Sample records for animal hect ubiquitin

  1. Evolution of Plant HECT Ubiquitin Ligases

    Ignacio Marín


    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. Regulation of EGFR Protein Stability by the HECT-type Ubiquitin Ligase SMURF2

    Dipankar Ray


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

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

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


    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.

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

    Komal Safdar


    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.

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

    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.

  6. RBR ubiquitin ligases: Diversification and streamlining in animal lineages.

    Marín, Ignacio


    The patterns of emergence and disappearance in animal species of genes encoding RBR ubiquitin ligases are described. RBR genes can be classified into subfamilies (Parkin, Ariadne, Dorfin, ARA54, etc.) according to sequence and structural data. Here, I show that most animal-specific RBR subfamilies emerged early in animal evolution, and that ancient animals, before the cnidarian/bilaterian split, had a set of RBR genes, which was as complex as the one currently found in mammals. However, some lineages (nematodes, dipteran insects) have recently suffered multiple losses, leading to a highly simplified set of RBR genes. Genes of a particular RBR subfamily, characterized by containing a helicase domain and so far found only in plants, are present also in some animal species. The meaning of these patterns of diversification and streamlining are discussed at the light of functional data. Extreme evolutionary conservation may be related to gene products having housekeeping functions. PMID:19526189

  7. Ubiquitin

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


    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...... 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......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...

  8. HectD1 E3 ligase modifies adenomatous polyposis coli (APC) with polyubiquitin to promote the APC-axin interaction.

    Tran, Hoanh; Bustos, Daisy; Yeh, Ronald; Rubinfeld, Bonnee; Lam, Cynthia; Shriver, Stephanie; Zilberleyb, Inna; Lee, Michelle W; Phu, Lilian; Sarkar, Anjali A; Zohn, Irene E; Wertz, Ingrid E; Kirkpatrick, Donald S; Polakis, Paul


    The adenomatous polyposis coli (APC) protein functions as a negative regulator of the Wnt signaling pathway. In this capacity, APC forms a "destruction complex" with Axin, CK1α, and GSK3β to foster phosphorylation of the Wnt effector β-catenin earmarking it for Lys-48-linked polyubiquitylation and proteasomal degradation. APC is conjugated with Lys-63-linked ubiquitin chains when it is bound to Axin, but it is unclear whether this modification promotes the APC-Axin interaction or confers upon APC an alternative function in the destruction complex. Here we identify HectD1 as a candidate E3 ubiquitin ligase that modifies APC with Lys-63 polyubiquitin. Knockdown of HectD1 diminished APC ubiquitylation, disrupted the APC-Axin interaction, and augmented Wnt3a-induced β-catenin stabilization and signaling. These results indicate that HectD1 promotes the APC-Axin interaction to negatively regulate Wnt signaling. PMID:23277359

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

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


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

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

    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

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

    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: [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden)


    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.

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

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


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

  13. Paip1, an effective stimulator of translation initiation, is targeted by WWP2 for ubiquitination and degradation.

    Lv, Yanrong; Zhang, Kai; Gao, Haidong


    Poly(A)-binding protein-interacting protein 1 (Paip1) stimulates translational initiation by inducing the circularization of mRNA. However, the mechanisms underlying Paip1 regulation, particularly its protein stability, are still unclear. Here, we show that the E6AP carboxyl terminus (HECT)-type ubiquitin ligase WW domain-containing protein 2 (WWP2), a homolog of the HECT-type ubiquitin ligase WWP1, interacts with and targets Paip1 for ubiquitination and proteasomal degradation. Mapping of the region including the WW domain of WWP2 revealed the interaction between WWP2 and the PABP-binding motif 2 (PAM2) of Paip1. The two consecutive PXXY motifs in PAM2 are required for WWP2-mediated ubiquitination and degradation. Furthermore, ectopic expression of WWP2 decreases translational stimulatory activity with the degradation of Paip1. We therefore provide evidence that the stability of Paip1 can be regulated by ubiquitin-mediated degradation, thus highlighting the importance of WWP2 as a suppressor of translation. PMID:25266661

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

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


    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

  15. Modulation of myocardin function by the ubiquitin E3 ligase UBR5.

    Hu, Guoqing; Wang, Xiaobo; Saunders, Darren N; Henderson, Michelle; Russell, Amanda J; Herring, B Paul; Zhou, Jiliang


    Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin

  16. In the family with ubiquitin

    Alexandru, Gabriela; Pariente, Nonia; Xirodimas, Dimitris


    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.

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

    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.

  18. Animals

    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)

  19. Animals



    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.



    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).

  1. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging.

    Jin, Jianping; Li, Xue; Gygi, Steven P; Harper, J Wade


    Modification of proteins with ubiquitin or ubiquitin-like proteins (UBLs) by means of an E1-E2-E3 cascade controls many signalling networks. Ubiquitin conjugation involves adenylation and thioesterification of the carboxy-terminal carboxylate of ubiquitin by the E1-activating enzyme Ube1 (Uba1 in yeast), followed by ubiquitin transfer to an E2-conjugating enzyme through a transthiolation reaction. Charged E2s function with E3s to ubiquitinate substrates. It is currently thought that Ube1/Uba1 is the sole E1 for charging of E2s with ubiquitin in animals and fungi. Here we identify a divergent E1 in vertebrates and sea urchin, Uba6, which specifically activates ubiquitin but not other UBLs in vitro and in vivo. Human Uba6 and Ube1 have distinct preferences for E2 charging in vitro, and their specificity depends in part on their C-terminal ubiquitin-fold domains, which recruit E2s. In tissue culture cells, Uba6 is required for charging a previously uncharacterized Uba6-specific E2 (Use1), whereas Ube1 is required for charging the cell-cycle E2s Cdc34A and Cdc34B. Our data reveal unexpected complexity in the pathways that control the conjugation of ubiquitin, in which dual E1s orchestrate the charging of distinct cohorts of E2s. PMID:17597759

  2. Origin and diversification of TRIM ubiquitin ligases.

    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.

  3. Ubiquitin domain proteins in disease

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


    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 their...... and cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb;

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

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


    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 ( 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

  5. The ubiquitin-proteasome system

    Dipankar Nandi; Pankaj Tahiliani; Anujith Kumar; Dilip Chandu


    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.

  6. Ubiquitin and proteasomes in transcription.

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


    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

  7. Non-degradative Ubiquitination of Protein Kinases.

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


    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...

  8. Ubiquitin-binding proteins: similar, but different

    Andersen, Katrine M; Hofmann, Kay; Hartmann-Petersen, Rasmus


    ubiquitin conjugation to endoplasmic reticulum degradation), UEV [ubiquitin E2 (ubiquitin-conjugating enzyme) variant] and NZF (nuclear protein localization gene 4 zinc finger) domain-containing proteins appear to have more specialized functions. Here we discuss functional and structural properties of...

  9. Functional assessment of ubiquitin-depended processes under microgravity conditions

    Zhabereva, Anastasia; Shenkman, Boris S.; Gainullin, Murat; Gurev, Eugeny; Kondratieva, Ekaterina; Kopylov, Arthur

    Ubiquitylation, a widespread and important posttranslational modification of eukaryotic proteins, controls a multitude of critical cellular processes, both in normal and pathological conditions. The present work aims to study involvement of ubiquitin-dependent regulation in adaptive response to the external stimuli. Experiments were carried out on C57BL/6 mice. The microgravity state under conditions of real spaceflight on the biosatellite “BION-M1” was used as a model of stress impact. Additionally, number of control series including the vivarium control and experiments in Ground-based analog were also studied. The aggregate of endogenously ubiquitylated proteins was selected as specific feature of ubiquitin-dependent processes. Dynamic changes of modification pattern were characterized in liver tissue by combination of some methods, particularly by specific isolation of explicit protein pool, followed by immunodetection and/or mass spectrometry-based identification. The main approach includes specific extraction of proteins, modified by multiubiquitin chains of different length and topology. For this purpose two techniques were applied: 1) immunoprecipitation with antibodies against ubiquitin and/or multiubiquitin chains; 2) pull-down using synthetic protein construct termed Tandem Ubiquitin Binding Entities (TUBE, LifeSensors). TUBE represents fusion protein, composed of well characterized ubiquitin-binding domains, and thereby allows specific high-affinity binding and extraction of ubiquitylated proteins. Resulting protein fractions were analyzed by immunoblotting with antibodies against different types of multiubiquitin chains. Using this method we mapped endogenously modified proteins involved in two different types of ubiquitin-dependent processes, namely catabolic and non-catabolic ubiquitylation, in liver tissues, obtained from both control as well as experimental groups of animals, mentioned above. Then, isolated fractions of ubiquitylated proteins

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

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


    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.

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

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


    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

  12. Non-degradative Ubiquitination of Protein Kinases.

    K Aurelia Ball


    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.

  13. Cold denaturation of encapsulated ubiquitin

    Pometun, Maxim S.; Peterson, Ronald W.; Babu, Charles R.; Wand, A. Joshua


    Theoretical considerations suggest that protein cold denaturation can potentially provide a means to explore the cooperative substructure of proteins. Protein cold denaturation is generally predicted to occur well-below the freezing point of water. Here NMR spectroscopy of ubiquitin encapsulated in reverse micelles dissolved in low viscosity alkanes is used to follow cold-induced unfolding to temperatures below −25 °C. Comparison of cold-induced structural transitions in a variety of reverse ...

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

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


    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.

  15. Sperm Ubiquitination Correlation with Human Semen Quality

    MR Sadeghi


    Full Text Available Background: Ubiquitin, an 8.5 kDa peptide that marks other proteins for proteasomal degradation, tags defective sperm during epididymal passage. Thus, sperm ubiquitination is a universal marker for sperm defects and can be used as a sperm function test. The objective of the present study was to examine the relationships between sperm ubiquitination and clinical semen parameters, using simplified immunofluorescence assays in order to establish ubiquitin as a biomarker of male infertility. Methods: Semen samples from 100 couples attending Avicenna Infertility Clinic, Tehran, Iran, were collected and analyzed according to WHO criteria. Each sample was washed and adjusted at 106 sperm/ml concentration. Sperm were coated on slides, using cytospin centrifugation and were fixed in buffered formaldehyde. Subsequently ubiquitinated spermatozoa were evaluated by direct immunofluorescence microscopy using FITC-labeled anti-ubiquitin antibodies. After counting at least 200 sperm per sample, while employing light microscopy, the percentage of ubiquitinated spermatozoa was recorded on the same fields through epifluorescence microscopy. Results: Negative correlations were obtained between sperm ubiquitination and sperm count (r=-0.278, P< 0.001, sperm concentration (r=-0.37, P< 0.001, viability (r=-0.407, P< 0.001, sperm morphology (r=-0.509, P< 0.001, rapid progressive motility (a (r=-0.246, P< 0.001 and slow progressive motility (b (r=-0.474, P< 0.001. There was a positive correlation between ubiquitinated sperm and the percentage of immotile spermatozoa (r=0.486, P< 0.000. Conclusion: Increased sperm ubiquitination is inversely associated with good semen quality parameters, supporting the use of ubiquitin as a biomarker for evaluation of human sperm quality.

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

    Ian R. Watson


    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.

  17. Pink1, the first ubiquitin kinase

    Zheng, Xinde; Hunter, Tony


    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.

  18. Regulation of nucleotide excision repair through ubiquitination

    Jia Li; Audesh Bhat; Wei Xiao


    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.

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

    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

  20. Mechanical unfolding of ubiquitin molecules

    Cieplak, Marek; Marszalek, Piotr E.


    Mechanical stretching of ubiquitin and of its several repeats are studied through molecular-dynamics simulations. A Go-type model [H. Abe and N. Go, Biopolymers 20, 1013 (1981)] with a realistic contact map and with Lennard-Jones contact interactions is used. The model qualitatively reproduces the experimentally observed differences between force-extension patterns obtained on polyubiquitins stretched by various linkages. The terminal-to-terminal stretching of polyubiquitin results in peak forces similar to those measured for titin-based polyproteins and of a magnitude that matches measurements. Consistent with the experimental measurements, the simulated peak forces depend on the pulling speed logarithmically when thermal fluctuations are explicitly introduced. These results validate the application of topology-based models in the study of the mechanical stretching of proteins.

  1. Role of ubiquitination in meiotic recombination repair


    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.

  2. Ubiquitination, intracellular trafficking, and degradation of connexins

    Su, Vivian; LAU, ALAN F.


    Gap junction channels provide a conduit for communication between neighboring cells. The function of gap junction channels is regulated by posttranslational modifications of connexins, the proteins that comprise these channels. Ubiquitination of connexins has increasingly been viewed as one mechanism by which cells regulate the level of connexins present in cells, as well as the corresponding intercellular communication. Here we review the current knowledge of connexin ubiquitination and the ...

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

    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: [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany)


    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.

  4. Ubiquitin-Mediated Response to Microsporidia and Virus Infection in C. elegans

    Bakowski, Malina A.; Desjardins, Christopher A; Smelkinson, Margery G.; Dunbar, Tiffany A.; Lopez-Moyado, Isaac F.; Rifkin, Scott A.; Cuomo, Christina A.; Troemel, Emily R.


    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 fin...

  5. Diversification and Specialization of Plant RBR Ubiquitin Ligases.

    Ignacio Marín

    Full Text Available BACKGROUND: RBR ubiquitin ligases are components of the ubiquitin-proteasome system present in all eukaryotes. They are characterized by having the RBR (RING - IBR - RING supradomain. In this study, the patterns of emergence of RBR genes in plants are described. METHODOLOGY/PRINCIPAL FINDINGS: Phylogenetic and structural data confirm that just four RBR subfamilies (Ariadne, ARA54, Plant I/Helicase and Plant II exist in viridiplantae. All of them originated before the split that separated green algae from the rest of plants. Multiple genes of two of these subfamilies (Ariadne and Plant II appeared in early plant evolution. It is deduced that the common ancestor of all plants contained at least five RBR genes and the available data suggest that this number has been increasing slowly along streptophyta evolution, although losses, especially of Helicase RBR genes, have also occurred in several lineages. Some higher plants (e. g. Arabidopsis thaliana, Oryza sativa contain a very large number of RBR genes and many of them were recently generated by tandem duplications. Microarray data indicate that most of these new genes have low-level and sometimes specific expression patterns. On the contrary, and as occurs in animals, a small set of older genes are broadly expressed at higher levels. CONCLUSIONS/SIGNIFICANCE: The available data suggests that the dynamics of appearance and conservation of RBR genes is quite different in plants from what has been described in animals. In animals, an abrupt emergence of many structurally diverse RBR subfamilies in early animal history, followed by losses of multiple genes in particular lineages, occurred. These patterns are not observed in plants. It is also shown that while both plants and animals contain a small, similar set of essential RBR genes, the rest evolves differently. The functional implications of these results are discussed.

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

    González González Humberto


    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.

  7. The recognition of ubiquitinated proteins by the proteasome.

    Grice, Guinevere L; Nathan, James A


    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

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

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


    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...

  9. Cellular contractility requires ubiquitin mediated proteolysis.

    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.

  10. Dengue Virus Genome Uncoating Requires Ubiquitination

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


    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

  11. Ubiquitination of specific mitochondrial matrix proteins.

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


    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

  12. Heat shock induced change in protein ubiquitination in Chlamydomonas

    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)

  13. Homocysteine thiolactone affects protein ubiquitination in yeast.

    Bretes, Ewa; Zimny, Jarosław


    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

  14. Targeting soluble proteins to exosomes using a ubiquitin tag.

    Cheng, Yong; Schorey, Jeffery S


    As "natural" antigen carriers in the body, exosomes are potential vaccine vectors. A number of animal studies indicate that antigen-containing exosomes can induce a specific immune response which can protect against tumor progression or various infections. Exosomes that carry the protective antigens can be purified from cells that release them including tumor cells, dendritic cells, and macrophages. However, this strategy is restricted to proteins that are naturally targeted to exosomes and is therefore limited in the number of antigens present within exosomes. Therefore, with the goal of developing an exosome-based vaccine that is more flexible in its antigen composition and has the potential to be scalable, we have developed a new approach where recombinant soluble proteins can be packaged into exosomes and released from a transformed cell line. In this study, we determined that a C-terminal fusion of ubiquitin to EGFP, tumor antigenic protein nHer2 and Mycobacterium tuberculosis proteins Ag85B and ESAT6 served as an efficient delivery sequence into exosomes when expressed in a human embryonic kidney (HEK 293) cell line, a cell line widely used in industrial recombinant protein production. Two stably transgenic HEK293 cell lines were generated using a retroviral vector to express the Ag85B-ESAT6 fusion protein either alone or tagged at the C-terminus with ubiquitin. Both transformants released exosomes containing the fusion proteins. However, the concentration of Ag85B and ESAT6 in exosomes was increased approximately 10-fold when they were coupled to ubiquitin. Moreover, when the exosomes were used for immunization, there was a direct correlation between the amount of fusion protein within the exosomes and the number of Ag85B and ESAT6 specific INFɣ-secreting T lymphocytes in the lung and spleen. This suggests that exosomes containing recombinant antigen can be used to elicit a T cell response. In summary our data indicates that a ubiquitin-based exosomal

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

    Malina A Bakowski


    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.

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

    Bakowski, Malina A; Desjardins, Christopher A; Smelkinson, Margery G; Dunbar, Tiffany L; Dunbar, Tiffany A; Lopez-Moyado, Isaac F; Rifkin, Scott A; Cuomo, Christina A; Troemel, Emily R


    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. PMID:24945527

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

    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)


    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.

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

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


    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.

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

    Banfield, Mark J.


    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...

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

    Minghua Nie


    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.

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

    Nie, Minghua; Boddy, Michael N


    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. PMID:26927199

  2. Radiation inhibits proteasomes and increases ubiquitinated proteins

    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

  3. The ubiquitin-proteasome system meets angiogenesis.

    Rahimi, Nader


    A strict physiological balance between endogenous proangiogenic and antiangiogenic factors controls endothelial cell functions, such that endothelial cell growth is normally restrained. However, in pathologic angiogenesis, a shift occurs in the balance of regulators, favoring endothelial growth. Much of the control of angiogenic events is instigated through hypoxia-induced VEGF expression. The ubiquitin-proteasome system (UPS) plays a central role in fine-tuning the functions of core proangiogenic proteins, including VEGF, VEGFR-2, angiogenic signaling proteins (e.g., the PLCγ1 and PI3 kinase/AKT pathways), and other non-VEGF angiogenic pathways. The emerging mechanisms by which ubiquitin modification of angiogenic proteins control angiogenesis involve both proteolytic and nonproteolytic functions. Here, I review recent advances that link the UPS to regulation of angiogenesis and highlight the potential therapeutic value of the UPS in angiogenesis-associated diseases. PMID:22357635

  4. The Ubiquitin System and Jasmonate Signaling

    Astrid Nagels Durand


    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.

  5. The Ubiquitin-Proteasome System Meets Angiogenesis

    Rahimi, Nader


    A strict physiological balance between endogenous pro-angiogenic and anti-angiogenic factors control endothelial cell functions, such that endothelial cell growth is normally restrained. However, in pathologic angiogenesis a shift occurs in the balance of regulators favoring endothelial growth. Much of control of angiogenic events is instigated through hypoxia-induced VEGF expression. Ubiquitin-proteasome system plays a central role in fine-tuning function of core pro-angiogenic proteins incl...

  6. Degradation of Activated Protein Kinases by Ubiquitination

    Lu, Zhimin; Hunter, Tony


    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.

  7. The increasing complexity of the ubiquitin code.

    Yau, Richard; Rape, Michael


    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

  8. Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)

    Hendriks, Ivo A; Schimmel, Joost; Eifler, Karolin; Olsen, Jesper V; Vertegaal, Alfred C O


    Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach......, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of...... RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO...

  9. Sch9 regulates intracellular protein ubiquitination by controlling stress responses

    Beibei Qie


    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.

  10. Chaperones, but not oxidized proteins, are ubiquitinated after oxidative stress

    Kästle, Marc; Reeg, Sandra; Rogowska-Wrzesinska, Adelina; Grune, Tilman


    After oxidative stress proteins which are oxidatively modified are degraded by the 20S proteasome. However, several studies documented an enhanced ubiquitination of yet unknown proteins. Since ubiqutination is a prerequisite for degradation by the 26S proteasome in an ATP-dependent manner this...... raises the question whether these proteins are also oxidized and, if not, what proteins need to be ubiquitinated and degraded after oxidative conditions. By determination of oxidized- and ubiquitinated proteins we demonstrate here that most oxidized proteins are not preferentially ubiquitinated. However......, we were able to confirm an increase of ubiquitinated proteins 16h upon oxidative stress. Therefore, we isolated ubiquitinated proteins from hydrogen peroxide treated cells, as well as from control and lactacystin, an irreversible proteasome inhibitor, treated cells, and identified some of these...

  11. Ubiquitin is part of the retrovirus budding machinery

    Patnaik, Akash; Chau, Vincent; Wills, John W.


    Retroviruses contain relatively large amounts of ubiquitin, but the significance of this finding has been unknown. Here, we show that drugs that are known to reduce the level of free ubiquitin in the cell dramatically reduced the release of Rous sarcoma virus, an avian retrovirus. This effect was suppressed by overexpressing ubiquitin and also by directly fusing ubiquitin to the C terminus of Gag, the viral protein that directs budding and particle release. The block to budding was found to be at the plasma membrane, and electron microscopy revealed that the reduced level of ubiquitin results in a failure of mature virus particles to separate from each other and from the plasma membrane during budding. These data indicate that ubiquitin is actually part of the budding machinery.

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

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


    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...

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

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


    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...

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

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


    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...

  15. Ubiquitin at the crossroad of cell death and survival

    Yu-Shan Chen; Xiao-Bo Qiu


    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.

  16. Crystallographic structure of ubiquitin in complex with cadmium ions

    Cheung Peter


    Full Text Available Abstract Background Ubiquitination plays a critical role in regulating many cellular processes, from DNA repair and gene transcription to cell cycle and apoptosis. It is catalyzed by a specific enzymatic cascade ultimately leading to the conjugation of ubiquitin to lysine residues of the target protein that can be the ubiquitin molecule itself and to the formation of poly-ubiquitin chains. Findings We present the crystal structure at 3.0 Å resolution of bovine ubiquitin crystallized in presence of cadmium ions. Two molecules of ubiquitin are present in the asymmetric unit. Interestingly this non-covalent dimeric arrangement brings Lys-6 and Lys-63 of each crystallographically-independent monomer in close contact with the C-terminal ends of the other monomer. Residues Leu-8, Ile-44 and Val-70 that form a hydrophobic patch at the surface of the Ub monomer are trapped at the dimer interface. Conclusions The structural basis for signalling by poly-Ub chains relies on a visualization of conformations of alternatively linked poly-Ub chains. This arrangement of ubiquitin could illustrate how linkages involving Lys-6 or Lys-63 of ubiquitin are produced in the cell. It also details how ubiquitin molecules can specifically chelate cadmium ions.

  17. Regulation and Cellular Roles of Ubiquitin-specific Deubiquitinating Enzymes

    Turcu, Francisca E. Reyes; Ventii, Karen H.; Wilkinson, Keith D.


    Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin or ubiquitin-like gene products, reverse the modification of proteins by a single ubiquitin (or ubiquitin-like protein), and remodel polyubiquitin (or ubiquitin-like) chains on target proteins. The human genome encodes nearly 100 DUBs with specificity for ubiquitin in five families: the UCH, USP, OTU, Josephin, and JAMM families. Four families are cysteine proteases, while the later is a family of metalloproteases. Most DUB activity is cryptic and active site rearrangements often occur during the binding of ubiquitin and/or scaffold proteins. DUBs with specificity for ubiquitin contain multiple domains with insertions and extensions modulating DUB substrate specificity, protein-protein interactions, and cellular localization. Binding partners and multi-protein complexes with which DUBs associate modulate DUB activity and substrate specificity. Quantitative studies of activity and protein-protein interactions, together with genetic studies and the advent of RNAi, have lead to new insights into the function of yeast and human DUBs. This review will discuss ubiquitin-specific DUBs, some of the generalizations emerging from recent studies of the regulation of DUB activity, and their roles in various cellular processes. Specific examples are drawn from studies of protein degradation, DNA repair, chromatin remodeling, cell cycle regulation, endocytosis, and modulation of signaling kinases. PMID:19489724

  18. E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation.

    Kumar, Yogesh; Kapoor, Isha; Khan, Kainat; Thacker, Gatha; Khan, Mohd Parvez; Shukla, Nidhi; Kanaujiya, Jitendra Kumar; Sanyal, Sabyasachi; Chattopadhyay, Naibedya; Trivedi, Arun Kumar


    Runx2, a master regulator of osteoblast differentiation, is tightly regulated at both transcriptional and post-translational levels. Post-translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3β are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7α. Fbw7α through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as in osteoblasts. GSK3β was also present in the same complex as determined by co-immunoprecipitation. Furthermore, overexpression of either Fbw7α or GSK3β was sufficient to down-regulate endogenous Runx2 expression and function; however, both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7α-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7α restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7α in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized (induction of surgical menopause) animals that show reduced Runx2 and enhanced Fbw7α, whereas this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7α negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3β-dependent manner and thus provides a plausible explanation for GSK3β-mediated bone loss as described before. PMID:26542806

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

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


    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

  20. The Ubiquitin-Proteasome Pathway and Synaptic Plasticity

    Hegde, Ashok N.


    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…

  1. A means to an end: ubiquitination of Mpl

    Tong, Wei


    In this issue of Blood, Saur and colleagues report that ubiquitin-mediated degradation of the Mpl receptor constrains Tpo-mediated cell proliferation, highlighting the importance of the E3 ubiquitin ligase c-Cbl in rapid down-regulation of Tpo/Mpl signaling.

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

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


    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.

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

    Chitra, Selvarajan; Nalini, Ganesan; Rajasekhar, Gopalakrishnan


    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

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

    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.

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

    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


    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. PMID:22427669

  6. Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4).

    Hendriks, Ivo A; Schimmel, Joost; Eifler, Karolin; Olsen, Jesper V; Vertegaal, Alfred C O


    Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR. PMID:25969536

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

    Matsumoto, Hotaru; Saitoh, Hisato


    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

  8. Enhanced detection of ubiquitin isopeptides using reductive methylation.

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


    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

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

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


    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

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

    Barbara Mojsa


    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.

  11. A UbcH5/Ubiquitin Noncovalent Complex is Required for Processive BRCA1-Directed Ubiquitination

    Brzovic, Peter S.; Lissounov, Alexei V.; Christensen, Devin; Hoyt, David W.; Klevit, Rachel E.


    Protein ubiquitination is a powerful regulatory modification that influences nearly every aspect of eukaryotic cell biology. The general pathway for ubiquitin (Ub) modification requires the sequential activities of a Ub-activating enzyme (E1), a Ub transfer enzyme (E2), and a Ub ligase (E3). The E2 must recognize both the E1 and a cognate E3 in addition to carrying activated Ub. These central functions are performed by a topologically conserved a/b-fold core domain ofw150 residues shared by all E2s. However, as presented herein, the UbcH5 family of E2s can also bind Ub noncovalently on a surface well removed from the E2 active site. We present the solution structure of the UbcH5c/ Ub noncovalent complex and demonstrate that this noncovalent interaction permits self-assembly of activated UbcH5cwUb molecules. Self-assembly has profound consequences for the processive formation of polyubiquitin (poly-Ub) chains in ubiquitination reactions directed by the breast and ovarian cancer tumor susceptibility protein BRCA1

  12. Biosynthetic Enzyme GMP Synthetase Cooperates with Ubiquitin-Specific Protease 7 in Transcriptional Regulation of Ecdysteroid Target Genes▿

    van der Knaap, Jan A.; Kozhevnikova, Elena; Langenberg, Karin; Moshkin, Yuri M.; Verrijzer, C. Peter


    Drosophila GMP synthetase binds ubiquitin-specific protease 7 (USP7) and is required for its ability to deubiquitylate histone H2B. Previously, we showed that the GMPS/USP7 complex cooperates with the Polycomb silencing system through removal of the active ubiquitin mark from histone H2B (H2Bub). Here, we explored the interplay between GMPS and USP7 further and assessed their role in hormone-regulated gene expression. Genetic analysis established a strong cooperation between GMPS and USP7, which is counteracted by the histone H2B ubiquitin ligase BRE1. Loss of either GMPS or USP7 led to increased levels of histone H2Bub in mutant animals. These in vivo analyses complement our earlier biochemical results, establishing that GMPS/USP7 mediates histone H2B deubiquitylation. We found that GMPS/USP7 binds ecdysone-regulated loci and that mutants display severe misregulation of ecdysone target genes. Ecdysone receptor (EcR) interacts biochemically and genetically with GMPS/USP7. Genetic and gene expression analyses suggested that GMPS/USP7 acts as a transcriptional corepressor. These results revealed the cooperation between a biosynthetic enzyme and a ubiquitin protease in developmental gene control by hormone receptors. PMID:19995917

  13. Ubiquitination of the common cytokine receptor γc and regulation of expression by an ubiquitination/deubiquitination machinery

    The common cytokine receptor γc is shared by the interleukin-2, -4, -7, -9, -15, and -21 receptors, and is essential for lymphocyte proliferation and survival. The regulation of γc receptor expression level is therefore critical for the ability of cells to respond to these cytokines. We previously reported that γc is efficiently constitutively internalized and addressed towards a degradation endocytic compartment. We show that γc is ubiquitinated and also associated to ubiquitinated proteins. We report that the ubiquitin-ligase c-Cbl induces γc down-regulation. In addition, the ubiquitin-hydrolase, DUB-2, counteracts the effect of c-Cbl on γc expression. We show that an increase in DUB-2 expression correlates with an increased γc half-life, resulting in the up-regulation of the receptor. Altogether, we show that γc is the target of an ubiquitination mechanism and its expression level can be regulated through the activities of a couple of ubiquitin-ligase/ubiquitin-hydrolase enzymes, namely c-Cbl/DUB-2

  14. Substrate specificity of the ubiquitin and Ubl proteases

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


    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

  15. A role for PCNA ubiquitination in immunoglobulin hypermutation.

    Hiroshi Arakawa


    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.

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

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


    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...

  17. FANCL ubiquitinates β-catenin and enhances its nuclear function

    Dao, Kim-Hien T.; Rotelli, Michael D.; Petersen, Curtis L.; Kaech, Stefanie; Nelson, Whitney D.; Yates, Jane E.; Hanlon Newell, Amy E.; Olson, Susan B.; Druker, Brian J.; Bagby, Grover C.


    Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-cat...

  18. Reconstitution of CHIP E3 Ubiquitin Ligase Activity

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


    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 ...

  19. Detection of ubiquitinated huntingtin species in intracellular aggregates

    Katrin eJuenemann


    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.

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

    Dong Long


    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.

  1. Autophagy and ubiquitin-mediated proteolysis may not be involved in the degradation of spermatozoon mitochondria in mouse and porcine early embryos.

    Jin, Yong-Xun; Zheng, Zhong; Yu, Xian-Feng; Zhang, Jia-Bao; Namgoong, Suk; Cui, Xiang-Shun; Hyun, Sang-Hwan; Kim, Nam-Hyung


    The mitochondrial genome is maternally inherited in animals, despite the fact that paternal mitochondria enter oocytes during fertilization. Autophagy and ubiquitin-mediated degradation are responsible for the elimination of paternal mitochondria in Caenorhabditis elegans; however, the involvement of these two processes in the degradation of paternal mitochondria in mammals is not well understood. We investigated the localization patterns of light chain 3 (LC3) and ubiquitin in mouse and porcine embryos during preimplantation development. We found that LC3 and ubiquitin localized to the spermatozoon midpiece at 3 h post-fertilization, and that both proteins were colocalized with paternal mitochondria and removed upon fertilization during the 4-cell stage in mouse and the zygote stage in porcine embryos. Sporadic paternal mitochondria were present beyond the morula stage in the mouse, and paternal mitochondria were restricted to one blastomere of 4-cell embryos. An autophagy inhibitor, 3-methyladenine (3-MA), did not affect the distribution of paternal mitochondria compared with the positive control, while an autophagy inducer, rapamycin, accelerated the removal of paternal mitochondria compared with the control. After the intracytoplasmic injection of intact spermatozoon into mouse oocytes, LC3 and ubiquitin localized to the spermatozoon midpiece, but remnants of undegraded paternal mitochondria were retained until the blastocyst stage. Our results show that paternal mitochondria colocalize with autophagy receptors and ubiquitin and are removed after in vitro fertilization, but some remnants of sperm mitochondrial sheath may persist up to morula stage after intracytoplasmic spermatozoon injection (ICSI). PMID:25513816

  2. Use of Fluorescence Spectroscopy for Quantitative Investigations of Ubiquitin Interactions with the Ubiquitin-Binding Domains of NEMO

    Dubosclard, Virginie; Fontan, Elisabeth; Agou, Fabrice


    Ubiquitin serves as a signal for a variety of cellular processes and its specifi c interaction with ubiquitin-binding domain (UBD) regulates key cellular events including protein degradation, cell-cycle control, DNA repair, and kinase activation. Several binding mechanisms for isolated UBDs have been reported in recent years. However, little is known about the mechanism through which proteins containing multiple-UBDs achieve specifi city for a particular oligomer of polyUb. The NF-κB essentia...

  3. " Animal, trop animal "

    Potestà, Andréa


    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...

  4. Protein Camouflage: Supramolecular Anion Recognition by Ubiquitin.

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


    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

  5. Autophagy collaborates with ubiquitination to downregulate oncoprotein E2A/Pbx1 in B-cell acute lymphoblastic leukemia

    B-cell acute lymphoblastic leukemia (B-ALL) accounts for the most cancer incidences in children. We present here that autophagy is downregulated in pediatric B-ALL, suggesting a possible link between autophagy failure and pediatric B-ALL leukemogenesis. With a pediatric t(1;19) B-ALL xenograft mouse model, we show here that activation of autophagy by preventive administration of rapamycin improved the survival of leukemia animals by partial restoration of hematopoietic stem/progenitor cells, whereas treatment of the animals with rapamycin caused leukemia bone marrow cell-cycle arrest. Activation of autophagy in vitro or in vivo by rapamycin or starvation downregulated oncogenic fusion protein E2A/Pbx1. Furthermore, E2A/Pbx1 was found to be colocalized with autophagy marker LC3 in autolysosomes and with ubiquitin in response to autophagy stimuli, whereas autophagy or ubiquitination inhibitor blocked these colocalizations. Together, our data suggest a collaborative action between autophagy and ubiquitination in the degradation of E2A/Pbx1, thereby revealing a novel strategy for targeted preventive or treatment therapy on the pediatric ALL

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

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


    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

  7. Alimento animal y energía de la caña de azúcar

    José Villar


    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.

  8. Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

    Suryadinata, Randy; Holien, Jessica K; Yang, George; Parker, Michael W.; Papaleo, Elena; Šarčević, Boris


    The attachment of ubiquitin (Ub) to lysines on substrates or itself by ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes results in protein ubiquitination. Lysine selection is important for generating diverse substrate-Ub structures and targeting proteins to different fates; however, the...... mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines....... Evaluation of the relative importance of different residues positioned -2, -1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the -1 and -2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter the...

  9. UHRF2, another E3 ubiquitin ligase for p53

    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: [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)


    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.

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

    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.

  11. The Challenge of Producing Ubiquitinated Proteins for Structural Studies

    Serena Faggiano


    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.

  12. Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes

    Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.


    Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

  13. High performance liquid chromatography resolution of ubiquitin pathway enzymes from wheat germ

    The highly conserved protein ubiquitin is involved in several cellular processes in eukaryotes as a result of its covalent ligation to a variety of target proteins. Here, we describe the purification of several enzymatic activities involved in ubiquitin-protein conjugate formation and disassembly from wheat germ (Triticum vulgare) by a combination of ubiquitin affinity chromatography and anion-exchange high performance liquid chromatography. Using this procedure, ubiquitin activating enzyme (E1), several distinct ubiquitin carrier proteins (E2s) with molecular masses of 16, 20, 23, 23.5, and 25 kilodaltons, and a ubiquitin-protein hydrolase (isopeptidase) were isolated. Purified E1 formed a thiol ester linkage with 125I-ubiquitin in an ATP-dependent manner and transferred bound ubiquitin to the various purified E2s. The ubiquitin protein hydrolase fraction was sensitive to hemin, and in an ATP-independent reaction, was capable of removing the ubiquitin moiety from both ubiquitin 125I-lysozyme conjugates (ε-amino or isopeptide linkage) and the ubiquitin 52-amino acid extension protein fusion (α-amino or peptide linkage). Using this procedure, wheat germ represents an inexpensive source from which enzymes involved in the ubiquitin pathway may be isolated

  14. Amazing Animals

    Al-Kuwari, Najat Saad


    "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…

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

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


    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 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...... can be caused by inactivating mutations in RNF216 or by the combination of mutations in RNF216 and OTUD4. These findings link disordered ubiquitination to neurodegeneration and reproductive dysfunction and highlight the power of whole-exome sequencing in combination with functional studies to unveil...

  16. E1AF degradation by a ubiquitin-proteasome pathway

    E1AF is a member of the ETS family of transcription factors. In mammary tumors, overexpression of E1AF is associated with tumorigenesis, but E1AF protein has hardly been detected and its degradation mechanism is not yet clear. Here we show that E1AF protein is stabilized by treatment with the 26S protease inhibitor MG132. We found that E1AF was modified by ubiquitin through the C-terminal region and ubiquitinated E1AF aggregated in nuclear dots, and that the inhibition of proteasome-activated transcription from E1AF target promoters. These results suggest that E1AF is degraded via the ubiquitin-proteasome pathway, which has some effect on E1AF function

  17. The Role of Ubiquitine Proteasome Pathway in Carcinogenesis

    N.Ceren Sumer Turanligil


    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

  18. The ubiquitin conjugating enzyme UbcH7, controls cell migration

    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 ...

  19. The role of the ubiquitin-proteasome proteolytic pathway in the anabolic drive of nutrition

    subunits (C9 and C2) were normalised by refeeding at 4 and 24h in muscle, and 24 and 6h in jejunum, respectively. Nutrients and/or hormones regulate the expression of critical components of the ubiquitin-proteasome pathway in a temporally and tissue-specific fashion. In striking contrast to protein synthesis, muscle proteolysis is not acutely responding in vivo to amino acids, nutrient intake and/or insulin in starved animals. The initial response to these anabolic stimuli is perhaps through inhibition of visceral proteolysis

  20. The Ubiquitin-like modifier FAT10 in tolerance induction

    Bürger, Stefanie


    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, ...

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

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


    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-...

  2. Ubiquitin B in Cervical Cancer: Critical for the Maintenance of Cancer Stem-Like Cell Characters

    Tian, Yuan; Ding, Wencheng; Wang, Yingying; Ji, Teng; Sun, Shujuan; Mo, Qingqing; Chen, Pingbo; Fang, Yong; Liu, Jia; Wang, Beibei; Zhou, Jianfeng; Ma, Ding; Wu, Peng


    Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate. Ubiquitin, which is a small, highly conserved protein expressed in all eukaryotic cells, can be covalently linked to certain target proteins to mark them for degradation by the ubiquitin-proteasome system. Previous studies highlight the essential role of Ubiquitin B (UbB) and UbB-dependent proteasomal protein degradation in histone deacetylase...

  3. Functional characterization of an E3 ubiquitin ligase involved in plant response to abiotic stress

    Guerra, Davide


    Protein ubiquitination is a post-translational modification that targets protein substrates for 26S proteasome-mediated degradation. It is based on the covalent attachment of the 76-amino acid eukaryotic molecule, ubiquitin, to substrate proteins. Protein ubiquitination plays a key role in a wide variety of cellular processes such as hormone signaling, DNA repair, biotic and abiotic stress response, cell cycle regulation. Ubiquitin conjugation is a multistep reaction, sequentially involvin...

  4. Weighing in on Ubiquitin: The Expanding Role of Mass Spectrometry-based Proteomics

    Kirkpatrick, Donald S.; Denison, Carilee; Gygi, Steven P.


    Mass spectrometry-based proteomics has become an essential tool for qualitative and quantitative analysis of cellular systems. The biochemical complexity and functional diversity of the ubiquitin system are well suited to proteomic studies. This review summarizes advances involving the identification of ubiquitinated proteins, the elucidation of ubiquitin-modification sites, and the determination of poly-ubiquitin chain linkages, while offering a perspective on the application of emerging tec...

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

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


    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...

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

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


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

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

    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


    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

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

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


    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

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

    Nicolas Massaly


    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.

  10. Role of Linear Ubiquitination in Health and Disease.

    Brazee, Patricia; Dada, Laura A; Sznajder, Jacob I


    The covalent attachment of ubiquitin to target proteins is one of the most prevalent post-translational modifications, regulating a myriad of cellular processes including cell growth, survival, and metabolism. Recently, a novel RING E3 ligase complex was described, called linear ubiquitin assembly complex (LUBAC), which is capable of connecting ubiquitin molecules in a novel head-to-tail fashion via the N-terminal methionine residue. LUBAC is a heteromeric complex composed of heme-oxidized iron-responsive element-binding protein 2 ubiquitin ligase-1L (HOIL-1L), HOIL-1L-interacting protein, and shank-associated RH domain-interacting protein (SHARPIN). The essential role of LUBAC-generated linear chains for activation of nuclear factor-κB (NF-κB) signaling was first described in the activation of tumor necrosis factor-α receptor signaling complex. A decade of research has identified additional pathways that use LUBAC for downstream signaling, including CD40 ligand and the IL-1β receptor, as well as cytosolic pattern recognition receptors including nucleotide-binding oligomerization domain containing 2 (NOD2), retinoic acid-inducible gene 1 (RIG-1), and the NOD-like receptor family, pyrin domain containing 3 inflammasome (NLRP3). Even though the three components of the complex are required for full activation of NF-κB, the individual components of LUBAC regulate specific cell type- and stimuli-dependent effects. In humans, autosomal defects in LUBAC are associated with both autoinflammation and immunodeficiency, with additional disorders described in mice. Moreover, in the lung epithelium, HOIL-1L ubiquitinates target proteins independently of the other LUBAC components, adding another layer of complexity to the function and regulation of LUBAC. Although many advances have been made, the diverse functions of linear ubiquitin chains and the regulation of LUBAC are not yet completely understood. In this review, we discuss the various roles of linear ubiquitin

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

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


    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

  12. Use of fluorescence spectroscopy for quantitative investigations of ubiquitin interactions with the ubiquitin-binding domains of NEMO.

    Dubosclard, Virginie; Fontan, Elisabeth; Agou, Fabrice


    Ubiquitin serves as a signal for a variety of cellular processes and its specific interaction with ubiquitin-binding domain (UBD) regulates key cellular events including protein degradation, cell-cycle control, DNA repair, and kinase activation. Several binding mechanisms for isolated UBDs have been reported in recent years. However, little is known about the mechanism through which proteins containing multiple-UBDs achieve specificity for a particular oligomer of polyUb. The NF-κB essential modulator (NEMO, also known IKKγ), which plays a key role in the NF-κB signaling pathway, belongs to the latter family of proteins since it contains two distal NOA (also known UBAN/CC2-LZ/NUB) and ZF UBDs, separated by an unstructured proline-rich linker of about 40 residues in length. Here, we show a new procedure for fast purification of this bipartite domain. We also describe the use of intrinsic fluorescence spectroscopy for quantitative investigations of ubiquitin interactions between two distal ubiquitin-binding domains of NEMO (NOA and ZF). This spectroscopic method has many advantages over other techniques like GST pulldown and Biacore's SPR for monitoring avid interactions between two UBDs, especially when UBDs are located at significant distance from each other within the protein. PMID:25736758

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

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


    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

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

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


    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. PMID:27049943

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

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


    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.

  16. Animal Farm



    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.

  17. Animal Bites

    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 ...

  18. Animal Bites

    ... 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 ...

  19. Animal Farm



    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.

  20. Animal ethics

    Palmer, Clare; Sandøe, Peter


    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...

  1. Quadruped Animation

    Skrba, Ljiljana; Reveret, Lionel; Hétroy, Franck; Cani, Marie-Paule; O'Sullivan, Carol


    Films like Shrek, Madagascar, The Chronicles of Narnia and Charlotte's web all have something in common: realistic quadruped animations. While the animation of animals has been popular for a long time, the technical challenges associated with creating highly realistic, computer generated creatures have been receiving increasing attention recently. The entertainment, education and medical industries have increased the demand for simulation of realistic animals in the computer graphics area. In...

  2. Thin Animals

    Johnston, D.


    Lattice animals provide a discretized model for the theta transition displayed by branched polymers in solvent. Exact graph enumeration studies have given some indications that the phase diagram of such lattice animals may contain two collapsed phases as well as an extended phase. This has not been confirmed by studies using other means. We use the exact correspondence between the q --> 1 limit of an extended Potts model and lattice animals to investigate the phase diagram of lattice animals ...

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

    Carlos Arturo Viveros Valens


    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

  4. Animal Deliberation

    Driessen, C.P.G.


    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

  5. Animal models

    Gøtze, Jens Peter; Krentz, Andrew


    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...

  6. Entry, Descent, Landing Animation (Animation)


    [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.

  7. Animal research

    Olsson, I.A.S.; Sandøe, Peter


    in science (as in any other human use that is not also in the animals’ best interest). These views are not compatible, and since all three views in more or less pure form are found in modern Western societies, use of animals for research is bound to cause controversy. However, there may be room for some kind......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...... 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...

  8. USP11 Deubiquitinates Hybrid SUMO-Ubiquitin Chains to Counteract RNF4

    Hendriks, Ivo A.; Schimmel, Joost; Eifler, Karolin; Olsen, Jesper V.; Vertegaal, Alfred C.O.


    Background RNF4 is a ubiquitin ligase targeted to SUMOylated proteins. Result USP11 co-purified with RNF4 and can remove ubiquitin polymers attached to SUMO chains. Conclusion USP11 is a ubiquitin protease with the ability to counteract RNF4 in the DNA damage response. Significance Identification of a ubiquitin protease to balance the activity of a SUMO-targeted ubiquitin ligase. RNF4 is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO Interaction Motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme USP11, a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into crosstalk between ubiquitin and SUMO, and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR. PMID:25969536

  9. Ubiquitin-aldehyde: a general inhibitor of ubiquitin-recycling processes

    The generation and characterization of ubiquitin (Ub)-aldehyde, a potent inhibitor of Ub-C-terminal hydrolase, has previously been reported. The authors examine the action of this compound on the Ub-mediated proteolytic pathway using the system derived from rabbit reticulocytes. Addition of Ub-aldehyde was found to strongly inhibit breakdown of added 125I-labeled lysozyme, but inhibition was overcome by increasing concentrations of Ub. The following evidence shows the effect of Ub-aldehyde on protein breakdown to be indirectly caused by its interference with the recycling of Ub, leading to exhaustion of the supply of free Ub: (i) Ub-aldehyde markedly increased the accumulation of Ub-protein conjugates coincident with a much decreased rate of conjugate breakdown; (ii) release of Ub from isolated Ub-protein conjugates in the absence of ATP (and therefore not coupled to protein degradation) is markedly inhibited by Ub-aldehyde. On the other hand, the ATP-dependent degradation of the protein moiety of Ub conjugates, which is an integral part of the proteolytic process, is not inhibited by this agent; (iii) direct measurement of levels of free Ub showed a rapid disappearance caused by the inhibitor. The Ub is found to be distributed in derivatives of a wide range of molecular weight classes. It thus seems that Ub-aldehyde, previously demonstrated to inhibit the hydrolysis of Ub conjugates of small molecules, also inhibits the activity of a series of enzymes that regenerate free Ub from adducts with proteins and intermediates in protein breakdown

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

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


    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

  11. Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy.

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


    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

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

    Hideya Ando


    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.

  13. Direct observation of silver nanoparticle-ubiquitin corona formation

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


    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...

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

    Marisa S Goo


    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.

  15. The ubiquitin proteasome system in human cardiomyopathies and heart failure

    Day, Sharlene M.


    Maintenance of protein quality control is a critical function of the ubiquitin proteasome system (UPS). Evidence is rapidly mounting to link proteasome dysfunction with a multitude of cardiac diseases, including ischemia, reperfusion, atherosclerosis, hypertrophy, heart failure, and cardiomyopathies. Recent studies have demonstrated a remarkable level of complexity in the regulation of the UPS in the heart and suggest that our understanding of how UPS dysfunction might contribute to the patho...

  16. Exploitation of the Ubiquitin System by Invading Bacteria

    Steele-Mortimer, Olivia


    A variety of bacterial intracellular pathogens target the host cell ubiquitin system during invasion, a process that involves transient but fundamental changes in the actin cytoskeleton and plasma membrane. These changes are induced by bacterial proteins, which can be surface-associated, secreted or injected directly into the host cell. Here the invasion strategies of two extensively studied intracellular bacteria, Salmonella enterica serovar Typhimurium and Listeria monocytogenes, are used t...

  17. Regulation of peroxisomal matrix protein import by ubiquitination

    Platta, Harald W; Brinkmeier, Rebecca; Reidick, Christina; Galiani, Silvia; Clausen, Mathias P; Eggeling, Christian


    Peroxisomes are organelles that play an important role in many cellular tasks. The functionality of peroxisomes depends on the proper import of their matrix proteins. Peroxisomal matrix proteins are imported posttranslationally in a folded, sometimes even oligomeric state. They harbor a peroxisom...... the first ubiquitinated peroxin 15years ago. Moreover, we discuss future tasks and the potential of using advanced technologies for investigating further details of peroxisomal protein transport. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann....

  18. Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis

    Jennifer Hurst-Kennedy


    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.

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

    Roberta Verciano Pereira


    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.

  20. Novel RING E3 Ubiquitin Ligases in Breast Cancer

    Angelika Burger


    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.

  1. Viral Mimicry to Usurp Ubiquitin and SUMO Host Pathways

    Peter Wimmer


    Full Text Available Posttranslational modifications (PTMs of proteins include enzymatic changes by covalent addition of cellular regulatory determinants such as ubiquitin (Ub and small ubiquitin-like modifier (SUMO moieties. These modifications are widely used by eukaryotic cells to control the functional repertoire of proteins. Over the last decade, it became apparent that the repertoire of ubiquitiylation and SUMOylation regulating various biological functions is not restricted to eukaryotic cells, but is also a feature of human virus families, used to extensively exploit complex host-cell networks and homeostasis. Intriguingly, besides binding to host SUMO/Ub control proteins and interfering with the respective enzymatic cascade, many viral proteins mimic key regulatory factors to usurp this host machinery and promote efficient viral outcomes. Advanced detection methods and functional studies of ubiquitiylation and SUMOylation during virus-host interplay have revealed that human viruses have evolved a large arsenal of strategies to exploit these specific PTM processes. In this review, we highlight the known viral analogs orchestrating ubiquitin and SUMO conjugation events to subvert and utilize basic enzymatic pathways.

  2. [Transgenic animals and animal welfare

    Reinhardt, Christoph


    Under the pressure of a public vote in Switzerland (7 June 1998) on an initiative to ban the production, use and patenting of transgenic animals, their value for biomedical research and development is intensely debated. In addition, the Swiss legislation has adopted (1992) a constitutional obligation to "take into account the dignity of creatures". The term "dignity of creatures", however, can be interpreted in anthropocentric or biocentric ways. The government has now formulated the legal implications of this term for transgenic animals and plants in various laws including the animal and environmental protection laws. This paper gives arguments for a fair evaluation of trangenic animals from an animal welfare point of view where not only the costs of animal suffering must be considered but also the probability of potential benefit for man. A self-confident research community should allow such an evaluation procedure even in view of an outcome which could ban many uses of transgenic animals PMID:11208266

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

    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.

  4. Animal Shelter


    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-

  5. Animal ethics

    Palmer, Clare; Sandøe, Peter


    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 possible to combine elements from the presented views, and how to make up one’s mind....

  6. Cullin 3 mediates SRC-3 ubiquitination and degradation to control the retinoic acid response

    Ferry, Christine; Gaouar, Samia; Fischer, Benoit; Boeglin, Marcel; Paul, Nicodeme; Samarut, Eric; Piskunov, Aleksandr; Pankotai-Bodo, Gabriella; Brino, Laurent; Rochette-Egly, Cecile


    SRC-3 is an important coactivator of nuclear receptors including the retinoic acid (RA) receptor α. Most of SRC-3 functions are facilitated by changes in the posttranslational code of the protein that involves mainly phosphorylation and ubiquitination. We recently reported that SRC-3 is degraded by the proteasome in response to RA. Here, by using an RNAi E3-ubiquitin ligase entry screen, we identified CUL-3 and RBX1 as components of the E3 ubiquitin ligase involved in the RA-induced ubiquitination and subsequent degradation of SRC-3. We also show that the RA-induced ubiquitination of SRC-3 depends on its prior phosphorylation at serine 860 that promotes binding of the CUL-3–based E3 ligase in the nucleus. Finally, phosphorylation, ubiquitination, and degradation of SRC-3 cooperate to control the dynamics of transcription. In all, this process participates to the antiproliferative effect of RA. PMID:22147914

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

    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

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

    Poulsen, Sara L.; Hansen, Rebecca K.; Wagner, Sebastian A.; van Cuijk, Loes; van Belle, Gijsbert J.; Streicher, Werner; Wikström, Mats; Choudhary, Chunaram; Houtsmuller, Adriaan B.; Marteijn, Jurgen A.; Bekker-Jensen, Simon; Mailand, Niels


    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), promoting their K48-linked ubiquitylation and degradation. Only a single mammalian STUbL, RNF4, has been identified. We show that human RNF111/Arkadia is a new STUbL, which used three adjacent SIMs for s...

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

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


    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...

  10. Regulation of mitophagy by the Gp78 E3 ubiquitin ligase

    Fu, Min; St-Pierre, Pascal; Shankar, Jay; Wang, Peter T. C.; Joshi, Bharat; Nabi, Ivan R.


    Glycoprotein 78 (Gp78) is a critical E3 ubiquitin ligase in endoplasmic reticulum–associated degradation. Overexpression of Flag-tagged Gp78 (Flag-gp78), but not Flag-gp78 mutated in its RING-finger domain (Flag-RINGmut) with deficient ubiquitin ligase activity, induces mitochondrial fragmentation and ubiquitination and proteasome-dependent degradation of the mitofusin (Mfn) mitochondrial fusion factors Mfn1/Mfn2. After mitochondrial depolarization with carbonyl cyanide m-chlorophenylhydrazon...

  11. Animal cytomegaloviruses.

    Staczek, J.


    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...

  12. ANIMAL code

    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

  13. Kindergarten Animation

    Hinshaw, Craig


    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…

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

    Zhang Hui


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

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

    Sun Yuan-Shui


    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.

  16. Cullin 3 mediates SRC-3 ubiquitination and degradation to control the retinoic acid response

    Ferry, Christine; Gaouar, Samia; Fischer, Benoit; Boeglin, Marcel; Paul, Nicodeme; Samarut, Eric; Piskunov, Aleksandr; Pankotai-Bodo, Gabriella; Brino, Laurent; Rochette-Egly, Cecile


    SRC-3 is an important coactivator of nuclear receptors including the retinoic acid (RA) receptor α. Most of SRC-3 functions are facilitated by changes in the posttranslational code of the protein that involves mainly phosphorylation and ubiquitination. We recently reported that SRC-3 is degraded by the proteasome in response to RA. Here, by using an RNAi E3-ubiquitin ligase entry screen, we identified CUL-3 and RBX1 as components of the E3 ubiquitin ligase involved in the RA-induced ubiquitin...

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

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


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

  18. Animal learning.

    Castro, Leyre; Wasserman, Edward A


    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

  19. Wild Animals



    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.

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

    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

  1. Animal performance

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


    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.

  2. Animation & Neurocinematics*

    Carpe Pérez, Inmaculada Concepción


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

    Germán Pabón


    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.

  4. Ubiquitin-proteasome system involvement in Huntington’s disease

    Zaira Ortega


    Full Text Available Huntington’s disease (HD is a genetic autosomal dominant neurodegenerative disease caused by the expansion of a CAG repeat in the huntingtin (htt gene. This triplet expansion encodes a polyglutamine stretch (polyQ in the N-terminus of the high molecular weight (348-kDa and ubiquitously expressed protein huntingtin (htt. Normal individuals have between 6 and 35 CAG triplets, while expansions longer than 40 repeats lead to HD. The onset and severity of the disease depend on the length of the polyQ tract: the longer the polyQ is, the earlier the disease begins and the more severe the symptoms are. One of the main histopathological hallmarks of HD is the presence of intraneuronal proteinaceous inclusion bodies (IBs, whose prominent and invariant feature is the presence of Ubiquitin (Ub; therefore, they can be detected with anti-ubiquitin and anti-proteasome antibodies. This, together with the observation that mutations in components of the Ubiquitin Proteasome system (UPS give rise to some neurodegenerative diseases, suggests that UPS impairment may be causative of HD. Even though the link between disrupted Ub homeostasis and protein aggregation to HD is undisputed, the functional significance of these correlations and their mechanistic implications remains unresolved. Moreover, there is no consistent evidence documenting an accompanying decrease in levels of free Ub or disruption of Ub pool dynamics in neurodegenerative disease or models thus suggesting that the Ub-conjugate accumulation may be benign and just underlie lesion in 26S function. In this chapter we will elaborate on the different studies that have been performed using different experimental approaches, in order to shed light to this matter.

  5. Groundwater animals

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


    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...

  6. Redox control of the ubiquitin-proteasome system

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


    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...

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

    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.


    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.

  8. Proteins containing the UBA domain are able to bind to multi-ubiquitin chains

    Wilkinson, C R; Seeger, M; Hartmann-Petersen, R; Stone, M; Wallace, M; Semple, C; Gordon, C


    The UBA domain is a motif found in a variety of proteins, some of which are associated with the ubiquitin-proteasome system. We describe the isolation of a fission-yeast gene, mud1+, which encodes a UBA domain containing protein that is able to bind multi-ubiquitin chains. We show that the UBA do...

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

    Guzmán, Plinio


    Major components of the ubiquitin proteasome system are the enzymes that operate on the transfer of ubiquitin to selected target substrate, known as ubiquitin ligases. The RING finger is a domain that is present in key classes of ubiquitin ligases. This domain coordinates the interaction with a suitable E2 conjugase and the transfer of ubiquitin from the E2 to protein targets. Additional domains coupled to the same polypeptide are important for modulating the function of these ubiquitin ligases. Plants contain several types of E3 ubiquitin ligases that in many cases have expanded as multigene families. Some families are specific to the plant lineage, whereas others may have a common ancestor among plants and other eukaryotic lineages. Arabidopsis Tóxicos en Levadura (ATLs) and BCA2 zinc finger ATLs (BTLs) are two families of ubiquitin ligases that share some common structural features. These are intronless genes that encode a highly related RING finger domain, and yet during evolutionary history, their mode of gene expansion and function is rather different. In each of these two families, the co-occurrence of transmembrane helices or C2/C2 (BZF finger) domains with a selected variation on the RING finger has been subjected to strong selection pressure in order to preserve their unique domain architectures during evolution. PMID:24388516

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

    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: [Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601 (Japan)


    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.

  11. Role of RNF4 in the ubiquitination of Rta of Epstein-Barr virus.

    Yang, Ya-Chun; Yoshikai, Yushi; Hsu, Shih-Wei; Saitoh, Hisato; Chang, Li-Kwan


    Epstein-Barr virus (EBV) encodes a transcription factor, Rta, which is required to activate the transcription of EBV lytic genes. This study demonstrates that treating P3HR1 cells with a proteasome inhibitor, MG132, causes the accumulation of SUMO-Rta and promotes the expression of EA-D. GST pulldown and coimmunoprecipitation studies reveal that RNF4, a RING-domain-containing ubiquitin E3 ligase, interacts with Rta. RNF4 also targets SUMO-2-conjugated Rta and promotes its ubiquitination in vitro. Additionally, SUMO interaction motifs in RNF4 are important to the ubiquitination of Rta because the RNF4 mutant with a mutation at the motifs eliminates ubiquitination. The mutation of four lysine residues on Rta that abrogated SUMO-3 conjugation to Rta also decreases the enhancement of the ubiquitination of Rta by RNF4. This finding demonstrates that RNF4 is a SUMO-targeted ubiquitin E3 ligase of Rta. Finally, knockdown of RNF4 enhances the expression of Rta and EA-D, subsequently promoting EBV lytic replication and virions production. Results of this study significantly contribute to efforts to elucidate a SUMO-targeted ubiquitin E3 ligase that regulates Rta ubiquitination to influence the lytic development of EBV. PMID:23504328

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

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


    Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING domain of rat RNF4 in complex with E2 (UbcH5A) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The carboxy-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilize the consequent tetrahedral transition-state intermediate. PMID:22842904

  13. Role of frameshift ubiquitin B protein in Alzheimer's disease.

    Chen, Xin; Petranovic, Dina


    Alzheimer's disease (AD) is the most common neurodegenerative disease and is characterized by accumulation of misfolded and aggregated proteins. Since the ubiquitin-proteasome system (UPS) is the major intracellular protein quality control (PQC) system in eukaryotic cells, it is likely involved in the etiology of AD. The frameshift form of ubiquitin (Ubb(+1) ) accumulates in the neuritic plaques and neurofibrillary tangles in patients with AD. Ubb(+1) accumulates in an age-dependent manner as a result of RNA-polymerase mediated molecular misreading during transcription, which allows the formation of mutant proteins in the absence of gene mutations. The accumulation of the Ubb(+1) protein may act as an endogenous reporter for proteasome dysfunction and a growing number of studies have shown that Ubb(+1) may play more important pathogenic roles in AD etiology than previously hypothesized. The yeast Saccharomyces cerevisiae shares many conserved biological processes with all eukaryotic cells, including human neurons. This organism has been regarded as a model system for investigating the fundamental intracellular mechanisms, including those underlying neurodegeneration. We propose here that yeast systems biology approaches, combined with cell and molecular biology approaches will increase the relevant knowledge needed for advancement and elucidation of mechanisms and complex traits, which could provide new targets for therapeutic intervention in AD. WIREs Syst Biol Med 2016, 8:300-313. doi: 10.1002/wsbm.1340 For further resources related to this article, please visit the WIREs website. PMID:27240056

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

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


    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.

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

    Full Text Available 16982211 Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. (.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

  16. Nickel compounds induce histone ubiquitination by inhibiting histone deubiquitinating enzyme activity

    Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis

  17. Biotecnologia animal

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


    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...

  18. Animated symbols

    Frølunde, Lisbeth


    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...

  19. Animal house

    Turka, Laurence A.


    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. Transgenic Animals.

    Jaenisch, Rudolf


    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.…

  1. Animated Symbols

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

    Scheuring David


    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

  3. Biotecnologia animal

    Luiz Lehmann Coutinho


    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

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

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


    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. PMID:24365593

  5. Structure of the Ubiquitin Hydrolase UCH-L3 Complexed with a Suicide Substrate

    Misaghi, S.; Galardy, P.J.; Meester, W.J.; Ovaa, H.; Ploegh, H.L.; Gaudet, R. (Harvard)


    Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 {angstrom} resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structure confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.

  6. Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation.

    Yamano, Koji; Queliconi, Bruno B; Koyano, Fumika; Saeki, Yasushi; Hirokawa, Takatsugu; Tanaka, Keiji; Matsuda, Noriyuki


    Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser(65) by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. PMID:26260794

  7. Structural basis for the RING catalyzed synthesis of K63 linked ubiquitin chains

    Branigan, Emma; Plechanovová, Anna; Jaffray, Ellis; Naismith, James H.; Hay, Ronald T.


    The RING E3 ligase catalysed formation of lysine 63 linked ubiquitin chains by the Ube2V2–Ubc13 E2 complex is required for many important biological processes. Here we report the structure of the RING domain dimer of rat RNF4 in complex with a human Ubc13~Ub conjugate and Ube2V2. The structure has captured Ube2V2 bound to the acceptor (priming) ubiquitin with Lys63 in a position that could lead to attack on the linkage between the donor (second) ubiquitin and Ubc13 that is held in the active “folded back” conformation by the RING domain of RNF4. The interfaces identified in the structure were verified by in vitro ubiquitination assays of site directed mutants. This represents the first view of the synthesis of Lys63 linked ubiquitin chains in which both substrate ubiquitin and ubiquitin-loaded E2 are juxtaposed to allow E3 ligase mediated catalysis. PMID:26148049

  8. Structural basis for the RING-catalyzed synthesis of K63-linked ubiquitin chains.

    Branigan, Emma; Plechanovová, Anna; Jaffray, Ellis G; Naismith, James H; Hay, Ronald T


    RING E3 ligase-catalyzed formation of K63-linked ubiquitin chains by the Ube2V2-Ubc13 E2 complex is required in many important biological processes. Here we report the structure of the RING-domain dimer of rat RNF4 in complex with a human Ubc13∼Ub conjugate and Ube2V2. The structure has captured Ube2V2 bound to the acceptor (priming) ubiquitin with K63 in a position favorable for attack on the linkage between Ubc13 and the donor (second) ubiquitin held in the active 'folded back' conformation by the RING domain of RNF4. We verified the interfaces identified in the structure by in vitro ubiquitination assays of site-directed mutants. To our knowledge, this represents the first view of synthesis of K63-linked ubiquitin chains in which both substrate ubiquitin and ubiquitin-loaded E2 are juxtaposed to allow E3 ligase-mediated catalysis. PMID:26148049

  9. Animal facilities

    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

  10. [Dangerous animals].

    Hasle, Gunnar


    As travellers seek ever more exotic destinations they are more likely to encounter dangerous animals. Compared to risks such as AIDS, traffic accidents and malaria, the risk is not so great; many travellers are, however, concerned about this and those who give pre-travel vaccines and advice should know something about it. This article is mainly based on medical and zoological textbooks. Venomous stings and bites may be prevented by adequate clothing and by keeping safe distance to the animals. Listening to those who live in the area is of course important. Travellers should not carry antisera with them, but antisera should be available at local hospitals. It should be borne in mind that plant eaters cause just as many deaths as large predators. In some cases it is necessary to carry a sufficiently powerful firearm. PMID:12555616

  11. Animal Locomotion

    Taylor, Graham K; Tropea, Cameron


    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.

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

    Vlachostergios Panagiotis J


    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.

  13. Ube2W conjugates ubiquitin to α-amino groups of protein N-termini.

    Tatham, Michael H; Plechanovová, Anna; Jaffray, Ellis G; Salmen, Helena; Hay, Ronald T


    The covalent attachment of the protein ubiquitin to intracellular proteins by a process known as ubiquitylation regulates almost all major cellular systems, predominantly by regulating protein turnover. Ubiquitylation requires the co-ordinated action of three enzymes termed E1, E2 and E3, and typically results in the formation of an isopeptide bond between the C-terminal carboxy group of ubiquitin and the ϵ-amino group of a target lysine residue. However, ubiquitin is also known to conjugate to the thiol of cysteine residue side chains and the α-amino group of protein N-termini, although the enzymes responsible for discrimination between different chemical groups have not been defined. In the present study, we show that Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity. Ube2W conjugates ubiquitin not only to its own N-terminus, but also to that of the small ubiquitin-like modifier SUMO (small ubiquitin-related modifier) in a manner dependent on the SUMO-targeted ubiquitin ligase RNF4 (RING finger protein 4). Furthermore, N-terminal mono-ubiquitylation of SUMO-2 primes it for poly-ubiquitylation by the Ubc13-UEV1 (ubiquitin-conjugating enzyme E2 variant 1) heterodimer, showing that N-terminal ubiquitylation regulates protein fate. The description in the present study is the first of an E2-conjugating enzyme with N-terminal ubiquitylation activity, and highlights the importance of E2 enzymes in the ultimate outcome of E3-mediated ubiquitylation. PMID:23560854

  14. Animal Drug Safety FAQs

    ... 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 ...

  15. Animal Testing

    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.

  16. Animated war

    Frølunde, Lisbeth


    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...

  17. Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats

    Lecker, S. H.; Solomon, V.; Price, S. R.; Kwon, Y. T.; Mitch, W. E.; Goldberg, A. L.


    Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of (125)I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2(14k) and E3alpha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E2(14k) inhibited this increase in ubiquitination rates. Both E2(14k) and E3alpha were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3alpha (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2(14k) and E3alpha content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2(14k) and E3alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.

  18. Identification of the receptor component of the IkappaBalpha-ubiquitin ligase

    Yaron, A; Hatzubai, A; Davis, M; Lavon, I; Amit, S; Manning, A M; Andersen, Jens S.; Mann, M; Mercurio, F; Ben-Neriah, Y


    integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. Here we isolate the pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF......-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIkappaBalpha degradation motif......(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation....

  19. Molecular piracy: manipulation of the ubiquitin system by Kaposi's sarcoma-associated herpesvirus.

    Fujimuro, Masahiro; Hayward, S Diane; Yokosawa, Hideyoshi


    Ubiquitination, one of several post-translational protein modifications, plays a key role in the regulation of cellular events, including protein degradation, signal transduction, endocytosis, protein trafficking, apoptosis and immune responses. Ubiquitin attachment at the lysine residue of cellular factors acts as a signal for endocytosis and rapid degradation by the 26S proteasome. It has recently been observed that viruses, especially oncogenic herpesviruses, utilise molecular piracy by encoding their own proteins to interfere with regulation of cell signalling. Kaposi's sarcoma- associated herpesvirus (KSHV) manipulates the ubiquitin system to facilitate cell proliferation, anti-apoptosis and evasion from immunity. In this review, we will describe the strategies used by KSHV at distinct stages of the viral life-cycle to control the ubiquitin system and promote oncogenesis and viral persistence. PMID:17688306

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

    Wasilko, David J; Mao, Yuxin


    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

  1. Ubiquitin-SUMO circuitry controls activated fanconi anemia ID complex dosage in response to DNA damage.

    Gibbs-Seymour, Ian; Oka, Yasuyoshi; Rajendra, Eeson; Weinert, Brian T; Passmore, Lori A; Patel, Ketan J; Olsen, Jesper V; Choudhary, Chunaram; Bekker-Jensen, Simon; Mailand, Niels


    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 core complex, and the SUMO E3 ligases PIAS1/PIAS4 and is antagonized by the SUMO protease SENP6. SUMOylation of the ID complex drives substrate selectivity by triggering its polyubiquitylation by the SUMO-targeted ubiquitin ligase RNF4 to promote its removal from sites of DNA damage via the DVC1-p97 ubiquitin segregase complex. Deregulation of ID complex SUMOylation compromises cell survival following replication stress. Our results uncover a regulatory role for SUMOylation in the FA pathway, and we propose that ubiquitin-SUMO signaling circuitry is a mechanism that contributes to the balance of activated ID complex dosage at sites of DNA damage. PMID:25557546

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

    Scaife Jes R


    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

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

    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


    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...

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

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


    -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....

  5. An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin.

    M. Kitagawa; Hatakeyama, S.; Shirane, M; Matsumoto, M; ISHIDA, N.; K. Hattori; Nakamichi, I; Kikuchi, A; Nakayama, K.


    beta-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of beta-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. beta-catenin levels are regulated by the ubiquitin-dependent proteolysis system and beta-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen syntha...

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

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


    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...

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

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina


    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

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

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


    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).

  9. Hrs Recognizes a Hydrophobic Amino Acid Cluster in Cytokine Receptors during Ubiquitin-independent Endosomal Sorting*

    Amano, Yuji; Yamashita, Yuki; Kojima, Katsuhiko; Yoshino, Kazuhisa; Tanaka, Nobuyuki; Sugamura, Kazuo; Takeshita, Toshikazu


    Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of the ESCRT-0 protein complex that captures ubiquitylated cargo proteins and sorts them to the lysosomal pathway. Although Hrs acts as a key transporter for ubiquitin-dependent endosomal sorting, we previously reported that Hrs is also involved in ubiquitin-independent endosomal sorting of interleukin-2 receptor β (IL-2Rβ). Here, we show direct interactions between bacterially expressed Hrs and interleukin-4 re...

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

    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.

  11. Animal Intuitions.

    Kaebnick, Gregory E


    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

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

    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)


    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.

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

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


    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.

  14. Optimising methods for the preservation, capture and identification of ubiquitin chains and ubiquitylated proteins by immunoblotting.

    Emmerich, Christoph H; Cohen, Philip


    Immunoblotting is a powerful technique for the semi-quantitative analysis of ubiquitylation events, and remains the most commonly used method to study this process due to its high specificity, speed, sensitivity and relatively low cost. However, the ubiquitylation of proteins is complex and, when the analysis is performed in an inappropriate manner, it can lead to the misinterpretation of results and to erroneous conclusions being reached. Here we discuss the advantages and disadvantages of the methods currently in use to analyse ubiquitin chains and protein ubiquitylation, and describe the procedures that we have found to be most useful for optimising the quality and reliability of the data that we have generated. We also highlight commonly encountered problems and the pitfalls inherent in some of these methods. Finally, we introduce a set of recommendations to help researchers obtain high quality data, especially those new to the field of ubiquitin signalling. The specific topics addressed in this article include sample preparation, the separation, detection and identification of particular ubiquitin chains by immunoblotting, and the analysis of ubiquitin chain topology through the combined use of ubiquitin-binding proteins and ubiquitin linkage-specific deubiquitylases. PMID:26325464

  15. Cullin 3 Ubiquitin Ligases in Cancer Biology: Functions and Therapeutic Implications

    Chen, Hsin-Yi; Chen, Ruey-Hwa


    Cullin-RING ubiquitin ligases are the largest E3 ligase family in eukaryotes and are multiprotein complexes. In these complexes, the Cullin protein serves as a scaffold to connect two functional modules of the ligases, the catalytic subunit and substrate-binding subunit. To date, eight members of the Cullin family proteins have been identified. In the Cul3 ubiquitin ligases, Bric-a-brac/Tramtrack/Broad complex (BTB) domain-containing proteins function as a bridge to connect Cul3 and substrates. While the BTB domain is responsible for Cul3 binding, these proteins usually contain an additional domain for substrate interaction, such as MATH, kelch, Zn finger, and PAM, Highwire, and RPM-1 (PHR domain). With the existence of a large number of BTB proteins in human, the Cul3 ubiquitin ligases ubiquitinate a wide range of substrates involving in diverse cellular functions. In this review, we will discuss recent advances on the functions of Cul3 ubiquitin ligases in cancer development, progression, and therapeutic response and the dysregulation of Cul3-mediated ubiquitination events in human malignancies. In particular, we will focus on three Cul3 substrate adaptors, kelch-like ECH-associated protein (Keap1), kelch-like family member 20 (KLHL20), and speckle type BTB/POZ protein (SPOP), with the intent to highlight novel targets in cancer therapy. PMID:27200299

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

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


    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

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

    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.

  18. Ubiquitin-specific protease 24 negatively regulates abscisic acid signalling in Arabidopsis thaliana.

    Zhao, Jinfeng; Zhou, Huapeng; Zhang, Ming; Gao, Yanan; Li, Long; Gao, Ying; Li, Ming; Yang, Yuhong; Guo, Yan; Li, Xueyong


    Abscisic acid (ABA) is an important plant hormone integrating environmental stress and plant growth. Protein ubiquitination and deubiquitination are reversible processes catalysed by E3 ubiquitin ligase and deubiquitinating enzyme, respectively. Lots of E3 ubiquitin ligase and transcriptional factors modified by ubiquitination were reported to modulate ABA signalling. However, no deubiquitinating enzyme has been identified that functions in ABA signalling until now. Here, we isolated an ABA overly sensitive mutant, ubp24, in which the gene encoding ubiquitin-specific protease 24 (UBP24, At4g30890) was disrupted by a T-DNA insertion. The ubp24 mutant was hypersensitive to ABA and salt stress in both post-germinative growth and seedling growth. However, stomata closure in the ubp24 mutant was less sensitive to ABA, and the ubp24 mutant showed drought sensitivity. UBP24 possessed deubiquitinating enzyme activity, and the activity was essential for UBP24 function. Additionally, UBP24 formed homodimer in vivo. UBP24 was genetically upstream of ABI2, and the phosphatase activity of protein phosphatase 2C was decreased in the ubp24 mutant compared with the wild type in the presence of ABA. These results uncover an important regulatory role for the ubiquitin-specific protease in response to ABA and salt stress in plant. PMID:26290265

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

    James J. Furniss


    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.

  20. Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes

    Grimsey, Neil J.; Aguilar, Berenice; Smith, Thomas H.; Le, Phillip; Soohoo, Amanda L.; Puthenveedu, Manojkumar A.; Nizet, Victor


    Protease-activated receptor 1 (PAR1) is a G protein–coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-β–activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4-2–mediated ubiquitination and TAB1–TAB2. TAB1–TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption. PMID:26391660

  1. Bioethical Problems: Animal Welfare, Animal Rights.

    March, B. E.


    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)

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

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


    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

  3. Ubiquitin-dependent regulation of Foxp3 and Treg function.

    Barbi, Joseph; Pardoll, Drew M; Pan, Fan


    Regulatory T (Treg) cells are crucial enforcers of immune homeostasis. Their characteristic suppressive function largely arises from an equally unique pattern of gene expression. A complex network of factors and processes contribute to this 'signature' Treg gene expression landscape. Many of these alter the level and activity of the Treg-defining transcription factor Foxp3. As stable expression of Foxp3 is important for the ability of Treg cells to successfully prevent excessive or inappropriate immune activation, uncovering the mechanisms regulating Foxp3 level is required for the understanding and therapeutic exploitation of Tregs. While transcriptional regulation of the Foxp3 gene has been studied in depth, additional regulatory layers exist controlling the expression and activity of this key transcription factor. These include less-defined mechanisms active at the post-translational level. These pathways are just beginning to be elucidated. Here, we summarize emerging evidence for distinct, post-translationally active, ubiquitin-dependent pathways capable of controlling the activation and expression of Foxp3 and the function of Tregs. These pathways offer untapped opportunities for therapeutic fine-tuning of Tregs and their all-important restraint of the immune system. PMID:26085205

  4. Role of the ubiquitin proteasome system in renal cell carcinoma

    Corn Paul G


    Full Text Available Abstract Renal cell carcinoma (RCC accounts for approximately 2.6% of all cancers in the United States. While early stage disease is curable by surgery, the median survival of metastatic disease is only 13 months. In the last decade, there has been considerable progress in understanding the genetics of RCC. The VHL tumor suppressor gene is inactivated in the majority of RCC cases. The VHL protein (pVHL acts as an E3 ligase that targets HIF-1, the hypoxia inducible transcription factor, for degradation by the ubiquitin proteasome system (UPS. In RCC cases with mutant pVHL, HIF-1 is stabilized and aberrantly expressed in normoxia, leading to the activation of pro-survival genes such as vascular endothelial growth factor (VEGF. This review will focus on the defect in the UPS that underlies RCC and describe the development of novel therapies that target the UPS. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb;

  5. Animated nature

    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

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

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


    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

  7. Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

    Solomon, V.; Baracos, V.; Sarraf, P.; Goldberg, A. L.


    The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin-proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3alpha, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3alpha-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

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

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


    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. PMID:26790995

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

    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.

  10. Stabilisation of an E3 ligase-E2-Ubiquitin complex increases cell surface MHC Class I expression

    Duncan, Lidia M; Nathan, James A.; Lehner, Paul J.


    The Kaposi’s sarcoma-associated herpesvirus (KSHV) encoded ubiquitin E3 ligase, K3 ubiquitinates cell surface MHC class I molecules (MHC I), causing the internalisation and degradation of MHC I via the endolysosomal pathway. K3 recruits the cellular E2 ubiquitin conjugating enzyme, Ubc13 to generate lysine-63 linked polyubiquitin chains on MHC I leading to the clathrin-mediated endocytosis and lysosomal degradation of MHC I. Here we identify a ubiquitin Ile-44-Ala mutant (I44A) which inhibits...