Ruiz; Salomé Prat; Javier Paz-Ares; Vicente Rubio

    2013-01-01

    Throughout evolution,plants have evolved sophisticated adaptive responses that allow them to grow with a limited supply of phosphate,the preferential form in which the essential macronutrient phosphorus is absorbed by plants.Most of these responses are aimed to increase phosphate availability and acquisition through the roots,to optimize its usage in metabolic processes,and to protect plants from the deleterious effects of phosphate deficiency stress.Regulation of these adaptive responses requires fine perception of the external and internal phosphate levels,and a complex signal transduction pathway that integrates information on the phosphate status at the whole-plant scale.The molecular mechanisms that participate in phosphate homeostasis include transcriptional control of gene expression,RNA silencing mediated by microRNAs,regulatory non-coding RNAs of miRNA activity,phosphate transporter trafficking,and post-translational modification of proteins,such as phosphorylation,sumoylation and ubiquitination.Such a varied regulatory repertoire reflects the complexity intrinsic to phosphate surveying and signaling pathways.Here,we describe these regulatory mechanisms,emphasizing the increasing importance of ubiquitination in the control of phosphate starvation responses.

  20. Housekeeping gene on the X chromosome encodes a protein similar to ubiquitin

    International Nuclear Information System (INIS)

    An X chromosome gene located 40 kilobases downstream from the G6PD gene, at Xq28, was isolated and sequenced. This gene, which the authors named GdX, spans about 3.5 kilobases of genomic DNA. GdX is a single-copy gene, is conserved in evolution, and has the features of a housekeeping gene. At its 5' end, a cluster of CpG dinucleotides is methylated on the inactive X chromosome and unmethylated on the active X chromosome. The GdX gene can code for a 157 amino acid protein, GdX. Residues 1-74 of GdX show 43% identity to ubiquitin, a highly conserved 76 amino acid protein. The COOH-terminal moiety of GdX is characterized in its central part (residues 110-128) by a sequence homologous to the COOH-terminal hormonogenic site of thyroglobulin. The structural organization of the GdX protein suggests the existence of a family of genes, in addition to the ubiquitin gene, that could play specific roles in key cellular processes, possible through protein-protein recognition

  1. Emerging Role of the Ubiquitin Proteasome System in the Control of Shoot Apical Meristem Function

    Institute of Scientific and Technical Information of China (English)

    Elisabetta Di Giacomo; Giovanna Serino; Giovanna Frugis

    2013-01-01

    The shoot apical meristem (SAM) is a population of undifferentiated cells at the tip of the shoot axis that establishes early during plant embryogenesis and gives rise to all shoot organs throughout the plant's life.A plethora of different families of transcription factors (TFs) play a key role in establishing the equilibrium between cell differentiation and stem cell maintenance in the SAM.Fine tuning of these regulatory proteins is crucial for a proper and fast SAM response to environmental and hormonal cues,and for development progression.One effective way to rapidly inactivate TFs involves regulated proteolysis by the ubiquitin/26S proteasome system (UPS).However,a possible role of UPS-dependent protein degradation in the regulation of key SAM TFs has not been thoroughly investigated.Here,we summarize recent evidence supporting a role for the UPS in SAM maintenance and function.We integrate this survey with an in silico analysis of publicly-available microarray databases which identified ubiquitin ligases that are expressed in specific areas within the SAM,suggesting that they may regulate or act downstream of meristem-specific factors.

  2. Differentiation of Th subsets inhibited by nonstructural proteins of respiratory syncytial virus is mediated by ubiquitination.

    Directory of Open Access Journals (Sweden)

    Ling Qin

    Full Text Available Human respiratory syncytial virus (RSV, a major cause of severe respiratory diseases, constitutes an important risk factor for the development of subsequent asthma. However, the mechanism underlying RSV-induced asthma is poorly understood. Viral non-structural proteins NS1 and NS2 are critically required for RSV virulence; they strongly suppress IFN-mediated innate immunity of the host cells. In order to understand the effects of NS1 and NS2 on differentiation of Th subsets, we constructed lentiviral vectors of NS1 or NS2 to infect 16 HBE and analyzed the expression of HLA-DR, CD80 and CD86 and differentiation of Th1, Th2 and Th17 by Flow Cytometric Analysis and real-time PCR. The results showed that NS1 inhibited expression of HLA-DR, CD80 and CD86 and differentiation of Th1, Th2 and Th17 lymphocytes, which could be reversed by deleting elongin C binding domain. NS2 inhibited the differentiation of Th2 and Th17, which was reversed by proteasome inhibitors of PS-341. Our results indicated that NS1 inhibited the differentiation of T lymphocytes through its mono-ubiquitination to interacted proteins, while NS2 inhibited differentiation of Th2 and Th17 through ubiquitin-proteasome pathway, which may be related with the susceptibility to asthma after RSV infection.

  3. Ubiquitin ligase gp78 targets unglycosylated prion protein PrP for ubiquitylation and degradation.

    Directory of Open Access Journals (Sweden)

    Jia Shao

    Full Text Available Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis.

  4. Ubiquitin ligase gp78 targets unglycosylated prion protein PrP for ubiquitylation and degradation.

    Science.gov (United States)

    Shao, Jia; Choe, Vitnary; Cheng, Haili; Tsai, Yien Che; Weissman, Allan M; Luo, Shiwen; Rao, Hai

    2014-01-01

    Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis. PMID:24714645

  5. Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity.

    Science.gov (United States)

    Belogurov, Alexey; Kuzina, Ekaterina; Kudriaeva, Anna; Kononikhin, Alexey; Kovalchuk, Sergey; Surina, Yelena; Smirnov, Ivan; Lomakin, Yakov; Bacheva, Anna; Stepanov, Alexey; Karpova, Yaroslava; Lyupina, Yulia; Kharybin, Oleg; Melamed, Dobroslav; Ponomarenko, Natalia; Sharova, Natalia; Nikolaev, Eugene; Gabibov, Alexander

    2015-05-01

    Recent findings indicate that the ubiquitin-proteasome system is involved in the pathogenesis of cancer as well as autoimmune and several neurodegenerative diseases, and is thus a target for novel therapeutics. One disease that is related to aberrant protein degradation is multiple sclerosis, an autoimmune disorder involving the processing and presentation of myelin autoantigens that leads to the destruction of axons. Here, we show that brain-derived proteasomes from SJL mice with experimental autoimmune encephalomyelitis (EAE) in an ubiquitin-independent manner generate significantly increased amounts of myelin basic protein peptides that induces cytotoxic lymphocytes to target mature oligodendrocytes ex vivo. Ten times enhanced release of immunogenic peptides by cerebral proteasomes from EAE-SJL mice is caused by a dramatic shift in the balance between constitutive and β1i(high) immunoproteasomes in the CNS of SJL mice with EAE. We found that during EAE, β1i is increased in resident CNS cells, whereas β5i is imported by infiltrating lymphocytes through the blood-brain barrier. Peptidyl epoxyketone specifically inhibits brain-derived β1i(high) immunoproteasomes in vitro (kobs/[I] = 240 M(-1)s(-1)), and at a dose of 0.5 mg/kg, it ameliorates ongoing EAE in vivo. Therefore, our findings provide novel insights into myelin metabolism in pathophysiologic conditions and reveal that the β1i subunit of the immunoproteasome is a potential target to treat autoimmune neurologic diseases.

  6. A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

    Science.gov (United States)

    Mulder, Monique P C; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C O; Schulman, Brenda A; Komander, David; Neefjes, Jacques; El Oualid, Farid; Ovaa, Huib

    2016-07-01

    Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades. PMID:27182664

  7. The roles of VHL-dependent ubiquitination in signaling and cancer

    Directory of Open Access Journals (Sweden)

    Qing eZhang

    2012-04-01

    Full Text Available The function of tumor suppressor VHL is compromised in the vast majority of clear cell Renal Cell Carcinoma (ccRCC, and its mutations or loss of expression was causal for this disease. pVHL was found to be a substrate recognition subunit of an E3 ubiquitin ligase, and most of the tumor-derived mutations disrupt this function. pVHL was found to bind to the alpha subunits of hypoxia inducible factor (HIF and promote their ubiquitination and proteasomal degradation. Proline hydroxylation on key sites of HIFα provides the binding signal for pVHL E3 ligase complex. Beside HIFα, several other VHL targets have been identified, including activated Epidermal Growth Factor Receptor (EGFR, RNA polymerase II subunits RPB1 and hsRPB7, atypical PKC, Sprouty 2, β-adrenergic receptor II and Myb-binding protein p160. HIFα is the most well studied substrate and has been proven to be critical for pVHL's tumor suppressor function, but the activated EGFR and PKC and other pVHL substrates might also be important for tumor growth and drug response. Their regulations by pVHL and their relevance to signaling and cancer are discussed.

  8. Integrated analysis of microarray data of atherosclerotic plaques: modulation of the ubiquitin-proteasome system.

    Directory of Open Access Journals (Sweden)

    Zhe Wang

    Full Text Available Atherosclerosis is a typical complex multi-factorial disease and many molecules at different levels and pathways were involved in its development. Some studies have investigated the dysregulation in atherosclerosis at mRNA, miRNA or DNA methylation level, respectively. However, to our knowledge, the studies that integrated these data and revealed the abnormal networks of atherosclerosis have not been reported. Using microarray technology, we analyzed the omics data in atherosclerosis at mRNA, miRNA and DNA methylation levels. Our results demonstrated that the global DNA methylation and expression of miRNA/mRNA were significantly decreased in atherosclerotic plaque than in normal vascular tissue. The interaction network constructed using the integrative data revealed many genes, cellular processes and signaling pathways which were widely considered to play crucial roles in atherosclerosis and also revealed some genes, miRNAs or signaling pathways which have not been investigated in atherosclerosis until now (e.g. miR-519d and SNTB2. Moreover, the overall protein ubiquitination in atherosclerotic plaque was significantly increased. The proteasome activity was increased early but decreased in advanced atherosclerosis. Our study revealed many classic and novel genes and miRNAs involved in atherosclerosis and indicated the effects of ubiquitin-proteasome system on atherosclerosis might be closely related to the course of atherosclerosis. However, the efficacy of proteasome inhibitors in the treatment of atherosclerosis still needs more research.

  9. 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.

  10. The replication stress response and the ubiquitin system: a new link in maintaining genomic integrity

    Directory of Open Access Journals (Sweden)

    Koepp Deanna M

    2010-03-01

    Full Text Available Abstract Maintenance of genomic integrity is important for cellular viability and proliferation. During DNA replication, cells respond to replication stress by activating checkpoint pathways that stabilize replication forks and prevent cell cycle progression. The Saccharomyces cerevisiae F-box protein Dia2 is a ubiquitin ligase component required for genomic stability and may help replication complexes negotiate damaged DNA or natural fragile sites. We recently implicated Dia2 in the replication stress response. We demonstrated that Dia2 is targeted for ubiquitin-mediated proteolysis and that activation of the S-phase checkpoint inhibits Dia2 protein turnover. S-phase checkpoint mutants fail to stabilize the Dia2 protein and checkpoint mutants that lack Dia2 exhibit increased sensitivity to replication stress. We also showed that Dia2 protein turnover is not the result of an autocatalytic mechanism. Instead, an N-terminal 20 amino acid motif that is also required for nuclear localization is necessary for Dia2 proteolysis. Dia2 mutants lacking this motif but modified with an exogenous strong nuclear localization signal are both nuclear and stable and disrupt cell cycle dynamics. In summary, our studies suggest that inhibition of Dia2 proteolysis is a novel target of the S-phase checkpoint. We think that this work will help to identify the mechanisms that function downstream of checkpoint activation and that intersect with cell cycle control pathways.

  11. Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

    Science.gov (United States)

    Brown, Nicholas G; VanderLinden, Ryan; Watson, Edmond R; Weissmann, Florian; Ordureau, Alban; Wu, Kuen-Phon; Zhang, Wei; Yu, Shanshan; Mercredi, Peter Y; Harrison, Joseph S; Davidson, Iain F; Qiao, Renping; Lu, Ying; Dube, Prakash; Brunner, Michael R; Grace, Christy R R; Miller, Darcie J; Haselbach, David; Jarvis, Marc A; Yamaguchi, Masaya; Yanishevski, David; Petzold, Georg; Sidhu, Sachdev S; Kuhlman, Brian; Kirschner, Marc W; Harper, J Wade; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A

    2016-06-01

    Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination. PMID:27259151

  12. Conformational Space and Stability of ETD Charge Reduction Products of Ubiquitin

    Science.gov (United States)

    Lermyte, Frederik; Łącki, Mateusz Krzysztof; Valkenborg, Dirk; Gambin, Anna; Sobott, Frank

    2016-08-01

    Owing to its versatility, electron transfer dissociation (ETD) has become one of the most commonly utilized fragmentation techniques in both native and non-native top-down mass spectrometry. However, several competing reactions—primarily different forms of charge reduction—occur under ETD conditions, as evidenced by the distorted isotope patterns usually observed. In this work, we analyze these isotope patterns to compare the stability of nondissociative electron transfer (ETnoD) products, specifically noncovalent c/z fragment complexes, across a range of ubiquitin conformational states. Using ion mobility, we find that more extended states are more prone to fragment release. We obtain evidence that for a given charge state, populations of ubiquitin ions formed either directly by electrospray ionization or through collapse of more extended states upon charge reduction, span a similar range of collision cross-sections. Products of gas-phase collapse are, however, less stabilized towards unfolding than the native conformation, indicating that the ions retain a memory of previous conformational states. Furthermore, this collapse of charge-reduced ions is promoted if the ions are `preheated' using collisional activation, with possible implications for the kinetics of gas-phase compaction.

  13. Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

    Science.gov (United States)

    Nakada, Shinichiro

    2016-01-01

    The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway. PMID:26983989

  14. Prenatal stress increased Snk Polo-like kinase 2, SCF β-TrCP ubiquitin ligase and ubiquitination of SPAR in the hippocampus of the offspring at adulthood.

    Science.gov (United States)

    Chutabhakdikul, Naunchan; Surakul, Pornprom

    2013-11-01

    Exposure to excessive glucocorticoids during fetal development period contributes to later life psychopathology. Prenatal stress decreases dendritic spine density and impair LTP in the hippocampus of rat pups, however, the mechanisms regulating these changes are still unclear. Glutamate receptors are localized in the postsynaptic density. PSD-95 is a postsynaptic scaffolding protein that plays a role in synaptic maturation and regulation of the synaptic strength and plasticity. PSD-95 interacts with other proteins to form the protein networks that promote dendritic spine formation. The present study investigated the effect of prenatal stress on the levels of scaffolding proteins of NMDA receptor in the hippocampus in order to explain how prenatal stress alters the amount of NMDA receptor in the pups' brain. Pregnant rats were randomly assigned to either the prenatal stress (PS) or the control group (C). The pregnant rats in the PS group were restrained in a plexiglas restrainer for 4h/day during the GD 14-21. Control rats were left undisturbed for the duration of their pregnancies. The amount of PSD-95, SPAR, NR2A and NR2B, as well as the levels of Snk Polo-like kinase 2 and the SCF β-TrCP ubiquitin ligase were measured in the hippocampus of the offspring. The results show that prenatal stress induces a reduction in the amount of NR2B and NR2A subunits in the hippocampus of rat pups, parallel to the decrease in PSD-95 and SPAR at P40 and P60. Moreover, prenatal stress increases Snk and β-TrCP in the hippocampus of rat pups, and the timing correlates with the decrease of SPAR and PSD-95. Prenatal stress also induces a significantly increases in the level of ubiquitinated SPAR in the hippocampus of rat pups at adulthood. The results suggest that degradation of SPAR via UPS system may contribute to the loss of PSD-95 and NMDA receptor subunits in the hippocampus of rat pups at adulthood. In conclusion, the present work demonstrates that the developing brain is

  15. Constitutive endocytosis and turnover of the neuronal glycine transporter GlyT2 is dependent on ubiquitination of a C-terminal lysine cluster.

    Directory of Open Access Journals (Sweden)

    Jaime de Juan-Sanz

    Full Text Available Inhibitory glycinergic neurotransmission is terminated by sodium and chloride-dependent plasma membrane glycine transporters (GlyTs. The mainly glial glycine transporter GlyT1 is primarily responsible for the completion of inhibitory neurotransmission and the neuronal glycine transporter GlyT2 mediates the reuptake of the neurotransmitter that is used to refill synaptic vesicles in the terminal, a fundamental role in the physiology and pathology of glycinergic neurotransmission. Indeed, inhibitory glycinergic neurotransmission is modulated by the exocytosis and endocytosis of GlyT2. We previously reported that constitutive and Protein Kinase C (PKC-regulated endocytosis of GlyT2 is mediated by clathrin and that PKC accelerates GlyT2 endocytosis by increasing its ubiquitination. However, the role of ubiquitination in the constitutive endocytosis and turnover of this protein remains unexplored. Here, we show that ubiquitination of a C-terminus four lysine cluster of GlyT2 is required for constitutive endocytosis, sorting into the slow recycling pathway and turnover of the transporter. Ubiquitination negatively modulates the turnover of GlyT2, such that increased ubiquitination driven by PKC activation accelerates transporter degradation rate shortening its half-life while decreased ubiquitination increases transporter stability. Finally, ubiquitination of GlyT2 in neurons is highly responsive to the free pool of ubiquitin, suggesting that the deubiquitinating enzyme (DUB ubiquitin C-terminal hydrolase-L1 (UCHL1, as the major regulator of neuronal ubiquitin homeostasis, indirectly modulates the turnover of GlyT2. Our results contribute to the elucidation of the mechanisms underlying the dynamic trafficking of this important neuronal protein which has pathological relevance since mutations in the GlyT2 gene (SLC6A5 are the second most common cause of human hyperekplexia.

  16. 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

  17. Animals as disgust elicitors

    DEFF Research Database (Denmark)

    Kasperbauer, Tyler Joshua

    2015-01-01

    This paper attempts to explain how and why nonhuman animals elicit disgust in human beings. I argue that animals elicit disgust in two ways. One is by triggering disease–protection mechanisms, and the other is by eliciting mortality salience, or thoughts of death. I discuss how these two types...... of disgust operate and defend their conceptual and theoretical coherence against common objections. I also outline an explanatory challenge for disgust researchers. Both types of disgust indicate that a wide variety of animals produce aversive and avoidant reactions in human beings. This seems somewhat odd......, given the prominence of animals in human lives. The challenge, then, is explaining how humans cope with the presence of animals. I propose, as a hypothesis for further exploration, that we cope with animals, and our disgust responses to them, by attributing mental states that mark them as inferior...

  18. Small Animal Retinal Imaging

    Science.gov (United States)

    Choi, WooJhon; Drexler, Wolfgang; Fujimoto, James G.

    Developing and validating new techniques and methods for small animal imaging is an important research area because there are many small animal models of retinal diseases such as diabetic retinopathy, age-related macular degeneration, and glaucoma [1-6]. Because the retina is a multilayered structure with distinct abnormalities occurring in different intraretinal layers at different stages of disease progression, there is a need for imaging techniques that enable visualization of these layers individually at different time points. Although postmortem histology and ultrastructural analysis can be performed for investigating microscopic changes in the retina in small animal models, this requires sacrificing animals, which makes repeated assessment of the same animal at different time points impossible and increases the number of animals required. Furthermore, some retinal processes such as neurovascular coupling cannot be fully characterized postmortem.

  19. Our love for animals.

    Science.gov (United States)

    Scruton, Roger

    2013-12-01

    Love does not necessarily benefit its object, and cost-free love may damage both object and subject. Our love of animals mobilises several distinct human concerns and should not be considered always as a virtue or always as a benefit to the animals themselves. We need to place this love in its full psychological, cultural, and moral context in order to assess what form it ought to take if animals are to benefit from it.

  20. Are ticks venomous animals?

    OpenAIRE

    Cabezas-Cruz, Alejandro; James J Valdés

    2014-01-01

    Introduction As an ecological adaptation venoms have evolved independently in several species of Metazoa. As haematophagous arthropods ticks are mainly considered as ectoparasites due to directly feeding on the skin of animal hosts. Ticks are of major importance since they serve as vectors for several diseases affecting humans and livestock animals. Ticks are rarely considered as venomous animals despite that tick saliva contains several protein families present in venomous taxa and that many...

  1. PRINCIPLES OF ANIMAL BREEDING

    OpenAIRE

    Sonja Jovanovac

    2014-01-01

    University textbook Principles of Animal Breeding is intended for students of agriculture and veterinary medicine. The material is the adapted curricula of undergraduate and graduate level studies in the framework of which the modules Principles of animal breeding as well as Basics of genetics and selection of animals attended are listened. The textbook contains 14 chapters and a glossary of terms. Its concept enables combining fundamental and modern knowledge in the ...

  2. The representative animal

    OpenAIRE

    Harrison, J. M.

    1994-01-01

    The anthropocentric approach to the study of animal behavior uses representative nonhuman animals to understand human behavior. This approach raises problems concerning the comparison of the behavior of two different species. The datum of behavior analysis is the behavior of humans and representative animal phenotypes. The behavioral phenotype is the product of the ontogeny and phylogeny of each species, and this requires that contributions of genotype as well as behavioral history to experim...

  3. Animal models of asthma

    OpenAIRE

    Bates, Jason H.T.; Rincon, Mercedes; Irvin, Charles G.

    2009-01-01

    Studies in animal models form the basis for much of our current understanding of the pathophysiology of asthma, and are central to the preclinical development of drug therapies. No animal model completely recapitulates all features of the human disease, however. Research has focused primarily on ways to generate allergic inflammation by sensitizing and challenging animals with a variety of foreign proteins, leading to an increased understanding of the immunological factors that mediate the in...

  4. Animal Violence Demystified

    OpenAIRE

    Natarajan, Deepa; Caramaschi, Doretta

    2010-01-01

    Violence has been observed in humans and animals alike, indicating its evolutionary/biological significance. However, violence in animals has often been confounded with functional forms of aggressive behavior. Currently, violence in animals is identified primarily as either a quantitative behavior (an escalated, pathological and abnormal form of aggression characterized primarily by short attack latencies, and prolonged and frequent harm-oriented conflict behaviors) or a qualitative one (char...

  5. Animal Model of Dermatophytosis

    OpenAIRE

    Tsuyoshi Shimamura; Nobuo Kubota; Kazutoshi Shibuya

    2012-01-01

    Dermatophytosis is superficial fungal infection caused by dermatophytes that invade the keratinized tissue of humans and animals. Lesions from dermatophytosis exhibit an inflammatory reaction induced to eliminate the invading fungi by using the host’s normal immune function. Many scientists have attempted to establish an experimental animal model to elucidate the pathogenesis of human dermatophytosis and evaluate drug efficacy. However, current animal models have several issues. In the presen...

  6. Thinking with animals

    OpenAIRE

    2015-01-01

    they also enlist them to symbolize, dramatize, and illuminate aspects of humans' experience and fantasy. Humans merge with animals in stories, films, philosophical speculations, and scientific treatises. In their performance on many stages and in different ways, animals move us to think." "Essays in the book investigate the changing patterns of anthropomorphism across different time periods and settings, as well as their transformative effects, both figuratively and literally, upon animals, h...

  7. 3D Animation Essentials

    CERN Document Server

    Beane, Andy

    2012-01-01

    The essential fundamentals of 3D animation for aspiring 3D artists 3D is everywhere--video games, movie and television special effects, mobile devices, etc. Many aspiring artists and animators have grown up with 3D and computers, and naturally gravitate to this field as their area of interest. Bringing a blend of studio and classroom experience to offer you thorough coverage of the 3D animation industry, this must-have book shows you what it takes to create compelling and realistic 3D imagery. Serves as the first step to understanding the language of 3D and computer graphics (CG)Covers 3D anim

  8. Political Communication with Animals

    OpenAIRE

    Meijer, E

    2013-01-01

    In this article I sketch the outlines of a theory of political human-animal conversations, based on ideas about language that I borrow from Ludwig Wittgenstein’s later work, in particular his notion of language-games. I present this theory as a supplement to the political theory of animal rights Sue Donaldson and Will Kymlicka present in Zoopolis (2011). I will argue their political theory is an important step forward in the debate about animal rights, because it proposes to see animals as po...

  9. Soy Glycinin Contains a Functional Inhibitory Sequence against Muscle-Atrophy-Associated Ubiquitin Ligase Cbl-b

    Directory of Open Access Journals (Sweden)

    Tomoki Abe

    2013-01-01

    Full Text Available Background. Unloading stress induces skeletal muscle atrophy. We have reported that Cbl-b ubiquitin ligase is a master regulator of unloading-associated muscle atrophy. The present study was designed to elucidate whether dietary soy glycinin protein prevents denervation-mediated muscle atrophy, based on the presence of inhibitory peptides against Cbl-b ubiquitin ligase in soy glycinin protein. Methods. Mice were fed either 20% casein diet, 20% soy protein isolate diet, 10% glycinin diet containing 10% casein, or 20% glycinin diet. One week later, the right sciatic nerve was cut. The wet weight, cross sectional area (CSA, IGF-1 signaling, and atrogene expression in hindlimb muscles were examined at 1, 3, 3.5, or 4 days after denervation. Results. 20% soy glycinin diet significantly prevented denervation-induced decreases in muscle wet weight and myofiber CSA. Furthermore, dietary soy protein inhibited denervation-induced ubiquitination and degradation of IRS-1 in tibialis anterior muscle. Dietary soy glycinin partially suppressed the denervation-mediated expression of atrogenes, such as MAFbx/atrogin-1 and MuRF-1, through the protection of IGF-1 signaling estimated by phosphorylation of Akt-1. Conclusions. Soy glycinin contains a functional inhibitory sequence against muscle-atrophy-associated ubiquitin ligase Cbl-b. Dietary soy glycinin protein significantly prevented muscle atrophy after denervation in mice.

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

    Science.gov (United States)

    Dantuma, Nico P; Pfeiffer, Annika

    2016-01-01

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

  11. 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.

  12. Ubiquitin Fusion Degradation Protein 1 as a Blood Marker for The Early Diagnosis of Ischemic Stroke

    Directory of Open Access Journals (Sweden)

    Laure Allard

    2007-01-01

    Full Text Available Background: Efficacy of thrombolysis in acute ischemic stroke is strongly related to physician’s ability to make an accurate diagnosis and to intervene within 3–6 h after event onset. In this context, the discovery and validation of very early blood markers have recently become an urgent, yet unmet, goal of stroke research. Ubiquitin fusion degradation protein 1 is increased in human postmortem CSF, a model of global brain insult, suggesting that its measurement in blood may prove useful as a biomarker of stroke.Methods: Enzyme-linked immunosorbent assay (ELISA was used to measure UFD1 in plasma and sera in three independent cohorts, European (Swiss and Spanish and North-American retrospective analysis encompassing a total of 123 consecutive stroke and 90 control subjects.Results: Highly significant increase of ubiquitin fusion degradation protein 1 (UFD1 was found in Swiss stroke patients with 71% sensitivity (95% CI, 52–85.8%, and 90% specificity (95% CI, 74.2–98% (N = 31, p < 0.0001. Significantly elevated concentration of this marker was then validated in Spanish (N = 39, p < 0.0001, 95% sensitivity (95% CI, 82.7–99.4%, 76% specificity (95% CI, 56.5–89.7% and North-American stroke patients (N = 53, 62% sensitivity (95% CI, 47.9–75.2%, 90% specificity (95% CI, 73.5–97.9%, p < 0.0001. Its concentration was increased within 3 h of stroke onset, on both the Swiss (p < 0.0001 and Spanish (p = 0.0004 cohorts.Conclusions: UFD1 emerges as a reliable plasma biomarker for the early diagnosis of stroke, and in the future, might be used in conjunction with clinical assessments, neuroimaging and other blood markers.Abbreviations: AUC: area under curve; BBB: blood–brain barrier; CO: cut-off; CSF: cerebrospinal fluid; CT: computerized tomography; H-FABP: heart-fatty acid binding protein; MMP9: matrix metalloproteinase 9; MRI: magnetic resonance imaging; NDKA: nucleotide diphosphate kinase A; OR: odds ratio; RFU: relative fluorescence

  13. Pax3 stimulates p53 ubiquitination and degradation independent of transcription.

    Directory of Open Access Journals (Sweden)

    Xiao Dan Wang

    Full Text Available BACKGROUND: Pax3 is a developmental transcription factor that is required for neural tube and neural crest development. We previously showed that inactivating the p53 tumor suppressor protein prevents neural tube and cardiac neural crest defects in Pax3-mutant mouse embryos. This demonstrates that Pax3 regulates these processes by blocking p53 function. Here we investigated the mechanism by which Pax3 blocks p53 function. METHODOLOGY/PRINCIPAL FINDINGS: We employed murine embryonic stem cell (ESC-derived neuronal precursors as a cell culture model of embryonic neuroepithelium or neural crest. Pax3 reduced p53 protein stability, but had no effect on p53 mRNA levels or the rate of p53 synthesis. Full length Pax3 as well as fragments that contained either the DNA-binding paired box or the homeodomain, expressed as GST or FLAG fusion proteins, physically associated with p53 and Mdm2 both in vitro and in vivo. In contrast, Splotch Pax3, which causes neural tube and neural crest defects in homozygous embryos, bound weakly, or not at all, to p53 or Mdm2. The paired domain and homeodomain each stimulated Mdm2-mediated ubiquitination of p53 and p53 degradation in the absence of the Pax3 transcription regulatory domains, whereas Splotch Pax3 did not stimulate p53 ubiquitination or degradation. CONCLUSIONS/SIGNIFICANCE: Pax3 inactivates p53 function by stimulating its ubiquitination and degradation. This process utilizes the Pax3 paired domain and homeodomain but is independent of DNA-binding and transcription regulation. Because inactivating p53 is the only required Pax3 function during neural tube closure and cardiac neural crest development, and inactivating p53 does not require Pax3-dependent transcription regulation, this indicates that Pax3 is not required to function as a transcription factor during neural tube closure and cardiac neural crest development. These findings further suggest novel explanations for PAX3 functions in human diseases, such

  14. [Biotechnology and animal health].

    Science.gov (United States)

    Desmettre, P

    1993-06-01

    The development of the first vaccines for use in animals, by Louis Pasteur at the end of the 19th Century, was an initial step in applying biotechnology to animal health. However, it is only much more recently that decisive progress has been made in finding applications for biotechnology, in both detecting and preventing infectious and parasitic diseases. This progress has shown the way to developing a range of procedures, the application of which will benefit the health of domestic and wild animals, enhance the well-being of companion animals, develop the performance of sporting animals and improve the productivity of farm animals, while also serving to protect human health. Such progress results from the increasingly rapid application of knowledge gained in the material and life sciences, all of which contribute to the multidisciplinary nature of biotechnology. Similarly, reagents and diagnostic techniques have been made more specific, sensitive, reproducible, rapid and robust by updating them through recent discoveries in immunology, biochemistry and molecular biology (monoclonal antibodies, nucleic probes, deoxyribonucleic acid amplification and many more). The development of new vaccines which combine efficacy, duration of protection, innocuity, stability, multivalence and ease of use (subunit vaccines, recombinant vaccines, synthetic vaccines and anti-idiotype vaccines) has resulted from recent progress in immunology, immunochemistry, molecular biology and biochemistry. Finally, the availability of new anti-infective, anti-parasitic agents and immunomodulatory therapeutic agents (capable of stimulating the specific and non-specific defence mechanisms of the body) demonstrates that biotechnology is continuing to find new applications in the field of animal health. New diagnostic techniques, vaccines and therapeutic substances are the most immediate applications of knowledge which may, in the future, extend to the development of transgenic animals of revised

  15. Suppression of Ubiquitin-Specific Peptidase 17 (USP17) Inhibits Tumorigenesis and Invasion in Non-Small Cell Lung Cancer Cells.

    Science.gov (United States)

    Zhang, Shengchao; Yuan, Jun; Zheng, Ruheng

    2016-01-01

    Recently, deubiquitinating enzymes (DUBs) are emerging as new regulators in cancer progression. However, understanding of the involvement of DUBs in non-small cell lung cancer (NSCLC) is just beginning. In this study, we investigated the expression and biological function of ubiquitin-specific peptidase 17 (USP17) in NSCLC progression in vitro and in vivo. We found that the expression of USP17 was higher than in a normal control. We further efficiently depleted USP17 expression in two different NSCLC cells, A549 and H1299. The anchorage-independent growth ability of these cells, estimated by soft agar colony formation assay, was significantly reduced after USP17 knockdown. Moreover, Matrigel-Transwell analysis showed that suppression of USP17 decreased cell migration and invasion capacity. Molecular mechanism studies found that USP17 silencing downregulated the expression of matrix metalloproteases (MMP3 and MMP9) in NSCLC cells. Furthermore, animal model results showed that USP17 suppression inhibited NSCLC tumorigenesis and growth. Altogether, this study illustrates the important functions of USP17 in NSCLC and suggests that USP17 might be an attractive target for NSCLC therapy. PMID:27656837

  16. 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.

  17. Political Communication with Animals

    NARCIS (Netherlands)

    E. Meijer

    2013-01-01

    In this article I sketch the outlines of a theory of political human-animal conversations, based on ideas about language that I borrow from Ludwig Wittgenstein’s later work, in particular his notion of language-games. I present this theory as a supplement to the political theory of animal rights Sue

  18. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... FDA Submit search Popular Content Home Food Drugs Medical Devices Radiation-Emitting Products Vaccines, Blood & Biologics Animal & Veterinary ... by Product Area Product Areas back Food Drugs Medical Devices Radiation-Emitting Products Vaccines, Blood & Biologics Animal & Veterinary ...

  19. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... Translation - Animation of Antimicrobial Resistance (WMV - 19.2MB) Chinese Translation - Animation of Antimicrobial Resistance (WMV - 19.2MB) ... FEAR Act Site Map Transparency Website Policies U.S. Food and Drug Administration 10903 New Hampshire Avenue Silver ...

  20. Humane Treatment of Animals.

    Science.gov (United States)

    Dawson, Joan Smithey

    This booklet is designed to give teachers resource information about the humane treatment of and care for animals. The topics are presented as springboards for discussion and class activity. Topics include the care of dogs, cats, birds, horses, and fish; wildlife and ecological relationships; and careers with animals. Illustrations on some pages…

  1. First Aid: Animal Bites

    Science.gov (United States)

    ... Story" 5 Things to Know About Zika & Pregnancy First Aid: Animal Bites KidsHealth > For Parents > First Aid: Animal Bites Print A A A Text Size ... For Kids For Parents MORE ON THIS TOPIC First Aid & Safety Center Infections That Pets Carry Dealing With ...

  2. The Classroom Animal: Snails.

    Science.gov (United States)

    Kramer, David S.

    1985-01-01

    Points out that snails are interesting and easily-managed classroom animals. One advantage of this animal is that it requires no special attention over weekends or holidays. Background information, anatomy, reproduction, and feeding are discussed, along with suggestions for housing aquatic and/or land snails. (DH)

  3. Companion Animals. [Information Packet.

    Science.gov (United States)

    National Anti-Vivisection Society, Chicago, IL.

    This collection of articles reprinted from other National Anti-Vivisection Society (NAVS) publications was compiled to educate the public on issues of importance to NAVS concerning companion animals. Topics covered include spaying and neutering, animal safety, pet theft, and the use of cats and dogs in research. The article on spaying and…

  4. Indian draught animals power

    Directory of Open Access Journals (Sweden)

    K. L. Phaniraja

    Full Text Available With the modernization of agriculture, the use of mechanical power in agriculture has increased but draught animal power (DAP continues to be used on Indian farms due to small holdings and hill agriculture. More than 55% of the total cultivated area is still being managed by using draught animals as against about 20% by tractors. India possessed the finest breeds of draught animals. Bullocks, buffaloes and camels are the major draught animals for field operations. Horses, mules, donkeys, yak and mithun are the pack animals for transport. The quality of work from the draught animals depends upon the power developed by them. The design of traditional implements is based on long experience and these have served the purpose of the farmers. However there is plenty of scope to improve the design based on animal-machine-environment interaction so as to have more output and increased efficiency without jeopardizing animal health. [Vet World 2009; 2(10.000: 404-407

  5. Animals in the Classroom

    Science.gov (United States)

    Roy, Ken

    2011-01-01

    Use of animals in middle school science classrooms is a curriculum component worthy of consideration, providing proper investigation and planning are addressed. A responsible approach to this action, including safety, must be adopted for success. In this month's column, the author provides some suggestions on incorporating animals into the…

  6. Ode to an Animal

    Science.gov (United States)

    Nelken, Miranda

    2008-01-01

    People know little about the non-domesticated animals that live around them. Somehow, they seem remote. In stories they hear about them, animals are often acting, speaking, and dressing like people. This article presents a lesson where students learn about the native species of their area while exploring the concept of interdependence through…

  7. Endangered Animals. Second Grade.

    Science.gov (United States)

    Popp, Marcia

    This second grade teaching unit centers on endangered animal species around the world. Questions addressed are: What is an endangered species? Why do animals become extinct? How do I feel about the problem? and What can I do? Students study the definition of endangered species and investigate whether it is a natural process. They explore topics…

  8. Regulation of HTLV-1 tax stability, cellular trafficking and NF-κB activation by the ubiquitin-proteasome pathway.

    Science.gov (United States)

    Lavorgna, Alfonso; Harhaj, Edward William

    2014-10-23

    Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%-5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-κB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-κB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-κB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis.

  9. Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3.

    Science.gov (United States)

    Chew, Boon Shang; Siew, Wee Leng; Xiao, Benjamin; Lehming, Norbert

    2010-11-01

    Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of the transcription defects of the TBP1E186D mutant strain. The TBP1R6S mutation stabilizes the Tbp1E186D mutant protein and suppresses the defects of the TBP1E186D mutant strain. Furthermore, we found that the overexpression of the de-ubiquitinating enzyme Ubp3 (ubiquitin-specific protease 3) also stabilizes the Tbp1E186D mutant protein and suppresses of the defects of the TBP1E186D mutant strain. Importantly, the deletion of UBP3 and its cofactor BRE5 lead to increased degradation of wild-type Tbp1 protein and to defects in transcriptional activation by Gal4 and Gcn4. Purified GST (glutathione transferase)-Ubp3 reversed Tbp1 ubiquitination, and the deletion of UBP3 lead to the accumulation of poly-ubiquitinated species of Tbp1 in a proteaseome-deficient genetic background, demonstrating that Ubp3 reverses ubiquitination of Tbp1 in vitro and in vivo. Chromatin immunoprecipitation showed that Ubp3 was recruited to the GAL1 and HIS3 promoters upon the induction of the respective gene, indicating that protection of promoter-bound Tbp1 by Ubp3 is required for transcriptional activation.

  10. Computational modeling of Repeat1 region of INI1/hSNF5: An evolutionary link with ubiquitin.

    Science.gov (United States)

    Bhutoria, Savita; Kalpana, Ganjam V; Acharya, Seetharama A

    2016-09-01

    The structure of a protein can be very informative of its function. However, determining protein structures experimentally can often be very challenging. Computational methods have been used successfully in modeling structures with sufficient accuracy. Here we have used computational tools to predict the structure of an evolutionarily conserved and functionally significant domain of Integrase interactor (INI)1/hSNF5 protein. INI1 is a component of the chromatin remodeling SWI/SNF complex, a tumor suppressor and is involved in many protein-protein interactions. It belongs to SNF5 family of proteins that contain two conserved repeat (Rpt) domains. Rpt1 domain of INI1 binds to HIV-1 Integrase, and acts as a dominant negative mutant to inhibit viral replication. Rpt1 domain also interacts with oncogene c-MYC and modulates its transcriptional activity. We carried out an ab initio modeling of a segment of INI1 protein containing the Rpt1 domain. The structural model suggested the presence of a compact and well defined ββαα topology as core structure in the Rpt1 domain of INI1. This topology in Rpt1 was similar to PFU domain of Phospholipase A2 Activating Protein, PLAA. Interestingly, PFU domain shares similarity with Ubiquitin and has ubiquitin binding activity. Because of the structural similarity between Rpt1 domain of INI1 and PFU domain of PLAA, we propose that Rpt1 domain of INI1 may participate in ubiquitin recognition or binding with ubiquitin or ubiquitin related proteins. This modeling study may shed light on the mode of interactions of Rpt1 domain of INI1 and is likely to facilitate future functional studies of INI1. PMID:27261671

  11. Computational modeling of Repeat1 region of INI1/hSNF5: An evolutionary link with ubiquitin.

    Science.gov (United States)

    Bhutoria, Savita; Kalpana, Ganjam V; Acharya, Seetharama A

    2016-09-01

    The structure of a protein can be very informative of its function. However, determining protein structures experimentally can often be very challenging. Computational methods have been used successfully in modeling structures with sufficient accuracy. Here we have used computational tools to predict the structure of an evolutionarily conserved and functionally significant domain of Integrase interactor (INI)1/hSNF5 protein. INI1 is a component of the chromatin remodeling SWI/SNF complex, a tumor suppressor and is involved in many protein-protein interactions. It belongs to SNF5 family of proteins that contain two conserved repeat (Rpt) domains. Rpt1 domain of INI1 binds to HIV-1 Integrase, and acts as a dominant negative mutant to inhibit viral replication. Rpt1 domain also interacts with oncogene c-MYC and modulates its transcriptional activity. We carried out an ab initio modeling of a segment of INI1 protein containing the Rpt1 domain. The structural model suggested the presence of a compact and well defined ββαα topology as core structure in the Rpt1 domain of INI1. This topology in Rpt1 was similar to PFU domain of Phospholipase A2 Activating Protein, PLAA. Interestingly, PFU domain shares similarity with Ubiquitin and has ubiquitin binding activity. Because of the structural similarity between Rpt1 domain of INI1 and PFU domain of PLAA, we propose that Rpt1 domain of INI1 may participate in ubiquitin recognition or binding with ubiquitin or ubiquitin related proteins. This modeling study may shed light on the mode of interactions of Rpt1 domain of INI1 and is likely to facilitate future functional studies of INI1.

  12. UEV-1 is an ubiquitin-conjugating enzyme variant that regulates glutamate receptor trafficking in C. elegans neurons.

    Directory of Open Access Journals (Sweden)

    Lawrence B Kramer

    Full Text Available The regulation of AMPA-type glutamate receptor (AMPAR membrane trafficking is a key mechanism by which neurons regulate synaptic strength and plasticity. AMPAR trafficking is modulated through a combination of receptor phosphorylation, ubiquitination, endocytosis, and recycling, yet the factors that mediate these processes are just beginning to be uncovered. Here we identify the ubiquitin-conjugating enzyme variant UEV-1 as a regulator of AMPAR trafficking in vivo. We identified mutations in uev-1 in a genetic screen for mutants with altered trafficking of the AMPAR subunit GLR-1 in C. elegans interneurons. Loss of uev-1 activity results in the accumulation of GLR-1 in elongated accretions in neuron cell bodies and along the ventral cord neurites. Mutants also have a corresponding behavioral defect--a decrease in spontaneous reversals in locomotion--consistent with diminished GLR-1 function. The localization of other synaptic proteins in uev-1-mutant interneurons appears normal, indicating that the GLR-1 trafficking defects are not due to gross deficiencies in synapse formation or overall protein trafficking. We provide evidence that GLR-1 accumulates at RAB-10-containing endosomes in uev-1 mutants, and that receptors arrive at these endosomes independent of clathrin-mediated endocytosis. UEV-1 homologs in other species bind to the ubiquitin-conjugating enzyme Ubc13 to create K63-linked polyubiquitin chains on substrate proteins. We find that whereas UEV-1 can interact with C. elegans UBC-13, global levels of K63-linked ubiquitination throughout nematodes appear to be unaffected in uev-1 mutants, even though UEV-1 is broadly expressed in most tissues. Nevertheless, ubc-13 mutants are similar in phenotype to uev-1 mutants, suggesting that the two proteins do work together to regulate GLR-1 trafficking. Our results suggest that UEV-1 could regulate a small subset of K63-linked ubiquitination events in nematodes, at least one of which is critical

  13. Ubiquitination by March-I prevents MHC class II recycling and promotes MHC class II turnover in antigen-presenting cells.

    Science.gov (United States)

    Cho, Kyung-Jin; Walseng, Even; Ishido, Satoshi; Roche, Paul A

    2015-08-18

    MHC class II (MHC-II)-dependent antigen presentation by antigen-presenting cells (APCs) is carefully controlled to achieve specificity of immune responses; the regulated assembly and degradation of antigenic peptide-MHC-II complexes (pMHC-II) is one aspect of such control. In this study, we have examined the role of ubiquitination in regulating pMHC-II biosynthesis, endocytosis, recycling, and turnover in APCs. By using APCs obtained from MHC-II ubiquitination mutant mice, we find that whereas ubiquitination does not affect pMHC-II formation in dendritic cells (DCs), it does promote the subsequent degradation of newly synthesized pMHC-II. Acute activation of DCs or B cells terminates expression of the MHC-II E3 ubiquitin ligase March-I and prevents pMHC-II ubiquitination. Most importantly, this change results in very efficient pMHC-II recycling from the surface of DCs and B cells, thereby preventing targeting of internalized pMHC-II to lysosomes for degradation. Biochemical and functional assays confirmed that pMHC-II turnover is suppressed in MHC-II ubiquitin mutant DCs or by acute activation of wild-type DCs. These studies demonstrate that acute APC activation blocks the ubiquitin-dependent turnover of pMHC-II by promoting efficient pMHC-II recycling and preventing lysosomal targeting of internalized pMHC-II, thereby enhancing pMHC-II stability for efficient antigen presentation to CD4 T cells.

  14. Nucleosome acidic patch promotes RNF168- and RING1B/BMI1-dependent H2AX and H2A ubiquitination and DNA damage signaling.

    Directory of Open Access Journals (Sweden)

    Justin W Leung

    2014-03-01

    Full Text Available Histone ubiquitinations are critical for the activation of the DNA damage response (DDR. In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, respectively. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a critical chromatin mediator of H2A/H2AX ubiquitination (ub. The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes containing a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a critical nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.

  15. Inhibition of spinal UCHL1 attenuates pain facilitation in a cancer-induced bone pain model by inhibiting ubiquitin and glial activation

    Science.gov (United States)

    Cheng, Wei; Chen, Yuan-Li; Wu, Liang; Miao, Bei; Yin, Qin; Wang, Jin-Feng; Fu, Zhi-Jian

    2016-01-01

    The present study examined alterations of spinal ubiquitin C-terminal hydrolase L1 (UCHL1), ubiquitin expression and glial activation in the cancer-induced bone pain rats. Furthermore, whether inhibition of spinal UCHL1 could alleviate cancer-induced bone pain was observed. The CIBP model was established by intrathecal Walker 256 mammary gland carcinoma cells in SD rats. The rats of CIBP developed significant pain facilitation in the Von Frey test. Double immunofluorescence analyses revealed that in the spines of CIBP rats, ubiquitin co-localized with NeuN, Iba-1 or GFAP; UCHL1 and NeuN were co-expressed and UCHL1 also co-localized with ubiquitin. The CIBP model induced up-regulation of ubiquitin and UCHL1 in the spines, as well as glial activation. Inhibition of spinal UCHL1 attenuated pain facilitation by down-regulation of ubiquitin expression and glial activation. in the CIBP rats. Our data suggests that UCHL1/ubiquitin distributed and increased in the spines of CIBP rats, that glial activation also increased in the CIBP model and that inhibition of spinal UCHL1 may be an effective method to alleviate cancer-induced bone pain. PMID:27508024

  16. Animal Diseases and Your Health

    Science.gov (United States)

    Animal diseases that people can catch are called zoonoses. Many diseases affecting humans can be traced to animals or animal products. You can get a disease directly from an animal, or indirectly, through the ...

  17. Interaction between animal personality and animal cognition

    Directory of Open Access Journals (Sweden)

    Claudio CARERE, Charles LOCURTO

    2011-08-01

    Full Text Available The study of animal personality has attracted considerable attention, as it has revealed a number of similarities in personality between humans and several nonhuman species. At the same time the adaptive value and evolutionary maintenance of different personalities are the subject of debate. Since Pavlov’s work on dogs, students of comparative cognition have been aware that animals display vast individual differences on cognitive tasks, and that these differences may not be entirely accounted for differences in cognitive abilities. Here, we argue that personality is an important source of variation that may affect cognitive performance and we hypothesise mutual influences between personality and cognition across an individual’s lifespan. In particular, we suggest that: 1 personality profiles may be markers of different cognitive styles; 2 success or failure in cognitive tasks could affect different personalities differently; 3 ontogenetic changes of personality profiles could be reflected in changes in cognitive performance. The study of such interplay has implications in animal welfare as well as in neuroscience and in translational medicine [Current Zoology 57 (4: 491–498, 2011].

  18. Cupper in animal tissues

    Directory of Open Access Journals (Sweden)

    Maximino Huerta Bravo

    2010-12-01

    Full Text Available Cupper is an essential element for plants, animals and humans. Under certain circumstances, cupper excessive consumption could result in animal and human intoxication. In order to ensure safe and innocuous and safe foods for Mexicans, government create legislation as Norma Oficial Mexicana to establish the maximum levels of residues, particularly cupper in liver, kidney and muscle of human consumption animals. Liver in Mexico ruminant animals regularly contain 60 mg Cu/kg, which is the legal limit for this metal. This demands a review of the actual legislation. The strict application of this Norma will limit the commercialization of these viscera, since approximately 50% will exceed the legal limit for cupper. A potential hazard for human health, especially young people, is found in the constant ovine liver consumption feed with animal excretes with higher amount of supplementary cupper.

  19. Becoming Sheep, Becoming Animal

    DEFF Research Database (Denmark)

    Grum, Charlotte; Svabo, Connie

    2016-01-01

    Proposal for Performance Research, in response to the call Turning Animal: As a part of a 2015 group exhibition exploring the history and local myths of a woman living in a Danish heath landscape 150 years ago, artist Charlotte Grum connected herself to a live sheep for 4 hours a day, 5 days a week......, for 5 weeks, turning the two into a hybrid relational assemblage, intra-acting and becoming with the heath habitat, the other by-passing human and non-human animals, the changing weather and their fluctuating biological needs. She wanted to explore the discursive and material effects of a site......-specific human-nonhuman animal intra-action, to challenge the gendered and anthropocentric reading of a particular historical subject and to explore the messy constituents of the very categories of women and animals. In general she is occupied with how to animate and perform the intra-active entanglement...

  20. Workshop on molecular animation.

    Science.gov (United States)

    Bromberg, Sarina; Chiu, Wah; Ferrin, Thomas E

    2010-10-13

    From February 25 to 26, 2010, in San Francisco, the Resource for Biocomputing, Visualization, and Informatics (RBVI) and the National Center for Macromolecular Imaging (NCMI) hosted a molecular animation workshop for 21 structural biologists, molecular animators, and creators of molecular visualization software. Molecular animation aims to visualize scientific understanding of biomolecular processes and structures. The primary goal of the workshop was to identify the necessary tools for producing high-quality molecular animations, understanding complex molecular and cellular structures, creating publication supplementary materials and conference presentations, and teaching science to students and the public. Another use of molecular animation emerged in the workshop: helping to focus scientific inquiry about the motions of molecules and enhancing informal communication within and between laboratories.